THECARDIOVASCULAR

SYSTEMPHYSIOLOGY,
DIAGNOSTICSAND
CLINICALIMPLICATIONS

EditedbyDavidC.Gaze

The Cardiovascular System Physiology, Diagnostics and Clinical Implications

Edited by David C. Gaze


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First published April, 2012
Printed in Croatia

A free online edition of this book is available at www.intechopen.com
Additional hard copies can be obtained from [email protected]

The Cardiovascular System Physiology, Diagnostics and Clinical Implications,
Edited by David C. Gaze
p. cm.
ISBN 978-953-51-0534-3

Contents

Preface IX
Section 1 Cardiovascular Physiology 1
Chapter 1 Control of Cardiovascular System 3
Mikhail Rudenko, Olga Voronova,
Vladimir Zernov, Konstantin Mamberger,
Dmitry Makedonsky, Sergey Rudenko
and Sergey Kolmakov
Chapter 2 Molecular Control of Smooth Muscle Cell
Differentiation Marker Genes by Serum Response
Factor and Its Interacting Proteins 23
Tadashi Yoshida
Chapter 3 Trans Fatty Acids and Human Health 43
Sebastjan Filip and Rajko Vidrih
Chapter 4 Control and Coordination
of Vasomotor Tone in the Microcirculation 65
Mauricio A. Lillo, Francisco R. Prez,
Mariela Puebla, Pablo S. Gaete
and Xavier F. Figueroa
Chapter 5 Hemodynamics 95
Ali Nasimi
Chapter 6 Adenosinergic System in the Mesenteric Vessels 111
Ana Leito-Rocha, Joana Beatriz Sousa
and Carmen Diniz
Chapter 7 Endothelial Nitric Oxide Synthase,
Nitric Oxide and Metabolic Disturbances
in the Vascular System 135
Grayna Lutosawska
VI Contents

Section 2 Cardiovascular Diagnostics 155
Chapter 8 The Diagnostic Performance of Cardiovascular System
and Evaluation of Hemodynamic Parameters Based
on Heart Cycle Phase Analysis 157
Mikhail Rudenko, Olga Voronova, Vladimir Zernov,
Konstantin Mamberger, Dmitry Makedonsky,
Sergey Rudenko, Yuri Fedossov, Alexander Duyzhikov,
Anatoly Orlov and Sergey Sobin
Chapter 9 Biophysical Phenomena in Blood Flow System in
the Process of Indirect Arterial Pressure Measurement 179
Mikhail Rudenko, Olga Voronova and Vladimir Zernov
Chapter 10 Interrelation Between the Changes
of Phase Functions of Cardiac Muscle Contraction
and Biochemical Processes as an Algorithm for
Identifying Local Pathologies in Cardiovascular System 195
Yury Fedosov, Stanislav Zhigalov, Mikhail Rudenko,
Vladimir Zernov and Olga Voronova
Chapter 11 Application of Computational Intelligence
Techniques for Cardiovascular Diagnostics 211
C. Nataraj, A. Jalali and P. Ghorbanian
Chapter 12 Analysis of Time Course Changes in the Cardiovascular
Response to Head-Up Tilt in Fighter Pilots 241
David G. Newman and Robin Callister
Section 3 Clinical Impact of Cardiovascular
Physiology and Pathophysiology 255
Chapter 13 Physical Activity and Cardiovascular Health 257
Raul A. Martins
Chapter 14 Cardiovascular Disease Risk Factors 279
Reza Amani and Nasrin Sharifi
Chapter 15 Cardiovascular and Cerebrovascular Problems
in the Development of Cognitive Impairment:
For Medical Professionals Involved
in the Treatment of Atherosclerosis 311
Michihiro Suwa
Chapter 16 French Paradox, Polyphenols and Cardiovascular Protection:
The Oestrogenic Receptor- Implication 319
Tassadit Benaissa, Thierry Ragot and Angela Tesse
Contents VII

Chapter 17 Importance of Dermatology in Infective Endocarditis 345
Servy Amandine, Jones Meriem and Valeyrie-Allanore Laurence
Chapter 18 Cardiovascular Risk Factors: Implications in Diabetes,
Other Disease States and Herbal Drugs 365
Steve Ogbonnia
Chapter 19 Morphology and Functional Changes
of Intestine, Trophology Status and Systemic
Inflammation in Patients with Chronic Heart Failure 383
G.P. Arutyunov and N.A. Bylova
Chapter 20 Evaluation and Treatment
of Hypotension in Premature Infants 419
Shoichi Ezaki and Masanori Tamura
Chapter 21 Role of Echocardiography in Research into Neglected
Cardiovascular Diseases in Sub-Saharan Africa 445
Ana Olga Mocumbi
Chapter 22 Psychophysiological Cardiovascular
Functioning in Hostile Defensive Women 465
Francisco Palmero and Cristina Guerrero

Preface

The cardiovascular system includes the heart located centrally in the thorax and the
vessels of the body which carry blood. The cardiovascular (or circulatory) system
supplies oxygen from inspired air, via the lungs to the tissues around the body. It is
also responsible for the removal of the waste product, carbon dioxide via air expired
from the lungs. The cardiovascular system also transports nutrients such as
electrolytes, amino acids, enzymes, hormones which are integral to cellular
respiration,metabolismandimmunity.
This book is not meant to be an all encompassing text on cardiovascular physiology
and pathology rather a selection of chapters from experts in the field who describe
recent advances in basic and clinical sciences. As such, the text is divided into three
mainsections:
1. Cardiovascular Physiology In this section, the control of the cardiovascular
system is discussed in particular the heaemodynamic mechanisms controlling
blood volume, flow and the regulation of systolic blood pressure. The next
chapter investigates the molecular control of smooth muscle cell (SMC)
differentiation marker genes by serum response factor (SRF) including the
interaction of myocardin as a potent cofactor of SRF in SMC differentiation. The
chapteralsodetailstheinteractionofGATA6,Klf4,LIMonlyproteinsCRP1and
2 and PIAS1 with SRF. The following chapter reports on trans fatty acids (TFA)
and human health, detailing the biochemistry of trans fats as well as
recommendeddailyintake.Thechapterdescribesbothanimalandhumanstudies
of TFA. There are details on the analytical determination of TFA as well as their
potential antioxidants. There is also a comprehensive overview of TFA and
legislative control in food production and consumption. This is followed by a
chapteronthecontrolandcoordinationofvasomotortoneinthemicrocirculation;
concentrating on the cellular membrane potential and potassium channels, the
role of prostaglandins, nitric oxide and endotheliumderived hyperpolarizing
factor as paracrine signalling in the wall of the vessel. There is also detail of the
roleofgapjunctionsinvascularsmoothmuscleandendotheliumcommunication
processes. The following chapter discusses the concept of hemodynamics,
detailing the relationship between physical factors and the effect on blood flow
through the vessel in laminar or turbulent flow patterns. The principles of
X Preface

velocity, elasticity and compliance are described. Furthermore the clinical
implications such as alteration to blood flow during atherosclerosis and
arteriosclerosis are described. The penultimate chapter of this section describes
the adenosinergic system in the mesenteric vessels which form the splanchnic
circulation.Thechapterdetailstheroleofadenosinefromitsproductiontotissue
concentration controlled by nucleoside transporter membrane proteins, namely
equilibrative and concentrative nucleoside transporters. The family member
subtypes are of these transporter proteins are described thoroughly. The final
chapter of section one concentrates on endothelial nitric oxide synthase (eNOS),
nitric oxide (NO) and subsequent metabolic disturbances within the vascular
system.Anoverviewofvasculardysfunctionisgivenalongwiththebiochemistry
of eNOS/NO. The endogenous eNOS and NO inhibitor asymmetric
dimethylarginineanditsroleinthevascularsystemisalsoreviewed.Thereader
is also given the importance of lifestyle on the vascular system, concentrating on
dietaryhabitsandphysicalactivityontheeNOS/NOsystem.
2. Cardiovascular Diagnostics Section 2 is concerned with modalities used in the
diagnosis and monitoring of parameters associated with the cardiovascular
system. The first chapter entitled the diagnostic performance of cardiovascular
system and evaluation of hemodynamic parameters based on heart cycle phase
analysis describes the development and use of the electrocardiogram (ECG) and
the rheogram. Furthermore the use of both the ECG and rheogram to assess
cardiovascular function in normal and diseased states are described. The second
chapter describes the biophysical phenomena of blood flow during indirect
arterialpressuremeasurement.Theroleoftheoscillograminmeasuringsystolic
and diastolic arterial pressure is well described compared to the practice of
auscultation of Korotkov sounds. The chapter also notes the peculiarities seen in
some oscillogram readings. The third chapter describes the interrelation between
changes of phase function of cardiac muscle concentration and the biochemical
processes as an algorithm for identifying pathological processes within the
cardiovascularsystem.Inthischaptertheauthorsoutlinetheirvisionofthemain
biochemical processes determining the clinical meaning of the pathology
diagnosed with the aid of the cardiac cycle analysis method. Selection of the
therapeuticagentsaimedatnormalizationofthediagnosedfunctionaldeviations
takingintoaccountthebiochemicalprocessesunderlyingthesefunctionsresulted
in the recovery of the functions. The next chapter investigates computational
intelligence techniques in cardiovascular diagnostics. Continual monitoring of
cardiac function in the acute care setting can allow the detection of cardiac
arrhythmias.Continuouswavelettransformandprincipalcomponentanalysisare
described in detail. The application of these techniques within a multi-layer
perceptron neural network is demonstrated. The final chapter of this section analysesthe
time course changes in the cardiovascular response to headup tilt in fighter
pilots. In this interesting chapter the authors describe the physiological
adaptations that occur following frequent exposure to Gforce acceleration. By
Preface XI

measuring mean arterial pressure, heart rate, stroke volume and total peripheral
resistance, the authors compare the cardiovascular responses in fighter pilots
comparedtononpilots.
3. Clinical Impact of Cardiovascular Physiology and Pathophysiology The final
section of this textbook relates physiology to pathophysiology, clinical
presentationandimplicationsofcardiovasculardiseases.Thefirstchapterofthis
section explores the relationship of cardiovascular health and exercise from both
the European and North American perspectives, detailing the relationship
betweenphysicalactivityandlifeexpectancyanddiscussestheproinflammatory
stateinrelationtoreducedphysicalactivityanditsrelationshiptocardiovascular
disease. The second chapter reviews the global burden of cardiovascular disease
and the associated risk factors, including lipid components, inflammatory
markers,fibrinogen,smokinganddietarymodificationtoreducetheincidenceof
cardiac disease. The next chapter details the associations between cardiac and
cerebral vascular issues in patients with neurodegenerative diseases such as
Alzheimers disease. Risk factors such as hyperlipidemia, hypertension, diabetes
and a history of smoking contribute to deterioration of cognitive function. A
reduction of cerebral perfusion following ventricular dysfunction can also
contributetotheadvancementofcognitivedecline.TheFrenchParadoxofalow
incidence of cardiovascular disease in people who consumed moderate red wine
irrespective of the quantity of saturated fatty acids and describes the
cardioprotectiveroleofpolyphenoliccompoundsisdiscussedinthenextchapter.
Thefifthchapterinsection3discussestheclinicalimplicationsofdermatological
findingsinpatientswhodevelopinfectiveendocarditis,inparticularthecausative
microorganisms, risk factors, the clinical signs and symptoms, and the clinical
tools to aid diagnosis. The next chapter details the cardiovascular risk factors
associatedwiththedevelopmentofdiabetesmellitusandtheroleofherbaldrugs
to control cardiac risk factors. The next chapter reviews the morphology and
functional changes of the intestine in patients with heart failure identifying the
systemic nature of heart failure. A comprehensive overview of the histological
patterns observed and the pro inflammatory state of the gastrointestinal tract is
presented. Chapter 20 describes the evaluation and treatment of hypotensive
prematureinfantswhichisacommonphenomenoninthefirstfewweeksoflife;
describing the interplay between hypovolaemia, tissue hypoxia and myocardial
dysfunction. The clinical presentation is described along with diagnostic
modalities used to detect hypotensive cardiac problems, followed by the
treatment regimens available to correct to the normotensive state. The next
interestingchapterdiscussestheroleofechocardiographyinSubSaharanAfrica.
Access to echocardiography is common place in the developed world. In the
remoteness of Africa, access to such diagnostic tests are rarely available due to
cost, logistical access and the lack of trained sonographers. This chapter reviews
the current usage of echocardiography to describe the epidemiology of
cardiovasculardiseaseinanotherwiseneglectedpopulation.Thefinalchapterto
XII Preface

this section and the whole book describes a study of cardiovascular functional
parameters such as heart rate and blood pressure along with a
psychophysiological assessment in females displaying defensive hostility
demonstrating such women have higher heart rates and blood pressures if they
weredefensivecomparedtothosewithlowhostility.
DavidC.Gaze
Dept of Chemical Pathology Clinical Blood Sciences,
St Georges Healthcare NHS Trust, London
United Kingdom
Acknowledgements
I would like to acknowledge the tremendous efforts of the contributing authors to
these chapters, especially when writing in English rather than their native tongue. I
would also like to thank Ms Iva Simcic of InTECH publishers for keeping the
productionofthisbookactiveandtoforsteeringmetocompletetheeditorialreview
bytheappropriatedeadlines.

Section 1
Cardiovascular Physiology

1
Control of Cardiovascular System
Mikhail Rudenko, Olga Voronova, Vladimir Zernov,
Konstantin Mamberger, Dmitry Makedonsky,
Sergey Rudenko and Sergey Kolmakov
Russian New University,
Russia
1. Introduction
The main method of cognition of the performance of biological systems is their
mathematical modeling. The essence of this method should reflect the principle of
optimization in biology[9]. Any biosystem cannot function if its energy consumption is
inadequately high.
The same is applicable to the blood circulatory system. Its main function is to transport
blood throughout the body in order to maintain the proper gaseous exchange, deliver
important substances to viscera and tissues in living body and remove decay products. It is
impossible to study this function without due consideration of hemodynamic features. But
how is the blood circulation provided? It is a question of principle, and so far no
unambiguous answer has been given thereto.
The conventional interpretation of blood circulation is that blood flows through blood
vessels under laminar flow conditions to which Poiseuille's law is applicable. But it is a
matter of fact that this conventional interpretation concept is inadequate because it is not in
compliance with the above principle of optimization in biology, according to which all
processes in bio systems show their best performance, i.e., their highest efficiency. It is just
the compliance with this principle that is the major criterion to be used for evaluation of
adequacy of any theoretical models describing various systems in living body and their
interactions both with each other and their external environment.
Significant progress in understanding of such phenomena is made after G. Poyedintsev and
O.Voronova discovered the so called mode of elevated fluidity, i.e., the third flow conditions
that show lesser losses of energy to overcome friction and that is noted for lesser friction
losses and specific pattern of the flow[4].
It has been proved that the blood flow through the blood vessels is provided in the third
flow mode that is the most efficient and therefore fully in compliance with the said principle
of optimization.
The theory of the third mode is a foundation for the development of new mathematical
models describing the performance of the blood circulation system. In addition, new
methods of quantitative determination of a number of hemodynamic parameters and

The Cardiovascular System Physiology, Diagnostics and Clinical Implications

4
qualitative evaluation of some processes occurring in the system have been elaborated. The
application of these methods in practice allows filling a lot of gaps in theoretical cardiology
and creates at the same time a system of analysis of the functions of the cardiovascular
system taking into account the relevant cause-effect relationship.
The detailed description of this theory is given in our book Theoretical Principles of Heart
Cycle Phase Analysis[3]. Our intention is to outline herein the general principles of the
performance of the cardiovascular system only.

2. Biophysical processes of formation of hemodynamic mechanism
2.1 Special features of hemodynamics and its regulation. Hemodynamic volumetric
parameters
There two types of liquid flow conditions described in the classical fluid mechanics: the first
type is the laminar flow, and the second one is the turbulent flow mode. In the 80
th
last
century, a new theory of a specific liquid flow mode was developed by G.M. Poyedinstev
and O.K. Voronova that was defined by them as the elevated fluidity mode[4]. Another
name the third flow mode was given by the above discoverers to differ it from the two
other modes well-known before. Being experts in solving technical problems of fluid
mechanics, the authors succeeded in modeling the above elevated fluidity mode in a rigid
pipe. For this purpose, hydraulic pulsators of specific design were used. It was established
that the energy used to transport liquid in the third flow mode is several times less than it is
the case under the laminar flow conditions[3]. Moreover, an efficiency of this process could
be considerably increased when liquid is pumped under certain conditions through an
elastic piping. The subsequent researches demonstrated that the physical processes
producing the elevated fluidity mode and those in the blood circulation are identical. The
mathematical tools used to describe the third flow mode was applied to describe the
hemodynamic processes.
It was established by the authors that there are processes which are always observed in a
rigid pipe at the initiation of a liquid flow from a quiescent state, as mentioned below.
Whilst particles of liquid are starting their moving in the rigid pipe due to a difference in the
static pressure, there a set of concentric waves of friction in the boundary layer is generating,
the front of propagation of which is directed towards the pipe axis[3] (Fig. 1). Amplitudes of
these waves depend on the diameter of the pipe, acoustic velocity in liquid and an initial
difference in pressures at the pipe ends. The length of these traveling waves during this
complex process continuously increases. The waves travel towards the axis of the pipe and
degenerate. Finally, there a single wave remains only close to the pipe wall, the profile of
which becomes parabolic that is typical for the laminar flow (s. Fig. 2 herein).
It should be noted that it is just within this short period of time, i.e., starting from the
moment of the motion initiation from a quiescent state till the moment of formation of the
laminar flow (s. positions E and F in Fig. 2 herein), when liquid flows in its optimum mode
of elevated fluidity, considering it from the point of view of energy consumption (s.
positions A, B, C, D in Fig 2 herein). The energy consumption under the laminar flow
conditions to transport liquid in the pipe is significantly higher due to increase in the flow
resistance.

Control of Cardiovascular System

5

Fig. 1. Formation of concentric waves of friction at initiation of flow in a pipe (according to
G.M. Poyedintsev and O.K.Voronova); t
1
- moment of pressure difference formation; V
1
-
velocity of plasma in stagnated layers; V
2
velocity of blood elements in accelerated layers
There is another phenomenon typical for the third flow mode. If liquid contains
suspended particles similar to those in blood, during the development of the above
mentioned wave process the particles are concentrated at the wave maxima, and the
particle-free liquid is delivered to their minima, correspondingly[3]. When the liquid,
patterned in such a way, flows along the pipe axis, the velocity of the concentric particle-
loaded layers is twice what the liquid pattern-free layers reach. Vectors of velocity are
parallel to the axis of the flow. And it is just a prerequisite to elevated fluidity of liquid with
reduced friction between the liquid layers and the pipe wall. Figure 2 herein shows the
locations of erythrocytes in the blood flow referring to each flow formation stage as
mentioned above. At the beginning of the formation of the third flow mode, there ring-
shaped alternating layers of the blood elements and plasma are available, while in the
laminar mode all elements are accumulated in the center of the flow. In this case they are
located very close to each other forming a thick mass. This process may result in an
aggregation of erythrocytes and hemolysis. In order to avoid such pathological
consequences, it is a must to manage the blood transportation in the third mode of flow,
avoiding its transformation into a laminar one.
The theory gives a clue that it can be obtained when transporting liquid in a pulsating mode
through an elastic pipe. According to this theory, the pipe clear width and the liquid flow
velocity should be changed with every impulse under certain laws[3]. The laws of
increasing in the pipe clear width and decreasing in the flow velocity with every impulse
take the form as follows[4].

The Cardiovascular System Physiology, Diagnostics and Clinical Implications

6

Fig. 2. Formation of two-phase pattern at the initiation of the flow from a quiescent state
(according to G.M. Poyedintsev and O.K. Voronova), A-F flow structure in corresponding
sections

5 1
0
0

=
t
t
r r
t

(1)

5 2
0
0

=
t
t
W W
t

(2)
where r
t
current radius of the pipe increasing;
r
0
initial radius (at t = t
0
);
t - current time (t t
0
);
t
0
time of acceleration of flow velocity up to maximum velocity in an impulse;
W
t
current value of liquid flow velocity;
W
0
maximum value of velocity in an impulse (at t = t
0
).
It is proved by the authors of this theory that the above conditions are met in the blood
circulation system.
This is provided by changing in the clear width of blood vessels in every cardiac cycle and
arterial pressure pulsating. The shape of the arterial pressure wave is given herein in Fig. 3
below.

Control of Cardiovascular System

7

Fig. 3. Arterial pressure wave shape reography-recorded. ECG recorded simultaneously
with Rheogram.
The foundation of hemodynamics is the phase mode of the heart performance. In one beat
the heart changes its shape ten times that corresponds to the heart cycle phases[4].
The most efficient way is to evaluate the status of hemodynamics not only by values of
integral parameters, i.e., stroke and minute volumes, but also phase-related volumes of
blood entering or leaving the heart in the respective phase in a cardiac cycle.
So, the final formulae for calculation the volumes of blood in the phase of rapid and slow
ejection, symbolized as PV3 and PV4, respectively, are as follows:
PV3=S (QR+RS)
2
f
1
( ) [f
2
( )+f
3
( , , , )] (ml); (3)
PV4=S (QR+RS)
2
f
1
( ) f
4
( , , , ) (ml), (4)
where S - cross-section of ascending aorta;
QR phase duration according to ECG curve;
RS phase duration according to ECG curve;
f
1
( )=
243 ) 2 5 (
] 27 ) 2 5 [( 5 , 22072
5
3

;
f
2
()= ;
2
1
5


f3
(

, , , )= )]; ( 2 ) ( 5 ) )( 4 (
3
10
[
8
1
5 5 2 4 4 3 3 2 2
+

The Cardiovascular System Physiology, Diagnostics and Clinical Implications

8
f
4
( , , , )= )]; ( 3 ) ( 5 , 7 ) )(
3
8
( 5 [
8
1
5 5 2 4 4 3 3 2 2
+ +
; ) 1 (
2 , 0
RS QR
Em
+
+ =

; ) 1 (
2 , 0
RS QR
Er Em
+
+
+ =

;
) 1 ( 2

=

) 2 ( + = .
Stroke volume SV is calculated by an equation as given below:
SV = PV3+ PV4=S (QR+RS)
2
f
1
( ) [f
2
( )+f
3
( , , , )+f
4
( , , , )] (ml) (5)
The minute stroke is computed as follows:
V = SV HR (l/min) (6)
In similar way calculated are other phase-related volumes of blood as listed below:
PV1 volume of blood entering the ventricle in premature diastole;
PV2 volume of blood entering the ventricle in atrial systole;
PV5 volume of blood pumped by ascending aorta as peristaltic pump.
So, the main parameters in hemodynamics are 7 volumes of blood entering or leaving the
heart in different heart cycle phases. They are as follows: stroke volume SV, minute volume
MV, two diastolic phase-related volumes PV1 and PV2, two systolic phase-related volumes
PV3 and PV4, and PV5 as volume of blood pumped by the aorta.
The authors of this theory in their researches utilized relative phase volumes denoted by
RV. Each relative phase volume is that expressed as a percentage of stroke volume SV.
These relative parameters demonstrate contributions of each phase process to the formation
of the stroke volume in general.
The above hemodynamic parameters should be used mainly in order to evaluate eventual
deviations from their normal values, if any. The limits of normal values of hemodynamic
parameters are not conditional, and they have their respective calculated values.
With respect to the normal values (the required parameters) in hemodynamics, they have
been taken on the basis of the known data on ECG waves, intervals and segments for adults
from the literature sources as given below:
1. The upper and lower limit of the QRS complex values:

Control of Cardiovascular System

9
QRS
max
= 0.1 s. ; QRS
min
= 0.08 s.
2. The upper and lower limit of the RS complex values:
RS
max
= 0.05 s. ; RS
min
= 0.035 s.
3. The normal value of interval QT in every specific cardiac cycle is determined from the
Bazett formula as follows:
QT = 0.37 RR
0.5
, s (male); (7)
QT = 0.4 RR
0.5
, s (female) . (8)
4. Normal value PQ
.
is calculated from a formula as indicated below:
PQ
.
= 1 / (10
-6
638,44 HR
2
+ 9,0787) s (9)
This equation has been produced according to the method of approximation of normal
values PQ
.
, as known from the sources, considering their dependence on heart rate (HR).
These values are used as initial values for calculations of an individual range of normal
values of volumetric parameters in hemodynamics considering individual patient cases. In
practice, for a better visualization of the data, it should be recommended to present them
not only numerically but also graphically, as bar charts, as shown in Figure 4 herein. In the
latter case, it is convenient to indicate the deviations from the normal value limits of the
actually calculated values of hemodynamic parameters as percentage.
For example. On Figure 4 a), b), c) the result of hemodynamic parameters PV2 measuring -
volume of blood entering the ventricle in atrial systole- is displayed as follows. Figure 4 a)
in column "Blood volumes" shows the result of measuring 18,31 (ml). The second column "%
of stroke volume" shows the deviation from the norm. It is 0% here. For quick associative
perception of both these values and rapid highlighting of going beyond the bounds of norm
parameter, there exists a dark green field with red light indicator to the right of this number
in the column "indicators of measurement results". On the left and right sides of the dark
green field we see the values of individual range of this hemodynamic parameter, calculated
using equation 7, 8, 9. In this case, it is from 15.26 to 35,13 ml. Measured parameter of 18,31
ml is in the middle of the range, which corresponds to the 0% deviation from the norm. And
the red light indicator that corresponds to this value is on the dark green background. Light
green field - is a bound of "norm - pathology". Sides of this field correspond to excess or
deficiency of more than 30% of norm. More than 30% excess requires special attention to the
patient. As a rule, such patients needs hospital care. Figure 4 b) shows another patients
result, PV2 = 12,85 ml, and this result goes 15.84% beyond patients individual norm 15,26 ...
35.13 ml. In this case red light indicates lack of blood volume, rather than redundancy.
Lower (upper) than 30% value, but lower (upper) than normal value corridor, denotes
further out-patient treatment for this patient. Fig. 4 c) shows a third patient with PV2 = 47,00
ml value, which goes 76.91% beyond his individual norm 10,72 ... 26.13 ml. Red light
indicates the redundancy of blood volume. This patient should be examined by cardiac cycle
phase analysis to identify the root causes of the disease. Its possible to identify these causes
using ECG and RHEO for phase compensation mechanism of the cardio-vascular system
determination.

The Cardiovascular System Physiology, Diagnostics and Clinical Implications

10

) b) c)
Fig. 4. Displayed measured phase-related values and their qualitative representation as bar
charts, with reference to normal values. This figure gives three different measuring cases
The values of phase-related blood volumes are influenced by the mechanism of
compensation existing in the cardiovascular system[6]. This mechanism is responsible for
the maintenance of the hemodynamic parameters within their respective norms. If any
parameter goes far beyond its norm, it means that it is an indication of physiological
problems of the respective phase process. In this case, the function in the next phase
compensates for the changes in the functioning of the problematic phase[6]. It is the just the
case with sportsmen whose cardiovascular system shows the proper performance.
Physical exercise may cause a deficiency in diastolic volumes of blood by more than 500
%.[4] Under the circumstances, the systolic phases undertake to compensate for the above
deficiency. For this purpose, the mechanisms may be involved, the manifestations of which
cannot be found even in a pathology case. Upon stress relieving, 1 minute later, all phase-
related volumes are normalized again. This kind of the performance of the cardiovascular
system hinders an identification of the cause of pathology at early stages for those who are
not professional athletes.
As a rule, deviations due to pathology exceed the norm by more than 30 %. Patients, who
receive their treatment at cardiology hospital, show sometimes deviations of 50 % and over.
The only way to find the primary cause of any pathology, based on the manifestation of the
compensation mechanism, can be a thorough analysis of the actual cause-relationship in
every individual case.
The phase-related volumetric parameters in hemodynamics are the most informative
characteristics of the performance of the cardiovascular system since they are capable of

Control of Cardiovascular System

11
reflecting the coordinated operation of the heart and the associated blood vessels. Knowing
their ratios and considering the actual anatomic and functional status of the heart and the
blood vessels in every phase, we can produce very reliably a diagnosis of the actual status of
the blood circulation system, reveal pathology and control the efficiency of therapy, if
required.
The above mentioned evidence is really of fundamental importance. It should be taken into
account when making diagnosis.
2.2 Mechanism of regulation of systolic pressure
The above mentioned main volumetric parameters should be complemented by another
one: it is arterial pressure (AP). The cardiovascular system has its own mechanism to
provide separate regulation of the systolic and diastolic pressures (AP)[8]. A narrowing in
sectional areas of the blood vessels in total leads to a displacement of a certain volume of
blood that is symbolized by V. The displacement volume enters the ventricles in premature
diastole phase T P. During myocardium contraction phase R S, the same volume is
displaced via the closed aortic valve into the aorta. Actually, before the ejection of stroke
volume SV into aorta, the total of displacement volume V enters the aorta. Therefore, it is
that the R S phase, when V can be ejected into the aorta, is preceded by that phase when
the motion of the entire mass of blood is actuated, and this preceding phase is the Q R
interval, when the contraction of the septum occurs. It is just the phase when the blood flow
becomes its directed vortex motion within the ventricle. Displacement volume V
contributes to moving against the total increased resistance of the blood vessels in the next
phase which shows rapid blood ejection.
The blood circulation scheme is shown in Figure 5 herein. The anatomy of the heart is
designed in such a way so that the displacement blood can penetrate without hindrance
through the closed arteric valve into the aorta. It is determined not only by the configuration
of the valves but also the mechanism of the contraction of the heart chambers that consists of
three phases. Phase one among them is the contraction of the septum. Phase two provides
for the contraction of the ventricle walls. Phase three is the phase of tension. The processes
occurring therein are responsible for spinning the blood flows so that the penetration of the
displacement blood through the closed valves into the aorta is assisted. Under normal
conditions, when there is no displacement volume V available, and, as a consequence, no
penetration is required, upon completion of the phase of tension, stroke volume SV residing
in the heart is supplied into the aorta. In this case, volume SV added to the volume of blood
residing in the aorta creates the systolic pressure that produces a difference in pressures
between the aorta and the periphery. Such mechanism required to overcome an increased
blood flow resistance operates cyclically till the cause of blood vessel constriction
disappears. The processes described above are typical for the mechanism of regulation of
the diastolic arterial pressure. Various Rheogram curve shapes reflect this mechanism.
The anatomy design of the heart is determined by the phase mechanism of hemodynamics,
i.e., the mechanism of the regulation of the diastolic pressure. This mechanism is responsible
for elimination of general vasoconstriction difficulties in blood circulation. Causes of the
said vasoconstriction cannot be diagnostically identified in this case.

The Cardiovascular System Physiology, Diagnostics and Clinical Implications

12

Fig. 5. ) Blood circulation scheme considering changes in blood vessel resistance. b) AP
changes in aorta; c) Changes in AP identifiable on Rheo curve in phase of tension S-L, in
proportion to displacement volume V in blood vessel constriction
With synchronous recording of an ECG and a Rheo from the ascending aorta, provided that
they are synchronized at wave point S on the ECG curve, the process of the regulation of
diastolic pressure may manifest itself as an early AP rise on the respective Rheo curve in
phases R S and S L.
2.3 Mechanism of regulation of systolic pressure
The mechanism of regulation of the systolic pressure differs significantly from that
responsible for the regulation of the diastolic pressure. It has the function to provide a pre-
requisite to the blood circulation in the blood vessels due to a difference in pressures
between the aorta and veins and manage the transportation of an oxygen quantity as
required by tissues and cells. For these purposes, several biophysical processes are engaged.
First and foremost, we should mention the process of myocardium contraction in tension
phase S L. The tension created in this phase presets the velocity of the blood flow during
the blood ejection phase. Therefore, the initial velocity of the blood flow in the aorta
depends on the degree of the myocardium tension.
The second important process is the phenomenon of an increase in the systolic pressure
during the propagation of the AP wave throughout the arteries[1]. The systolic pressure in
the aorta and that in the brachial artery may considerably differ from each other. On the

Control of Cardiovascular System

13
normal conditions, the pressure increase is provided by the pumping function of the blood
vessels and their increasing resistance.
An additional point to emphasize is that there is another biophysical phenomenon
connected with hemodynamics. It is cavitation in blood that promotes blood volume
expansion[2]. It may spread over very quickly within one heart cycle and is capable of
considerably expanding the blood volume.
The cause of the systolic pressure buildup is a reduction in blood supply of some viscera. The
pressure buildup is aimed at elimination of hindrances in blood supply in order to maintain
the proper blood circulation. The blood supply mechanism of some viscera provides for
protection from arterial overpressures. In the first place, the protection of the cerebral blood
supply system should be mentioned. The cerebral blood vessels are anatomically connected
with veins. During an increase in AP, the venous drainage is hindered, affecting the blood
vessel constriction and limiting in such a way an excessive AP increase.
If for some reason a viscus is not sufficiently supplied with blood, it leads to a systolic AP
growth. The venous drainage will be hindered. The first symptoms of this problem could be
edema of legs. To solve this problem, required should be elimination of the cause of the
improper blood supply to the affected viscus that should decrease the AP and,
subsequently, normalize the venous drainage.
3. Phase structure of heart cycle according to ECG curve
Every heart cycle consists of 10 phases. Each phase undertakes its own functions[7].
The complete phase structure of an ECG is shown in Figure 6 herein.
Phase of atrial systole P

;
Phase of closing of atrioventricular valve P

Q;
Phase of contraction of septum Q R;
Phase of contraction of ventricle walls R S;
Phase of tension of myocardium S L;
Phase of rapid ejection L j;
Phase of slow ejection j - T

;
Phase of buildup of maximum systolic pressure in aorta T

- T
,
;


Phase of closing of aortic valve T

- U

;
Phase of premature diastole of ventricles U

- P

.
Each phase serves its purpose. But the phases may be grouped in a manner as follows:
Group of diastol4ic phases which are responsible for blood supply to the ventricles:
Phase of premature diastole of ventricles U

- P

;
Phase of atrial systole P

;
Phase of closing of atrioventricular valve P

Q.
The phase of premature diastole contains a period of time equal to the duration of wave U
which reflects an intensive filling of the coronary vessels with blood. It occurs in
synchronism with filling of the ventricles.
The diastolic phases are described as hemodynamic values PV1 and PV2.

The Cardiovascular System Physiology, Diagnostics and Clinical Implications

14

Fig. 6. Phase structure of ECG recorded from ascending aorta; Phase of atrial systole P

;
Phase of closing of atrioventricular valve P

Q; Phase of contraction of septum Q R;

Phase of contraction of ventricle walls R S; Phase of tension of myocardium S L; Phase of
rapid ejection L j; Phase of slow ejection j - T

; Phase of buildup of maximum systolic
pressure in aorta T

- T
,
; Phase of closing of aortic valve T

- U

; Phase of premature diastole

of ventricles U

- P

Group of systolic phases which provide for the conditions for the proper blood circulation.
They can be divided into subgroups undertaking certain functions as given below:
Subgroup responsible for diastolic AP regulation:
Phase of contraction of septum Q R;
Phase of contraction of ventricle walls R S;
Phase of tension of myocardium S L (partially).
Subgroup responsible for systolic AP regulation:
Phase of tension of myocardium S L,
Phase of rapid ejection L j.
Subgroup responsible for aorta pumping function control:
Phase of slow ejection j - T

;
Phase of buildup of maximum systolic pressure in aorta T

- T
,
;



Control of Cardiovascular System

15
Phase of closing of aortic valve T

- U

;
The given systolic phases are characterized by hemodynamic values PV3, PV4 and SV.
Hemodynamic value MV is an indication of a blood flow rate.
Hemodynamic parameter PV5 shows what share of blood is pumped by the aorta operating
as a peristaltic pump during the ejection of blood from the ventricles.
It should be noted that phase of slow ejection j - T

is a time when the stroke volume of
blood is distributed throughout the large blood vessel, i.e., the time of the aorta expansion.
As our investigations demonstrate, in case of improper elasticity of the aorta this period of
time is prolonged.
4. Phase structure of heart cycle on RHEO curve
An electrocardiogram reflects the most important hemodynamic processes. According to an
ECG curve, it is possible to identify an intensity of the contraction of the muscles of the
respective segment in the cardiovascular system by analyzing inflection points in the
respective heart cycle phase and considering the respective phase amplitudes. However, it is
required to understand how the flow of blood changes. For this purpose, rheography should
be used. A rheogram shows changes in the arterial pressure. An ECG and a RHEO are
produced by using signals of different nature. To record an ECG used is electric potential,
and for RHEOgraphy employed are changes in amplitudes of high-frequency AC under the
influence of changing blood volumes in blood circulation, which produce changes in the
conductivity within the space between the recording electrodes.
There is no AP increase in myocardium tension phase S L. The aortic valve opens at the
moment denoted as L. The slope ratio of RHEO in phase of rapid ejection L j is descriptive
of the velocity of stroke volume travel, and, finally, decisive in governing the systolic AP.
5. Criteria for recording phases on ECG, Rheo and their derivatives
When considering an ECG as a complex signal, it should be pointed out that it consists of a
number of single-period in-series sinusoidal signals connected. It is referred to a re-
distribution of energy in bio systems in a not a stepwise, but sinusoidal way, showing half-
periods as follows: energy increase, retardation, attenuation and development. Transition
points of these processes should be at the same time the points of inflection of energy
functions which are shown by the first derivative at their extrema. Similar processes occur in
the cardiovascular system control. Figure 8 represents a schematic model of an ECG
comprising the said in-series single-period sinusoidal waves.
Should an ECG curve be differentiated, 10 extrema on the derivative can be identified which
correspond to the boundaries of the respective phases of the heart cycle. It should be
mentioned that each phase shall be determined by the same criterion, i.e., by the respective
local extremum on the derivative curve. Since a wavefront steepness varies, the respective
amplitudes of the derivative extrema differ. The ECG phases are equivalent to those of
energy variations responsible for the heart control. For illustration purposes, it is better to
use graphic differentiation.

The Cardiovascular System Physiology, Diagnostics and Clinical Implications

16

Fig. 7. Phase structure of RHEO recorded from ascending aorta

Control of Cardiovascular System

17

Fig. 8. Schematic model of ECG comprising in-series single-period sinusoidal variations
It is just the graphic differentiation that is capable of clearly illustrating all specific points of
such complex signal like an ECG signal. Whereas it is practically impossible to detect
visually on an ECG curve the inflection points, they can be easy identified on the derivative
by local extrema without error. Figure 9 gives an ECG curve and its first derivative. It is
evident that point P on the ECG curve corresponds to point on the derivative that is found
by the respective local extremum. In the same way point T should be identified. It is of great
importance to localize point S. There are no other methods capable of identifying this point.

Fig. 9. Graphic differentiation of ECG curve. Shown are an ECG and its first derivative.
Wave points on the ECG curve are its inflection points that correspond to the local extrema
on the derivative

The Cardiovascular System Physiology, Diagnostics and Clinical Implications

18
It is just the derivative that is capable of recognizing point S very clearly by the respective
local positive extremum. The proposed procedure of identifying the above mentioned key
points makes possible to develop a computer-assisted technology for measuring durations
of every heart cycle phase.
For the same purpose, the second derivative may be used, too, but in this case there is no
need to do it since the informative content of the heart cycle phase identifiable criteria with
utilization of the first derivative is quite sufficient.
Some real ECG curves recorded from the aorta are given in Figure 10 herein. Wave points P,
Q, S and T are marked on the curves which are reliably found according to the first
derivative.
Figure 11 herein illustrates real ECG signals and the first derivative of this ECG. The ECG
shape shown in this Figure is close to an ideal one. It is the matter of fact that in practice we
deal with such ECG curves that significantly differ from the ideal ECG type represented
herein. Therefore, it is the differentiation only that can very reliably identify the boundaries
of every phase in every heart cycle.



point P point Q


point S point T
Fig. 10. Key points P, Q, S and T on ECG curve, characterizing the respective phases of the
heart cycle and corresponding to the respective local extrema on the derivative

Control of Cardiovascular System

19

Fig. 11. Identification of phases on an ECG curve with use of the first derivative graph
6. Functions of cardiovascular system to be evaluated on the basis of heart
cycle phase analysis
The complex of the functions of the cardiovascular system is a combination of the functions
in every individual heart cycle phase. There is a certain logic design available explaining
this. Every phase has its own significance but the basis of all phases is the mechanism of
contraction or relaxation of muscles. Should metabolic disturbance in a muscle occur, its
contraction or relaxation will be diminished. In this case, every next phase will undertake to
compensate for this malfunction by enhancing its activity. The phase analysis gives us a clue
to clearly identifying such imbalances.
In this connection, the following functions of the cardiovascular system should be
mentioned:
Function Regulated parameter
1 Contraction of septum diastolic AP in the aorta
2 Contraction of myocardium; diastolic AP in the aorta
3 Tension of myocardium muscles systolic AP in the aorta
4 Elasticity of aorta Maintain blood flow structure
5 condition of venous flow
6 condition of pulmonary function
7
whether pre-stroke conditions
are available or not

8
problems with coronary blood
flow

Table 1. Main functions and regulated parameters of cardiovascular system

The Cardiovascular System Physiology, Diagnostics and Clinical Implications

20
Figure 12 given below demonstrates the relations between the heart cycle phases on an ECG
& RHEO and the respective functions of the cardiovascular system. Although it seems that
the hemodynamic mechanism as a whole and the performance of the cardiovascular system
are very complicated, the heart cycle phase analysis allows establishing of cause-effect
relationship of any pathology in every individual case within the shortest time. It is very
important that it makes possible to detect the primary cause of a cardiac disease.
Figure 13 displays anatomic segments of the heart and their respective functions in every
heart cycle phase.

Fig. 12. Diagnosable heart segments with their functions and their relations to heart cycle
phases on ECG and RHEO
7. Conclusion
Making progress in research of biophysical processes of the formation of the hemodynamic
mechanism is possible only when theoretical models are tested for their compliance in
practice, i.e., a model to be validated should show in practice its compliance with the
requirements for all simulated functions. The results of many years researches accumulated
by our R & D team made it possible not only to develop an innovative, radically new theory
of the heart cycle phase analysis but also provide metrology for such field of medical science
as cardiology[4]. We have succeeded in solving the problem of indirect measuring
technologies for hemodynamic parameters, including phase-related volumes, by the
mathematical modeling.

Control of Cardiovascular System

21

Fig. 13. Anatomical design of the heart predetermined by the required functions in every
heart cycle
Our clinical studies offer a clearer view of how many difficult issues associated with
biochemical reactions responsible for the stable maintenance of the hemodynamic and the
entire performance of the cardiovascular system can be answered. This is a pre-requisite to
developing and validating of new high-efficient therapy methods.
Hereby the authors would like to express their hope that within the nearest future we shall
deal with a new research field, which is cardiometry. The basis of this science should create
mathematical modeling and instrumentation technology.
8. Acknowledgements
The well-known recipe for success in any work is to create a team of like-minded researches
working for the same cause. If the concept of their work is that the point of life is work, and
if the work results encourage and motivate them, then success is assured. But our life is able
to make its corrections. We regret to say that, one of the authors of our discovery, who
originated the idea of the third mode of flow, died. We speak about Gustav M.
Poyedintsev, a great mathematician and scientist. Our last book Theoretical Principles of Heart
Cycle Phase Analysis published in 2007 was devoted to the memory of him and his work.
The other sad news has been received by us when we were working on this Chapter: Jaana
Koponen-Kolmakova, another member of our R & D team, has departed this life. She was
really an outstanding person! She was the General Manager of the Company
CARDIOCODE-Finland. She remains in our memory for ever.

The Cardiovascular System Physiology, Diagnostics and Clinical Implications

22
During our work we meet a lot of people who dedicate their life to science. We always enjoy
communicating with them. This is a sort of people who deserve our special recognition and
respect.
9. References
[1] Caro, C.; Padley, T.; Shroter, R. & Sid, W. (1981) Blood Circulation Mechanics. Mir. M.
(fig.12.14).
[2] Goncharenko, A. & Goncharenko, S. (2005) Extrasensory Capabilities of Heart. Magazin
Technika Molodyozhi, No. 5, ISSN 0320 331 Rosen, P. (1969) The Principle of
Optimization in Biology. Mir.
[3] . Rudenko, M.; Voronova, O. & Zernov. V. (2009). Theoretical Principles of Heart Cycle
Phase Analysis. Fouqu Literaturverlag. ISBN 978-3-937909-57-8, Frankfurt a/M.
Mnchen London - New York.
[4] Voronova, O. (1995). Development of Models & Algorithms of Automated Transport Function
of The Cardiovascular System. Doctorate Thesis. Prepared by Mrs. O.K. Voronova,
Ph.D., VGTU, Voronezh.
[5] Voronova, O. & Poyedintsev, G. Patent 94031904 (RF). Method of Determination of the
Functional Status of the Left Sections of the Heart & their Associated Large Blood Vessels.
[6] Rudenko, M.; Voronova, O. & Zernov. V. Innovation in cardiology. A new diagnostic
standard establishing criteria of quantitative & qualitative evaluation of main
parameters of the cardiac & cardiovascular system according to ECG and Rheo
based on cardiac cycle phase analysis (for concurrent single-channel recording of
cardiac signals from ascending aorta). (npre.2009.3667.1). Nature Precedings.
Available from: http://precedings.nature.com/documents/3667/version/1/html
[7] Rudenko, M.; Voronova, O. & Zernov. V. (2009) Study of Hemodynamic Parameters
Using Phase Analysis of the Cardiac Cycle. Biomedical Engineering. Springer New
York. ISSN 0006-3398 (Print) 1573-8256 (Online). Volume 43, Number 4 / July, 2009.
. 151 -155.
[8] Rudenko, M.; Voronova, O. & Zernov. V. (2010) Innovation in theoretical cardiology.
Phase mechanism of regulation of diastolic pressure. Arrhythmology Bulletin
(Appendix B) . - P. 133.
[9] Rosen, P. (1969) The Principle of Optimization in Biology. Mir. M.
2
Molecular Control of Smooth Muscle Cell
Differentiation Marker Genes by Serum
Response Factor and Its Interacting Proteins
Tadashi Yoshida
Apheresis and Dialysis Center
School of Medicine, Keio University
Japan
1. Introduction
Vascular smooth muscle cells (SMCs) exhibit a wide range of different phenotypes at
different stages of development (Owens, 1995; Owens et al., 2004; Yoshida & Owens, 2005).
Even in mature animals, SMCs retain the capability to change their phenotype in response to
multiple local environmental cues. The plasticity of SMCs enables them to play a critical role
in physiological processes in the vasculature, as well as the pathogenesis of numerous
vascular diseases including atherosclerosis, re-stenosis after percutaneous coronary
intervention, aortic aneurysm, and hypertension. Thus, it is important to understand the
precise mechanisms whereby SMCs exhibit different phenotypes under distinct conditions.
Because one of the most remarkable differences among SMC subtypes is the difference in
expression levels of SMC-specific/-selective genes, elucidation of the molecular mechanisms
controlling SMC differentiation marker gene expression may shed light on this issue.
Most of SMC differentiation marker genes characterized to date, including smooth muscle
(SM) -actin (Mack & Owens, 1999), SM-myosin heavy chain (SM-MHC) (Madsen et al., 1998),
SM22 (Li et al., 1996), and h1-calponin (Miano et al., 2000), have multiple highly conserved
CC(A/T-rich)
6
GG (CArG) elements in their promoter-enhancer regions. Results of studies in
vivo have shown that expression of these genes is dependent on the presence of CArG
elements (Li et al., 1997; Mack & Owens, 1999; Manabe & Owens, 2001a). For example,
expression of the SM -actin gene requires a promoter-enhancer region from -2.6 kb to +2.8
kb to recapitulate the expression patterns of the endogenous gene, and mutation of any one
of three conserved CArG elements within the regions abolishes the expression (Mack &
Owens, 1999). Likewise, SMC-specific expression of the SM-MHC gene requires 4.2 kb of the
5-flanking region, the entire first exon, and 11.5 kb of the first intronic sequence, and
mutation of CArG elements in the 5-flanking region abolishes the expression (Manabe &
Owens, 2001a). These results indicate the critical roles of CArG elements in the regulation of
SMC differentiation marker gene expression. Currently, it is reported that over 60 of SMC-
specific/-selective genes possess CArG elements in the promoter-enhancer regions by in-
silico analysis (Miano, 2003), although it is not fully determined how many CArG elements
of them are functional.

The Cardiovascular System Physiology, Diagnostics and Clinical Implications

24
The binding factor for CArG elements is the ubiquitously expressed transcription factor,
serum response factor (SRF) (Norman et al., 1988). Knockout of the SRF gene in mice
resulted in early embryonic lethality due to abnormal gastrulation and loss in key
mesodermal markers (Arsenian et al., 1998), precluding the evaluation of requirement of
SRF for SMC differentiation. Instead, conditional knockout of the SRF gene in the heart and
SMCs exhibited the attenuation in cardiac trabeculation and the compact layer expansion, as
well as decreases in SMC-specific/-selective genes including SM -actin in aortic SMCs
(Miano et al., 2004). Moreover, SRF has been shown to be required for differentiation of
SMCs in an in vitro model of coronary SMC differentiation (Landerholm et al., 1999). Indeed,
over-expression of dominant-negative forms of SRF inhibited the induction of SMC
differentiation marker genes including SM22, h1-calponin, and SM -actin in proepicardial
cells excised from quail embryos. As such, the preceding studies provide evidence
indicating that the CArG-SRF complex plays an important role in the regulation of SMC
differentiation marker gene expression. However, SRF was first cloned as a binding factor
for the core sequences of serum response element (SRE) in the c-fos gene (Norman et al.,
1988). Because the c-fos gene is known as one of the growth factor-inducible genes, major
unresolved issues in the field are to identify the mechanisms whereby: (1) the CArG-SRF
complex can simultaneously contribute to two disparate processes: induction of SMC
differentiation marker gene expression versus activation of growth-regulated genes; and (2)
the ubiquitously expressed SRF can contribute to SMC-specific/-selective expression of
target genes.
To date, a number of factors have been reported to interact with SRF. Several recent studies
suggest that these interactions are responsible for multiple actions of SRF. Therefore, this
review article will summarize recent progress in our understanding of the transcriptional
mechanisms involved in controlling expression of SMC differentiation marker genes by
focusing on SRF and its interacting factors.
2. Myocardin is a potent co-factor of SRF for SMC differentiation marker gene
expression
One of the major breakthroughs in the SMC field was the discovery of myocardin (Wang et
al., 2001). Myocardin was cloned as a co-factor of SRF by a bioinformatics-based screen and
found to be exclusively expressed in SMCs and cardiomyocytes (Chen et al., 2002; Du et al.,
2003; Wang et al., 2001; Yoshida et al., 2003). It has two isoforms, and smooth muscle-
enriched isoform consists of 856 amino acids (Creemers et al., 2006). Myocardin has several
domains including three RPEL domains, a basic domain, a glutamine-rich domain, a SAP
(Scaffold attachment factors A and B, Acinus, Protein inhibitor of activated STAT) domain,
and a leucine zipper-like domain. It has been shown that leucine zipper-like domain is
required for homodimerization of myocardin (Figure 1) (Wang et al., 2003), but the function
of the other domains is not well understood. Transcriptional activation domain, TAD, is
localized at the carboxy-terminal region, and deletion mutants that lack TAD behaved as
dominant-negative forms (Wang et al., 2001; Yoshida et al., 2003). Over-expression of
myocardin potently induces transcription of virtually all CArG-dependent SMC
differentiation marker genes, including SM -actin, SM-MHC, SM22, h1-calponin, and
myosin light chain kinase (MLCK) (Chen et al., 2002; Du et al., 2003; Wang et al., 2001; Wang et
al., 2003; Yoshida et al., 2003). Mutation of CArG elements in the SMC promoters abolished
Molecular Control of Smooth Muscle Cell Differentiation
Marker Genes by Serum Response Factor and Its Interacting Proteins

25
the responsiveness to myocardin, suggesting that myocardin activates the transcription in a
CArG-dependent manner. However, myocardin showed no DNA binding activity, but
showed interaction with SRF. In addition, myocardin failed to activate the transcription of
CArG-dependent genes in the absence of SRF (Du et al., 2003), demonstrating that
myocardin is a co-activator of SRF. Over-expression of myocardin also induced the
endogenous expression of SMC differentiation marker genes in cultured SMCs and non-
SMCs, including 3T3 fibroblasts, L6 myoblasts, 3T3-L1 preadipocytes, COS cells, and
undifferentiated embryonic stem cells (Chen et al., 2002; Du et al., 2003; Du et al., 2004;
Wang et al., 2001; Wang et al., 2003; Yoshida et al., 2003; Yoshida et al., 2004b). However,
forced expression of myocardin in non-SMCs was not sufficient to induce the full SMC
differentiation program, because some SMC-enriched genes, which do not contain CArG
elements in their promoter-enhancer region, were not induced (Yoshida et al., 2004b).
Nevertheless, it was sufficient to establish a SMC-like contractile phenotype (Long et al.,
2008). Either dominant-negative forms of myocardin or siRNA-induced suppression of
myocardin decreased the transcription of SMC differentiation marker genes in cultured
SMCs (Du et al., 2003; Wang et al., 2003; Yoshida et al., 2003). In addition, myocardin-
deficient mice exhibited no vascular SMC differentiation and died by embryonic day 10.5 (Li
et al., 2003), although this may have been secondary to the defect in the extra-embryonic
circulation. Moreover, mice lacking the myocardin gene in neural crest-derived cells died
prior to postnatal day 3 from patent ductus arteriosus, and neural crest-derived SMCs in
these mice exhibited a cell-autonomous block in expression of SMC differentiation marker
genes (Huang et al., 2008). Taken together, the preceding results provide compelling
evidence that myocardin plays a key role in the regulation of expression of SMC
differentiation marker genes.

Fig. 1. Myocardin potently induces the transcription of CArG-element containing SMC
differentiation marker genes. Myocardin preferentially activates SMC differentiation marker
genes which contain multiple CArG elements in their promoter-enhancer regions.
Homodimerization of myocardin through the leucine zipper-like domain efficiently
activates the transcription. In contrast, myocardin does not induce the transcription of the
growth factor-inducible gene, c-fos, because it only contains a single CArG element in the
promoter.

The Cardiovascular System Physiology, Diagnostics and Clinical Implications

26
2.1 Transcriptional mechanism for myocardin-dependent SMC differentiation marker
genes
Although myocardin is a powerful transcriptional co-activator of SRF, there are still some
questions for the mechanisms whereby myocardin induces SMC differentiation marker
genes. One of these questions is: what cis-elements and transcriptional co-activators other
than SRF are required for the function of myocardin? Initial studies (Wang et al., 2001)
suggested that myocardin activated the transcription through the formation of complex with
SRF and multiple CArG elements, based on the findings that: (1) the single CArG-containing
c-fos gene had no responsiveness to myocardin; and (2) myocardin could activate an
artificial promoter consisting of 4x c-fos SREs coupled to the basal promoter. Such a 2-
CArG model, in which multiple CArG elements are required for myocardin-induced
transactivation, is strengthened by the results showing that homodimerization of myocardin
extraordinary augmented the transcriptional activity of SMC differentiation marker genes
(Figure 1) (Wang et al., 2003). However, several SMC-specific genes that only contain single
CArG element in their promoter, such as the telokin gene and the cysteine-rich protein-1 (CRP-
1) gene, have also been shown to be activated by myocardin (Wang et al., 2003; Yoshida et
al., 2004b). These results raised a question as to how myocardin distinguishes these single
CArG-containing SMC differentiation marker genes from the c-fos gene. One hypothesis is
that the presence of a ternary complex factor (TCF)-binding site in the c-fos promoter
regulates the binding of myocardin to SRF. In support of this, it has been shown that one of
the TCFs, Elk-1, could compete for SRF binding with myocardin on the SMC promoters
(Wang et al., 2004; Yoshida et al., 2007; Zhou et al., 2005). Such a possibility will be discussed
in detail in a later section.
An additional possibility is that degeneracy within CArG elements, i.e. conserved base pair
substitutions that reduce SRF binding affinity, contributes to the promoter selectivity of
myocardin. Consistent with this idea, the majority of SMC differentiation marker genes
including SM -actin and SM-MHC have degenerate CArG elements in their promoter-
enhancer regions (Miano, 2003). For example, both of CArG elements located within 5-
flanking region of the SM -actin gene contain a single G or C substitution within their A/T-
rich cores that is 100% conserved between species as divergent as humans and chickens
(Shimizu et al., 1995). Results of our previous studies showed that substitution of SM -actin
5 CArGs with the c-fos consensus CArGs significantly attenuated injury-induced
downregulation of SM -actin expression (Hendrix et al., 2005). In addition, of interest, over-
expression of myocardin selectively enhanced SRF binding to degenerate SM -actin CArG
elements compared to c-fos consensus CArG element in SMCs, as determined by
quantitative chromatin immunoprecipitation assays. These results raise a possibility that the
degeneracy in the CArG elements is one of the determinants of promoter selectivity of
myocardin. However, it should be noted that there is a difference not only in the sequence
context of CArG elements, but also in the number of CArG elements between the SM -actin
gene versus the c-fos gene. Moreover, there is no G or C substitution in the CArG elements
of several SMC differentiation marker genes including the SM22, telokin, and CRP-1 genes
(Miano, 2003), although previous studies showed that the binding affinity of SRF to SM22
CArG-near element was lower than that to the c-fos CArG element by electromobility shift
assays (EMSA) (Chang et al., 2001). It is interesting to determine whether CArG elements in
the telokin gene and the CRP-1 genes also exhibit lower binding affinity to SRF than the c-fos
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consensus CArG element. If this is the case, it is likely that reduced SRF binding to CArG
elements, which does not necessarily have G or C substitutions, is one of the mechanisms for
target gene selectivity of myocardin. If this is not the case, it is still possible that the
degeneracy in CArG elements may explain a part of the promoter selectivity of myocardin,
but this mechanism cannot be applicable to all of the SMC differentiation marker genes.
Regarding the mechanism of myocardin-induced transcription of SMC differentiation
marker genes, the physical interaction of myocardin with histone acetyltransferase, p300,
and class II histone deacetylases, HDAC4 and HDAC5, has been reported (Cao et al., 2005).
Indeed, results showed that over-expression of myocardin induced histone H3 acetylation in
the vicinity of CArG elements at the SM -actin and SM22 promoters in 10T1/2 cells (Cao
et al., 2005). In addition, they showed that p300 augmented the stimulatory effect of
myocardin on the transcription of the SM22 gene, whereas either HDAC4 or HDAC5
repressed the effect of myocardin by co-transfection/reporter assays. Moreover, they
demonstrated that p300 and HDACs, respectively, bound to distinct domains of myocardin
simultaneously, suggesting that the balance between p300 and HDACs is likely to be one of
the determinants of the transcriptional activity of myocardin.
These results are of significant interest in that they provided evidence that transcription of
SMC differentiation marker genes is regulated by the recruitment of chromatin modifying
enzymes by myocardin. Previous studies showed that SMC differentiation was associated
with increased binding of SRF and hyperacetylation of histones H3 and H4 at CArG-
containing regions of the SM -actin and SM-MHC genes in A404 SMC precursor cells
(Manabe & Owens, 2001b). In addition, we showed that over-expression of myocardin
selectively enhanced SRF binding to CArG-containing region of the SM -actin gene, but not
to that of the c-fos gene in the context of intact chromatin in SMCs (Hendrix et al., 2005).
Results of studies by another group (Qiu & Li, 2002) also showed that HDACs reduced the
transcriptional activity of the SM22 gene in a CArG-element dependent manner. These
findings are consistent with the results showing the association of myocardin with p300 or
HDACs (Cao et al., 2005). However, it remains unknown how the association between
myocardin and p300 or HDACs regulates the accessibility of SRF to CArG elements, as has
been observed during the induction of SMC differentiation in A404 cells (Manabe & Owens,
2001b). It is possible that particular histone modifications by the myocardin-p300 complex
enable SRF to bind to CArG-elements within the SMC promoters. It is also possible that the
association between myocardin and chromatin modifying enzymes including p300 may
alter the binding affinity of myocardin to SRF. Because regulation of SMC differentiation
marker genes by platelet-derived growth factor-BB (PDGF-BB) or oxidized phospholipids
has been shown to be accompanied by the recruitment of HDACs and thereby changes in
acetylation levels at the SMC promoters (Yoshida et al., 2007, 2008a), it is interesting to
determine if these changes are caused by the modulation of association between myocardin
and these chromatin modifying enzymes.
2.2 Role of the myocardin-related family in SMC differentiation
Two factors were identified as members of the myocardin-related transcription factors:
MKL1 (also referred to as MAL, BSAC, and MRTF-A) (Cen et al., 2003; Miralles et al., 2003;
Sasazuki et al., 2002; Wang et al., 2002) and MKL2 (also referred to as MRTF-B) (Selvaraj &
Prywes, 2003; Wang et al., 2002). It has been shown that expression of MKL1 mRNA is

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ubiquitous, whereas expression of MKL2 mRNA is restricted to several tissues including the
brain and the heart (Cen et al., 2003; Selvaraj & Prywes, 2003; Wang et al., 2002). Co-
transfection studies revealed that both MKL1 and MKL2 were capable of inducing the
transcription of multiple CArG-containing promoters including atrial natriuretic factor (ANF),
SM22, SM -actin, and cardiac -actin. A truncated MKL2 protein that lacks both amino-
terminal region and carboxy-terminal region (MKL2NC700) behaved as a dominant-
negative manner for both MKL1 and MKL2, and over-expression of MKL2NC700 inhibited
skeletal muscle differentiation in C2C12 skeletal myoblasts (Selvaraj & Prywes, 2003). In
addition, MKL1 strongly induced SMC differentiation marker gene expression in
undifferentiated embryonic stem cells, even in the absence of myocardin (Du et al., 2004).
Moreover, a truncated form of MKL1, which behaved as a dominant-negative form of MKL1
and myocardin, inhibited MKL1-induced transcription of the SM22 gene (Du et al., 2004).
Taken together, MKL factors appear to be important regulators of SMC differentiation marker
gene expression as well as myocardin, and they appear to exhibit the redundant function with
myocardin as SRF co-factors. However, the precise roles of MKL factors in SMC differentiation
marker gene expression in SMCs are still unclear, because most of these studies analyzing the
function of MKL factors have been performed by over-expression experiments. Regarding this
point, there are several interesting studies as described below. First, MKL1 knockout mice
were viable, but were unable to effectively nurse their offspring due to a failure in
maintenance of the differentiated state of mammary myoepithelial cells during lactation (Li et
al., 2006; Sun et al., 2006). Second, conditional knockout of the MKL2 gene in neural crest-
derived cells exhibited a spectrum of cardiovascular defects including abnormal patterning of
the branchial arch arteries (Li et al., 2005; Oh et al., 2005). The abnormalities in MKL2 knockout
mice were accompanied by a decrease in SM -actin expression in SMCs within the branchial
arch arteries. Based on the results of these studies, MKL1 is unlikely to play an important role
in expression of SMC differentiation marker genes in vivo. In addition, role of MKL2 for SMC
differentiation in SMCs derived from other origins is still unknown. A biggest issue is how
broadly expressed MKL factors regulate SMC-specific/-selective CArG-dependent genes.
Recently, several studies suggest the importance of intracellular localization of MKL factors in
SMCs and non-SMCs (Hinson et al., 2007; Nakamura et al., 2010; Yoshida et al., 2007). Further
studies are required to address this issue.
In summary, it is clear that myocardin plays a critical role in SMC differentiation in concert
with the CArG-SRF complex. However, myocardin is not a SMC-specific gene in that it is
also expressed in cardiomyocytes, suggesting that myocardin alone is not enough to
coordinate expression of SMC differentiation marker genes. It is highly likely that
cooperative interaction of the SRF-myocardin complex with other transcription factors is
necessary for expression of SMC differentiation marker genes in SMCs. Further studies are
needed to clarify these combinatorial mechanisms.
3. Ternary complex factors exhibit dual roles in the transcription of SRF-
dependent CArG-Containing genes
TCFs are a subfamily of the Ets domain transcription factors (Buchwalter et al., 2004). TCF
was first described as 62 kD nuclear fractions (p62) that form a ternary complex with SRF on
the c-fos SRE (Shaw et al., 1989). Three members, Elk-1, Sap-1/Elk-4, and Net/Sap-2/Elk-3,
have been identified as TCFs. Previous studies demonstrated that TCFs are present on SREs
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of the c-fos gene with SRF dimers both before and after growth factor stimulation, and that
after the stimulation with growth factors, TCFs are phosphorylated and activate
transcription of the c-fos gene (Buchwalter et al., 2004).
Although it has been believed, for a long time, that most of SMC differentiation marker
genes lack the TCF-binding site in their promoter regions (Miano 2003), results of recent
studies by multiple laboratories including our own (Wang et al., 2004; Yoshida et al., 2007;
Zhou et al., 2005) suggest the involvement of Elk-1 in the regulation of SMC differentiation
marker genes. They presented evidence that repression of SMC differentiation marker genes
including SM -actin and SM22 by PDGF-BB was due to the displacement of myocardin
from SRF by phosphorylated Elk-1 in cultured SMCs (Figure 2). Indeed, they showed that
treatment with PDGF-BB induced phosphorylation of Elk-1 through the activation of the
MEK1/2-Erk1/2 pathway and increased the association between Elk-1 and SRF, whereas
the association between myocardin and SRF was decreased at the same time. By extensively
mapping the domain of myocardin and Elk-1, they found that both factors have a
structurally related SRF-binding motif and thereby compete for the common docking region
of SRF. These results are very interesting in that phosphorylation of Elk-1 simultaneously
exhibits the dual roles in the regulation of CArG-dependent genes: transcriptional activation
of the c-fos gene versus transcriptional repression of SMC differentiation marker genes.

Fig. 2. Phosphorylation of Elk-1 competes for SRF binding with myocardin. The myocardin-
SRF-CArG complex activates the transcription of SMC differentiation marker genes in the
absence of growth factors as shown in Fig. 1. Activation of the Erk1/2 pathway by growth
factors such as PDGF-BB induces phosphorylation of Elk-1. Phosphorylated Elk-1 displaces
myocardin from SRF and binds to SRF, thereby suppressing the transcription of SMC
differentiation marker genes. It has been reported that phosphorylated Elk-1 is able to bind
to the TCF-binding site within the SM22 promoter (Wang et al., 2004), although the TCF-
binding site is not present within the promoter region of most SMC differentiation marker
genes.
However, the mechanisms responsible for these dual effects have not been clearly
understood yet. That is, although the binding of Elk-1 on the putative TCF-binding site (5-
TTCCCG-3) adjacent to the CArG-far element at the SM22 promoter was detected by

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30
EMSA and chromatin immunoprecipitation assays (Wang et al., 2004), this sequence is not
the consensus binding site for Elk-1 (Treisman et al., 1992). By using the site selection
method to purify DNA capable of forming ternary complexes from a pool of randomized
oligonucleotides, the consensus binding motif for Elk-1 and Sap-1 was determined as 5-
(C/A)(C/A)GGA(A/T)-3 previously (Treisman et al., 1992). The putative TCF-binding site
within the SM22 gene (sense: 5-TTCCCG-3 and antisense: 5-CGGGAA-3) does not
match this sequence completely. In addition, although over-expression of Elk-1
downregulated the SM22 promoter-luciferase activity through the competition with
myocardin, this competition was still observed when the mutational SM22-luciferase
construct, in which the putative TCF-binding site was abolished, was used. Furthermore,
there is no putative Elk-1 binding site near the CArG elements within the SM -actin
promoter (Mack & Owens, 1999). Because chromatin immunoprecipitation assays can detect
not only the direct binding of protein to DNA sequence, but also the binding of protein to
protein, it is highly possible that the attachment of Elk-1 to the TCF-binding site may not be
absolutely required for the competition with myocardin for SRF binding. Nevertheless, the
SM22 promoter with a mutation in the TCF-binding site has been reported to direct ectopic
transcription in the heart in a later embryonic stage, as compared with the wild-type SM22
promoter in vivo (Wang et al., 2004). Further studies are needed to determine if these
findings are applicable to multiple SMC differentiation marker genes.
It is also of interest to determine whether the activation of Elk-1 can recruit histone
deacetylases to the promoter regions of SMC differentiation marker genes. Elk-1 contains
two transcriptional repression domains, an N-terminal transcriptional repression domain
and an R motif located in the C-terminal transcriptional activation domain (Buchwalter et
al., 2004). It has been shown that HDAC1 and HDAC2 were recruited to the N-terminal
transcriptional repression domain of Elk-1 on the c-fos promoter followed by the activation
of the MEK1/2-Erk-1/2 pathway, and this recruitment kinetically correlated with the
shutoff of the c-fos gene expression after growth factor stimulation (Yang et al., 2001; Yang &
Sharrocks, 2004). We previously showed that repression of SMC differentiation marker
genes after stimulation with PDGF-BB was accompanied by the recruitment of multiple
HDACs, HDAC2, HDAC4, and HDAC5 in cultured SMCs (Yoshida et al., 2007). It is
possible that the association between Elk-1 and these HDACs on the SMC promoters is one
of the mechanisms for repression of SMC differentiation marker gene expression. Moreover,
it was reported that SUMO modification of the R motif in Elk-1 could antagonize the
MEK1/2-Erk1/2 pathway and repress the transcription of the c-fos gene (Yang et al., 2003).
Thus, it is also possible that PDGF-BB can induce sumoylation of Elk-1 and exhibit the
repressive effects on SMC differentiation marker genes.
In summary, the preceding results indicate that Elk-1 plays dual roles in the transcription of
CArG-dependent genes as both an activator and a repressor. However, there are still some
questions as discussed above. Clearly, one of the most fascinating questions is to determine
if knockdown of Elk-1 abolishes PDGF-BB-induced repression of SMC differentiation
marker genes both in vivo and in vitro.
4. Multiple homeodomain proteins regulate SMC differentiation
Homeodomain proteins are a family of transcription factors with a highly conserved DNA-
binding domain that regulate cell proliferation, differentiation, and migration in many cell
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types during embryogenesis (Gorski & Walsh, 2003). This family is comprised of over 160
genes, and it has been reported that several homeodomain proteins are able to regulate
differentiation of SMCs by interacting with the CArG-SRF complex.
One of these factors is Prx-1 (Paired-related homeobox gene-1), which is also known as MHox
and Phox (Cserjesi et al., 1992; Grueneberg et al., 1992). Expression of Prx-1 is completely
restricted to mesodermally derived cell types during embryogenesis and to cell lines of
mesodermal origin including cultured aortic SMCs (Blank et al., 1995; Cserjesi et al., 1992).
Previous studies from our laboratory and others showed that Prx-1 was capable of inducing
the transcription of the CArG-SRF dependent genes (Grueneberg et al., 1992; Hautmann et al.,
1997; Yoshida et al., 2004a). Indeed, we found that angiotensin II increased expression of
multiple SMC differentiation marker genes including SM -actin, as well as Prx-1 expression in
cultured SMCs (Hautmann et al., 1997; Turla et al., 1991; Yoshida et al., 2004a). Of major
interest, we provided evidence that siRNA-induced suppression of Prx-1 dramatically reduced
both basal and angiotensin II-induced transcription of the SM -actin gene (Yoshida et al.,
2004a). In addition, Prx-1 increased the SRF binding to degenerate CArG B element within the
SM -actin gene by EMSA (Hautmann et al., 1997). Similarly, Prx-1 enhanced the binding of
SRF to c-fos CArG element by EMSA (Grueneberg et al., 1992). However, the formation of a
stable higher order complex comprised of Prx-1, SRF, and CArG element was not detected by
EMSA. Rather, Prx-1 enhanced both the rate of association and the rate of dissociation between
SRF and CArG element, thereby increasing the rate of exchange of SRF on the CArG element.
Although further studies are required to clarify these mechanisms in detail, results thus far
suggest that Prx-1 plays a key role in the transcription of CArG-dependent genes through
regulating the binding of SRF to CArG elements.
Although the preceding results suggest that Prx-1 is involved in the regulation of SMC
differentiation marker gene expression (Hautmann et al., 1997; Yoshida et al., 2004a), it also
plays a role in proliferation of SMCs. Prx-1 expression was induced during the development
of pulmonary vascular disease in adult rats, and Prx-1 enhanced the proliferation rate of
cultured rat A10 SMCs via the induction of tenascin-C expression (Jones et al., 2001). Taken
together, results suggest that Prx-1 plays multiple roles in the regulation of differentiation
status and the regulation of proliferation status in SMCs. This is consistent with the idea that
differentiation and proliferation are not necessarily mutually exclusive processes (Owens &
Thompson, 1986; Owens et al., 2004). However, it remains unknown whether Prx-1 exhibits
these two roles simultaneously or Prx-1 exhibits distinct roles in a developmental stage-
specific manner. Of interest, Prx-1 knockout mice have been made and shown to exhibit
major defects in skeletogenesis and die soon after birth (Martin et al., 1995). Mice null for
both Prx-1 and its homologue, Prx-2, showed a vascular abnormality with an abnormal
positioning and awkward curvature of the aortic arch and a misdirected and elongated
ductus arteriosus (Bergwerff et al., 2000). Moreover, expression of endothelial markers such
as Flk-1 and VCAM-1 and von Willebrand factor-positive cells were decreased in the lung of
Prx-1 null newborn mice (Ihida-Stansbury et al., 2004), suggesting that Prx-1 is required for
lung vascularization in vivo. It will be of interest to directly test the role of Prx-1 in CArG-
dependent SMC differentiation marker gene expression in these mice.
Another homeodomain protein related to SMC differentiation is Hex. Hex was originally
isolated from hematopoietic tissues by PCR using degenerate oligonucleotide primers
corresponding to the conserved homeodomain sequences and has been shown to play an

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important role in inducing differentiation of vascular endothelial cells (Thomas et al., 1998).
In SMCs, Hex protein expression was induced in the neointima after balloon injury of rat
aorta, while it was undetectable in normal aorta (Sekiguchi et al., 2001). The expression
pattern of Hex was similar to that of SMemb/NMHC-B, a marker of phenotypically
modulated SMCs. Hex induced the transcription of the SMemb promoter, and cAMP-
responsive element (CRE) located at -481 bp within the promoter was critical for Hex
responsiveness. However, Hex failed to bind to CRE directly, thus the precise mechanisms
whereby Hex activated the SMemb promoter are still unclear. Of interest, subsequent studies
showed that Hex also induced expression of a subset of SMC differentiation marker genes
including SM -actin and SM22, but not SM-MHC and h1-calponin (Oyama et al., 2004).
Hex induced the transcription of the SM22 gene in a CArG-dependent manner, and it
enhanced the binding of SRF to CArG-near element within the SM22 promoter, as
determined by EMSA. In addition, immunoprecipitation assays revealed the physical
association between SRF and Hex. As such, the mechanisms whereby Hex induces SMC
differentiation marker genes seem to be similar to those of Prx-1. However, results showing
that Hex simultaneously activated expression of both SMC differentiation marker genes and
those characteristic of phenotypically modulated SMCs are paradoxical, and further studies
are clearly needed to precisely define the pathophysiological role of Hex in SMCs.
Nkx-3.2 is also a homeodomain protein that regulates expression of SMC differentiation
marker genes (Nishida et al., 2002). It has been demonstrated that a triad of SRF, GATA-6,
and Nkx-3.2 formed a complex with their corresponding cis-elements and cooperatively
transactivated SMC differentiation marker genes including 1-integrin, SM22, and
caldesmon. Because co-localization of GATA-6, Nkx-3.2, and SRF was exclusively observed in
SMCs, SMC-specific gene expression does not appear to be the result of any single
transcription factor that is unique to SMCs, but rather is due to unique combinatorial
interactions of factors that may be expressed in multiple cell types but only found together
in SMCs.
Furthermore, we recently identified Pitx2 as a homeodomain protein which is required for
the initial induction of SMC differentiation by using a subtraction hybridization screen
(Shang et al., 2008). Over-expression of Pitx2 induced expression of CArG-dependent SMC
differentiation marker genes, whereas knockdown of Pitx2 attenuated retinoic acid-induced
differentiation of SMCs from undifferentiated SMC precursor cells. Furthermore, Pitx2
knockout mouse embryos exhibited impaired induction of SMC differentiation markers in
the dorsal aorta and branchial arch arteries. We identified three mechanisms for Pitx2-
induced transcription of SMC differentiation marker genes (Figure 3). First, Pitx2 bound to
its consensus TAATC(C/T) element in the promoter region of SMC differentiation marker
genes. Second, Pitx2 physically associated with SRF. Third, Pitx2 mediated exchange of
HDACs with p300 to increase acetylation levels of histone H4 at the SMC promoters. These
results provide compelling evidence that Pitx2 plays a critical role in the induction of SMC
differentiation during the early embryogenesis. Further studies are needed to determine if
Pitx2 also contributes to the pathogenesis of vascular diseases including atherosclerosis.
As such, several homeodomain proteins are involved in the regulation of CArG-SRF
dependent SMC differentiation marker gene expression, and some of the mechanisms
appear to be mediated by common pathways. Further studies are needed to clarify the
temporal and spatial roles of each of these homeodomain proteins in SMC differentiation.
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Fig. 3. Pitx2 transactivates SMC differentiation marker genes through three mechanisms.
Pitx2 induces expression of SMC differentiation marker genes by: (1) binding to a consensus
TAATC(C/T) cis-element; (2) interacting with SRF; and (3) mediating exchange of HDACs
with p300 at the promoter region of SMC differentiation marker genes. These mechanisms
are important for the initial induction of SMC differentiation during the early embryonic
development.
5. A number of factors associate with SRF
In addition to the factors described above, there are a number of transcription factors known
to interact with SRF. These factors also play key roles in the control of SMC differentiation
marker gene expression. In this section, some of these transcription factors will be discussed
briefly.
5.1 GATA-6
GATA proteins are a family of zinc finger transcription factors, and play essential roles in
development through their interaction with a DNA consensus element, WGATAR
(Molkentin, 2000). Six GATA transcription factors have been identified in vertebrates, and
GATA-4, GATA-5, and GATA-6 are thought to be involved in the formation of the heart,
gut, and vessels. During the early murine embryonic development, expression patterns of
GATA-6 and GATA-4 were similar, with expression being detected in the precardiac
mesoderm, the embryonic heart tube, and the primitive gut (Morrisey et al., 1996). However,
during the late development, GATA-6 became the only GATA factor to be expressed in
vascular SMCs. Knockout of the GATA-6 gene in mice resulted in embryonic lethality
between embryonic day 6.5 and 7.5, precluding the evaluation of the role of GATA-6 in SMC
differentiation and maturation (Morrisey et al., 1998).
As described in a previous section, GATA-6 has shown to interact with SRF and Nkx-3.2
and to induce SMC differentiation marker gene expression (Morrisey et al., 1998; Nishida et
al., 2002). GATA-6 expression in SMCs was rapidly downregulated after vascular injury in
rat carotid arteries, and adenovirus-mediated transfer of GATA-6 to the vessel wall after the
balloon injury partially inhibited the formation of intimal thickening and reversed the
downregulation of SMC differentiation marker genes including SM -actin and SM-MHC
(Mano et al., 1999). These results suggest the important role of GATA-6 in regulating SMC

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34
differentiation. Of interest, results of studies (Yin & Herring, 2005) showed that GATA-6
increased the transcriptional activity of the SM -actin and SM-MHC genes, whereas it
reduced the transcriptional activity of the telokin gene. They found that the GATA-6 binding
site was located adjacent to CArG element in the telokin promoter and that over-expression
of GATA-6 interfered the interaction between myocardin and SRF by mammalian two-
hybrid assays. However, it is unclear why GATA-6 has positive and negative effects on
CArG-dependent SMC differentiation marker genes. It is possible that these opposite effects
are due to the number of CArG elements or the distance between the GATA-6 binding site
and the CArG element. Further studies are needed to test these possibilities.
5.2 Klf4
Klf4 is a member of Krppel-like transcriptional factors that have recently received
increased attention. Previously, Klf4 was identified as a binding factor for the transforming
growth factor-1 control element (TCE) found in the promoter region of the SM -actin and
SM22 genes, based on a yeast one-hybrid screen (Adam et al., 2000). Klf4 exhibited a
profound inhibitory effect on expression of SMC differentiation marker genes via a TCE-
dependent and a CArG-SRF-dependent manner (Liu et al., 2003, 2005). For example,
adenovirus-mediated over-expression of Klf4 repressed endogenous expression of SM -
actin and SM-MHC genes, as well as expression of myocardin, in cultured SMCs as measured
by real-time reverse transcription-PCR (Liu et al., 2005). In addition, over-expression of Klf4
completely abolished myocardin-induced activation of SMC differentiation marker genes.
Co-immunoprecipitation assays revealed that Klf4 physically interacted with SRF, and
chromatin immunoprecipitation assays showed that over-expression of Klf4 markedly
reduced the binding of SRF to CArG elements on the SM -actin promoter in intact
chromatin of cultured SMCs (Liu et al., 2005). Moreover, PDGF-BB treatment induced Klf4
mRNA expression in cultured SMCs, and siRNA-induced suppression of Klf4 partially
blocked PDGF-BB-induced suppression of SMC differentiation marker genes (Liu et al.,
2005). Of significant interest, we demonstrated that conditional knockout of the Klf4 gene in
mice exhibited a delay in suppression of SMC differentiation markers, and an enhanced
neointimal formation following vascular injury (Figure 4) (Yoshida et al., 2008b).
Additionally, we showed that Klf4, Elk-1, and HDACs cooperatively suppress oxidized
phospholipid-induced suppression of SMC differentiation marker genes in cultured SMCs
(Yoshida et al., 2008a). Taken together, these results suggest that Klf4 plays a key role in
mediating phenotypic switching of SMCs.
5.3 Cysteine-rich LIM-only proteins, CRP1 and CRP2
The members of the cysteine-rich LIM-only protein (CRP) family, CRP1 and CRP2, are
expressed predominantly in SMCs and contain two LIM domains in the structure
(Henderson et al., 1999; Jain et al., 1996). It is known that the functions of LIM domains are
to mediate protein-protein interactions, to target proteins to distinct subcellular locations,
and to mediate assembly of multimeric protein complexes. One of the functions of CRP1 and
CRP2 is to interact with both the actin crosslinking protein, -actinin, and the adhesion
plaque protein, zyxin, and to regulate the stability and structure of adhesion complexes
(Arber & Caroni, 1996; Schmeichel & Beckerle, 1994). In addition to such a cytoplasmic role,
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Fig. 4. Conditional knockout of the Klf4 gene in mice accelerates neointimal formation
following vascular injury. Klf4 is a potent repressor of SMC differentiation marker genes.
Interestingly, conditional knockout of the Klf4 gene in mice delays downregulation of SMC
differentiation markers, but also accelerates neointimal formation after vascular injury
(Yoshida et al., 2008).
it has been reported that CRP1 and CRP2 are also able to function as transcriptional co-
factors (Chang et al., 2003). Over-expression of three factors, SRF, GATA-6, and CRP1/CRP2
strongly activated the transcription of SMC differentiation marker genes including SM -
actin, SM-MHC, SM22, h1-calponin, and h-caldesmon. The N-terminal LIM domain of
CRP1/2 interacted with SRF, and that the C-terminal LIM domain of CRP1/2 interacted
with GATA-6, and that SRF and GATA-6 also interacted each other. These results suggest a
critical role of CRP1/2 in organizing multiprotein complexes onto the SMC promoters for
SMC differentiation. However, it is still unclear how CRP1 and CRP2 are translocated from
the cytoplasm to the nucleus and what signaling pathways control their nuclear localization.
Moreover, there is a lack of evidence that these factors play a role in control of SMC
differentiation marker gene expression in vivo in SMCs. Indeed, results of recent studies
showed that SMC differentiation in CRP1 knockout mice or CRP2 knockout mice appeared
to be normal, although neointimal formation was altered after vascular injury (Lilly et al.,
2010; Wei et al., 2005). Results raised a question as to the role of CRP1/2 in SMC
differentiation.
5.4 PIAS-1
Results of previous studies showed that over-expression of class I basic Helix-Loop-Helix
proteins, E2-2, and SRF exhibited a synergistic effect on the transcription of the SM -actin
promoter-enhancer in BALBc/3T3 cells (Kumar et al., 2003). However, direct interaction
between E2-2 and SRF was undetectable by EMSA using the recombinant proteins. We
isolated PIAS-1 (protein inhibitor of activated STAT-1) as an interacting protein for E2-2 by a
yeast two-hybrid screen (Kawai-Kowase et al., 2005). We also found that PIAS-1 interacted
with SRF, suggesting that PIAS-1 works as a bridging molecule between E2-2 and SRF.
Interestingly, PIAS-1 belongs to a family of E3 ligases which promote SUMO modifications
of target proteins (Schmidt & Mller, 2002). Indeed, recent studies showed that transcription
factors involved in SMC differentiation, such as myocardin and Klf4, were sumoylation

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targets of PIAS-1. Myocardin sumoylation by PIAS-1 transactivated cardiogenic genes in
10T1/2 fibroblasts (Wang et al., 2007), whereas sumoylation of Klf4 by PIAS-1 promoted
transforming growth factor- induced activation of SM -actin expression in SMCs (Kawai-
Kowase et al., 2009). Further studies are needed to determine effects of PIAS-1 knockout on
SMC differentiation as well as phenotypic switching of SMCs.
6. Conclusion and perspectives
As discussed above, it is clear that the CArG-SRF complex plays a central role in the
regulation of SMC differentiation marker gene expression. However, it is also clear that
expression of SMC differentiation marker genes is not controlled by the CArG-SRF complex
alone, nor by any single transcription factor that is expressed exclusively in SMCs. Rather,
SMC-selective gene expression appears to be mediated by complex combinatorial
interactions of multiple transcription factors and co-factors, including some that are
ubiquitously expressed like SRF and PIAS-1, as well as others that are selective for SMCs
like myocardin, Prx-1, CRP-1/2, and GATA-6. In addition to the transcription factors
described above, several novel factors, including Fhl2 (Philippar et al., 2004), HERP1 (Doi et
al., 2005) and lupaxin (Sundberg-Smith et al., 2008), have also been identified as factors
interacting with SRF.
However, our knowledge is immature regarding the overall connection among multiple
transcription factors and co-factors that can modify the activity of SRF. Most of studies
analyzing the protein-protein interaction thus far have been focused on the relationship
among two or three proteins. However, a number of factors should be coordinately
regulated and interacted by a single environmental cue. It is of interest to determine
whether all of SRF-interacting factors are simultaneously required for SMC differentiation
marker gene expression or these factors independently contribute to SMC differentiation
marker gene expression in time- and position-specific manner. Thus, in the long term, future
studies in the SMC field are needed not only to screen out other key transcription factors,
but also to map out the connection networks of these factors.
During the past decade, there is a tremendous progress in our understanding of the roles
of chromatin modifying enzymes and chromatin structure in gene transcription in all cell
types. Accumulating evidence indicates that the N-terminal tails of histones are the target
of numerous modifications, including acetylation, methylation, phosphorylation,
ubiqutination, and ADP ribosylation, and that these modifications control gene
transcription (Fischle et al., 2003). However, this issue in the SMC field is obviously in its
infancy. Thus far, only several transcription factors have been reported to be involved in
chromatin remodeling. Clearly, more detailed studies are required to determine the
mechanisms whereby SRF and its interacting factors coordinately contribute to chromatin
remodeling.
Finally, although much progress has been made in our understanding of the role of
transcription factors in the control of SMC differentiation marker gene expression, some of
these studies are performed only in cultured SMCs or SM-like systems. Studies of these
factors in vivo will provide more compelling information to enhance our knowledge about
SMC differentiation and development.
Molecular Control of Smooth Muscle Cell Differentiation
Marker Genes by Serum Response Factor and Its Interacting Proteins

37
7. Acknowledgments
This work was supported in part by Keio Gijuku Academic Development Funds and Takeda
Science Foundation.
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3
Trans Fatty Acids and Human Health
Sebastjan Filip and Rajko Vidrih
Biotechnical Faculty,
Department of Food Science and Technology,
University of Ljubljana,
Slovenia
1. Introduction
According to various studies, fats of animal and vegetable origins satisfy 22% to 42% of the
daily energy demands of human beings (Srinivasan et al., 2006; Wagner et al., 2008; Willet,
2006). Some fats, and especially those that are hydrogenated, contain trans fatty acids
(TFAs), i.e. unsaturated fatty acids with at least one double bond in a trans configuration
(Craig-Schmidt, 2006). This trans-double-bond configuration results in a greater bond angle
than for the cis configuration, thus producing a more extended fatty-acid carbon chain that
is more similar to that of the saturated fatty acids (SFAs), rather than to that of the cis-
unsaturated double-bond-containing fatty acids (Fig. 1) (Moss, 2006; Oomen et al., 2001).

Fig. 1. Structure of different isomers of C16 (Willett, 2006)

The Cardiovascular System Physiology, Diagnostics and Clinical Implications

44
Fat is a thus major source of energy for the body, and it also aids in the absorption of
vitamins A, D, E and K, and of the carotenoids. Both animal-derived and plant-derived food
products contain fat, and when eaten in moderation, fat is important for correct growth and
development, and for the maintenance of good health. As a food ingredient, fat provides
taste, consistency and stability, and helps us to feel full. In addition, parents should be
aware that fats are an especially important source of calories and nutrients for infants and
toddlers (up to 2 years of age), who have the highest energy needs per unit body weight of
any age group.
However, SFAs and TFAs raise low-density lipoprotein (LDL; or bad) cholesterol levels in
the blood, thereby increasing the risk of heart disease. Indeed, prospective epidemiological
studies and case-control studies support a major role for TFAs in the risk of cardiovascular
disease, and therefore dietary cholesterol can also contribute to heart disease (see below).
Unsaturated fats, which can be mono-unsaturated or polyunsaturated, do not raise LDL
cholesterol and are beneficial to health when consumed in moderation.
Hydrogenated oils tend to have a higher TFA content than oils that do not contain
hydrogenated fats. In the partially hydrogenated soybean oil, which is the major source of
TFAs worldwide, the main isomer is trans-10 C18:1. In the European countries with the
highest TFA intake (The Netherlands and Norway), consumption of partially
hydrogenated fish oils was common until the mid-1990s, after which they largely
disappeared from the dietary fat intake. These partially hydrogenated fish oils included a
variety of very-long-chain TFAs. Recent findings from Asian countries (India and Iran)
have indicated a very high intake of TFAs from partially hydrogenated soybean oil (4% of
energy). Thus, TFAs appear to be a particular problem in developing countries where
soybean oil is used.
Formation of these trans double bonds thus impacts on the physical properties of a fatty
acid. Fatty acids that contain a trans double bond have the potential for closer packing and
alignment of their acyl chains, which will result in decreased molecular mobility (Willett,
2006). Therefore, the oil fluidity will be reduced when compared to that of fatty acids that
contain a cis double bond. Partial hydrogenation of unsaturated oils results in the
isomerisation of some of the remaining double bonds and the migration of others,
producing an increase in the TFA content and a hardening of the fat. It has been shown that
foods that contain hydrogenated oils tend to have a higher TFA content than those that do
not contain hydrogenated oils (Moss, 2006; Oomen et al., 2001). Nevertheless, the
hydrogenation of oils, such as corn oil, can result in both cis and trans double bonds, which
are generally located anywhere between carbon 4 and carbon 16 of the fatty acids. One of
the major TFAs is elaidic acid (trans-9 C18:1), although during hydrogenation of
polyunsaturated fatty acids (PUFAs), small amounts of several other TFAs are produced,
including: trans-9,cis-12 C18:2; cis-9,trans-12 C18:2; cis-9,cis-12,trans-15 C18:3; and cis-5,cis-
8,cis-11,cis-14,trans-17 C20:5 (Craig-Schmidt, 2006; Wagner et al., 2008). Conversely, one way
to produce zero levels of TFAs is through the trans-esterification reaction between
vegetable oils and solid fatty acids, like C8:0, C12:0, C14:0 and C16:0.
Correlations between high intake of industrially produced TFAs (IP-TFAs) and increased
risk of coronary heart disease (CHD) have been reported (Stender et al., 2006; Tarrago-Trani

Trans Fatty Acids and Human Health

45
et al., 2006), and lowering the intake of TFAs can also reduce the incidence of CHD (Willett,
2006). Estimates based on changes in plasma concentrations of LDL and high-density
lipoprotein (HDL) indicate around a 4% reduction in CHD incidence, while based on
epidemiological associations, when TFA intake is lowered by 2% (5 g/day), the estimates
indicate a >20% reduction in CHD incidence (Katan, 2006; Moss, 2006). In The Netherlands,
a major reduction in the TFA content of retail foods was achieved in the 1990s through the
efforts of the industry and with minimal government intervention. Society pressure is also
now helping to reduce the TFA content of fast foods. This illustrates the feasibility of
reducing TFAs in fast foods without increasing the saturated fats, with the daily intake kept
as low as possible, to minimise the health risks (Stender et al., 2006).
Comparison of the different recommendations for macronutrients in some European
countries, for the World Health Organisation/ Food and Agriculture Organisation of the
United Nations (WHO/FAO), and in the USA and Canada, are given in Table 1. Most of the
recommendations are the same, or are in similar ranges. The recommendations for protein,
however, are expressed differently, either as grams per day or grams per kilogram per day,
and usually without any indication of a representative weight at each age to allow
conversion of one to the other. The Joint FAO/WHO/United Nations University (UNU)
Expert Consultation of 1985 (WHO, 1985) defined the protein requirement of an individual
as the lowest level of dietary protein intake that will balance the losses of nitrogen from the
body in persons maintaining an energy balance at modest levels of physical activity. The
human body can synthesise both SFAs and mono-unsaturated fatty acids (MUFAs) from
acetate, whereas PUFAs (in both the n6 linoleic acid and n3 linolenic acid series) are
required in the diet, and they are therefore known as essential fatty acids. These essential
fatty acids are important for various cell-membrane functions, such as fluidity, permeability,
activity of membrane-bound enzymes and receptors, and signal transduction. Linoleic and
linolenic acids can be elongated and desaturated in the body, and transformed into
biologically active substances, like prostaglandins, prostacyclins and leukotrienes. These
substances participate in the regulation of blood pressure, renal function, blood coagulation,
inflammatory and immunological reactions, and many other functions (Nordic Nutrition
Recommendations, 2004). The DACH Reference Values for Nutrient Supply (DACH, 2000)
for total fat intake in adults (not more than 30% of the energy intake) are related to light
work, heavy muscle work (not more than 35% of energy intake) and extremely heavy work
(not more than 40% of energy intake). SFAs should not exceed 10% of energy intake. PUFAs
should provide about 7%, and up to 10% if SFAs provide more than 10% of energy intake.
MUFAs should constitute the rest. TFAs should contribute not more than 1% of the daily
energy. The ratio of n6 linoleic acid to n3 linolenic acid should be about 5:1 (WHO/FAO,
2002). These fatty acids compete for the metabolic enzymes, and it is therefore important to
maintain a balance between them (Nordic Nutrition Recommendations, 2004). The Nordic
Nutrition Recommendations indicate the limiting of the intake of SFAs plus TFAs to about
10% of the daily energy and the total fat intake to 30% of the daily energy (25%-30%) (Filip
et al., 2010). The recommendations for carbohydrate intake are from 50%of the daily energy
in the DACH (2000) reference values, to 55% (50%-60%) in the Nordic Nutrition
Recommendations (2004), 55%-75% by WHO/FAO, and 45%-65% in the USA/ Canada
recommendations, as detailed in Table 1.

The Cardiovascular System Physiology, Diagnostics and Clinical Implications

46
Component
NNR
(2004)
DACH
(2000)
WHO/FAO
(2002)
Euro Diet
(2000)
USA/Canada
AMDR
(2002)
Total energy from fat
(%)
30 (25-35) 30 15-30 <30 20-35
SFAs (%) 10 10 <10 <10 Minimise
PUFAs (%) 5 (10) 7-10 6-10 - -
n-6 FAs (%) 4 (9) 2.5 5-8 4-8 5-10 (linoleic)
n-3 FAs (%) 1 0.5 1-2 2 (linolenic) 0.6-1.2
TFAs (%)
Included
in SFAs
1 <1 <2 Minimise
MUFAs (%) 10-15 The rest of the total -
Total energy from
carbohydrates (%)
55 (50-60) 50 55-75 >55 45-65
Energy from sugars
(%)
<10 30 <10 <25
Fibre (g/day) 25-35 (3 g/MJ) (12.5 g/1000 kcal)
25-38
(14 g/1000
kcal)
Energy from proteins
(%)
15 (10-20) 8-10 10-15 - 10-35
Cholesterol (mg/day) 300 <300 Minimise
Salt (sodium) (g/day) 56 (2.3-2.7) <5 (2)
NNR, Nordic Nutrition Recommendations; DACH, AustriaGermanySwitzerland Reference Values
for Nutrient Supply; WHO, World Health Organisation; FAO, Food and Agriculture Organisation of the
United Nations; AMDR; acceptable macronutrient distribution; FAs, fatty acids; SFAs, saturated fatty
acids; PUFAs, polyunsaturated fatty acids; TFA, trans fatty acids; MUFAs, mono-unsaturated fatty
acids.
Table 1. Comparison of reference daily intakes for adults according to different
recommendations around the World (Pavlovic et al., 2007)
As indicated above, prospective epidemiological studies and case-control studies using
adipose-tissue analyses have confirmed a major role for TFAs in the risk of CHD. The
magnitude of the association with CHD is considerably stronger than for SFAs, and it is
stronger than that predicted for the effects of TFAs on LDL and HDL cholesterol (Katan,
2006; Tarrago-Trani et al., 2006). In this context, it needs to be considered that data for the
Russian Federation show that every year 1,005 people per 100,000 of the population between
25 and 64 years of age die because of circulatory system diseases (WHO, 2008). As a
consequence influence of TFAs on CHD, in 2003, the United States FDA issued a ruling that
required food manufacturers to list the TFAs in the nutritional facts labels of all packaged
food products (FDA, 2003), with the food industry being given until 1 January, 2006 to
comply. Along with these growing health concerns about TFAs, this mandate led to marked
changes in the fat and oil industries, with newer technologies developed to reduce the TFA
contents of fats and oils used in the manufacture of food products. Conversely, given the
labelling mandate and these technological advances, it is possible that food products
traditionally considered to be sources of TFAs are now much lower in, or indeed do not

Trans Fatty Acids and Human Health

47
contain, TFAs (Borra et al., 2007). Then in late 2006, New York City became the first major
city in the United States to pass a regulation limiting IP-TFAs in restaurants. This has served
as a model for others to follow, with these regulations including: a maximum level per
serving size of 0.5 g TFAs; a distinction between frying and baking, with a phased-in
implementation; a help centre to assist restaurants to make the switch to more healthy
options; and plans to evaluate the regulation and its impact on CHD (Borra et al., 2007).
Accurate quantification of C18:1 TFAs in food products is thus an important issue, with
policies recently implemented in different countries to limit their consumption and their
occurrence in food products because of their relationship with CHD (Carriquiry et al., 2008;
Chen et al., 2007).
2. History
Margarine was invented in 1869 by Hippolyte Mge Mouris, a French food research
chemist, in response to a request by Napoleon III for a wholesome alternative to butter. It is
not entirely clear whether the primary aim was the betterment of the working classes or the
economics of the food supply to the French army. In the laboratory, Mge Mouris solidified
purified fat, after which the resulting substance was pressed in a thin cloth, which formed
stearine and discharged oil. This oil formed the basis of the butter substitute. For the new
product, Mge Mouris used margaric acid, a fatty-acid component isolated in 1813 by the
Frenchman Michel Eugne Chevreuil. While analysing the fatty acids that are the building
blocks of fats, he singled out this one and named it margaric acid, because of the lustrous
pearly drops that reminded him of the Greek word for pearls, i.e. margarites (Chen et al.,
2008; Craig-Schmidt, 2006).
In 1871, Mge Mouris sold this know-how to the Dutch firm Jurgens, which is now part of
Unilever. In the early days, margarine contained two types of fat: a large proportion of
animal fat and a small proportion of vegetable fat. As time passed, the small vegetable-fat
element increased, through two specific stages in the process. First, by improving the
process of refining vegetable oils, use could be made of a greater variety of liquid oils and a
higher proportion of solid vegetable fats. Secondly, through the development of processes
for turning liquid oils into solid fats on a commercial scale, use could be made of larger
quantities of liquid vegetable oils (Filip, 2010).
During the early years of this period, in the late 1800s, TFA intake from partially hydrogenated
vegetable oils was minimal. Indeed, it was not until the late 1800s that the process of partial
hydrogenation of oils was invented in Europe. These partially hydrogenated oils apparently
entered the United States food supply by 1920. Although the rate of increase before 1950 is not
completely clear, by 1950 the amount of IP-TFAs in the food supply was quite substantial.
Partly because of economic effects during World War II, margarine production rose rapidly as
a replacement for butter (Chen et al., 2007). Then during the 1960s, margarine became viewed
as a healthy alternative to butter because of its absence of cholesterol and its low content of
SFAs. Thus, consumption increased further, and so margarine, which was heavily
hydrogenated at that time, became widespread in the food supply and was the major source of
IP-TFAs. This phenomenon is illustrated in Figure 2. The total TFAs consumption was
approximately 2% to 3% of the food energy. Since then, the sources of TFAs have changed,
from mainly margarine to mainly deep-fried fast foods and commercially baked products,
although per capita, the intake has remained roughly the same (Willett, 2006).

The Cardiovascular System Physiology, Diagnostics and Clinical Implications

48

Fig. 2. Relative food energy supplied by the different fatty acids, and the predicted changes
for the food industry and fat hydrogenation (Simopoulos, 2004)
After World War II, the process of making hydrogenated and hardened fats from cheaper
sources of vegetable oils was widely adopted. Margarines were developed and marketed as
alternatives to butter, and vegetable shortening increasingly replaced animal fats in cooking
(Albers et al., 2008). However, as early as 1975, at what is now the University of Glamorgan
in South Wales, a group of scientists led by Leo Thomas suspected that deaths from CHD
were connected with this eating of partially hydrogenated fats. It is now generally accepted
that TFAs are actually worse for health than the SFAs that they were designed to replace
(Blake, 2009).
3. Studies of trans fatty acids
Increases in civilization diseases in the developed world led scientists to investigate why
this was happening. While there is enough food that is also cheaper and more accessible
than ever before in the developed world, we are witnessing more and more overweight and
obese populations. Modern populations have worse nutrition habits than ever before, except
for some specific small social groups e.g. through religion, ecology and ethnic aspects
(WHO, 2008). Obesity is a severe health issue that is characterised by fat accumulation and
defined by means of the body mass index (BMI), as body weight [kg]/ (height [m])
2
.
According to this index, different obesity levels have been described, ranging from
overweight (BMI, 25.0-29.9), through obese (BMI, 30.0-40.0) to the most detrimental stage,
morbid obesity (BMI, 40) (Garaulet et al., 2011). The relevance of this classification is that as
the BMI increases, the morbidity and mortality risks also increase (Bray, 2003). Furthermore,
regional fat accumulation is an important factor in the development of obesity-related
alterations. It has been suggested that excess visceral fat is more detrimental than excess

Trans Fatty Acids and Human Health

49
subcutaneous fat, because visceral deposits release free fatty acids directly into the portal
vein (Bray, 2003). The fatty acid pattern carried to the portal circulation is of great
importance, because different fatty acids show distinct atherogenicities, depending on the
chain length and degree of unsaturation. Here, SFAs have been associated with increased
cardio-metabolic risk, while n-3 and n-9 unsaturated fatty acids have been proposed as
protective agents against these alterations (Garaulet et al., 2011).
3.1 Studies in animals
In milk fat, TFAs are produced by anaerobic fermentation of PUFAs in the rumen of
lactating cows (Destaillats et al., 2007; Fournier et al., 2006). This fermentation process is
called biohydrogenation, and it results in TFAs that can be further metabolised in the
mammary gland. Accurate estimations of fatty-acid compositions are vital not only for the
definition of the nutrient composition of foods, but also to accurately determine treatment
effects that can alter the fatty-acid composition of the foods (Ascherio, 2002; Burdge et al.,
2005; Kummerow et al., 2004; Murrieta et al., 2003; Triantafillou et al., 2003).
There is a considerable overlap of TFA isomers in fats of ruminant origin and in partially
hydrogenated vegetable oils, as they have many isomers in common. However, there are
considerable differences in the amounts of individual TFAs in these sources. While there is
evidence of unfavourable effects of TFAs from hydrogenated vegetable oils on LDL and
other risk factors for atherosclerosis, at present it is not certain which of the component(s) of
the TFAs created by chemical hydrogenation are responsible for these a negative metabolic
effects (Ascherio, 2002). Prospective studies addressing the effects of TFA intake on CHD
risk, where estimates of TFA intake were based on dietary protocols, have mostly been
carried out in populations with a relatively low intake of dairy or ruminant TFAs (Pfeuffer
& Schrezenmeir, 2006). Nevertheless, the biggest effects of fatty-acid composition and the
nutritive quality of foods of animal origin, like meat and milk products, depend on the feed
quality and the health of the animals.
3.2 Studies in humans
These TFA-containing fats can be incorporated into both foetal and adult tissues, although
the transfer rate through the placenta continues to be a contradictory subject. In preterm
infants and healthy term babies, the trans isomers have been inversely correlated with infant
birth mass (Koletzko & Mller, 1990). Maternal milk reflects precisely the mothers daily
dietary intake of TFAs, with presence of 2% to 5% total TFAs in human milk. The levels of
linoleic acid in human milk are increased by a high trans diet, although long-chain
polyunsaturated TFAs remain mostly unaffected (Koletzko, 1992; Koletzko & Desci, 1994).
Alterations in the maternal dietary intake of PUFAs cause similar changes in the PUFA
content of their milk. Several investigations have shown that supplementation of the
consumed fat with fish oils increases the amounts of C20:5n-3 and C22:6n-3 in the milk and
in the maternal and infant erythrocyte lipids. Likewise, infant tissues incorporate the TFAs
from the maternal milk, increasing the levels of linoleic acid and decreasing arachidonic acid
and docosahexaenoic acid. This suggests an inhibitory effect of TFAs on the liver n-6 fatty-
acid-desaturase activity (Jensen et al., 1992).

The Cardiovascular System Physiology, Diagnostics and Clinical Implications

50
As opposed to blood and liver, the brain appears to be protected from TFA accumulation in
experimental animals, although no data have yet been reported for newborn humans
(Larqu, 2001). A significant interaction between diet and pregnancy was shown for the
activities of 6-desaturase and glucose 6-phosphatase in liver microsomes: dietary TFAs
decreased the activities of both of these enzymes, although only in pregnant rats (Larqu et
al., 2000; Larqu & Zamora, 2000; Larqu et al., 2003). In Spain, TFAs in human milk were
investigated by Boatella et al. (Boatella et al., 1993), and they showed that the average
content of TFAs in 38 samples was 0.98% of the milk fatty acids. This value is lower than
that for human milk from other developed countries, where consumption of hydrogenated
fats is higher. In a study by Chen et al. (Chen et al., 1995) on TFAs in human milk in Canada,
the mean total TFA content was 7.19% (3.03%) of the total milk fatty acids, with a range
from 0.10% to 17.15%.
The compelling data linking dietary TFAs to increased risk of CHD have originated from
large, prospective, population-based studies, which included from 667 to 80,082 men and
women across different age groups who were monitored for six to 20 years. This link has
also been seen in controlled feeding trials (Oomen et al., 2001). Among these studies, there
are: the United States Health Professionals follow-up study; the Finnish alpha-tocopherol,
-carotene Cancer Prevention Study; the United States nurses health study (with 14-year
and 20-year follow-up) (Willett, 2006); and the Dutch Zutphen elderly study (Oomen et al.,
2001). These studies are consistent in their finding of a strong positive association between
TFA intake and the risk of CHD. Interestingly, a weaker correlation between SFA intake and
the risk of CHD also has been reported (Willettt, 2006).
The Zutphen elderly study included 667 men from 64 to 84 years of age who were free of
CHD at baseline (Oomen et al., 2001). Dietary surveys were used to establish the food
consumption patterns of the participants. Information on risk factors and diet were obtained
in 1985, 1990 and 1995. After a 10-year follow-up, from 1985-1995, there were 98 cases of
fatal or non-fatal CHD. The findings showed that over this period, the mean TFA intake
decreased from 4.3% to 1.9% of the food energy. After adjustments for age, BMI, smoking
and dietary covariates, TFA intake at baseline was positively associated with 10-year risk of
CHD. Thus, a high intake of TFAs, which included all types of isomers, contributed to the
risk of CHD. A substantial decrease in TFA intake, which was mainly due to the lowering of
the TFA content in edible fats in the Dutch industry, therefore had a large impact on public
health (Craig-Schmidt, 2006; Larqu et al., 2001).
In multiple and rigorous randomised trials, the intake of TFAs has been consistently shown
to have adverse effects on blood lipids, and most notably on the LDL/HDL cholesterol ratio,
which is a strong marker of cardiovascular risk. When a mixture of TFA isomers obtained by
partial hydrogenation of vegetable oils is used to replace oleic acid, there is a dose-
dependent increase in the LDL/HDL ratio. The relationship between the levels of TFAs as
the percentage of energy and the increase in the LDL/HDL ratio appears to be
approximately linear, with no evidence of a threshold at low levels of TFA intake, and with
a slope that is twice as steep as that observed by replacing oleic acid with a SFA (Borra et al.,
2007; Mensink & Nestel, 2009). Studies comparing animal and vegetable TFAs have shown
similar effects on the total/HDL cholesterol ratio. The effects of TFAs on lipoproteins from
both sources appeared at doses exceeding 2% of energy (Mensink & Nestel, 2009). The
average impact of TFA-induced changes in the LDL/HDL ratio corresponds to tens of

Trans Fatty Acids and Human Health

51
thousands of premature deaths in the United States alone (Mensink & Nestel, 2009).
Although dramatic, this effect is substantially smaller than the increase in cardiovascular
mortality associated with TFA intake in epidemiological studies, suggesting that other
mechanisms are likely to contribute to the toxicity of TFAs (Ascherio, 2006). Thus, although
there is accumulating evidence linking inflammatory proteins and other biomarkers to
CHD, lipid concentrations in the blood remain one of the strongest and most consistent
predictors of risk. Therefore, the LDL/HDL cholesterol ratio is probably the best marker to
date for estimating the effects of TFAs on plasma lipids, which are most likely relevant to
CHD incidence and mortality (Larqu & Zamora, 2001).
Further rigorous randomised trials to establish the effects of hydrogenated fats and TFA
intake on individual lipoprotein classes started in 1990, when a report from The Netherlands
suggested that a diet enriched in elaidic acid (trans-9 C18:1) increases the total and LDL
cholesterol concentrations and decreased HDL cholesterol concentrations, compared to a
diet enriched in oleic acid. In contrast, enrichment of the diet with SFAs increases LDL
cholesterol, but has no effect on HDL cholesterol, thus resulting in a smaller adverse change
than in the case of elaidic acid (Mensink & Katan, 1993; Mensink & Nestel, 2009).
3.3 Studies of antioxidant effects
In one study (Filip et al., 2011), the effects of natural antioxidants on formation of TFAs
during heat treatment of sunflower oil was investigated. The data from the fatty acid
analyses are summarized in Table 2. Here, the non-treated control sunflower oil had a 7.5%
palmitic acid content, with 4.5% stearic acid, 25.0% oleic acid, and 60.5% linoleic acid, as is
usual for the common (not high in oleic acid) sunflower oils; these data compare well with
those of other studies (Snchez-Gimeno et al., 2008; Bansal, Zhou, Tan, Neo, & Lo, 2009).
This sunflower oil was purchased directly from a supplier of oils that are used mainly by
small food enterprises (Zvijezda d.d., Zagreb, Croatia). The natural antioxidant extract of
rosemary (Rosmarinus officinalis L.) that was added to this sunflower oil (SOR) was
purchased directly from Vitiva d.d., Markovci, Slovenia (INOLENS4; Product N 301770;
Batch N. LAB. 09-779004), and had a carnosic acid content of 4.30%. Similarly, the lutein
added to this sunflower oil (SOL) was from pelargonium (2.2% mixture), as obtained from
Etol, d.o.o., Celje, Slovenia (NovaSoL

Lutein; Aquanova AG, Birkenweg 8-10, Germany).
The initial levels of the total TFAs in the samples was 0.91% (0.01%). This compares with
the range from 0.15% to 6.03% reported by Bansal et al. (2009) for TFAs in refined oils
(soybean, corn, sunflower, high oleic sunflower, low erucic rapeseed and high erucic
rapeseed oils). The aim in this study with the sunflower oil was to evaluate the effects of
heat on this TFA composition of the oil when subjected to treatment representative of deep-
fat frying (185 5C). Since sunflower oil is in common use for deep-fat frying, it is
particularly important to know what species and levels of TFA isomers appear during such
heat treatment (Filip et al., 2011; Martin et al., 2007).
In this study, we focussed mainly on these effects of heat on the TFAs with 18 carbon atoms,
which were the most represented. Prior to the treatment, the content of trans C 18:1, t-9 was
0.67% (0.08%). At the end of the heat treatment (120 h at 185 5C), in the control sunflower
oil the trans C 18:1, t-9 increased to 1.12% (0.14%), in SOR, to 0.99% (0.04%), and in SOL, to
0.91% (0.01%). Within each treatment, these increases were significantly different from the

The Cardiovascular System Physiology, Diagnostics and Clinical Implications

52
Component Time (h)
0 24 48 72 96 120
Sunflower oil (control)
SFAs (%) 12.43 0.13
b
12.54 0.12
b
12.77 0.58
b
14.02 0.49
ab
14.62 2.63
b
14.76 0.35
a
MUFAs (%) 26.13 0.68
d
28.56 1.22
c
29.40 1.06
cb
29.84
0.85
abc
31.18 0.96
ab
31.59 0.35
a
PUFAs (%) 61.44 0.75
a
58.50 1.23
b
57.83 1.33
b
56.14 1.15
bc
56.20 3.01
bc
53.64 2.08
c
n6 PUFAs
(%)
61.13 0.76
a
58.20 1.23
b
57.82 1.34
b
55.64 1.17
bc
55.66 3.03
bc
53.05 2.08
c
n3 PUFAs
(%)
0.31 0.02
b
0.30 0.00
b
0.31 0.02
b
0.34 0.01
a
0.35 0.02
a
0.36 0.01
a
n6/n3
199.93
15.84
a
192.92
4.71
a
185.41
11.56
a
163.68
7.73
b
159.78
16.01
b
149.37
6.99
b
TFAs (%) 0.91 0.03
d
0.99 0.06
d
1.25 0.07
c
1.46 0.15
b
1.56 0.09
b
1.71 0.07
a
Sunflower oil with rosemary extract (SOR; 1.0g/kg oil)
SFAs (%) 12.39 0.57c 12.70 0.48c 12.74 0.41c 13.80 0.29
b
14.02 0.70
ab
14.68 0.61
a
MUFAs (%)
25.72
1.92bc
25.37 1.67c
28.73
0.81ab
25.69 3.46
bc
28.87 0.40
ab
30.25 2.24
a
PUFAs (%) 61.88 1.67a 61.93 1.43a
58.53
0.57bc
60.51 3.33
ab
57.11 0.55
cd
55.07 1.71
d
n6 PUFAs
(%)
61.58 1.66a 61.62 1.43a
58.13
0.56bc
60.07 3.32
ab
56.56 0.57
cd
54.45 1.72
d
n3 PUFAs
(%)
0.31 0.02b 0.31 0.01b 0.31 0.01b 0.33 0.01
a
0.33 0.01
a
0.35 0.01
a
n6/n3
201.53
10.73a
198.56
6.72a
186.36
5.67b
179.91
9.26
bc
169.54
7.89
c
154.65
3.96
d
TFAs (%) 0.91 0.09d 0.82 0.02d 1.02 0.05c 1.24 0.20
c
1.35 0.10
b
1.55 0.16
a
Sunflower oil with lutein (SOL; 0.1g/kg oil)
SFAs (%) 12.51 0.72
c
12.36 0.35
c
13.06 0.36
bc
13.21 0.65
bc
13.91 0.54
ab
14.84 0.96
a
MUFAs (%) 26.25 2.60
b
25.05 0.64
b
28.22 2.49
b
27.55 2.79
b
28.07 0.85
b
32.79 1.63
a
PUFAs (%) 61.24 2.82
ab
62.59 0.72
a
58.72 2.47
b
59.24 3.15
b
58.02 1.37
b
52.37 0.74
c
n6 PUFAs
(%)
60.93 2.83
ab
62.29 0.72
a
58.31 2.48
bc
58.80 3.15
bc
57.51 1.40
c
51.80 0.73
d
n3 PUFAs
(%)
0.31 0.02
b
0.31 0.01
b
0.32 0.01
ab
0.33 0.02
ab
0.33 0.02
ab
0.35 0.03
a
n6/n3
199.10
16.69
ab
203.99
5.54
a
180.24
9.67
bc
177.99
19.87
c
174.65
12.92
c
149.07
10.26
d
TFAs (%) 0.91 0.06
c
0.84 0.03
c
1.01 0.02
b
1.23 0.16
b
1.28 0.11
b
1.43 0.04
a
SFAs, saturated fatty acids; MUFAs, mono-unsaturated fatty acids; PUFAs, polyunsaturated fatty acids;
TFAs, trans fatty acids;
a, b, c, d
Values followed by a different letter are significantly different along each
row according to the Duncan test (P <0.05);
Table 2. Effect of cooking heat (185 5C) on the fatty acids composition of sunflower oil,
with the addition of the natural antioxidants of a rosemary extract (SOR) and of lutein (SOL)
(Filip et al., 2011)

Trans Fatty Acids and Human Health

53
start to the end of the treatment (P <0.001), and also the decreases in trans C 18:1, t-9
production with the addition of rosemary oil and lutein were statistically significant in
comparison with the control (SOR vs. sunflower oil: 0.32% vs. 0.45%; SOL vs. sunflower oil:
0.24% vs. 0.45%; P <0.001 for both). These data are consistent with an earlier report where
there were reductions in trans-isomerisation and polar compounds in model oils when -
tocopherol (1%) was added as an antioxidant (Tsuzuki et al., 2008).
When the content of the total TFAs is expressed as the sum of the unsaturated FAs with at
least one trans double bond, these increased significantly from the initial control sunflower
oil of 0.91% (0.03%), to 1.71% (0.07%) at 120 h, with significantly lower increases for SOR
and SOL, to 1.55% (0.16%) and 1.43% (0.04%), respectively (Table 2). Indeed, these
differences among treatments were statistically significant (P <0.001) at each step of the heat
treatment (24, 48, 72, 96, 120 h). These data relating particularly to the increases in TFAs are
comparable to those of Gamel et al. (1999), where they looked at the effects of phenol
extracts on TFA formation during frying. A linear relationship between the amounts of
elaidic acid and the number of frying cycles has also been reported (Bansal et al., 2009).
According to the nutritional recommendations of the various health authorities, the content
of SFAs should not exceed 30% in dietary fats. Sunflower oil thus fits into this
recommendation, even though its content in the control sunflower oil increased from 12.43%
(0.13%) to 14.76% (0.35%), and in the SOR and SOL to 14.68% (0.61%) and 14.84%
(0.96%), respectively (Table 2).
The initial PUFA:SFA ratio here was 4.94 (0.10), and after the full time of the heat exposure
for the control sunflower oil, this was significantly decreased to 3.64 (0.14) (P 0.05).
Meanwhile, , for the SOR and SOL at 120 h of heat treatment, the PUFA:SFA ratio decreased
to 3.75 (0.09; P <0.001) and 3.54 (0.18; P <0.001). As higher PUFA/SFA ratios are more
nutritionally appropriate, these data confirm that the heat treatments of this sunflower oil
also worsened this nutritional factor.
4. Trans fatty acids and legislation
Governments are increasingly recognising that the risks to consumers from the increased
consumption of TFAs cannot be ignored. In 2003, Denmark became the first country to
introduce laws to control the sale of foods containing TFAs. This started with the
publication of a study in The Lancet by Willett in 1993. Then the Danish Nutrition Council,
which was established in 1992, was the driving force behind the campaign that convinced
Danish politicians that IP-TFAs can be removed from foods without any effects on their
taste, price or availability. The Nutrition Council argued that as no positive health effects of
IP-TFAs had ever been reported, then just the suspicion that a high intake has harmful
effects on health justified the ban (Astrup, 2006; Mjs, 2003). The Danish success story might
be interesting for other countries, where this unnecessary health hazard could also be
eliminated from the foods.
Then in January 2006, it became law in the United States that the contents of TFAs have to be
specifically listed on food labels. There is a complication to this, however, because there
were two reasons why the consumers might not see a TFA content on the label of a food
product. First, although products entering interstate commerce on or after 1 January, 2006,
had to be labelled, the FDA realized that it would take some time for food products to move

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54
through the distribution chain to a store shelf. Then, foods that contain less than 0.5 g TFAs
per serving can be labelled as being free from TFAs. Furthermore, in Europe, the declaring
of TFAs on food labels is still not obligatory in many countries. At the same time, these
regulations only applied to food that was labelled; food sold in restaurants and canteens
was not covered by this law (FDA, 2003; Moss, 2006; Stender et al., 2006). Thus many still
feel that foods that contain more than 4 g/100 g SFAs and TFAs together should not be
claimed to be healthy food. Indeed, Danish law prohibits the sale of foods that contain more
than 2 g TFAs per 100 g of fat, excluding food that naturally contains more TFAs (Filip et al.,
2010). Denmark decided to impose this maximum level of IP-TFAs as labelling was deemed
insufficient to protect consumers, and especially for risk groups like children and adults
with a high intake of fast foods (Garchs & Mancha, 1993; Leth et al., 2006).
Then, in December 2006, the Board of Health of New York City banned many TFAs from
restaurants in the city, prompting similar moves in Philadelphia, Montgomery County in
Maryland, and the Boston suburb of Brooklyn. The first phase of the regulation applies to
oils, shortening and margarine, used in cooking and as spreads, and for recipes that contain
more than 0.5 g TFA per serving. Since 1 July, 2007, New York City officials have also called
for restaurants to clearly display calorie counts next to their menu items, in a bid to increase
consumer awareness of the nutritional content of their food. By 1 July, 2008, the ban had
been extended to include TFAs used in baked goods, including bread and cakes, in prepared
foods, salad dressings and oils used for deep frying, and in dough and cake batter. Similar
bans are being proposed in Chicago and in the state of Illinois; other cities may follow suit,
most likely in California (Albers et al., 2008; Blake, 2009).
The American Heart Association recommends a healthy dietary pattern and lifestyle to
combat heart disease, limiting TFA consumption to less than 1% (or approximately 2 g on a
2,000-calorie diet), and saturated fat consumption to less than 7% of the total daily calories
(Borra et al., 2007). This is consistent with the TFA recommendations made by the American
Dietetic Association and the Dietitians of Canada (ADA, 2007).
The benefits of adding TFAs on food Nutrition Facts labels in the United States means that
consumers now know the levels of SFAs, TFAs and cholesterol in the foods that they choose
to eat. This enables them to make heart-healthy food choices, to help them to reduce their
risk of CHD. This labelling is also of particular interest to those concerned about high blood
cholesterol. However, to gain the full benefit of this system, all of the consumers need be
aware of the risk posed by consuming too high levels of SFAs, TFAs and cholesterol.
At the same time, about half of the convenience products on the Austrian market that have
been tested contained less than 1% TFAs, and one third less than 5% (Wagner et al., 2008).
However, almost 5% of the products tested contained more than 20% TFAs. A similar level
was seen for fast food products, with the highest TFA levels of 8.9%, while the total TFAs of
household fats were significantly lower (1.45% 1.99%) than fats for industrial use (7.83%
10.0%; P <0.001). Compared to investigations in Austria (and Germany) around 10 years
ago, the TFA contents of foods have decreased significantly. About half of the investigated
products contained less than 1% of TFAs or total fatty acids, although very high levels of
TFAs (>15%) are still detected, and an intake of more than 5 g TFA per portion is possible,
which has been shown to significantly increase the risk of CHD (Oomen et al., 2001; Wagner
et al., 2008; Wilett, 2006).

Trans Fatty Acids and Human Health

55
5. Analytical methods for trans fatty acid determination
The fatty acid composition of food is usually determined using gas-liquid chromatography
of the corresponding fatty acid methyl esters (FAMEs) (Baggio et al., 2005; Bondia-Pons et
al., 2004; Chen et al., 1999; Ratnayake, 1995; Ulberth & Henninger; 1992). Usually, the
FAMEs can be conveniently prepared by heating lipids with a large excess of either acid-
catalysed or base-catalysed reagents. However, most of the analytical methods are time
consuming and impractical for the processing of large numbers of samples, because the
lipids have to be extracted prior to preparation of the FAMEs. For this reason, some
procedures have been developed that can be used to prepare FAMEs directly from fresh
tissue (Park & Goins, 1994; Garchs & Mancha, 1993).
6. Consumption of trans fatty acids
Vaccenic acid (trans-11 C18:1) accounts for over 60% of the natural TFAs, whereas with IP-
TFAs, a broad mixture of TFAs is produced, with elaidic acid (trans-9 C18:1) as the main
product (Oomen et al., 2001). In recent years, new technologies have been developed to
reduce the TFA content in fats and oils used in the manufacture of food products. As
indicated above, the content of TFAs in Danish food has been monitored for the last 30
years. In margarine and shortening, the TFA content has steadily declined, from about 10 g
per 100 g of margarine in the 1970s, to practically no TFAs in margarine in 1999, to
efficiently reduce the health risk related to TFAs.
In North America, the daily TFA intake has been estimated using food frequency
questionnaires, and it was found to be 3-4 g per person (ADA, 2007), while by extrapolation
of human milk data, it was said to be greater than 10 g per person (Chardigny et al., 1995).
The data also show that the levels of TFAs can vary considerably among foods within any
specific category, reflecting the differences in the fats and oils used in the manufacturing or

preparation processes. For example, the range of TFAs in 17 brands of crackers was from
23% to 51% of the total fatty acids, which represents differences of 1 g to 13 g TFAs per 100 g
of crackers. These data thus show that the wide variability in the TFA content of different
foods can result in large errors

in the estimation of the TFA intake of individuals,

and
potentially, of groups (Innis, 2006).
TFA consumption in European countries varies considerably. The diet in northern European
countries traditionally contains more TFAs than that in the Mediterranean countries, where
olive oil is commonly used. The diet in France has always been relatively low in TFAs, because
France has traditionally used predominantly ruminant fats, as compared to hydrogenated
vegetable oils. A more recent decrease in dietary TFAs has been seen due to the modification
of commercial fats and changes in consumer choice (Larqu et al., 2001). In the TRANSFAIR
study (Poppel et al., 1998), which was based on a market basket analysis of diets across 14
European countries, the mean daily intake of TFAs in European countries ranged from the
lowest in Greece (1.4 g TFA per day) to the highest in Iceland (5.4 g TFA per day) (Fig. 3).
The lover daily intake of TFAs was recorded in Greece where 1.4 g of TFAs are consumed
per day what represent 0.6 % of daily energy intake. The highest daily intake of TFAs was
recorded in Iceland where 5.4 g of TFAs are consumed per day what represent 2.0 % of daily
energy intake. As shown by researches (Innis et al., 1999; Leth et al., 2006; Poppel et al.,
1998) the lowest TFA intake is more often in countries with Mediterranean type of nutrition
habits (Mediterranean diet).

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56

Fig. 3. Mean daily intake of TFAs across the European countries (Innis et al., 1999; Leth et al.,
2006; Poppel et al., 1998)
6.1 Dairy products and trans fatty acids
Milk fat is also the most abundant source of conjugated linoleic acids (CLAs), which are a
group of geometrical and positional isomers of linoleic acid (LA cis-9,cis-12 C18:2). The
major isomer of the CLAs in milk fat is cis-9, trans-11, and it represents 80 g to 90 g per 100 g
of the total CLAs (Chardigny et al., 1995; Ledoux et al., 2005; Sekim et al., 2005). Some of
these fatty acids have biological, physiological and nutritional properties that are very
interesting for consumer health, as especially seen for butyric acid and CLAs (Pandya &
Ghodke, 2007). The CLAs are synthesised in ruminants both from dietary linoleic acid (cis-
9,cis-12 C18:2) in the rumen by the microbial flora, and from vaccenic acid (trans-11 C18:1) in
the mammary glands during de-novo synthesis (Bauman & Griinari, 2001).
6.2 Industrially produced fat and trans fatty acids
Brt and Pokorn (Brt & Pokorn, 2000) investigated a series of 20 margarines, nine
cooking fats, and butter that were available on the Czech market. They used the American
Oil Chemistry Society standard analysis methods, with capillary gas chromatography. The
margarines contained 15.2% to 54.1% cooking fats, and 16.5% to 59.1% SFAs, which was less
than the butter. The content of linoleic acid varied between 3.7% and 52.4% in the
margarines; small amounts of linolenic acid were present in most samples, while oleic acid
prevailed in the cooking fats. Monoenoic TFAs were present only in trace amounts in 10
samples, and trans-polyenoic acids were present only in small amounts. Most cooking fats
had a high content of TFAs. They summarised these data by indicating that the number of
trans-free margarines had rapidly increased over a few years.
More recently, Ceni-Kodba (Ceni-Kodba, 2007) examined 13 margarines and fatty food
samples in Slovenia, which were selected according to the frequency of use among the

Trans Fatty Acids and Human Health

57
population group in the community. All of the fried food and bakery food samples included
in this study contained TFAs, the levels of which varied from less than 0.5% to 6.8%. The
highest TFA content in the margarines was 5.2%, with 0.3% as the lowest, and a mean
margarine TFA content of 2.3%. The main TFAs were the trans isomers of mono-unsaturated
octadecenoic acid (C18:1).
Similarly, the findings of Larqu et al. (Larqu et al., 2003) suggest that Spanish margarines
have moved to becoming products with a potentially healthier distribution of fatty acids.
Even so, the great variability shown in the fatty-acid compositions of margarines and the
poor labelling continue to highlight the importance of greater consumer information to
avoid detrimental changes to the traditional Mediterranean diet in Spain.
7. Conclusions and future trends
It can be concluded at present that the reduction of TFAs in the food supply is a complex
issue that has involved, and still involves, interdependent and interrelated stakeholders.
Any further actions taken to reduce TFAs need to be carefully considered, regarding both
the intended and unintended consequences related to nutrition and public health. As shown
above, the WHO (WHO/FAO, 2002) has already included TFA levels in their recommended
daily food intake (Table 1). Many different options of alternative oils and fats can now be
used to replace TFAs, as many of these are already available, while others are still being
developed. However, decisions on which alternatives to use are complicated and often time
consuming, and they involve considerations of health effects, food availability, quality and
taste, research and development investments, supply-chain management, operational
modifications, consumer acceptance, and cost (Borra et al., 2007; FDA, 2003).
As industry responses are now well underway following the policy actions over the past
few years, it is possible to take a present-day snapshot of industry activities that provide
preliminary answers to these considerations. The first results of most of the anti-trans fat
campaigns can be seen as modifications that have been made to the fatty-acid compositions
of industrial fats. In these fats, there are significantly higher levels of SFAs and possibly a
higher index of atherogenicity. Several major food companies have announced efforts to
remove TFAs from their leading brands over the past two decades, starting with Unilever in
the 1990s, and then more recently with Nestl in 2002, Kraft in 2003, Campbells in 2004 (for
Goldfish crackers), Kelloggs in 2005, and Frito-Lay in 2006 (for chips). It is of note that the
earliest announcements came from European firms, where the use of partially hydrogenated
soy was not as common as it was in the United States, and thus this reformulation process
has not been as onerous.
The announcements over the last three years or so have reflected the attention brought to
this issue through lawsuits and debates about nutritional labelling regulations. Many
companies even chose to implement the disclosure of these trans-fat contents earlier than the
January 1, 2006, deadline, particularly when they were able to advertise zero trans fats on
their products (Crisco, 2008).
One aspect for producing such zero TFAs lies in the transesterification reactions between
vegetable oils and the SFAs of C8:0, C12:0, C14:0 and C16:0. These reactions can be catalysed
by an immobilised sn-1,3 specific Rhizomucor miehei lipase. When considering a TFA-free or

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58
low TFA fat that is suitable for use as a confectionery fat, a non-hydrogenated vegetable fat
composed of an inter-esterified fat can be used: this can be obtained by subjecting a blend of
at least one fat rich in lauric acid and at least one fat without lauric acid to inter-
esterification (Farmani et al., 2007).
For all of the products introduced in 2005 and 2006 that have claimed to contain no trans
fats, the most commonly used oil ingredients have been canola, sunflower and soybean oils.
Palm oil, which is high in saturated fat, also appears among the commonly used ingredients,
but not as an alternative to reducing TFAs. Eleven percent of food producers in the United
States still use partially hydrogenated oils as ingredient, because the regulations allow 0.5 g
per serving of trans fats in products that claim to contain no trans fat, while the use of small
amounts of partially hydrogenated oils has facilitated the reformulation of some products
(Unnevehr & Jagmanaite, 2008).
Between 2006 and 2007, consumer awareness of trans fats increased and attained levels
similar to those for saturated fats. This increased awareness has been associated with
improved self-reporting behaviour in consumer shopping for groceries (Eckel et al., 2009).
However, food labels and food claims that accompany packed foods are still largely
incomprehensible for consumers, and therefore they appear to be of very little use at
present. Moreover, in Europe, consumers still cannot identify the content of TFAs in the
labelling of food products, particularly as the only legislation that restricts the content of
TFAs in Europe is in Denmark.
At the same time, we have to be aware that indicators are showing that the world
population is still increasing and is expected to reach nearly 8.9 thousand million
(8,900,000,000) by the year 2050 (UN, 2004). Knowing of some of the problems that are
associated with this increasing population, we are now combating the need that will arise
for more and more potential food products to be used for biofuels (Fink & Medved, 2011).
Thus, in the future, it will become increasingly difficult to assure food security and food
safety, as well as the nutritional quality of food. Indeed, it is the nutritional quality of food
and its distribution all over the World that are the main factors that will have a huge impact
on human health. In this way, human health is more than just of personal value, as it is also
part of the welfare of the whole of our society.
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4
Control and Coordination
of Vasomotor Tone in the Microcirculation
Mauricio A. Lillo, Francisco R. Prez, Mariela Puebla,
Pablo S. Gaete and Xavier F. Figueroa
Departamento de Fisiologa, Facultad de Ciencias Biolgicas,
Pontificia Universidad Catlica de Chile, Santiago,
Chile
1. Introduction
The blood vascular system consists in a complex network of vessels that is mainly intended
to provide oxygen and nutrients to all individual cells of peripheral tissues and help to
dispose metabolic wastes. Several distinct functional compartments can be distinguished in
the vascular network: arteries, arterioles, capillaries, venules and veins. Conduit arteries
(diameter, 1 to several millimeters) carry blood away from the heart through a divergent
arborescence that reaches and penetrates into the tissues via the feed arteries (diameter, 100
to 500 m) (Davis et al., 1986; Segal, 2000, 2005). These muscular vessels give rise to the
arterioles (diameter, < l00 m), which control and coordinate the blood flow distribution in
such a way that each capillary is correctly supplied at the proper pressure (Mulvany, 1990;
Segal, 2005). This part of the vascular network composed of arterioles, capillaries and
venules is embedded within the organ irrigated and is called microcirculation (Davis et al.,
1986; Segal, 2005; Lockhart et al., 2009). Finally, veins carry blood back to the heart through a
convergent arborescence.
In general, the vascular wall of arteries consists of an outer tunica adventitia, a central tunica
media, and an inner tunica intima. The adventitia mainly contains connective tissue,
fibroblasts, mast cells, macrophages, and nerve axons. Although the amount of the wall
taken up by adventitia varies with the vascular territory, it is directly proportional to the
size of the vessel (Gingras et al., 2009). The media is comprised of circumferentially arranged
smooth muscle cells and is bounded on the luminal side by a well-defined internal elastic
lamina. An external elastic lamina may also be present between the media and the
adventitia or even within the media in larger vessels such as the aorta, but this structure is
fragmented in small arteries and absent in arterioles (Mulvany, 1990; London et al., 1998).
The number of smooth muscle cell layers decreases with decreasing vessel diameter and, in
arterioles, only an unbroken monolayer of smooth muscle cells is found (Davis et al., 1986;
Mulvany, 1990; Segal, 2005). In contrast, the structure of the intima is similar in all blood
vessels and is formed by a smooth, continuous single layer of endothelial cells that lines the
inner surface of the vessels (Mulvany, 1990). These cells are very thin (2 m thick) and
elongated (10 to 20 m wide and 100 to 150 m long, in arterioles), and are oriented parallel
to the longitudinal axis of the vessel (Haas & Duling, 1997).

The Cardiovascular System Physiology, Diagnostics and Clinical Implications

66
Correct supply of blood to the tissues relies on the ability of the vascular system to adjust
the resistance of each vessel by controlling its lumen diameter, which is, in turn, a function
of the level of tone of the vascular smooth muscle (i.e. vasomotor tone). As blood vessels are
complex structures that must work as an unit, control of vasomotor tone depends on the fine
synchronization of function of the different cellular components of the vessel wall, mainly
smooth muscle cells and endothelial cells (Segal, 2000; Figueroa et al., 2004; Segal, 2005;
Figueroa & Duling, 2009). Such synchronization and coordination is accomplished by an
intricate system of radial and longitudinal cell-to-cell communication (Beach et al., 1998;
Figueroa et al., 2004; Rummery & Hill, 2004; Segal, 2005; Figueroa & Duling, 2009; Bagher &
Segal, 2011). In addition, arterioles in the microcirculation form a complex network, and
then, the changes in the luminal diameter of different arteriolar segments must also be
coordinated to regulate blood flow distribution and peripheral vascular resistance (Figueroa
et al., 2004; Rummery & Hill, 2004; Segal, 2005; Figueroa & Duling, 2008). It has typically
been assumed that most of the total resistance to blood flow resides on the arterioles.
However, it has become apparent that as much as 50% of the precapillary resistance lies
proximal to the arterioles (Davis et al., 1986; Mulvany, 1990; Segal, 2000), which situates the
feed arteries at a key point for controlling vascular function and highlights the importance
of the functional communication between arterioles and feed arteries in the regulation of
blood flow distribution.
It is widely recognized that the endothelium plays a critical role controlling function of the
vessel wall by the release of paracrine molecules such as nitric oxide (NO), prostaglandins
(PGs) and also by the activation of the signaling mechanism known as endothelium-derived
hyperpolarizing factor (EDHF) (Moncada et al., 1991; Busse et al., 2002; Feletou & Vanhoutte,
2007; Vanhoutte et al., 2009). However, another mechanism of communication that has
emerged as a key pathway to command and coordinate the vascular wall function is the
direct cell-to-cell communication via gap junctions (Sandow et al., 2003; Figueroa et al., 2004,
2006). In addition, it is important to note that K
+
channels expressed in the endothelium and
smooth muscle cells play a central role in the control of vasomotor tone by paracrine or gap
junction-mediated signaling mechanisms (Jackson, 2005).
2. Membrane potential and vascular K
+
channels
In contrast to endothelial cells, Ca
2+
is a signal for contraction in smooth muscle cells. In
smooth muscle cells of blood vessels the L-type voltage-dependent Ca
2+
channels play a
central role controlling the vasomotor tone (Jackson, 2000). Changes in membrane potential
modulate the opening of these Ca
2+
channels. Thereby, depolarization produces a Ca
2+

influx that leads to vasoconstriction and, on the contrary, hyperpolarization leads to a
reduction in intracellular Ca
2+
concentration and, subsequently, vasodilation (Jackson, 2000,
2005). In this context, K
+
channels play a pivotal role in vascular function by controlling the
membrane potential of both endothelial and smooth muscle cells. The main K
+
channels
expressed in resistance vessels, from a functional point of view, are: the ATP-sensitive K
+

channels (K
ATP
), inward rectifying K
+
channels (K
ir
) and Ca
2+
-activated K
+
channels (K
Ca
) of
small (SK
Ca
), intermediate (IK
Ca
) and large (BK
Ca
) conductance (Jackson, 2000, 2005). K
ATP

and K
ir
are expressed in both endothelial and smooth muscle cells (Quayle et al., 1996;
Jackson, 2000, 2005; Ko et al., 2008), whereas BK
Ca
are mostly found in smooth muscle cells
(Jackson, 2005; Ko et al., 2008), but, on occasion, these K
+
channels have also been described

Control and Coordination of Vasomotor Tone in the Microcirculation

67
in endothelial cells (Papassotiriou et al., 2000; Wang et al., 2005). In contrast, SK
Ca
and IK
Ca

are expressed exclusively in endothelial cells (Jackson, 2000; Kohler et al., 2000; Nilius &
Droogmans, 2001; Eichler et al., 2003; Taylor et al., 2003; Brahler et al., 2009).
All these K
+
channels play critical roles in the regulation of vascular function. K
ATP
channels
are opened at rest, and then, are very relevant in the control of smooth muscle membrane
potential and vasomotor tone in basal unstimulated conditions (Jackson, 1993, 2000).
Interestingly, K
ir
are typically closed at resting conditions, but are activated by
hyperpolarization of membrane potential and by increments in extracellular K
+

concentration ([K
+
]
o
) smaller than 20 mM (Jackson, 2005; Jantzi et al., 2006; Smith et al., 2008).
Although BK
Ca
channels are involved in the response to several vasomotor stimuli, the most
relevant function of these K
+
channels is the tonic control of vasomotor tone by buffering the
smooth muscle cell depolarization. The increase in intracellular Ca
2+
concentration
associated to smooth muscle depolarization activates local Ca
2+
transients (i.e. Ca
2+
sparks)
that result from the opening of tightly clustered ryanodine receptor channels located at
extensions of sarcoplasmic reticulum. Ca
2+
sparks activate a BK
Ca
-dependent
hyperpolarizing current that opposes the smooth muscle depolarization, and thereby,
regulates the magnitude of the vasoconstriction (Jaggar et al., 1998; Gollasch et al., 2000;
Gordienko et al., 2001; Lohn et al., 2001). SK
Ca
and IK
Ca
channels play a central role in the
endothelial cell control of vasomotor tone and peripheral vascular resistance (Busse et al.,
2002; Eichler et al., 2003; Taylor et al., 2003; Si et al., 2006; Brahler et al., 2009). However,
probably the most recognized function of these K
+
channels is their participation in the
EDHF signaling (see below) (Busse et al., 2002; Vanhoutte, 2004).
3. Paracrine signaling in the vessel wall
One of the most well-characterized mode of communication in the vessel wall is the
production of paracrine signals by endothelial cells such as PGs, NO and EDHF (Vanhoutte,
2004; Vanhoutte et al., 2009). The role of these signaling pathways in vascular physiology has
been extensively studied and there are several recent reviews that address their involvement
in vascular function in normal conditions and disease (Feletou & Vanhoutte, 2009;
Vanhoutte et al., 2009; Rafikov et al., 2011). In this section, we will address the most relevant
aspects of these signals in relation to the control of vasomotor tone in physiological
conditions.
3.1 Prostaglandins
PGs are a family of bioactive lipids derived from arachidonic acid (AA or 5,8,11,14-
eicosatetraenoic acid), which, in turn, is generated by the enzyme phospholipase A
2
(PLA
2
)
from phospholipids of the cell membrane in a Ca
2+
-dependent manner (Simmons et al., 2004;
Fortier et al., 2008). The metabolism of PGs is complex and depends on the hydrolysis of AA
by the enzymes cyclooxygenase-1 (COX-1) or cyclooxygenase-2 (COX-2) to form the
unstable endoperoxide derivative, prostaglandin G
2
(PGG
2
), and subsequently,
prostaglandin H
2
(PGH
2
) (Simmons et al., 2004). PGH
2
is the parent compound of all PGs,
which are synthesized by specific enzymes: prostaglandin I
2
synthase (PGIS), prostaglandin
E
2
synthase (PGES-1), prostaglandin D
2
synthase (PGDS), prostaglandin F
2
synthase
(PGES-2), and thromboxane A
2
synthase (TBXAS-1) that catalyze the production of

The Cardiovascular System Physiology, Diagnostics and Clinical Implications

68
prostacyclins (PGI
2
), PGE
2
, PGD
2
, PGF
2
and thromboxane A2 (TXA
2
), respectively
(Simmons et al., 2004; Gryglewski, 2008). The presence of the different PG synthases varies
from tissue to tissue. Finally, PGs are released to the extracellular space and exert their
physiological effects by acting on specific membrane receptors (Norel, 2007), as depicted in
Figure 1. Then, the production of prostanoids is triggered by an increase in intracellular Ca
2+

concentration and the key reaction of this complex enzymatic cascade is catalyzed by the
enzymes COXs (Figure 1).

Fig. 1. Biosynthetic pathway of prostaglandins (PGs). An increase in intracellular Ca
2+

concentration activates the production of arachidonic acid (AA) by phospholipase A2
(PLA2) from cell membrane phospholipids. The enzymes cyclooxygenase-1 (COX-1) or
cyclooxygenase-2 (COX-2) convert AA into the endoperoxide PGH2, which is then
metabolized by several synthases to PGs PGD
2
, PGE
2
, PGF
2
, TXA
2
and PGI
2
(prostacyclin).
Each PG acts on specific membrane receptors located in endothelial and/or smooth muscle
cells. The transduction pathways activated by PGs are also depicted in the figure.
COX-1 and COX-2 are very similar and show a 60% homology. However, COX-1 is
expressed constitutively, whereas the expression of COX-2 is inducible, since the levels of
this COX isoform are very low in normal conditions and its expression increases in response
to pro-inflammatory stimuli (Simmons et al., 2004). Consistent with this, in normal
physiological conditions, vascular endothelial and smooth muscle cells express COX-1
(Vanhoutte, 2009). In these cells, COX-1 mainly leads to the production of PGI
2
, which

Control and Coordination of Vasomotor Tone in the Microcirculation

69
induces the relaxation of smooth muscle cells by the stimulation of IP receptors (Figure 1)
(Gryglewski, 2008; Vanhoutte, 2009). In contrast to COX-1, expression of COX-2 in normal
blood vessels is very low (Crofford et al., 1994; Schonbeck et al., 1999). However, Topper et
al. (Topper et al., 1996) found that laminar shear stress, but not turbulent flow, up-regulates
the levels of COX-2 expression in cultures of vascular endothelial cells. Laminar shear stress
is a highly relevant stimulus that is involved in the tonic control of vasomotor tone, which
highlights the participation of COXs and PGs in the regulation of vascular function.
3.2 Nitric oxide
Probably, the most relevant intercellular communication signal in vascular physiology is the
endothelium-dependent NO production. NO is a potent vasodilator synthesized by the
enzyme NO synthase (NOS) (Moncada et al., 1991). The substrates for NOS-mediated NO
production are the amino acid L-arginine, molecular oxygen and nicotinamide adenine
dinucleotide phosphate (NADPH). Three isoforms of NOS have been described: endothelial
NOS (eNOS), neuronal NOS (nNOS) and inducible NOS (iNOS) (Moncada et al., 1991;
Alderton et al., 2001). The enzyme expressed in endothelial cells (eNOS) is the main NOS
isoform found in the vascular system in normal conditions. The NO released by endothelial
cells elicits the relaxation of the underlying vascular smooth muscle cells mainly through the
initiation of the signaling cascade cGMP/PKG by activation of soluble guanylate cyclase,
which has been ascribed as the primary receptor of NO (Moncada et al., 1991). Although
certainly the cGMP-dependent signaling pathway has several targets in the vessel wall, the
relaxation induced by NO is mainly associated with a reduction in the Ca
2+
sensitivity of
smooth muscle contractile machinery (Bolz et al., 1999; Bolz et al., 2003).
Consistent with the importance of NO in vascular function, the activity of eNOS is finely
regulated at transcriptional and posttranscriptional level (Fleming & Busse, 2003). Although
eNOS was initially characterized as a Ca
2+
-dependent enzyme and binding of the complex
Ca
2+
-calmodulin plays a central role in the activation of eNOS, NO production is also
modulated by phosphorylation and protein-protein interactions (Mount et al., 2007; Rafikov
et al., 2011). In this context, the sub-cellular targeting of eNOS is a key process in the
regulation of NO production. Two functional pools of eNOS have been identified in
vascular endothelial cells: one associated to Golgi complex and other located at caveolae, a
subset of invaginated plasmalemmal rafts where the function of key signaling proteins is
coordinated (Govers & Rabelink, 2001; Goligorsky et al., 2002; Michel & Vanhoutte, 2010),
which provides eNOS with a special proximity to signaling molecules, such as calmodulin,
Ca
2+
channels, BK
Ca
channels and plasma membrane Ca
2+
pumps (Darby et al., 2000; Wang
et al., 2005). Although both pools of eNOS have been demonstrated to be functional, it is
widely recognized that the integrity of caveolae is critical for the control of Ca
2+
-mediated
activation of NO production. In caveolae, eNOS is found in an inhibitory association with
caveolin-1, an integral membrane protein of this signaling microdomain, and the interaction
of eNOS with calcium-calmodulin releases the enzyme from its inhibitory association with
caveolin-1 (Govers & Rabelink, 2001; Goligorsky et al., 2002; Michel & Vanhoutte, 2010).
The eNOS localization at caveolae seems to be essential for the regulation of eNOS function
by controlling L-arginine substrate supply. Typically, regulation of L-arginine availability
has been under-appreciated, since intracellular L-arginine concentration is saturating from

The Cardiovascular System Physiology, Diagnostics and Clinical Implications

70
the perspective of eNOS kinetics (Km = ~5 M) (Harrison, 1997). However, several reports
indicate that increments in extracellular L-arginine levels can enhance NO production in
endothelial cells (Zani & Bohlen, 2005; Kakoki et al., 2006), despite a saturating intracellular
L-arginine concentration, which was termed as the Arginine Paradox (McDonald et al.,
1997). This control of NO production by substrate suggests that intracellular L-arginine is
not fully available for eNOS, whereas extracellular L-arginine is preferentially delivered to
the enzyme. Consistent with this notion, NO production seems to be coupled to L-arginine
uptake, because the main carrier that transports 60 80% of L-arginine across the plasma
membrane of endothelial cells, the cationic amino acid transporter-1 (CAT-1), was found to
co-localize with eNOS in caveolae (McDonald et al., 1997) (Figure 2). Interestingly, it was
reported that eNOS interacts directly with CAT-1 in bovine aortic endothelial cells (BAECs),
and apparently, the eNOSCAT-1 association in addition to facilitate the delivery of
extracellular L-arginine for NO generation, also enhances the eNOS enzymatic activity by
increasing the activating phosphorylation of the enzyme at serine 1179 and 635, and by
decreasing the association of eNOS with caveolin-1 (Li et al., 2005).

Fig. 2. Local control of eNOS activity by L-arginine. eNOS synthesizes nitric oxide (NO) and
the byproduct L-citrulline from L-arginine. The eNOS localization at signaling
microdomains known as caveolae provides to this enzyme with a direct, local source of L-
arginine. In caveolae, eNOS is in direct association with the main carrier of L-arginine in
endothelial cells, the cationic amino acid transporter-1 (CAT-1), and is also associated with
the enzymes argininosuccinate synthase (ASS) and argininosuccinate lyase (ASL) that
regenerate L-arginine from L-citrulline.

Control and Coordination of Vasomotor Tone in the Microcirculation

71
Another mechanism that has emerged as an important source of L-arginine supply for NO
production is the regeneration of L-arginine from the other product of the eNOS-catalyzed
reaction, L-citrulline. This regeneration is catalyzed by the enzymes argininosuccinate
synthase (ASS) and argininosuccinate lyase (ASL), which are mostly expressed in caveolae
in endothelial cells (Flam et al., 2001; Solomonson et al., 2003) (Figure 2). Interestingly,
addition of exogenous L-citrulline results in a larger increase in endothelial NO production
than that observed with exogenous L-arginine, without a proportional increase in
intracellular L-arginine (Solomonson et al., 2003), suggesting that recycling of L-citrulline to
L-arginine is channeled directly to synthesize NO (Figure 2). In addition, it was estimated
that under maximum stimulation of NO production with bradykinin, but not in
unstimulated conditions, approximately 80% of the eNOS-catalyzed L-arginine was
supplied by the recycling of L-citrulline (Solomonson et al., 2003). These findings indicate
that eNOS activation is functionally coupled with the L-citrulline recycling system (ASS and
ASL) in caveolae (Figure 2). Therefore, NO production seems to be regulated by a complex
interaction between different pools of L-arginine, where direct channeling to eNOS of the L-
arginine regenerated from L-citrulline by the coordinated action of the enzymes ASS and
ASL is likely to play a central role (Figure 2).
3.3 Endothelium-derived hyperpolarizing factor
Although the development of knockout animals has demonstrated the importance of the
multiple functions of NO along the whole vascular system, it has become apparent that the
relevance of NO in the control of vasomotor tone depends on vessel size. Accordingly, NO
is the primary endothelium-dependent vasodilator signal in large, conduit vessels
(Shimokawa et al., 1996). However, an additional vasodilator component has also been
identified in small resistance arteries and arterioles (Suzuki et al., 1992; Murphy & Brayden,
1995). In these vessels, blockade of NO and PG production only attenuates the response to
endothelium-dependent vasodilators such as acetylcholine (ACh) or bradykinin (Vanhoutte,
2004). The relaxant pathway resistant to NOS and COX blockers is associated with smooth
muscle hyperpolarization, and thereby, it was attributed to the release of an endothelium-
derived hyperpolarizing factor (EDHF). The chemical nature of EDHF remains controversial
and seems to depend on vessel size, vascular territory, and species (Vanhoutte, 2004). In this
context, several EDHF candidates have been proposed, such as K
+
ions (Edwards et al.,
1998), epoxyeicosatrienoic acids (EETs) (Archer et al., 2003; Fleming, 2004), hydrogen
peroxide (Shimokawa & Morikawa, 2005), and C-type natriuretic peptide (CNP) (Chauhan
et al., 2003; Ahluwalia & Hobbs, 2005). However, in most cases, the EDHF-mediated smooth
muscle hyperpolarization and vasodilation has been shown to be sensitive to simultaneous
blockade of SK
Ca
and IK
Ca
(Doughty et al., 1999; Ghisdal & Morel, 2001; Crane et al., 2003;
Eichler et al., 2003; Hilgers et al., 2006). Interestingly, these K
+
channels have been reported to
be located in two different subcellular domains. While SK
Ca
channels are found in caveolae
(Absi et al., 2007; Rath et al., 2009), IK
Ca
channels were proposed to be expressed in the
abluminal side of endothelial cells (Figure 3), facing Na
+
pumps and K
ir
channels situated in
smooth muscle cells (Edwards et al., 1998; Dora et al., 2008). Then, the opening of IK
Ca
channels
may increase the K
+
ion concentration in the myoendothelial space, which may couple
endothelial cell IK
Ca
signaling to Na
+
pump- and K
ir
channel-mediated smooth muscle
hyperpolarization (Edwards et al., 1998; Dora et al., 2008) (Figure 3).

The Cardiovascular System Physiology, Diagnostics and Clinical Implications

72

Fig. 3. K
+
channel distribution and endothelium-dependent vasodilation. Ca
2+
-activated K
+

channels of small (SK
Ca
) and intermediate (IK
Ca
) conductance may contribute to the
vasodilation associated with an endothelium-mediated smooth muscle hyperpolarization
(response typically attributed to an endothelium-derive hyperpolarizing factor, EDHF) by
two pathways. First, the hyperpolarization induced by activation of SK
Ca
and IK
Ca
is
transmitted electrotonically to the underlying smooth muscle cells (SMC) through the gap
junctions located at discrete points of contact between endothelial and smooth muscle cells,
structure known as myoendothelial junctions (MEJ). In addition, the small increase in
extracellular K
+
concentration (<20 mM) resulting from the opening of the IK
Ca
found at the
abluminal side of endothelial cells (EC) may activate inward rectifying K
+
channels (K
ir
) and
Na
+
pump in smooth muscle cells. Between SMC and EC is found the internal elastic lamina
(IEL).
Notwithstanding the smooth muscle hyperpolarization is considered to be the hallmark of
EDHF action (Vanhoutte, 2004), it is important to note that hyperpolarization of the vessel
wall is not a unique characteristic of EDHF. In several vessel preparations, in addition to a
reduced Ca
2+
sensitivity of the contractile machinery, the NO-dependent vasodilation has
also been associated with smooth muscle hyperpolarization (Cohen et al., 1997; Lang &
Watson, 1998). Furthermore, consistent with a NO-mediated hyperpolarization, NO has
been reported to activate BK
Ca
, K
ir
and K
ATP
channels on the smooth muscle cells and
endothelial cells directly or through the activation of cGMP production (Bolotina et al., 1994;
Abderrahmane et al., 1998; Lee & Kang, 2001; Si et al., 2002; Schubert et al., 2004). Therefore,
NO and EDHF are not only complementary, but also additive and the effect of both

Control and Coordination of Vasomotor Tone in the Microcirculation

73
vasodilator components may be confounded. In this context, it is interesting to note that, as
mentioned above, the EDHF-mediated response is typically studied in presence of NOS and
COX blockers. However, NOS inhibition with analogues of L-arginine is a slow, time-
dependent process and, on occasion, blockade of NO production with these drugs has been
observed to be incomplete (Vanheel & Van de Voorde, 2000; Figueroa et al., 2001; Chauhan
et al., 2003; Stoen et al., 2003; Stankevicius et al., 2006), and then, the residual NO production
observed in presence of NOS inhibitors may contribute to the vasodilation associated with
the smooth muscle hyperpolarization attributes to EDHF. In addition, the findings reported
recently by Gaete et al. (Gaete et al., 2011) are another important point to take into account in
the interaction between EDHF and NO. In this work Gaete et al., demonstrated that SK
Ca

and IK
Ca
channels control the Ca
2+
-dependent NO release, and thereby, the inactivation of
these K
+
channels is associated with an increase in NAD(P)H oxidase-mediated superoxide
production, which leads to the inhibition of eNOS primarily by its phosphorylation at
threonine 495 (Gaete et al., 2011). These findings highlight the relevance of these K
+
channels
in the control of vascular function and indicate that the participation of superoxide in the
EDHF-mediated response associated to SK
Ca
and IK
Ca
channels must be evaluated.
Furthermore, the regulation of NO and EDHF is different depending on gender. In male
animals, NO is the major endothelium-dependent vasodilator signal, but in female EDHF
prevails over NO or PGI
2
(Scotland et al., 2005). In this context, it is interesting to note that
estrogen enhances the EDHF-mediated vasodilation in response to flow (Huang et al., 2001),
which suggests that the EDHF-dependent signaling pathway may be more important in the
control of blood pressure in female than in male animals. This idea was confirmed using an
eNOS/COX-1 double knockout. Deletion of eNOS and COX-1 did not alter the mean arterial
blood pressure in female mice, whereas the double knockout resulted in hypertension in
male mice (Scotland et al., 2005). In these animals, the endothelium-dependent relaxation
was intact in resistance vessels of female mice and was mediated by the smooth muscle
hyperpolarization (Scotland et al., 2005), strongly supporting that EDHF plays a
predominant role in the tonic control of blood pressure in female. These data suggest that
EDHF rather than NO may underlie the higher resistance of premenopausal females to
cardiovascular diseases such as hypertension.
4. Gap junction communication in the vascular function
Gap junctions are intercellular channels that directly connect the cytoplasm of neighboring
cells, allowing the passage of current or molecules smaller than ~1.4 nm of diameter such as
metabolites (e.g., ADP, glucose, glutamate and glutathione) or second messengers (e.g.,
Ca
2+
, cAMP and IP
3
) (Evans & Martin, 2002; Saez et al., 2003). These intercellular channels
are made up by a protein family known as connexins (Cx), which are named according to
their predicted molecular mass expressed in kDa. Connexin proteins have four
transmembrane domains with the N- and C-termini located on the cytoplasmic membrane
face. The radial arrangement of six connexins around a central pore makes a connexon or
hemichannel, and the association in the plasma membrane of two hemichannels provided
by adjacent cells forms an intercellular gap junction channel (Evans & Martin, 2002; Saez et
al., 2003). It is noteworthy that independent hemichannels can also remain unpaired and
functional, which have been recognized to release paracrine signals such as ATP, PGE
2
or
NAD
+
(Goodenough & Paul, 2003; Cherian et al., 2005; Saez et al., 2005). The importance of

The Cardiovascular System Physiology, Diagnostics and Clinical Implications

74
this mode of communication in the vasculature is just starting to be evaluated and,
consistent with the participation of vascular hemichannels in paracrine signaling, human
microvascular endothelial cell (HMEC-1) monolayers were found to release ATP through
Cx43-formed hemichannels (Faigle et al., 2008).
At least twenty connexin isoforms have been described in mammals and one cell type may
express more than one connexin (Saez et al., 2003). However, the expression of several
connexins in one cell does not seem to be redundant, because gap junctions are not just
simple channels that offer a low-resistance intercellular pathway, but connexins mediate
highly specific cell-to-cell signaling pathways, and the molecular selectivity as well as
subcellular localization differs among connexins (Saez et al., 2003; Figueroa et al., 2004; Locke
et al., 2005). Thus, although these proteins may have some overlap in function, they work in
concert (Simon & Goodenough, 1998; Figueroa et al., 2004, 2006; Haefliger et al., 2006) and,
consequently, it has been observed that many times the function of one connexin cannot be
replaced by other connexin isoform (White, 2003; Haefliger et al., 2006; Zheng-Fischhofer et
al., 2006; Wolfle et al., 2007). In addition, hemichannels can be composed by one or a mixture
of connexin proteins, which provides an additional mechanism for fine regulation of gap
junction-mediated signaling processes (White & Bruzzone, 1996; He et al., 1999; Beyer et al.,
2000; Cottrell et al., 2002; Moreno, 2004).
Five connexin proteins have been found to be expressed in the vasculature: Cx32, Cx37, Cx40,
Cx43, and Cx45 (Severs et al., 2001; Figueroa et al., 2004; Haefliger et al., 2004; Okamoto et al.,
2009). The expression of connexins in the different cell types of the vessel wall is not uniform
and vary with vessel size, vascular territory, and species (van Kempen et al., 1995; van Kempen
& Jongsma, 1999; Hill et al., 2002). In most cases, Cx45 is only observed in smooth muscle cells
and has mainly been detected in brain vessels (Kruger et al., 2000; Li & Simard, 2001). In
contrast, the expression of Cx32 and Cx37 seems to be restricted to the endothelium (Gabriels
& Paul, 1998; van Kempen & Jongsma, 1999; Severs et al., 2001; Okamoto et al., 2009), but Cx37
has also been detected in smooth muscle cells (Rummery et al., 2002). Although Cx40 and Cx43
may be expressed in both cell types (Little et al., 1995; Gabriels & Paul, 1998; van Kempen &
Jongsma, 1999; Severs et al., 2001), Cx40 is located predominately in endothelial cells (Gabriels
& Paul, 1998; van Kempen & Jongsma, 1999) and Cx43 is the most prominent gap junction
protein found in smooth muscle cells (van Kempen & Jongsma, 1999). It should be noted,
however, that in mouse, Cx40 is expressed exclusively in the endothelium (de Wit et al., 2000;
Figueroa et al., 2003; Figueroa & Duling, 2008).
In addition to connexins, another family of three members of membrane proteins named
pannexins (Panxs 1-3) has been documented (Bruzzone et al., 2003). Apparently, pannexins
only form hemichannels, and then, the main function of pannexin-based channels is paracrine
or autocrine communication (Locovei et al., 2006). Although connexins and pannexins share a
similar membrane topology, their amino acid sequences present only a 16% homology
(Bruzzone et al., 2003). Only the expression of Panx-1 has been identified in blood vessels at the
moment and recently this pannexin was found to be involved in the activation of the
vasoconstrictor response mediated by 1-adrenoceptor stimulation (Billaud et al., 2011).
4.1 Gap junctions in vascular smooth muscle
Coordination of vasomotor signals among smooth muscle cells is critical for the function of
blood vessels. As mentioned above, the contractile state of smooth muscle cells depends on

Control and Coordination of Vasomotor Tone in the Microcirculation

75
the cytoplasmic Ca
2+
concentration and Ca
2+
sensitivity of the contractile apparatus.
Intracellular Ca
2+
concentration is controlled by the smooth muscle cell membrane potential.
Then, gap junctions play a central role integrating the smooth muscle cell function because
these intercellular channels synchronize changes in both membrane potential and
intracellular Ca
2+
between adjacent

smooth muscle cells (Christ et al., 1991; Christ et al., 1992;
Christ et al., 1996).
In addition, gap junction communication of vascular smooth muscle cells seems to be
involved in the development of myogenic vasomotor tone in resistance arteries (Lagaud et
al., 2002; Earley et al., 2004). Interestingly, the participation of gap junction in this process is
not related to synchronization of Ca
2+
signaling, but rather to earlier signaling events such
as coordination of the smooth muscle cell-depolarization or directly the mechanosensitivity
of the vascular smooth muscle. This notion is supported by the fact that the gap junctions
and connexin hemichannels inhibitors Gap27 (a connexin mimetic peptide) or 18-
glycyrrhetinic acid, in addition to block Ca
2+
influx and vasoconstriction in mesenteric
resistance arteries, also prevented the pressure-induced smooth muscle cell depolarization
(Earley et al., 2004). It is important to note that Gap27 and 18-glycyrrhetinic acid are two
well-known gap junction blockers, but they also block connexin-formed hemichannels,
which indicates that hemichannels may also be involved in the development of the
myogenic response. In any case, the involvement of Cx43-based channels in the control of
vasomotor tone is consistent with the finding that tensile stretch increased the expression of
this connexin as well as gap junction intercellular communication in vascular smooth
muscle cells (Cowan et al., 1998). Interestingly, this response was mediated by the formation
of reactive oxygen species (Cowan et al., 1998; Cowan et al., 2003), which has been reported
to contribute to the initiation of the myogenic constriction in mouse-tail arterioles (Nowicki
et al., 2001).
Cx43 has also been involved in the regulation of cell proliferation and migration in the
vasculature (Polacek et al., 1997; Yeh et al., 1997; Kwak et al., 2001), which can be appreciated
in Cx43-deficient smooth muscle cells. Damage of carotid artery by vascular occlusion or
wire injury resulted in an increase in neointima and adventitia formation in smooth muscle
cell Cx43 specific knockout mice as compared to wild type animals (Liao et al., 2007),
suggesting an accelerated growth of smooth muscle cell with the Cx43 deletion, which was
further confirmed using cultured cells. Nevertheless, in apparent opposition to these
findings, Chadjichristos et al. (Chadjichristos et al., 2006) show that in heterozygous Cx43
knockout mice the neointimal formation was reduced. However, in those animals, Cx43 was
reduced from all cell types expressing Cx43 and the experiments included a high-fat diet,
which may have influenced the result by either vascular adaptive response to the diet or
complex interactions between different cell types. Although the participation of Cx43 in
neointimal formation demands further investigation, these data highlight the relevance of
Cx43 in the feedback control pathways necessary for vascular morphogenesis.
4.2 Gap junctions in vascular endothelium
The endothelium plays a key role in the tonic control of blood pressure and the
development of knockout animals of vascular connexins has disclosed that gap junction
communication of endothelial cells is essential in the coordination and integration of

The Cardiovascular System Physiology, Diagnostics and Clinical Implications

76
microvascular function. Vascular endothelial cells-specific deletion of Cx43 (VEC Cx43
-/-
)
results in hypotension (Liao et al., 2001) and, in contrast, ablation of Cx40 produces a
hypertension associated with an irregular vasomotion (de Wit et al., 2000; de Wit et al., 2003;
Figueroa & Duling, 2008) and a dysregulation of renin production (Krattinger et al., 2007;
Wagner et al., 2007). Although deletion of Cx37 does not appear to alter vascular function or
blood pressure (Figueroa & Duling, 2008), several polymorphisms of this connexin have
been associated with myocardial infarction, coronary artery disease and atherosclerosis
(Boerma et al., 1999; Yamada et al., 2002; Hirashiki et al., 2003; Yamada et al., 2004). In mice,
Cx40 and Cx37 are primarily expressed in the endothelium, which emphasizes the
importance of the endothelial cell-gap junction communication in the control of
cardiovascular homeostasis.
Although the mechanistic bases of the hypotension observed in VEC Cx43-/- are still
unknown, the plasma levels of angiotensin I and II as well as NO were elevated in these
animals (Liao et al., 2001), suggesting that a dysregulation of NO production may have been
the responsible of the hypotension with the subsequent activation of the renin-angiotensin
system. Also, it is interesting to note that shear stress up-regulates the expression of Cx43 in
cultured endothelial cells (DePaola et al., 1999; Bao et al., 2000) and in the endothelium of rat
cardiac valves (Inai et al., 2004), which suggests that Cx43 may be involved in the response
to mechanical stimuli.
4.3 Gap junctions in smooth muscle-endothelium communication
Smooth muscle cells and endothelial cells have also been found to be electrically and
metabolically connected by gap junctions located at discrete points of contact between the two
cell types at the myoendothelial junction (MEJ) (Beny & Pacicca, 1994; Little et al., 1995;
Emerson & Segal, 2000; Sandow et al., 2003). This heterocellular communication seems to play
a pivotal role in the Ca
2+
-mediated responses induced by endothelium-dependent
vasodilators, such as ACh. As mentioned above, these vasodilator responses are typically
paralleled by hyperpolarization of the underlying smooth muscle cells (Emerson & Segal, 2000;
Goto et al., 2002; Griffith, 2004), which has been attributed to the release of an EDHF
(Vanhoutte, 2004; Feletou & Vanhoutte, 2009). However, the direct electrotonic transmission of
a hyperpolarizing current from the endothelial cells to the smooth muscle cells via
myoendothelial gap junctions may explain the EDHF pathway (Busse et al., 2002; Dora et al.,
2003; Griffith, 2004). In this perspective, the increase in endothelial cell intracellular Ca
2+

concentration activates SK
Ca
and IK
Ca
channels leading to the endothelium-dependent
hyperpolarization of smooth muscle cells via gap junctions located at the MEJ (Busse et al.,
2002; Crane et al., 2003; Eichler et al., 2003; Feletou et al., 2003) (Figure 3). Consistent with this
hypothesis, the EDHF-dependent vasodilation has been reported to be prevented by connexin-
mimetic peptides that are thought to specifically block gap junctions (De Vriese et al., 2002;
Karagiannis et al., 2004; Chaytor et al., 2005) as well as endothelial cell-selective loading of
antibodies directed against the carboxyl-terminal region of Cx40 (Mather et al., 2005).
Interestingly, the gap junction-mediated EDHF signal might be controlled by NO through S-
nitrosylation. Cx43-based channels can be activated by S-nitrosylation (Retamal et al., 2006).
Cx43 and eNOS has been found to be express at MEJ and the activation of NO production in
this microdomains leads to a S-nitrosylation-associated opening of Cx43-formed
myoendothelial gap junction (Straub et al., 2011), which support the idea that EDHF and NO
are not parallel, independent vasodilator components, but in contrast, they work in concert.

Control and Coordination of Vasomotor Tone in the Microcirculation

77
Flow (i.e. shear stress) is one of the most important stimuli involved in the tonic regulation
of vasomotor tone. Although the response to shear stress is thought to be mediated
primarily by NO, shear stress has also been reported to activate an EDHF-dependent
vasodilator response (Watanabe et al., 2005), which suggests that a gap junction-mediated
EDHF pathway may be involved in the tonic control of peripheral vascular resistance.
Consistent with this idea, intrarenal infusion of connexin-mimetic peptides homologous to
the second extracellular loop of Cx43 (
43
Gap 27) or Cx40 (
40
Gap 27) not only decreased basal
renal blood flow, but also increased mean arterial blood pressure of rats, either in presence
or absence of NOS and COX blockers (De Vriese et al., 2002), suggesting that connexin-
mimetic peptides induced vasoconstriction by disrupting or reducing the response to a tonic
vasodilator stimulus such as shear stress.
5. Conduction of vasomotor responses
Longitudinal conduction of vasomotor responses provides an essential means of
coordinating changes in diameter and flow distribution among vessels of the
microcirculation. Vasomotor signals spread along the vessel length through gap junctions
connecting cells of the vessel wall, and thereby, participate in the minute-to-minute
coordination of vascular resistance by integrating function of proximal and distal vascular
segments in the microcirculation (de Wit et al., 2000; Figueroa et al., 2004, 2006). Although
vasoconstrictor responses are thought to be conducted by smooth muscle cells (Welsh &
Segal, 1998; Bartlett & Segal, 2000; Budel et al., 2003), the cellular pathway for conduction of
vasodilator signals is more controversial and may be either exclusively by the endothelium
(Emerson & Segal, 2000; Segal & Jacobs, 2001) or by both smooth muscle and endothelial
cells (Bartlett & Segal, 2000; Budel et al., 2003). The cellular pathway for conduction of
vasomotor responses has been studied by selectively damaging a short segment of
endothelial cells or smooth muscle cells by injection of an air bubble via a side branch
(Bartlett & Segal, 2000; Figueroa et al., 2007) or with a light-dye (fluorescein-conjugated
dextran) treatment (Emerson & Segal, 2000). In feed arteries, selective damage of the
endothelium completely blocked the ACh-induced conducted vasodilation (Emerson &
Segal, 2000; Segal & Jacobs, 2001), but in arterioles, either damage of the endothelium or the
smooth muscle did not affect the ACh-induced conducted responses (Bartlett & Segal, 2000;
Budel et al., 2003), which led to the proposal that the cellular pathway for conduction of
vasodilations depends on the functional location of the vessel in the microvascular network
(Segal, 2005). However, the cellular pathway of vasodilator signals may also depend on the
stimulus that initiated the response, because, in contrast to ACh, selective damage of the
endothelium blocked the vasodilation induced by bradykinin in arterioles (Welsh & Segal,
1998; Budel et al., 2003).
Direct measurements of membrane potential have shown that conducted vasomotor
responses are associated with rapid propagation (milliseconds) of an electrical signal along
the vessel length (Xia & Duling, 1995; Welsh & Segal, 1998; Emerson & Segal, 2000). Because
many observations have revealed an exponential decay of the conducted electrical signal, it
was proposed that longitudinal spread of vasomotor responses reflects the passive,
electrotonic conduction of changes in membrane potential via gap junctions connecting cells
of the vessel wall (Pacicca et al., 1996; Welsh & Segal, 1998; Gustafsson & Holstein-Rathlou,
1999). Therefore, the decay of the conducted vasomotor responses along the vessel length

The Cardiovascular System Physiology, Diagnostics and Clinical Implications

78
should be consistent with the length constant estimated from electrotonic potentials
produced by current injection into the smooth muscle or endothelial cells of arterioles,
which is between 0.9 and 1.6 mm (Hirst & Neild, 1978; Hirst et al., 1997; Emerson et al., 2002).
Conduction of vasoconstrictor responses typically behaves as predicted by the electrotonic
model. However, a simple electrotonic model often fails to predict conduction of vasodilator
signals initiated by endothelium-dependent stimuli, such as ACh or bradykinin. These
signals have been reported to propagate for many millimeters without showing noticeable
decay in magnitude (Emerson & Segal, 2000; Figueroa & Duling, 2008). In addition, the
electrical length constant of ACh-induced hyperpolarization has been shown to be longer
than that measured for current injection (Emerson et al., 2002) and the hyperpolarizing
signal activated by ACh has been also reported to increase during the first 1000 m of
longitudinal conduction (Crane et al., 2004). The lack of decay of these responses suggests
that a regenerative, energy-dependent mechanism underlies the conduction process, similar
to that described in neurons. Consistent with this idea, electrical stimulation also activates a
conducted, non-decremental endothelium-dependent vasodilation that was hypothesized to
be mediated by a complex interplay between voltage-gated Na
+
channels (Na
v
) and T type,
voltage-gated Ca
2+
channels (T-Ca
v
) (Figueroa et al., 2007). In this hypothetic model, Na
v

channels underlie the conduction of the signal and T-Ca
v
mediates the vasodilation.
Interestingly, deletion of Cx40 selectively eliminates the regenerative component of the
conducted vasodilation induced by ACh (Figueroa & Duling, 2008), bradykinin (de Wit et
al., 2000) or electrical stimulation (Figueroa et al., 2003), leaving a decaying component
consistent with the electrotonic model (Figueroa & Duling, 2008), which suggests that Cx40-
based gap junctions provide the pathway for the intercellular propagation of the
regenerative conducted component of vasodilator signals. Deletion of Cx37 did not affect
conduction of vasodilator responses (Figueroa & Duling, 2008) and replacement of Cx40 by
Cx45 did not restore the non-decremental component of the conducted vasodilation
activated by ACh or bradykinin (Wolfle et al., 2007), supporting the idea that individual
connexins have different functions.
The opening of K
ir
channels induced by the smooth muscle hyperpolarization may be an
alternative hypothesis to explain the extended conduction of vasodilator responses. An
intrinsic biophysical property of K
ir
channels is that they increase their activity upon cell
hyperpolarization and it has been proposed that the activation of these K
+
channels in the
smooth muscle cells amplify the hyperpolarizing current initiated by ACh, thereby
facilitating the conduction of this signal (Jantzi et al., 2006). However, as mentioned above,
current-induced hyperpolarization decays faster than the response induced by ACh
(Emerson et al., 2002), which argues against the participation of K
ir
alone in the non-
decremental component of the conducted vasodilation, and suggests that further
investigation is needed to elucidate the mechanisms involved in the conduction of
vasomotor responses.
6. Neurovascular coupling
The brain has a very high metabolic demand and its activity depends on the communication
between brain cells and local microvessels (i.e. neurovascular unit). Then, the function of

Control and Coordination of Vasomotor Tone in the Microcirculation

79
cerebral microcirculation must be coupled to neuronal activity, which is known as
neurovascular coupling (Hawkins & Davis, 2005; Leybaert, 2005). In this case, however,
vasomotor signals seem to be conducted by astrocytes as opposed to smooth muscle or
endothelium (Anderson & Nedergaard, 2003; Zonta et al., 2003; Mulligan & MacVicar, 2004;
Koehler et al., 2006; Metea & Newman, 2006; Takano et al., 2006). Tight spatial and temporal
coupling between neuronal activity and blood flow is essential for brain function (Anderson
& Nedergaard, 2003; Hawkins & Davis, 2005; Leybaert, 2005) and astrocytes are found in a
strategic location between neurons and the microvasculature, with the astrocytic endfeet
ensheathing the vessels. This spatial organization places the astrocytes in a key position to
orchestrate the neurovascular coupling and an increasing body of evidence shows that the
astrocyte transduces and conducts to the local microvasculature vasomotor signals
generated by an increase in synaptic activity (Anderson & Nedergaard, 2003; Zonta et al.,
2003; Mulligan & MacVicar, 2004; Metea & Newman, 2006; Takano et al., 2006) (Figure 4). As
a result, astrocytes couple neuronal activation to vasodilation of local parenchymal
arterioles (Figure 4), which, in turn, leads to an increase in blood-borne energy substrate that
rapidly matches the enhanced metabolic demand (Anderson & Nedergaard, 2003; Hawkins
& Davis, 2005; Leybaert, 2005).
Calcium seems to be the intracellular vasomotor signal of the astrocyte-mediated
neurovascular coupling. Astrocytes express receptors for several neurotransmitters such as
glutamate, GABA and ATP (Anderson & Nedergaard, 2003; Leybaert, 2005; Koehler et al.,
2009), which can initiate Ca
2+
signals (Figure 4). Then, the increase in neuronal activity
results in an astrocytic calcium signaling that propagates through the astrocytic processes
into the endfeet (Anderson & Nedergaard, 2003; Zonta et al., 2003; Filosa et al., 2004;
Mulligan & MacVicar, 2004; Straub et al., 2006). The increase in cytosolic calcium
concentration in the endfeet ultimately causes the release of vasoactive factors and arteriolar
dilation (Anderson & Nedergaard, 2003; Zonta et al., 2003; Mulligan & MacVicar, 2004; Filosa
et al., 2006; Straub et al., 2006) (Figure 4). Interestingly, astrocytes express gap junctions
(Martinez & Saez, 2000; Saez et al., 2003; Retamal et al., 2006) and a calcium signal may
propagate between neighboring astrocytes in a wave-like manner (Cornell-Bell et al., 1990;
Nedergaard, 1994; Cai et al., 1998; Nedergaard et al., 2003), coordinating the neurovascular
coupling in the local cerebral microcirculation (Anderson & Nedergaard, 2003; Zonta et al.,
2003; Filosa et al., 2004; Mulligan & MacVicar, 2004). Some of the Ca
2+
-dependent vasodilator
mechanisms that may be activated at the astrocytic endfeet facing the vessel wall are the
production of epoxyeicosatrienoic acid (EETs) by the cytochrome P450 epoxygenase and PGs
by the COX enzyme (Anderson & Nedergaard, 2003; Zonta et al., 2003; Zonta et al., 2003; Filosa
et al., 2004; Straub et al., 2006; Koehler et al., 2009), and also ATP release (Shi et al., 2008) via
connexin or pannexin hemichannels (Figure 4). In addition, astrocytic endfeet express BK
Ca

and Girouard et al. (Girouard et al., 2010) recently showed in mouse cortical brain slices that
these K
+
channels play a central role in neurovascular coupling through the release of K
+
ion
into the perivascular space (Figure 4). The small increase in local [K
+
]
o
(<20 mM) activates the
K
ir
channels located in the smooth muscle cell membrane facing the endfeet, which leads to
hyperpolarization, and subsequently, vasodilation (Girouard et al., 2010) (Figure 4). It is
noteworthy that a higher increase in [K
+
]
o
would produce smooth muscle cell depolarization
and vasoconstriction (Girouard et al., 2010).

The Cardiovascular System Physiology, Diagnostics and Clinical Implications

80

Fig. 4. Astrocytes-mediated neurovascular coupling. Neurotransmitters may exit the
synaptic cleft and activate receptors on astrocytes, which couple neuronal activity with
astrocyte signaling. The activation of astrocyte receptors triggers a Ca
2+
wave that reaches
the astrocytic endfeet, leading to the opening of large conductance Ca
2+
-activated K
+

channels (BK
Ca
). The K
+
ion release via BK
Ca
elicits a small increase in extracellular K
+

concentration (<20 mM) in the perivascular space that activates the K
ir
channels located in
the smooth muscle cell membrane facing the endfeet, which, in turn, leads to
hyperpolarization, and subsequently, vasodilation. The vessel wall hyperpolarization-
mediated vasodilation is conducted to upstream arterioles, coupling function of proximal
and distal vessels.
As described in the peripheral microcirculation (Segal & Kurjiaka, 1995; Segal, 2000), local
vasodilation of cerebral arterioles must be communicated to upstream vascular segments to
produce a functional increase of blood flow supply and effectively match the local metabolic
demand (Cox et al., 1993; Iadecola et al., 1997). Although vasomotor responses have been
observed to be conducted by the wall of cerebral arterioles (Dietrich et al., 1996; Horiuchi et
al., 2002), it seems to be that astrocytes also play a central role in integrating function of local
arterioles with upstream cerebral vessels involved in the neurovascular coupling. Pial
arterioles are important upstream vessels of the parenchymal cerebral arterioles. It is
important to note that pial arterioles overlie a thick layer of astrocytic processes, known as

Control and Coordination of Vasomotor Tone in the Microcirculation

81
the glia limitans, which isolate these arterioles from the neurons that are located right below.
Vasodilation of pial arterioles associated with neuronal activation was blocked by either
selective elimination of astrocytes with L- aminoadipic acid treatment or the inhibition of
Cx43-based channels with the specific connexin mimetic peptide gap-27 (Xu et al., 2008). In
astrocytes, Cx43 may be found forming unpaired hemichannels or gap junction intercellular
channels (Stout et al., 2002; Saez et al., 2003; Retamal et al., 2006). Thus, astrocytic Cx43-based
channels could be involved in the coordination of calcium waves between astrocytes, or in
the release of vasoactive factors such as ATP that can be metabolized to the potent
vasodilator, adenosine (Shi et al., 2008)
7. Conclusion
Control of vasomotor tone relies on a complex interplay between NO, PGs, K
+
channels and
gap junction communication. It is typically thought that NO is the most relevant
endothelium-dependent vasodilator signal, but, in resistance vessels and arterioles, K
+

channels and gap junction communication between the cells of the vessel wall have emerged
as major players in the tonic control and coordination of vascular function. While several K
+

channels (e.g. BK
Ca
, K
ir
and K
ATP
channels) may contribute to the vasodilator response
induced by NO, the endothelial cell K
+
channels, SK
Ca
and IK
Ca
, seem to be involved in the
fine regulation of eNOS activation. In addition, it has become apparent that NO production
is also modulated by a delicate caveolar control of L-arginine supply. Although
myoendothelial gap junction communication probably contributes to the EDHF signaling
mediated by SK
Ca
and IK
Ca
channels, the strategic spatial organization of IK
Ca
and K
ir
may
also be involved in the intercellular transmission of an endothelium-initiated smooth muscle
hyperpolarization. A similar organization, but between BK
Ca
and K
ir
channels, is observed
in the astrocyte-mediated neurovascular coupling. Connexin- and pannexin-based
hemichannels are an attractive signaling mechanism that may be involved in the control of
vascular function, but the study of hemichannels in resistance vessels is just beginning.
8. Acknowledgment
This work was supported by Grant Anillos ACT-71 from Comisin Nacional de
Investigacin Cientfica y Tecnolgica CONICYT and Grant #1100850 and #1111033 from
Fondo Nacional de Desarrollo Cientfico y Tecnolgico FONDECYT.
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5
Hemodynamics
Ali Nasimi
Isfahan University of Medical Sciences,
Iran
1. Introduction
Hemodynamics is the study of the relationship among physical factors affecting blood flow
through the vessels. In this chapter these factors and their relationship were discussed.
2. Blood flow is a function of pressure difference and resistance (Darcys
law)
Blood flow (F) through a blood vessel is determined by two main factors: (1) pressure
difference (AP) between the two ends of the vessel and (2) the resistance (R) to blood flow
through the vessel (Fig. 1).

Fig. 1. Blood flow through a blood vessel.
The equation relating these parameters is:
F = AP/R (1)
This equation is called Darcys law or Ohms law.
Flow (F) is defined as the volume of blood passing each point of the vessel in one unit time.
Usually, blood flow is expressed in milliliters per minute or liters per minute, but it is also
expressed in milliliters per second.
Pressure which is the force that pushes the blood through the vessel is defined as the force
exerted on a unit surface of the wall of the tube perpendicular to flow. Pressure is expressed
as millimeters of mercury (mmHg). Since the pressure is changing over the course of the
blood vessel, there is no single pressure to use; therefore the pressure parameter used is
pressure difference (AP), also called pressure gradient, which is the difference between the
pressure at the beginning of the vessel (P
1
) and the pressure at the end of the vessel (P
2
), i.e.

The Cardiovascular System Physiology, Diagnostics and Clinical Implications

96
AP = P
1
- P
2
. As seen in the Darcys law, AP is the cause of the flow; with no pressure
difference there would be no flow. The pressure energy is produced by the ventricle and it
drops throughout the vessel due to resistance. In other words, resistance is the cause of the
pressure drop over the course of a vessel.
Resistance is how difficult it is for blood to flow from point 1 to point 2. Resistance impedes
flow and it is a measure of interactions between flowing particles (including molecules and
ions) themselves and interactions between flowing particles and the wall of the vessel. As
seen Darcys law, resistance is the impeding cause of the flow; the bigger the resistance the
lesser the flow. If the resistance is (complete closure of the vessel) there will be no flow.
The resistance equation is:

8
4
L
R
r
q
t
=
(2)
Where q = fluid viscosity
L = vessel length
r = inside radius of the vessel
Viscosity represents the interactions between flowing particles themselves and radius
represents the interactions between flowing particles and the wall of the vessel. The units of
viscosity are Pas = Ns/m
2
, or Poise (dyness/cm
2
), with 1 Pas = 10 Poise.
The red blood cells, erythrocytes (RBCs), constitute 99% of the suspending particle volume
of blood. Therefore viscosity of blood depends on the concentration of various constituents
of plasma and volume % of red blood cells (hematocrit), as well as size, shape and
deformability of RBCs. In a healthy individual all these parameters are constant, therefore
blood viscosity is constant and viscosity is not a mean of control (regulation) of the
resistance. In abnormal situations viscosity abnormally affects the total resistance. Low
hematocrit, as in anemia, decreases viscosity of blood. Inversely, polycythemia increases
viscosity and lowers blood flow. In sickle cell anemia the erythrocytes are misshapen and
inflexible causing serious disturbances of regional blood flow.
In the resistance equation, L represents the vessel length. Since the length of the vessels of
the body is constant, L could not be used for control of the resistance.
Resistance has an inverse relation with the 4
th
power of r (inside radius of the vessel);
therefore radius of the vessel has the most powerful effect on the resistance, so that with
small changes in radius, resistance will change dramatically. Radius is the main factor for
control of the resistance by the cardiovascular system. Radius of the vessels of the body is
controlled by the sympathetic system.
Resistance is mainly located in the arterioles. Assuming an aortic radius of 15 mm and an
(arbitrary) length of 50 cm and an arteriole with a radius of 7.5 m and a length of 1 mm.
The ratio of the radius is 2000 and the length ratio is ~500, therefore the resistance ratio
would be (2000)
4
/500, i.e., ~310
10
. It means that the resistance of a single arteriole is 310
10

as large as that of a 50 cm long aorta. Since there are 310
8
parallel arterioles, their total
resistance is about 310
10
/310
8
~ 100 times as large as the resistance of the aorta (Westerhof
et al. 2010).

Hemodynamics

97
Even though all vessels except metarterioles and capillaries are innervated by the
sympathetic system, the arterioles receive the most profound innervations and play the
main role in the control of the total peripheral resistance by the sympathetic system.
The resistance of any vessel can be calculated by having AP and F. For systemic circulation,
if mean aortic pressure (P
1
) is taken to be 100 mmHg and mean right atrial pressure (P
2
) is 0
mmHg, the pressure difference (AP) is 100 mmHg. With a cardiac output of 6 l/min (100
ml/s), the total resistance is 100/100 = 1 mmHg/ml/s. This unit is called peripheral
resistance unit (PRU). Other physical units are used in the clinic and resistance is expressed
in dynscm
5
or Pas/m
3
. The total peripheral resistance of the systemic circulation may
change from 4 PRU in very strong constriction to 0.2 PRU in great dilation of the vessels. In
the pulmonary system, the mean pulmonary arterial pressure is 16 mm Hg and the mean
left atrial pressure is 2 mm Hg, giving a AP of 14 mm. With a cardiac output of 100 ml/sec,
the total pulmonary vascular resistance is 0.14 PRU, about one seventh of that in the
systemic circulation.
In the body, blood vessels arranged in series and in parallel. The arteries, arterioles,
capillaries, venules and veins are arranged in series. The total resistance of a series of vessels
is equal to the sum of the resistances of each vessel:
R
total
= R
1
+ R
2
+ R
3
+ (3)
Blood vessels branch extensively to form parallel circuits in all organs and tissues of the
body. The total resistance of parallel vessels is calculated by:

...
1 2 3
1 1 1 1
R R R R total
= + + +
(4)
As a result, adding a parallel vessel to a circuit will reduce the total resistance. This is the
reason that the resistance of each organ alone is far greater than the total peripheral
resistance. For example in renal circulation, if blood pressure in the renal artery is taken to
be 100 mmHg and that of the renal vein be 10 mmHg and renal flow is taken to be 20 ml/s
(1200 ml/min), then R = 90/20 = 4.5 PRU, 4.5 times as much as the total resistance of the
systemic circulation.
2.1 Poiseuilles law
In equation F= AP/R if we substitute R with its equation results in:

8
4
P
F
L
r
t
q
A
=
(5)
This is called Poiseuilles law. As seen, flow is proportional to AP which is the main cause of
flow. Flow is also proportional to the 4
th
power of internal radius of the vessel indicating the
great importance of the radius for flow.
2.2 Physiological and clinical applications of Darcys law
In equation F= AP/R if P
1
is increased, since AP = P
1
- P
2
, AP will increase which results in an
increase in blood flow (F) and P
2
. For example, during exercise, contractility of the left ventricle

The Cardiovascular System Physiology, Diagnostics and Clinical Implications

98
increases and produces more pressure energy which results in the increase of aortic pressure
(P
1
), causing blood flow to various organs and capillary pressure (P
2
) to increase. On the
contrary, a decrease of P
1
results in a decrease of flow and capillary pressure.
An increase in P
2
results in a decrease of AP and blood flow. For example if the venous
resistance increases or atrial pressure (P
2
) increases, such as in heart failure, AP will be lower
than that of the normal and blood flow will decrease. This subsequently results in a small
increase in the arterial pressure (P
1
). Therefore a change in either one of the P
1
or P
2
causes a
similar change in the corresponding P which is smaller than the first one. It is smaller, since
resistance always causes a pressure drop between the two points of the vessel. For example,
|P
1
|AP (1st) |F |P
2
+AP (2nd), but flow is still higher, since due to resistance,
the magnitude of the increase of P
2
is smaller than the increase of P
1
, therefore the
magnitude of the second change (decrease) of AP is smaller than the first change (increase)
of AP.
Changing the resistance by adjusting the radius of the vessels is the main mechanism of
controlling blood flow to each tissue and organ, called local control of blood flow. It is also
one of the two major mechanisms (control of the heart and the resistance) to control arterial
blood pressure.
Darcys equation (F = AP/R) could be rewritten as: AP = P
1
-

P
2
=

FR. If R is increased by
decreasing the radius, other three parameters of the equation will change. The first thing
that happens is the reduction of flow, exactly as expected from the Darcys equation.
Reduction of flow then causes a pile up of flowing materials (such as blood) before
resistance and a decrease in blood volume after the resistance; thus P
1
will increase and P
2

will decrease and subsequently AP will get bigger, exactly as expected from the 2
nd
form of
Darcys equation. The resultant increase in AP is always quantitatively smaller than the
primary increase of R; therefore F is always less than before. The steps could be summarized
as follows:
R| F+ = AP/R| P
1
| & P
2
+ AP| F+ = AP|/R|
Exactly opposite will happen if R is decreased. As seen, any change in R will change both P
1

and P
2
in

opposite to each other. For example when some one turns the valve of a tap clock
wise, the radius of the outlet is decreased, flow decreases, output pressure (P
2
) decreases
and the pressure of pre-valve water (P
1
) increases.
Based on Darcys law, if the cardiovascular system is to increase blood flow, it could either
increase AP by increasing the heart work, or decrease R by decreasing sympathetic outflow
to the vessels, especially to arterioles, resulting in a decrease of resistance. Also it could do
both (AP| and R+). In local control mechanism, only local resistance is adjusted to control
blood flow to a tissue. High metabolism of a tissue changes the concentration of some
chemical factors including oxygen. These factors make metarterioles and precapillary
sphincters dilate. Based on Darcys law, blood flow to that tissue increases so that the blood
supply to that tissue will be proportional to its metabolism.
When arterial pressure is low, baroreflex stimulates the heart by increasing contractility and
heart rate which results in higher P
1
. Baroreflex also increases the total peripheral resistance
which results in higher P
1


Hemodynamics

99
Resistance causes pressure drop across the vascular system (Fig. 2). In the large arteries,
resistance is relatively small and pressure drop is small. The small arteries have moderate
resistance to blood flow. Resistance is highest in the arterioles, which are sometimes referred
to as the stopcocks of the vascular system. Therefore, the pressure drop is greatest across the
terminal part of the small arteries and the arterioles (Fig. 2).
Resistance vessels make pressure drop from ~100 to 30 mmHg. Based on Darcys law, high
resistance of these vessels increases the P
1
(arterial pressure) and decreases the P
2
(capillary
pressure). Both effects are absolutely necessary for survival. High arterial pressure

makes it
possible for blood to reach all parts of the body especially to the head which is at a higher
level than the heart. The second effect, low capillary pressure, is also very useful because it
prevents the capillaries from being damaged and it is necessary for stable transport between
the capillaries and the interstitial fluid. High capillary pressure makes capillaries too
permeable so that proteins can cross the endothelium which results in edema.

Fig. 2. Pressure drop across the vascular system in the hamster cheek pouch. AP, mean
arterial pressure; VP, venous pressure (From Bern et al., Physiology, 2007, with permission).
Anaphylaxis is an allergic condition in which the cardiac output and arterial pressure often
decrease drastically. It results from an antigen-antibody reaction after an antigen to which
the person is sensitive enters the circulation, causing secretion of histamine by basophile and
mast cells. Histamine dilates the arterioles, resulting in greatly reduced arterial pressure that
could result in coma and death. Too much vasodilator drugs also could produce similar
effect.
In aneurysm disease, part of an artery dilates abnormally. Based on Darcys law, blood flow
to the zone perfused by that artery is increased (F|) which results in a higher pressure in
microcirculation of that zone (P
2
|), which may produce pain and damage.



The Cardiovascular System Physiology, Diagnostics and Clinical Implications

100
In coarctation of descending aorta, a local malformation marked by deformed aortic media,
causes narrowing of the lumen. As expected from Darcys law, blood flow to the lower parts
of the body is seriously decreased (F+). As a consequence, the arterial pressure in the lower
part of the aorta decreases (P
2
+) and of the upper part of the aorta may be 40-50 per cent
higher (P
1
|) than the lower aorta. Due to the low renal blood pressure, water and salt
retention occurs that eventually returns the blood pressure of the lower part of the body to
normal and produces hypertension in the upper part of the body.
In heart ischemic diseases, narrowing or obstruction of one or more coronary arteries,
decreases or ceases blood flow to the regions supplied by the affected arteries.
In aortic stenosis, the diameter of the aortic valve opening is reduced significantly, and the
aortic pulse pressure (difference between systolic and diastolic pressure) is decreased
significantly because of great decrease of systolic pressure. Based on Darcys law, due to
high resistance of aortic valve, P
1
(ventricular pressure) increases and P
2
(aortic systolic
pressure) decreases. This is exactly what we see in the disease. Due to high ventricular
pressure, ventricle hypertrophy may occur.
Migraine, is a symptom complex of periodic headaches, often with irritability, nausea,
vomiting, constipation or diarrhea and photophobia. It is preceded by constriction of some
cranial arteries, which results in low blood flow to the affected regions and consequently
results in prodromal sensory, especially occular symptoms. Then remarkable vasodilation of
those cranial arteries occurs resulting in overperfusion of the affected regions which
produces other symptoms, especially headache.
3. Laminar or turbulent flow
Blood flow in the straight vessels, is normally laminar. Blood moves in smooth parallel
concentric layers. As flow increases, the fluid motion becomes wavy, leading to vortices in
different seemingly random directions. This irregular fluid motion is called turbulence (Fig. 3).

Fig. 3. Laminar and turbulent flow

Hemodynamics

101
In turbulent flow the resistance to flow is higher and energetically is more costly than
laminar flow, since part of the mechanical energy is lost in the erratic motion between the
fluid particles. The probability of turbulence is related to blood density, velocity, the
diameter of the vessel and the viscosity of the blood. To judge whether a fluid flow is
laminar or turbulent, the Reynolds number (Re, a dimensionless parameter: has no unit) is
often used. Re is defined as:
Re = vD/q (6)
is the fluid density, v is the mean fluid velocity; D is the tube inner diameter and q is the
fluid viscosity. The Reynolds number reflects the ratio of inertia and viscous effects. The
critical Reynolds number is 2200. For low Reynolds numbers (<2200) the viscous effects are
dominant and flow is laminar, but for high Reynolds numbers (>2200), flow is turbulent.
For transitional numbers around the critical Reynolds number of 2200 flow is neither strictly
laminar nor strictly turbulent (Westerhof et al. 2010, p- 22).
At normal resting conditions, arterial flows are laminar. But in heavy exercise, where flow
may increase as much as five-folds, the Reynolds number may get higher than the critical
value and turbulence occurs.
Laminar flow can be disturbed at the branching points of arteries resulting in turbulence
which may deposit the atherosclerotic plaques.
Turbulence is delayed in accelerating flow whereas occurs faster in decelerating flows. For
example turbulence occurs distal to a stenosis. Fluid particles accelerate through the narrow
part of the stenosis and decelerate fast in the distal expanding part resulting in turbulence.
Turbulence in severe stenosis can be initiated for Reynolds numbers as low as 50 (Westerhof
et al. 2010, p- 23). This turbulence also widens the vessel after the stenotic part. Constriction
of an artery likewise produces turbulence and sound beyond the constriction. This is the
reason that murmurs are heard over arteries constricted by atherosclerotic plaques and the
sounds of Korotkoff heard when measuring blood pressure (Barrett et al. 2010, p- 540). In
severe anemia, because of low viscosity, functional cardiac murmurs are often heard.
4. Bernoullis principle
Regarding Darcys law alone, some aspects of hemodynamics seem puzzling. For example,
mean arterial pressure of aorta is about 100 mmHg while it is 180 mmHg in the foot arteries
during standing. The very high arterial pressure of the foot is due to gravitational force, as a
column of blood with an altitude of ~ 130 cm produces a high pressure in the foots arteries.
Based on Darcys law, since the pressure in the foots arteries is higher than aorta, blood
should move upward from the foot arteries to aorta, which is not the case. Also blood
pressure in the venous sinuses of the brain is highly negative, while the right atrial pressure
is ~0. Again based on Darcys law, blood should move upward from right atrium to venous
system of brain, which is not the case. Such problems are solved by Bernoullis principle.
Bernoullis theory states that flow between point A and point B is dependent on the total
mechanical energy difference between A and B, not on pressure difference alone. Total
mechanical energy consisted of pressure energy, potential energy and kinetic energy. The
pressure energy equals pressure volume (P V). The potential energy equals fluid mass

The Cardiovascular System Physiology, Diagnostics and Clinical Implications

102
(m) gravitational force (g) height (h). Kinetic energy equals mass (m) velocity squared
(v
2
) divided by 2 (m v
2
/2). Thus:
Total mechanical energy = PV + mgh + mv
2
(7)
Based on the conditions, these pressures could easily convert to each other. For example
consider the model presented in figure 4 (Burton 1972). This figure demonstrates an
experiment showing some basic hydraulic points. In this experiment, flow in the tube is
constant. As seen in figure 4, flow is driven by the gradient of total mechanical energy. At
the first part, cross-sectional area (A) is 6 and velocity (v) is 1. Based on the equation V =
F/A (V: velocity, F: flow, A: cross-sectional area), as at the middle of the tube cross-sectional
area gets smaller, velocity increases with the same ratio.

Fig. 4. Flow is driven by the total mechanical energy difference. In the middle of the tube,
the cross-sectional area (A) gets smaller resulting in an increase of velocity (v). In other
words, pressure energy is converted to kinetic energy. In the third part of the tube, opposite
will happen. Pr.E., pressure energy; K.E., kinetic energy. (Data from Burton 1972).
It means that pressure energy is converted to kinetic energy. This is shown by the numbers
at the bottom of the figure and is displayed on the middle vertical tube. Since there are both
pressure and total mechanical gradients, flow from the first part to the second part of the
tube is consistent with both Darcys and Bernoullis equations. The third part of the tube
gets wider again resulting in an increase of pressure energy and a decrease of kinetic energy.
Here kinetic energy is converted to pressure energy. This shows that these three mechanical
energies can readily convert to each other. Flow from the middle part to the third part is not
expected from Darcys law, but is consistent with Bernoullis principle. Another important
point shown in this experiment is that due to resistance, total mechanical energy is
decreasing over the course of the tube.

Hemodynamics

103
Now we can explain the puzzling examples mentioned above. In the upright posture, the
aortic blood possesses much more gravitational potential energy than the foot arteries, so
that the total mechanical energy in the aorta is higher than the foot arteries and makes blood
flow from aorta to the foot.
When someone lies down, Darcys law is sufficient for explaining blood flow, but in sitting
or standing positions, the gravitational potential energy gets quite large and Bernoullis law
should be applied for more accurate explanation of the blood flow. For example in the
upright posture, blood flow to the lung could not be explained well without using
Bernoullis principle. Since the pressure in the pulmonary arteries is low, the gravitational
energy is comparatively large and greatly affects the pulmonary blood flow, so that during
diastole, blood does not reach the apex of the lung.
5. Law of Laplace
The law of Laplace gives the relation between transmural pressure, wall tension, radius and
wall thickness in a vessel (Fig. 5) as:
T = Pr/w (8)
Where T is the force per unit length tangential to the vessel wall called wall tension
(dynes/cm), P is transmural pressure, intravascular pressure minus extravascular pressure,
in dynes/cm
2
, r is radius of the vessel in cm, and w is thickness of the vessel in cm.

Distending force (Pr) tends to pull apart a theoretical slit in the vessel, while the wall
tension (T) will keep the parts together.

Fig. 5. Transmural pressure (P) and wall tension (T) in a vessel.
Thin-walled capillaries can withstand high internal blood pressure since even though their
wall thickness is very small, their radius is also very small and their internal pressure is
much smaller than that of the arteries. In aneurysm (local widening of an artery) since
radius gets bigger the distending pressure gets higher and makes the vessel more prone to
rupture. In eccentric hypertrophy where a ventricle dilates, due to increase in radius,

The Cardiovascular System Physiology, Diagnostics and Clinical Implications

104
distending force is higher and the ventricle must work harder to pump the normal stroke
volume and it will deteriorates the already diseased ventricle.
6. Velocity is inversely related to cross-sectional area
Velocity (V) is related to flow (F) and inversely related to cross-sectional area (A) of a vessel
as follows:
V = F/A (9)
As blood vessels branch extensively from aorta to capillaries, cross-sectional area of each
vessel decreases while the total cross-sectional area increases.

Fig. 6. Velocity and total cross-sectional area in the systemic circulation. There is the
maximal cross-sectional area and minimal velocity in the capillaries. AO, Aorta; LA, large
arteries; SA, small arteries; ART, arterioles; CAP, capillaries; VEN, venules; SV, small veins;
LV, large veins; VC, venae cavae. (Bern et al. 2007, p-267, with permission)
As seen in figure 6, capillaries have the maximal total cross-sectional area resulting in the
lowest blood velocity. This low velocity provides ample time for exchange between blood
and interstitial fluid.
7. Elasticity and compliance
When a strip of material with cross-sectional area A, and length l
0
, is subjected to a force (F)
it will lengthen by Al (Fig. 7). For a specimen with a larger cross-sectional area the same
force will produce a smaller change of the length. Also if the starting length (l
0
) is longer, the
same force causes a larger length change. To have a unique characterization of the material,
independent of the sample primary length and thickness, force is normalized by starting

Hemodynamics

105
cross-sectional area, o = F/A called stress, and length is normalized by starting length c =
Al/l
0
called strain. Elasticity is defined as E = o /c (Westerhof et al. 2010, p-49).
The relation between stress and strain for biological material is given in the right part of the
figure 7. As seen the relationship between stress and strain for biological material almost
always is nonlinear. This nonlinearity implies that a biological material cannot be
characterized by a single E. Therefore we should get the local slope of the stress-strain
relation for the desired point. This point elasticity is called incremental elasticiy (E
inc
). E
inc

increases with strain, i.e., the biological materials become stiffer with increasing stress and
strain.

Fig. 7. Stress-strain relationship for biological materials (taken from snapshot p-49 with
permission)
For a vessel or a heart, increasing its blood volume results in an increase in the internal
pressure and increasing the internal pressure results in an increase in the volume.
Pressure is comparable to stress and volume is comparable to strain. Therefore in
cardiovascular physiology, pressure-volume relation (Fig. 8) is normally used instead of
stress-strain relation. An advantage of pressure-volume relation is that it can be measured
in vivo. It is important to note that pressure-volume relation does not characterize the
material alone but includes the structure of the organ as a whole ((Westerhof et al. 2010, p-
58). The change of volume per one unit change of pressure is called compliance (C =
AV/AP). The change of pressure per one unit change of volume is called elastance (E =
AP/AV). For biological organs like vessels and heart, the pressure-volume relation is
curved toward volume axis indicating that by increasing volume or pressure stiffness
increases (Fig. 8). Therefore there is not a single compliance or elastance and for a
working point, the tangent of the pressure-volume curve is used. Thus, when comparing
compliance or elastance the chosen working point, the pressure at which compliance or
elastance was determined, should be reported.

The Cardiovascular System Physiology, Diagnostics and Clinical Implications

106

Fig. 8. Pressure-volume relationship for biological organs (Westerhof et al. 2010, p-57, with
permission)
Compliance and elastance depend on the original volume (V
0
) of the organ under study. To
compare properties of different blood vessels, or hearts, compliance and elastance should be
normalized with respect to the original volume of the organ. Normalized compliance is
called distensibility [distensibility = C/V
0
= AV/(APV
0
) ]. Normalized elastance is called
volume elasticity [ volume elasticity = EV
0
= (APV
0
) /AV ].
7.1 Physiological and clinical applications
Distensibility of the veins is 8 times as much as the arteries and the original volume of the
veins is 3 times as much as the arteries, thus compliance of each vein is 24 times as much as
its corresponding (parallel artery and vein which have the same flow) artery. It means that
perfusing a vein and its corresponding artery with the same volume of blood, increases the
arterys pressure 24 times as much as the vein. Therefore veins can store large amount of
blood with little increase in pressure. Veins are called capacitance vessels storing 60-70
percent of the total blood volume.
Figure 9 shows the effect of blood pressure on blood flow through an isolated vessel. As
expected from Darcys law (F = AP/R) increasing pressure results in an increase of flow, but
in fact, the effect of pressure on blood flow is greater than expected from Darcys law (Fig.
9b), as shown by the upward curving lines in Figure 9a. This is because due to vascular
distensibility, increased arterial pressure not only increases the force that pushes blood
through the vessels but it also distends the elastic vessels, actually decreasing vascular
resistance. Therefore elasticity makes the heart work less to pump normal cardiac output,
resulting in longer survival.

Hemodynamics

107

Fig. 9. Effect of blood pressure on blood flow through an isolated vessel (a), and calculated
from Darcys law (b). (Modified from Guyton and Hall, 2011, p-166, with permission).
In arteriosclerosis, blood vessels are less distensible, therefore they extend less which results
in a higher resistance causing hypertension, high pulse pressure and high work load of the
heart. These symptoms have serious deteriorating effects on the cardiovascular system.
During systole, due to vascular distensibility, high blood pressure distends the arteries, i.e.
some pressure energy is stored in the walls of the arteries as potential energy. During
diastole the wall of the arteries return to their diastolic position releasing the stored
potential energy to the blood as pressure energy. This function attenuates systolic pressure
and increases diastolic pressure resulting in normal pulse pressure (difference between
systolic pressure and diastolic pressure) of 40 mmHg. Keeping diastolic pressure reasonably
high, keeps blood flowing during diastole. In arteriosclerosis, due to stiffness of the arteries,
less pressure energy is stored in the wall of the arteries causing systolic pressure to get
abnormally high, resulting in a high pulse pressure which has a deteriorating effect on the
arteries.
Another physiological benefit of elasticity is damping of the pulse pressure in the smaller
arteries, arterioles, and capillaries. Figure 10 shows typical changes in the pulse pressure as
the pulse travels into the peripheral vessels. The intensity of pulsation becomes
progressively less in the smaller arteries and eventually disappears in the capillaries. In fact,
only when the aortic pulsations are extremely large or the arterioles are greatly dilated can
pulsations be observed in the capillaries. Lack of pulsation in the capillaries guarantees
stable pressure thus stable permeability and stable transport across the capillaries wall.

The Cardiovascular System Physiology, Diagnostics and Clinical Implications

108

Fig. 10. Damping of the pulse pressure in the smaller arteries, arterioles, and capillaries
(Guyton and Hall, 2011, p-170, with permission).
The cause of progressive diminution of the pulsations in the periphery is twofold: (1)
resistance and (2) elasticity of the vessels. Resistance is the cause of pressure drop
throughout of the vessels, thus decreases the pulse pressure. Elasticity continuously
decreases the systole and adds to diastole pressure bringing them closer to each other.
8. References
Badeer H.S., Hemodynamics for medical students, Adv Physiol Educ, 2001, 25: 4452.
Barrett Kim E., Boitano Scott, Barman Susan M. and Brooks Heddwen L. Ganongs Riview
of Medical Physiology, 2010, The McGraw-Hill Companies, New York.
Baun J., Hemodynamics: Physical Principles in: Physical Principles of General and Vascular
Sonography, 2009, ProSono publishing, San Francisco, 149-158.
Bern et al., Physiology, 2007, Elsevier ltd.
Burton A.C., physiology and biophysics of the circulation, 1972, Year Book Medical
Publishers, Chicago.
Glaser R., Biophysics, 2001, Springer-Verlag Berlin Heidelberg
Guyton and Hall, Textbook of Medical Physiology, 2011, Saunders
Westerhof N., Stergiopulos N. and Noble M.I.M. Snapshots of Hemodynamics, An Aid for
Clinical Research and Graduate Education, 2010, Springer, New York.
6
Adenosinergic System
in the Mesenteric Vessels
Ana Leito-Rocha, Joana Beatriz Sousa and Carmen Diniz
REQUIMTE/FARMA, Department of Drug Science,
Laboratory of Pharmacology, Faculty of Pharmacy,
University of Porto,
Portugal
1. Introduction
1.1 Adenosinergic pathways in the cardiovascular system
Adenine-based purines, such as adenosine, and adenosine triphosphate (ATP), are
ubiquitous signalling molecules that mediate diverse biological actions and physiological
processes. Adenosine is an important signalling molecule in the brain, lungs, kidneys, heart,
blood vessels and immune systems (Lu et al., 2004), that exerts a potent action on many
physiological processes including vasodilation, hormone and neurotransmitter release, platelet
aggregation, and lipolysis (Baldwin et al., 2004; Podgorska et al., 2005). Reports of adenosine
and adenosine monophosphate (AMP), effects on the heart and blood vessels (Drury &
Szent-Gyorgyi, 1929), were the first in a major line of research concerning the physiological
actions of purines. Since then, the list of biological processes in which extracellular purines
participate has dramatically increased. Insights into the physiological roles of purines came
from studies of their biological sources and the stimuli for their release.
Adenosine is composed of an adenine base, consisting of two carbon-nitrogen rings, bound
to a ribose sugar group via a beta glycosidic link (Fig. 1); it is considered a nucleoside due to
the absence of phosphate groups in its structure. It presents a short half-life due to its rapid
conversion into inosine by adenosine deaminase, phoshorylation by adenosine kinase and
rapid uptake by adenosine transporters into tissues (Thorn & Jarvis, 1996).

Fig. 1. Adenosine molecule.

The Cardiovascular System Physiology, Diagnostics and Clinical Implications 110
Adenosine is a plurisystem mediator/modulator, influencing responses in various cell and
tissue types, and via numerous receptor and cell signalling pathways. Adenosine can be
generated by intracellular and extracellular enzyme pathways depending upon the specific
and unique conditions, giving rise to elevated extracellular concentrations. Both
equilibrative and concentrative adenosine transport proteins can move adenosine across
cellular membranes, influencing extracellular adenosine concentrations (Conlon et al., 2005).
There are several pools of adenosine which arise from different sources. Firstly, there is the
existing adenosine being transported in and out of cells via transporters. ATP present in the
cytosol is dephosphorylated to AMP which can be dephosphorylated further by the action
of adenosine kinase to produce adenosine. Alternatively, ATP can be released from the cell
by exocytosis, which can then be acted upon by nucleotidases to form adenosine
diphosphate (ADP), then AMP and finally adenosine. It can then be transported between the
inside of the cell and the interstitial fluid via transporters. Another pool of adenosine is
generated by neurons. ATP, as a neurotransmitter can be released into the interstitial fluid
when carrying a nerve impulse. As before, ATP is acted on by nucleotidases to ADP which
is further hydrolysed to AMP and then adenosine (Rang et al., 2007).
Under physiological conditions, adenosine is produced intracellularly (Fig. 2) by AMP
dephosphorylation, and extracellularly (Fig. 2) by dephosphorylation of released adenine
nucleotides (Brunton et al., 2006; Rang et al., 2007), mainly ATP (Conlon et al., 2005; Meghji
et al., 1992).
1.2 Adenosine receptors
The intra and extracellular concentration of adenosine is determined, nearby their receptors,
by the existence and function of the transporters. Adenosine is a potent modulator of
cardiovascular function and when administered systemically, adenosine produces
hypotension and bradycardia (Barraco et al., 1987; Evoniuk et al., 1987). These effects are
thought to be mediated at adenosine receptors localized centrally (central nervous system)
and in the periphery (heart and vasculature), through different receptor subtypes,
particularly the adenosine A
1
and A
2A
subtypes (Dhalla et al., 2003; Shryock & Belardinelli,
1997; Spyer & Thomas, 2000; Tabrizchi & Bedi, 2001). In the periphery, A
1
receptors are
located primarily in the heart and mediate negative inotropic and chronotropic effects
(Shryock & Belardinelli, 1997). Adenosine A
2A
receptors are located primarily in the
vasculature and mediate vasodilation (Tabrizchi & Bedi, 2001). In the central nervous
system, adenosine A
1
receptors are widely distributed, while adenosine A
2A
receptors are
found in limited regions of the brain, most prominently in the striatum (Dunwiddie &
Masino, 2001). However, high levels of A
2A
receptors are also found in the cardiovascular
regulation regions of the hindbrain, including the nucleus tractus solitarius and the rostral
ventral lateral medulla (Thomas et al., 2000). In fact, adenosine A
2A
receptors are thought to
play a neuromodulatory role in baroreceptor reflex control (Barraco et al., 1988; Schindler et
al., 2005; Thomas et al., 2000).
Adenosine receptors activation may alter vascular tonus in normotensive rats (Cox, 1979;
Fresco et al., 2002; Fresco et al., 2004; Fresco et al., 2007), and its modulation differs in
hypertensive rats. Thus, it is conceivable that the availability of adenosine may be altered in
pathological conditions (Karoon et al., 1995), such as hypertension (Rocha-Pereira et al., 2009).

Adenosinergic System in the Mesenteric Vessels 111

Fig. 2. Metabolism of Adenosine: extracellular. Partial schema of enzyme pathways involved
in the regulation of extracellular adenosine concentrations. Cyclic adenosine
monophosphate (cAMP) can be transported out of cells upon activation of adenylate
cyclase. The actions of an ecto-phosphodiesterase on cAMP results in the formation
of AMP. AMP can also be directly released by some cell types. AMP is acted upon by
an ecto-5'-nucleotidase to form adenosine; it can then be transported into the cell, or
deaminated to inosine by adenosine deaminase. Hypoxanthine is formed after removal
of ribose from inosine by the actions of purine nucleoside
phosphorylase. Hypoxanthine enters the xanthine oxidase pathway to sequentially
form xanthine and uric acid, generating oxyradicals as a byproduct; Metabolism of
Adenosine: intracellular. Partial schema of enzyme pathways involved in the regulation of
intracellular adenosine concentrations. Adenosine monophosphate (AMP) can be directly
deaminated to inosine monophosphate (IMP) by AMP deaminase, or acted upon by an
endo-5'-nucleotidase to form adenosine; it can be rephosphorylated to AMP by adenosine
kinase, or deaminated to inosine by adenosine deaminase. IMP can also be a source of
inosine by the same endo-5'-nucleotidase. Hypoxanthine is formed after removal
of ribose from inosine by the actions of purine nucleoside phosphorylase. Hypoxanthine can
be salvaged to IMP by hypoxanthinephospho-ribosyltransferase, or by entering the xanthine
oxidase pathway to sequentially form xanthine and uric acid, generating oxyradicals as a
byproduct. Intracellular adenosine can be transported into and out of the cell by membrane-
associated transporter proteins. Being an endogenous purine nucleoside, adenosine is
constitutively present in the extracellular spaces at low concentrations. However, its levels
increase dramatically in blood and interstitial fluids (extracellular level), in response to cell
injury and metabolically-stressful conditions such as tissue damage, hypoxia, ischemia and
inflammation. Extracellular adenosine levels have been observed to increase by
dephosphorylation of ATP and so a large amount of adenosine is produced from the
breakdown of adenine nucleotides by ecto-5-nucleotidase (Fig. 2) (Cronstein, 1994; Li et al.,
2009; Li et al., 2011); and then to be released through the action of specialized nucleoside
transporters (Pastor-Anglada et al., 2001).

The Cardiovascular System Physiology, Diagnostics and Clinical Implications 112

Fig. 3. Adenosine binding to purinergic receptors in smooth muscle tissue. Adenosine can
bind to purinergic receptors in different cell types where it can produce diverse
physiological actions. One important action is vascular smooth muscle relaxation, which
leads to vasodilation. This mechanism is particularly important for matching coronary blood
flow to the metabolic needs of the heart. In coronary vascular smooth muscle, adenosine
binds to adenosine receptors A
2A
, which are coupled to the Gs-protein. Activation of this G-
protein stimulates adenylate cyclase, increases cAMP and causes protein kinase activation.
This stimulates K
ATP
channels, which hyperpolarize the smooth muscle, causing relaxation.
Increased cAMP also causes smooth muscle relaxation by inhibiting myosin light chain
kinase, which leads to decreased myosin phosphorylation and a decrease in contractile
force. There is also evidence that adenosine inhibits Ca
2+
entry into the cell through L-type
Ca
2+
channels. Since Ca
2+
regulates smooth muscle contraction, reduced intracellular Ca
2+

causes relaxation. In some types of blood vessels, there is evidence that adenosine produces
vasodilation through increases in cGMP, which leads to inhibition of Ca
2+
entry into the cells
as well as opening of K
+
channels.
It is well established that adenosine effects occur via activation of specific membrane
receptors, known as A
1
, A
2A
, A
2B
and A
3
(Olsson & Pearson, 1990; Ralevic & Burnstock, 1998)
that are currently accepted to be coupled to G
i/o
, G
s
, G
s/Gq
and G
i/o
/G
q
, respectively
(Fredholm et al., 2001). Adenosine receptors are broadly grouped into two categories:
A
1
and A
3
receptors, which couple to inhibitory G proteins, and A
2A
and A
2B
receptors,
which couple to stimulatory G proteins. However, adenosine receptors are pleiotropic; they
can couple with various G proteins and transduction systems according to their degree of
activation and their particular cellular or subcellular location (Cunha, 2005).
1.2.1 Adenosine receptors and vasodilation
Adenosine receptors are present in many areas of the organism including the smooth
muscle cells of blood vessels - these subtypes of receptors have been found to be distributed

Adenosinergic System in the Mesenteric Vessels 113
in different blood vessels such as the coronary artery, pulmonary artery, mesenteric artery,
renal vasculature and aorta (Olah et al., 1995; Olah & Stiles, 1995).
The importance of the adenosine induced vasodilatation (Fig. 3), is known in the coronary
artery of many species including rats. The vasodilatory effect appears to be mediated by A
2

receptors on vascular smooth muscle cells, thus increasing blood flow and oxygenation;
also, adenosine released during preconditioning by short periods of ischemia followed by
reperfusion can induce cardioprotection to subsequent sustained ischemia (Li et al., 2011).
There are two pathways which can result in relaxation. The first pathway is via activation of
A
2
receptors located on smooth muscle cells, which are linked to K
ATP
sensitive channels,
and the second is through activation of A
2
receptors located on nitric oxide associated
endothelial cells. Alternatively there are blood vessels such as the pulmonary artery in
which vascular control is mediated via both A
1
and A
2
receptor activation, with
vasoconstriction occurring via the activation of A
1
receptors and vasodilatation mediated by
the activation of A
2
receptors (Tabrizchi & Bedi, 2001). A
2
receptor agonists, including 5'-N-
ethylcarboxamide-adenosine, were investigated on porcine coronary artery by King and co-
workers (King et al., 1990), and their findings showed that these compounds caused
vasodilation. These results support the idea that the activation of adenosine A
2
receptors on
smooth muscle results in adenosine-induced relaxation. On the other hand, no evidence has
been linked to A
3
receptor activation producing relaxation of blood vessels. Similar findings
were obtained from a study by Hiley and co-workers (Hiley et al., 1995), which tested the
effects of adenosine analogues on rat mesenteric artery which showed that adenosine
analogue, 5'-N-ethylcarboxamide-adenosine acts on adenosine A
2
receptors on the
mesenteric bed to produce relaxation. In addition, relaxation mediated by adenosine
receptors in the mesenteric bed was sensitive to inhibition by 8-(3-chlorostyryl)caffeine, a
selective adenosine A
2A
receptor antagonist.
Signalling of the adenosine receptor occurs via a G-protein coupled mechanism, with
differences between the subtypes: the A
1
subtype is thought to be coupled to G
i
or G
o

proteins via inhibition of adenylate cyclase or activation of phospholipase C, respectively,
and conducing to the opening of K
+
channels and inhibition of Ca
2+
channels; the A
2A

subtype (A
2A
R) interacts with the G protein G
s
and the A
2B
subtype (A
2B
R) interacts with the
G proteins G
s
or G
q
to induce adenylate cyclase activity and elevate cAMP levels and
consequently, activating calcium channels; the A
3
subtype couples to the G
i
or G
q
proteins
through activation of phospholipase C/D or inhibition of adenylate cyclase, respectively
(Olah & Stiles, 1995).
It is conceivable that adenosine cardioprotective effect is mediated through the activation of
adenosine receptors A
1
and A
3
in cardiomyocytes, and involves protein kinase C and
mitochondrial K
ATP
channels. However, a recent study has shown that A
2B
receptors may
also be involved since adenosine A
2B
receptor-deficient mice are more susceptible to acute
myocardial ischemia and the treatment of normal mice with an agonist of the receptor A
2B

significantly attenuated the infarct size after ischemia (Li et al., 2011).
It has been established that adenosine receptor activation occurs via a series of signalling
pathways as a result of the binding of adenosine. The affinity of these receptors for
adenosine varies; thus their activation depends on the adenosines concentration.
Metabolism and transport across the plasma membrane are the main factors influencing the
adenosine level (Podgorska et al., 2005).

The Cardiovascular System Physiology, Diagnostics and Clinical Implications 114
In summary, when adenosine binds to these receptors it can cause vascular smooth muscle
relaxation leading to vasodilatation of blood vessels. As mentioned above, this occurs via a
G-protein coupled protein mechanism. On activation of the G-protein, adenylate cyclase is
activated causing an increase in cAMP concentration. This then leads to protein kinase A
activation which stimulates K
+
channels, hyperpolarizing smooth muscle, causing relaxation
(Tawfik et al., 2005).
1.3 Nucleoside transporters
Membrane transporters are responsible for the uptake of essential nutrients, modulation of
concentrations of physiologically relevant chemicals, and active release of substances such
as signaling molecules (Hyde et al., 2001). Transmembrane transport is a critically important
physiological process in all cells and, is likely to have evolved early to allow for controlled
uptake and release of nonlipophilic compounds. Nucleoside Transporters constitute a
family of membrane proteins with different pharmacological and kinetic properties
(Fredholm, 2003), recently identified and characterized in humans. These transport proteins
were initially purified from human blood red cells for more than two decades ago, and the
lack of abundance of nucleoside transporters proteins in the membranes of mammalian
cells, has hampered the analysis of the relationship between its structure and function
(Endres et al., 2009; Molina-Arcas et al., 2008; Molina-Arcas et al., 2009).
As previously discussed, there are several ways in which adenosine can be produced and
made available for adenosine receptors. One of such, being the transport of adenosine across
the plasma membrane, through nucleoside transporters, which determine the intra and
extracellular levels of nucleosides, including adenosine (Baldwin et al., 2004; Lu et al., 2004).
Generally, nucleoside transporters facilitate the movement of nucleosides and nucleobases
across cell membranes but their distribution is not homogeneous among tissues, and their
expression can be regulated by various physiological and pathophysiological conditions
(Baldwin et al., 2004; Lu et al., 2004; Molina-Arcas et al., 2008). Over the past two decades
important advances in the understanding of nucleoside transporters functioning have been
achieved. One of nucleoside transporters functions is to salvage extracellular nucleosides for
intracellular synthesis of nucleotides; besides, they also control the extracellular
concentration of adenosine in the vicinity of its cell surface receptors and regulate processes
such as neurotransmission and cardiovascular activity (Anderson et al., 1999; Cass et al.,
1999). Other function of nucleoside transporters is vital for the synthesis of nucleic acids in
cells that lack de novo purine synthesis: carrier-mediated transport of this nucleoside plays
an important role in modulating cell function, because the efficiency of the transport
processes determines adenosine availability to its receptors or to metabolizing enzymes.
Therefore, nucleoside transporters may be key elements as therapeutic targets in the
cardiovascular disorders as they are, for example, in anticancer and antiviral therapy where
nucleoside analogues are successfully used (Huber-Ruano & Pastor-Anglada, 2009; Lu et al.,
2004; Molina-Arcas et al., 2005; Yao et al., 2002).
To date it is accepted that there are two types of transporters (Fig. 6) (Baldwin et al., 2004;
Podgorska et al., 2005):
Equilibrative Nucleoside Transporters (ENT) equilibrative bidirectional transport
processes driven by chemical gradients by facilitated diffusion. ENT are present in

Adenosinergic System in the Mesenteric Vessels 115
most, possibly all, cell types (Cass et al., 1998). They might mediate adenosine
transporter in both directions, depending on the concentration gradient of adenosine
across the plasma membrane. Until the present day, there are four subtypes described:
ENT1, ENT2, ENT3 and ENT4 (Baldwin et al., 2004; Molina-Arcas et al., 2009;
Podgorska et al., 2005).
Concentrative Transporters (CNT) active inwardly directed concentrative processes,
driven by the Na
+
electrochemical gradient: Na
+
-dependent. CNT are expressed in a
tissue-specific fashion (Cass et al., 1998). Three subtypes were described: CNT1, CNT2
and CNT3 (Hyde et al., 2001; Kong et al., 2004; Molina-Arcas et al., 2009).
Identication and molecular cloning of the ENT and CNT families from mammals and
protozoan parasites have provided detailed information about the structure, function,
regulation, tissue and cellular localization (Baldwin et al., 2004; Molina-Arcas et al., 2008).
Comparing these different types of transporters, CNT and ENT, some differences become
evident. Whereas the CNT transport processes are present primarily in specialized epithelia,
the ENT transport processes are found in most mammalian cell types (Cass et al., 1998).
Both types of transporters are tightly regulated, both by endocrine and growth factors and by
substrate availability. They transport endogenous substrates such as adenosine, thymidine,
cytidine, guanosine, uridine, inosine, and hypoxanthine (Lu et al., 2004). They are both
involved in the transport of adenosine, but ENT have higher affinity for adenosine than CNT
(Molina-Arcas et al., 2009), a reason why the present study focused exclusively on ENT.
ENT play an important role in the provision of nucleosides, derived from the diet or
produced by tissues such as the liver, for salvage pathways of nucleotide synthesis in those
cells deficient in de novo biosynthetic pathways. The latter include erythrocytes, leukocytes,
bone marrow cells and some cells in the brain. The co-existence in many cell types of both
ENT1 and ENT2, which exhibit similar nucleoside specificities, may reflect the importance
of the ENT2 substrate hypoxanthine as a source of purines for salvage. Similarly, this ability
to transport hypoxanthine and the higher apparent affinity of ENT2 for inosine have been
suggested to reflect a role in the efflux or uptake of these adenosine metabolites during
muscle exercise and recovery respectively (Baldwin et al., 2004; Endres et al., 2009). Several
polymorphisms have been described in ENT proteins that could affect nucleoside
homeostasis, adenosine signalling events or nucleoside-derived drug cytotoxicity or
pharmacokinetics (Kong et al., 2004; Molina-Arcas et al., 2009). Although the transport of
adenosine involves a simple carrier system, it is a complex process.
1.3.1 Equilibrative nucleoside transporters isoforms
The first example of the ENT family was characterized in human tissues at the molecular
level only 10 years ago. Since that time, the identification of homologous proteins by
functional cloning and genome analysis has revealed that the family is widely distributed in
eukaryotes. The SLC29 family of integral membrane proteins, is part of a larger group of
equilibrative and concentrative nucleoside and nucleobase transporters found in many
eukaryotes. ENT are a unique family of proteins with no apparent sequence homology to
other types of transporters, which enable facilitated diffusion of nucleosides, such as
adenosine, and nucleoside analogues across cell membranes (Hyde et al., 2001). Studies
performed over the past thirty years have revealed that most mammalian cells exhibit low-

The Cardiovascular System Physiology, Diagnostics and Clinical Implications 116
affinity, ENT processes, now known to be mediated by members of the SLC29 family. Some
mammalian ENT have been well characterized at the molecular and pharmacological levels
(Crawford et al., 1998), and currently, four isoforms are known: ENT14 (Hyde et al., 2001).
Human (h) and rat (r) ENT1 and ENT2 (456457 amino acid residues) transport both purine
and pyrimidine nucleosides, including ADO. They also differ in their sensitivity to
vasodilator drugs (hENT1 > hENT2 > rENT1 > rENT2) and by the ability of hENT2 and
rENT2 to transport nucleobases as well as nucleosides (Hyde et al., 2001).
ENT family members are predicted to possess 11 transmembrane helices, with a cytoplasmic
N-terminus and an extracellular C-terminus experimentally confirmed for ENT1 (Baldwin et
al., 2004). The number of molecules present of each ENT subtype depends on both the cell
and the tissue type. The intra and extracellular concentration of adenosine is determined,
nearby their receptors, by the existence and function of the transporters, and the four
isoforms although structurally similar, show differences in their ability to regulate
adenosine concentrations, which may be due to slight modifications in configuration
(Baldwin et al., 2004).
Whilst the name of the family reflects the properties of its prototypical member ENT1, some
family members can also transport nucleobases and some are proton-dependent,
concentrative transporters. Therefore, the transporters play key roles in nucleoside and
nucleobase uptake for salvage pathways of nucleotide synthesis, and are also responsible for
the cellular uptake of nucleoside analogues. In addition, by regulating the concentration of
adenosine available to cell surface receptors, they influence many physiological processes
ranging from cardiovascular activity to neurotransmission (Baldwin et al., 2004). ENT are
targets, for example, for coronary vasodilator drugs, are responsible for the cellular uptake
of nucleoside analogues used in the treatment of cancers and viral diseases (Elwi et al., 2006;
Young et al., 2008) and they can also act as routes for uptake of cytotoxic drugs in humans
and protozoa (Hyde et al., 2001).
The best-characterized members of the family, ENT1 and ENT2, are cell surface proteins that
possess similar broad substrate specificities for purine and pyrimidine nucleosides
regulating, eventually, the access of adenosine to its receptors. ENT1 plays a primary role
mediating adenosine transport while ENT2, in addition, efficiently transport nucleobases
(Baldwin et al., 2004). More recently, the ENT3 and ENT4 isoforms have been shown to be
also genuine nucleoside transporters, they are both pH sensitive, and optimally active under
acidic conditions. ENT3 has a similar broad permeant selectivity for nucleosides and
nucleobases and appears to function in intracellular membranes, including lysosomes. ENT4
is uniquely selective for adenosine, but yet present a low affinity to this nucleoside, and it
may also transport a variety of organic cations (Baldwin et al., 2004; Kong et al., 2004).
All four isoforms are widely distributed in mammalian tissues, although their relative
abundance varies. In polarised cells ENT1 and ENT2 are found in the basolateral membrane
and, in tandem with CNT of the SLC28 family, may play a role in transepithelial nucleoside
transport. ENT2 is known to be particularly abundant in skeletal muscle while the ENT3
isoform seems to be widely distributed and the most abundant ENT in the heart.
Nevertheless, since ENT3 is a lysosomal transporter functioning in intracellular membranes,
is unlikely to contribute to a direct regulation of interstitial adenosine concentrations in
tissues. Finally, in what concerns the ENT4, it presents low sequence identity to the other

Adenosinergic System in the Mesenteric Vessels 117
members of the family (due to differences in its structure), is highly selective for adenosine
and is also widely distributed. For instance, ENT4 is present in vascular endothelial cells
and contributes to regulate the extracellular concentration of adenosine in these structures
but only at acidic pH (Baldwin et al., 2004; Barnes et al., 2006).
In summary, all four members of the family share an ability to transport adenosine, but
differ in their abilities to transport other nucleosides and nucleobases.
The human gene encoding the human ENT1 (hENT1) protein has been localized to region
p21.1-21.2 on chromosome 6 (Baldwin et al., 2004). hENT1 protein consists of 456-residue
protein and its sequence displays about 78% identity to the 457-residue rat homologue
(rENT1) and 79% identical to the 460-residue mouse protein (mENT1.1) homologues. Splice
variants of hENT1 have not been reported, but a 458-residue variant of the mouse
homologue (mENT1.2), generated by alternative splicing at the end of exon 7, is widely
distributed (Abdulla & Coe, 2007).
The two forms of mENT1 protein appear to be functionally identical, although mENT1.2
lacks the potential casein kinase II phosphorylation site. Both rENT1 and hENT1 proteins
display broad substrate specicity for pyrimidine and purine nucleosides with Km values
ranging from 50 mM (adenosine) to 680 mM (cytidine), but are unable to transport the
pyrimidine base uracil (Yao et al., 1997). hENT1 and mENT1, which are sensitive to
nitrobenzylthioinosine (NBMPR), are also inhibited by the coronary vasodilators
dipyridamole, dilazep, and draazine. In contrast, rENT1 although presenting sensitivity to
NBMPR is essentially insensitive to inhibition by the coronary vasodilators dipyridamole
and dilazep (Baldwin et al., 2004; Podgorska et al., 2005; Ward et al., 2000; Yao et al., 1997).
The messenger ribonucleic acid (mRNA) for hENT1 is widely distributed in different tissues,
including erythrocytes, liver, heart, spleen, kidney, lung, intestine, and brain (Endres et al.,
2009; Griffith & Jarvis, 1996; Lum et al., 2000; Pennycooke et al., 2001). mENT1.2 protein was
shown to be commonly co-expressed with mENT1.1 (460 aminoacids) and the highest level
was found in the liver, heart and testis. Moreover, studies at both the mRNA and protein
levels have revealed that ENT1 is almost ubiquitously distributed in human and rodent
tissues, although its abundance varies between tissues (Baldwin et al., 2005).
Human ENT2 (hENT2) protein, responsible for the ei type nucleoside transport, is encoded
by a gene localized at position 13q on chromosome 11. hENT2 consists of 456 aminoacids
and their sequence displays 88% identity to mouse (mENT2) and rat (rENT2) homologues.
In humans, besides the 456-aminoacid ENT2 protein, exists at least, two shorter forms of
ENT2, generated from mRNA splice variants. The 326 aminoacid protein, termed hHNP36,
lacks the rst three transmembrane domains and is inactive as a nucleoside transporter.
Inactive is also the second splice variant, a 301-aminoacid protein named hENT2A that lacks
the C-terminal domain (Crawford et al., 1998).
The ENT2 protein accepts a broad range of substrates, including purine and pyrimidine
nucleosides and nucleobases. It has been postulated that hENT2 plays a role in the efflux
and reuptake of inosine and hypoxanthine generated from adenosine during and after
strenuous physical exercise. ENT2 (both rat and human), is much less susceptible to inhibition
by NBMPR and the coronary vasodilators dipyridamole and draazinethan ENT1 (Baldwin et
al., 2004; Crawford et al., 1998; Podgorska et al., 2005; Ward et al., 2000; Yao et al., 1997, 2002).

The Cardiovascular System Physiology, Diagnostics and Clinical Implications 118
The mRNA for ENT2 was reported to be present in several tissues including heart, kidney,
brain, placenta, thymus, pancreas, intestine and prostate, but the highest expression level was
found in skeletal muscle (Crawford et al., 1998; Lum et al., 2000; Pennycooke et al., 2001).
The gene encoding the human ENT3 (hENT3) protein is located at position q22.1 on
chromosome 10. hENT3 is a 475-residue protein displaying 73% identity to the mouse
homologue (mENT3) (Baldwin et al., 2004, 2005; Kong et al., 2004). ENT3 has a
characteristic, long (51 aminoacids), hydrophilic N-terminal region preceding the rst
transmembrane (TM1) domain. The N-terminal region of ENT3 consists of two di-leucine
motifs characteristic for endossomal, lysosomal targeting motifs. This architectural design
distinguishes the ENT3 protein from other members of the equilibrative transporters family.
Indeed, it was demonstrated that hENT3 protein is predominantly localized intracellularly
and that mutation of the dileucine motif to alanine triggers the relocation of ENT3 protein to
the cell surface (Baldwin et al., 2004, 2005).
In comparison with ENT1, the ENT3 protein is much less susceptible to inhibition by
NBMPR and coronary vasodilatory drugs (dipyridamole and dilazep). hENT3 demonstrates
a broad selectivity for nucleosides, but does not transport hypoxanthine. Moreover, the
hENT3 protein facilitates transport of several adenosine analogues like cordycepin (3-
deoxyadenosine) (Baldwin et al., 2004; Podgorska et al., 2005). hENT3 and hENT4, which are
mainly located in the intracellular organelles, are not prominent nucleoside transporters like
hENT1 and hENT2 (Endo et al., 2007).
The mRNA for ENT3 has been detected in a variety of mouse and human tissues, including
brain, kidney, colon, testis, liver, spleen, placenta (highest level), and in a number of
neoplastic tissues (Baldwin et al., 2004, 2005; Hyde et al., 2001).
The gene encoding the human ENT4 (hENT4) protein is located on chromosome 7, at
position p22.1. Interestingly, the hENT4 is more closely related to the products of the
Drosophila melanogaster gene CG11010 (28% identity) and the Anopheles gambiae gene
agCG56160 (30% identity), than to hENT1 (18% identity), indicating an ancient divergence
from the other members of the SLC29 family (Acimovic & Coe, 2002). hENT4 is a 530-
residue protein 86% identical in sequence to its 528-residue mouse homologue (mENT4)
(Baldwin et al., 2004). The substrate specicity of hENT4 has not yet been established in
detail, but among the ENT proteins, hENT4 has the lowest affinity for adenosine (Kong et
al., 2004). The mRNA for hENT4 was detected in several human tissues. However, recent
characterisation of the complementary deoxyribonucleic acids (cDNAs) encoding h/mENT4
has confirmed that these proteins are indeed nucleoside transporters, capable of low-affinity
adenosine transport. Analysis of multiple tissue RNA arrays indicates that hENT4 is likely
to be ubiquitously expressed in human tissues (Baldwin et al., 2005; Podgorska et al., 2005).
1.3.2 Equilibrative nucleoside transporters in the cardiovascular system
There are currently no reports implicating ENT - SLC29 transporters family, in the
pathogenesis of human disease (Baldwin et al., 2004). Still, as mentioned above, adenosine
transporters contribute to the intra and extracellular concentration of adenosine, modulating
its concentration in the vicinity of its receptors (Li et al., 2011; Tawfik et al., 2005). It is
therefore conceivable that the availability of adenosine may be altered in pathological states.

Adenosinergic System in the Mesenteric Vessels 119
Adenosine exerts vasodilatory and cardioprotective effects, and also reduces the
proliferation of vascular smooth muscle cells, inhibits platelet aggregation and attenuates
the inflammatory response. Apart from adenosine receptors and ecto-5-nucleotidase,
transporter proteins can regulate adenosine function by modulating extracellular levels of
adenosine. The extracellular adenosine is rapidly taken up into cells by nucleoside
transporters and is, subsequently, metabolized to inosine by adenosine deaminase and
phosphorylated to AMP by adenosine kinase. Nucleoside transporters are supposed to play
an integral part in adenosine functions by fine-tuning local levels of adenosine in the
vicinity of adenosine receptors (Li et al., 2011).
Recent studies have proposed the occurrence of a greater degree of adenosine release from
cells that are metabolically stressed. In other words, cells with a high oxygen demand such
as the vascular smooth muscle cells in the hypertensive state (Conlon et al., 2005; Tabrizchi
& Bedi, 2001). Several studies have been conducted in order to further understand the role
of ENT in cardiovascular diseases (Chaudary et al., 2004; Li et al., 2011; Reyes et al., 2010;
Rose et al., 2010). Adenosine seems to be a cardioprotective metabolite. Hypoxia and
ischemia lead to a large increase in extracellular adenosine, which is released by
cardiomyocytes. Extracellular adenosine activates G-protein coupled adenosine receptors
linked to various signalling pathways, which initiate compensatory responses. Intracellular
and extracellular levels of adenosine fluctuate, considerably, depending on the metabolic
state of the heart, the flux of adenosine (down its concentration gradient), across the
cardiomyocyte cell membrane, is facilitated by the ENT. These transporters are highly
expressed in the cardiovascular system but very little is known about their role in
cardiomyocyte physiology (Baldwin et al., 2004; Chaudary et al., 2004; Reyes et al., 2010).
As previously mentioned, ENT are bidirectional, allowing adenosine to be released from
cells (to act as an autocrine/paracrine hormone), or transported into the cell (to terminate
receptor activation, or restore adenosine metabolite pools). Thus, cardiomyocyte adenosine
physiology is dependent on the adenosine receptor profile, and on the presence and activity
of the ENT. In the past years, ENT have been shown to be important in modulating the
effects of adenosine in human epithelial cells. Moreover, a correlation was found between
ENT1 and A
1
adenosine receptor distribution in the brain, suggesting potential interactions
and/or feedback between receptors and transporters. Nevertheless, there is an extensive
literature on adenosine and adenosine receptor physiology in the cardiovasculature,
whereas very little is known about ENT (Baldwin et al., 2004; Chaudary et al., 2004).
ENT inhibitors, by virtue of their effect on extracellular adenosine concentrations, can also
modulate a variety of physiological processes, potentially leading to therapeutic benefits.
For example, by inhibiting nucleoside uptake into endothelial and other cells the coronary
vasodilator draflazine substantially increases and prolongs the cardiovascular effects of
adenosine. The latter exerts beneficial, cardioprotective effects in the ischaemic/reperfused
myocardium mediated, at least in part, via activation of A
1
and possibly also A
3
receptors,
probably involving the protein kinase C and mitochondrial K
ATP
channels. Transport
inhibitors have also potential value in the context of ischaemic neuronal injury: pre-
ischaemic administration of the pro-drug NBMPR phosphate has been shown to increase
brain adenosine levels and reduce ischaemia-induced loss of hippocampal neurons in the
rat. In a clinical setting, pharmacological inhibition of ENT, using drugs such as

The Cardiovascular System Physiology, Diagnostics and Clinical Implications 120
dipyridamole, dilazep and draflazine, is used to promote cardiovascular health. However,
despite the clinical relevance of ENT as drug targets, very little is known about them
(Baldwin et al., 2004; Tabrizchi & Bedi, 2001; Takahashi et al., 2010).
Recent studies have challenged the role of ENT in purine nucleoside-dependent physiology
of the cardiovascular system. Rose and co-workers (2010), investigated whether the ENT1-
null mouse heart was cardioprotected in response to ischaemia. In that study, the authors
observed that ENT1-null mouse hearts showed significantly less myocardial infarction
compared with wild-type littermates, demonstrating that ENT1 activity may contribute to
cardiac injury. A posterior study (Reyes et al., 2010), confirmed that isolated wild-type adult
mouse cardiomyocytes express predominantly ENT1, which is primarily responsible for
purine nucleoside uptake in these cells. However, ENT1-null cardiomyocytes exhibit
severely impaired nucleoside transport and lack ENT1 transcript and protein expression.
Adenosine receptor expression profiles and expression levels of ENT2, ENT3, and ENT4
were similar in cardiomyocytes isolated from ENT1-null adult mice compared with
cardiomyocytes isolated from wild-type littermates. Moreover, small interfering RNA
knockdown of ENT1 in the cardiomyocyte cell line, mimics findings in ENT1-null
cardiomyocytes. Taken together, the data from the study conducted by Rose and co-workers
(2010), demonstrated that the absence of ENT1 plays an essential role in cardioprotection,
most likely due to its effects in modulating purine nucleoside-dependent signalling and that
the ENT1-null mouse is a powerful model system for the study of the role of ENT in the
physiology of the cardiomyocyte.
Other authors, determined that adenosine and inosine accumulate extracellularly during
hypoxia/ischaemia and that both may act as neuroprotectors (Takahashi et al., 2010). In the
spinal cord, there was pharmacological evidence for an extracellular adenosine levels
increase during hypoxia, but no direct measurements of purine release have been done;
furthermore, the efflux pathways and origin of extracellular purines are still not defined.
Therefore, to characterize hypoxia-evoked purine accumulation, Takahashi and co-workers
(2010), examined the effect of acute hypoxia on the extracellular levels of adenosine and
inosine in isolated spinal cords from rats, and these authors found that both inhibitors of
adenosine deaminase or ENT, abolished the hypoxia-evoked increase in inosine but not
adenosine: extracellular level of inosine was about 10-fold higher than that of adenosine.
These data suggest that hypoxia releases adenosine itself from intracellular sources, on the
other hand, inosine formed intracellularly may be released through ENT (Takahashi et al.,
2010).
Gestational diabetes has been associated with increased L-arginine transport and nitric
oxide (NO) synthesis as well as a reduced adenosine transport in human umbilical vein
endothelial cells. Adenosine increases endothelial L-arginine/NO pathway via A
2
adenosine
receptors in human umbilical vein endothelial cells, in normal pregnancies (Vasquez et al.,
2004; Vega et al., 2009) compared to the reduction in adenosine transport observed in veins
of women with gestational diabetes. Additionally, an association between L-arginine
transport and NO synthesis was also found. In fact, Vsquez and co-workers (2004),
demonstrated that in gestational diabetes, stimulation of L-arginine transport and NO
synthesis occurs with a reduction in adenosine transport in human umbilical vein
endothelial cells.

Adenosinergic System in the Mesenteric Vessels 121
The effect of gestational diabetes on the L-arginine/NO pathway may result from an
increased extracellular adenosine level, due to low adenosine uptake as a consequence of a
reduced hENT1mRNA expression. Accumulation of extracellular adenosine could activate
A
2A
adenosine receptors, which leads to an increased expression of cationic amino acid
transporter-1 (hCAT-1) mRNA, and of endothelial nitric oxide synthase (eNOS), mRNA or
protein expression, an increased L-arginine transport activity, as well as, of the NO
synthesis. The effect of gestational diabetes on adenosine and L-arginine transport involves
activation of protein kinase C, and p42/44 MAPK pathways and increased the NO levels.
Thus, the authors hypothesized the establishment of a functional link between adenosine
transport and the L-arginine/NO pathway, governing the normal function of human fetal
endothelium from gestational diabetic pregnancies (Vasquez et al., 2004; Vega et al., 2009).
These results also highlight the physiological effects of purinoceptors, particularly of
adenosine receptors, in the umbilical vein endothelium, in pathologies, where alterations of
blood flow from the mother to the fetus (via the umbilical vein may occur), altering the
normal supply of nutrients to the developing fetus, such as in intrauterine growth
restriction, fetal hypoxia or gestational diabetes. Finally, these findings also demonstrate
that gestational diabetes induces alterations in the phenotype of human fetal endothelium
(Vasquez et al., 2004).
It has been demonstrated that insulin inhibited elevated ENT1 expression in human
umbilical arterial smooth muscle cells from pregnancies in diabetic subjects (Aguayo et al.,
2001). However this is probably due to the activation of adenylate cyclase rather than the
effect of insulin on glucose metabolism. The effects of oral anti-diabetic agents on nucleoside
transporters are rarely reported: Li and co-workers (2011), studied the effects of different
oral anti-diabetic agents such as metformin, sulfonyureas, meglitinides and
thiazolidinediones on nucleoside transporters; among them, only the thiazolidinedione
troglitazone showed inhibitory effects on nucleoside transporters, but unfortunately it was
withdrawn because of hepatic toxicity.
To our knowledge, until the present date, only one study has been carried out to investigate
the relationship between hypertension and nucleoside transporters. The binding of a ENT1
probe [
3
H]NBMPR in membranes prepared from platelets, as well as renal, pulmonary, cardiac
and brain tissues of Spontaneously Hypertensive Rats (SHR), was compared to those of age
matched Wistar-Kyoto (WKY) controls (Williams et al., 1990). The number of [
3
H]NBMPR
binding sites were higher in the kidneys of SHR but lower in platelets, whereas no difference
was found in the heart, lung or brain. Age-dependent decreases were also observed in the
heart and platelets of SHR and WKY. The results indicated that the expression of ENT1
changed with age as well as with the pathogenesis of hypertension. Li and co-workers (2011),
compared the expressions of nucleoside transporters in basilar arteries in SHR and WKY rats
and they found that ENT1 and ENT2 were unaffected by hypertension. Interestingly, the
mRNA expression of CNT2 was higher than that seen in WKY; nevertheless, whether the
upregulation of CNT2 is a primary or secondary event in the development of hypertension is
questionable. It has been speculated that the increase in the activities of ENT1 and CNT2 may
reduce the availability of adenosine to its receptors, thereby weakening the vascular functions
of adenosine. It may explain why patients with diabetes and hypertension suffer greater
morbidity from ischemia and atherosclerosis (Li et al., 2011).

The Cardiovascular System Physiology, Diagnostics and Clinical Implications 122
2. Mesenteric vessels
The branches of the abdominal aorta are divided into parietal and visceral parts. The
visceral arteries are in turn divided into paired and unpaired branches. The mesenteric
artery is an elasto-muscular resistance vessel. In adult rats, for example WKY, the
mesenteric artery branches from the abdominal aorta and is composed of five to seven
concentric layers of smooth muscle cells, separated by three to four medial laminae. The
medium is separated from the endothelial cells of the intima by the continuous internal
elastic lamina and from the adventitia, which contains a few fibroblasts and nerve terminals,
by the external elastic lamina (McGuire et al., 1993; Sullivan et al., 2002).
Three major unpaired branches exist: the celiac trunk, the superior mesenteric artery and the
inferior mesenteric artery. Each has several major branches supplying the abdominal organs:
the superior mesenteric artery, supplies the pancreas, small intestine and the colon, whereas
the inferior mesenteric artery supplies the descending colon and rectum (Fig. 4).

Fig. 4. Schematic representation of splanchnic circulation. Under normal resting conditions
in humans, total hepatic blood flow is 1200 to 1400 mL/min (~100 mL/min/100g), which
represents about 25% of cardiac output. Blood flow to the four lobes of the liver is derived
from two major sources, the portal vein and the hepatic artery. The hepatic artery is a
branch of the celiac axis and accounts for 25 to 30 percent of total hepatic blood flow and 45
to 50 percent of the oxygen supply. The portal vein is a valveless afferent nutrient vessel of
the liver that carries blood from the entire capillary system of the stomach, spleen, pancreas,
and intestine (McGuire et al., 1993).

Adenosinergic System in the Mesenteric Vessels 123
The mesenteric circulation plays an important role in maintenance of systemic blood
pressure, and regulation of tissue blood flow. Actually, the entire splanchnic circulation can
receive up to 60% of cardiac output and contains about one third of the total blood volume.
Mesenteric arteries and veins have significant resistance and capacitance functions in the
systemic circulation, respectively. In comparison to the associated veins, the mesenteric
artery has a high resting basal tone mediated in part by a thicker layer of vascular smooth
muscle (Kreulen, 2003). Constriction of the mesenteric artery is thought to increase total
peripheral resistance in the systemic circulation greatly. In contrast, the mesenteric vein,
contains fewer layers of vascular smooth muscle cells and are more compliant vessels. The
function of these low pressure vessels is to store significant quantities of blood that can be
utilized to maintain the central venous pool of blood and cardiac output. As such, the
degree of vascular tone in mesenteric vasculature plays a major role in the regulation of
systemic blood pressure and overall body hemodynamics (Ross & Pawlina, 2006).
2.1 Vascular tonus regulation and physiology in mesenteric vessels
The tone of the mesenteric artery and resistance blood vessels are mainly regulated by
sympathetic adrenergic nerves through the release of neurotransmitter noradrenaline. It is also
controlled by nonadrenergic noncholinergic nerves, and possibly by parasympathetic
cholinergic nerves. Noradrenaline and adrenergic cotransmitters including neuropeptide Y,
and ATP, act as a vasoconstrictor neurotransmitter for sympathetic nerves. While, dopamine,
calcitonin gene-related peptide and acetylcholine act as a vasodilator neurotransmitter for
adrenergic, nonadrenergic noncholinergic and cholinergic nerves, respectively. In the
mesenteric circulation, these nerves containing various neurotransmitters and cotransmitters
interact and modulate each other via feedback autoregulatory mechanisms and
neuromodulation of various vasoactive substance to regulate vascular resistance (Takenaga &
Kawasaki, 1999). In fact, net vascular tone in the mesenteric vasculature is under the influence
of several key factors. These factors include locally acting and circulating hormones, intrinsic
myogenic properties of the vessel, as well as neurotransmitters released from perivascular
post-ganglionic sympathetic neurons. In general, the arteries and veins of the splanchnic
circulation are richly innervated with sympathetic nerves that act to constrict these vessels.
Maximal activation of the sympathetic constrictor nerves can produce an 80% reduction in
blood flow to the splanchnic region (Morhrman & Heller, 2006).
In vivo, sympathetic neurogenic influence of vascular tone is mediated by three
neurotransmitters: neuropeptide Y, noradrenaline, and ATP make up the sympathetic triad
of neurotransmitters. Perivascular neurons store the sympathetic neurotransmitters in
synaptic vesicles and release these neurotransmitters from varicosities to act on
postjunctional receptors on the vascular smooth muscle cells. The arrangement of the
sympathetic neurons differs between arteries and veins. The nerve plexus for the mesenteric
artery consists of a bundle of axons arranged in a mesh-like network with nerve fibres
equally likely to run parallel or perpendicular to the longitudinal axis of the vessel. In
contrast, in mesenteric vein the nerve plexus consists of single axons with a circumferential
nerve fibre arrangement about the vessel. In both cases, the sympathetic neurotransmitters
released cause depolarization of the nearby vascular smooth muscle cells. As a whole,
activation the sympathetic postjunctional receptors mediates contraction of the vascular
smooth muscle cells and, therefore, constriction of the artery or vein (Park et al., 2007).

The Cardiovascular System Physiology, Diagnostics and Clinical Implications 124
In vitro, electrical field stimulation studies have found that upon stimulation of mesenteric
perivascular nerves a measurable amount of noradrenaline is released (Bobalova &
Mutafova-Yambolieva, 2001). Once released, noradrenaline can act on a variety of receptors
on the vascular smooth muscle cells. Previous in vitro studies have found contractile
responses to be mediated by activation of the
1
adrenoceptor in mesenteric arteries and
both
1
and
2
adrenoceptors in the mesenteric vein (Perez-Rivera et al., 2007). These
adrenoceptors are G-protein linked receptors that are coupled to an intracellular increase of
inositol 1,4,5-triphosphate (IP3). Contraction of the smooth muscle is mediated by IP3 acting
on sarcoplasmic reticulum receptors to release intracellular Ca
2+
stores. Additional Ca
2+
is
taken up into the vascular smooth muscle cells following depolarization via L-type voltage-
gated calcium channels (Lee et al., 2001).
Like noradrenaline, a measurable amount of ATP is released from perivascular sympathetic
neurons when activated by electrical field stimulation (Bobalova & Mutafova-Yambolieva,
2001). In vivo, ATP is thought to mediate neurogenic contractions of vascular smooth muscle
by acting on various purinergic receptors present on the smooth muscle cells. The two
subtypes of purinergic receptors are the P2X and P2Y receptors. The P2X receptors are ATP-
gated ion channels that cause an influx of Ca
2+
into the smooth muscle cells from the
extracellular environment (Donoso et al., 2004). Though there are several P2X receptor
isoforms, there is evidence that vascular smooth muscle cells primarily express the P2X1
receptors (Wang et al., 2002). P2X receptors are thought to be responsible for the excitatory
junction potentials (rapid and short depolarization of vascular smooth muscle) present in
mesenteric artery (Kreulen, 2003). In contrast, excitatory junction potentials are not present
in mesenteric vein. This is largely thought to be the result of selective expression of only the
P2Y receptor subtype in mesenteric vein (Mutafova-Yambolieva et al., 2000). The P2Y
receptors mediate slower contractile responses than the P2X receptors, and are G-protein
linked receptors that have similar intracellular effects as the -adrenoceptors. The isoforms
of the P2Y receptors that are thought to be expressed in vascular smooth muscle cells are the
P2Y2 and the P2Y4 receptors (Galligan et al., 2001).
Noradrenaline and ATP contract the mesenteric artery and the mesenteric vein through the
activation of adrenergic and purinergic vascular smooth muscle receptors respectively. The
use of selective adrenoceptor agonists and antagonists suggests that the
1
adrenoceptor is
the primary adrenoceptor mediating responses to noradrenaline in these vessels. In
addition, the data collected suggests that the P2X and/or the P2Y1 receptors contract the
mesenteric artery, but do not mediate substantial contractile responses in rat mesenteric
vein. Therefore, this data suggests that other purinergic receptors, such as the P2Y2 and the
P2Y4 receptor subtypes, mediate vasoconstriction in these vessels in response to ATP. The
Sympathetic Nervous System, is an important modulator of net vascular tone in mesenteric
arteries and veins, and that sympathetic modulation of these vessels is an important
regulator of the resistance function of mesenteric artery and the capacitance function of
mesenteric vein.
Splanchnic veins and venules account for most of the active capacitance responses in the
circulation and are richly innervated by the Sympathetic Nervous System. In fact, it has been
estimated that innervation to the non hepatic splanchnic organs accounts for half of the total
noradrenaline released in the entire body. Therefore, the recent observations in Angiotensin
II salt hypertension of neurogenically mediated increases, in whole body venous tone,

Adenosinergic System in the Mesenteric Vessels 125
would best be explained by increased Sympathetic Nervous System activity to the splanchnic
circulation. The splanchnic vascular resistance rises in proportion to the blood pressure, and
the transvascular escape rate of plasma proteins is increased. Vascular resistance increases in
the hepatosplanchnic circulation before any other bed in humans with borderline
hypertension. Therefore, increased sympathetic activity to the splanchnic circulation may
represent a common stage in the development of hypertension (King et al., 2007).
The various animal models of hypertension show variable results, but in general support the
concept that vascular resistance changes in the splanchnic organs are similar in direction
and magnitude to pressure changes. These resistance changes appear to result from
increased responsiveness of the arterioles to a variety of constrictor influences, and they
may result from either structural or functional changes. Hypertension appears to alter
splanchnic arteriolar permeability via a pressure-dependent mechanism. These vessels may
also undergo degenerative histological changes. In addition to the resistive and exchange
alterations, the capacitance function of splanchnic veins is reduced, probably via a structural
change (Nyhof et al., 1983).
Also, chronic hypertension is associated with resistance artery remodelling and mechanical
alterations. A study by Briones and co-workers (2003), evaluated the role of elastin in
vascular remodelling of mesenteric artery from SHR. When compared with WKY, the
mesenteric artery of SHR showed: smaller lumen, decreased distensibility at low pressures,
a leftward shift of the stressstrain relationship, redistribution of elastin within the internal
elastic lamina leading to smaller fenestrae but no change in fenestrae number or elastin
amount. Elastase incubation fragmented the structure of internal elastic lamina in a
concentration-dependent fashion, abolished all the structural and mechanical differences
between strains, and decreased distensibility at low pressures. Mesenteric artery
remodelling and increased stiffness are accompanied by elastin restructuring within the
internal elastic lamina and elastin degradation reverses structural and mechanical
alterations of SHR mesenteric artery. Differences in elastin organisation are, therefore, a
central element in small artery remodelling in hypertension (Briones et al., 2003).
The rat superior mesenteric vein, which drains blood from the intestine, or the splenic vein,
which drains from the spleen, is a capacitance vein. The blood flow in the superior mesenteric
vein is the primary source of irrigation of the rat liver and this vein plays an important role in
maintaining bile flow, bile acid excretion, and bilirubin conjugation and in preventing the
precipitation of bile (possibly preventing hepatolithiasis) (Adachi et al., 1991).
The splanchnic venous bed is the largest vascular bed in terms of capacitance because 50%
of the intestinal blood volume is in the venules and small mesenteric veins (Dunbar et al.,
2000). Also, many animal studies have shown that the splanchnic bed is very responsive to
baroreceptor and sympathetic stimulation (Haase & Shoukas, 1991, 1992; Shoukas & Bohlen,
1990), pointing to this vascular bed as a primary source of blood volume changes. These
studies have demonstrated that sympathetic stimulation of splanchnic veins and venules
will cause them to constrict, leading to signicant volume shifts out of this vascular bed.
Consequently, changes in splanchnic venous capacitance can have large effects on venous
lling pressure. Capacitance changes can occur through changes in both vessel compliance
and unstressed vascular volume. One way to experimentally assess these changes is to

The Cardiovascular System Physiology, Diagnostics and Clinical Implications 126
examine changes in the pressure-diameter relationships of individual vessels, particularly in
the splanchnic regions of the body. The mesenteric veins are also critical in modulating
cardiac lling through venoconstriction.
The mesenteric circulation is regulated by multiple mechanisms and there is sufficient
amount of aspects described in the literature that support the suspicion that local metabolic
factors are especially important in the control of intestinal vasculature. Of these, adenosine,
which is a mesenteric vasodilator, may be the messenger of the intestinal tissue to signal
appropriate responses of the intestinal vessels. The evidence supporting the candidacy of
this nucleoside as a local regulator of mesenteric circulation may be summarized, as follows:
adenosine is present in the tissue of the gut in measurable quantities; exogenous adenosine
is a powerful dilator of mesenteric resistance vessels; blockade of adenosine receptors in the
mesenteric circulation interferes significantly with three autoregulatory phenomena, i.e.,
postprandial hyperaemia, pressure-flow autoregulation, and reactive hyperaemia (Jacobson
& Pawlik, 1992).
3. Adenosinergic system and hypertension
Some lines of investigation have already used both these models to study the role of
adenosine in hypertension. For example, in 1987, Jackson performed an interesting assay,
where the author compared the in vivo role of adenosine, as a modulator of noradrenergic
neurotransmission, in the SHR and WKY. In the in situ blood-perfused rat mesentery,
vascular responses to sympathetic periarterial nerve stimulation, and to exogenous
noradrenaline, were enhanced in SHR compared with WKY. In both SHR and WKY,
vascular responses to periarterial nerve stimulation were more sensitive to inhibition by
adenosine, than were responses to noradrenaline. At matched base-line vascular responses,
compared with WKY, SHR were less sensitive to the inhibitory effects of adenosine on
vascular responses to periarterial nerve stimulation, but SHR and WKY were equally
sensitive with respect to adenosine-induced inhibition of responses to noradrenaline.
Antagonism of adenosine receptors with 1,3-dipropyl-8-p-sulfophenylxanthine, shifted the
dose-response curve to exogenous adenosine six-fold to the right, yet did not influence
vascular responses to periarterial nerve stimulation or noradrenaline in either SHR or WKY.
Furthermore, periarterial nerve stimulation did not alter either arterial or mesenteric venous
plasma levels of adenosine in SHR or WKY, and plasma levels of adenosine in both strains
were always lower than the calculated threshold level required to attenuate
neurotransmission. According to these findings, the author concluded that in vivo
exogenous adenosine interferes with noradrenergic neurotransmission in both SHR and
WKY; SHR are less sensitive to the inhibitory effects of exogenous adenosine on
noradrenergic neurotransmission than are WKY; endogenous adenosine does not play a role
in modulating neurotransmission in either strain under the conditions of this study; and
enhanced noradrenergic neurotransmission in the SHR is not due to defective modulation of
neurotransmission by adenosine (Jackson, 1987).
Other studies have found differences in blood vessels when comparing SHR and WKY (Cox,
1979; Gisbert et al., 2002; Leal et al., 2008; Lee, 1987); Rocha-Pereira et al., 2009). Findings
from the study by Gisbert and co-workers (2002), showed that the population of
constitutively active
1D
-adrenoceptors is significantly increased in aorta and mesenteric

Adenosinergic System in the Mesenteric Vessels 127
artery from adult SHR when compared to WKY - these results verify that the vessels in
question, from hypertensive animals, have an increased population of constitutively active
receptors as well as an increased functionality of the
1D
-subtype, with respect to
normotensive animals. Other studies (Villalobos-Molina & Ibarra, 1999; Villalobos-Molina et
al., 1999; Xu et al., 1998), highlighted the importance of the
1D
-adrenoceptor in the pathology
of hypertension, suggesting that, it appears first in the vasculature, followed by a rise in blood
pressure.
1D
-adrenoceptors can be found on smooth muscle cells and the activation of these
receptors by noradrenaline (released by sympathetic postganglionic terminals), conduce
mainly to vasoconstriction. These can lead to an increase in blood pressure playing, therefore,
a role in the development of hypertension observed in SHR animals. The opposite effect to the
contraction of smooth muscle can occur as a result of adenosine acting on A
2
adenosine
receptors, thus causing smooth muscle relaxation. The levels of extracellular adenosine, which
can act on those receptors to produce this effect, can be altered by ENT. These transporters
play a role in determining the levels of adenosine available extracellularly and hence the extent
of vasodilation which can occur (Rang et al., 2007).
On the other hand, previous studies have shown that the vasodilatory response to
adenosine and its analogues is weakened in hypertension and in other pathological
conditions affecting blood vessels (Lockette et al., 1986; Luscher et al., 1987). Lscher and
co-workers (1987) investigated the effects of antihypertensive therapy on hypertensive
rats and their findings demonstrated a prevention or reversal of decreased endothelium-
dependent relaxations in response to agonists, suggesting that antihypertensive treatment
normalizes endothelium-dependent relaxations. It was, therefore, proposed that
antihypertensive treatment may be important in preventing cardiovascular complications
in hypertensive individuals. A possible explanation for the attenuated vasodilatory
response to adenosine in hypertension can be linked to the levels of adenosine receptors
and their functionality in hypertensive models. Vasodilation of blood vessels occurs via
the activation of adenosine receptors by adenosine and so an alteration in these receptors
can result in a modified vasodilatory response (Rocha-Pereira et al., 2009). This hypothesis
can be associated with adenosine transporters, which are partly responsible for making
adenosine available to adenosine receptors. It is, therefore, legitimate to hypothesize that
an increase or decrease in transporter population could indirectly alter the physiology of
the adenosinergic system and, contributing indirectly to the contractility of blood vessels
and conducing to an elevated blood pressure (correspondent to the pathologic situation of
hypertension).
4. Nucleoside transporters as therapeutic tools Future perspectives
Nucleoside derived drugs or nucleobase analogues are being developed and investigated
for a number of different applications, in order to pursue a better and more efficient way of
treating several diseases. The studies conducted in several other conditions might shed
some light in this challenging pathway, as promising results have already been published,
for example in cancer and infection by Human Immunodeficiency Virus (Cano-Soldado et
al., 2008; Damaraju et al., 2003; Endo et al. 2007; Hyde et al., 2001; Kong et al., 2004; Molina-
Arcas et al., 2005, 2009; Ritzel et al., 2001; Yao et al., 2002), chronic pain and inflammation
(Eltzschig et al., 2005; Li et al., 2009, 2011; Reyes et al., 2010).

The Cardiovascular System Physiology, Diagnostics and Clinical Implications 128
The amount of research being done in these past years is indicative of the interest and
potential that the area of ENT have in many scientific fields, especially in what concerns its
role in a pharmacological perspective. Hopefully, in the future, as the knowledge increases
and the mechanisms involving these transporters are better understood, the application of
ENT will have an impact, particularly, in cardiovascular diseases, such as hypertension.
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7
Endothelial Nitric Oxide
Synthase, Nitric Oxide and Metabolic
Disturbances in the Vascular System
Grayna Lutosawska
University of Physical Education, Warsaw,
Poland
1. Introduction
It is well documented that hyperlipidemia, obesity and diabetes increase the risk for the
development of atherosclerosis and subsequent cardiovascular disease (Vinik, 2005, Ritchie
& Connell 2007, Stapleton et al. 2010). However, until now the precise mechanism by which
the above mentioned metabolic perturbations contribute to atherosclerosis has not been
fully elucidated.
Numerous studies have focused on the detrimental effects of excessive body fat stores as a
possible reason for both insulin resistance and disturbed lipoprotein metabolism with special
attention paid to the adverse effects of visceral fat (Sharma et al., 2002, Matsuzawa, 2005). In
overweight and/or obesity, free fatty acids (FFA) are released into the circulation and their
availability for lipoprotein synthesis in the liver is markedly elevated (Jensen, 2006).
Moreover, high circulating FFA negatively affects whole body insulin sensitivity and
disturbs carbohydrate and lipid metabolism (Kohen-Avramoglu et al., 2006). Furthermore,
body fat excess brings about increased secretion of adipokines which depress insulin
sensitivity (e.g. leptin, resistin), and decreased secretion of insulin-sensing adiponectin.
Additionally, IL-6 and TNF-, derived from adipose tissue, on the one hand induce
inflammation, and on the other stimulate adipose tissue lipolysis and augment FFA
availability for lipid and lipoprotein synthesis (Lago et al., 2009).
In addition, both insulin resistance and adipokines affect endothelial nitric oxide synthase
(eNOS) and nitric oxide (NO) production and in consequence deteriorate blood vessel
contractility (Muniyappa et al., 2008). Moreover, there are data indicating an adverse effect
of LDL-cholesterol and positive action of HDL-cholesterol on eNOS expression and NO
production (Stepp et al., 2002, Rmet et al., 2003).
All the above-mentioned metabolic disturbances have pronounced consequences for the
cardiovascular system due to inflammation, atherosclerotic plaque formation and structural
alterations in the endothelium and subsequently lead to its dysfunction.
Thus, in this sequence of metabolic perturbations the endothelium was recognized rather as
a target of unfavorable events related to excessive body fat stores, insulin resistance and

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136
dyslipidemia, but not as an independent player contributing to dysfunction of the
cardiovascular system.
2. Vascular dysfunction Primary or secondary target
However, there were also data suggesting that endothelial dysfunction was a major
mechanism involved in the development of metabolic disturbances and subsequent
atherogenesis (Yang & Ming, 2006).
Recently this hypothesis has been the focus much attention mostly as a consequence of data
concerning a wide spectrum of metabolic eNOS/NO action. It has been recognized that
eNOS itself is indispensable for physiological insulin action and glucose disposal in the
working muscle (Roberts et al., 1997, Kingwell et al., 2002, Ross et al., 2007). Moreover, in
vitro NO markedly increases glucose transporter (GLUT 4) expression in the muscle and
regulates AMP- kinase (AMPK) signaling (Lira et al., 2007). Taking into account the special
role of AMPK in the regulation of substrate utilization it is clear that eNOS activity and NO
production markedly affect energetic processes in the muscle (Smith A.C., et al., 2005).
In contrast, eNOS deficiency in eNOS
-/-
mice depresses oxidative processes and brings
about defective mitochondrial fatty acid oxidation (Momken et al., 2002, Le Gouill et al.,
2007). Recent data have shown that the ablation of eNOS in mice accelerates glucose and
free fatty acid uptake by muscles and increases liver and muscle glycogenolysis (Lee-Young
et al., 2010). In consequence, eNOS knockout animals exhibit hypoglycemia and limited
exercise capacity during exercise.
It is well documented that in vitro NO contributes to the regulation of lipid metabolism in
the liver by inhibiting acetyl-CoA carboxylase (ACC) activity and de novo free fatty acid
synthesis (Garcia-Villafranca et al., 2003). There are also data suggesting that both in vitro
and in vivo NO exerts a hypocholesterolemic effect, since stimulation of NO synthesis in
rabbits decreases circulating LDL-cholesterol (Kurowska & Carrol, 1998).
At present eNOS/NO system contribution to the regulation of metabolism is far from being
fully elucidated. However, it is accepted that the vascular endothelium is not exclusively a
target responding to metabolic disturbances accompanying cardiovascular disease, but is an
important and independent player in the complicated relationships between cardiovascular
disease, obesity and diabetes.
This assumption is partially supported by research indicating that adverse changes in
vasculature in response to high fat diet (inflammation, insulin resistance, reduced NO
production) precede detrimental effects in muscle, liver, or adipose tissue (Kim et al., 2008).
3. Endothelial Nitric Oxide Synthase (eNOS) and Nitric Oxide (NO) system
coupling and uncoupling
It should be pointed out that the endothelium is one of the largest systems in human body
spread throughout the capillaries and arterioles in all tissues, forming a selectively
permeable barrier between the outer vascular wall and the bloodstream. It also the tissue
producing nitric oxide (NO) responsible for vasorelaxation, platelet aggregation, leukocyte-
endothelium adhesion and vascular smooth muscle cell migration and proliferation (Michel
& Vanhoutte, 2010).
Endothelial Nitric Oxide Synthase,
Nitric Oxide and Metabolic Disturbances in the Vascular System

137
The mechanism of endothelial eNOS regulation is not fully elucidated due to its complexity.
However, there are data indicating that enzyme activity is subjected to complicated
regulation by many intracellular factors including heat shock protein (HSP90), different
phosphatases, kinases, but also by enzyme location in the cell and potentially motor proteins
(Dudzinski & Michel, 2007).
On the other hand, it is well documented that eNOS activity is also regulated by factors
generated outside the endothelium - negatively by resistin, TNF-, and leptin and positively
by estrogen ((Dai et al., 2004, Kougias et al., 2005, Valerio et al., 2006, Korda et al,. 2008,
LeBlanc et al. 2009).
Nitric oxide is synthesized from L-arginine in a reaction catalyzed by the endothelial eNOS
(Moncada et al., 1991) (Fig. 1). Thus, any factors decreasing eNOS activity and/or increasing
NO degradation i.e. affecting the eNOS/NO system have been recognized as a potential
source of disturbed endothelium function.
Under physiological conditions and optimal eNOS activity L-arginine in the presence of O
2

is converted to NO and citrulline with minor production of superoxide (Alp & Channon,
2004). In consequence, NO production is coupled with eNOS activity.
In contrast, inadequate L-arginine intake and deficiency of the eNOS cofactor -
tetrahydropterin (BH4) brings about depressed NO synthesis, and promotes superoxide and
peroxynitrite generation - a phenomenon named eNOS uncoupling (Huang, 2009).
Taking into account that L-arginine is the exclusive substrate for NO synthesis it is clear that
its metabolism catalyzed by arginase has the potential to decrease eNOS activity and NO
production (Wu et al., 2009).
In mammals there are two types of arginase, encoded by two genes arginase I and II.
Arginase I is expressed mostly in the liver catalyzing L-arginine conversion into urea and
ornithine and in this way participating in ammonia detoxication. Arginase II is a
mitochondrial enzyme of extrahepatic tissues contributing to biosynthesis of amino acids
(glutamate, proline and ornithine) and polyamines, but also playing a fundamental role in
the depression of endothelial NO production decreasing L-arginine availability for eNOS
action. In addition, arginase II overexpression seems to induce superoxide and peroxynitrite
generation per se harmful for the endothelium. There are data suggesting increased
arginase activity in atherosclerosis and hypertension, thus diseases characterized by
endothelial dysfunction (Ryoo et al., 2011)
BH4 bioavailability within the endothelium plays a fundamental role in eNOS/NO
coupling. It has been demonstrated that the inhibition of the rate-limiting enzyme
responsible for de novo BH4 synthesis - GTP cyclohydrolase 1 - brings about eNOS/NO
uncoupling and elevated superoxide production in isolated bovine or mouse aortic
endothelial cells. Moreover, superoxide production was reduced by the sepiapterin BH4
precursor (Tiefenbacher et al. ,2000, Wang et al., 2008).
However, recent data have indicated that the regulation of BH4 levels in the endothelium is
even more complicated since it is oxidized to 7,8-dihydrobiopterin (BH2) which in turn is
recycled into BH4 in the reaction catalyzed by dihydrofolate reductase (DHFR). Moreover, a
genetic DHFR knockout or pharmacological inhibition of the enzyme suppresses BH4
synthesis and causes eNOS uncoupling (Crabtree et al., 2009) (Fig.2).

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138

Fig. 1. L-arginine as a source of nitric oxide (NO) under physiological condition and minor
superoxide production.

Fig. 2. eNOS/ NO uncoupling in response to metabolic disturbances resulting in increased
superoxide and peroxynitrite production
synthesis/degradation
Endothelial Nitric Oxide Synthase,
Nitric Oxide and Metabolic Disturbances in the Vascular System

139
It should be pointed out that regulation of cardiovascular system is not limited to eNOS
action. Numerous research focus on neuronal (nNOS) and inducible (iNOS) nitric oxide
synthase role in the cardiovascular system. It has been postulated that nNOS expressed
outside of the vascular system might protect mice from diet-induced atherosclerosis through
indirect action on hormonal and/or nervous system and blood pressure regulation.
(Lowenstein, 2006). On the other hand, iNOS is expressed in a wide range of cells in
response to cytokines and is overexpressed in macrophage and cardiovascular system of
diabetic rats (Soski et al.,2011). However, much more studies are needed to fully elucidate
the relationship between three isoforms of NO in vascular system dysfunction.
4. Asymmetrical dimethylarginine (ADMA) and the vascular system
Recently numerous studies have focused on the role of endogenous inhibitor of eNOS
activity and NO production asymmetrical dimethylarginine (ADMA). ADMA is
synthesized in many tissues, including the endothelium, by the methylation of L-arginine
released from proteins which undergo regular turnover. The methylation process is
catalyzed by arginine methyltransferase type I (PRMT I) and ADMA production is related to
both protein turnover and enzyme activity (Pope et al., 2009) (Fig.3). However, about 90% of
ADMA is metabolized to citrulline and dimethylamine by dimethylarginine
dimethylaminohydrolase (DDAH), with the remainder partially excreted with urine (Tran et
al., 2003). Numerous studies have indicated a substantial role for DDAH in ADMA
turnover. DDAH is expressed as two isoforms (DDAH I and DDAH II) encoded by different
genes (Leiper et al,. 1999). Animal studies have revealed that in mice overexpressing DDAH
I plasma ADMA levels are reduced with concomitant increase in tissue NOS activity.
(Dayoub et al., 2003). Moreover, in humans genetic variants of DDAH I and DDAH II genes
are significantly associated with plasma ADMA levels (Abhary et al., 2010). Moreover,
ADMA concentration in tissues and plasma is also affected by cationic amino acid
transporter (CAT) in exchange for arginine and other cationic amino acids (Teerlink et al.,
2009). Reference values of circulating ADMA in healthy subjects vary widely, even when
similar analytic methods are used (Meinitzer et al., 2007). However, the risk of acute
coronary events and mortality increases with elevated plasma ADMA concentrations
(Valkonen et al., 2001, Zoccali et al., 2001). Moreover, it is well documented that circulating
ADMA is inversely related to endothelial function in hypertensive and healthy subjects
(Perticone et al., 2003, Bger et al., 2007). Furthermore, it has been established that the
intima-media thickness of the carotid artery and aortic stenosis are related to circulating
ADMA (Furuki et al., 2007, Ngo et al., 2007). Additionally, circulating ADMA has been
recognized as an independent factor determining flow mediated dilatation in cardiac
syndrome X (Haberka et al., 2010).
The mechanism of detrimental ADMA action in the vascular system is not fully established.
It is still under debate whether ADMA represents a novel risk factor for the development of
endothelial dysfunction or its production reflects endothelium response to other metabolic
disturbances such as oxidative stress (Sydow & Mnzel, 2003). This latter hypothesis could
not be excluded since in vitro oxidative stress decreases ADMA-demethylating enzyme
(DDAH) activity and causes elevated ADMA levels (Leiper et al., 2002).

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Fig. 3. Asymmertical dimethyl arginine (ADMA) synthesis and action on eNOS/NO system
On the other hand, the analysis of 131 cases with coronary heart disease (CHD) and 131
controls matched for age, sex and body mass index has revealed that plasma ADMA
concentrations in patients were higher than in controls and ADMA is an independent risk
factor for CHD (Schultze et al., 2006). Similarly, in 138 patients with acute myocardial
infartion ADMA was recognized as a marker of cardiovascular risk independent of
traditional risk factors (Korandji et al., 2007).
Despite these doubts the detrimental effects of ADMA on the endothelium are well
documented. First of all ADMA is a potent inhibitor of eNOS inducing eNOS/NO
uncoupling (Jin & Loscalzo, 2010). Moreover, it has been found that ADMA is an
endogenous inhibitor of mobilization, differentiation and function of endothelial progenitor
cells which participate in continuous endothelial renewal and neovascularization of
ischemic tissues (Thum et al., 2005). Additionally, in vitro pathological concentrations of
ADMA are sufficient to elicit marked changes in coronary artery endothelial cell gene
expression of bone morphogenic protein receptor, and PRMT the enzyme responsible for
methylation of arginine to ADMA Moreover, in mice treated with high ADMA doses
(2 M) more than 50 genes in endothelium were significantly altered (Smith C.L., et al.,
2005). Some data also data suggest proinflammatory ADMA action in human endothelial
cells (Chen et al., 2007)

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Thus, it should be pointed out that ADMA-mediated pathological processes are not
exclusively due to eNOS uncoupling, however, eNOS inhibition is most likely being the
dominant ADMA vascular effect (Cooke, 2004).
5. Lifestyle and vascular system
There is no doubt that lifestyle has a pronounced effect on health, decreasing body fat stores,
improving insulin sensitivity, lipid and lipoprotein metabolism and positively affecting the
cardiovascular system (Lamon-Fave et al., 1996, Lee et al., 2005, Takahashi et al., 2011).
Numerous data have revealed that both eNOS and NO production are the target of lifestyle
interventions such as dietary habits and physical activity.
5.1 Dietary habits, eNOS and NO
Dietary habits are associated with both acute and chronic effects on the vascular system. In
healthy, normolipidemic young and middle-aged men a single high fat meal has been found
to adversely affect endothelial function depressing the flow-mediated vasodilation of the
brachial artery (Vogel et al., 1997, Marchesi et al., 2000). Moreover, a decrease in endothelial
function has been observed in response to both glucose and fat load, with a more
pronounced effect when high fat and glucose were combined (Ceriello et al., 2002). Thus,
postprandial state has to be taken into consideration as a possible reason for diet-induced
depression in vascular reactivity.
The mechanism of the effects of postprandial state on vascular function is not fully
elucidated, however it seems that oxidative stress due to elevated plasma remnant
lipoproteins, triglycerides, and glucose concentrations contributes to the adverse effects of a
single meal on vascularity (Doi et al., 2000, Bae et al., 2001, Ceriello et al,. 2004).
Moreover, recent data have suggested that in addition to oxidative stress, oral fat load
enhances metalloproteinase-2 and metalloproteinase-9 activity which in turn bring about
unfavorable vascular remodeling (Derosa et al., 2010).
However, it should be pointed out that adverse effects of fat load on the vascular system are
mostly due to saturated fat (Vogel et al., 2000, Cortz et al,. 2006, Berry et al., 2008). In
contrast, an exchange of saturated for unsaturated fat load has been found to improve
postprandial vascular function probably due to the positive effect of the latter on endothelial
eNOS/NO system (Armah et al., 2008, Masson & Mesink, 2011).
Numerous experimental studies have focused on chronic effects of dietary habits on
endothelium function and vasoreactivity, however, their results are inconsistent. In
patients with coronary artery disease a long-term (6 weeks) treatment with purified
eicosapentaenoic acid (EPA) markedly improved NO-mediated forearm vasodilatation
(Tagawa et al., 1999). Similarly, improved forearm microcirculation has been noted in
hyperlipidemic, overweight subjects following a 6 week treatment with purified
docosahexaenoic acid (DHA), but not with EPA (Mori et al., 2000). On the contrary,
positive action of longer (7 weeks) EPA and DHA supplementation on systemic arterial
compliance has been demonstrated in dyslipidemic elderly men (Nestel et al., 2002).
Additionally, it has been noted that 32 weeks EPA and DHA-rich fish oil supplementation
improve endothelial function and vascular tone in healthy middle-aged men and women
(Khan et al., 2003).

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Thus, it seems that duration of supplementation possibly contributes to discordant results
concerning the response of the vascular system to polyunsaturated fatty acid (PUFA)
treatment.
There are also data suggesting that EPA and DHA-rich fish oil exert a more pronounced
effect on vascular function than other oils In rats fed a fish-oil rich diet the aortic content of
eNOS protein and enzyme activity are markedly (by 70% and 102 %, respectively) higher
than in rats fed corn oil (Lopez et al., 2004). Moreover, improved vascular reactivity and
enhanced eNOS expression have been indicated in aortic rings of spontaneously
hypertensive rats fed diet rich in pomace olive oil, but not refined olive or corn oil
(Rodriguez-Rodriguez et al., 2007). Thus, the positive effect of unsaturated fat provision
seems to be related to its composition.
Recent data have indicated a positive effect of conjugated linoleic acid (CLA) on vascularity
in obese fa/fa rats due to CLA-induced elevation in adiponectin production and subsequent
eNOS phosphorylation increasing enzyme activity and NO production (DeClerq et al.,
2011). Therefore, it seems feasible that well-known beneficial effects of oil consumption on
health are at least partially due to its action on the eNOS/NO system.
Much attention has been paid to effects of dietary protein on vascular function. It has been
demonstrated that in hypertensive men there is an inverse relationship between blood
pressure and protein consumption with more pronounced action of soy and fish than
animal protein intake. Further studies have shown that this effect is due to various amino
acids such as cysteine, glutamate, and arginine which decrease oxidative stress, improve
renal function and insulin resistance (Vasdev & Stuckles, 2010). However, numerous studies
have focused on L-arginine contribution to vascular system regulation since, as was
mentioned earlier, L-arginine serves as a substrate for NO synthesis.
In young hypercholesterolemic adults after 4 week L-arginine supplementation (7 grams x
3/day) marked improvement in endothelium-dependent vasodilation has been noted
(Clarkson et al.,1994). Similarly, it has been observed that in patients with heart failure 6 weeks
L-arginine treatment (5.5 to 12.6 g/ day) positively affects vascular system (Rector, et al. 1996).
Growing evidence indicates that L-arginine supplementation brings about improved insulin
sensitivity and decreases circulating free fatty acids and triglycerides in chemically induced
diabetic and genetically obese rats (Kohli et al., 2004, Fu et al., 2005). Moreover, similar
effects have been observed in obese and type II diabetic patients receiving oral/or
intravenous L-arginine (Lucotti et al,. 2006). Furthermore, it has been documented that
postprandial lipemia-induced endothelial dysfunction is neutralized by addition of proteins
to the fatty meals due to increased L-arginine to ADMA ratio (Westphal et al.,2006).
Moreover, in healthy volunteers addition of 2.5 g L-arginine to fatty meal prevents the
lipemia-induced endothelial dysfunction (Borucki et al., 2009).
The above data suggest a possible beneficial effect of L-arginine treatment in cardiovascular
dysfunction. However, it should be pointed out that some studies do not show any
beneficial effect of L-arginine treatment (Chin-Dusting et al., 1996, Oomen et al., 2000). It
could not be excluded that this discrepancy is due to individual variability in the response
to L-arginine treatment (Evans et al., 2004). Recently it has been postulated that beneficial L-
arginine action in vascular system is related to circulating ADMA with no effect in subjects
with low metabolite levels (Bger, 2007).
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On the other hand, it should be stressed that the acute provision of exogenous L-arginine
possibly depresses NO production due to induction of arginases which metabolize L-
arginine to urea and in consequence divert it from eNOS and in this way adversely affects
cardiovascular system (Dioguardi, 2011).
Data concerning dietary carbohydrate effects on the eNOS/NO system are fragmentary. In
obese Zucker rats a low carbohydrate diet (10 %) improves vascular function with no effect
on NO production in comparison with that containing 59 % carbohydrates (Focaroli et al.,
2007).
However, it is well documented that in the rat excessive fructose supply adversely affects
endothelium-dependent vasodilation both in vitro and in vivo and this effect is probably due
to inhibition of NO synthesis (Verma et al., 1997, Rickey et al., 1998, Kamata et al., 1999).
Similarly, high glucose concentration iv vitro decreases eNOS protein expression and
enzyme activity as a result of destroyed enzyme interaction with HSP-90 (Noyman et al,.
2002, Mohan et al., 2009).
On the other hand, many diet components have the potential to reduce detrimental effects of
poor dietary habits.
Consumption of antioxidant rich products such as fruits and vegetables in humans
prevents the detrimental action of a saturated fat load due to their positive effect on the
eNOS/NO system (Plotnick et al., 2003, Traber & Stevens, 2011). Similarly, low cholesterol ,
walnut-enriched and the Mediterranean diets are effective in improving the eNOS/NO
system and vascular function (Winkler- Mbius et al., 2010).
Data concerning diet effects on ADMA an endogenous eNOS inhibitor and risk factor are
scarce. Piv et al., (2004) have indicated that in a middle-aged population with mild
hypercholesterolemia circulating ADMA is inversely related to carbohydrate consumption.
Additionally, Puchau et al. (2009) have demonstrated that in healthy young men circulating
ADMA is inversely related to zinc and selenium status.
In elderly subjects polyunsaturated fatty acid (PUFA) supplementation markedly elevates
circulating L- arginine and in this way decreases L-arginine/ADMA ratio what might be
discussed as an improvement of endothelial function (Eid et al., 2006). However, there are
also data which question the fat contribution to increased ADMA level in the blood.
Recently Engeli et al. (2011) have revealed that the variation in fat consumption (20% and
above 40% of energy) exerts divergent effect on circulating ADMA. In obese subjects higher
fat consumption slightly (by 4%) decreases ADMA level. In contrast, in lean subjects both
low and high fat consumption causes 6% elevation in ADMA concentration. The authors
have postulated that contradictory data concerning dietary fat intake on ADMA levels are
mostly due to methodological issues concerning ADMA determination.
Thus, the effects of dietary habits on ADMA plasma levels are far from being elucidated.
Moreover, in analysis of the effect of the diet on the eNOS/NO system not only diet
composition but also total caloric intake has to be taken into consideration. Animal studies
have demonstrated that caloric restriction for 3 or 13 months significantly improves the
expression of eNOS protein in various tissues (Nisoli et al., 2005).

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5.2 Physical activity and eNOS/NO system
For many years physical activity which decreases body fat stores, improves lipid and
lipoprotein metabolism and insulin sensitivity and positively affects cardiovascular system
has been recommended in the therapy of obesity, hypertension, type 2 diabetes and
cardiovascular disease (Shephard & Balady, 1999).
Assuming the importance of the eNOS/ NO system in the regulation of many metabolic
processes in recent years numerous studies have focused on the relationship between
endothelial function and physical activity. This issue seems to be of special importance since
animal studies have indicated that physical inactivity induces endothelial dysfunction due
to decreased eNOS activity (Suvarova et al., 2004).
It is well documented that in rats both acute exercise and regular physical activity (2-4
weeks) markedly enhance eNOS activity and endothelial NO synthesis in skeletal muscle
arterioles (Sun et al., 1994, Roberts et al., 1999). Similarly, in dogs following exercise elevated
NO synthesis has been noted in coronary circulation being responsible for of the
vasodilation response (Bernstein et al., 1996, Ishibashi et al., 1998). Moreover, in active
animals eNOS phosphorylation and activity is significantly elevated after 12 weeks of
training (Touati et al., 2011).
In apparently healthy young men and women acute aerobic exercise markedly counteracts
detrimental effects of a high-fat meal on flow-mediated dilatation (FMD), but also improves
FMD in participants consuming a low-fat meal possibly due to reduction of circulating
lipids, insulin resistance and oxidative stress (Padilla et al,. 2006, Silvestre et al., 2008,
Tyldum et al.,2009). Thus, it has been postulated that physical activity can attenuate adverse
postprandial changes in vascular function (Johnson et al., 2011).
It should be pointed out that a positive effect of physical activity on the eNOS/NO system
has also been noted in patients with stroke, chronic heart failure, and myocardial infarction
(Gertz et al., 2006, Mendes-Ribeiro et a.,2009, De Waard et al., 2010).
The mechanism of exercise-induced positive changes in the eNOS/NO system is not fully
elucidated. However, it is well documented that physical activity brings about hyperemia
and subsequently endothelial shear stress (ESS) defined as a fractional force exerted by
blood flow (Boushei et al,. 2000, Taylor et al., 2002, Boo & Jo, 2003).
It is well documented that shear stress markedly affects a myriad of intracellular events in
endothelial cells including remodeling, inflammation and NO production with low ESS
inducing plaque formation (Harrison, 2005, Koskinas et al., 2010).
Early studies have demonstrated that in bovine aortic endothelial cells the elevation of shear
stress causes elevation in eNOS phosphorylation and expression which in turn increases
enzyme activity (Corson et al., 1996, Malek et al. 1999). Furthermore, in human vessels
increase in shear stress inhibits lipid peroxidation induced by high glucose and arachidonic
acid in the medium (Mun et al., 2008). Thus, direct effects of physical activity on eNOS/NO
system and inhibition of oxidative processes contribute to exercise induced improvement
in endothelium function. However, it is worth noting that positive action of physical activity
is limited to moderate intensity, since it has been demonstrated that high intensity exercise
(90 % VO
2
max) enhances platelet reactivity to shear stress and induces coagulation which
in turn increases the risk of thrombosis (Ikaguri et al., 2003).
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Taking into account all data cited in this review it is clear that eNOS/NO system undergoes
complicated regulation by both genetic and lifestyle factors (Fig. 4).
eNOS/NO system
Genetic factors
eg. arginase and DDAH
variants affecting
circulating ADMA
Lifestyle factors
adequate protein intake
low fructose and saturated intake
high intake of oils
high intake of antioxidant-rich food
moderate physical activity

Fig. 4. Interplay between genetic and lifestyle factors affecting eNOS/NO system
6. Conclusion
Our present knowledge about eNOS and NO effects on overall metabolic processes at least
partially supports the hypothesis concerning a special and possibly central role of
endothelium as an active tissue, and not only the target of metabolic disturbances.
Moreover, circulating ADMA seems to be a risk factor of endothelial disturbances and
disturbed cardiovascular system. In consequence, further research is required on strategies
improving the eNOS/NO system and decreasing ADMA synthesis, including both
pharmacological and lifestyle interventions.
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Section 2
Cardiovascular Diagnostics

8
The Diagnostic Performance
of Cardiovascular System and
Evaluation of Hemodynamic Parameters
Based on Heart Cycle Phase Analysis
Mikhail Rudenko et al.
*

Russian New University,
Russia
1. Introduction
The heart cycle phase analysis based on the mathematical equations by G. Poyedinstev and
O. Voronova is a foundation for practically obtaining new data on normal performance of
the human cardiovascular system, cardiovascular pathology, and therapy control aimed at
recovery processes [1]. It provides a way to establish cause-effect relationship between the
mechanism and the behavior of pathological processes.
Considering the fact that the application of this method in clinical practice has been producing
further novel data and ideas, it is obvious that even the results already achieved can radically
change the conventional approaches in electrophysiology. This gives us an opportunity to
utilize electrocardiography in a more efficient way in solving practical problems.
This implies the following:
1. Screening to reveal risk groups.
2. Establishing diagnosis and deciding on treatment strategy.
3. On-line monitoring of therapy efficiency.
4. On-line acute and surgical monitoring.
5. Monitoring of age-related changes.
6. Evaluation of efficiency of training procedures for conditioning in sports.
For these purposes, an electrocardiogram (ECG) is recorded according to an innovative
technology developed by the authors hereof in order to identify the phase pattern of a heart
cycle. This technology is easier in use than the existing one and can delivers data of higher
informative value.
There are certain difficulties which exist in early diagnosis of the cardiovascular diseases
since it is very often the case when variations of hemodynamic parameters of a person, who

*
Olga Voronova
1
, Vladimir Zernov
1
, Konstantin Mamberger
1
, Dmitry Makedonsky
1
, Sergey Rudenko
1
,
Yuri Fedossov
1
, Alexander Duyzhikov
2
, Anatoly Orlov
2
and Sergey Sobin
2

2
Rostov Cardiology & Cardiovascular Surgery Center

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is absolutely healthy but who stands under exercise load, may be even far beyond the scope
of pathology changes.
Many questions might come to mind of how age-related changes affect the performance of
the cardiovascular system. Of great importance is an evaluation of the coronary flow.
Another subject treated by the authors in their researches is the problem of sudden cardiac
death. The authors succeeded in establishing criteria for early diagnosis for the said death
cases that makes possible now to forecast and avoid such potential risks by taking adequate
preventive measures.
All results of the researches described herein have been clinically verified and validated.
Contrary to many conventional well-known methods of diagnosis, the informative
potentialities of which have been already exhausted, the method of the heart cycle phase
analysis is well under way
2. Development of innovative ECG recording technology
As mentioned above, one of the key issues in the heart cycle phase analysis is an ECG
recording technology. Beginning with W. Einthoven, the challenges to research was how to
record electrical activity of different parts of the cardiac muscle. In more exact terms, the
final goal of those investigations was to develop methods of diagnostics of the structural
features and the performance of the individual heart segments (left & right ventricles and
atria) by interpreting an ECG curve. Making step by step on the road to the said goal, the
investigators have come to their conclusion that there is a phase mechanism in existence,
which is responsible for the proper performance of our heart. Therefore, most attention has
been concentrated on this subject in further research.
That has become a driving force for an increase in ECG channels, the number of which reaches
one hundred. Then, computer-assisted equipment offered new opportunities in an advanced
mathematical modeling. In particular, as a consequence, that gave rise to a radically new
method of ECG recording. Next step in the history of electrocardography was the EASI
method [1] (fig.1). Thereupon, a new trend made its appearance: to reduce the number of the
recording electrodes and provide at the same time a greater volume of information.

Fig. 1. Development of ECG recording methods
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System and Evaluation of Hemodynamic Parameters Based on Heart Cycle Phase Analysis

159
But all that has not assisted in the development of the heart cycle phase analysis. In the 1980s,
the mode of elevated fluidity of liquid was discovered by G. Poyedintsev and O. Voronova
(the so called third mode of flow), an innovative mathematical model of the blood flow
through blood vessels and new methods how to calculate hemodynamic parameters, based on
durations of the respective phases of every heart cycle were offered by the above scientists [2].
By this means the theoretical foundation was created in order to develop the phase analysis at
a new level. But the only way to implement the above mathematics was an elaboration of a
new reliable method of recording of the phase pattern of the heart cycle.
At that time there was no unambiguous interpretation available how to identify the heart
cycle phase boundaries on an ECG curve. Different research schools gave their different
descriptions of criteria of how to properly record the phases. First of all, it was applicable to
key wave point S on an ECG curve. For instance, each channel in 6-lead ECG recording
delivers different values of the same R-S interval.
The EASI method at its core delivers additional sources of errors in ECG processing. In
order to properly record all phases, it was required to minimize the number of the channels
for error reduction. At the beginning of the 2000s, medical scientists succeeded in
identifying those areas on the human body where ECG recording electrodes are to be placed
to obtain all fine points of electric activity of the heart [1]. It has been detected that the area
delivering the most informative signals is located within the zone of the ascending aorta
(Fig.1). It should be mentioned, that it is important that the second electrode is not neutral
but an active one, contrary to other known methods. This electrode should be located within
the area of the heart apex. As a result, using one ECG channel only, we obtain full
information about electric activity within the area located between the aorta and the apex of
the heart. Principally, it is essential that we deal with a signal that is not integrated because
of parallel influence of conductivity of the close-located tissues, as it may be the case with
other conventional methods where the second electrode is used as the neutral zone (Fig.2).
In particular, it is critical for recording of interval S T, which includes 4 periods of the
phase pattern of the heart cycle.
Searching for criteria of how to record point S was successfully completed by the authors on
the basis of the equtions by G.Poyedintsev O.Voronova. It follows from the equiations that
the sum of diastolic phase volumes PV1 and PV2, should be equal and that of systolic phase
volumes PV3 and PV4 as well as stroke volume SV that can be expressed as follows [1]:
PV1+PV2 = PV3 + PV4 = SV (1)
Taking into account the fact that the above equations include several phases of the heart cycle,
to make this exactly equal is possible only when all phases are recorded in the absolutely
proper way. By experiment, the required criteria for the appropriate recording of every phase
have been found by local extrema on the first order derivative of an ECG. It is of importance
that first time a universal criterion has been established to record any phase at all.
In the course of the investigations, another thing has been revealed: the widely used
conventional electronic filters are not substantiated from the scientific point of view, when
selecting the proper pass bands, so that they produce signal distortions. Of special note is in
this case the lower cut-off frequency of the filters. It is just the frequency that is favorably
used in Cardiocode technology based on the many years experience.

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Fig. 2. A clear ECG signal according to Cardiocode single-channel method versus standard
V3 lead ECG signal modified due to integration. A difference between the durations of the
same R S interval is about 25 %
Finally, according to equation (1), only the Cardiocode technology is capable of recording an
ECG from the aorta with identification of every phase at local extrema of the first derivative.
Any other methods or procedures are not acceptable for making heart cycle phase analysis.
But it was found that recording of an ECG curve alone is not sufficient for analysis of the
performance of the cardiovascular system. Therefore, it was required to develop the so
called pin-point rheography, when a rheogram is recorded from the ECG electrodes
simultaneously with the ECG. Two signals of different nature that are recorded at the same
time give a comprehensive idea of how the cardiovascular system performs.
3. Single-channel recording of ECG from ascending aorta, supplemented by
simultaneous recording of aortic pin-point RHEOgram (Cardiocode
technology)
A synchronous recording of a RHEOgram from the ECG electrodes is possible when an
additional external sinusoidal high-frequency signal, supplied by a generator, passes the
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161
electrode area. This frequency is amplitude-modulated by blood circulation. The modulation
shape is equivalent to changes in blood filling within the given area. By detecting a signal, we
obtain a RHEO signal, the shape of which is equivalent to changes in arteric pressure.
According to the Cardiocode technology, the RHEO signal is picked off the ECG electrodes,
therefore the generating electrodes for RHEO recording should be placed adjacent to the ECG
electrodes. A scheme of electrode arrangement is shown in Figure 3 below.

Fig. 3. Scheme of electrode arrangement for synchronous recording of ECG and RHEO from
ascending aorta
An ECG and a RHEO produced in such a way contain full information of hemodynamics
and the performance of the cardiovascular system. Figure 4 displays ECG and RHEO
signals recorded synchronously.
The said figure consists of two parts. A exhibits actual ECG and RHEO curves
demonstrated on the instrument display after recording. The first derivative of the ECG
curve is located in between them. Local extremes on the derivative which are used for
identification of the heart cycle phases are clearly marked. For instance, phase S L is
identified in such a manner. There is no other way available to detect this phase with a high
accuracy. For convenience, in order to properly analyze the relations between the phases on
the ECG and RHEO curves, their ideal models are presented in figure B.
Specific criteria established for identification of the phase boundaries make it easy to
identify wave point j. Little is known about this wave from the literature: it is called M.
Osborn wave. Phase L j refers to the phase of rapid ejection, and it is characterized by
hemodynamic parameter PV3. The systolic pressure can be evaluated by a slope ratio of the
RHEO curve in this phase.
Of particular interest is segment j T (initiation of wave ), that is an integral part of slow
ejection phase. This interval has never been identified or described in the electrophysiology
literature. This period of time is required to distribute stroke volume SV throughout the
space within the aorta, expanding the latter. The duration of this segment depends on elastic

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properties of the aorta, so that it increases with loss of its elasticity. Following this way, we
can produce a criterion for evaluation of the aorta elasticity status.
The distinctive feature of our innovative technology and methodology is that it is now possible
to evaluate the coronary flow qualitatively. For this purpose, wave U is analyzed. The said
wave appears in premature diastole phase (wave decay) P (wave initiation). The authors
think that the appearance of this wave is associated with the coronary flow features. But many
other questions remain to be answered in this connection. At present, some preliminary
conclusions can be made only. We are carrying out our further investigations in this area, and
there are good grounds to believe that they will be successful.

Fig. 4. A: real ECG and RHEO curves recorded from ascending aorta; B: ideal ECG and
RHEO curves theoretically constructed
In order to analyze an ECG in combination with a RHEO, both curves should be
synchronized. This step is of great importance. To do this, provided should be that the
RHEO curve meets the isoelectric line at a point corresponding to point S on the respective
ECG. In this case, it becomes possible to analyze arterial pressure development in the aorta
both before and after opening of the aortic valve as shown in Figure 5 below.
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Fig. 5. Ideal ECG and RHEO curves. An interval is marked where a diastolic AP buildup can
be evaluated
The RHEO isoelectric line meets point S on the ECG curve. It makes possible to evaluate an
AP increase both before and after opening of aortic valve. Normally, the RHEO curve in
phase S L should be horizontal, and the AP buildup should be started at point L.
4. Classification of ECG curve shapes by reference criteria
The long-time researches of the performance of the cardiovascular system by the
Cardiocode technology result in identification of such ECG and RHEO curve shapes that can
be considered to be the reference curves. Figure 6 displays some recorded curves which are
accepted by us as the references. Considering the fact that a reference is a matter of
convention, such axiomatic approach, as it is often the case in practice, can solve a lot of
problems in introducing the phase analysis theory.

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Fig. 6. ECG and RHEO reference curves applied in practice for phase analysis
The recorded curves should be classified by changes in the contraction function of the
respective heart muscle area in each phase. On an ECG curve we can find the contraction
function being expressed as phase amplitudes. Let us denote the respective maxima and
minima on an ECG by conventional letters P; - Q; R; - S; L; j; T and U (s. Fig.7). It should be
noted that it is our own legend since the same lettering is typically used for the conventional
ECG waves but in our case the same letters carry other information, and, in order to avoid
any confusion, they are underlined herein.
Let us denote the amplitudes of waves on the reference ECG curve as follows:
P1; - Q1; R1; - S1; L1; j1; T1; U1
If amplitudes of the waves on a real ECG differ from their reference, numerical coefficients
should be other, too. For instance, if amplitude R is greater, we obtain R1,5 or R2 . With a
decrease in the amplitude, we have R0,5 or R0 (for the Brugada syndrome).
Information about the performance of the cardiovascular system presented in such a way is
suitable to be processed automatically. The only thing for a doctor is in this case to analyze
the obtained data in the context of the actual cause-effect relations and establish the primary
cause of the changes in the performance. To make it easier, the changed amplitudes may be
marked only. As an example, a recorded curve indicating an increased pumping function of
the aorta and a diminished function of the myocardium contraction should be presented as
follows:
2 ; - S 0,1
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Fig. 7. Maxima and minima on ECG, which correspond to the respective heart cycle phases
and characterize the contraction function of the muscle groups in the given phase. An
indication is amplitude displacement of maxima or minima
The investigations carried out by us create a basis for classification of 19 most significant
cases of functional changes that may occur in a staged manner and lead to some
cardiovascular pathology cases. These cases are as given below:
1. Increased function of contraction of interventricular septum (IVS).
2. Diminished function of contraction of IVS.
3. Diminished function of contraction of IVS and myocardium
4. Diminished function of contraction of myocardium.
5. Reduced level of relaxation of heart in premature diastole (appearance of multiple
waves).
6. Condition of coronary flow.
7. Condition of function of regulation of diastolic AP.
8. Condition of function of regulation of systolic AP.
9. Effect of reverse contraction of IVS (at 100% passivity of myocardium).
10. Q wave dip
11. No-S-wave and P-variation effect.
12. No-premature-diastole effect.
13. Regurgitation of aortic and mitral valves.
14. R-wave-bifurcation effect.
15. T-wave inversion effect.
16. No-P-wave effect.
17. P Q phase changes
18. Respiratory arrhythmia (QRS after wave).
19. and wave bifurcation.
It is impossible to present here all possible variations of the functional changes. Maybe, it
should be treated separately in another book. Therefore, it is reasonable to outline general
approaches to the proposed classification only and give some exemplary cases herein.

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In practice, we always deal with a great variety of ECGs and RHEO curves so that no two
curves are alike. It depends on individual features of the performance of the cardiovascular
system of everybody. Therefore, it is expedient to consider a certain scope of functional
changes and their peculiarities which may be typical for any pathology case.
The significance of the above classification is based on its practical effect. It allows for
evaluating a deviation of a function from its conventional norm and detecting primary cause
of the changes. Moreover, this approach makes possible early diagnostics in case of a
pathology developing process well in advance so that the most favorable conditions are met
to apply the most efficient ways to improve the functions.
4.1 Increased function of contraction of interventricular septum (IVS)
Table 1 illustrates one of 19 cases of the functional changes. It is a staged increase of the
function of the contraction of the IVS up to its limiting critical level.
Temporal
development
(stage)

R

- S

L
Associated features

Symptom

Clinical aspect

Increased Q S
width
1 R1,5
-
S0,5

2 R2 - S2 -L2 Increased
3 R3 - S2 -L3 Increased
Periodical short-
time vertigo Manifestation
not in every
heart cycle 4 R4 - S4 -L4
High probability of
IVS attenuation in
contraction
Periodical loss of
consciousness
5
Sudden cardiac
death
Table 1. Increased function of contraction of IVS up to its limiting critical level
Figures 8, 9 and 10 display the recorded curves to be classified.

Fig. 8. Stage 2: R2; S2; L2
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Fig. 9. Stage: R3; S3; L3

Fig. 10. Stage 4: R4; S4; L4
4.2 Diminished function of contraction of interventricular septum (IVS)
Item number two in the list of the significant functional changes is diminished function of
contraction of the IVS (s. Table 2 below).
Energetical processes which occur in the muscle cells of the septum, the myocardium and
the atria play a decisive role in the performance of the heart. The energetics depends on
biochemical processes that maintain the functioning of mitochondria in tissue cells. The cell
membranes and the transport elements are key factors in the said processes. Changes in
mitochondria energetics are directly proportional to the function of the muscle contraction.
The authors have recorded in practice a complete range of ECG changes of one patient from
the extremely pathological Brugada syndrome before therapy up to the normal condition
after the required treatment received. The recovery of the functions of the cardiovascular
system was provided by re-establishing of functioning of mitochondria and restoring the
carbon dioxide oxygen balance in blood. Figures 11 - 16 show the ECG and RHEO curves
recorded in orthostatic testing within the period of time from the beginning the therapy up
to achieving the acceptable treatment results. The said figures in the above case illustrate the

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curves arranged in the reverse order in order to provide insight into the development of the
ECG characteristics, beginning with the achieved normal status and ending with the initial
extreme pathology, i.e., the Brugada syndrome.
The represented history can be described on the basis of the classification as mentioned
above. The exemplary curves illustrate how the compensation mechanisms start their
operation. It should be noted that the compensation mechanism takes effect at MV > 4,5
l/min.

Temporal
development
(stage)

R

- S

L

T
Associated
features


Symptom

Clinical
aspect
Increased
R S width
1 R0,75 L1,5 T1,5
Increased diastolic
AP
Manifesta
tion in
every
heart
cycle
2 R0,5 L2 T1,75 Increased
Manifestation of
periodical
extrasystoles.
Increased systolic
AP at increased
diastolic AP
3 R0,25 - S1,25 L2 T1,75 Wide
Increased systolic
AP at increased
diastolic AP
4 R0,25 - S1,25 L2 T2 Wide
S-wave double
contraction at
normalization of
its width.
Instability of this
process is
recorded
5 R0,25 - S1,5 L2 T2 Wide
Increased systolic
AP at increased
diastolic AP
6
R0,1

- S1,5 L2,5 T2,5
High systolic AP
at high diastolic
AP
7
R-
wave
dip


Table 2. Diminished function of contraction of IVS
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Fig. 11. Stage 1: R0,75; L1,5; T1,5

Fig. 12. Stage 2: R0,5; L2; T1,75

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Fig. 12b. Stage 2: Appearance of extrasystoles after exercise stress or poor sleep

Fig. 13. Stage 3: R0,25; - S1,25; L2; T1,75
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Fig. 14. Stage 4: R0,25; - S1,25; L 2; T2. S wave recovering after double contraction is
observable. The record was produced in orthostatic testing

Fig. 15. Stage 5: R0,25; - S1,25; L2; T2

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Fig. 16. Stage 6: R0,1; - S1,5; L1,25; T2,5. Brugada syndrome manifestation
Next to last stage 6 offers ECG curves where R wave is not available at all and where we
observe a considerable widening of the S-wave and a significant increase in amplitude of
wave S L. This ECG curve shape is typical for the Brugada syndrome.
The suggested classification can be very effectively used in practice. Table 2 given above
shows that every stage has its own risk level, considering changes in the function of
contraction of the IVS. It enables to identify in the very efficient way certain risk groups
among patients to be examined during either their routine periodic health examination or
under emergency conditions.
It should be mentioned that the Cardiocode technology requires an orthostatic testing. That
means that cardiac signals should be recorded both in lying and sitting positions. Recording
in lying position lasts over 20 seconds, then the patient should change his/her position to
sitting, and another 20 second-recording should be carried out within next 1 minute
thereupon. It is also advisable to offer to the patient to squat 10 15 times, and thereupon to
record the curves the third time in a session with the same patient in standing position.
4.3 Diminished function of interventricular septum and myocardium
Cases of diminished contraction function of the interventricular septum and the
myocardium are observed in clinical practice (Fig. 17). In these cases, the QRS complex
shows very small amplitudes. It indicates that the contraction function of the IVS and the
myocardium is diminished. The relaxation of the heart in the premature diastole phase is
weakened. In order to provide the proper blood filling of the heart, the contraction function
of the atria increases, as evidenced by the high amplitude of the wave. In the given case,
the pressure, actually built up by the heart, is not high enough due to the diminished
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function of the myocardium. As a consequence, in order to reduce the resistance to the
blood flow, the aorta is expanded and exceeds its normal volume that is reflected on the
ECG curve in an increasing of amplitude of wave .
According to the lettering used for the suggested classification, the ECG can be described as
follows:
R0,25; - S0,25; 3; T3
Considering the fact that the reference ECG curves, as shown in Fig.6, are taken as the basis,
we can obtain more precisely coefficients in automatic measuring of the actual ECG version
given in Figure 17. These coefficients assist in understanding of what group is applicable to
the given record. In order to qualify the primary cause of any cardiovascular pathology, it is
also required to involve the respective RHEO into the phase analysis. It should be
mentioned that it is not our intention to treat this issue in this Chapter.

Fig. 17. Diminished contraction function of IVS and myocardium
4.4 Condition of functioning of regulation of diastolic AP (R-wave bifurcation effect)
The process of regulation of diastolic pressure, as described in Chapter 1 above, in case of
pathology, is provided by different compensation mechanisms. In the instance illustrated in
Figure 18, the problem with the myocardium contraction is connected with the coronary
flow. The compensation mechanism manifested as R-wave bifurcation makes possible to
maintain the blood flow unhindered in the ventricles and provide the blood flow with the
valves closed due to additional vibration of the IVS.
The classification can be expressed as follows: (R1,5; R1,5); - S0,25.

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4.5 Condition of function of regulation of diastolic AP. IVS reverse contraction effect
at 100 % passivity of myocardium
Another case of the operation of the compensation mechanism, when the myocardium is
passive, is an IVS reverse contraction effect (s. Fig. 19). It can be treated as an extreme case of
R-wave bifurcation. But it is caused by a pathology problem other than the coronary flow.
As a rule, the case history of such patients contains records of close-spaced respiratory
diseases in infancy.

Fig. 18. Condition of function of regulation of diastolic AP (R-wave bifurcation effect)
This curve version can be classified as given below: (-R1,5; R1,5); S0,25.
One more case of the manifestation of the IVS reverse effect was recorded during the
orthostatic testing of one of the patients. Figure 20 demonstrates the R wave bifurcation for
horizontal position of the patient. When changing to the vertical position, appeared is
permanently the said IVS reverse effect (s. Fig. 20 b). By analyzing the respective RHEO
curve shape we can detect an aortic dilatation since there is no increase in the AP in the slow
ejection phase for the patients vertical position available.
4.6 No-S-wave & P-variation effect
The authors recorded and investigated the case of no-S-wave & P-variation effect (s. Fig. 21).
On these conditions, the compensation mechanisms are not capable of providing the proper
hemodynamics, therefore, it is the septum only which is in operation. It is evident from
Figure 21 that hemodynamics is maintained due to the second P-wave periodically
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appearing on the ECG. Thereupon, the arterial pressure reaches its norm but in subsequent
cycles it rapidly drops and remains at its low level, and the P-wave is not available. Such
fluctuations are synchronized by respiratory rhythm. It is remarkable that this patient
visited the doctor unaccompanied, and before visiting the doctor he had not received any
treatment at hospital.
The record of this type can be classified as given below: (R3); -S0,25.

Fig. 19. IVS reverse contraction effect at 100 % passivity of myocardium. It appears at all
times irrespective of the patients position in orthostatic testing

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Fig. 20. The classified curve can be presented as follows: (R1,25; R1,5); -S0,25

Fig. 20b. The curve can be classified as follows: (-R0,5; R1,5); -S0,25
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Fig. 21. No-S-wave & P-variation effect
5. Conclusion
The materials presented herein create primarily a bridge for applying theory to practice.
Based on the concept of the heart cycle phase analysis, the authors have developed their
own innovative diagnostics equipment Cardiocode that is now in production. All cardiac
signal records contained herein were produced with this equipment. We expect that its
merits like easy-in use and high informative value will be recognized by practicing
physicians. But to justify the expectations is always not easy. New knowledge is finding the
proper way accompanied with great difficulties. To overcome every difficulty, our team
carried out a large body of research work the results of which are reflected herein.
We have focused on the basic definitions. Well-defined is also the range of problems solved
by the heart cycle phase analysis theory. The way offered by the authors which is the way of
systematization, unification and associated perception of new knowledge should support
the medical experts in phase analysis application by them. It is commonly supposed that
every innovation goes through three stages in order to be generally recognized which are as
follows: it is stage one when everybody says that it is in principle impossible; then stage two
comes when people say that there's something in it, and last stage three appears, when it is
believed that it seems to be very simple! The authors adhere to an opinion that the heart
cycle phase analysis theory is now between stage two and three from the point of view of its
recognition procedure.
6. Acknowledgements
We would like to express our gratitude to all doctors who participated in the clinical
testing of our methodology. We have completed a 30- year investigation cycle that
includes last five years of very hard work. All opinions, comments and recommendations
submitted to us by our colleagues during our researches have been considered and
embodied in this Chapter.

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7. References
[1] . Rudenko, M.; Voronova, O. & Zernov. V. (2009). Theoretical Principles of Heart Cycle
Phase Analysis. Fouqu Literaturverlag. ISBN 978-3-937909-57-8, Frankfurt a/M.
Mnchen London - New York
[2] Voronova O. (1995) Development of Models & Algorithms of Automated Transport Function of the
Cardiovascular System. Doctorate Thesis Prepared by Mrs O. K. Voronova, PhD, VGTU,
Voronezh
[3] Rudenko, M.; Voronova, O. & Zernov. V. (2009) Study of Hemodynamic Parameters
Using Phase Analysis of the Cardiac Cycle. Biomedical Engineering. Springer New
York. ISSN 0006-3398 (Print) 1573-8256 (Online). Volume 43, Number 4 / July, 2009.
. 151 -155.
[4] Caro, C.; Padley, T.; Shroter, R. & Sid, W. (1981) Blood Circulation Mechanics. Mir. M.
[5] Eman .. Biophysical principles of arterial pressure measurement. ., 1983.
9
Biophysical Phenomena
in Blood Flow System
in the Process of Indirect
Arterial Pressure Measurement
Mikhail Rudenko, Olga Voronova
and Vladimir Zernov
Russian New University,
Russia
1. Introduction
Diagnostic parameter arterial pressure known in medical practice from the earliest times
is now widely used for assessment of body state. Indirect occlusive methods are the most
popular measurement techniques. Although the indirect method of measurement is more
than one hundred years old there is no precise understanding of biophysical processes
taking place in compressed blood flow.
The idea of indirect arterial pressure measurement with the help of occlusive
cuff belongs to Riva-Rocci. However, the phenomenon of noise appearing and
disappearing in the blood flow distal of the brachial artery compression during the
equality moments of occlusive, systolic and diastolic pressure was called Korotkov
sounds. The origin of the sounds is considered from different viewpoints [1]. But it is
important to mention that their identification has no valid criteria. If their appearance
corresponds to the systolic pressure, their disappearance is not always characteristic for
the diastolic pressure. Thus, during the Olympic Games in Mexico continuous sounds
were recorded with the swimmers. But this fact didnt mean that they had zero diastolic
pressure.
In case of measurement method computerization more reliably recorded biosignals in the
form of oscillogram are used [1]. Nowadays Korotkov sounds are out of use in case of
computerization of the arterial pressure measurement. The oscillometrical method of
measurement is more reliable. But this method has no explanation from the point of view of
biophysics as well.
The authors of the present research work have studied biophysics of the processes in the
occlusive blood flow for a long time. The study resulted in discovery of the objective law
concerning the origin of arterial pressure waves interference in occlusive blood flow. It
enabled to understand the processes taking place in occlusive blood flow and find the
criteria which systolic pressure and diastolic pressure correspond to.

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2. Biophysical processes of origin of the arterial pressure waves interference
in occlusive blood flow
Let us consider the occlusive method of the arterial pressure measurement. Big blood
vessels are compressed with occlusive pressure artificially produced in the rubber cuff put
as a rule on one of the patients arms. Then the pressure is measured at the moments of its
balance with the arterial pressure using the corresponding criteria. This method enables the
measurement of two parameters systolic arterial pressure and diastolic arterial pressure.
Which criteria can be used for accurate measurement?
In practice the method of Korotkov sounds and the oscillometrical method are used. In case
of the measurement process computerization the sounds method is not used. It is not
reliable for noise recording. Oscillometrical method seems to be more reliable. Oscillogram
is the signal of pulse wave oscillations modulated by the occlusive pressure. When recorded,
these oscillations are extracted from the pressure signal in the cuff as a variable component
with the help of the filtering method. This process is technically simple and reliable.
To understand the biophysics of occlusive blood flow it is necessary to have at least its
hypothetical model. We propose to study the model living bodymechanic system. It can
help to define the real biophysical processes. In this case the model is represented by the
arterycuff system.
Electronic converters quite accurately register the processes taking place in the system.
However different existing theories of biophysical processes give ambiguous characteristic
of the criteria of arterial and occlusive pressure balance [3]. This prevents the provision of
electronic arterial pressure measuring instruments with the corresponding metrology.
Practically metrology of the indirect method of arterial pressure measurement does not
exist.
Although the problem is quite serious we will try to study it. Let us consider the biophysical
phenomena in the system of the proposed arterycuff hypothetical model in the process of
indirect arterial pressure measurement [4].
Figure 1 shows the simplified version of this model. For convenience only a half of the cuff
is shown, it is conventionally in contact with the artery. Pulsating blood flow contacting
with the cuff influences it which is recorded in the form of the corresponding signals.
a. beginning of phase 1;
b. end of phase 1, beginning of phase 2;
c. transitory moment of phase 2;
d. end of phase 2, beginning of phase 3;
e. the first inflection moment in phase 3;
f. the second inflection moment in phase 3.
travel direction of the arterial pressure wave;
- - changed travel direction of the arterial pressure wave influenced by the occlusive
pressure.
Figure 2 shows the synchronous record of decompression occlusive pressure. The
oscillogram (Fig.2, b) is received by means of filtration in the frequency band and increase of
the oscillations which exist against the background of occlusive pressure as pulsations with
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181

Fig. 1. Changes in the arterycuff contact profile during different phases of arterial
pressure measurement.
small amplitudes (Fig.2, a). Several heart cycles of the oscillogram and its derivatives can be
more closely seen in Fig.3. The extreme values of one cardiac cycle and the corresponding
derivatives are marked by points 1, 2 and 3.

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Fig. 2. Synchronous record of decompression occlusive blood pressure (a); oscillogram of
first-order derivative (b); oscillogram of second-order derivative (c); oscillogram of the part
of the second derivative (d); oscillogram of the plethysmogram (e). The explanation is to be
found in the text.
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Fig. 3. Synchronous record of several cardiac cycles in the first phase of occlusive pressure
(a); oscillogram (b); first-order derivative (c); second derivative (d); part of the second
derivative (e) and photoplethysmogram (f). 1, 2, 3 are extreme values of one cardiac cycle
and their corresponding derivatives.

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In the process of modulation of blood flow by occlusive pressure additional characteristic
vessel impedance is needed. Its value is calculated in compliance with the formula:
c
Z
S

where:
blood density; c arterial pressure wave velocity; S vessel area.
Increase of the additional characteristic vessel impedance causes the appearance of standing
wave proximal the occlusive place. Designed in such a manner interference pattern is
registered by the cuff against the background of the occlusive blood pressure (see Fig. 2, a).
We should mention that reflected waves emergence in physiological blood flow is
considered from a perspective of characteristic impedance disagreement in vessel branching
and curving points [2]. In scientific literature this approach is based on research in rigid
pipes imitating blood vessels. The received results are not proved by the experiments over
the living bodies [3]. Theoretic calculations of the arterial wave reflection level in case of
vessel branching for physiological blood flow can be found in [2]. It is marked that even in
case of 10% mismatch of the vessel impedances which significantly exceeds the real one, the
reflected waves are imperceptible and can not considerably influence the falling pressure
wave. Scientific literature does not provide the investigation of the process in case of local
increase of the characteristic vessel impedance which cuff occlusion under condition of
arterial pressure measurement is. Thus, the study of the phenomena in case of maximum
impedance change range arouses interest.
Deviation in extreme points amplitude by the falling wave is characteristic for interference
[5]. This phenomenon is presented on the second derivative (see Fig. 2,d; 3,d).
For convenience we shall divide the process of arterial pressure measurement into three
phases (see Fig. 1).
During the first phase (see Fig.1, a, b) the occlusive pressure exceeds the systolic pressure
and the characteristic impedance is maximum. In case of decompression in one phase it
remains constant. Increase of the cuff-artery contact area leads to cuff elasticity growth (see
Fig. 1, a, b). As a result the amplitudes of the recorded oscillations on the oscillogram rise
(see Fig. 2, b). Herewith the deviations of the extreme points remain maximum. This fact is
proved by the first and second derivatives character (see Fig. 2, c, d). Distal of the occlusion
place the arterial pressure oscillations are not to be found (see Fig. 2, f).
The second measurement phase starts from the moment of occlusive and systolic pressure
balance when the oscillating part of the arterial pressure wave begins to recover distal of the
occlusion place (see Fig., b, c; Fig.2, f) and finishes at the moment of occlusive and systolic
pressure balance (see Fig.1, d; Fig.2, b, d).
There exist two characteristic features of this phase. Firstly, the elasticity and area of the
arterycuff contact continue to increase when the occlusive pressure falls. As a result
condition for continuous oscillation amplitudes growth on the oscillogram is created.
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185
Secondly, characteristic impedance remains maximum for the arterial pressure values that
are lower than those of the occlusive pressure. For the values that exceed the occlusive
pressure ones the impedance is proportional to difference of the current occlusive and
systolic pressure values. This fact is related to the alteration of the straight-line travel
direction of arterial pressure wave influenced by the occlusion (see Fig.1). The reflected
wave amplitude will decrease proportionately with the decrease of the vessel characteristic
impedance for the arterial pressure wave layers the values of which exceed the occlusive
pressure. This will lead to decrease of the corresponding layers amplitude offset against the
overall interference background. As a result offset of the indicated extreme points will
proportionally decrease.
For the described process of layer-by-layer vessel characteristic impedance alteration for the
arterial pressure falling waves the corner of triangle (marked with 2 in Fig.2, d, e) with a
definite error can serve as a criterion for systolic pressure measurement. In Fig. 2 ,e a part of
the second derivative shown in Fig.2 ,d can be seen. Point 2 characterizes the amplitude
offset of the dicrotic oscillogram part in case of interference of falling and reflected arterial
pressure waves during the occlusion. Figure 3 shows that point 2 being extreme corresponds
with the dicrotic oscillogram part.
Along the same line consideration of the extreme values of the oscillogram and its
derivatives indicated with 1 and 3 in Fig. 3 enables to mark similar triangles with the
corresponding corners (see Fig.2, d).
The triangle corner indicated with point 1 corresponds to the moment of diastolic pressure and
occlusive pressure balance (see Fig.1, d and Fig.2, d). At this moment the oscillating blood flow
part is fully recovering distal of the occlusion (Fig. 2,f). On the oscillogram the oscillation with
maximum amplitude and maximum leading edge steepness which corresponds to the
maximum of the first derivative conforms to the described process (see Fig.2, c).
The considered model of biophysical processes enables the pressure measurement of
different arterial wave layers according to characteristic maximums of oscillogram
derivatives, in particular, systolic and diastolic pressure.
The beginning of the third measurement phase is the moment of diastolic and occlusive
pressure balance (see Fig.1, d). This phase is characterized by the two inflections of
enveloping oscillogram, defined by the alteration of the cuff shape and elasticity as a result
of occlusive pressure fall (see Fig.1, e, Fig.2, b).
For the oscillating part of arterial pressure characteristic impedance is defined only by the
alteration of the arterial wave travel direction influenced by the occlusive pressure. At the
moment of enveloping oscillogram inflection (see Fig.2, b) the cuff loses its shape rigidness
(see Fig.1, d). At the same time characteristic impedance changes rapidly. During the period
preceding the second inflection (see Fig.1, f) the cuff takes almost the same shape as it would
have without the creation of the occlusive pressure. After the second inflection the cuff
perceives the arterial pressure oscillations at the expense of the occlusive pressure. Herewith
the cuff elasticity does not change.
The described biophysical phenomena allow concluding that indirect method can be used to
measure systolic and diastolic pressure with a definite error in the process of the oscillogram
and its derivatives recording. To accomplish this it is necessary to search and record the first

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186
oscillogram derivative maximum (see Fig.2,b) and a part of the second oscillogram
derivative maximum (see Fig.2, e).
The described process of falling and reflected arterial pressure waves interference is
accompanied by turbulence distal of the occlusion place. Let us consider the equation of
continuity:

1
S
1
=
2
S
2
=
3
S
3

where:

1
stands for velocity proximal of the occlusion place;
2
is velocity at the place of occlusion;

3
is velocity distal of the occlusion place; S
1
,
2,3
artery cross-section area at the
corresponding places.
Considering the equation of continuity it is possible to state that if S
2
at the place of
occlusion tends to zero velocity
2
should tend to infinity. Velocity
2
in Reynolds number
equation defining the interrelation of inertial and viscous forces in the blood flow is found
in numerator:
.
Re
d
v

where:
- velocity; d - vessel diameter; v - kinematic viscosity coefficient.
As velocity 2 is found in the numerator of the above equation then during the systolic time
interval Reynolds number will exceed the value of 2500 which corresponds to turbulence
emersion. Turbulence promotes the acoustic noise called Korotkov sounds.
Synchronous record of occlusive decompression pressure is shown in Figure 4. According to
considered above criteria of occlusive and arterial pressure balance Korotkov sounds
appearance corresponds to the moment of systolic pressure measurement. The peak
amplitude of the sounds corresponds to the moment of diastolic pressure measurement and
the disappearance of sounds occurs during the second presented inflection of the
enveloping oscillogram. The connection of Korotkov sounds disappearance with the
moment of their maximum amplitude and the diastolic pressure measurement criterion is
possible using the cuffartery contact measurement. To achieve this, the cuff should not
bear against the patients arm. As a result the second inflection almost matches the peak
amplitude and a drastic decrease of the enveloping oscillation and the sounds is registered.
3. Measurement criteria of systolic and diastolic arterial pressure based on
recording by the oscillogram derivative extreme point
3.1 The peculiarities of oscillogram recording
In the first part of this chapter the biophysical processes forming the oscillogram were
considered. It was proved that the oscillogram is a reflection of arterial pressure waves
interference process in the place of artery occlusion. The process of falling and reflected
Biophysical Phenomena in Blood Flow
System in the Process of Indirect Arterial Pressure Measurement

187

Fig. 4. Synchronous record of decompression occlusive pressure (a); oscillogram (b); the first
derivative (c); the second derivative (d) and Korotkov sounds (e).
waves interference modulated by external pressure can be investigated only with the help of
mathematical derivatives. In this case we used the first-order and second-order derivatives.
We should notice that the first derivative reflects the process of the object alteration. The
extreme points of the first derivative always indicate the moment of transformation of an
objects state or its function to a different state or function. The second derivative is a result
of interaction of the object or its functions with ambient environment. Here the extreme

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points are also informative. But their amplitude indicates the end result i.e. the fact of
interaction and its result.
In the process of investigation the authors faced an interesting problem. We would name it
take something not known what. The fact is that both the engineers and the doctors
worked on the development of ECG and other bioelectric signals recording. The engineers
tried to provide the doctors with the instruments that show a fine signal. They were
unaware of the degree of distortion during the filtration process and its difference from the
real processes. Our research showed that these distortions are significant and reach 25% [5].
This situation could be improved but the received distorted ECG have for a long time
served for creation of cardiological standards in diagnostics. Moreover, the theory which
was formed had many blank spaces.
That is why here we shall reveal a secret. It is essential that the lower cut-off band in the
filter should be equal:
F
H
= 0,35 Hz.
The signal upper this value is differentiated, the signal lower the value is integrated. If the
frequencies differ from the indicated ones it would be very difficult to understand what
happens in the occlusive blood flow. The same could be said about rheography.
For the engineers we should note that for the filtration process the rate of signal increase is
important as well. Different ECG phases have different amplitudes. In case of incorrect
choice of filtration band R deflection can be integrated increasing the RS phase to a
considerable extent. This process is influenced by the upper cut-off band. Inserting this
phase time in G.PoedintsevO.Voronova hemodynamics equation we shall obtain
fantastic results which will be different from the real results.
3.2 Criteria of systolic and diastolic arterial pressure measurement
The considered above criteria of systolic and diastolic arterial pressure measurement using the
oscillometric method and Korotkov sounds method allow to obtain identical values. But these
methods have considerable discrepancies concerning diastolic pressure measurement. When
the oscillometric method is used diastolic pressure is measured by the maximum of the first
oscillogram derivative. When Korotkov sounds method is used diastolic pressure is measured
by the sounds disappearance. Figure 5,c shows these discrepancies in points 2 and 3.
The study showed that the difference approximately accounts for 15 mm.Hg (Fig.5, points 2
and 3). How do the engineers solve this problem when developing commercial arterial
pressure measuring instruments?
1. The oscillogram is recorded (Fig.5, b). At the beginning of its amplitudes increase
comparator threshold is selected. Thus, systolic pressure is recorded.
2. Diastolic pressure is recorded when the oscillogram amplitude decreases below the
comparator threshold.
3. In automatic devices the oscillogram resembles the first derivative. It occurs due to
minimization of transient phenomena influence in the process of pumping pressure into
cuff.
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189

Fig. 5. Diastolic pressure measurement using the moment of Korotkov sounds
disappearance: a occlusive pressure; b oscillogram; c first-order derivative; d second
derivative; e Korotkov sounds.
Thus, all automatic machines with comparator threshold of signal amplitude do not
measure diastolic pressure accurately. The measuring instrument developed by the authors
of the present research work makes it possible to solve the problem in the following way.
Figure 6 shows the signal records received from the commercially produced instrument.
These signals are used to record systolic and diastolic pressure by derivatives maximum.
After the measurement 15 mm.Hg. are deducted from diastolic pressure value.

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Fig. 6. Synchronous record of processed oscillogram derivatives and signals of amplitudes
relation analyses unit. The amplitudes were extracted from the commercially produced
measuring instrument based on arterial pressure waves interference. a first-order
derivative; b search of diastolic pressure measurement criterion; c - search of systolic
pressure measurement criterion; d second-order derivative.
The end result will correlate with Korotkov sounds method and will not leave the doctors
asking questions. Thus, the measuring instrument provides two variants of the
measurement results presentation: 1) systolic pressure and average pressure (average
pressure conforms to the time of maximum of the first derivative and maximum sounds
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191
amplitude i.e. true diastolic pressure (point 2 on Fig.5,b); 2) systolic pressure and diastolic
pressure (diastolic pressure conforms to the second inflection of the enveloping oscillogram
or the moment of Korotkov sounds disappearance (point 3 on Fig. 5,b). It is close to the
value of comparator threshold in the measuring instruments produced by different firms.
4. Conclusion
1. The conditions of local increase of vessel characteristic impedance leading to
interference of falling and reflected arterial pressure waves are considered. Criteria of
systolic and diastolic pressure measurement compared with the used in practice
Korotkov sounds are revealed.
2. The described method enables to measure systolic and diastolic arterial pressure
accurately.
3. Commercially produced automatic arterial pressure measuring instruments using
comparator functioning as criteria for diastolic arterial pressure measurement during
oscillogram amplitude decrease do not measure diastolic pressure accurately; they
rather show the value which is 15 mm.Hg lower than the true value.
5. Acknowledgement
The problem of arterial pressure measurement is given much attention in the world
scientific literature. Specialized magazines are published. Scientific subpanels formed at
symposiums study the results of research in this scientific field. Having studied in Radio-
technical institute the authors of the present research work took interest in the popular at
that time idea of computerization of arterial pressure measurement process. In the 1970s the

Fig. 7. M. Rudenko is in the hostel soldering the pressurevoltage transformation junction.
The strain indicator is glued to the shoe cream box. Thus, the Big Science started (1978).

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192
problem of pressure measurement in the place of brachial artery and temporal artery was
set. Their magnitude relation should be equal to two. This coefficient was supposed to
indicate good physical fitness of the sportsmen. The young authors started to work
enthusiastically. We could not know then that the study of this problem would become a
foundation for considerable scientific research the authors would devote their life to. A
group of professionals would carry on research and develop medical instruments. Many of
the researchers would found their schools of thought and achieve good results in business.
Thirty years later the authors would receive a diploma for scientific discovery Objective
laws of arterial pressure waves propagation in blood-vessels in the areas of their impedance
local increase.
An outstanding school of thought with distinguished scientists was formed. Over the past
twenty years more than 150,000 copies of books were published. There emerged a need in
system research of human biophysics. All of the research works found practical use.


Fig. 8. The first in the world commercial instrument for indirect arterial pressure
measurement based on artery pressure waves interference (1986).
Biophysical Phenomena in Blood Flow
System in the Process of Indirect Arterial Pressure Measurement

193



Fig. 9. The documents of the official registration of scientific discovery Objective laws of
arterial pressure waves propagation in blood-vessels in the areas of their impedance local
increase (2005).
6. References
[1] Savitsky N.N. Biophysical principles of circulation and clinical methods of hemodynamic
study. 3-d ed. ., 1974.
[2] Caro, C.; Padley, T.; Shroter, R.& Sid,W. (1981). Blood Circulation Mechanics. - Mir. .
[3] Eman .. Biophysical principles of arterial pressure measurement. ., 1983.

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[4] Rudenko, M.; Voronova, O.; & Zernov, V. (2009). Study of Hemodynamic Parameters
Using Phase analyses of the Cardiac Cycle. Biometrical Engineering. Springer New
York. ISSN 0006-3398 (Print) 1573-8256 (Online). Volume 43, Number 4 / July 2009.
P. 151-155.
[5] Rudenko, M.; Voronova, O.; & Zernov, V. (2009). Theoretical Principles of Heart Cycle
Phase Analyses. Fouqu Literaturverlag. ISBN 978-3-937909-57-8, Frankfurt a/M.
Mnchen London New York.
10
Interrelation Between the Changes of Phase
Functions of Cardiac Muscle Contraction and
Biochemical Processes as an Algorithm
for Identifying Local Pathologies
in Cardiovascular System
Yury Fedosov, Stanislav Zhigalov, Mikhail Rudenko,
Vladimir Zernov and Olga Voronova
New Russian University,
Russia

1. Introduction
Investigations of cardiovascular system based on mathematical models of hemodynamics
developed by the authors allowed studying in details the cardiac cycle functions
of different parts of the heart during different phases of the cardiac cycle. The proposed
fundamentally novel diagnostic method based on phase analysis of cardiac cycle made
it possible to track any functional and hemodynamic changes in the cardiovascular
system. However, treatment of patients was always an issue after the diagnosis was
established.
The existing understanding of the interrelations between the shape of the ECG an clinical
meaning of the pathology were often in conflict with the insights gained from the phase
analysis of cardiac cycle. New knowledge was needed about the processes occurring in the
normal and pathological cardiovascular systems at the cellular level. The unique method of
cardiac cycle phase analysis allowed verifying all the theoretical concepts based on the
biochemical processes underlying development of the pathology, affecting functions of each
segment of the cardiovascular system. Moreover, it proved possible to establish a number of
recurring patterns of the influence of biochemical processes in the heart cells upon the
observed shape of ECG and RHEOgrams.
In this chapter the authors outline their vision of the main biochemical processes
determining the clinical meaning of the pathology diagnosed with the aid of the cardiac
cycle analysis method. Selection of the therapeutic agents aimed at normalization of the
diagnosed functional deviations taking into account the biochemical processes underlying
these functions resulted in the recovery of the functions.

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2. Interrelation between the contraction functions of myocardial muscles and
biochemical processes in the cardiovascular system
2.1 Cardiac muscle contraction function and cell energy balance
Investigations with the aid of cardiac cycle phase analysis have revealed a compensatory
mechanism for maintaining normal hemodynamics [1]. The essence of the mechanism is that
a decrease of the contraction phase function of one segment of the heart entails an increase
of the contraction phase function in an adjacent segment. E. g., decrease of amplitude of
contraction of the ventricular septum causes the amplitude of contraction of ventricles to
increase. E. g., decrease of amplitude of contraction of the ventricular septum causes the
amplitude of contraction of ventricles to increase. Such transformations of cardiovascular
system can only be diagnosed with the aid of cardiac cycle phase analysis.
Without knowing the compensatory mechanisms, neither a precise localization of the
pathology nor its controlled treatment is possible. Phase analysis taking into account the
compensatory mechanisms and cause-and-effect relation logics also allows identifying the
origin of the pathology. Elimination of the original cause of the disease results in
normalization of functions of other segments that used to perform compensatory functions
for the affected segment.
In this manner, the authors attempted to control the process of influencing local pathological
zones. Assessment of the recovery of the affected segments revealed that the cause of the
change of function was not in the degradation of conductivity of the cardiac electrical
system, but in the biochemical processes taking place within the myocardial cells.
According to publications of other authors, there is a number of various factors affecting the
effectiveness of myocardial cell recovery in terms of their energy supply functions and
further normalization of the muscle contraction function. [2] I. Leontieva and V. Sukhorukov
have introduced a new term mitochondrial cardiomyopathy.
Mitochondria are the major consumers of oxygen in the body. Hypoxia resulting from
insufficient saturation of blood with oxygen is causing tissue damage up to necrosis. The
primary symptom of hypoxia is swelling of mitochondria. The mitochondria of heart muscles
have anatomic specificities. These are associated with the increased intensity of oxidation
processes occurring in the cardiovascular system. The main function of mitochondria is ATP
synthesis based on the uptake of fatty acids, pyruvate, glucose and amino acids from cell
cytoplasm and their oxidative cleavage with generation of 2 CO2. Fatty acids can only
be delivered to mitochondria upon interaction with carnitine. Importantly, the quantative
content of carnitine depends on the amount of secreted endorphins, thus regulating ATP
synthesis. Besides that, carnitine regulates the exchange of phospholipids, essential substances
required for normal function of the peripheral and central neural system. Its active form, L-
carnitine is used for treating anorexia, extreme exhaustion.
It is due to effective functioning of mitochondria that muscle contraction occurs. They are,
however, the weakest link in the cell functioning. Hypoxia substantially alters their energy
budget. Oxidative phosphorylation is inhibited, transferring the mitochondria into free
operation mode. Normally, oxidation in mitochondria takes place aerobically. In case of
ischemia, this process becomes anaerobic. Anaerobic processes also start to become
predominant at the heart rates above 150 beats per minute.
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2.2 Stress and functional phase changes
In order to elucidate the influence of stress upon the work of heart and associated changes,
normal energy supply to cardiac myocytes should be considered.
Contractility is the main function of cardiomyocytes. This is an energy dependent process
requiring sufficient amount of ATP and
2+
. Energy supply to heart cells is a complex of
sequential processes, such as binding by carnitine and transportation into mitochondria of
the oxidation products, ATP generation, its transportation and consumption in various
energy-dependent reactions.
Following are the main specific features of the cardiomyocyte metabolism:
1. The metabolism is predominantly aerobic. The main route of energy generation is
oxidative phosphorilation.
2. The main substrates of oxidation are fatty acids.
3. High rate of energy-dependent processes in the myocard.
4. Minimal inventory of high-energy compounds.
Metabolism of cardiomyocytes is predominantly aerobic. Thus, they receive most of the
energy through electron transfer from organic substrates to molecular oxygen. Therefore,
contraction function of the cardiac muscle is a linear function of the oxygen uptake rate [3,4].
Synthesis of molecular ATP occurs in the process of oxidative phosphorylation in
mitochondria. The amount of ATP generated depends on the amount of acetyl-CoA (EC
6.4.1.2), which gets oxidized in the tricarbonic acid cycle. When myocard is normally
supplied with oxygen, 60 to 80% of the acetyl-CoA is generated due to -oxidation of fatty
acids, and 20-30 % - in the course of aerobic glycolysis. As a result of one loop of tricarbonic
acid cycle, one molecule of acetyl-CoA gets decomposed to
2
and
2
, 38 molecules of
ATP being formed. Protons enter the mitochondrial respiratory chain in the form of reduced
nicotineamides (NAD+ and NADF+). The main sources of reducing agents and their
interrelation with mitochondrial respiratory chain are illustrated in figure 1.

Fig. 1. The main sources of reducing agents and their interrelation with mitochondrial
respiratory chain (NAD nicotineamides).

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The main transporter of ATP in cardiomyocytes is creatine phosphate. ATP-ADP translocase
transports ATP to the outer side of the inner mitochondrial membrane, where creatine is
phosphorylated under the action of creatine kinase (EC 2.7.3.2). Thus, creatinine phosphate
and ADP are generated. Thereafter, ADP is transported inside the mitochondrial membrane.
The most energy-consuming process in the cardiomyocyte is contraction of myofibrils.
Translocation of counter-lateral actin filaments against myosin filaments towards the center
of sarcomeres and formation of actin-myosin bridges in the myofibrils occurs when
sufficient amount of ATP is present.
Having considered the energy balance of cardiomyocytes, let us move to the metabolic
processes occurring under the conditions of local stress.
From the standpoint of heart muscle, stress primarily results in hypoxia. Lack of oxygen
affects all the stages of the cell energy supply (synthesis, transportation and consumption of
ATP). In order to compensate for this, cardiomyocyte mobilizes energy from the
intracellular inventories and reduces energy consumption. The inventories of the energy-
rich substances creatine phosphate, glucose and triglycerides are insignificant, and the cell
soon starts to experience energy shortages. Anaerobic glycolysis is then activated to
overcome the energy shortage.
Changes to fatty acid metabolism during hypoxia is characterized by disruption of -
oxidation of fatty acids, which is associated with the decrease of L-carnitine level caused by
stress. Intracellular accumulation of fatty acids, acyl-carnitine and acyl-CoA (EC 6.2.1.3)
occurs. The increase of acyl-CoA concentrations suppresses transportation of adenine
nucleotides in mitochondria.
Development of hypoxia decreases the share of aerobic glycolysis to 5%. Thus, under
conditions of stress caused by lack of oxygen energy, energy supply in cardiomyocytes is
reduced by 65-95% of its normal value. Anaerobic glycolysis is then activated to compensate
for the energy deficiency. Generation of ATP is reduced to 2 molecules per a molecule of
glucose (as compared to 38 molecules under normal conditions). Increase of the share of the
anaerobic glycolysis covers about 60-70% of the energy consumption. However, if this
compensation occurs for an extensive period, it becomes dangerous.
In the course of anaerobic glycolysis, lactate builds up causing lactic acidosis. Against this
background, accumulation of ATP hydrolysis products, and free fatty acids causes
intracellular acidosis. This is accompanied by the loss of integrity of lysosomal membranes,
release of lysosomal ferments, which, under conditions of energy deficiency, results in the
damage of mitochondria ultra structure.
The energy deficiency also contributes to loss of ion balance. Reduced concentration of ATP
inhibits the Na
+
/K
+
pump of the cellular membranes. Consequentially, sodium and
potassium ion concentration gradients start to decrease. Accumulation of sodium ions in the
cardiomyocytes along with the increase of concentration of potassium ions in the
extracellular solution result in the decrease of the resting potential and reduced duration of
the action potential. Such deviations from the normal concentrations of ions in the
intracellular and extracellular solutions cause hyperosmia, i.e. cell swelling, disrupting
calcium homeostasis in the cardiomyocytes. Permittivity and contractility of certain sections
of the cardiac muscle degrade, whereas neighboring parts of the cardiac muscle take
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199
additional load in a compensatory manner. These processes are clearly reflected in the
cardiac cycle phases on the ECG. Relevant examples are given in the end of the chapter.
These abnormalities can be tracked with the aid of detecting functional phase contractions of
the heart muscle.
2.3 Neural pulse Interaction with cells
The influence of neural pulse on cardiac cells is associated primarily with initiation of
sequential interrelated processes supporting cardiac muscle contraction.
Normal rhythmic contractions of cells occur as a result of spontaneous activity opf the
pacemaker cells located in the sinoatrial node (SA node). Time interval between the heart
contractions is determined by the time needed by the membranes of the pacemaker cells to
reach the threshold level due to depolarization. Autonomous frequency of heart
contractions is about 100 beats per minute without external impacts. An external impact is
needed in order to increase or decrease this heart rate.
Vegetative neural system produces two most significant impacts on the heart beat rate. The
fibers of both sympathetic and parasympathetic parts of the vegetative neural system
terminate on the cells of the SA node and affect the heart rate beat. The impact is caused by a
change of the process of spontaneous (autonomous) depolarization of the resting potential
in the pacemaker cells of the sinoatrial node.
Acetylcholine released by parasympathetic neural fibers going to the heart as a part of
branches of vagus nerve increases permeability of the membranes at rest to
+
and
decreases diastolic permeability for Na
+
. These changes of permeability have two effects on
the resting potential of the pacemaker cells. Firstly, they cause initial hyperpolarization of
the membrane resting potential, making it closer to the potassium equilibrium potential.
Secondly, they decrease the rate of spontaneous depolarization of the membrane at rest.
Both these effects tend to increase the lag between heart contractions due to increased
period of depolarization of the resting membrane to the threshold value.
Sympathetic neural fibers release noradrenalin. The most essential effect of noradrenalin is
the increase of the Na
+
and Ca
2+
intake by the cell during the diastole. These changes
increase heart beat rate due to increased rate of diastolic depolarization.
Besides the influence on the heart beat rate, vegetative neural fibers affect the rate of
conduction of action potentials through heart tissues. Enhanced sympathetic influence
increases the conduction rate, whereas the enhanced parasympathetic influence decreases
the conduction rate of action potentials.
Cardiomyocyte contraction is initiated by the action potential signal to the intracellular
organelles, resulting in increased tension and contraction of the cell. This process is known
as excitation-contraction coupling. The key element of these processes is an abrupt increase
of intracellular concentration of free
2+
. Concentration of
2+
changes from less than 0.1
mkm at rest to 100 mkm during maximal activation of the contraction machinery.
If we now recall the influence of local stress on cardiomyocytes and mechanisms of
occurrence of this influence, the reasons behind and abnormalities in conduction and
contraction of heart muscle become clear.

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When the energy transformation processes in the mitochondria become abnormal, parts of
the respiratory chain are inhibited by specific therapeutic agents, chemical reagents or
antibiotics, decrease of the amplitude of cardiomyocyte contraction due to lack of ATP is the
first consequence to be observed. Thereafter, due to accumulation of free fatty acids,
hydrolysis products, and lactate, due to development of internal acidosis and loss of ion
balance of cell, conductance of action potential starts degrading, resulting not only in
degraded conductivity of heart muscle and disturbance of the regulatory influence of the
neural system on the work of heart as a whole.
2.4 Endorphin stimulation as a natural way of enhancing stress resistance
Having considered the specifics of biochemical processes taking place in stressed cardiac
muscle, we can touch upon another important question: How does the body fight stress?.
It is a common knowledge that when stress factors appear, all the systems of the body are
activate. These processes are aimed at maintaining integrity, normal operability and
survival of an organism. Regulation of the cascades of biochemical reactions occurring in
response to stress factors is mediated by interactions of neural and endocrinal systems.
As shown in figure 2, as a result of stress the central neural system activates the following
pathway of endocrinal regulation: hypothalamus corticoliberin pituitary gland -
adrenocorticotropic (AcTH) hormone suprarenal gland cortisol. Besides the
adrenocorticotropic (AcTH) hormone, -lipotropic hormone (LPH) is generated from the C-
terminal part of the protein. LPH proteolysis results in generation of either -LPH and -
endorphin, or -melanotropin and -endorphin. Beside that, LPH can decompose to -
endorphin and met-enkephalin. Simultaneous production of all these hormones causes the
following effects:
Enhancement of carbohydrate metabolism (glucocorticoids)
Enhancement of lipid metabolism (lipotropins)
Reduced pain sensibility and euphoric sensation (endorphins and enkephalines)
Stimulation of immune system (melanotropin).
Thus, there is a system of multiple regulatory signals initiated by a single stimulus
regulating simultaneously a number of metabolic processes and receptor systems.
2.5 Example of application of phase analysis of cardiac cycle for controlling recovery
of the function of cardiovascular system in the course of treatment
Based on our understanding of biophysical processes and having a tool for investigating
phase processes of the heart function, we attempted to influence in an integrated manner the
metabolic processes occurring in the myocardium and track the associated changes of the
phase functions of heart contraction.
In order to influence the metabolism in an integrated manner, we performed normalization
of the acid-base balance. L-carnitine and octolipen were used to affect lipid metabolism.
Transcranial electrostimulation method was used in order to increase production of the
pituitary gland hormones (adrenocorticotropic (AcTH) hormone, LPH, melanotropin,
endorphins and enkephalines).
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Fig. 2. Stimulation of the neuroendocrine regulation mechanism by stress

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During this study, we tracked not only changes of the cardiac phase functions, but also the
phase hemodynamics parameters.
The results presented below were obtained in the course of integrated impact on the patient
organisms.
The figure 3 illustrates the initial results. These are ECG and RHEO records of the ascending
aorta and the table of the phase hemodynamics parameters. ECG and RHEO records
correspond to the same cardiac cycle. For the sake of convenience, only one cardiac cycle is
represented on the figure. The table summarizes results for 18 cardiac cycles. The number of
cycles is not fixed during the recording. The duration of the record is about 20 seconds. This
period is sufficient to obtain information for assessing hemodynamics parameters of several
cardiac cycles.
The shape of ECG corresponds to Brugada syndrome. Interventricular septum lost its
contraction function. This is evidenced by minimal amplitude of the R deflection. Expansion
of the S deflection is a compensatory function. Having assumed increased contraction load,
myocardial muscle increased its volume. Raise of SL wave on the ECG is indicative of
increased arterial pressure. In this case, there is a continuous stress of myocardium since the
amplitude of the SL phase is above the isoline in each cardiac cycle
The identified factors allow making conclusions and selecting the treatment strategy. The
original cause is the issue with the interventricular septum, and it is this problem that has to
be addressed. Widening of the S deflection and high amplitude of the SL phase are
secondary factors caused by the compensatory mechanism of substitution of its lost
function. In case of successful recovery of the function of the interventricular septum, other
function are to normalize on their own.
There was an assumption that the problem of the loss of contractility function is based on
mitochondrial cardiomyopathy. It was therefore decided that the patient should take L-
carnitine simultaneously with octolipen. In addition to that, daily use of the breathing
exerciser was prescribed in order to normalize the balance of carbon dioxide and
oxygen in blood. These procedures were performed domiciliary. In the outpatient
conditions, he was undergoing electrical treatment, excitation of specific cranial zones
with small current pulses in order to stimulate release of endorphins. No limitations in
diet were imposed.
According to the table on the figure 3, in the beginning of the treatment the average value of
the cardiac output (minute blood volume) of the patient ws MV = 9.71 liters. In the course of
treatment, MV variations from 7.63 to 10.93 liters were recorded.
In two months, the results presented in figure 4 were recorded. The record corresponds to
the upright position of the patient body during orthostatic test. Splitting of the deflection S
is clearly visible. This is not a pathology, but rather a reaction of myocardium to overload.
When the patient was in horizontal position, no splitting/vibrations were observed.
However, already in the next cycle the ECG assumes fairly normal shape, though the shape
is not yet stable. This is also evidenced both by the parameters of hemodynamics, namely
the minute volume MV.
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Fig. 3. September 2010

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Fig. 4. November 2010
Another month later ECG remained unstable, but the average value of MV decreased to 9.06
liters.
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205


Fig. 5. December 2010
In two months, hemodynamics parameters grew somewhat. The patient continued to
receive the treatment, having only excluded the octolipen.

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Fig. 6. February 2011
In another month the patient stated that he had gone through a medical exam in the regional
clinics, where he was offered surgery to narrow the interventricular septum. The patient
rejected the surgery. Coronary angiography was also performed, having indicated that the
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207
coronary arteries were clear. The patient was concerned with premature beats (extra
systole). The figure 6 illustrates the original record made during investigation of phase
parameters.


Fig. 7. March 2011
After a series of sit-ups, extra systole was detected (see Fig. 8). Minute volume MV increased
to 13.66 liters.

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Fig. 8. March 2011, after sit-ups having caused extra systole.
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209
After relaxation of the patient, the extra systoles disappeared. MV = 13.32.


Fig. 9. March 2011, Relaxation after extra systole.
The treatment course was continued. In two months, ECG was almost normal. No extra
systoles were detected. MV = 7.72 liters.

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Fig. 10. May 2011
3. Conclusion
Cardiac cycle phase analysis method allows tracking any changes of hemodynamics and
functions of the cardiovascular system. It can be used to identify the original cause of
pathologies and to efficiently monitor the treatment progress.
4. References
[1] Rudenko, M.; Voronova, O. & Zernov. V. Innovation in cardiology. A new diagnostic
standard establishing criteria of quantitative & qualitative evaluation of main
parameters of the cardiac & cardiovascular system according to ECG and RHEO
based on cardiac cycle phase analysis (for concurrent single-channel recording of
cardiac signals from ascending aorta).
http://precedings.nature.com/documents/3667/version/1/html
[2] Leontieva I. & Sukhorukov V. The implications of metabolic disorders in the genesis of
cardiac myopathia and possible use of L-carnitine for therapeutic correction. . Saint
Petersburg. Manuscript -2006.
[3] Vasilenko V. Kh., Feldman S. B., Khotrov N.N., Miocardyodistrophia. - Moscow.
Medicine. 1989. -272.
[4] Kushakovsky M.S. Metabolic cardiac diseases. Saint Petersburg. Manuscript -2000. 128.
11
Application of Computational
Intelligence Techniques for
Cardiovascular Diagnostics
C. Nataraj, A. Jalali and P. Ghorbanian
Department of Mechanical Engineering,
Villanova University, Villanova, Pennsylvania,
USA
1. Introduction
Cardiovascular disease, including heart disease and stroke, remains the leading cause of
death around the world. Yet, most heart attacks and strokes could be prevented if it were
possible to provide an easy and reliable method of monitoring and diagnostics. In
particular, the early detection of abnormalities in the function of the heart, called
arrhythmias, could be valuable for clinicians.
Hemodynamic instability is most commonly associated with abnormal or unstable blood
pressure (BP), especially hypotension, or more broadly associated with inadequate global or
regional perfusion. Inadequate perfusion may compromise important organs, such as heart
and brain, due to limits on coronary and cerebral auto regulation and cause life-threatening
illnesses, or even death. Therefore, it is crucial to identify patients who are likely to become
hemodynamically unstable to enable early detection and treatment of these life-threatening
conditions (Cao, Eshelman et al. 2008). Modern intensive care units (ICU) employ
continuous hemodynamic monitoring (e.g., heart rate (HR) and invasive arterial BP
measurements) to track the state of health of the patients. However, clinicians in a busy ICU
would be too overwhelmed with the effort required to assimilate and interpret the
tremendous volumes of data in order to arrive at working hypotheses. Consequently, it is
important to seek to have automated algorithms that can accurately process and classify the
large amount of data gathered and to identify patients who are on the verge of becoming
unstable (Cao, Eshelman et al. 2008).
Modern ICUs are equipped with a large array of alarmed monitors and devices which are
used to try to detect clinical changes at the earliest possible moment so as to prevent any
further deterioration in a patients condition. The effectiveness of these systems depends on
the sensitivity and specificity of the alarms, as well as on the response of the ICU staff to the
alarms. However, when large numbers of alarms are either technically false, or true, but
clinically irrelevant, response efficiency can be decreased, reducing the quality of patient
care and increased patient (and family) anxiety (Laramee, Lesperance et al. 2006).
It is patently obvious that physiological time series such as hemodynamic and
electrophysiological data represent the physiological state of subjects in a medical

The Cardiovascular System Physiology, Diagnostics and Clinical Implications 212
environment. These time series are collected over long periods of time and are usually a
source of a large number of interesting behaviors or features which have the potential to be
used in identifying and predicting a subjects current and future state of health. However,
the high dimensionalities and complexity of the measured physiological signals make the
interpretation and analysis difficult, if not impossible. Hence, although they clearly contain
useful information, these signals cannot be used directly. Extraction of such hidden
information can be addressed using the concept of feature extraction. Essentially, feature
extraction is focused on dimensionality reduction and on revealing information from the
different time scales that underlie physical phenomena. Also of importance is the concept of
classification, where the features are employed in an intelligent algorithm to classify the
patient, for example, as healthy or sick. Clearly, this is a broad area with an increasingly
diverse set of applications. In order to illustrate the power and utility of these methods, and
given the limited space, we limit ourselves to two examples both of which illustrate feature
extraction and classification approaches.
The first application discussed in this chapter is the detection of cardiac arrhythmia
detection. In this application, we apply continuous wavelet transform (Daubechies 2006)
and principal component analysis (Jolliffe 2002) as feature extraction tools and artificial
neural network algorithm as a classifier (Caudill 1989).
The second application discussed concerns the identification of ICU patients. In this
example, we apply some novel feature extraction techniques to highlight the differences
between healthy and patient subjects. Then we apply fuzzy decision theory (Zadeh 1968) as
a final classifier.
2. An improved procedure for detection of heart arrhythmias
The electrocardiogram (ECG) plays an important role in the process of monitoring and
preventing heart attacks. The typical ECG, shown in Figure 1, consists of three basic waves:
P, QRS, and T. These waves correspond to the far field induced by specific electrical
phenomena on the cardiac surface, namely, the atrial depolarization, P, the ventricular
depolarization, QRS complex, and the ventricular repolarization, T. It should be noted
however that the ECG signal does not look the same in all the leads of the standard 12-lead
system used in clinical practice.
There is increasing recognition that computer-based analysis and classification of diseases
could be very helpful in diagnostics and several algorithms have been reported in the
literature for detection and classification of ECG beats using artificial neural networks (ANN).
It has indeed been shown that neural networks are particularly able to recognize and classify
ECG signals more accurately than other classification methods (Ozbay and Karlk 2001).
The techniques, developed for automated detection of changes in electrocardiographic
signals, work by transforming the mostly qualitative diagnostic criteria into a more objective
quantitative signal feature classification problem. This transformation of the ECG signals
has been carried out in the past using techniques such as autocorrelation function, time
frequency analysis, and wavelet transforms (WT) (Maglaveras, Stamkopoulos et al. 1998;
Addison, Watson et al. 2000; Kundu, Nasipuri et al. 2000; Dokur and Olmez 2001; Saxena,
Kumar et al. 2002). Results of these and other studies in the literature have demonstrated
that WT is the most promising method to extract features that characterize the behavior of
ECG signals in an effective manner.

Application of Computational Intelligence Techniques for Cardiovascular Diagnostics 213

Fig. 1. The components of the ECG signal.
A study of the nonlinear dynamics of electrocardiogram signals for arrhythmia
characterization was presented by Owis (Owis, Abou-Zied et al. 2002). They selected the
correlation dimension and the largest Lyapunov exponent as two features for characterizing
five different classes of ECG signals. The statistical analysis of the calculated features indicated
that they differ significantly between the normal heart rhythm and the different arrhythmia
types and, hence, can be somewhat useful in ECG arrhythmia detection. However, their study
is limited by the fact that the discrimination between different arrhythmia types is difficult
using those features. Application of the wavelet transform, principal component analysis
(PCA) and several types of artificial neural network structures to detect and classify different
kinds of heart arrhythmias have also been reported (Silipo and Marchesi 1998); this study
compared results of different neural network structures in order to find the best one for the
classification of specific types of arrhythmias. A neural network classifier was used by
(Christov and Bortolan 2004) to recognize premature ventricular contraction arrhythmia beats
in an ECG signal database. A combination of neural network and discrete wavelet transform
(DWT) has also been applied for detecting four types of heart arrhythmias (Guler and Ubeyli
2005). Another application of a combination of wavelet transform and ANN in arrhythmia
detection is proposed in the study by Vikas (Vikas and Sahambi 2004). In the first step, a set of
discrete wavelet transform coefficients which contain the maximum information about the
arrhythmia is selected from the wavelet decomposition. Then, these coefficients, in addition to
the information about the RR interval, QRS duration, and amplitude of the R-peak, are fed into
a multi-layer perceptron algorithm. They reach an overall accuracy of 98% in the classification
of 47 patient records.
Papaloukas, et al. (Papaloukas, Fotiadis et al. 2002) used a neural network classifier to detect
and classify ischemic arrhythmia episodes in the ECG signal. They also used PCA to select

The Cardiovascular System Physiology, Diagnostics and Clinical Implications 214
and extract features from the ECG signal. Lee (Lee, Park et al. 2005) applied linear
discriminant analysis to 17 input features, which were based on wavelet coefficients, to
reduce the feature dimension from 17 to 4, for arrhythmia detection. Then, a multi-layer
perceptron classifier was applied to detect 6 types of arrhythmia beats from a 4-dimensional
input feature. Foo (Foo, Stuart et al. 2002) compared and evaluated different types of
multilayer neural network structures as the ECG pattern classifiers and finally settled on a
two-layer feed-forward neural network. However, their work is limited to detecting only
two types of patterns including normal beats and premature ventricular contractions (PVC).
Acharya, et al. (Acharya, Bhat et al. 2003) proposed an algorithm based on a neural network
classifier and fuzzy cluster to analyze ECG signals. They compared these two classifiers and
reported the fuzzy cluster as a better classifier in comparison with the neural one. They
classified 4 types of ECG signals including ischemic cardiomyopathy beat, complete heart
block beat, atrial fibrillation beat, and normal beat. Also, Ozbay (Ozbay, Ceylan et al. 2006)
proposed a comparative study of the classification accuracy of ECG signals using a well-
known neural network architecture, a multi-layered perceptron (MLP) structure, and a new
fuzzy clustering neural network architecture (FCNN) for early diagnosis; They used these
two classifiers to classify 10 types of ECG signals. Based on their test results they suggested
that a new proposed FCNN architecture can generalize better than ordinary MLP
architecture and could also learn better and faster. The advantage of their proposed
structure was a result of reduction in the number of segments by grouping similar segments
in training data with fuzzy C-means clustering.
Zhang (Zhang and Zhang 2005) developed an algorithm for recognizing and classifying four
types of ECG signal beats including normal beat, left bundle branch block beat, right bundle
branch block beat and premature ventricular contraction PVC beat. They extracted the
principal characteristics of the signals by means of the PCA technique and they showed that
out of 100 principal components, the first 30 principal components have most of the total
energy of the data set and hence used it as the input vector for the classifier. Among different
types of classifiers, they used the support vector machine (SVM), which has exhibited very
good success compared to other classification methods in complicated problems. A
comparison between different classifiers is also presented in their research. A comparison
between different structures for heart arrhythmia detection algorithms based on neural
network, fuzzy cluster, wavelet transform and principal component analysis, was carried out
by Ceylan (Ceylan and Ozbay 2007). Kutlu (Kutlu, Kuntalp et al. 2008) applied a K-nearest
neighborhood algorithm for the purpose of classification. They extracted features from the
electrocardiograph signals by using higher order statistics. They achieved an accuracy of 97.3%
in classifying 5 types of heart arrhythmias. Cvikl (Cvikl and Zemva 2010) designed a field-
programmable gate array-based (FPGA) system for ECG signal processing. Their system
performs QRS complex detection and beat classification into either normal or PVC. They
reached a sensitivity of 92.4% for PVC detection.
The most difficult problem faced by todays automatic ECG analysis is the large variation in
the morphologies of ECG waveforms, not only of different patients or patient groups but
also within the same patient. The ECG waveforms may differ for the same patient to such an
extent that they could be unlike each other, and at the same time, alike for different types of
beats. This is the main reason that the beat classifiers, which were reviewed in this study,
perform well on the training data, while generalizing poorly when presented with the ECG

Application of Computational Intelligence Techniques for Cardiovascular Diagnostics 215
waveforms of different patients (Ozbay, Ceylan et al. 2006). We address this problem of beat
classifier performance by using a combination of continuous wavelet transform (CWT) and
principal component analysis in order to prepare a more effective input data for the artificial
neural network classifier. Since this would lead to a better input vector structure for the
neural network classifier, we expect to obtain a better and more accurate performance of the
classifier. Moreover, we propose to use a signal filtering method in order to remove ECG
signal baseline wandering which can be further expected to improve classification.
This section is not focused on improving the processing techniques such as CWT and PCA
or on improving the neural network structure. It is instead focused on designing an
innovative algorithm which is a combination of these techniques in order to achieve
reasonably accurate classification results in the field of heart arrhythmia detection. Although
we address a better classification performance in the field of heart arrhythmia detection,
another interesting achievement of this study is that the classifier in this study detects 6
types of ECG signals including the normal signal and 5 types of arrhythmia beats. This
quantity of ECG signal types studied here is a much larger number in comparison with
other studies in this field. The structure proposed in this section is composed of three sub
stages: (a) continuous wavelet transform, which provides feature extraction; (b) principal
component analysis, which performs elimination of inconsiderable features; and finally, (c)
multilayer perceptron neural network, working as a final classifier.
The outline of this section is as follows; a basic definition of CWT is presented in Section 2.1.
In Section 2.2 the procedure of computing principal components of a data set is provided. In
Section 2.3, the designed algorithm of our study is presented with a detailed explanation.
Finally, in Section 2.4, the results of our study are presented.
2.1 Continuous wavelet transform
The wavelet transform (WT) provides very general techniques, which can be applied to
many tasks in signal processing. Wavelet transform can be thought of as an extension of the
classic Fourier transform; the difference is that, instead of working on a single scale (time or
frequency), it works on a multi-scale basis and describes the signals frequency content at
given times. This multi-scale feature of the WT allows the decomposition of a signal into a
number of scales, each scale representing a particular coarseness of the signal under study.
Continuous wavelet transform (CWT) is a time-frequency analysis method which differs
from the more traditional short time Fourier transform (STFT) by having a variable window
width, which is related to the scale of observation. Another important distinction from the
STFT is that the CWT is not limited to using sinusoidal analyzing functions (Osowski and
Linh 2001); a large selection of localized waveforms can be employed as the analyzing
function. The wavelet transform of a continuous time signal, x (t), is defined as

where *(t) is the complex conjugate of the analyzing wavelet function (t), a is the dilation
parameter of the wavelet, which is called scale, and b is the location parameter of the
wavelet (Osowski and Linh 2001).
1
( , ) ( ) ( )
t b
T a b x t dt
a
a

+

The Cardiovascular System Physiology, Diagnostics and Clinical Implications 216
2.2 Principal component analysis
Principal component analysis (PCA) has become a well-established technique for feature
extraction and dimensionality reduction. An assumption made for feature extraction and
dimensionality reduction by PCA is that most of the information of the observation vectors,
with the dimension p, is contained in the subspace spanned by the first m principal axes,
where . Therefore, each original data vector can be represented by its principal
component vector with dimensionality m (Ceylan and Ozbay 2007). This procedure
decreases the data dimensionality without significant loss of information (Addison 2005).
Principal components analysis has been used in a wide range of biomedical problems,
including the analysis of ECG data (Silipo and Marchesi 1998; Wang and Paliwal 2003;
Addison 2005; Ceylan and Ozbay 2007).
In order to apply PCA on a data set, X, the following five steps are required (Zhang and
Zhang 2005; Ceylan and Ozbay 2007):
1. Subtract the mean value, , from each of the data dimensions.
2. Calculate the covariance matrix, S.

where, , is the sample mean, and N is the number of samples.
3. Calculate the eigenvectors and eigenvalues of the covariance matrix.
4. Choose the components and form a feature vector.
In general, once the eigenvectors are found from the covariance matrix, the next step is to
order them by decreasing order of the magnitude of the eigenvalue. Then the feature vector
is constructed by taking the corresponding eigenvectors.
Feature Vector = (eig 1 eig 2 eig 3 eig n)
5. Derive the new data set.
Once the components (or eigenvectors) have been chosen and the feature vector is
constructed, the final data is constructed by pre-multiplying by the transpose of the feature
vector as shown below.
Final Data = Row Feature Vector x Row Data Adjust
where, Row Feature Vector is the transpose of the matrix with the eigenvectors in the
columns, Row Data Adjust is the transpose of the mean-adjusted data matrix, and Final
Data is the final data set, with data items in columns.
2.3 Methodology
A schematic of the designed algorithm in this study is shown in Figure 2. This algorithm
consists of three stages: pre-processing, main process and finally, classification of the ECG
beats. The data of ECG signals used in this study are taken from the MIT-BIH ECG signal
p m<

=
=
N
i
i
T
i
x x
N
S
1
) ( ) (
1

X x
i


Application of Computational Intelligence Techniques for Cardiovascular Diagnostics 217
database, including normal beats and five types of different arrhythmia beats. MIT-BIH
ECG signal database is a well-known standard database which has been used in many
research projects reported in the literature (Silipo and Marchesi 1998; Owis, Abou-Zied et al.
2002; Zhang and Zhang 2005; Ceylan and Ozbay 2007; Cvikl and Zemva 2010). For this
study, the selected types of arrhythmias are atrial premature beats (A), right bundle branch
block beats (R), left bundle branch block beats (L), paced beats (P), and premature
ventricular contraction beats (PVC or V).

Fig. 2. Schematic of the designed algorithm

The Cardiovascular System Physiology, Diagnostics and Clinical Implications 218
2.3.1 Pre-processing
This stage includes four levels of data processing: signal filtering, sample selection, feature
extraction, and dimensionality reduction.
In the stage of signal filtering, a mathematical method presented by Ghaffari (Ghaffari,
SadAbadi et al. 2006) is employed to remove baseline wandering of the ECG signal. Figures
3.a and 3.b show raw ECG signal of records 232 and 208 from the MIT-BIH database, each of
which clearly exhibit baseline wandering. Figures 3.c and 3.d show the same ECG signals
after applying the filtering method. It is clear that the baseline wandering has been
removed, leading to a better performance of the neural classifier.
For the stage of sample selection, the suitable range of samples from the raw ECG signal
was found experimentally to be 150 samples after the R wave for all types of signals,
which together comprise what we call a segment. These segments are found to be an
appropriate range of ECG signals which represent morphological differences between
different types of ECG beats and include sufficient amount of data needed for
classification of heart arrhythmias. For three types of ECG signals under study, the
morphologies of ECG beats are shown in Figures 4.a - 6.a; Figures 4.b - 6.b show the
selected segments of these beats.


Fig. 3a. Raw ECG signal from record 232 Fig. 3b. Raw ECG signal from record 208

Fig. 3c. Filtered ECG signal from record 232 Fig. 3d. Filtered ECG signal from record 208
6 6.5 7 7.5 8 8.5
x 10
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Application of Computational Intelligence Techniques for Cardiovascular Diagnostics 219

Fig. 4. (a) Normal beat, (b) selected segment for Normal beat.

Fig. 5. (a) Atrial beat, (b) selected segment for Atrial beat.

Fig. 6. (a) Right Bundle beat, (b) selected segment for Right Bundle beat.

The Cardiovascular System Physiology, Diagnostics and Clinical Implications 220
The choice of the analyzing function in wavelet transform, which is called the mother
wavelet, has a significant effect on the result of analysis and should be selected carefully
based on the nature of the signal (Addison 2005). Several mother wavelets, such as Morlet
and Mexican-hat, have been used in ECG signal analysis for component detection and
disease diagnosis (Stamkopoulos, Diamantaras et al. 1998). Because of the harmonic nature
of Morlet and Mexican-hat, they are often used for analysis of harmonic signals. These
mother wavelets are not likely to be suitable options in the case of ECG signal classification.
In fact, the simplicity of the computed CWT coefficients can be used as a convenient
criterion to help in the selection of the mother wavelet as shown below.
Figure 7 shows a normal signal and its CWT with different mother wavelets in the scale
a=10. Figure 7.a shows a normal signal beat, which has three picks. Figure 7.b shows CWT
of the same signal beat with Haar mother wavelet. This figure is very simple and the effects
of the raw signal picks are obvious and observable. These effects can be analyzed easily and
the extracted features would be suitable and appropriate for the data classification. Also,
these computed coefficients can represent morphological differences very well. Figure 7.c
shows CWT of the signal with Mexican-hat mother wavelet. The effect of raw signal picks
is not obvious in this figure and cannot be analyzed easily. Although this figure is not
complicated, the extracted features do not seem to be useful for classification of the data
since they are similar to each other. Figure 7.d, 7.e, and 7.f show CWT of the signal with
Morlet, Daubechies8 (db8) and Symlet6 (sym6) mother wavelets, respectively. It is
obvious in these figures that the computed CWT coefficients are similar to each other.
Moreover, these figures are quite complicated, and the effects of raw signal picks are not
obvious and cannot be analyzed easily. Therefore, the computed CWT coefficients are not
suitable features for data classification, since they are similar to each other and cannot
represent morphological differences very well. Hence, in this study, Haar mother wavelet
has been selected for feature extraction.
To compute the CWT of signals, it is not necessary to use scales in the range of 1 through
100. In view of the fact that computing CWT of signals in this range of scales will lead to a
huge volume of data as extracted features, it is not advisable to use it. Instead, a specific
range of scales, which is suitable and appropriate for feature extraction, is needed. The
following is an analysis to determine the appropriate range of scales for the current study.
Figure 8 shows 200 samples of a raw normal signal from record 208 from MIT-BIH database
and its CWT in different scales, with the Haar mother wavelet. In Figure 8.a, the raw
normal signal beat is shown. This signal has 3 picks, which are numbered on the figure;
these picks are related to P, R, and T waves. Figure 8.b shows CWT of the signal in scale a=5.
In this figure, the noise of the signal has been highlighted; however, the extent of noise is not
so large as to interfere with the performance of the neural classifier, and as a result, it is
possible to analyze the effect of noise of the raw signal. Moreover, the effect of picks number
1 and 3 can be analyzed to some extent. Figure 8.c shows CWT of the signal in scale a=10. In
this figure the effect of the three picks is fully observable and can be analyzed completely;
note that there is little noise in the figure. Figure 8.d, which shows CWT of the signal in scale
a=20, has no noise and only the effect of three picks can be analyzed according to it. Figures
8.e, 8.f, and 8.g show CWT of signal in scales a=50, 80 and 100, respectively. These figures
are similar to each other and neither the noise of the raw signal nor the effect of its picks can
be analyzed from these figures; therefore, these figures are not useful for the analysis. It is

Application of Computational Intelligence Techniques for Cardiovascular Diagnostics 221
obvious that morphological differences, which are useful and necessary for neural classifier
performance, have been eliminated in these figures. Hence, these extracted features are not
appropriate for the neural classifier.

Fig. 7. (a) Normal signal beat, (b) CWT of signal with Haar mother wavelet, (c) CWT of
signal with Mexican hat mother wavelet, (d) CWT of signal with Morlet mother wavelet,
(e) CWT of signal with db8 mother wavelet, (f) CWT of signal with sym6 mother wavelet.

The Cardiovascular System Physiology, Diagnostics and Clinical Implications 222

Fig. 8. (a) Raw normal signal beat, (b) CWT of signal in scale a=5, (c) CWT of signal in scale
a=10, (d) CWT of signal in scale a=20, (e) CWT of signal in scale a=50, (f) CWT of signal in
scale a=80, (g) CWT of signal in scale a=100.
From the above analysis, it is clear that computing CWT of the signals in the range of scales
from a=5 to 20 can lead to a complete and useful analysis. Since both noise of signals and the
effect of morphological differences can be analyzed in this range, the extracted features
would be useful for classification of the signals under study.

Application of Computational Intelligence Techniques for Cardiovascular Diagnostics 223
In this study and for the stage of feature extraction, scales in the range of a = 6 through 15
are used that lead to matrices with 10 X 150 dimension for each segment, where each row
includes the CWT coefficients in each scale. Using this range of scales has two advantages.
First, by computing CWT in the range of a = 6 through 9, the ECG signal can be analyzed in
detail. Second, by using the range of a = 10 through 15, the general morphology of the signal
and its differences with other types of ECG signals can be highlighted.
It should be noted that computing CWT of signals in ten scales can represent morphological
differences between several types of ECG signals better than computing CWT of signals in
one scale only because of the fact that the differences are analyzed 10 times. This would
hence be expected to result in a better performance of the neural classifier.
It would not be efficient to use a huge amount of data to perform a pattern recognition
process. Hence, in the final level of pre-processing of our algorithm, PCA is applied on the
computed matrices of wavelet coefficients, where each of them is a 10x150 matrixes,
resulting in 10 principal component (PC) vectors.
In this study and for the stage of dimensionality reduction, the first three PC vectors have been
selected and arranged as the neural network classifier input vector. This number of PC vectors
was chosen according to the results which are presented in Table 1. In this table the accuracy of
the neural network classifier with respect to the selected number of PC vectors is shown.
According to Table 1, the accuracy of the neural network classifier increases as the number of
selected PC vectors increases from 1 to 5, since, by increasing the size of data in this level and
this range, the classifier will have a more appropriate set of data for classification. The
accuracy of the neural network classifier decreases as the number of selected PC vectors
increases from 5 to 10, since at this level, the size of the data is too much for the classifier to
have a good performance. Since the difference between classification accuracy in the case of 3
PC vectors and 5 PC vectors is not that significant, we chose 3 PC vectors in order to have a
reasonable accuracy, while reducing the computational effort. As a result, by selecting only
three PC vectors, dimensionality reduction without significant loss of data information is
achieved, leading to a better performance of the neural classifier. These results, which are
based on a trial and error method, are not necessarily identical for all kinds of data and all
types of algorithm structures. For any change in the algorithm, this analysis should be carried
out again in order to find the appropriate number of PC vectors as a classifier input.
The prepared vectors, which are the principal components, are used as the neural network
classifier input vector. The analysis for providing the input vector structure is the same for
both the training and testing database.
Number of Selected PC
Vectors
Classification Accuracy (%)
1 98.41 %
2 98.83 %
3 99.17 %
5 99.28 %
8 98.53 %
10 98.94 %
Table 1. Variation of classification accuracy with respect to the number of selected PC
vectors

The Cardiovascular System Physiology, Diagnostics and Clinical Implications 224
2.3.2 Main process
After finishing the pre-processing stages, data is ready as the input vector for the neural
network classifier. In this study, a classical multi-layer perceptron neural network (MLPNN)
structure (Silipo and Marchesi 1998; Guler and Ubeyli 2005) is used as the neural network
classifier structure. This MLPNN is trained with the back propagation method of error.
Selection of the neural network inputs is the most important component of designing the
neural network based pattern classification since even the best classifier will perform poorly
if the inputs are not selected well (Guler and Ubeyli 2005). The inputs of neural network in
this study are constructed in the way which was described in previous section.
In our algorithm, we used a classical MLPNN structure with 2 hidden layers and with 60
nodes in the first hidden layer and 15 nodes in the second hidden layer for 160 iterations.
The structure of this MLPNN classifier with input, hidden, and output layers is shown in
Figure 9. For this structure, the training error was selected to be 0.01 in order to have precise
neural network training. From all 6 types of ECG beats under study and for neural network
training data, two segments have been selected and processed in the way that was described
in previous section.

Fig. 9. MLPNN structure used as the neural classifier
2.3.3 Classification
When the neural network has been trained, it is ready as a classifier to detect and classify
different types of ECG signals into one of six ECG beat groups under study. The classifier
has been tested by 100 segments from each group of ECG signals. These testing segments
are processed and prepared exactly like the input vector of the neural network; this means

Application of Computational Intelligence Techniques for Cardiovascular Diagnostics 225
that all four levels of pre-processing stage have been applied to each segment in order to
prepare it as a testing segment. These segments are used to test and evaluate the trained
neural network classifier.
2.4 Results
As stated earlier, the MIT-BIH arrhythmia database is used to evaluate the proposed
algorithm. To assess the accuracy of the classifier, sensitivity, positive predictive accuracy
and total accuracy have been calculated. These are defined as follows:



Here, TP is the number of true positive detections, FN stands for the number of false
negative detections, and FP stands for the number of false positive misdetections.
Table 2 shows the result of classification by the neural network. It can be seen from this table
that from the whole testing data base, the classification fails only in 5 cases. According to
this table, the algorithm achieves a good performance with 99.5 % Se, 99.66% PPA and
99.17% TA.
Normal
Atrial
premature
beats
Right
bundle
branch
block
Left
bundle
branch
block
Paced
Premature
ventricular
contraction
Sum
All 100 100 100 100 100 100 600
TP 100 99 99 98 100 99 595
FN 0 0 0 2 0 1 3
FP 0 1 1 0 0 0 2
Se (%) 100 100 100 98 100 99 99.5
PPA (%) 100 99 99 100 100 100 99.66
TA (%) 100 99 99 98 100 99 99.17
Table 2. Results of the algorithm on MIT- BIH database
A comprehensive comparison between results from different studies in the field of specified
ECG beat classification is very difficult since the database, signals under study, the number
of arrhythmias in classification, the algorithm structure, and the data processing methods
are not the same in the various studies. However, in order to present an estimate of the
performance of our algorithm and our classifier we show the results of this study versus the
reported results of other well-known studies in the area of selected heart arrhythmias
detection in Table 3. As seen from this table, the algorithm in the present study shows
) ( FN TP
TP
Se
+
=
) ( FP TP
TP
PPA
+
=
) ( FP FN TP
TP
TA
+ +
=

The Cardiovascular System Physiology, Diagnostics and Clinical Implications 226
reasonably accurate results, and compare favorably with other studies. The goal of this
study, which was classification of ECG beats and detection of heart arrhythmias, has clearly
been achieved.
TA (%) PPA (%) Se (%)
Silipo et al. 1998 85 % 77 %
Papaloukas et al. 2002 89 % 90%
Foo et al. 2002 92 % - -
Vikas et al. 2004 - - 98.02 %
Christov et al. 2004 - - 99.3 %
Guler et al. 2005 96.94 % - 96.37 %
Lee et al. 2005 - - 98.59 %
Kutlu et al. 2008 97.3 % - -
Cvikl et al. 2010 - - 92.36 %
This Study 99.17 % 99.66 % 99.5 %
Table 3. Comparison of several classifier performances on MIT-BIH database (Blank boxes
have not been reported
3. A Novel technique for identifying patients with ICU needs using
hemodynamic features
Modern ICUs are equipped with a large array of alarmed monitors and devices which are
used in an attempt to detect clinical changes at the earliest possible moment, so as to prevent
any further deterioration in a patients condition. The effectiveness of these systems
depends on the sensitivity and specificity of the alarms, as well as on the responses of the
ICU staff to the alarms. However, when large numbers of alarms are either technically false,
or true, but clinically irrelevant, response efficiency can be decreased, reducing the quality
of patient care and increased patient (and family) anxiety (Laramee, Lesperance et al. 2006).
Medical and technical progress has extended the therapeutic possibilities of ICUs
tremendously. A multitude of devices is available for monitoring and treatment in an
individual assembly according to the requirements of the situation (Friesdorf, Buss et al.
1999). Due to limited physiological monitoring and a patient's individual pathophysiology,
intensive care medicine has to cope with a high amount of uncertainty. Unusual
circumstances caused by patients, clinicians and technology occur frequently and must be
controlled and managed adequately to prevent a bad outcome and to achieve system
reliability (Friesdorf, Buss et al. 1999).
Cao et al. (Cao, Eshelman et al. 2008) have used ICU minute-by-minute heart rate (HR) and
invasive arterial blood pressure (BP) monitoring trend data collected from the MIMIC II
database to predict hemodynamic instability at least two hours before a major clinical
intervention. They derived additional physiological parameters of shock index, rate
pressure product, heart rate variability, and two measures of trending based on HR and BP
and they applied multi-variable logistic regression modeling to carry out classification and
implemented validation via bootstrapping, resulting in 75% sensitivity and 80% specificity.
Eshelman et al. (Eshelman, Lee et al. 2008) have developed an algorithm for identifying ICU
patients who are likely to become hemodynamically unstable. Their algorithm consists of a

Application of Computational Intelligence Techniques for Cardiovascular Diagnostics 227
set of rules that trigger alerts and uses data from multiple sources; it is often able to identify
unstable patients earlier and with more accuracy than alerts based on a single threshold. The
rules were generated using the machine learning techniques of support vector machines and
neural network, and were tested on retrospective data in the MIMIC II ICU database,
yielding a specificity of approximately 90% and a sensitivity of 60%.
Several investigations have been reported in the literature in the area of cardiovascular fault
diagnosis using hemodynamic features. Javorka et al. (Javorka, Lazarova et al. 2011)
compared heart rate and blood pressure variability among young patients with type I
diabetes mellitus (DM) and control subjects by using Poincare plots, which are the standard
tools of nonlinear dynamic analysis. They found significant reduction of all HRV Poincare
plot measure in patients with type I diabetes mellitus, indicating heart rate dysregulation.
The study carried out by Pagani et al. (Pagani, Somers et al. 1988) concerned patients
suffering from hypertension. They showed that baroreflex gain decreases with the presence
of hypertension. Blasi et al. (Blasi, Jo et al. 2003) studied the effects of arousal from sleep on
cardiovascular variability. They performed time-varying spectral analyses of heart rate
variability (HRV) and blood pressure variability (BPV) records during acoustically induced
arousals from sleep. They found that arousal-induced changes in parasympathetic activity
are strongly coupled to respiratory patterns, and that the sympathoexcitatory cardiovascular
effects of arousal are relatively long lasting and may accumulate if repetitive arousals occur
in close succession.
Advances in knowledge-based systems have also enhanced the functionality of intelligent
alarm systems and ICU needed patient detection. Using the knowledge of a domain expert
to formulate rules or an expertly classified data set to train an adaptive algorithm has
proven useful for intelligent processing of clinical alarms (Laramee, Lesperance et al. 2006).
Expert systems such as neural network (Westenskow, Orr et al. 1992), knowledge based
decision trees (Muller, Hasman et al. 1997; Tsien, Kohane et al. 2000) and neuro-fuzzy
systems (Becker, Thull et al. 1997) that encode the decisions of an expert clinician all show
significant statistical improvement in the classification of alarms and ICU needed patients.
Singh et al. (Singh and Guttag 2011) proposed a classification algorithm based on a decision
tree method for cardiovascular risk stratification. They have shown that the decision tree
method can improve performance of the classification algorithm. They have reported that
the decision tree models outperform the radial basis function (RBF) kernel-based support
vector machine (SVM) classifiers. Timms et al. (Timms, Gregory et al. 2011) have used a
Mock circulation loop for hemodynamic modeling of the cardiovascular system in order to
test cardiovascular devices, which are used in the ICU and can provide a better indication of
patients condition for nursing staff. Also, Laramee et al. (Laramee, Lesperance et al. 2006)
have described an integrated systems methodology to extract clinically relevant information
from physiological data. Such a method would aid significantly in the reduction of false
alarms and provide nursing staff with a more reliable indicator of patient condition.
Several studies have focused on an effort to find a suitable classifier structure. Ghorbanian
et al. (Ghorbanian, Jalali et al. 2011) proposed an algorithm based on a neural network
classifier for heart arrhythmias detection. Their results show that the multi-layer perceptron
neural network (MLPNN) structure is a strong and precise classifier. However, they used
several pre-processing techniques in their algorithm to improve the performance of the NN
classifier. Acharya et al. (Acharya, Bhat et al. 2003) proposed an algorithm based on a neural

The Cardiovascular System Physiology, Diagnostics and Clinical Implications 228
network classifier and fuzzy cluster for classification of heart arrhythmias. They compared
these two classifiers and they reported that the fuzzy cluster is a better classifier in
comparison with the neural one. Also, Ozbay et al. (Ozbay, Ceylan et al. 2006) proposed a
comparative study of the classification accuracy cardiovascular diseases using a well-known
neural network architecture, MLP structure, and a new FCNN for early diagnosis. Based on
their test results they suggested that a new proposed FCNN architecture can generalize
better than ordinary MLP architecture and also learn better and faster.
The method for classification of subjects into two categories of normal and abnormal
subjects, as described in this paper, is based on the hypothesis that there should be
differences between the hemodynamic data collected from normal subjects and abnormal
patients. This hypothesis is constructed on the same foundation as all developed scoring
methods for ICU patients. The idea behind all patient scoring methods in ICU is that
critically ill patients in ICU are typically characterized by disturbance of the bodys
homeostasis. These disturbances can be estimated by measuring to what extent one or many
physiologic variables differ from the normal range (Lacroix and Cotting 2005).
3.1 Methodology
While the proposed method in this paper shares some fundamental ideas with traditional
scoring methods, it differs from them in two key areas. The first difference comes from fact
that the patient scoring methods are based on the wide variety of data ranging from
cardiovascular and respiratory systems to neurologic and renal systems variables. However,
in our method we use a small subset of hemodynamic data, namely, HR and systolic blood
pressure (SBP). The principal objection to this could be that such a small amount of data
could be insufficient for identifying the patient state; the answer to this objection leads us to
the second major difference of the proposed method with the scoring methods. Scoring
methods just look at the data as they are being collected in the ICU, and ignore information
hidden in the different time scales. In our proposed method on the other hand, this hidden
information is extracted which can be expected to give us better insight into the patients
physiological condition.
The data used in this study is collected from the Physionet database. Data are collected from
two databases: MIT-BIH Polysmonographic and MIMIC II databases within Physionet
archive. Twenty five subjects from these databases were collected for training. For each
subject, ECG signal and blood pressure waveform, in a five-hour range of the total data
were collected. For the first part of the study, the HR and SBP series for each subject are
derived from ECG and arterial pressure waveforms respectively.
The algorithm of the developed method of this study is shown in Figure (10). According to
the proposed algorithm, in the first step and after collecting the data, four features which
highlight the differences between normal subjects and patients, are extracted from data. We
then define four criteria based on the extracted features. These four criteria which form the
basis of our classification algorithm are: circle criterion, estimation error criterion, Poincare
care plot deviation, and autonomic response delay criterion. In the next step and for the task
of classification, we define three groups; namely, healthy, high risk and patient. Then we
design three fuzzy membership functions for each criterion to find the subject degree of
membership to each group. Finally, a scoring method is developed based on the degree of
membership of each case, and subjects are classified based on this scoring method.

Application of Computational Intelligence Techniques for Cardiovascular Diagnostics 229
In the following sections, we provide a step by step description of our method, beginning
with the definition of the proposed criteria.

Fig. 10. Schematic of the proposed algorithm. The proposed algorithm consists of two
stages: training and testing. In the training stage 25 subjects data are used to extract features
to classify patients from healthy subjects. In the test stage subjects will be divided into three
predefined groups of healthy, high risk and patient, based on their assigned score.
3.1.1 Circle criterion
To evaluate the differences between healthy and patients, the SBP against HR diagram for
each subject is plotted. Figure 11 shows these plots for healthy and patient cases. Clearly, the

The Cardiovascular System Physiology, Diagnostics and Clinical Implications 230
plots show a significant difference between normal subjects and abnormal patients: the data
for normal subjects are concentrated, while those of the patients are scattered.
The mean value of SBP and HR for each normal subject and abnormal patient is then
calculated and plotted in one diagram. Figure 12 shows the mean values for all the subjects
in one diagram. The principal difference between the two groups is quite clear. This

Fig. 11. SBP against HR for a healthy (left) and an abnormal (right) case

Fig. 12. Mean values of SBP versus HR for all subjects
diagram reveals the fact that there are differences between the HR and SBP data in normal
subjects and abnormal ones. The plot shows that the data for the normal subjects is clustered
and limited in a specific area, while those of the patients are spread out through the whole
plot. The first criterion is named the "circle criterion". The center of the circle is located at

Application of Computational Intelligence Techniques for Cardiovascular Diagnostics 231
point "O" where its coordinates are the mean values of HR and SBP of normal patients and,
in this case, is (83, 120). The radius of this circle is calculated based on Euclidian distance
between the center and the outer limit of the circle.
A given subject would be considered to be a patient if its corresponding means (HR, SBP)
point is out of the healthy subject's circle (the limited area).
3.1.2 Estimation error criterion
As the second feature, a system identification method is used for the prediction of the next
HR based on the current and previous HR and SBP data. A Nonlinear ARX or NARX model
is employed to estimate HR series (Jalali, Ghaffari et al. 2011). NARX models in general are
represented by the following equation:
y(t) = F(y(t 1), y(t 2), , y(t n
a
), u(t n
k
), , u(t n
k
n
b
+ 1))
where, y(t) and u(t) are the output and input of the system, respectively. In Eq. (1) the
matrix |n
a
n
b
n
k
] is the same as the order of the model. Model order is selected by use of
the A-Information Criterion (AIC) method. This is the traditional method for model order
selection in cardiovascular system identification research. Model order for data in this
research has been calculated to be |
9 6 S
].
In this criterion, Artificial Neuro Fuzzy Inference System (ANFIS) structure is employed for
the identification. The model has 15 inputs and one output. Membership functions for inputs
are designed based on physiological facts. Since the nervous system consists of sympathetic
and parasympathetic nerves, for each input, two generalized bell-shaped membership
functions are assigned to designate the sympathetic and parasympathetic functions.
The system identification results are described in Table 4. The results in this table show that
differences exist in the normalized root mean square error (NRMSE) with respect to the
estimation of the HR for the two groups under study. In particular, the results indicate that
NRMSE is smaller for normal subjects than for patients. These differences are due to the fact
that the model is designed for normal subjects; thus, the output of the model for patients
have higher errors than for normal subjects.
Group Mean Max Min
Normal 0.193 0.238 0.119
abnormal 0.367 0.473 0.263
Table 4. Error estimation for identification of HR baroreflex
Based on these results and noting that the maximum error for healthy subject is 0.238, while
the minimum error for patient is 0.263, we define a second criterion called "estimation error
criterion". According to this criterion, the subject would be flagged as abnormal if the
calculated error in HR estimation raise is more than 0.25.
3.1.3 Poincare plot deviation
A Poincare plot, named after Henri Poincare, is used to quantify self-similarity in processes
which are usually characterized by periodic functions. This plot is commonly used in heart

The Cardiovascular System Physiology, Diagnostics and Clinical Implications 232
rate variability (HRV) analysis. The Poincare plot is a graph in which each heart rate episode
is plotted as a function of previous HR, and then the line y = x is fitted to the data. In
(Javorka, Lazarova et al. 2011) this method is also applied to classify patients with type I DM
from healthy subjects. Drawing the Poincare plot for healthy and abnormal subjects, it is
found that the deviation from the mentioned line in healthy subjects is less than in abnormal
subjects. These plots are shown in Figure 13.
The deviation from the line y=x in the Poincare plot for the two groups under study is
shown in Table 5. Therefore, we define the third criterion using this deviation to
characterize abnormality. Based on this criterion, subjects would be called abnormal If
deviation from line y=x is more than 15%.
Group Mean Max
Healthy 8% 13%
Patient 19% 24%
Table 5. Deviation from line y=x in Poincare plot


Fig. 13. Poincare plots of HR for two healthy (up) and two abnormal (down) cases. The
Poincare plot is a plot of HR(n+1) vs. HR(n). Line y=x is illustrated in all pictures.
3.1.4 Autonomic response delay criterion
The normally occurring delay in the autonomic response to a stimulus has its origins in the
parasympathetic nervous system. Calculating the delay for healthy subjects and patients we

Application of Computational Intelligence Techniques for Cardiovascular Diagnostics 233
can infer that response delays in abnormal subjects are remarkably higher than healthy
subjects. The results of calculating the delay in the autonomic response are shown in Figure
14. Fifteen abnormal patients and ten healthy subjects were involved in the training group.

Fig. 14. Delay in autonomic response
The results of the delay calculations in the autonomic response are also represented in Table
6. Based on the above results, we define the fourth criterion where the subject is
characterized as abnormal if the calculated delay in the autonomic response increases to
more than 0.021 second.
Group Mean Delay (sec) Max Delay (sec)
Healthy 0.015 0.02
Patient 0.038 0.06
Table 6. delay in autonomic response for two groups
After deriving the four criteria discussed above, an algorithm is designed to classify healthy
subjects from patients. In the following section we describe the proposed algorithm.
3.2 Scoring method and classification algorithm
Based on the evaluated criteria from training data, an algorithm is developed to
automatically distinguish patients from healthy subjects. The algorithm is based on a fuzzy
decision making method. First, for each criterion, three Gaussian bell membership functions
are designed as an indicator of three major groups: healthy, high risk and patient. Since this
algorithm is designed for clinical use and since there exists a high degree of uncertainty in
clinical applications, we added the high risk groups to our predefined healthy and patient
groups to account the cases that do not completely belong to the healthy or patient groups.
For the training part we first made a general guess for the shape of the membership
functions. The membership functions during the training round then adapt their shape
parameters to the incoming data for best classification performance. Now the classifier is

The Cardiovascular System Physiology, Diagnostics and Clinical Implications 234
designed and ready for the testing stage. Figure 15 represents the adapted membership
functions for each criterion based on the training data.
To test the developed algorithm, in the first step for each subject, all the mentioned features
that form the basis of four criteria are extracted and used as an input for the four
abnormality criteria. Then, for each criterion, the subject's degree of membership to all
groups is evaluated. In this step, for each subject, we have 12 degrees of membership to the
designed three groups, meaning four degrees of membership for each group. After
evaluating the degree of memberships, the cumulative sum of the four degrees of
membership of each group will be calculated. In this stage we have three numbers
indicating subjects degree of membership to each group. We call these numbers the
subjects score for each group. A given subject will belong to the group whose score is the
largest.


Fig. 15. The designed membership functions for each criterion
3.3 Results
From a total of seventy subject data which were collected from MIMIC II database, the
algorithm was first trained with twenty five subjects including ten healthy and fifteen
patients. The training data was selected randomly to avoid bias toward a specific disease.
Then, three groups of subjects were tested, each group with four healthy individuals and
eleven patients.

Application of Computational Intelligence Techniques for Cardiovascular Diagnostics 235
The proposed method was applied to 45 cases from Physionet database, containing 12
healthy subjects and 33 patients. From all cases, 37 cases were accurately detected, while
there was one false detection. Furthermore, in five cases, a patient subject was classified as
high risk and, in two cases, a healthy subject was classified as high risk.
Here, TP is the number of true positive detections, FN stands for the number of false
negative detections, and FP stands for the number of false positive misdetections. Table (7)
shows the overall result of the classification for all 45 cases of the 3 groups. The FP is the
healthy subject who is misclassified as a high risk subject and FN is the patient who is
misclassified as a high risk subject. According to this table, the scoring method of the
proposed algorithm results in 86% sensitivity, 94.8% positive predictive accuracy and 82.2%
total accuracy.

Group I II III All
All 15 15 15 45
TP 12 13 12 37
FN 3 1 2 6
FP 0 1 1 2
Se (%) 80 92.8 85.7 86
PPA (%) 100 92.8 92..3 94.8
TA (%) 80 86 80 82.2
Table 7. Results of testing the algorithm on Physionet database
A comprehensive comparison between the results of different studies in the field of
identifying ICU needed patients by the use of hemodynamic features is very difficult since
the database, signals under study, the algorithm structure, and the data processing methods
are not the same in the various studies. However, in order to present an estimate of the
performance of our algorithm and our classifier we show the results of this study versus the
reported results of two other well-known studies in the area of ICU needed patients
identifying in Table 8. As seen from this table, the algorithm in the present study shows
reasonably accurate results, and compares favorably with other studies. The goal of this
study, which was identifying patients with ICU needs by use of the hemodynamic features,
has clearly been achieved.
Study Se (%)
Cao et al. [1] 75
Eshelman et al. [4] 60
This study 86
Table 8. Comparison of several classifier performances on MIMIC II ICU database (Blank
boxes have not been reported)
4. Conclusion
Physiological time series, including hemodynamic and electrophysiological data clearly
represent the physiological state of subjects in a medical environment. Automatic detection
of heart arrhythmias could be very important in clinical usage and lead to early detection of

The Cardiovascular System Physiology, Diagnostics and Clinical Implications 236
a fairly common malady and could help contribute to reduced mortality as cardiovascular
disease remains the leading cause of death around the world. Hemodynamic instability is
most commonly associated with abnormal or unstable blood pressure (BP), especially
hypotension, or is more broadly associated with inadequate global or regional perfusion.
Inadequate perfusion may compromise important organs, such as heart and brain, due to
limits on coronary and cerebral autoregulation and cause life-threatening illnesses or even
death. Therefore, it is crucial to identify patients who are likely to become hemodynamically
unstable for an early detection and treatment of these life-threatening conditions.
In the first example of this study, the use of neural networks for classification of the ECG
beats is presented. Several stages of pre-processing have been used in order to prepare the
most appropriate input vector for the neural classifier. ECG signal baseline wandering is one
of the most critical problems for neural classifiers, since it causes virtual morphological
differences between same types of ECG beats. In this example, this wandering is removed
by application of a signal filtering method which leads to better results. As the performance
of the computerized ECG classification algorithms depends on the selection of the ECG
features, continuous wavelet transform, which performs better than other methods, is used
to extract appropriate features. Also, data dimensionality reduction is one of the most
important ways of improving neural classification, since large volume of data causes
problems for neural network classifier performance, and reduction in the data size is
necessary for better performance of the classifier. Therefore, principal component analysis is
used to achieve dimensionality reduction. Results show that PCA is more effective than
other reported methods. The performance of the proposed algorithm has been shown to be
reasonably acceptable and ECG beat detection and classification has been achieved.
Compared to other reported work in this field, the presented algorithm shows reasonably
accurate results in the field of heart arrhythmia detection.
The main advantage of this example is that, by using ten scales in computing CWT of
signals, the morphological differences between several types of ECG signal are highlighted
and the extracted features show the differences more clearly. Another advantage of this
example is that the reduction of the dimension of data by applying PCA led to the most
appropriate input vector for neural network classifier which improved the performance of
the neural network classifier significantly. The main achievement of this algorithm is that
the classifier in this example detects 6 types of ECG signals which include normal beats and
5 types of arrhythmia beats. Even though the number of ECG signal types considered in this
example is much larger than the typical number of ECG signal types in other studies in this
field, the classification results lead to a reasonably good performance.
In the second example of this study, a scoring method based on fuzzy logic and feature
extraction is proposed to distinguish patients from healthy subjects. The method is based on
the same principle that the ICU scoring methods follow: that of finding differences between
hemodynamic data of healthy subjects and patients. Four different criteria are proposed to
detect and identify patients from a group of subjects. For each criterion a fuzzy classifier is
designed such that the individuals are classified into the healthy, high risk and patient fuzzy
groups. In other words, a given person may have a membership grade in all three classes. A
score is assigned to the subject for that group which is defined as the sum of degree of
memberships to one group for different criteria. The algorithm calculates a combined

Application of Computational Intelligence Techniques for Cardiovascular Diagnostics 237
criterion based on the results of the four criteria to arrive at a classification decision for each
individual.
It is shown that the algorithm is highly reliable and has been able to detect correctly all
members of the first group. It is also been able to detect all eleven patients in each of the
next two groups correctly. Only one of the healthy members in the second and third was
classified as high risk. In this example, four different criteria were proposed and used in the
proposed algorithm in order to detect the abnormalities in testing subjects. From each
testing subject, various features were extracted and used as input for the criteria, and based
on the results of all four criteria, a decision was made about the type of subject, as to
whether he/she is normal, high risk or a patient. The proposed algorithm gave reliable
results in detecting the ICU needed patients but still needs to be improved. The difference
between the proposed method in this example and other similar research in this field of
study is that by using the presented algorithm in this example, existence of any abnormality
in a patient will be found, while in most similar studies in this area, a specific abnormality is
found in a patient or among a database of subjects. Therefore, our results are more general
and more useful from the point of view of clinical applications. This method tends to be
more detective rather than predictive, and this could be one drawback of the algorithm.
Further investigations need to be carried out to render the algorithm more predictive.
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12
Analysis of Time Course Changes
in the Cardiovascular Response
to Head-Up Tilt in Fighter Pilots
David G. Newman
1
and Robin Callister
2

1
Aviation Discipline, Faculty of Engineering and Industrial Sciences,
Swinburne University, Melbourne,
2
Human Performance Laboratory, Faculty of Health,
University of Newcastle,
Australia
1. Introduction
Fighter pilots are exposed to significant levels of +Gz acceleration on a frequent
occupational basis (Newman & Callister, 1999). There is an emerging body of experimental
research that suggests that they physiologically adapt to this frequent +Gz exposure
(Convertino, 1998; Newman & Callister, 2008, 2009; Newman et al, 1998, 2000). Our previous
work has shown that fighter pilots are able to maintain their cardiovascular function to a
much greater extent than non-pilots when exposed to an orthostatic stimulus such as head-
up tilt (Newman & Callister, 2008, 2009; Newman et al, 1998, 2000).
To further examine the mechanisms underlying these differences in cardiovascular response
to +Gz, a beat-to-beat analysis of the time course of dynamic cardiovascular responses to
head-up tilt (HUT) was conducted. The hypothesis was that the time course of acute
changes in mean arterial pressure (MAP), heart rate (HR), stroke volume (SV) and total
peripheral resistance (TPR) in +Gz-adapted fighter pilots would be different from that of
non-pilots. Such differences would provide further evidence of cardiovascular adaptation to
repetitive high +Gz exposure, and help to further our understanding of how this adaptation
is mediated.
2. Methods
The subjects were 20 male volunteers drawn from personnel of Royal Australian Air Force
(RAAF) Base Williamtown. No female subjects were recruited as the RAAF did not have any
female fighter pilots at the time of the study. The control group consisted of 12 non-pilots
(NP). The second group consisted of 8 current operational jet fighter pilots (FP) from RAAF
Base Williamtown.
The two groups were closely matched in terms of age, height, weight, aerobic fitness level,
resting blood pressure and heart rate (Newman et al, 1998, 2000). All subjects gave their

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242
written informed consent before being tested. The study was approved by both the
Australian Defence Medical Ethics Committee and the Human Research Ethics Committee
of the University of Newcastle. All subjects were asked to refrain from eating for 2 hours
and from drinking caffeinated beverages for 4 hours prior to the test for standardisation
purposes. Subjects were assigned an alpha-numeric code to maintain confidentiality.
Each subject was non-invasively instrumented for the beat-to-beat measurement of stroke
volume via impedance cardiography. Four impedance cardiograph metallic band electrodes
were applied to the thorax of the subject in the manner described previously (Newman et al,
1998, 2000). The leads were then attached to the impedance cardiography unit
(Instrumentation for Medicine, Model 400, Greenwich, CT). Heart rate was determined via
an electrocardiogram (ECG) signal generated by the impedance cardiography unit.
Data from the impedance cardiograph and other recording instruments were stored on
video tape via a digital video cassette recorder (Vetter, Model 4000A, Rebersburg, PA). The
video tape data were analysed using a MacLab/8s 8-channel digital chart recorder and
analysis system (ADInstruments, Model ML 780, Castle Hill, Australia). MacLab Chart
software (ADInstruments, Version 3.5.2/s, Castle Hill, Australia) was used to capture and
analyse the digital video data.
Four cardiovascular parameters were examined in this analysis: MAP, HR, SV and TPR. MAP
was calculated according to the formula MAP = DP + 1/3 (SP-DP). SV was determined using
the Kubicek equation (Newman et al, 1998, 2000). TPR was calculated as MAP/(HR x SV).
The data were divided into Control (C), Anticipation (A) and Tilt (T) periods. C consisted of
data from the beginning of recording until the start of A, which was defined as the 5 heart
beats immediately prior to the tilting event. T consisted of the 30 heart beats from the onset
of tilt. For the purposes of tracking changes across time, and for ease of description, the T
period data were divided into 6 phases (I-VI) consisting of 5 heart beats each. The transition
from the supine to the full +75
0
head-up tilt position occurred during Phase I.
Analysis of the data was performed using a statistical software package (SuperANOVA,
Abacus Concepts, Inc., v1.1). Repeated measures analysis of variance with one within factor
(time) and one between factor (group) was used as the test of statistical significance. An
alpha level of p<0.05 was considered significant at the 95% confidence interval for all effects.
3. Results
Figure 1 shows the mean T period values (+ SEM) on a beat-to-beat basis for each of the four
variables for both experimental groups. The mean values (+ SEM) for each groups C and A
periods are shown as the first two data points. The data are divided into phases for ease of
reference during description.
3.1 Responses to tilt
The NP data show an early rise (Phase I) in MAP, which then decreases to values
significantly below control levels in Phase III. MAP then progressively rises to levels slightly
above but not significantly different from C during the late part of the tilt (Phases IV to VI).
In the FP group, MAP also rose initially during Phase I and decreases towards C values in

Analysis of Time Course Changes in the Cardiovascular Response to Head-Up Tilt in Fighter Pilots

243

Fig. 1. Comparison of the time course of the non-pilot (left columns) and fighter pilot
responses to 75
0
HUT across time. The first two data points on each plot are C and A values.
The bracketed areas on each curve represent areas of significant difference (p<0.05) from C.
Phase II. Phase III of the FP MAP response is clearly different from that of the NP group,
with MAP plateauing and never falling below the C level. Early in Phase IV, MAP rises
progressively, reaching values in Phases V and VI significantly greater than the C level.
HR is elevated significantly above C in both groups within four heartbeats of tilt. In NP, HR
rises immediately during Phase I, is sustained at this level during Phase II and then
progressively decreases slowly towards C levels in Phases III to VI. HR is significantly
different from C for most of the tilt period. In FP, HR also increases in Phase I, begins to
decrease in the early part of Phase II but then increases again by the end of Phase II, and
remains significantly elevated throughout Phases III and IV, reaching maximum elevation at
the junction of Phases IV and V, then begins to decrease back towards C values.
In NP, SV falls precipitously at the onset of tilt, then increases slightly during the later part
of Phase II and the early part of Phase III. SV then progressively decreases again, although at
a slower rate in Phases III to VI. SV in the FP group falls in Phase I, but not as immediately
or to the same extent as the NP group. It recovers a little in Phase II, then progressively
decreases in later phases. Like the NP group, this late-phase decrease occurs at a slower rate
than in Phase I.

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In NP, TPR increases initially during Phase I, then decreases to levels below C values by the
middle of Phase III. Phases IV to VI are marked by progressive increases in TPR to values
significantly above C values by Phase VI. In FP, TPR rises during Phase I, then decreases
below C values during Phase II. Phase III is marked by a small recovery in TPR, which is not
evident in the same phase in the NP group. TPR increases throughout Phases IV to VI,
becoming significantly different from C values earlier than in the NP group.
3.2 Group comparison
Figure 2 plots the T deviation from C values for each of the four cardiovascular variables,
again divided into the same 6 phases. The analysis was performed on individual data
points, although these and the error bars have been removed from the figure, purely for ease
of visualising the comparison between the groups across time. This series of curves
demonstrates the relative contribution of these variables to the observed time-based changes
in cardiovascular dynamics.
The MAP curves show a similar overall pattern of response to tilt, although a significantly
greater response is seen in the FP group to the same gravitational stimulus (p<0.05). The FP
group maintains MAP above C values at all times, and in the second half of tilt MAP values
are significantly higher than those of the NP group.
The HR curves are similar for each group, although in the later phases the group responses
tend to diverge, with the FP group demonstrating a more sustained elevation. In this group,
HR is maintained at its peak level until the early part of Phase V, when it begins to decrease.
In the NP group HR begins to decrease in Phase II, although it remains elevated above
control levels throughout tilt.
There is no statistically significant difference in the SV response to tilt of the two groups,
although the initial rate and magnitude of decrease in SV appears less in the FP group.
The TPR curves show similar patterns, rising initially, then decreasing and rising again in
Phase V in both groups. The FP group shows a more marked late-phase rise in TPR, which is
of greater magnitude than that in the NP group, and coincides with the FPs fall in HR
during Phase V. This rise in TPR becomes significantly different (p<0.05) from C values
earlier in the FP group.
4. Discussion
The results of this analysis show similar overall patterns of response between the two
groups. There are some key differences, however, in terms of the timing and magnitude of
the responses. These are just sufficiently different that they produce statistically significant
and physiologically meaningful differences in the MAP response between the two groups.
The NP response to HUT is the normal, well-documented human response to upright
posture. On assuming the upright position, there is an initial, transient HR- and TPR-
mediated rise in MAP, then both MAP and venous return fall in accordance with the
applied hydrostatic force. The fall in these parameters activates the baroreceptors, both the
high-pressure arterial baroreceptors and the low-pressure cardiopulmonary baroreceptors.
This leads to activation of these negative feedback regulating systems and a subsequent
restoration of MAP and venous return towards normal levels (Mancia & Mark, 1983).

Analysis of Time Course Changes in the Cardiovascular Response to Head-Up Tilt in Fighter Pilots

245

Fig. 2. Comparison of the change from control values across time of the non-pilots (thin line)
and fighter pilots (thick line) in response to 75
0
HUT. Data are mean values. SEM bars have
not been drawn. The time course has been divided into 6 phases of 5 beats each, labelled I to
VI (details in text).
The FP response is an adapted or modified version of the NP response. Analysis of the
different phases of the groups responses to tilt provides important information as to the
mechanisms that are active during the sequence of events. The focus of this discussion will
be on the integration of cardiovascular control inputs.
4.1 Cardiovascular regulation
There are four possible inputs used in the regulation of the cardiovascular system under
conditions of orthostatic stress such as HUT. Firstly, there may well be some cognitive or
psychological input to the autonomic nervous system at the onset of a rapid tilt or postural
change, and in anticipation of this impending event. This heightened sense of arousal or

The Cardiovascular System Physiology, Diagnostics and Clinical Implications

246
alerting reaction would produce an increase in HR, vasodilation in some vascular beds (e.g.,
skeletal muscle) and vasoconstriction in others (e.g., gastrointestinal tract and kidneys). The
rapid, almost immediate increase in HR (due to parasympathetic withdrawal) will shorten
cardiac ejection time, which will in turn contribute to a fall in SV. These changes reflect an
overall shift in the autonomic balance in favour of the sympathetic system. The net effect is
an increase in arterial pressure (Mancia & Mark, 1983).
The arterial baroreceptors also have a well established influence on the cardiovascular
system under orthostatic stress (Mancia & Mark, 1983). The overall effect is also a shift in the
autonomic balance, with the sympathetic system becoming more dominant. HR increases
due to parasympathetic withdrawal, while cardiac contractility and total peripheral
resistance both increase due to greater sympathetic drive. A more forceful, rapid ejection of
blood with higher vascular resistance results in an overall boost in mean arterial pressure.
The time taken for cardiac contractility and vascular resistance to increase is much longer
than that for HR, due to these sympathetically-innervated tissues taking longer to respond
to neural command signals.
During HUT the aortic and carotid baroreceptors will be stimulated to different extents,
based on their respective distances from the heart. In this experiment, arterial pressure was
recorded effectively at aortic level, and as such does not reflect the changes occurring at the
level of the carotid baroreceptors. HUT to +75
0
would lead to a decrease in carotid
distending pressure providing a stimulus for cardiovascular compensation to drive up mean
arterial pressure.
The third input source is from the cardiopulmonary baroreceptors, on the low-pressure side
of the circulation. Changes in hydrostatic force will affect not only the arterial baroreflexes
but also the cardiopulmonary reflexes. On standing (i.e., on exposure to the +Gz axis)
central venous pressure, venous return, stroke volume and cardiac output all decrease. The
drop in central venous pressure and venous return leads to activation of the
cardiopulmonary baroreflexes, and subsequent reflex increases in HR and TPR. Again, HR
changes will be rapid (within 1 to 2 seconds) while vascular resistance changes will take
several seconds to become evident after the stimulus.
Fourthly, the vestibular system may also be involved in regulation of the cardiovascular
system via the vestibulosympathetic reflex (Doba & Reis, 1974; Essandoh & Duprez, 1998;
Ray et al, 1997; Shortt & Ray, 1997; Yates, 1992; Yates & Miller, 1998). The vestibular system
will signal the dynamic postural change taking place, which may be supplemented by
ocular inputs (the vestibulo-ocular reflex). The state of the cardiovascular system may then
be altered by the action of the VSR, which may provide feed-forward adjustment of arterial
pressure during dynamic postural change.
The efferent output of the vestibulosympathetic reflex will be reflected in changes in
vascular resistance, based on experimental findings in animals and humans (Doba & Reis,
1974; Essandoh & Duprez, 1998; Ray et al, 1997; Shortt & Ray, 1997; Yates, 1992; Yates &
Miller, 1998). The time course of changes in vascular resistance will be in the order of several
seconds. A change in HR is not likely, given that this has not been reported as a feature of
VSR activity.

Analysis of Time Course Changes in the Cardiovascular Response to Head-Up Tilt in Fighter Pilots

247
4.2 Experimental findings
The phases seen in Figures 1 and 2 are in 5-beat intervals, which amount to approximately 4
to 6 seconds. Due to the inherent time lags in the tissue response to efferent signals of the
neural control mechanisms responsible for cardiovascular regulation, the effect in a
particular phase is generally a response to a stimulus that occurred in the previous one to
two phases.
4.2.1 Anticipation period
During the 5-beat anticipation period, there may be changes occurring in the cardiovascular
system due to an alerting response to the impending postural challenge. These changes will
result in an increase in HR and changes in regional vascular resistance. While the HR
change will occur rapidly, the changes in vascular resistance will take longer to develop. As
such, changes in TPR due to arousal prior to tilt are likely to be seen in the tilt period phases
rather than within the anticipation period itself.
4.2.2 Phase I
Phase I coincides with the dynamic phase of tilt, in which the postural change is made from
0
0
to +75
0
. During this phase MAP rises almost immediately in both groups, and reaches a
maximum at the conclusion of this phase. This rise in MAP is due to observed increases in
both HR and TPR, since SV falls immediately in both groups during this phase.
Which of the four control inputs discussed above is responsible for driving the increase in
HR during Phase I? An increase in arousal at the onset of HUT could account for this
observed increase in HR, given that the temporal characteristics of this increase closely
mirror the time taken to achieve the full HUT position (approximately 4 seconds). The HR
changes seen in this early phase of HUT may be due to these arousal effects alone, and
mediated by withdrawal of parasympathetic control. The fact that the FP group
experienced a smaller increase in HR during Phase I could reflect a lower level of
psychological arousal than in the NP group, due to the formers frequent exposure to a
dynamic motion environment. This is supported by the FP group having little
anticipatory rise in HR compared with the NP group, whose HR increased in anticipation
of impending tilt.
The change in HR could be due to the action of the arterial or cardiopulmonary baroreflexes.
However, these reflex arcs must be stimulated first, and as such some postural change must
take place before baroreflex-mediated increases in HR occur. There is not likely to be a
stimulus to the high- or low-pressure receptors until at least midway through this phase.
Baroreflex-mediated HR increases are thus unlikely to be seen until the end of Phase I. HR
increases immediately in both groups, well before the full head-up tilt position is reached,
which suggests that other inputs such as arousal are responsible for the early Phase I HR
increases.
Since there is no established connection between vestibular control of the cardiovascular
system and HR changes, the action of the VSR is not likely to be responsible for the increase
in HR.

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248
The increase in TPR in this phase is interesting, given that changes in vascular resistance
take time to occur after the initiating stimulus. The stimulus for this increase must be
something that occurred prior to tilt, such as the alerting response to impending postural
change.
This increase in TPR in both groups during Phase I is important, as it combines with the HR
increase to boost MAP. There are several speculative explanations for this phenomenon. The
first reflects the changes in vascular resistance effected by the increase in arousal during the
anticipation period. Since these changes take time to develop, they may not be evident until
Phase I. Vasoconstriction of some regional vascular beds (such as renal and splanchnic
regions) occurs as a consequence of increased arousal. Due to the low level of skeletal
muscle vasoconstrictor drive in the horizontal resting position of the anticipation period,
there is likely to be little additional vasodilation occurring in these vascular beds as a result
of arousal. The net result of these changes would be an increase in TPR due to the
anticipatory stimulus, which is seen in Phase I.
The second explanation involves the vestibular system and its influence on the
cardiovascular response to HUT. The activation of a vestibulosympathetic reflex due to the
dynamic postural changes as HUT proceeds may facilitate the observed increases in TPR
during Phase I. The vestibular system is in effect responding in a dynamic fashion to the
postural change stimulus. The time course of this phenomenon is in accord with
experimental findings that vestibular stimulation can evoke sympathetic discharges within
100 milliseconds (Yates, 1992). However, the response of vascular smooth muscle will take
longer to occur, and changes in resistance values will take longer again (in the order of
several seconds). The vestibular system could initiate vascular resistance changes, but these
would probably not occur until late in Phase I at the earliest.
The third possible explanation may be a mechanical feature of the blood vessels themselves.
As HUT proceeds, the hydrostatic force will progressively dump more blood into the
dependent lower limb vessels. This sudden increase in vascular volume as HUT occurs may
initiate a smooth muscle reflex in the blood vessels, in keeping with the length-tension
relationship of muscle. Such a short-lived response may lead to the transient increase in TPR
seen during Phase I.
The postural changes in Phase I will eventually lead to stimulation of the arterial and
cardiopulmonary baroreceptors, particularly late in Phase I when the full HUT position is
reached. However, the time interval involved during Phase I is too short for arterial and
cardiopulmonary baroreceptor activity to have much effect in this phase. Efferent output
from these baroreflexes will be seen in later phases.
What is responsible for the precipitous fall in SV during Phase I? In the NP group, SV falls in
the anticipation period, reflecting a shortened ejection time as a consequence of increased
HR. HR continues to increase throughout Phase I, which will exacerbate the fall in SV. As
the tilt progresses, more hydrostatic force is generated. This is unlikely to be a significant
input to the cardiovascular system until the second half of Phase I, and it is only at the end
of the phase that it becomes maximal, once the full HUT position is achieved. The dramatic
falls in SV observed in Phase I are thus due to the combination of HR changes due to the
arousal effects from the anticipation period (early in Phase I) and progressive increases in
hydrostatic force reducing venous return.

Analysis of Time Course Changes in the Cardiovascular Response to Head-Up Tilt in Fighter Pilots

249
SV falls less in the FP group during Phase I than it does in the NP group. As a result, MAP
reaches a higher peak value for the same effective increase in TPR as the NP group, while
the increase in HR is slightly slower. What could account for this better SV performance in
the FP group? There are two possibilities. The FP group did not have a significant fall in SV
or a rise in HR during the anticipation period. As a group they begin Phase I in a better
cardiovascular state. This would help defend SV against further falls due to a developing
hydrostatic force. Another explanation may be an expanded circulating blood volume in the
FP group. An expanded blood volume would also help to preserve SV in the face of an
orthostatic challenge. There is emerging evidence that such blood volume expansion does
occur in +Gz-trained individuals (Convertino, 1998). In the FP group, the important effect of
even slightly improved SV performance is a greater value of MAP during this early dynamic
postural change.
Therefore, it appears that the changes in HR and TPR seen in Phase I are due to the effects of
a prior alerting reaction in anticipation of an impending postural change. Although the FP
group has less HR rise during this phase, it is able to generate a higher level of MAP due to
enhanced SV performance.
4.2.3 Phases II and III
Phase II begins with the full HUT position having been achieved. Phases II and III are
marked by the progressive effects of the hydrostatic force on the cardiovascular system, and
the systems attempts to compensate for these effects.
MAP falls in both groups from the peak value in Phase I towards C values. In the NP group
it falls well below the C value, reaching a minimum in the late part of Phase III. In contrast,
the FP response to tilt in these phases is clearly different from that of the NP group. MAP
plateaus, and remains at or slightly above C levels during both phases. The difference in
MAP response in Phase III is the most striking and fundamental difference between the
responses of the two groups. During Phases II and III, the two groups MAP responses
diverge considerably from each other, whereas in Phase I they tracked relatively closely.
What is driving MAP down during these two phases? Heart rates in both groups during
Phase II are similar, remaining at the elevated levels achieved in Phase I for most of Phase II.
HR then tends to decrease during Phase III in the NP group, but increases slightly in the FP
group during this phase. If the Phase I rise in HR was due to the autonomic effect of
increased psychological arousal, the fact that HR tends to remain at the same elevated level
during Phase II in both groups suggests that the arousal effect cannot increase HR any
further. This is especially true given that arousal levels tend to be higher in the upright
position compared with the supine or prone positions. HR presumably decreases towards C
values in the NP group due to arousal no longer being the dominant stimulus to the
cardiovascular system. The FP group, however, goes on to a further sustained HR increase
during Phase III. What is responsible for this rise, which is quite different from the NP
response? Further increases in HR may be due to the developing action of the arterial and
cardiopulmonary baroreflexes, as a result of the ongoing effect of hydrostatic pressure. The
fact that this occurs in the FP group and not in the NP group may well reflect a difference in
the operating characteristics of the baroreflex in the FP group. This would suggest an
enhanced level of baroreflex activity on modulation of HR.

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SV continues to decrease in both groups during Phases II and III, despite a transient
recovery in SV which occurs at a similar point in both groups, around the junction of Phases
II and III. This temporary increase in SV may well reflect an increase in cardiac contractility,
as a countermeasure against the orthostatic challenge of HUT. There is little difference in
either the time course or magnitude of this contractility change between groups. This
increase in contractility is mediated by the baroreflexes (arterial and cardiopulmonary).
Assuming that the stimulus for this is the consequence of the full HUT position, the time
course for this contractility increase would fit with the operating characteristics of cardiac
tissue. Eventually, of course, this increase in contractility is unable to effectively counteract
the ongoing deterioration in VR due to the upright position, and SV continues to fall.
TPR falls in both groups back to C values during Phase II after peaking in Phase I. It then
effectively plateaus during Phase III. This is likely to be a reflection of the changes occurring
due to the alerting reaction developed in the anticipation period. The lack of significant
vasoconstrictor drive generated by the alerting reaction in the supine position is now being
realised in Phases II and III. Although there may be a small contribution from vasodilation
of skeletal muscle beds to this fall in TPR, it is the time lag in developing adequate
vasoconstriction that is more likely to be responsible for this overall reduction in TPR. As
vasoconstriction develops in Phase III, further decline in TPR is arrested. This considerable
time lag between afferent input and efferent output is consistent with the operating
characteristics of vascular resistance changes. The effect of arousal-induced changes in
regional vascular resistance is the most likely explanation for the observed decline in TPR.
While the arterial and cardiopulmonary baroreceptors would clearly be stimulated by the
decreases in MAP and VR, especially in the NP group, their ability to effect a change in
vascular resistance is not evident for some time due to their inherent inertia and latency of
operation. The baroreflexes are likely to contribute towards arresting further decline in both
MAP and TPR and driving them up again by the very end of Phase III, but will exert their
efferent effects predominantly in subsequent phases of tilt.
In both groups, the fall in MAP appears to be due to a decrease in TPR, despite the sustained
increase in HR. TPR plateaus in both groups presumably due to the developing action of the
baroreflexes that were initiated in Phase I. In the FP group, the fall in MAP that occurs
during Phase II is arrested during Phase III by the combination of a sustained increase in HR
and an increase in cardiac contractility. These increases compensate for any vasodilation-
induced decrease in TPR and the ongoing deterioration in SV. Phase III demonstrates that
the FP group is much better able to defend MAP against the fall in VR and SV caused by
sudden exposure to an orthostatic challenge than the NP group.
4.2.4 Phases IV to VI
Phases IV to VI, the late stages of HUT, show a progressively stabilised picture, with no
dynamic postural changes occurring. Hydrostatic force is constant, and the efferent outputs
of all the stimulated control mechanisms are now operative. MAP rises in both groups
throughout these three phases, largely due to increases in TPR. In the NP group, it is not
until the end of Phase IV that MAP is restored to C levels, mediated largely by increases in
TPR. In the FP group, MAP is boosted in mid-Phase IV via a combination of HR and TPR
increases. HR reaches its maximum value in FP during Phase IV, but as these last three

Analysis of Time Course Changes in the Cardiovascular Response to Head-Up Tilt in Fighter Pilots

251
phases progress, HR decreases. TPR increases significantly and as such assumes the
dominant role in maintaining MAP.
The rise in TPR is almost certainly due to the activity of the arterial and cardiopulmonary
baroreflexes. These reflexes were initiated during Phase I, with the onset of the dynamic
postural change. Another reflex that will have been stimulated is the vestibulosympathetic
reflex. The VSR is likely to respond to the dynamic inputs of postural change, as these may
have cardiovascular consequences that the VSR is presumably designed to modulate and
counter.
Clearly it has taken a long time for the vascular resistance changes to occur following this
initial stimulation. This is consistent with what is known about the operating characteristics
of the sympathetically-mediated vascular resistance changes. The FP groups rise in TPR
occurs basically at the same time as that of the NP group. This suggests that any adaptation
to +Gz does not extend to shortening the time lag involved in effecting a change in vascular
resistance. This may reflect a mechanical limitation in the system. Indeed, this fact helps
explain why fighter pilots continue to rely on the anti-G suit, which will boost peripheral
resistance almost immediately after the onset of +Gz acceleration. The FP groups TPR rise
is, however, steeper than the NP group, and reaches a maximum value earlier. This reflects
an increased gain.
Another contributing factor to the increase in TPR may be the putative feed-forward function
of the VSR. After detecting a postural change, the vestibular system may send an excitatory
signal to the medullary vasomotor centre to effect a change in vascular resistance before the
efferent arm of the arterial baroreflexes becomes fully active. Such a feed-forward mechanism
would clearly be an advantage to the pilot operating in the high +Gz environment. A point
worthy of note is that although the vestibular input has changed from the dynamic input of
Phase I to a stable static input in the full HUT position, it is likely that this static input
continues to act as a command signal for the vestibulosympathetic neural link.
While both groups in this experiment presumably had some vestibulosympathetic input, it
is possible that the VSR in the FP group could adapt to the demands of the high +Gz
environment (and its cardiovascular effects) leading to enhancement of this feed-forward
mechanism. This phenomenon would better protect the pilot from circulatory compromise
due to high +Gz, and may contribute to the gain increase in vascular resistance changes
observed in the FP group.
The HR and TPR changes in the FP group are very closely related. The sustained elevation
in HR is effectively switched off only when TPR begins to increase substantially. This effect
is not seen in the NP group, with HR progressively decreasing well before TPR rises to any
great extent. It seems reasonable to suggest that this pattern of response in the FP group
indicates an adaptation strategy. The +Gz-adapted baroreflexes are able to increase HR and
sustain it at higher levels until such time as the increase in TPR is sufficiently established for
it to assume the dominant position. Knowing that TPR increases will take a finite amount of
time, the only other protective option is to keep HR up. Only when the vascular resistance
changes are safely underway will the increased HR be allowed to switch off. This effect is
not seen in the NP group. As such, it is highly suggestive of enhanced baroreflex function as
a result of adaptation to repetitive +Gz acceleration.

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252
4.3 Significance of the findings
Previous studies have demonstrated the existence of a difference in the cardiovascular
response to an applied +Gz load in the FP group compared with the NP group (Newman et
al, 1998, 2000). MAP, SP and DP all increased significantly, with PP being maintained in the
FP group, whereas in the NP group MAP and SP were unchanged, DP increased and PP fell
dramatically. HR, SV and TPR all demonstrated some degree of enhanced performance in
the FP group relative to the NP group. These findings suggested that the FP group had more
effective activation of their baroreflexes in response to a given accelerative stimulus. The FP
group appeared to have enhanced baroreflex function due to their frequent and repetitive
exposure to high +Gz loads.
The findings in this time course analysis support these earlier results. Indeed, from this
analysis it is apparent that in fact the time course of changes in the cardiovascular response
to dynamic postural change is similar between the groups, but that adaptation to +Gz
appears to lead to a greater magnitude of response. The +Gz-adapted pilot demonstrates
increased sensitivity of the arterial and cardiopulmonary baroreflex arcs, which in turn
reflects an increase in the gain of these reflexes. This enhanced function is demonstrated by
a sustained increase in HR and a more marked increase in TPR relative to the NP group.
It is likely that both arterial and cardiopulmonary baroreflexes contribute to the rise in HR
and TPR seen in both groups, and that their enhanced function in the FP group acts to drive
HR up (and to sustain it for longer) and to increase TPR to a greater extent over a similar
time course.
Both the arterial and cardiopulmonary baroreflexes have been shown to be capable of a
certain degree of functional plasticity and altered function. The central fluid shifts
accompanying long-duration spaceflight have been shown to cause attenuation of both
cardiopulmonary and arterial baroreflexes (Billman et al, 1981; Bungo & Johnson, 1983;
Fritsch-Yelle et al, 1994; Thompson et al, 1990). Significantly, changes in cardiovascular
parameters with resultant orthostatic intolerance have been observed after only 5 hours
exposure to the microgravity environment. Microgravity analogue experiments, such as 60
head-down bedrest studies, have produced similar results. These studies confirm that
removal of the normal gravitational gradient results in impaired baroreflex function, with
these important mechanisms becoming less sensitive and as such less effective in dealing
with transient changes in arterial pressure (Convertino et al, 1990).
In contrast, the research reported in this paper involving increased levels of +Gz suggests an
opposite effect, with the baroreflexes becoming more effective at reacting to transient
changes in cardiovascular dynamics. Other researchers have also shown enhanced
baroreflex function in different settings (Krieger, 1970). It seems logical to argue that if a)
both low- and high-pressure baroreflexes can develop attenuated function, and b) high-
pressure baroreflexes can develop enhanced function, then the low-pressure
cardiopulmonary baroreflexes must also be capable of enhanced function. The findings in
this analysis would tend to support this.
These results confirm the findings in previous studies that the cardiovascular response of
fighter pilots to a mild accelerative stimulus is different from that of a group of non-pilots
(Newman et al, 1998, 2000). Furthermore, this analysis shows that this difference is mediated

Analysis of Time Course Changes in the Cardiovascular Response to Head-Up Tilt in Fighter Pilots

253
by differences in the magnitude-time course balance of the dynamic cardiovascular response
to applied +Gz, specifically in terms of HR and TPR. These results provide some additional
insight into the mechanisms involved in postural baroreflex adaptation to high +Gz in
fighter pilots. In addition, this adaptation may not be limited to the arterial baroreflexes
alone; the cardiopulmonary baroreflexes may similarly adapt to the same stimulus. Indeed,
it seems likely that all reflex arcs involved in the regulation of arterial pressure undergo
some form of adaptation to repetitive +Gz exposure.
The roles of the vestibular system in cardiovascular control in general and in adaptation to
+Gz in particular have also been highlighted in this analysis. It is quite possible that the
vestibular system also adapts to frequent exposure to high +Gz, by enhancing its normal
feed-forward vestibulosympathetic action. The enhanced function of the baroreflexes may
well be aided by earlier signals of changing hydrostatic force being sent via the vestibular
system as a means of early alerting and correction of potentially deleterious postural
changes. This certainly warrants further research attention.
5. Conclusion
The findings in this analysis support the results of previous studies, in that repetitive
occupational exposure to the high +Gz environment is capable of inducing a degree of
physiological adaptation. This adaptation appears to be due in part to enhanced arterial and
cardiopulmonary baroreflex sensitivity, which in this analysis is illustrated by sustained
rises in HR and more marked elevations in TPR. The effect of this magnitude-time course
balance shift is to produce a more marked elevation in MAP in the +Gz-adapted pilot. The
analysis also suggests that an increase in effective circulating blood volume may also make a
contribution to the adaptation process. In addition, the results point indirectly to the
possibility of a vestibulosympathetic input into the regulation of arterial pressure during an
orthostatic challenge.
6. References
Billman GE, Dickey DT, Teoh KK, Stone HL. (1981). Effects of central venous blood volume
shifts on arterial baroreflex control of heart rate. Am J Physiol, Vol. 241 (Heart Circ.
Physiol. 10): pp. H571-H575.
Bungo MW, Johnson PJ. (1983). Cardiovascular examinations and observations of
deconditioning during space shuttle orbital flight test program. Aviat Space Environ
Med, Vol. 54, pp. 1001-4.
Convertino VA, Doerr DF, Eckberg DL, Fritch JM, Vernikos -Danellis J. (1990). Head-down
bed rest impairs vagal baroreflex responses and provokes orthostatic hypotension.
J Appl Physiol, Vol. 68, pp. 1458-64.
Convertino VA. (1998). High sustained +Gz acceleration: physiological adaptation to high-G
tolerance. J Grav Physiol, Vol. 5, No. 1, pp. P51-4.
Doba N, Reis DJ. (1974). Role of the cerebellum and the vestibular apparatus in regulation of
orthostatic reflexes in the cat. Circ Res, Vol. 34, pp. 9-18.
Essandoh LK, Duprez DA, Shepherd JT. (1998). Reflex constriction of human resistance
vessels to head-down neck flexion. J Appl Physiol, Vol. 64, pp. 767-70.

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254
Fritsch-Yelle JM, Charles JB, Jones MM, Beightol LA, Eckberg DL. (1994). Spaceflight alters
autonomic regulation of arterial pressure in humans. J Appl Physiol, Vol. 77, pp.
1776-83.
Krieger, EM. (1970). Time course of baroreceptor resetting in acute hypertension. Am J
Physiol, Vol. 218, p. 486.
Mancia G, Mark AL. (1983). Arterial baroreflexes in humans. In: Handbook of Physiology. The
Cardiovascular System, Sect. 2, Vol III, Ch. 20, pp. 755-793, American Physiological
Society, Bethesda, MD.
Newman DG, Callister R. (1999). Analysis of the +Gz environment during air combat
manouevring in the F/A-18 fighter aircraft. Aviat Space Environ Med, Vol. 70, pp.
310-15.
Newman DG, Callister R. (2008). Cardiovascular training effects in fighter pilots induced by
occupational high G exposure. Aviat Space Environ Med, Vol. 79, pp. 774-778.
Newman DG, Callister R. (2009). Flying experience and cardiovascular response to rapid
head-up tilt in fighter pilots. Aviat Space Environ Med, Vol. 80, pp. 723-726.
Newman DG, White SW, Callister R. (1998). Evidence of baroreflex adaptation to repetitive
+Gz in fighter pilots. Aviat Space Environ Med, Vol. 69, pp. 446-51.
Newman DG, White SW, Callister R. (2000). The effect of baroreflex adaptation on the
dynamic cardiovascular response to head-up tilt. Aviat Space Environ Med, Vol. 71,
pp. 255-259.
Ray CA, Hume KM, Shortt TL. (1997). Skin sympathetic outflow during head-down neck
flexion in humans. Am J Physiol, Vol. 273 (Regulatory Integrative Comp. Physiol.
42), pp. 1142-46.
Shortt TL, Ray CA. (1997). Sympathetic and vascular responses to head-down neck flexion
in humans. Am J Physiol, Vol. 272 (Heart Circ. Physiol. 41), pp. H1780-1784.
Thompson CA, Tatro DL, Ludwig DA, Convertino VA. (1990). Baroreflex responses to acute
changes in blood volume in humans. Am J Physiol, Vol. 259 (Regulatory Integrative
Comp. Physiol. 28), pp. R792-R798.
Yates BJ. (1992). Vestibular influences on the sympathetic nervous system. Brain Res Rev,
Vol. 17, pp. 51-9.
Yates BJ, Miller AD. (1998). Physiological evidence that the vestibular system participates in
autonomic and respiratory control. J Vestibular Res, Vol. 8, pp. 17-25.
Zoller RP, Mark AL, Abboud FM, Schmid PG, Heistad DD. (1972). The role of low pressure
baroreceptors in reflex vasoconstrictor responses in man. J Clin Invest, Vol. 51, pp.
2967-2972.
Section 3
Clinical Impact of Cardiovascular
Physiology and Pathophysiology

13
Physical Activity and
Cardiovascular Health
Raul A. Martins
University of Coimbra,
Faculty of Sport Science and Physical Education,
Portugal
1. Introduction
The approach of this chapter makes the assumption that relationships between levels of
physical activity and cardiovascular health are complex. The theoretical framework
considers that physical activity can influence cardiovascular health by itself but can also
influence health-related fitness, which in turn may be able to influence cardiovascular health
and the level of habitual physical activity. To add more complexity, all these relationships
are thought to occur in a reciprocal manner.
Cardiovascular disease corresponds to a group of disorders occurring in the heart and in the
blood vessels. The various manifestations of the disease include sudden death, myocardial
infarction, angina pectoris, stroke (ischemic or hemorrhagic), or peripheral vascular disease.
The risk factors for the cardiovascular diseases are classified usually considering the positive
or negative association with the disease and the modifiable or non-modifiable nature. On
the other hand, the definition of criterions to some risk factors could be dependent of the
context of prevention primordial, primary, secondary, tertiary or even quaternary.
Additionally, it is necessary the plausibility theoretic and biological, and the reversibility of
the effect by the reduction or suspension of the risk factor. Modifiable risk factors like
dyslipidemia, hypertension, diabetes, excess of adipose tissue, pro-coagulant state, pro-
inflammatory state, ignorance, sedentariness or low fitness play alone or, more frequently,
in conjunction with each others, augmenting exponentially the risk of disease.
White Paper on Sport (CEC, 2007) was released by the European Union (EU) to give
strategic orientation on the role of sport in Europe. The document use the definition of
"sport" established by the Council of Europe: "all forms of physical activity which, through
casual or organized participation, aim at expressing or improving physical fitness and
mental well-being, forming social relationships or obtaining results in competition at all
levels." With the ratification of the Lisbon Treaty in late 2009, sport was assumed as
contributing to the EU strategic objectives of solidarity and prosperity. This follows the
Olympic ideal, born in Europe, of developing sport to promote peace and understanding
among nations and cultures as well as the education of young people. Member States are
encouraged to implement evidence-based policies in order to improve their provision of
sporting facilities and opportunities. This means that for the first time the EU is actively

The Cardiovascular System Physiology, Diagnostics and Clinical Implications 258
aiming to promote sport and physical activity at the policy level not only with a view to
improving physical wellbeing and health across the EU, which is the main focus of this
chapter, but also to enhance the role of sport in boosting social cohesion and its educational
value. This chapter will explore the relationship of cardiovascular health with sport, as the
European understanding, or physical activity in the North American understanding.
Physical activity concept, physical activity epidemiology and cardiovascular health, physical
activity guidelines, prevalence of sedentariness in Europe and USA, physical activity and
life expectancy, and pro-inflammatory state and physical activity are topics explored in this
chapter.
2. Physical activity
Physical activity comprises any body movement produced by the skeletal muscles that
results in a substantial increase over the resting energy expenditure (Bouchard & Shephard,
1994). Included in this large umbrella is considered the leisure-time physical activity
(LTPA), daily physical activities, intentionally practiced exercise (frequency, intensity, type,
time) and sport, or occupational work, together with other physical expressions that modify
the total energy expenditure. Within the concept of physical activity, physical exercise is a
narrow concept, usually defined as planned and repeated movements intending to maintain
or to improve one or more components of the health-related fitness or of the performance-
related fitness. Physical activity has been understood as a behavior that could also change
health-related or performance-related fitness. However, it is also taken in account as a
determinant behavior to health and functionality. When one is talking about the potential
benefits on health, obviously, all determinants of human energy expenditure should be
under careful consideration. Contrarily, sedentariness refers that people remain sitting
much of the labor and leisure times.
There are a lot of methods to characterize and measure the behavior physical activity
including calorimetry (direct and indirect), physiologic markers (heart rate or maximal
oxygen uptake) mechanical and electronic devices (pedometers and accelerometers), the
observation of behaviors, or the caloric intake (Welk, 2002). Independently of the selected
method, the investigator should consider the complex nature of the behavior physical
activity and the errors derived from the method usually used with largest number of
participants the self-reported questionnaires. The use of questionnaires imply low costs
but sometimes introduces considerable error because, for instance, could exist social
tendency to associate physical activity to sport participation. It means that it is desirable to
use direct methods as pedometers or accelerometers. Pedometers count the number of steps
but are not able to distinguish different levels of activity. This limitation is overcome by the
accelerometry, and the last National Health and Nutrition Examination Survey (NHANES)
realized in 2003-2004 evaluated physical activity of around 4867 American citizens with
accelerometry (Troiano et al., 2008).
3. Prevalence of sedentariness
A national representative study of Portuguese people has measured directly physical
activity by accelerometry (Baptista et al., 2011). Volunteered to participate 5231 adolescents
(10-17 years-old; 1456 males; 1755 females), adults (18-64 years-old; 441 males; 803 females),

Physical Activity and Cardiovascular Health 259
and older adults (65+ years-old; 303 males; 473 females). All participants were measured
during four days (two week days and two weekend days). The cut-off points for moderate
intensity were 3-5,9 METs (adults and older adults) and 4-6,9 METs (adolescents), and for
vigorous intensity were 6+ METs (adults and older adults) and 7+ METs (adolescents).
Adolescents with less than 60 minutes/day of moderate/vigorous physical activity on 5
days/week, and adults or older adults with less than 30 minutes/day on 5 days/week were
classified as insufficiently active.

Source: Baptista et al., 2011.
Fig. 1. Prevalence of insufficiently active Portuguese people measured by accelerometry.
As illustrated by Figure 1, the prevalence of insufficiently active people was particularly
high in the adolescents (80%) but also in the older adults (65%). The adult people (18-64
years old) attained only 32%, which represents the group with higher volume of
moderate/vigorous weekly physical activity. The prevalence of insufficiently active in all
the people evaluated was 67%. Males are more active than females, in each one of the three
groups, with higher difference (21%) among adolescents and lower difference among adults
(13%). One can speculate that the indirect methods like self-reported questionnaires tend to
overestimate physical activity when compared with a direct measure (accelerometry), as
seems to result when one compares these overall data (67%) with data provided from
Eurobarometer 2003 (30%) and Eurobarometer 2010 (52%) on Figure 2. However, the high
prevalence of insufficiently active, particularly in adolescents and older adults, claim for the
adoption of specific strategies to change these sedentary behaviors.
90%
36%
72%
69%
23%
55%
80%
32%
65%
0
20
40
60
80
100
Adolescents Adults Older adults
P
r
e
v
a
l
e
n
c
e

(
%
)
Females (F)
Males (M)
F & M

The Cardiovascular System Physiology, Diagnostics and Clinical Implications 260


USA data is No Leisure-Time Physical Activity in 2003, and 2008.
http://www.cdc.gov/nccdphp/dnpa/physical/stats/leisure_time.htm. Extracted on 24/Oct/2011.
Fig. 2. Prevalence of the sedentariness in some EU Member States measured by self-reported
questionnaires.
Data from the 2003 Eurobarometer (EC, 2003) are presented in Figure 2. Sedentary people are
those not meeting the threshold for low activity. Cut-off points for low physical activity
participation were 30 minutes of walking or moderate-intensity activity on at least 5
days/week, or 20 minutes of vigorous-intensity activity on at least 3 days/week. Participated
people from each one of the EU Member States with 15+ years-old (N=16230), randomly
sampling with probability proportional to population size (for a total coverage of the country)
and to population density (metropolitan, urban, and rural areas). It was used the International
Physical Activity Questionnaire (IPAQ) to characterize physical activity in a face-to-face
interview in peoples home and in the appropriate national language. Frequency of 5+
days/week of moderate intensity physical activity was not achieved by 72% of EU Member
States citizens (equal for women and men) while 74% did not achieve 3+ days/week of
vigorous intensity physical activity (81% on women, and 68% on men). Prevalence of people
who do not practiced 3+ days/week of vigorous intensity increases with age from 66% (15-25
years) to 69% (26-44 years), to 76% (45-64 years), and to 89% (65+ years). Prevalence of people
not engaged on 5+ days/week of moderate intensity also increases with age: 70% (15-25
years), 70% (26-44 years), 72% (45-64 years), and 79% (65+ years).
10
20
30
40
50
60
2003 2010
P
r
e
v
a
l
e
n
c
e

(
%
)
USA

Physical Activity and Cardiovascular Health 261
The 2010 Eurobarometer (EC, 2010) analyzed people with 15+ years-old of 27 EU Member
States (N=26788), with a different self-reported questionnaire than IPAQ, and revealed a
prevalence of 27% for people saying they engage in physical activity regularly at least 5
times/week, while 38% answered that exercising with some regularity (1-4 times/week)
(Figure 2). The other 35% EU citizens never engage in any physical activity or engage below
the desirable level (1-3 times/month). By analyzing data from some EU Member States it is
possible to observe clear discrepancies in the values from 2003 to 2010. These discrepancies
maybe is reflecting partially the utilization of different instruments with different self-
reported answers. Sedentariness was considered in Eurobarometer 2010 to the people that
engage only 1-2 times/month or even less in physical activities. With these cut-off points,
sedentariness was respectively in 2003 and 2010: Portugal - 30% and 52%; France - 43% and
25%; Germany 24% and 22%; UK 37% and 27%; Netherlands 19% and 16%; All EU
31% and 35%.

Source: Church et al., 2011.
Fig. 3. Trends in the prevalence of sedentary, light and moderate intensity occupations from
1960 to 2008.
Troiano and colleagues (2008) have described physical activity levels of children (6-11 years
old), adolescents (12-19 years old), and adults (20+ years old), using objective data obtained
with accelerometers from a representative sample of the U.S. population. The results were
attained from the 2003-2004 National Health and Nutritional Examination Survey
(NHANES), a cross-sectional study of a complex, multistage probability sample of the
civilian, noninstitutionalized population. Data are described from 6329 participants who
provided at least 1 day of accelerometer data and from 4867 participants who provided 4+
days of accelerometer data. Males were more physically active than females. Authors
observed that physical activity declines dramatically across age groups between childhood
(42% obtained the recommended 60 minutes/day) and adolescence (8% achieve 60

The Cardiovascular System Physiology, Diagnostics and Clinical Implications 262
minutes/day) and continues to decline in adults (less than 5% attained 30 minutes/day).
Objective and subjective measures of physical activity gave qualitatively similar results
regarding gender and age patterns of activity. However, adherence to physical activity
recommendations according to accelerometer-measured activity is substantially lower than
according to self-reported questionnaire. Occupational work is also an important expression
of physical activity contributing to energy expenditure and to the energy balance.

Source: Church et al., 2011.
Fig. 4. Predicted mean U.S. body weight based on change in occupation related daily energy
expenditure since 1960 compared to mean U.S. weight gain based on the NHANES
examination periods for 4050 year old.
Trends in occupational physical activity during the past 5 decades (Figure 3), and the
concurrent changes in body weight in the U.S. were explored by Church and colleagues
(2011). Authors observed that in 1960 almost half the jobs (48%) in private industry in the

Physical Activity and Cardiovascular Health 263
U.S. required at least moderate intensity physical activity whereas in 2008 less than 20%
demand this level of energy expenditure. While there has been a steady increase in the
prevalence of sedentary and light intensity physical activity occupations since 1960, the
prevalence of moderate intensity physical activity occupations has decreased. At the same
period (1960-2008) there was a drop in occupation-related daily energy expenditure of about
142 calories for men and 124 calories for women. Authors estimate that the decrease of 142
calories in men would result in an increase in mean weight from 76.9 kg (1960-62) to 89.7 kg
(2003-06), with the results having similar pattern for women (Figure 4).
Over the last 50 years the prevalence of Americans in the labor force has increased
approximately 40% to 50%, with women assuming a growing prevalence in the work force
from 43% in 1970 to 60% in 2007. This fact helps to explain the decrease in the pattern of
occupation-related energy expenditure (Lee & Mather, 2008). Given this, it is unlikely a return
to occupations demanding moderate levels of physical activity, which addresses further strong
evidence of the public health importance of promoting physically active lifestyles outside of
the work day. The reduction of 124 (women) and 142 calories (men) per day in occupation-
related energy expenditure over the last 50 years would have been adequately compensated
for by meeting the 2008 Physical Activity Guidelines of 150 minutes/week of moderate
intensity activity or 75 minutes/week of vigorous intensity activity (USDHHS, 2011). While it
is often noted that the prevalence of Americans who achieve this recommendation has been
constant over recent decades, the fact remains that based on self-report data only 25% adults
achieve this level (CDC, 2008), but when physical activity is assessed with accelerometers the
number of adult people achieving the recommendations drops dramatically to less than 5%
(Troiano et al., 2008). Therefore, since energy expenditure of the labor activities has largely
been removed, the relative importance of LTPA has increased and should be considered as a
major focus of public health interventions and research.
Brownson and colleagues (2005) developed a revision to describe current patterns and long-
term trends (up to 50 years when possible) related to (i) physical activity, (ii) employment
and occupation, (iii) travel behavior, (iv) land use, and (v) related behaviors (e.g., television
watching). Available data allows the following trends: relatively stable or slightly increasing
levels of LTPA, declining work-related activity, declining transportation activity, declining
activity in the home, and increasing sedentary activity. These reflect an overall trend of
declining total physical activity, with large differences noted in the rates of walking for
transportation across metropolitan areas, and a strong linear increase in vehicle miles
traveled per person, coupled with a strong and consistent trend toward people living in
suburbs. Authors concluded that although difficult to quantify, it appears that a
combination of changes to the built environment and increases in the proportion of the
population engaging in sedentary activities put the majority of the population at high risk of
physical inactivity.
4. Physical activity guidelines
Vigorous activity was centrally considered to health promotion until 1995 when
recommendations of the Center for Disease Control (CDC) and the American College of
Sports Medicine (ACSM) pointed out for adults to accumulate at least 30 minutes of
moderate-intensity physical activity on most days of the week (Pate et al., 1995). These

The Cardiovascular System Physiology, Diagnostics and Clinical Implications 264
recommendations were described in the 1996 U.S. Surgeon Generals Report on Physical
Activity and Health (USDHHS, 1996), and served as cornerstone for the Healthy People
2010 (HP 2010) goals on physical activity (USDHHS, 2000), inspiring public policies and
programs over the next years. The HP 2010 objectives stated that adults should engage in
vigorous LTPA (60-84%VO
2Res
or %HR
Res
; 77-93%HR
max
; >60%VO
2max
; >6 METs) for at least
20 minutes, at least 3 times/week, or moderate LTPA (40-59%VO
2Res
or %HR
Res
; 64-
76%HR
max
; 40-60%VO
2max
; 3-6 METs) for at least 30 minutes, at least 5 times/week.
For the purposes of HP 2010, lesser amounts of vigorous and moderate activities could not be
combined. In 2007, the CDC/ACSM recommendations published in 1995 were updated and
clarifed, emphasizing the potential health benefts of combinations of moderate and vigorous-
intensity activities and of strengthening activities (Haskell et al., 2007). Meantime, the Healthy
People 2020 (HP 2020) goals on physical activity were released (USDHHS, 2010) introducing
some modifications, and establishing and encouraging to increase the prevalence of
suffciently active adults engaging in moderate-intensity aerobic physical activity of at least
150 minutes/week or vigorous-intensity aerobic activity of at least 75 minutes/week or an
equivalent combination. The HP 2020 also pursue to increase the prevalence of highly active
adults engaging in aerobic physical activity of at least moderate intensity for more than 300
minutes/week or more than 150 minutes/week of vigorous intensity or an equivalent
combination. And, finally, to increase the prevalence of adults who perform muscle-
strengthening activities on 2 or more days/week of 7 large muscle groups.
5. Physical activity and life expectancy
Mortality differentials by level and intensity of physical activity have been documented,
with Lollgen and colleagues (2009) obtaining significant association of lower all-cause
mortality for active individuals comparing with sedentary persons. Highly active men had a
22% lower risk of all-cause mortality (RR=0.78; 95% CI: 0.72 to 0.84), and women had 31%
(RR=0.69; 95% CI: 0.53 to 0.90) comparing to mildly active men and women, respectively.
The authors also found a similar and significant association of activity to all-cause mortality
in older participants.
Schoenborn and Stommel (2011) studying the benefits of accomplish the 2008 Physical
Activity Guidelines for Adults (USDHHS, 2011), which are similar to the HP 2020 goals,
achieved 27% lower risk of all-cause mortality among people without existing chronic
comorbidities, and by almost half among people with chronic comorbidities (such as heart
disease, stroke, diabetes, cancer, respiratory conditions, or any functional limitation),
regardless of age and obesity levels. Assuming several limitations present on causal
interpretations, when examining for interactions of physical activity with smoking and
alcohol consumption, data suggest that relative survival benefts associated with physical
activity are largest among current smokers and light-moderate drinkers.
Figure 5 shows the survival curves associated with four types of adherence to the 2008
Guidelines for all adults: meeting both the aerobic and muscle-strengthening guidelines, the
aerobic only, the muscle-strengthening only, and neither of the minimum recommendations.
This figure suggests that meeting the 2008 Guidelines is associated with survival benefts,
with stronger benefits for both the aerobic and muscle-strengthening exercises; also suggests
that aerobic activity alone promotes stronger benefts than muscle strengthening alone.

Physical Activity and Cardiovascular Health 265


Note: U.S. adults aged 18 years (weighted); respondents not linked to death records were considered
censored, meaning they were presumed to be alive as of December 31, 2006. NHIS, National Health
Interviews Survey, 1997-2004.
Adapted from Schoenborn and Stommel (2011)
Fig. 5. Survival probabilities by levels of adherence to 2008 Physical Activity Guidelines.
Figure 6 illustrates that higher volumes of aerobic exercise are associated with higher
increase in survival probabilities. Those who engage in none aerobic leisure-time activity
attained lower survival probability while people that engaged in more than 300
minutes/week have the higher survival probability. In other words, it means that additional
survival benefts can be achieved with higher levels of aerobic leisure-time activity.
6. Epidemiology of physical activity and cardiovascular health
Epidemiology has been defined as the study of the distribution and determinants of health-
related states or events in specified populations, and the application of this study to the
prevention and control of health problems (Last, 2001). When this definition considers
health-related states or events instead of the former disease frequency is having in
account the contemporary definition of health that considers positive health states, as a good
quality of life, or well succeeded aging, and not only the absence of disease. The word
epidemiology is derived from the Greek words: epi upon, demos people, and logos
study. In fact, epidemiology origin based on Hippocrates observation, made more than
2000 years ago, that environmental factors could be determinant for the occurrence of a
disease. However, it was only in second half of the XIX century when the first truly
epidemiologic investigations appeared (Bonita et al., 2006).

The Cardiovascular System Physiology, Diagnostics and Clinical Implications 266

Note: U.S. adults aged 18 years (weighted); respondents not linked to death records were considered
censored, meaning they were presumed to be alive as of December 31, 2006. NHIS, National Health
Interviews Survey, 1997-2004.
Adapted from Schoenborn and Stommel (2011).
Fig. 6. Survival probabilities by levels of adherence to 2008 aerobic physical activity
guidelines.
Physical activity and the relationship with cardiovascular health was firstly studied in an
epidemiologic basis by Morris and colleagues (1953a,b). Works were conducted to
understand how both vocational and LTPA relate to fitness and risk of coronary heart
disease (CHD). Authors studied London transit workers, and other occupations as postal
service employees and civil servants. Initially they found bus conductors on Londons
double-decker omnibuses to be at lower risk than bus drivers; what disease the conductors
did develop was less severe, and they were more likely to survive an attack. The conductors,
who walked up and down stairs in a daily basis, often for decades, experienced roughly half
the number of heart attacks and sudden death as the drivers. After that, Morris and
colleagues (1990) studied a random sample of 3591 British civil servants during a follow-up
of 8-year period ending in 1977, during which time 268 men died. Subjects were classified as
having engaged in vigorous activities (>6 METs), or not. Of the subjects 22% reported some
kind of vigorous exercise and their death rate was 4,2%. The remaining 78% reported no
vigorous exercise and their mortality rate was 8,2%, i.e., twice as high. This differential in
death rates persisted when controlling for age, smoking, obesity and successive intervals of
follow up.
Several other populations have been studied for physical activity and physiological fitness
in relation to health and specifically cardiovascular health (USDHHS, 1996). One of the most
remarkable studies was conducted with 17549 men who entered Harvard College between

Physical Activity and Cardiovascular Health 267
1916 and 1950 (Paffenbarger et al., 1978), and when aged 5584 years responded to a
questionnaire on their personal characteristics, health status and lifestyle habits like current
and former physical activity, as participation in student sport whilst at university. These
patterns have been related to cardiovascular disease mortality over a 16-year follow-up
period (1962 to 1978), during which 1413 men died (Paffenbarger et al., 1986a,b).
Among Harvard alumni there were strong significant inverse associations between death
rates and levels of each of the following physical activity: walking, stair climbing, sports
play and combinations of these activities, measured in kJ/week. Gradients of benefit from
more active lifestyles were consistent throughout, and maintained after controlling for age,
smoking, hypertension and obesity. As compared with the one-third of least active men, the
middle third experienced a 23 % reduction in death rate during follow-up and the one-third
of most active men, a 32 % reduction. Light activities (<4METs), moderate activities (4
5METs), and vigorous activities (>6METs) each predicted lower death rates. Physical
activity related inversely to total mortality, primarily to death due to cardiovascular or
respiratory causes. Death rates declined steadily as energy expended on such activities
increased from less than 500kcal/week to 3500kcal/week, beyond which rates increased
slightly. This relationship was independent of the presence or absence of hypertension,
cigarette smoking, extremes or gains in body weight, or early parental death (Figure 7).

Source: Paffenbarger et al., 1986a.
Notes: Participated 16936 men. A total of 1413 alumni died during 12 to 16 years of follow-up (1962-
1978). Exercise reported as walking, stair climbing, sports play, and combinations of these activities.
Fig. 7. Inverse association between weekly energy expenditure and RR of all-cause
mortality.

The Cardiovascular System Physiology, Diagnostics and Clinical Implications 268
Those men were studied for the effect on all-cause mortality from changing physical activity
habits. Men who had increased or decreased their activity by less than 250kcal/week
between the 1960s and 1977 were considered in an unchanged category. Compared with
their death rates, gradient reductions in mortality were observed with increased levels of
physical activity, and gradient increases in mortality with decreased levels of activity. At the
extremes of this gradient, men who had increased their energy expenditure by
1250kcal/week had a 20% lower risk of death than men in the unchanged category; men
who decreased their activity by 1250kcal/week had a 26% higher risk (Paffenbarger et al.,
1993, 1994).
Vigorous activity should be encouraged. Not only because in todays world, where time is a
precious commodity, a short period of vigorous exercise expends as much energy as does
moderate activity carried out for two or three times as long (Figures 8 and 9), but also
because the kind of stimulation over tissues and systems could be more benefic to
compensate lost, asymptomatic in an initial stage, that tends to occur with aging.

Fig. 8. Duration (min) of daily walking sessions, with moderate intensity (velocity of
80m/min; 3,3METs), for people with different body masses, to gain cardiovascular health
(500kcal/week: RR=1,00; 2000kcal/week: RR0,62; 3499kcal/week: RR0,46).
Figure 8 illustrates time spent with walking at moderate intensity (velocity of 80m/min, or
4,8km/h) by people of different body masses, within the range 500-3499kcal/week
(Paffenbarger et al., 1986a). A person weighting 75kg will needs to walk 116 minutes per
134
124
116
109
102
97
76
71
66
62
58
55
19
18
17
16
15
14
0
20
40
60
80
100
120
140
65 70 75 80 85 90
D
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r
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a
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s
e
s
s
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(
m
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)
Body mass (kg)
3499 Kcal/week
2000 Kcal/week
500 Kcal/week

Physical Activity and Cardiovascular Health 269
each one of the 7 days of the week to maximize the potential benefits of physical activity on
cardiovascular health, i.e. to spend 3499kcal/week (RR=0,46). In other words, and taking in
account the work of Paffenbarger and colleagues (1986a) illustrated by the Figure 1, a person
of 75kg will obtain progressive cardiovascular gain from 17 minutes of horizontal walking
(RR=1,0) to 116 minutes of daily horizontal walking (RR=0,46). However, if that same
person of 75kg of body mass decides to exercise at vigorous intensity (Figure 9), 40 minutes
of horizontal running at 150m/min (9km/h) will be enough to maximize the potential gains
on cardiovascular health.

Fig. 9. Duration (min) of daily running sessions, with vigorous intensity (velocity of
150m/min; 9,6METs), for people with different body masses, to gain cardiovascular health
(500kcal/week: RR=1,00; 2000kcal/week: RR0,62; 3499kcal/week: RR0,46).
7. Pro-inflammatory state and physical activity
Inflammation has emerged some years ago as a key pathophysiological event in vascular
diseases and the consequent cardiovascular and cerebral injury. Inflammation is a
complex process involving multiple cellular and molecular components, and is triggered
by different pro-inflammatory mediators generated directly and indirectly by microbial
invasion, endotoxins, immune complexes, and cytokines. Vascular endothelium is
subjected to pro-inflammatory insults but fortunately is awarded with strong anti-
inflammatory molecules that confer resistance to damage by transient pro-inflammatory
attacks.
46
43
40
37
35
33
26
24
23
21
20
19
7
6 6
5 5 5
0
10
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(
m
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)
Body mass (kg)
3499 Kcal/week
2000 Kcal/week
500 Kcal/week

The Cardiovascular System Physiology, Diagnostics and Clinical Implications 270
Inflammation is a natural response to infection or damage that intends to destroy or to
inactivate the foreign agents permitting tissues repairing. Inflammation could be a local or
systemic response, and the key mediators are the cells that act as phagocytes, with the most
important being neutrophils, macrophages, and macrophages-like cells. The sequence of
local events in a typical nonspecific inflammatory response includes: (i) vasodilatation of the
microcirculation in the infected area, leading to increased blood flow; (ii) large increase in
protein permeability of the capillaries and venules in the infected area, with resulting
diffusion of protein and filtration of fluid into the interstitial fluid; (iii) chemotaxis:
movement of leukocytes from the venules into the interstitial fluid of the infected area; (iv)
destruction of bacteria in the tissue either through phagocytosis or by other mechanisms; (v)
tissue repair (Widmaier et al., 2011). The events of inflammation, such as vasodilation, are
induced and regulated by several chemical mediators including kinins, complement,
products of blood clotting, histamine, eicosanoids, platelet-activating factors, cytokines,
nitric oxide, C-reactive protein (CRP). CRP is an acute phase protein produced by the liver,
always found at some concentration in the plasma, and act to minimize the extent of local
tissue damage. CRP can bind nonspecifically to carbohydrates or lipids in the cell wall of
microbes and facilitate opsonization to enhance phagocytosis.


Source: OECD Health Data 2011. Extracted on 25 Oct 2011 from:
http://stats.oecd.org/Index.aspx?DataSetCode=HEALTH_LVNG
Notes: Data of Portugal are self-reported, in 1999 and 2006; data of France are self-reported, in 1990,
2000 and 2008; data of Germany are self-reported, in 1999 and 2009; data of UK are measured in 1991,
2000 and 2009; data of Netherlands are self-reported, in 1990, 2000 and 2009; data of USA are measured
in 1991, 2000 and 2008.
Fig. 10. Decennial evolution of obesity (BMI 30kg/m
2
) in % of total population.
13
15
6 9
11
12
14
21
23
12
23
31
34
5
10
15
20
25
30
35
1990 2000 2009
P
r
e
v
a
l
e
n
c
e

o
f

o
b
e
s
i
t
y

(
%
)

Physical Activity and Cardiovascular Health 271
There is scientific evidence indicating to atherosclerosis as an inflammatory disease (De
Haro et al., 2008; Hamer & Stamatakis, 2008; Virani et al., 2008). In fact, some of the most
prevalent risk factors for cardiovascular diseases have been shown to have a pro-
inflammatory action including hypertension (Imatoh et al., 2007; Hamer & Stamatakis,
2008), diabetes (Porrini et al., 2007; Hwang et al., 2008), dyslipidemia (Kim et al., 2007), and
overweight or obesity (Hamer & Stamatakis, 2008; Piestrzeniewicz et al., 2008). As high-
sensitivity C-reactive protein (hs-CRP) is a sensitive marker of inflammation, it has been
pointed as the golden marker of inflammation, with Berk and colleagues (1990) establishing
for the first time a positive association between hs-CRP and angina pectoris. Other authors
have also found positive association of the hs-CRP with risk of vascular disease (Koenig et
al., 1999; Kuller et al., 1996; Ridker et al., 1998a; Ridker et al., 2002; Ridker et al., 2003). Since
then, various investigations have concentrated on the effects of physical activity on hs-CRP
(Church et al., 2002; Martins et al., 2010a; Martins et al., 2010b; Mora et al., 2006;
Wannamethee et al., 2002).
Figure 10 illustrates decennial evolution of obesity, self-reported or measured, in different
countries of Europe and North America. United States of America and United Kingdom
attained higher values of obesity (BMI 30kg/m
2
) prevalence, according to the
Organization for Economic Co-Operation and Development (OECD) 2011 health data, with
34% and 23% in 2009, respectively. The importance of this risk factor in this context is
related with pro-inflammation action, as referred above. One would yet speculate that self-
reported data by people in Portugal, France, Germany, and Netherlands are below to the
real prevalence, which addresses for a rise of prevalence of obesity in these countries to
values close to the measured ones in the USA and UK.

Before After
Body weight (kg)
Waist circumference (cm)
Body mass index (kg/m
2
)
Blood pressure (mm Hg)
Systolic
Diastolic
Triglycerides (mmol/l)
Total cholesterol (mmol/l)
HDL-cholesterol (mmol/l)
LDL-cholesterol (mmol/l)
Total Cholesterol/HDL-cholesterol
hs-CRP (mg/l)
6-minute walk distance (m)
73 (11)
94 (10)
30.6 (5.0)

149 (21)
77 (10)
1.35 (0.58)
5.64 (0.86)
1.31 (0.25)
2.36 (0.77)
4.40 (0.92)
5.4 (3.9)
387 (76)
72 (11)*
91 (10)**
30.3 (4.9)*

150 (19)
74 (9)*
1.20 (0.54)*
5.29 (1.03)*
1.37 (0.32)*
2.05 (0.86)**
4.02 (0.81)**
4.0 (2.0)*
437 (83)**
Values are mean (SD). *p<0.05, **p<0.01 compared with before.
Source: Martins RA et al., (2010b).
Table 1. Exercising group.
Measurement of cholesterol by itself do not allow the recognition of about of the individuals
who will present later with myocardial infarctions (Rifai & Ridker, 2001), and a number of
studies (Ridker et al., 1998b; Ridker et al., 2001; Ridker et al., 2002; Onat et al., 2001; Torres &
Ridker, 2003) reinforce the idea that introducing markers of inflammation in the models of

The Cardiovascular System Physiology, Diagnostics and Clinical Implications 272
diagnosis, beyond the lipid profile, result in more accuracy to predict atherogenic events,
comparing with lipid-based models only. High serum levels of CRP have also been found
not only in patients with elevated blood pressure but also in those with congestive heart
failure (Barbieri et al., 2003; Torre-Amione, 2005), type 2 diabetes, metabolic syndrome and
obesity (Das, 2001; Pradham et al., 2001; Ridker et al., 2003). Therefore, factors that may
impact negatively hs-CRP levels, like physical activity, should be further studied,
particularly in populations at increased risk of the above diseases.

Before After
Body weight (kg)
Waist circumference (cm)
Body mass index (kg/m
2
)
Blood pressure (mm Hg)
Systolic
Diastolic
Triglycerides (mmol/l)
Total cholesterol (mmol/l)
HDL-cholesterol (mmol/l)
LDL-cholesterol (mmol/l)
Total Cholesterol/HDL-cholesterol
hs-CRP (mg/l)
6-minute walk distance (m)
71 (12)
93 (10)
29.0 (4.4)

146 (20)
76 (9)
1.10 (0.35)
5.14 (0.94)
1.33 (0.28)
2.39 (0.86)
3.99 (0.98)
5.5 (3.5)
342 (126)
70 (13)
91 (10)**
28.8 (4.7)

142 (24)
75 (13)
1.15 (0.35)
5.27 (1.02)
1.32 (0.29)
2.27 (0.61)
4.07 (0.83)
5.1 (2.3)
343 (170)
Values are mean (SD). *p<0.05, **p<0.01 compared with before.
Source: Martins RA et al., (2010b).
Table 2. Control group.
Inflammatory processes have been positively associated with aging (Pedersen et al., 2003),
with studies suggesting that physical activity would benefit atherosclerotic disease, at least
partially, by reducing the inflammatory level (Wannamethee et al., 2002; Reuben et al.,
2003). Serum CRP levels has been negatively associated with physical activity or physical
fitness, but also with BMI and other adiposity measures (Church et al., 2002; Wannamethee
et al., 2002; Mora et al., 2006; Martins et al., 2010b). These studies suggest that regular
physical exercise might lower CRP levels, acting as an anti-inflammatory agent, by the
effects over adipose tissue and/or by the effects on the muscle mass.
Martins and colleagues (2010b) present results (Table 1) showing beneficial effects of two
exercising programs (i.e., aerobic and strength-based) in older adults with significant
differences on body weight (-1%), waist circumference (-3%), BMI (-1%), diastolic blood
pressure (-4%), triglycerides (-11%), total cholesterol (-6%), HDL-cholesterol (5%), LDL-
cholesterol (-13%), total cholesterol/HDL-cholesterol relationship (-9%), hs-CRP (-26%), and 6-
minute walk distance (13%), while the control group (Table 2) only had significant differences
on waist circumference (-2%). At baseline, BMI correlated with total cholesterol (r=0.35,
p=0.007), triglycerides (r=0.38, p=0.004), and hs-CRP (r=0.46, p=0.001). Waist circumference
correlated with total cholesterol (r=0.30, p=0.022), triglycerides (r=0.35, p=0.010), hs-CRP
(r=0.38, p=0.010), and total cholesterol/HDL-cholesterol (r=0.38, p=0.005). Finally, body
weight also correlated with total cholesterol (r=0.33, p=0.011), triglycerides (r=0.27, p=0.044),
hs-CRP (r=0.40, p=0.006), and total cholesterol/HDL-cholesterol (r=0.33, p=0.016).

Physical Activity and Cardiovascular Health 273
Studies examining the effects on cardiovascular health by endurance and strength training
have generally found either positive changes in lipid profile or no changes at all. More
pronounced dyslipidemia at baseline has been pointed has having more favorable changes
after training (Laaksonen et al., 2000), mediated by the reduction of body fat (Leon &
Sanchez, 2001). On the other hand, older people are known to be under the effects of
sarcopenia, which is characterized by loss of skeletal muscle mass and strength weakness.
Sarcopenia has been associated not only with functional fitness impairment (Reid et al.,
2008) but also with systemic inflammation (Visser et al., 2002). Resistance training (Marini et
al., 2008) has been suggested to be an effective way to prevent the adverse outcomes of
sarcopenia whereas the effects of aerobic training are not as clear.
The mechanisms underlying the positive effects of the physical activity on inflammation
remain under discussion, being considered the hypothesis of reduction of body fat, and/or
the increase of muscle mass. Some have hypothesized about changes in circulating
inflammatory cytokine levels alter hs-CRP hepatic production. Reductions of serum IL-18,
IL-6 and CRP have been reported after 10 months of aerobic exercise but not after
flexibility/resistance exercise (Kohut et al., 2006). However, others failed to obtain exercise-
induced effects in plasmatic inflammatory cytokines, including IL-6, TNF- and IL-1 after
12 weeks of combined aerobic/resistance and flexibility training (Stewart et al., 2007).
Additionally, TNF- may contribute directly to sarcopenia once can disrupt the
differentiation process in cultured muscle cells and promotes catabolism in mature muscle
cells. Muscle mass is a primary site for glucose and triglyceride disposal (Dinneen et al.,
1992) and the major determinant of metabolic rate (Zurlo et al., 1994). Age-related muscle
loss may contribute to insulin resistance, dyslipidemia and increased adiposity. IL-6 protein
is expressed in contracting muscle fibers and released from skeletal muscle during exercise
whereas this is not the case for TNF- (Steensberg et al., 2002). IL-6 is able to inhibit TNF-,
and IL-1 production stimulates the production of IL-1ra and IL-10 and the release of soluble
TNF-receptors (Steensberg et al., 2003). In synthesis, a chronic training-induced reduction on
hs-CRP concentrations in older adults is supported by various studies having as key factors
increase in muscular mass and reduction in body fat.
8. Summary
Exercise and physical activity, or a wide concept of sport as defined by the Council of
Europe, are cornerstones to act at different levels of prevention for cardiovascular health.
Physical activity comprises any voluntary movement that substantially increases oxygen
uptake above the resting level. Prevalence of sedentariness should be considered as a key
point for public health initiatives across all ages, with particular emphasis in older adults
because not only they have the higher prevalence of inactivity, but also the higher costs of
health services. On the other side, energy expenditure related with occupational work has
been diminishing, which addresses more importance to the leisure-time physical activity to
the energy balance. Physical activity and cardiovascular fitness, i.e. oxygen uptake capacity,
are both risk factors for cardiovascular health. Sometimes, questions arise about the most
appropriate kind of exercise to burn energy and enhance oxygen uptake. However, the
question seems to be easily answered since all fuel used in the body is ultimately processed
by the aerobic energy pathways. This means that we can use the amount of oxygen
consumed during the activity to calculate caloric burn. The impact of physical activity on fat

The Cardiovascular System Physiology, Diagnostics and Clinical Implications 274
mass is a sensitive point, and the question about the most appropriate intensity to burn fat
also arises occasionally. Again, the answer seems to be very easy. Each individual should
practice with the higher possible intensity, according to their risk stratification. The time
necessary to reach the same level of energy expenditure is about one third when comparing
vigorous intensity with moderate intensity, addressing for the lack of importance to discuss
about the right zone to burn fat. Moreover, it is very well known that after about 2 minutes
of exercising at high intensity the aerobic pathway (using fat free acids as fuel) becomes
predominant over glycolytic pathway. Recent risk factors, as inflammation, have been
considered. Again, exercise seems to be very promising in reducing the inflammatory
processes, with the actual discussion centered on the acute and chronic effects of different
modes of exercise, and on the underlying mechanisms.
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14
Cardiovascular Disease Risk Factors
Reza Amani and Nasrin Sharifi
Ahvaz Jondishapour University of Medical Sciences,
Iran
1. Introduction
Cardiovascular disease (CVD) is the leading cause of death not only in industrialized and
developed countries but also in developing societies (WHO, 2008a). Changes in lifestyle of
the population living in developing countries, which is due to the socioeconomic and
cultural transition, are important reasons for increasing the rate of CVD. This observation
has led to extensive research on prevention. Diagnosis the risk factors and predictors of
CVD can help us detect high risk patients and prevent the disease, effectively.
Nowadays with a rapid progress in medical technology and diagnostic tools, more predictors
are being added to the previous list of CVD risk factors. Therefore, we need to design updated
risk assessment methods to screen high risk individuals early in their life span.
This chapter defines cardiovascular risk factors, classifies them, briefly describes how they
interact, and discusses what strategies Should be implemented to prevent CVD progression.
2. Definitions
2.1 Coronary Heart Disease (CHD)
Coronary heart disease (CHD) is a condition in which the walls of arteries supplying blood
to the heart muscle (coronary arteries) become thickened. This thickening, caused by
development of lesions in the arterial wall, is called atherosclerosis; the lesions are called
plaques. It can restrict the supply of blood to the heart muscle (the myocardium) and may
manifest to the patient as chest pain on exertion (angina) or breathlessness on exertion.
(Frayn 2005).
2.2 Cerebrovascular disease
Cerebrovascular disease involves interruption of the blood supply to part of the brain and
may result in a stroke or a transient ischemic attack. The loss of blood supply to part of the
brain may lead to irreversible damage to brain tissue. The blockage most commonly arises
from the process of thromboembolism, in which a blood clot formed somewhere else (e.g. in
the heart or in the carotid artery) becomes dislodged and then occludes an artery within the
brain (cerebral arteries). Narrowing of the intracerebral arteries with atherosclerotic plaque
may increase the risk, and may also lead to local formation of a blood clot. The etiology is
similar to that of CHD (Frayn 2005).

The Cardiovascular System Physiology, Diagnostics and Clinical Implications 280
2.3 Peripheral Vascular Disease (PVD)
Peripheral vascular disease (PVD) involves atherosclerotic plaques narrowing the arteries
supplying other regions apart from the myocardium and brain. A common form involves
narrowing of the arteries supplying blood to the legs. The result may be pain on exercise. In
more severe cases, impaired blood supply leads to death of leg tissues, which requires
amputation (Frayn 2005).
3. Epidemiology
3.1 Global and regional trends in CVD burden
In recent years, the dominance of chronic diseases as major contributors to total global
mortality has emerged (WHO, 2008a). By 2005, the total number of cardiovascular disease
(CVD) deaths (mainly coronary heart disease, stroke, and rheumatic heart disease) had
increased globally to 17.5 million from 14.4 million in 1990(WHO, 2009a).
The World Health Organization (WHO) estimates there will be about 20 million CVD deaths
in 2015, accounting for 30 percent of all deaths worldwide (WHO, 2005). Thus, CVD is today
the largest single contributor to global mortality and will continue to dominate mortality
trends in the future (WHO, 2009a).
Globally, there is an uneven distribution of age-adjusted CVD mortality that is mapped in
Figure 1. The lowest age-adjusted mortality rates are in the advanced industrialized
countries and parts of Latin America, whereas the highest rates today are found in Eastern
Europe and a number of low and middle income countries(WHO, 2008a). The broad causes
for the rise and, in some countries, the decline in CVD over time are well described. The key
contributors to the rise across countries at all stages of development include tobacco use and
abnormal blood lipid levels, along with unhealthy dietary changes (especially related to fats
and oils, salt, and increased calories) and reduced physical activity(Hu, 2008; ). Key
contributors to the decline in some countries include declines in tobacco use and exposure,
healthful dietary shifts, population-wide prevention efforts, and treatment interventions
(Shafey et al, 2009; Davies et al, 2007).
4. Pathophysiology
Cardiovascular diseases, whether affecting the coronary, cerebral or peripheral arteries,
share a common pathophysiology involving atherosclerosis and thrombosis (or clotting).
4.1 Atherosclerosis
Atherosclerosis is the most common cause of CVD and related mortality. The first
observable event in the process of atherosclerosis is the accumulation of plaque
(cholesterol from low-density lipoproteins [LDLs], calcium, and fibrin) in large and
medium arteries.
This plaque can grow and produce ischemia either by insufficient blood flow if there is a high
oxygen demand or by rupturing, forming a thrombus and occluding the lumen (Rudd et al.,
2005). Only high-risk or vulnerable plaque forms thrombi. Characteristics of vulnerable

Cardiovascular Disease Risk Factors 281

NOTE: Rates are age-standardized to WHOs world standard population.
SOURCES: WHO, 2009a
Fig. 1. Age-standardized deaths due to cardiovascular disease (rate per 100,000), 2004.

The Cardiovascular System Physiology, Diagnostics and Clinical Implications 282
plaque are lesions with a thin fibrous cap, few smooth muscle cells, many macrophages
(inflammatory cells),and a large lipid core( Figure 2 ) (Rudd et al., 2005). The site of plaque
formation or atherogenesis is the endothelium in the artery wall. Normally the endothelium
promotes dilation of the blood vessel ,less smooth muscle cell growth, and prevention of an
anti-inflammatory response (Davignon and Ganz, 2004). In atherosclerosis the endothelium
becomes dysfunctional before an atheroma or plaque, a more serious lesion, develops. This
endothelial dysfunction results in the production of less nitric oxide, a key vasodilator, and
the blood vessel becomes more constricted .It also becomes more permeable and allows LDL
cholesterol to be taken up by macrophages, which then accumulate and form foam cells and
eventually an early lesion known as a fatty streak.
5. Risk factors for cardiovascular disease
This section described the major risk factors for CVD in more detail. The section begins with
lipid and inflammation-related factors, behavioral risk factors, including tobacco use,
dietary factors, alcohol, and physical activity. This is followed by the major biological risk
factors that mediate the role of these behaviors leading to CVD, including obesity, blood
pressure, blood lipids, and diabetes.
5.1 Modifiable cardiovascular risk factors
5.1.1 Lipid-related factors
Lipid-related cardiovascular risk factors have attracted enormous attention over the past
years, and consensus documents have been produced to implement treatment and
preventive strategies. Essentially, this applies to the conventional risk factors such as high
plasma total and low-density lipoprotein (LDL) cholesterol, low plasma high density
lipoprotein (HDL) cholesterol and elevated plasma triglycerides.

Fig. 2. Natural Progression of atherosclerosis

Cardiovascular Disease Risk Factors 283
5.1.1.1 Atherogenic lipoproteins
A number of factors determine whether a cholesterol-containing lipoprotein particle
resident in plasma has atherogenic properties. The size of the particle determines the ease by
which the endothelium can be penetrated; small particles are more likely to be deposited in
the arterial wall than large particles. The binding to the subendothelial matrix is also
dependent on size, in which the smaller particles bind more avidly to proteoglycans (Anber
et al., 1997). The apoB protein, present as one molecule per lipoprotein particle, seems to be
crucial. Firstly, lipoprotein particles without apoB are not atherogenic; secondly, apoB has
multiple proteoglycan binding domains which enhance the retention of the particle in the
subendothelial matrix (Skalen et al., 2002). Finally, physicochemical and compositional
characteristics, such as resistance factors against oxidative stress, are likely to be important
in reducing the modification of lipoprotein particles.
5.1.1.2 Small, dense Low-Density Lipoprotein (LDL)
The formation of small, dense LDL particles is complex and can be seen as a genetic trait,
but the major gene(s) responsible remain unknown. Environmental factors also play a major
role, in that dietary factors can influence triglyceride as a major determinant. In vitro studies
have shown that small, dense LDL particles are formed by sequential exchange of lipids
between LDL and triglyceride-rich lipoproteins. The cholesteryl esters contained in the core
of the LDL particle are exchanged for triglycerides by the cholesteryl ester transfer protein
(CETP). Triglycerides entering the LDL particle are hydrolyzed by hepatic lipase and the
core volume of the particle is reduced. The formation of small, dense LDL is limited by the
availability of triglyceride-rich lipoproteins, as evidenced by the close positive correlation
between plasma triglycerides and small, dense LDL. It is assumed that these processes take
quite some time and the end product is therefore an aged particle that has lost its defense
against free radical attack. The retention in plasma of the particle is partly due to the fact
that small, dense LDL has a lower affinity for the LDL receptor than normal buoyant LDL
(Nigon et al., 1991). It is thought that a consequence of the altered chemical composition of
the small, dense LDL particle is that it more avidly binds to the subendothelial matrix and
upon challenge more easily undergoes oxidative modification thereby triggering foam cell
formation (Tribble et al., 1992; Chait et al., 1993; Dejager et al., 1993;).
The presence of triglyceride-rich lipoproteins is a principal modulator of small, dense LDL;
the plasma concentration of the latter is strongly and positively related to the concentration
of plasma triglycerides. In fact, all examples in which the triglyceride concentration has been
altered to observe a change in the LDL profile are consistent: elevation of triglycerides leads
to higher abundance of small, dense LDL; the opposite is observed when triglycerides are
lowered, treated by diet or pharmacological agents. Low fat diets may lead to increased
plasma triglyceride concentration; consequently a reduction in LDL size was observed in a
study of 105 men switching from a high fat (46%) to a low fat (24%) diet (Dreon et al., 1994).
The total LDL cholesterol concentration was, however, reduced simultaneously, so the net
effect on cardiovascular risk is not entirely clear.
In the Quebec Cardiovascular Study the cholesterol concentration in small dense LDL
particle may give even more precise information. Again, in the Quebec heart study, the
cholesterol concentration in small dense LDL particles showed the strongest association
with the risk of CHD. These data suggest that the cholesterol within small dense LDL is

The Cardiovascular System Physiology, Diagnostics and Clinical Implications 284
particularly harmful. Therefore measurement of LDL particle size and possibly cholesterol
content within these particles may enhance our capability to predict cardiovasular events
(Lamarche et al., 2001).
5.1.1.3 High-Density Lipoprotein (HDL)
HDL is another lipid profile fraction which is associated to the risk of CVD. Decreased levels
of HDL-c correlated to increased risk of CVD. The atheroprotective role for HDL is mainly
due to the pathway named reverse cholesterol transport (RCT). RCT is defined as the uptake of
cholesterol from peripheral tissues back to the liver by HDL. Apo A1 is an apolipoprotein of
the HDL that activates the enzyme lecithin-cholesteryl ester acyl-transferase (LCAT). The
main function of LCAT is transferring cholesterol from cell to HDL. Both apo C and apo E
on HDL are transferred to chylomicrons.
Apo E helps receptors metabolize chylomicron remnants and also inhibits appetite (Gotoh et
al., 2006). Therefore high HDL levels are associated with low levels of chylomicrons; very
low density lipoprotein (VLDL) remnants; and small dense LDLs and subsequently lower
atherosclerotic risk. HDL has other potentially atheroprotective properties. The anti-
oxidative activity of HDL is typically characterized by its ability to inhibit LDL oxidation. It
has also been shown to inhibit the formation of reactive oxygen species. HDL can help to
protect endothelial cells from apoptosis induced by mildly oxidized LDL. It can affect
platelet function through the promotion of nitric oxide production (Chen et al, 1994; Suc et
al 1997) and coagulation by the inhibition of several coagulation factors. There is
considerable evidence for a direct protective role of HDL in inflammatory, oxidative,
apoptotic, and thrombotic processes.
Major factors that increase HDL cholesterol level are exogenous estrogen, intensive exercise,
loss of excess body fat, moderate consumption of alcohol and triglyceride lowering drugs
such as fibrates and niacin. Treatment of low serum levels of HDL-C in at risk patients is
an important therapeutic intervention and impacts rates of disease progression as well as
cardiovascular events (Scanu and Edelstein , 2008).
5.1.2 Inflammation-related factors
Inflammation is a part of atherosclerosis process, beginning with the formation of fatty
streak underlying the endothelium of large arteries. The infiltration of monocytes and
lymphocytes occurs as a result of the expression of adhesion molecules by endothelial cells
lining the artery wall. Several stimuli for the inflammatory response in atherosclerosis have
been proposed in which oxidised low-density lipoprotein (LDL), is of most importance.
Monocytes that have infiltrated the arterial intima and differentiated into macrophages take
up oxidized LDL through scavenger receptors in an unregulated manner, accumulating
large amounts of cholesterol and becoming foam cells. Macrophages eventually die, through
necrosis and apoptosis, the lipid is deposited within the core of the developing plaque
(Figure 3). Cytokines secreted by both lymphocytes and macrophages within the plaque
exert pro- and anti-atherogenic effects on components of the vessel wall. Smooth muscle
cells migrate from the medial portion of the arterial wall towards the intima and secrete
extracellular matrix proteins that form a fibrous cap. The cap separates the highly
thrombogenic contents of the plaque lipid core from the potent coagulation system
contained within the circulating blood.

Cardiovascular Disease Risk Factors 285
This chronic, low-grade inflammation is likely to be the result of cytokines secreted by
monocytes and soluble adhesion molecules from the vessel wall into the circulation, where
they subsequently act on the liver to induce the secretion of acute phase proteins, including
C-Reactive Protein(CRP), fibrinogen and serum amyloid A (Frayn, 2005).

From Frayn 2005
Fig. 3. Schematic representation of the development of an atherosclerotic lesion, showing
plaque rupture and platelet aggregation .
5.1.2.1 C-Reactive protein
Multiple studies have demonstrated that elevated levels of high-sensitivity C-reactive
protein (hs-CRP) are associated with increased CVD risk (Buckley et al.,2009; Musunuru et
al., 2008). hs-CRP, previously considered to be an indicator of systemic inflammation, has
recently received much attention in the scientific literature, not only as a potential marker of
increased atherosclerotic risk, but also as a potential target of therapy for the prevention of
atherosclerotic CVD. Evidence derived mainly from statin trials, supports the potential
value of CRP as a therapeutic target for both primary and secondary prevention of CVD and
CHD. The largest study to suggest an integral role for CRP as a target for therapy in primary
prevention of CVD is the recent Justification for the Use of Statins in Prevention: An
Intervention Trial Evaluating Rosuvastatin (JUPITER) trial. The investigators randomized
17,802 men 50 years of age and women 60 years of age with low LDL cholesterol levels <
130 mg/dL and hs-CRP 2 mg/L and no history of CVD or diabetes to 20 mg rosuvastatin
daily or placebo. The primary end point was the first occurrence of MI, stroke,
hospitalization for unstable angina, arterial revascularization, or CV death (Ridker et al;
2008). During the 1.9-year median follow-up duration (maximum follow-up period 5 years),
rosuvastatin reduced LDL cholesterol by 50% and hs-CRP by 37%. Thus, JUPITER
demonstrated a magnitude of effect larger than that of almost all prior statin trials (Ridker
et al; 2009). Based on the results of the JUPITER study, the U.S. Food and Drug
Administration (FDA) in February 2010 agreed to broader labeling for rosuvastatin.
Rosuvastatin is currently approved for the reduction of risk for stroke, MI, and
revascularization procedures in individuals who have normal LDL cholesterol levels and no
clinically evident CHD but who do have an increased risk based on age, CRP levels, and the

The Cardiovascular System Physiology, Diagnostics and Clinical Implications 286
presence of at least one additional CVD risk factor. Accordingly, JUPITER not only
demonstrated that hs-CRP successfully identified a population with hidden risk for CVD
but also provided additional evidence for the potential utility of hs-CRP as a target for
therapy in primary prevention of CVD disease.
Other inflammatory states such as obesity produce elevations in CRP even in young obese
children (Blum et al., 2005). Body mass index (BMI) is moderately correlated (r = 0.5) to CRP
levels (Rawson et al., 2003). Recently it was demonstrated that weight loss lowers CRP
(Tchernof et al., 2002), which provides another physiologic benefit for weight management
as a preventive strategy for CHD reduction. Currently it is known that elevated insulin
levels in overweight children affect CRP. Physical activity did not appear to be related
(Rawson et al., 2003). To date few studies have investigated the effects of dietary variables
on CRP. In a cross-sectional study, higher intakes of fruits and vegetables were associated
with lower CRP levels (Gao et al., 2004).
5.1.2.2 Fibrinogen
Fibrinogen is the precursor of fibrin. As the major clotting factor in the blood and a pro-
inflammatory molecule, fibrinogen plays role in atherosclerosis. It is synthesized in the liver
and, like CRP, is an acute phase protein, whose circulating levels can change during acute
responses to tissue damage or infection. Thus Fibrinogen (Fg) is a biomarker of
inflammation (Ross 1999), which, when elevated, indicates the presence of inflammation
and identifies individuals with a high risk for cardiovascular disorders. Increased plasma Fg
concentration typically accompanies hypertension development (Lominadze et al. 1998) and
stroke (D'Erasmo et al. 1993). Factors associated with an elevated fibrinogen are smoking,
diabetes, hypertension, obesity, sedentary lifestyle, elevated triglycerides, and genetic
factors. Recent studies indicate that increased Fg content affects microcirculation by
increasing plasma viscosity, RBC aggregation and platelet thrombogenesis (Lominadze et al;
2010). These changes lead to vascular dysfunction and exacerbate microcirculatory
complications during cardiovascular diseases (figure 4).

From Lominadze et al. 2010
Fig. 4. Schematic representation of fibrinogen-induced vascular dysfunction.

Cardiovascular Disease Risk Factors 287
5.1.2.3 Serum amyloid A
Serum amyloid A (SAA) is a precursor of amyloid A protein and comprises both
constitutive (apoSAA1, apoSAA2) and acute phase (apoSAA4) isoforms. The serum amyloid
A proteins are a family of inflammatory apolipoproteins with a high affinity for HDL, and
their production by the liver and other tissues is thought to be induced by IL-1 and IL 6.
Their role in lipid metabolism is unclear, although they may be involved in HDL trafficking.
A small number of studies have investigated the association between SAA and the incidence
of CHD ( Ridker et al., 1998, 2000; Danesh et al., 2000b). Taken together, these studies show
that a comparison of individuals with values in the top third with those in the bottom third
gives a combined risk ratio of 1.6 for CHD. However, further studies are required to
determine whether the association is independent of possible confounders.
5.1.3 Behavioral and lifestyle risk factors
5.1.3.1 Tobacco
There are currently more than 1 billion smokers worldwide. Although use of tobacco
products is decreasing in high income countries, it is increasing globally, with more than 80
percent of the worlds smokers now living in low and middle income countries (Jha and
Chaloupka, 1999).
In the Global Burden of Disease study, Lopez et al. (2006) estimated that in 2000, 880,000
deaths from CHD and 412,000 deaths from stroke were attributable to tobacco. Smoking
cessation has been shown to have significant impacts on reducing CHD. In a major review
of the evidence, Critchley and Capewell (2003) determined that successful smoking
cessation reduced CHD mortality risk by up to 36 percent. Smoking cessation leads to
significantly lower rates of reinfarction within 1 year among patients who have had a
heart attack and reduces the risk of sudden cardiac death among patients with CHD
(Gritz et al., 2007).
Two major trends are of real concern with respect to the future of tobacco-related CVD. First,
in most parts of the world, the smoking rates are higher among the poorest populations
(WHO, 2008b). The second worrisome trend is in smoking among girls (IOM, 2010).
In addition to active smoking, it has become increasingly apparent that exposure to
secondhand smoke significantly increases cardiovascular risk. A recent IOM review of the
effects of secondhand smoke exposure concluded that exposure to secondhand smoke
significantly increases cardiovascular risk and that public smoking bans can significantly
reduce the rate of heart attacks. The report concluded that secondhand smoke exposure
increases cardiovascular risk by 25 to 30 percent and that there is sufficient evidence to
support a causal relationship between secondhand smoke exposure and acute myocardial
infarction (AMIs). This causality was reinforced by the reports conclusion that smoking
bans significantly reduce the rate of AMIs, with declines ranging from 6 to 47 percent (IOM,
2009).
5.1.3.2 Dietary factors
The relationship between CVD and diet is one of the most studied relationships in
epidemiology. Although nutritional research has traditionally focused on the effect of

The Cardiovascular System Physiology, Diagnostics and Clinical Implications 288
individual food groups or nutrients on CVD, there has been a shift in recent years toward
comparing how different types of dietary patterns in their entirety affect CVD risk (IOM,
2010). The following sections reflect this shift by discussing research on dietary factors that
have clear impacts on CVD risk.
5.1.3.2.1 Dietary fat
Healthy oils are those that contain no commercially introduced trans fatty acids, are low in
saturated fatty acids, and are high in mono- and polyunsaturated fatty acids.
There is accumulating evidence that it is fat quality (the type of dietary fat), rather than the
total amount of fat, that is particularly important for cardiovascular disease (Astrup, 2002).
5.1.3.2.1.1 Saturated Fatty Acids (SFA)
The predominant sources of SFAs are animal foods (meat and dairy). SFAs have the most
potent effect on LDL cholesterol, which rises in a dose response fashion when increasing
levels of SFAs are consumed. In our study, consumption of habitual hydrogenated fats and
full-fat yoghurts (fat content more than 2.5%) increased the risk of CVD (OR = 2.12(1.23-
3.64) and 2.35(1.32-4.18), respectively) (Amani et al, 2010). These foods are the major sources
of SFAs. SFAs intake is the principal determinant of total cholesterol(TC) and CVD and
substitution of 1% carbohydrate calories by SFAs increases TC by 1.5 mg/dL (Joseph et al,
2000). Of all the added fats in the diet, the most hypercholesterolemic promoting are palm
kernel, coconut, and palm oils; lard; and butter. SFAs raise serum LDL cholesterol by
decreasing LDL receptor synthesis and activity. Regardless of form, all fatty acids lower
fasting triglycerides if they replace carbohydrate in the diet. In secondary prevention trials
replacement of SFAs with MUFA, -linolenic acid, and increased fruits and vegetables
prevented fatal and nonfatal CVD events in persons with established disease (de Lorgeril,
1999). Thus fatty acids affect disease progression through lipids and other mechanisms and
possibly through inflammation and thrombosis (Krummel, 2008).
5.1.3.2.1.2 Monounsaturated Fatty Acids (MUFA)
The American Heart Association (AHA) does not have any recommendation for the cis form
of MUFAs (Lichtenstein et al., 2006). Oleic acid (C18:1) is the most prevalent MUFA in the
American diet. Substituting oleic acid for carbohydrate has almost no appreciable effect on
blood lipids; however, replacing SFAs with MUFA lowers serum cholesterol levels, LDL
cholesterol levels, and triglyceride levels to about the same extent as polyunsaturated fatty
acids (PUFAs ) . The effects of MUFAs on HDL cholesterol depend on the total fat content of
the diet. When intakes of both MUFA (>15% of total kilocalories) and total fat (>35% of
kilocalories) are high, HDL cholesterol does not change or increases slightly compared with
levels with a lower-fat diet (Krummel , 2008). Oleic acid as a part of the Mediterranean diet
has been shown to have antiinflammatory effects. In epidemiologic studies high-fat diets of
people in Mediterranean countries have been associated with low blood cholesterol levels
and CHD incidence (Trichopoulou et al., 2003). Among other factors, the main fat source is
olive oil, which is high in MUFA. Although higher-fat diets (low in SFA with MUFAs as the
predominant fat) can lower blood cholesterol, they should be used with caution because of
the caloric density of high-fat diets and the results of clinical trials, which have shown new
atherosclerotic lesions in men who consume higher-fat diets. The negative association
between the Mediterranean diet and CHD could be the result of factors other than MUFA

Cardiovascular Disease Risk Factors 289
intake. For example, these populations consume more fruits and vegetables, bread, cereals,
fish, and nuts, and less red meat than many populations. Olive oil is the primary source of
fat, and eggs are consumed from zero to four times per week (krummel, 2008).
5.1.3.2.1.3 Polyunsaturated Fatty Acids (PUFA)
The essential fatty acid linoleic acid (LA) is the predominant PUFA consumed in the
American diet (Krummel, 2008). Population studies have demonstrated a negative
correlation between LA intake and CHD rates (Wijendran and Hayes, 2004). Similarly, a
meta analysis of 60 controlled human trials found that replacing PUFA for carbohydrate in
the diet resulted in a decline in serum LDL cholesterol (Mensink et al., 2003). When SFAs are
replaced with PUFAs in a low-fat diet, LDL and HDL cholesterol levels will be lowered. The
lipid lowering effects of LA depend on the total fatty acid profile of the diet (Wijendran and
Hayes, 2004). When added to study diets, large amounts of LA diminished levels of HDL
cholesterol serum levels (Karmally, 2005). Studies suggest that high intakes of n-6 PUFAs
may exert adverse effects on the function of vascular endothelium or stimulate production
of proinflammatory cytokines. A low ratio of omega-6:omega-3 PUFA is recommended
(Basu et al., 2006; Gibauer eial., 2006).
5.1.3.2.1.4 Omega-3 fatty acids
Fish oils, fish oil capsules, and ocean fish are rich source of the two main omega-3 fatty acids
(i.e., eicosapentaenoic acid (EPA) and docosahexaenoic acid [DHA]). Many studies have
shown that eating fish is associated with a decreased CVD risk. The recommendation for the
general population for fish consumption is to eat fish high in omega-3 fatty acids (salmon,
tuna, mackerel, sardines) at least twice a week (Psota et al., 2006). For patients who have
CVD, 1 g of EPA and DHA combined is recommended from fish if possible but, if not, then
from supplements (Lichtenstein et al., 2006). Patients who have hypertriglyceridemia need 2
to 4 g of EPA and DHA per day for effective lowering (Lichtenstein et a1.,2006). Omega-3
fatty acids lower triglyceride levels by inhibiting VLDL and apo B-100 synthesis and by
decreasing postprandial lipemia.
-Linolenic acid (ALA), an omega-3 fatty acid from vegetables, has anti-inflammatory
effects. CRP levels were reduced when male patients consumed 8 g of ALA daily; similar
results have not been observed for fish oil supplementation (Basu et a1.,2006). Omega-3 fatty
acids also interfere with blood clotting by altering prostaglandin synthesis (Krummel, 2008).
5.1.3.2.1.5 Trans fatty acids
Trans-fatty acids are produced in the hydrogenation process used in the food industry to
increase shelf life of foods and to make margarines, firmer (Krummel ,2008). The AHA
(Lichtenstein et al., 2006) recommends no more than 1% of calories (about 1-3 g/day) from
trans-fatty acids. These fatty acids raise LDL cholesterol; however, effects on inflammation
have been conflicting (Basu et a1.,2006). Most trans-fatty acids intake comes from partially
hydrogenated vegetable oils (krummel, 2008).
Mozaffarian et al (2007) showed that partially hydrogenated oils are extensively being used
for cooking in Iranian homes with average per-person intake of 14 g/1000 kcal. Trans fatty
acids (TFAs) accounted for 33% of fatty acids in these products, or 4.2% of all calories
consumed (12.3 g/day). Consumption of hydrogenated fats was associated with higher

The Cardiovascular System Physiology, Diagnostics and Clinical Implications 290
CAD risk (OR = 2.12(1.23-3.64)) in a study performed by Amani et al (2010). On the basis of
TC:HDL-cholesterol effects alone, 9% of CHD events would be prevented by replacement of
TFA in homes with cis-unsaturated fats (8% by replacement with saturated fats). On the
basis of relationships of TFA intake with CHD incidence in prospective studies, 39% of CHD
events would be prevented by replacement of TFA with cis-unsaturated fats (31% by
replacement with saturated fats).
5.1.3.2.1.6 Dietary cholesterol
Dietary cholesterol raises total cholesterol and LDL cholesterol but to a lesser extent than SFAs.
The AHA dietary patterns contain no more than 200 mg of cholesterol each day (Krummel;
2008). There is a threshold beyond which addition of cholesterol to the diet has minimal
effects. When cholesterol intakes reach 500 mg/day, only small increments in blood
cholesterol occur. Cholesterol responsiveness also varies widely among individuals. Some
people are hyporesponders (i.e., their plasma cholesterol level does not increase after dietary
cholesterol challenge), whereas others are hyperresponders (i.e., their plasma cholesterol level
responds more strongly than expected to a cholesterol challenge). It has been suggested that
hyperresponders may have the apo E-4 allele and poor rates of conversion of cholesterol to bile
acids, which causes elevated LDL cholesterol. Feedings cholesterol to animals enriches
lipoproteins, which are atherogenic beyond just the rise in serum cholesterol (Krummel, 2008).
SFAs and cholesterol synergistically affect LDL cholesterol level, decrease LDL receptor
synthesis and activity, increase VLDLs enriched with apo E, increase all lipoproteins, and
decrease chylomicron size ( which is associated with CHD risk). The effect of dietary
cholesterol on inflammatory factors has been inconsistent (Basu et a1.,2006).
5.1.3.2.2 Dietary sodium
There is evident that excessive sodium intake significantly increases CVD risk and that
reduction in sodium intake at the population level decreases CVD burden (He and
MacGregor, 2009, IOM, 2010 ). The most well-established mechanism by which sodium
intake increases CVD risk is increasing blood pressure (BP). Numerous studies have
found that there is a continuous and graded relationship between salt intake and blood
pressure. In their recent major review of sodium trends and impact, He and MacGregor
concluded that a reduction in salt from the current global intake of 9 to 12 g/day to the
recommended levels of 5-6 g/day would have a major impact on BP and CVD (He and
MacGregor, 2009; IOM, 2010). Salts impact on CVD, however, extends beyond blood
pressure. Animal and epidemiological studies have found that a diet high in sodium may
directly increase the risk of stroke, which is independent and additive to salts effect on
BP (He and MacGregor, 2009 ; IOM, 2010).
5.1.3.2.3 Soy protein
In recent years, a great deal of interest has emerged in the role of soy-bean isoflavones in
reducing heart diseases, and isoflavones might be responsible, in part, for the ability of
soybean to lower the risk of CVD and atherosclerosis (Anderson et al, 1995) . Anderson
suggested that about 60-70% of the cholesterol lowering effect of soy protein may be due to
its isoflavone content (Anderson et al, 1995). Isoflavones are a group of phytoestrogens
which occur mainly in soy and it is consumed for the purpose of both promoting health and
preventing several chronic diseases, including coronary heart disease, cancers of

Cardiovascular Disease Risk Factors 291
reproductive organs and osteoporosis ( Lichtenstein, 1998 ; Anerson et al, 1999). Aglycone
forms of soy isoflavones especially genistein and daidzein have greatly been studied because
of their greater estrogenic and antioxidant activities (Arora et al, 1998). Soy isoflavones have
been shown to decrease total, VLDL and LDL cholesterol levels while increasing HDL
cholesterol levels in peripubertal rhesus monkeys fed soy protein-based diets (Antony et al,
1996). It is claimed that purified isoflavones have no effect on plasma lipid and lipoprotein
concentrations in normolipidemic subjects (Nestel et al, 1997; Hodgson et al, 1998). At present,
there is no general agreement about the effect of soy protein isoflavones (SPI) on lipid profiles
and moreover, it is not clear that which part of the soy protein has lipid-lowering effects. In a
study, we designed animal model to assess the effect of SPI on serum lipid, lipoprotein profile,
and blood sugar of experimentally- induced hypercholesterolemic rabbits,and to detect any
dose-response effect of SPI on the above mentioned variables. In this research, the effect of soy
protein containing 200 mg, 100 mg and a trace amount of both glycoside and aglycone forms
of soy isoflavones were assessed in hypercholesterolemic male rabbits. Although the rabbits
had a cholesterol-rich diet, the serum total and LDL-cholesterol remained unchanged in the
SPI+ group (i.e. intact soy protein diet). The results have indicated that soy protein isoflavones
maintained the serum lipid and lipoprotein levels in hypercholesterolemic rabbits kept on a
high cholesterol diet, but alcohol-extracted (even half-dose isoflavones) soy protein diets do
not have positive effect. Moreover, the hypocholesterolemic effect of isoflavones is not in a
dose-response manner and it is suggested that isoflavones activity is closely related to soy
protein (Amani et al, 2005).
5.1.3.2.4 Fiber
One of the potential ways by which soy protein might exert its effect on blood cholesterol is
via its fiber content (about 6 g as non-starch polysaccharide per 100 g boiled beans), which is
primarily soluble fiber. Soluble fiber (e.g. from oats) has been shown to lower plasma total
and LDL-cholesterol, although the effect is small for those consuming moderate amounts
(Truswell, 2002). In the meta-analysis by Brown et al. (1999) 210 g/day of soluble fiber was
associated with a small but significant fall in total cholesterol (0.045 mmol/l per g fiber) and
LDL-cholesterol (0.057 mmol/l per g fiber). Three apples or three (28 g) servings of oatmeal,
providing 3 g soluble fiber, decreased total and LDL-cholesterol by about 0.13 mmol/l. The
mechanism of this effect remains undefined. Suggestions include bile acid binding, resulting
in an up-regulation of LDL receptors and thus increased clearance of LDL-cholesterol;
inhibition of hepatic fatty acid synthesis byproducts of fermentation in the large bowel (e.g.
propionate, acetate, butyrate); changes in motility or satiety; or slowed absorption of
macronutrients resulting in improved insulin sensitivity (Brown et al., 1999).
Consumption of diets rich in whole-grain cereals (e.g. whole-wheat cereals, whole meal
bread and brown rice) has been associated with a lower risk of cardiovascular disease
(Pietinen et al., 1996; Jacobs et al., 1999; Liu et al., 1999; Truswell, 2002). Vitamin E, dietary
fibre (Richardson, 2000), resistant starch and oligosacchrarides (Cummings et al., 1992), as
well as plant sterols (Jones et al., 1997) are some of the components of whole-grain cereals
that may contribute to a reduced risk of heart disease (Mc Kevith (2004) .
5.1.3.2.5 Antioxidant
Vitamins C, E, and B-carotene have antioxidant roles in the body. Vitamin E is the most
concentrated antioxidant carried on LDLs and its major function is to prevent oxidation of

The Cardiovascular System Physiology, Diagnostics and Clinical Implications 292
PUFA in the cell membrane (Krummel, 2008). Epidemiologic studies suggest that vitamin E
and carotenoids are inversely related to CVD, but randomized trials have not supported
these observations (Lee et al., 2005; Lichtenstein et al., 2006). Because data have not shown
vitamin E to be protective, the AHA does not recommend vitamin E supplementation for
CVD prevention (Lichtenstein et al., 2006). However, RRR-a-tocopherol, the natural form of
vitamin E, shows promise as an antiinflammatory agent (Gasu et al., 2006). Foods with
concentrated amounts of the phytonutrients catechins, have been found to improve vascular
reactivity. These foods are red grapes, red wine, tea (especially green tea), chocolate, and
olive oil, and should be worked into any CVD preventive eating plan (Kay et al., 2006). In
our case control study, drinking tea was significantly associated with lower risk of
coronary events (Amani et al, 2010).
5.1.3.2.6 Stanols and sterols
Since the early 1950s plant stanols and sterols isolated from soybean oils or pine tree oil
have been known to lower blood cholesterol (Lichtensteine et al., 2001). Recently they have
been esterified and made into margarines. Consuming between 2 to 3 g/day lowers
cholesterol by 9% to 20% (Lichtenstein et al., 2001). The mechanism for cholesterol lowering
is by inhibiting absorption of dietary cholesterol. Adult Treatment Panel III (ATP-III)
includes stanols as part of dietary recommendations for lowering LDL cholesterol in adults.
Because these esters can also affect the absorption of and cause lower -caroten , -
tocopherol, and lycopene levels, further safety studies are needed for use in
normocholesterolemic individuals, children, and pregnant women (Krummel, 2008).
5.1.3.2.7 Dietary patterns
The effect on CVD risk of diets rich in whole grains and low in processed foods that are high in
fat, sodium, and sugars has increasingly been investigated in both developed and developing
countries. In parallel with economic development, radical dietary shifts toward Westernized
diets that are high in animal products and refined carbohydrates and low in whole grains and
other plant-based foods have occurred in many developing countries. For example in Iran, the
results of Amani et al (2010) study showed that daily consumption of vegetable oils, tea and
fish is significantly associated with lower risk of coronary events (odds ratio = 0.55(0.31-0.91),
0.3(0.15-0.65), 0.23(0.13-0.42), respectively). On the other hand, it was indicated that
consumption of hydrogenated fats and full-fat yoghurt is associated with higher risk of
coronary artery disease (OR = 2.12(1.23-3.64) and 2.35(1.32-4.18), respectively).
Substantial evidence has accumulated to support the notion that the traditional
Mediterranean dietary pattern is protective against CVD . This pattern is characterized by
an abundance of fruits, vegetables, whole grain cereals, nuts, and legumes; olive oil as the
principal source of fat; moderate consumption of fish and lower consumption of red meat. It
is important to note, however, that the dominance in research on the Mediterranean diet has
come at the cost of research on other diets commonly consumed around the world that may
also have heart health benefits (IOM , 2010).
5.1.3.2.8 Therapeutic life style change dietary pattern (TLC)
The ATP-III recommends the TLC dietary pattern for primary and secondary prevention of
CHD. AHA recommends diet and lifestyle changes to reduce CVD risk in all people over the
age of 2 years (Table 1) (Lichtenstein et al., 2006). SFA recommendations are less than 7% of
calories; total fat content has a range of 25% to 35% of calories.

Cardiovascular Disease Risk Factors 293
Consuming 30% to 35% of calories from fat while maintaining a low SFA and trans-fatty
acid intake is the dietary pattern recommended for individuals with insulin resistance or
metabolic syndrome. This higher fat intake, emphasizing PUFAs and monounsaturated fatty
acids (MUFA), can be beneficial in lowering triglycerides and raising HDL cholesterol. Also,
with a more liberal fat intake, LDL cholesterol can be lowered without exacerbating blood
glucose levels. Increasing physical activity and decreasing energy intake to facilitate weight
loss are critical to normalize multiple risk factors. Behavioral strategies for weight
management to reduce cardiovascular risk have been provided by the AHA (Klein et al.,
2004). Learning outcomes include planning meals that fit the TLC plan, reading food labels,
modifying recipes, preparing or purchasing appropriate foods, and choosing healthier
choices when dining out. Along with the TLC dietary pattern, the Dietary Approaches to
Stop Hypertension (DASH) pattern is also appropriate for CVD prevention and treatment .
Both of these dietary patterns emphasize grains, cereals, legumes, vegetables, fruits, lean
meats, poultry fish, and nonfat dairy products.
Because animal fats provide about two thirds of the SFAs in diet, these foods are limited.
High-fat choices are omitted, but low-fat choices can be included. Meat is limited to 5
oz/day, and eggs to four or fewer per week. Lean meats are high in protein, zinc, and iron;
thus, patients who wish to consume meat, a 5-oz portion or less can be fit into the dietary
plan if other low SFA choices are made. Neither food group has to be omitted; it is a matter
of choice. Most people need to add the recommended two servings of fatty fish per week.
Meeting sodium guidelines (1500 to 2300 mg daily) can be a challenge because lower-fat
processed foods often contain salt to increase palatability. Patients may need to limit
processed foods (Krummel 2008).
5.1.3.3 Alcohol
The global burden of diseases attributable to alcohol has recently been summarized; leading
to the conclusion that alcohol is one of the largest avoidable risk factors in low and middle
income countries (Rehm et al., 2009). Indeed, WHO estimates that the harmful use of alcohol
was responsible for 3.8 percent of deaths and 4.5 percent of the global burden of disease in
2004 (WHO, 2009b). Excessive alcohol intake is associated with increased risk for
hypertension, stroke, coronary artery disease, and other forms of CVD; however, there is
also a robust body of evidence in a range of populations suggesting light to moderate intake
of alcohol may reduce the risk of CHD. Indeed, research suggests that the relationship
between alcohol intake and CVD outcomes follows a J curve, with the lowest rates being
associated with low to moderate intakes of alcohol (Beilin and Puddey, 2006; Lucas et al.,
2005). It is important to recognize that, as with any discussion of alcohol and health, the key
issues are the quantity of alcohol consumed and the risk or benefit conferred by
consumption. Although evidence indicates that low to moderate alcohol use can reduce the
risk of CHD, excessive and harmful use clearly increases CVD risk (Beilin and Puddey, 2006;
Lucas et al., 2005). It is important that approaches to reduce the burden of CVD not neglect
the importance of reducing excessive alcohol consumption.
5.1.3.4 Physical activity
WHO and FAO have highlighted the importance of physical activity as a key determinant of
obesity, CVD, and diabetes (Joint WHO/FAO Expert Consultation, 2003). For decades,
evidences of the relationship between physical activity and CVD, independent of effects on

The Cardiovascular System Physiology, Diagnostics and Clinical Implications 294
weight and obesity, have been strengthened. Increasing physical activityincluding brisk
walkinghas been shown to decrease the risk of chronic diseases such as CHD, stroke,
some cancers (e.g., colorectal and breast cancer), type 2 diabetes, osteoporosis, high blood
pressure, and high cholesterol (Physical Activity Guidelines Advisory Committee, 2008)

Table 1.
Regular physical activity and higher cardiorespiratory fitness are associated with lower
overall mortality from CVD. Men and women who are physically active experience a lower
risk of cardiovascular disease in general and CHD in particular (US Department of Health

Cardiovascular Disease Risk Factors 295
and Human Services, 1996; Wannamethee & Shaper, 2002). Furthermore, in the Nurses
Health Study, a large prospective study in the USA, both brisk walking and regular
vigorous exercise were associated with a reduction in risk of coronary events by 3040%
(Manson et al., 1999), and sedentary women who became active in middle age or later had a
lower risk than their counterparts who remained sedentary . In one study performed among
145 women with central obesity, serum concentration of HDL-c was significantly higher in
women who do more physical activity (Sharifi et al,2008). With overall mortality, the
epidemiological literature for CHD indicates an inverse association and a dose response
gradient between physical activity level or cardiorespiratory fitness and incidence of CHD.
It helps to improve several risk factors for cardiovascular disease, including raised blood
pressure, adverse blood lipid profile and insulin resistance.
5.1.4 Related diseases/syndrome
5.1.4.1 Overweight and obesity
Traditionally, obesity is defined as a BioPsychoSocial problem but in this chapter we rather
intend to present it as an EcoBioPsychoSocioCutural issue.
Overweight and obesity have reached epidemic proportions, not only in developed but also
in developing countries (Sassi et al., 2009). Even in low and middle income countries where
undernutrition is still highly prevalent, overweight and obesityespecially among
womenis a public health problem (Caballero, 2005; Hosseinpanah et al 2009). WHO and
FAO reviewed the evidence on the relationship between obesity and the risk of CVD and
concluded that overweight and obesity confer a significantly elevated risk of CHD (Joint
WHO/FAO Expert Consultation, 2003). Increased body mass index (BMI) is also associated
with greater risk of stroke in both Asian and Western populations (WHO/FAO, 2003). The
association between obesity and CVD is partly mediated through hypertension, high
cholesterol, and diabetes. Abdominal or central obesity measured by waist-to-hip ratio or
waist circumference is associated with both CHD and stroke independent of BMI and other
cardiovascular risk factors. Even in university educated women, obesity and central fat are
also prevalent which increase the risk of heart disease. We studied 101female staff of the
university, aged 20-45 years. Based on the bioelectrical impedance analysis (BIA) method,
overweight and obesity rates were determined in 34.6 and 40.6 percent of women,
respectively, and central obesity was prevalent in 27% of them (Amani, 2007a). It is worthy
to note that to prevent erroneous classification, localization of cut-off points can be a more
practical way to detect individuals at greater risk of chronic disease. In a sample of 637
married females, it was indicated that subjects in low BMI range tend to have higher fat
percentages and they might represent a different category as overfat thin other than normal
weight obese in Iranian women. (Amani, 2007b).
Obesity is also an independent risk factor for other cardiovascular outcomes, such as
congestive heart failure and sudden cardiac death. Excess energy intake is one of the key
contributors to obesity. The lack of data limits policy makers abilities to focus attention on
which dietary components lend to effective interventions that would reduce total calorie
intake. One category that has been well studied in developed countries relates to sugar
consumption, primarily in the form of sugar-sweetened beverages (including soft drinks,

The Cardiovascular System Physiology, Diagnostics and Clinical Implications 296
juice drinks, and energy and vitamin drinks). Recent NHANES data shows that up to 5.5
percent of dietary calories come from sugar-sweetened beverages in the United States
(Bosire et al., 2009), which has led the American Heart Association to recommend an upper
limit of 100 calories per day for women and 150 calories per day for men from added sugars,
including soft drinks (Johnson et al., 2009). In some developing countries, consumption of
sugar-sweetened beverages has increased dramatically in recent decades. Because of its
excess caloric and sugar content, increasing consumption of sugar sweetened beverages may
have important implications for obesity and cardiometabolic risk.
5.1.4.2 Hypertension
Hypertension is a risk factor for CHD, stroke, and heart failure. A recent review of the
global burden of high blood pressure found that approximately 54 percent of stroke, 47
percent of IHD and 25 percent of other CVDs were attributable to hypertension. Among the
major underlying risks for hypertension are sodium, body weight, and access to treatment.
Primary prevention focused on sodium reduction, fruit and vegetable intake, weight
control, and avoidance of excessive alcohol intake has been shown to make a difference
(Krummel, 2008).
5.1.4.3 Diabetes
Around the world, diabetes is increasingly growing and is a significant contributor to CVD
risk. People with diabetes have more than two-fold greater risk of CVD compared to non-
diabetics (Asia Pacific Cohort Studies Collaboration, 2003). In fact, CVD is the leading cause
of morbidity and mortality in people with diabetes (Booth et al., 2006; Kengne et al., 2007,
2009). Individuals without established clinical diabetes, but who are at increased risk of
developing diabetes in the future, also have a higher risk of CVD (Asia Pacific Cohort
Studies Collaboration, 2007). Women and younger individuals with diabetes have greater
risk of CVD. Obesity is the single most important risk factor for type 2 diabetes, but
unhealthy diet and physical inactivity also independently raise the population risk for
diabetes (Schulze and Hu, 2005). According to the International Diabetes Federations
Diabetes Atlas 2010, the global estimated prevalence of diabetes for 2010 among people aged
20 to 79 years will be approximately 285 million people (6.4 percent of the global
population), of which some 70 percent will be living in developing countries (International
Diabetes Federation, 2010).
Diabetes is emerging as a particular concern in Asia, where more than 110 million
individuals were living with diabetes in 2007, a large proportion of whom were young and
middle aged. Asians tend to develop diabetes at a relatively young age and low BMI, and by
2025 the number of individuals with diabetes in the region is expected to rise to almost 180
million (Chan et al., 2009). Intensive glucose control reduces the risk of major cardiovascular
events by approximately 10 percent, compared with standard treatment in people with
diabetes. Interestingly, this benefit appeared to be independent of other cardiovascular risk
factors (Kelly et al., 2009; Turnbull et al., 2009).
To sum up, as with the raising obesity epidemic, the prevalence of diabetes has increased
dramatically worldwide. It is associated with serious health consequences and is a major
risk factor for CHD and stroke. Therefore, prevention and management of diabetes are
critical in reducing the global burden of CVD.

Cardiovascular Disease Risk Factors 297
5.1.4.4 Psychosocial risk and mental health
Of all the psychosocial stressors associated with CVD, the link between depression and CVD
is probably the best documented. There have been many published reviews and numerous
meta-analyses have consistently found that depression and depressive symptoms are
associated with an increased likelihood of developing CVD, a higher incidence of CVD
events, poorer outcomes after CVD treatment and prevention efforts, and increased
mortality from CVD. These associations remain consistent even after controlling for other
CVD risk factors (Everson-Rose and Lewis, 2005; Frasure-Smith and Lesperance, 2006;
Lesperance and Frasure-Smith, 2007; Lichtman et al., 2008).
Behaviors that increase CVD risks are more common in depressed patients. They are more
likely to smoke, have poor diets, and be physically inactive. Furthermore, depression has
been found to associate with the risk of non adherence to medical treatment regimens and
lifestyle changes, making depressed patients with CVD or high CVD risk less likely to
adhere to prevention efforts (Lichtman et al., 2008; Ziegelstein et al., 2000).
Chronic stress, most often studied by examining work-related stress, has been associated
with negative behaviors such as low physical activity and poor diet, increased likelihood of
recurrent CVD, as well as physiological consequences such as decreased heart rate
variability. For instance, we found that the prevalence of overweight and obesity was higher
in a sample of firefighters who may have chronic stress. Moreover they had high TC, TG
and lipoprotein (a) and low HDL-C concentrations (Azabdaftari et al, 2009).
Acute stress from traumatic life events such as the death of a relative, earthquakes, or
terrorist attacks have all been associated with significant temporal increases in the incidence
of MI (Everson-Rose and Lewis, 2005; Figueredo, 2009).
It is clear that psychosocial factors play an important role in increasing CVD risk through both
direct and indirect pathways. Continued research is needed to further explain the mechanisms
by which psychosocial stressors and mental illness affect CVD risk. It is also important that
clinicians are made aware of the effect of psychosocial factors on CVD risk, prognosis, and
adherence to prevention efforts through improved training and knowledge expanding.
5.2 Nonmodifiable factors
5.2.1 Menopausal status
Loss of estrogen following natural or surgical menopause is associated with increased CVD
risk. Endogenous estrogen has a protective role against CVD in premenopausal women,
probably by preventing vascular injury. Rates of CHD in premenopausal women are low
except in women with multiple risk factors. During the menopausal period total cholesterol,
LDL cholesterol, and triglyceride levels increase; and HDL cholesterol level decreases,
especially in women who are overweight or obese (Regitz-Zagrosek, 2006).
5.2.2 Age and gender
Age is a nonmodifiable risk factor for CHD. The increased risk for CHD parallels increase
in age. Higher mortality rates from CHD are seen in both genders with increasing age.

The Cardiovascular System Physiology, Diagnostics and Clinical Implications 298
Being older than 45 years of age is considered a risk factor for men (NCEP, 2002). For
women the increased risk comes after the age of 55 years, which is after menopause for
most women.
CVD prevalence, incidence, and mortality rates tend to be higher for men than for women.
This finding has remained consistent historically (Lawlor et al., 2001) and across countries
and regions (Allen and Szanton, 2005; Pilote et al., 2007; WHO, 2009).
Estrogen has a protective effect on the development of CVD risk factors and consequently is
the reason most often cited for these gender differences (Regitz-Zagrosek, 2006). Estrogen is
thought to contribute to premenopausal womens tendency to have lower systolic blood
pressure, higher levels of HDL cholesterol, and lower triglyceride levels than men ( Pilote et
al., 2007).
The lower prevalence of smoking among women is another factor that could contribute to
their decreased CVD incidence and mortality rates. Around the world, the prevalence of
female smoking is lower than that of men (Pilote et al., 2007). Although rates of smoking,
dyslipidemia, and hypertension are generally lower among women than men, women tend
to have less favorable profiles for other key CVD risk factors. Worldwide, women are more
likely to be sedentary than men (Guthold et al., 2008). Some researchers have suggested that
womens social status in many cultures and their lack of leisure time due to childcare and
other familial responsibilities likely contribute to their lower levels of physical activity
(Brands and Yach, 2002; Pilote et al., 2007).
Another troubling gender difference is the increased prevalence of obesity among women.
WHO data indicate that although overweight (BMI 25 kg/m2) is more common among
men globally; obesity (BMI 30 kg/m2) is more common among women.
A number of different reasons have proposed by CVD researchers that why women might
delay seeking medical attention, receive delayed treatment, and experience poorer outcomes
during and after an MI or stroke. One often-cited reason that women tend to wait longer to
seek treatment is that many do not perceive themselves as being at risk (Jensen and Moser,
2008).
Because of the robust evidence indicating gender differences in CVD incidence, morbidity,
and outcomes, these differences, as well as the unique needs of women, should be
considered when developing CVD research priorities, policies, and health service
interventions.
6. Association between early life factors and subsequent risk for CVD
6.1 Low birth weight and adult cardiovascular disease
Ther is growing evidence in developing and developed countries ,based on cohort studies,
that fetal and early childhood periods is important in the onset of CVD later in life (Victora
et al., 2008; Walker and George, 2007; WHO, 2009c). The influences during this period
include maternal factors during pregnancy, such as smoking, obesity, and malnutrition, and
factors in infancy and early childhood, such as breastfeeding, low birth weight, and
undernutrition.

Cardiovascular Disease Risk Factors 299
Maternal smoking during pregnancy has been linked to CVD-related risk factors. It has been
consistently associated with increased childhood obesity independent of other risk factors
(Oken et al., 2008). A number of studies have examined the effects of maternal obesity on the
body weight of their children; however, the evidence is inconsistent. Two cohort studies in the
United States found that excessive weight gain or maternal obesity during pregnancy was
associated with overweight and obesity in the children at ages 3 and 4 years (Gillman et al.,
2008; Whitaker,2004). Similarly, a cohort study in Finland found that mothers body mass
index (BMI) was positively associated with their sons BMI in childhood (Eriksson et al., 1999).
Another factor that appears to influence risk for long-term cardiovascular health is
breastfeeding. Breastfeeding has been found to not only reduce childhood morbidity and
mortality but also to be weakly protective against obesity later in life (Bhutta et al., 2008;
Gluckman et al., 2008).
Undernutrition in infancy, especially when followed by rapid weight gain, is associated
with increased risk of CVD and diabetes in adulthood (Barker and Bagby, 2005; Caballero,
2005; Gluckman et al., 2008 ). This phenomenon is known as the developmental origins
theory of CVD. It means that if disruptions to the nutritional, metabolic, and hormonal
environment at critical stages of development are happened, it may lead to permanent
programming of the bodys structure, physiology, and metabolism that translate into
pathology and disease, including CVD, later in life (Barker, 1997, 1998, 2007). The exact
physiological mechanisms through which this programming occurs are not yet fully
elucidated; however, there is evidence that fetal and early postnatal undernutrition can
cause metabolic, anatomic, and endocrine adaptations that affect the hypothalamic-
pituitary-adrenal axis, lipoprotein profiles, and end organ glucose uptake, among other
processes (Prentice and Moore, 2005). Support for the developmental origins theory of CVD
comes from a number of retrospective, and more recently prospective, cohort studies in
various populations. Studies in the United Kingdom, the United States, Finland, and India
found that fetal undernutrition followed by a rapid catch-up growth from childhood to
early adolescence was significantly associated with the later development of CVD in both
men and women (Barker et al., 2005; Eriksson et al., 1999; Osmond and Barker, 2000). Early
undernutrition followed by catch-up growth during childhood has also been associated
with subsequent hypertension and type 2 diabetes (Barker, 1998; Osmond and Barker, 2000)
This emerging data on the effects of rapid weight gain after early undernutrition have
prompted some researchers to suggest a shift from the original fetal origins hypothesis to
an accelerated postnatal growth hypothesis of CVD (Singhal et al., 2003, 2004).
The emerging evidence on the association between low birth weight followed by rapid
growth in childhood and subsequent risk for CVD raises important considerations for
addressing global CVD because low birth weight and exposure to undernutrition in utero
and in infancy are common in many developing countries (Caballero, 2009; Kelishadi, 2007).
The acquisition and accumulation of risk for CVD continues in childhood and adolescence
(Celermajer and Ayer, 2006 ). Unhealthful lifestyle practices such as consumption of high
calorie and high fat foods, tobacco use, and physical inactivity begin in childhood,
introducing major behavioral risks for CVD. Childhood adversity also influences adult
cardiovascular health. In addition, there is also an emerging body of evidence on the

The Cardiovascular System Physiology, Diagnostics and Clinical Implications 300
presence of biological risk factors in children and youth, including pathophysiological
processes associated with heart disease that can be seen as early as childhood.
6.2 Childhood obesity and CVD risk
Childhood obesity is associated with multiple risk factors for CVD, which are amplified in
the presence of overweight and persist from childhood into adulthood. These risk factors
include hyperlipidemia, high blood pressure, impaired glucose tolerance and high insulin
levels, as well as metabolic syndrome. It has been estimated that 60 percent of overweight
children possess at least one of these risk factors that can lead to CVD in adulthood
(Freedman et al., 1999). This is especially important in terms of implications for global CVD
because the prevalence of childhood obesity is increasing in developing countries (WHO,
2008a).
7. Public health approach to cardiovascular disease risk reduction
7.1 Cardiovascular disease as a public health problem
The prevention of cardiovascular disease is a major public health challenge for a number of
years around the world. Although death rates have been falling in many westernized
countries (e.g. USA, Australia, UK), rates are rising rapidly elsewhere.
7.2 Current dietary recommendations for primary prevention
Dietary recommendations tend to be country specific and are based on the available
evidence.
The ATP-III recommends the TLC dietary pattern for primary and secondary prevention of
CHD. In agreement, the AHA recommends diet and lifestyle changes to reduce CVD risk in
all people over the age of 2 years (Lichtenstein et al., 2006).
As is evident, knowledge about the role of diet in risk factor reduction and reducing the risk
of cardiovascular events themselves continues to expand. It is now recognized that CVD risk
can be mediated through multiple biological pathways other than only serum total and LDL
cholesterol or dietary factors. With this in mind, it is necessary to modify the dietary advice
offered to those with an increased risk of CVD.
7.3 Health promotion in children and other subgroups of the population
Health aspects present in childhood, such as blood lipids, body weight and blood pressure
may track into adulthood. Therefore, a useful health strategy is the adoption of sensible
eating habits and an active lifestyle early in childhood. It is important to promote
cardiovascular health in childhood by increasing physical activity and preventing or
treating obesity, raised blood pressure, insulin resistance and type 2 diabetes (Williams et al.,
2002). Current nutritional recommendations for the general population are applicable for
most children over 5 years and can be gradually applied from the age of 2 years. It is also
recommended that all children and adolescents participate in physical activity for 1 hour
daily which should be of at least moderate intensity (Fox & Riddoch, 2000). The
implementation of guidelines and success of health strategies require input from the

Cardiovascular Disease Risk Factors 301
governments, health professionals, the food industry and teachers, as well as the children
themselves and their parents. Moreover, social and cultural influences must be recognized
when designing and implementing strategies.
8. Summary
Cardiovascular disease is the leading cause of death worldwide, accounting for around
18 million deaths each year.
Modifiable risk factors for cardiovascular disease include atherogenic lipoproteins,
inflammatory related factors behaviors, lifestyle and chronic diseases such as obesity,
diabetes and hypertension.
Non modifiable risk factors include menopause, age and gender.
There is evidence that a chronic, low-grade inflammation underlies atherosclerosis,
although it is not clear whether this is a cause or effect phenomenon.
The acute phase proteins, C-reactive protein (CRP), fibrinogen and serum amyloid A,
appear to be associated with risk for cardiovascular disease.
The advantages of a dietary pattern approach rather than individual dietary
components can influence plasma cholesterol levels and may also affect other emerging
risk factors.
Physical activity has great impact on CVD risk reduction when it is accompanied by
dietary pattern changes.
Low birth weight and low weight gain during infancy are associated with an increased
risk of adult cardiovascular disease, hypertension, type 2 diabetes and the insulin
resistance syndrome.
9. Future research
Future research is required to establish the strength of the associations between the
emerging risk factors described in this chapter and cardiovascular disease, in order to
compare their predictive value with the established risk factors. For example, further work
is required to evaluate the independence of many of the novel risk factors for cardiovascular
disease and whether these associations are causal. In addition, more information is needed
about how these novel risk factors might be modified by different aspects of the diet. As
indicated in ancient Traditional Persian Medicine (TPM), understanding the effect of
individual foods on the trend of heart disease and hyperlipidemias can be leading fields of
study in the near future.
Moreover, local modified risk factors should be defined and addressed to track the patients
at greater risks in more applicable ways.
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15
Cardiovascular and Cerebrovascular Problems
in the Development of Cognitive Impairment:
For Medical Professionals Involved in
the Treatment of Atherosclerosis
Michihiro Suwa
Department of Cardiology,
Hokusetsu General Hospital, Takatsuki, Osaka,
Japan
1. Introduction
When cognitive function declines at a rate greater than that expected based on the actual
age, life circumstances and educational level, we define it as cognitive impairment (Blennow
et al., 2006; Gauthier et al 2006). In the older generation, the neurodegenerative process is
considered to occur several years before the development of clinically detectable cognitive
impairment. Although aging is the most clear factor in the development of this disease
process, several epidemiological studies have elucidated that cardiovascular risk factors, i.e.,
hyperlipidemia, hypertension, smoking and diabetes, are also associated with
cerebrovascular disease processes that may deteriorate cognitive function and advance
dementia (Casserly & Topol, 2004; Nash & Fillit, 2006; Whitmer et al., 2005). In this chapter, I
would like to introduce the current positioning of cognitive impairment as a consequence of
cardiovascular diseases as well as a form of cerebrovascular disease, to neurologists or
psychiatrists as well as to general physicians or cardiologists.
2. Cognitive impairment
Currently, Alzheimers disease is the most common form of dementia or cognitive
impairment, but less than 20% of dementia patients exhibit the isolated form of Alzheimers
disease and around 50% of such patients exhibit a combination of Alzheimers disease and
intracerebral vascular disease. By contrast, the isolated vascular type only contributes 20%
(Meguro et al., 2002). Therefore, various problems related to metabolic syndrome, i.e.,
hypertension, hyperlipidemia, diabetes, and smoking habit, contribute to the development
or deterioration of cognitive impairment.
3. Cardiovascular problems in cognitive impairment
Furthermore, recent investigations have suggested that the incidence of cognitive
impairment is higher in patients with congestive heart failure and that treatment for left
ventricular (LV) systolic dysfunction may prevent or delay the development of dementia in

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elderly patients (Zuccala et al., 2003, 2005). An Italian investigation indicated that the incidence
of congestive heart failure was markedly higher in subjects with Mini-Mental State
Examination (MMSE) scores <24 (20.2%), compared with those with scores 24 (4.6%)
(Zuccala et al., 2003). Also, the presence of cognitive impairment has been associated with
increased in-hospital mortality in older patients with heart failure (Zuccala et al., 2005). These
data may indicate that low cardiac output due to heart failure is related to the deterioration of
cognitive function, although there have been no reports regarding the pathophysiology or
etiology of heart failure in relation to cognitive impairment. Another investigation indicated an
independent relation between the levels of b-type natriuretic peptide (BNP) and the degree of
cognitive impairment in older subjects (>55years of age) with cardiovascular disease; despite
the fact that the mechanism was unclear (Gunstad et al., 2006).
In patients with heart failure, more than half of them, especially in females, showed normal or
preserved LV ejection fraction (EF), i.e., exhibited heart failure resulting from LV diastolic
dysfunction (Hogg et al. ,2004). Also, in heart failure patients with cardiovascular risk factors,
i.e., hyperlipidemia, hypertension, smoking and diabetes, heart failure with preserved EF is
also common. Therefore, we evaluated the relationship between LV diastolic dysfunction and
cognitive impairment in Japanese patients with cardiovascular diseases (Suwa & Ito, 2009).
In our study, patients were divided into 2 groups; those patients with normal cognitive
function or mild cognitive impairment (MMSE>=24; n=68: group N) and those with
depressed cognitive function (MMSE<24, n=13). Diastolic function was evaluated based
upon the ratio of the early diastolic mitral flow velocity (E) by pulse-wave Doppler
echocardiography to the early diastolic mitral annular myocardial velocity (e) by tissue
Doppler echocardiography (Diastolic Doppler index: E/e). BNP was also evaluated as an
index of heart failure. Consequently, in depressed cognitive function, diastolic Doppler
index, E/e, was deteriorated (6.11.3 vs. N: 4.61.3, p<0.0003) and BNP was higher
(137142 pg/ml vs. N: 6049 pg/ml, p<0.007), compared with those with normal cognitive
function. Furthermore, the number of patients with diabetes was also higher in depressed
cognitive function than in normal cognitive function (46% vs. N: 18%). From these studies
evaluating the relation between cardiac and cognitive function, heart failure due to LV
systolic and diastolic dysfunction can affect the development and the deterioration of
cognitive impairment.

Poor cerebral circulation: Left ventricular systolic dysfunction
Left ventricular diastolic dysfunction
Stroke: Atrial fibrillation, Atherosclerotic vascular
diseases including of carotid artery stenosis
Deep white matter hyperintensity
in brain MRI:
Atherosclerotic abnormalities, due to
hypertension, diabetes, hyperlipidemia, and
smoking
Hippocampal atrophy in brain: Senile process
MRI: magnetic resonance imaging
Table 1. Relationship between cardiovascular abnormalities and cerebrovascular diseases
affecting the decline of cognitive function
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The occurrence of atrial fibrillation (AF) is a risk of stroke, and stroke increases the risk of
cognitive decline and dementia. Therefore, AF has been reported to be associated with
cognitive decline and dementia (Jozwiak et al., 2006). Recently, even in stroke-free patients it
has been shown that AF is a risk for cognitive impairment and hippocampal atrophy
(Knecht et al., 2008). For these reasons, it was considered that AF was associated with
abnormalities of hemostasis, endothelial damage, platelet dysfunction, and low cardiac
output.
As another measurable index in the vascular system, the ankle to brachial index is also
related to incidence of total dementia, vascular dementia and Alzheimers disease, especially
in carriers of the apolipoprotein E gene abnormality (Laurin et al. 2007).
4. Common interest between cardiovascular and cerebrovascular diseases
Brain magnetic resonance imaging is conducted to screen for cerebrovascular disease as well
as cerebral disease, and hyperintensities in the deep white matter on T2-weighted images
can be incidentally detected in 10-20% in adults aged 64 to 94% at age 82 in the general
population (Fig.1 and Fig.2). At present, these white matter lesions are related to chronic
hypoperfusion and disruption of the blood brain barrier due to small vessel disease in the
lesion area (Debette & Markus, 2010). Also, white matter hyperintensities are more common
in patients with cerebrovascular disease as well as cardiovascular disease, with risk factors
affecting atherosclerosis, inclusive of hypertension (Hajjar et al., 2011). Furthermore, meta-
analysis reveals that the white matter lesions predict an increased risk of stroke, dementia,
and death. Although data that treatment for these risk factors reduces the progression of
white matter hyperintensities are limited, it is also reported that antihypertensive therapy
reduced the progression in patients with stroke (Saxby et al., 2008). Therefore, white matter
hyperintensities may be important markers to not only detect the risk of stroke and
dementia but also diagnose the atherosclerosis in cerebro-cervical vascular system. The
progression of white matter lesions is independently related to baseline cerebral lesion,
higher age, hypertension, and current smoking. Also, atherosclerotic processes in the carotid
artery, connecting to the cerebral artery, are associated with the cerebral small vessel disease
(Romero et al., 2009).
Modified Mini-Mental State Examination scale falls more according to the worsening of
white matter grade (prominently in grade Two +). (Fig.3. Longstreth et al. 2005) (with
permission, License No.2776831264727)
To date, some angiotensin receptor blockers have been reported to significantly reduce the
incidence and progression of Alzheimers disease and dementia, compared with the use of
angiotensin converting enzyme inhibitors or other cardiovascular drugs, in a predominant
male population (Saxby et al., 2008). Therefore, when prescribing antihypertensive drugs we
may have to consider the contribution of such medicines.
At present, the CHADS2 score is widely used to validate the risk for stroke in patients with
AF. A newer clinical study has shown that the CHADS2 score is useful to predict ischemic
stroke in patients with stable coronary artery disease, even in those without baseline atrial
fibrillation (Welles et al., 2011)

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Fig. 1. Brain magnetic resonance imaging on T2 weighted images obtained from a 77 year
old female without cognitive impairment and being under medication for hypertension.
These images show somewhat brain atrophy but no white matter hyperintensities.
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Fig. 2. White matter hyperintensities on brain magnetic resonance imaging from a 73 year
old female with advanced cognitive impairment (score 18 on MMSE). Extensive
hyperintensities can be seen in deep white matter, especially in periventricular region. She is
under medication for hypertension and hyperlipidemia

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Fig. 3. Scores on modified Mini-Mental State Examination (vertical axis) for each year of
study from initial to follow-up brain MRI scans (horizontal axis) by groups of participants
(people aged 65 years and older) defined by worsening grade of white matter hyperintensity
(three grade: None, grade One, and grade Two+).
Left ventricular systolic dysfunction: Low left ventricular ejection fraction
Left ventricular diastolic dysfunction: Reduced e, Increased E/e ratio
B-type natriuretic peptide (BNP): Increased BNP or N-terminal pro-BNP
Rhythm disturbance: Development of atrial fibrillation
Ankle-brachial index (ABI): Low ABI
Carotid ultrasonography Internal carotid artery stenosis
e: early diastolic mitral annular myocardial velocity by tissue Doppler echocardiography
E: early diastolic mitral flow velocity by pulse-wave Doppler echocardiography
Table 2. Cardiovascular indexes possibly to detect cognitive impairment
5. Relationship between echocardiographic parameters and age
Currently, LV diastolic function is evaluated based upon the following indices. Using pulse-
wave Doppler echocardiography, the ratio of E velocity and the late diastolic transmitral flow
velocity (A): (E/A ratio), and early diastolic flow deceleration time are measured. By tissue
Doppler echocardiography of mitral annular motion, e myocardial velocity can be evaluated.
Also, diastolic function was evaluated by the diastolic Doppler index (E/e), and this index is
also useful to evaluate LV end-diastolic pressure or left atrial pressure in patients with heart
failure with depressed or normal LV EF (Ommen et.al., 2000).
Cardiovascular and Cerebrovascular Problems in the Development of
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Among the healthy subjects, the previous reports have evaluated the changes with age in
various parameters. LV systolic function, obtained from LV EF by standard
echocardiography or LV myocardial performance index using Doppler echocardiography,
have shown minimum increments with age. However, LV diastolic function declined with
age on echocardiographic parameters, i.e., E/A ratio and e velocity decreased, and E/e
ratio increased (Munagala et al., 2003). Furthermore, LV wall thickness and LV mass
increased gradually with age, and suggested depression of LV myocardial compliance
(Daimon et al., 2008).
In some reports discussing the relationship between cognitive impairment and
cardiovascular dysfunction, LV diastolic function was depressed with the deterioration of
cognitive function, which may be more progressive than that with aging alone (Hogg et al.
,2004). Furthermore, in patients with heart failure, cognitive function is depressed, but there
is no clear data as to whether LV systolic function is depressed with the decline of cognitive
function and its decline is improved with the correction of systolic dysfunction.
6. Conclusion
While age is the most significant contributing factor to the development of cognitive
impairment, several epidemiological studies have elucidated that cardiovascular risk
factors, i.e., hyperlipidemia, hypertension, smoking and diabetes contribute to the
deterioration of cognitive function. Furthermore, LV systolic and diastolic dysfunction, have
been reported to cause cognitive impairment, likely as a result of the development of poor
cerebral perfusion. Brain white matter hyperintensitiy and thromboembolic stroke due to
atherosclerotic factors and atrial fibrillation are also related to producing cognitive
impairment. Therefore, doctors and medical professionals, who are involved with medical
treatment for cerebrovascular and cardiovascular diseases must keep in mind that
atherosclerotic disease processes are also related to the development of cognitive
impairment and progression of dementia.
7. References
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Daimon M et al. (2008). Normal values of echocardiographic parameters in relation to age in
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Debette S & Markus HS. (2010). The clinical importance of white matter hyperintensities on
brain magnetic resonance imaging: Systemic review and meta-analysis. BMJ
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Gauthier, S et al (2006). Mild cognitive impairment. Lancet, Vol. 367: No. No.9518, pp.1262-
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Gunstad J et al. (2006). Relation of brain natriuretic peptide levels to cognitive dysfunction
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Hajjar I et al. (2011). Hypertension, white matter hyperintensities, and concurrent
impairments in mobility, cognition, and mood: The cardiovascular health study.
Circulation Vol. 123, No.8, pp. 858-865.
Hogg K et al. (2004). Heart failure with preserved left ventricular systolic function:
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3, pp.317-327
Jozwiak A et al. (2006) Association of atrial fibrillation and focal neurologic deficits with
impaired cognitive function in hospitalized patients 65 years of age. Am J Cardiol
Vol. 98, No.9, pp.1238-41.
Knecht S et al. (2008). Atrial fibrillation in stroke-free patients is associated with memory
impairment and hippocampal atrophy. Eur Heart J Vol. 29, No.17, pp. 2125-2132.
Laurin D et al. (2007) Ankle-to-brachial index and dementia. Honolulu-Asia aging study.
Circulation Vol. 116, No.20, pp. 2269-2274.
Longstreth WT Jr. et al. (2005). Incidence, manifestation, and predictors of worsening white
matter on serial cranial magnetic resonance imaging in the elderly. The
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Meguro K et al. (2002). Prevalence of dementia and dementing diseases in Japan: the Tajiri
project. Arch Neurol. Vol. 59, No. 7, pp. 1109-1114
Munagala VK et al. (2003). Association of newer diastolic function parameters with age in
healthy subjects: A population-based study. J Am Soc Echocardiogr Vol. 16, No.10,
pp. 1049-56
Nash DT & Fillit H. (2006). Cardiovascular disease risk factors and cognitive impairment.
Am J Cariol, Vol. 97, No.8, pp.1262-65.
Ommen SR et.al (2000). Clinical utility of Doppler echocardiography and tissue Doppler
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Saxby BK et al. (2008). Candesartan and cognitive decline in older patients with
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16
French Paradox, Polyphenols
and Cardiovascular Protection:
The Oestrogenic Receptor- Implication
Tassadit Benaissa
1
, Thierry Ragot
2
and Angela Tesse
1

1
INSERM, UMR 915, Institut de recherche thrapeutique (IRT), Nantes,
2
CNRS, UMR 8203, Institut de Cancrologie Gustave Roussy,Villejuif,
France
1. Introduction
Several epidemiological and clinical studies confirm an inverse correlation between a diet
rich in vegetables, fruits, and red wine, in cancer development and chronic diseases such as
cardiovascular diseases. This is linked to the presence in these aliments of high levels of
nutrients of vegetal origin called phytonutrients. They are natural phytochemical
compounds contained in plant food; they are not vitamins or minerals but they have
beneficial effects on the health, sometimes acting in association with other essential
nutrients. Phytonutrients are divided in three families: the terpenes, the sulfuric
compounds, and the polyphenols which are the subject of this chapter.
Polyphenols are the most important group of phytonutrients. They are not only present in
fruits and vegetables but also in seeds, spices, herbs and teas, at different concentrations and
molecular structures in correlation with the aliment involved. The most studied
polyphenolic compounds for their vascular action are resveratrol, delphinidin, quercetin
and tannins contained in red wine. Indeed, the red wine is the beverage the most correlated
to cardiac and vascular protection. It could reduce of 40% the risk of myocardial infarction,
and of 25% the risk of vascular thrombotic events in brain.
Elevated content in polyphenols of red wine seems to play a benefic role in the mechanism
of vascular and cardiac protection, not only by its anti-oxidant but also by its anti-
thrombotic properties. Thus, more recently, research works were focused to study the
vascular and cardiac effects of non-alcoholic fractions of red wine and have identified the
oestrogenic receptor- (ER), as the preferential endothelial target of these molecules.
First, this chapter is focused on the French Paradox history. Then, we have described
successively the composition and content of these compounds in food and beverages, and
the epidemiological and fundamental studies showing how red wine polyphenolic
compounds (RWPC) are able to improve endothelial function and cardiovascular protection.
Finally, we explain the effects of oestrogens on the cardiovascular system and the
implication of ER in the beneficial cardiovascular effects of these natural molecules that
could be used to prevent or treat cardiovascular diseases.

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2. French paradox history
For a long time, it was suggested that a high fat intake is associated with an elevated risk of
mortality for cardiovascular diseases in Anglo-Saxon populations. In contrast, several
epidemiological studies have revealed a relatively low incidence of coronary heart diseases
(CHD) in the French population, despite a high dietary intake of saturated fats. This was
potentially attributable to the consumption of red wine (Renaud et al., 1999).
One of the first epidemiological studies conducted on 100,000 subjects in 1970 by Doctor
Arthur Klatsky, a cardiologist of the Oakland Hospital in California, clearly evidenced that
people following a diet with moderated consumption of red wine (1-3 glasses per day),
showed a very little risk of death by CHD (Renaud et al., 1999). This was confirmed in 1979
by Doctor Saint-Lger which evidenced a negative correlation between wine consumption
and mortality for CHD in men and women (from 55 to 64 years old), in more than 18
developed countries. Furthermore, Italy and France showed a lower level of mortality by
myocardial infarction (about 3 or 5 folds less) compared to Anglo-Saxon populations such as
Irish, North-American, and Scottish (Renaud et al., 1999). On the other hand, it has been
demonstrated that to drink one glass of wine per day reduced death risk by CHD, but to
drink more than three glasses of wine per day was associated with an increased death rate
(Thun et al., 1997). In wine drinkers, the lower all-cause mortality was associated with a
significant reduction in mortality from CHD, for about 45-48%, and other cardiovascular
diseases (CVD), for about 39-40% (Renaud et al., 1999). Other studies have also suggested
that both non-drinkers and heavy-drinkers have a higher risk of cardiovascular mortality
than those who drink wine moderately (Providencia, 2006).
Then, numerous correlation studies concerning the strict relation between consumption of
fat and CHD mortality have been conducted in various countries. In one of the most
interesting ones, Artaud-Wild and colleagues examined the relation between CHD mortality
and the intake of foodstuffs and nutrients in 40 different countries. After they have defined a
cholesterolsaturated fat index (CSI), they studied this correlation in 100,000 men (aged 55
to 64 years) in all the countries studied. The findings of this epidemiological study
evidenced that France had a CSI of 24 per 1000 kcal and a CHD mortality rate of 198;
whereas Finland had a CSI of 26 per 1000 kcal and a CHD mortality rate of 1031 (Ferrires,
2004). The high consumption of saturated fatty acids suggests that French subjects could be
exposed to a high risk of CHD (Renaud 1992), but it is in fact not the case considering the
low rate of CHD mortality observed. Then, much attention has been focused on the possible
superior protective effect of red wine consumption relative to those of other alcoholic
beverages. So, the differential effects of wine, beer, and spirits have been examined.
European research carried out in France and Denmark has shown that wine consumption
was associated with a decrease of 24 up to 31% of all cause mortality; little to moderate wine
drinking leads to a lower mortality rate from CVD than having an equivalent consumption
of beer or spirits (Ferrires, 2004).
Nevertheless, alcohol consumption, from whatever sources, appears to have a J-shaped
curve, whereby a modest intake is beneficial and either no intake or excess is harmful. This
is confirmed by several studies: the Framingham study (Fuchs et al., 1995), the British
Doctors study (Doll et al., 1994), the Cancer Prevention study of Thun and coworkers,
conducted on about 490,000 persons (Thun et al., 1997), the Nurse Health study (Emberson
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321
et al., 2005), and other epidemiological investigations (Gaziano et al., 2000; Suh et al., 1992).
It would take too long to report on all the studies dealing with the relations between alcohol
and CHD.
The mechanisms involved in the protective role of red wine include anti-platelet, anti-
coagulatory, improved glucose control, and anti-inflammatory effects as shown in MONICA
(multinational MONItoring of trends and determinants in CArdiovascular disease) study
(Imhof et al., 2004). The World Health Organization had collected all the results of these
data, evidencing the protective role of moderated red wine consumption in cardiovascular
disease development. Despite the high consumption of saturated fatty acids, why the French
people do not develop a high CHD risk? This is the central question behind the French
Paradox concept. The French epidemiologist Serge Renaud evidenced for the first time this
Paradox, which is defined as the light level of incidence of CVD in people following a diet
containing a high quantity of saturated fatty acids, but also having a moderated red wine
consumption (Pechanova et al., 2006, Renaud et al., 1999).
The results of Criqui and colleagues (Criqui & Ringel, 1994) were found in agreement with
the French Paradox. In 21 developed countries, subjects in an age range of 35 to 74, without
differences linked to gender, were studied and assessed at four time periods: 1965, 1970,
1980, and 1988, respectively. The independent variables chosen were: consumption of wine,
beer, spirits, animal fats, vegetables, and fruits. Ischemic heart disease and all-cause
mortality were finally assessed. Wine was the beverage most strongly negatively correlated
with coronary diseases. Animal fat had a tendency to positive correlation, while fruits were
negatively correlated. On the light of the numerous epidemiological studies, a protective
activity of wine against CVD has been widely described, suggesting that moderated
consumption of wine could reduce the risk of myocardial infarction and the risk of vascular
thrombosis of brain vessels.
So many questions arose next. What were the elements that differentiate the wine (especially
red wine) of other spirits? What were the processes responsible for the beneficial effect of
wine consumption? What, in wine, promoted this effect?
3. Differences in polyphenolic compositions in food and beverages
Polyphenolic compounds are the biggest group of phytochemicals characterized by one or
more phenolic rings associated with one or more hydroxyl groups, free or implicated in an
ester, ether or eteroside function (Richter, 1993). This family of substances includes more
than 8000 phenolic structures currently known, and among them, over 4000 flavonoids have
been yet identified in plants and the list is constantly growing (Bravo, 1998; Cheynier, 2005;
Harborne & Williams, 2000). Flavonoids contain a structural backbone C6-C3-C6,
characterized by two C6 units of phenolic nature; while the non-flavonoids are phenolic
acids divided in two main types, benzoic acid and cinnamic acid derivatives, based on C1-
C6 and C3-C6 backbones, respectively (Tsao, 2010). The phenolic acids are usually contained
as free molecules in fruits and vegetables. Phenolic acids could be also found in the bound
form in grains and seeds (Chandrasekara & Shahidi, 2010).
Polyphenols are enrolled in numerous physiological functions in vegetal organisms: cell
development, latent buds, blooming, and tuber formation. These substances are involved in

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the color of fruits, in particular they play a main role to confer the red color of ripe fruits,
and in the savor and properties of food (Bahorun, 1997). Polyphenols include yellow,
orange, red and blue pigments and various compounds implicated also in bitterness and
astringency of unripe fruits, resulting from interaction of tannins with salivary proteins.
Moreover, some volatile polyphenols, in particular vanillin and eugenol, are potent
odorants and are responsible of the characteristic odor of cloves (Cheynier, 2005).
The content of polyphenolic compounds is particularly elevated in red wine but also in skin
of red grapes, red fruits, cereals, several vegetables such as red onions, chocolate, tea, and
coffee with different polyphenolic composition and percentage according to the kind of
vegetal food or beverage (see Table 1) (Bravo, 1998; Tsao, 2010). Considering the diversity
and wide distribution of polyphenols, they have been classified by their source of origin,
biological function, and chemical structure. In plants, the majority of polyphenols exists as
glycosides associated to sugar units or acylated sugars linked at different positions of the
polyphenolic skeletons (Tsao, 2010).
Food Total polyphenols Food Total polyphenols
(mg/100 g of dry mutter) (mg/100 g of fresh mutter)
Cereals: Vegetables:
Barley 1200-1500 Onion 100-2025
Millet 590-1060
Legumes: Fruits:
Black gram 540-1200 Apple 27-298
Green gram 440-800 Blackcurrant 140-1200
Pigeon peas 380-1710 Grapes 50-490
Raspberry 37-429
Beverages Total polyphenols Beverages Total polyphenols
(mg/L) (mg/L)
Orange juice 370-7100 Tea 750-1050
Red wine 1000-6500 Coffee 1330-3670
White wine 200-300

Table 1. Plant food and beverages with high levels of total polyphenolic compounds (from
Bravo, 1998).
Some flavonoids such as the isoflavones are mostly found in plants of the leguminous
family. Genistein and daidzein are the two main isoflavones found in soybeans and red
clovers (Tsao et al., 2006). The flavonoid subgroup of the neoflavonoids is rarely present in
food plants, but the open-ring chalcones are still found in fruits, in particular in apples and
hops of beers (Tsao et al., 2003; Zhao et al., 2005). In contrast, other flavonoid subgroups
such as flavones, flavonols, flavanones and flavanonols are most common and ubiquitous in
the plant kingdom and in particular quercetin and kaempferol (Tsao, 2010). Flavanols or
flavan-3-ols, also called catechins, are found in many fruits, the skin of grapes, apple and
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323
blueberries (Tsao et al., 2003). Catechin, epicatechin (isomer of catechin with cis
configuration), and their derivatives, gallocatechins, are the major flavonoids contained in
tea leaves and cacao beans and thus in chocolate (Si et al., 2006; Prior et al., 2001).
The red, blue and purple pigments of the majority of flower petals, fruits and vegetables and
certain varieties of grains, for instance black rice, mainly contain anthocyanidins and in
particular cyanidin, delphinidin, pelagonidin, and their methylated derivatives (composed
up to 90% of anthocyanins). The color of these kinds of molecules can change with the pH
and temperature: they are red in acidic and blue in basic conditions (Tsao, 2010). In grapes
and apples, anthocyanins are found only in the red varieties (Cheynier, 2005).
Polyphenols are highly unstable species and, accordingly, their chemical structure can
change during food and beverage processing and storage, leading to new compounds with
different properties compared to their precursors (Xu et al., 2011). In particular, total
catechin contents of fresh fruits can decrease of about 26% up to 58% after home preparation
or industrial transformations (Cheynier, 2005).
Wine is a hydro-alcoholic acid solution. Indeed, its major component is water (80-90%) and
ethanol (10-14%) implicated in the solubilization of polyphenols. The fraction of
polyphenolic compounds contained in wine is high in red wine and its composition
depends of the kind of wine. More precisely, generally red wine contains 1.2 gr/L of
polyphenolic compounds while white wine contains only 0.2 gr/L of these compounds and,
besides, does not contain the molecules involved in the red color such as the anthocyanidins
and in particular delphinidin (see Table 2) (Pellegrini et al., 2000; Soleas et al., 1997).
Interestingly, the level of these compounds in red wine is modified by the fermentation
process used during wine production. Vinification variations and techniques are known to
affect the

phenolic composition of red wines. The fermentation of grape juice into wine is a
Compounds White young White aged Red young Red aged
(mg/L) wine wine wine wine
Total phenols 215 190-290 1300 955-1215
Non flavonoides 175 160-260 235 240-500
Flavonoides 30 25 1060 705
Catechins 25 15 200 150
Anthocyanins 0 0 200 20
Soluble tannins 5 10 550 450

Table 2. Polyphenolic compound contents in several types of wine (from Soleas et al., 1997).
complex microbial reaction, traditionally due to the sequential development of various
species of yeast and lactic acid bacteria. In the past, wine was produced by natural
fermentation of grape juice by yeasts originating from grapes and winery equipment
(Ribereau-Gayon et al., 2000). Nowadays, another kind of fermentation process, the carbonic
maceration, is more and more used to produce wine. With this method, freshly harvested
bunches of grapes are allowed to ferment in carbonic anaerobiosis, in an atmosphere
saturated with carbon dioxide (Navarro et al., 2000). The absence of oxygen is important to

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324
reduce the oxidation of polyphenolic compounds, especially the monomeric anthocyanidins
such as malvidin and delphinidin. The preservation of these molecules by this new carbonic
process increases their final levels in wine compared to the traditional maceration of grapes
(Pellegrini et al., 2000). Furthermore, the wine ageing could modify polyphenol composition
and levels in white and red wines with a time-dependent reduction of catechins and
anthocyanidins contents (see Table 2) (Pellegrini et al., 2000; Soleas et al., 1997).
It is interesting to note that, after food or beverage intake, the degradation and absorption of
polyphenols within the gastrointestinal tract depend on the nature of the polyphenolic
compound but also of the intestinal microflora, with subsequent fermentative effect on other
dietary components. Thus, these molecules are modified by intestinal bacteria but they can
influence in return microflora and its fermentative capacity (Bravo, 1998). Several recent
studies are focused in how processing and beverage composition might influence phenolic
profiles and bioavailability of an individual polyphenol. Specifically, they showed the
impact of beverage formulations and the influence of digestion on stability, bioavailability,
and metabolism of bioactive polyphenolic compounds from food and beverages. For
example, the co-formulation with ascorbic acid and other phytochemicals may improve
absorption of these health-promoting phytochemicals (Ferruzzi, 2010). Thus, it is critical to
develop beverage products designed to deliver specific health benefits.
4. Beneficial effects of RWPC in cardiac and vascular functions
Evidences from different experimental studies has suggested the presence of molecules with
anti-oxidant properties in red wine, such as tannins and other flavonoids. These molecules
could be key factors in the protective effects observed (Vidavalur et al., 2006). Red wine,
might provide, through the polyphenols (non-flavonoids and flavonoids), an anti-oxidant
role, leading to additional protection mechanisms in coronary arteries (Liu et al., 2007).
Thus, RWPC are able to decrease oxidative stress, enhance cholesterol efflux from the
vascular wall, and inhibit lipoprotein oxidation. These components may also increase nitric
oxide (NO) bioavailability, thereby antagonizing the development of endothelial
dysfunction. Thus, RWPC are able to modify several factors involved in the development of
CDV by a direct action on vascular cells and in particular in endothelium, thus playing a
preventive role in the development of atherosclerosis, hypertension and myocardial
infarction. One of the most studied molecules, the resveratrol, found in grapes and wine in
significant amounts, is implicated in this beneficial action because of its ability to act as an
anti-oxidant and an inhibitor of platelet aggregation (Kopp, 1998; Providencia, 2006).
On the light of several recent major studies, the consumption of RWPC reduces the
incidence of CVD probably by their ability to change many factors and intermediate
markers implicated in these diseases. A beneficial association between consumption of food
rich in polyphenols, especially flavonoids, and other chronic diseases was also investigated.
People with very low consumption of flavonoids showed a higher risk to develop chronic
and degenerative diseases including cardiovascular disorders, diabetes, obesity and
neurodegenerative disorders compared to people with a diet rich in polyphenols (Mojzisova
and Kuchta, 2001). Thus, it is important to better identify factors that may affect the
bioavailability of specific phenolic components from food and beverages and to better
understand how these molecules are able to act positively on organism.
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4.1 Role on nitric oxide production
RWPC are able to improve NO production and vascular endothelium-dependent relaxation.
This is possible through their action to increase endothelial nitric oxide synthase (eNOS)
expression and activation in vitro on endothelial cells and ex vivo on rodent vessels.
One of the earliest works on this purpose was conducted in 1993 by Fitzpatrick and
coworkers. They found that extracts from grapes and wine containing polyphenols were
able to induce an endothelial-dependent vasorelaxation, probably by NO production and
elevated accumulation of guanosine 3,5-cyclic monophosphate (cGMP) (Fitzpatrick et al.,
1993). The mechanisms and the identification of the molecules involved in these vascular
effects were still unknown. These findings were confirmed later by another study, in which
it was evidenced an endothelial and NO-dependent relaxation induced by a non-alcoholic
red wine extract, RWPC, and leucocyanidol administrated directly at low concentrations
(from 10
-4
to 10
-2
g/L) ex vivo on noradrenaline pre-contracted rat aortic rings
(Andiambeleson et al., 1997). This was associated with an enhanced NO generation and a
seven-fold increase in cGMP accumulation. A non-relevant relaxant effect was found using
the structurally closely related polyphenol, catechin, at the same concentrations on the same
vessels. To better determine which group(s) of polyphenols were able to cause endothelial-
dependent vasorelaxation, the same team separated RWPC by chromatography in 10
fractions. These fractions were tested separately for their capacity to induce the vascular
relaxation on rat aortic rings with and without endothelium. In this study, it was shown that
fractions containing high polymeric condensed tannins produced a moderate
vasorelaxation, at relatively high concentrations (10
-2
to 10
-1
g/L) and flavan-3-ol,
(+)-epicathtechin, also failed to produce endothelium-dependent vasorelaxation. In contrast,
oligomeric condensed tannins and fractions containing anthocyanins, and in particular
delphinidin, displayed strong vasorelaxant properties (maximal relaxation in the range of
5977%) comparable to the original RWPC mixture (Andriambeloson et al., 1998).
The same endothelial-dependent relaxation was also found in small mesenteric arteries, but
it was due to both NO and endothelium-derived hyperpolarizing factor (EDHF) and it was
absent in vessels without endothelium. The NO component of the relaxation was linked to
eNOS activity and absent when the NOS inhibitor, the N
G
-nitro-L-arginine methyl ester (L-
NAME), was used, while the EDHF component was abolished by partial depolarization
with KCl. Thus, NO and EDHF are both required to promote endothelium-dependent
relaxation produced by RWPC in mesenteric resistance arteries (Duarte et al. 2004).
Several studies conducted in vitro confirmed these results. In bovine aortic endothelial cells
(BAECs) treated with RWPC (10
-2
g/L), it was found an increased Ca
2+
-dependent eNOS
activation and a subsequent increased NO production. These required the presence of
extracellular Ca
2+
, although polyphenolic compounds were able to mobilize Ca
2+
from
intracellular stores and were also able to activate phospholipase C (PLC) and tyrosine kinase
(TK) pathways. Provinols
TM
, which contain similar types of polyphenols compared to the
RWPC used by Andriambeloson and coworkers, and delphinidin displayed differences in
the process leading to this increase in endothelial intracellular Ca
2+
, thus illustrating
multiple cellular targets of natural dietary polyphenolic compounds (Martin et al., 2002).
This effect of RWPC in this cell model is associated with an increased superoxide ion (O
2
-
)
production in order to promote Ca
2+
signaling (Duarte et al., 2004). Most recently, it was
found that resveratrol, a stilbenoid contained in wine, used at nanomolar concentrations,

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326
rapidly activated extracellular-signal-regulated kinase (ERK)1/2 in BAECs and, in turn,
activated eNOS (Klinge et al., 2005). The same effect of resveratrol was confirmed later in
another model of endothelial cells, the human umbilical endothelial cells (HUVECs). The
implication of ER in the eNOS-pathway activation by resveratrol was also evoked (Klinge
et al., 2008).
Interestingly, beneficial effects on hemodynamic parameters and on endothelial function
were confirmed in vivo after a short-term oral administration of RWPC in normotensive rats
at the dose of 20 mg/kg for 7 days. Indeed, these rats, after only 4 days of treatment,
showed a significant decrease in blood pressure (129 4 mmHg versus 141 2 mmHg for
control non-treated rats). This effect was associated, ex vivo, with an increased endothelium-
dependent relaxation to acetylcholine in aortic rings, that was related to the enhanced
endothelial NO activity. Nevertheless, RWPC induced at the same time gene expression of
inducible NOS (iNOS) and inducible cyclooxigenase (COX-2), with subsequent endothelial
thromboxane A
2
release in the arterial wall, maintaining unchanged agonist-induced
contractility (Diebolt et al., 2001). The in vivo effects of Provinols
TM
(40 mg/kg per day) on
hemodynamic and functional cardiovascular changes were also investigated during the
inhibition of NO synthesis by L-NAME (40 mg/kg per day for 4 weeks) in rats. This model
of hypertension evidenced that RWPC partially prevent L-NAME-induced hypertension,
cardiovascular remodeling, and vascular dysfunction or accelerate the decrease of systolic
blood pressure after L-NAME administration. These beneficial effects were mediated by the
increased NO-synthase activity and the oxidative stress prevention (Bernatova et al., 2002;
Pechanova et al., 2004). Nevertheless, most recently, the anti-hypertensive effects of RWPC,
orally administered for 5 weeks at the dose of 40 mg/kg by gavage, was confirmed in
female spontaneously hypertensive rats (SHR). The authors suggested that a chronic
treatment with RWPC reduced hypertension and vascular dysfunction in this model of
hypertension, rather through reduction in vascular oxidative stress (Lopez-Sepulveda et al.,
2008). This findings revealed a major preventive role of these substances in cardiovascular
complications linked to hypertension.
Polyphenol vascular activity in human vessels after food or beverage intake was confirmed
by several studies that detected these molecules in human plasma at individual levels in the
range of 0.5 to 1.6 mol/L, comparable to the concentration required to induce 50% of the
maximal relaxation, comprised between 1 and 10 mol/L of active fractions (Paganga and
Rice-Evans, 1997). Polyphenols detected in human plasma are in the range of 2.5 g/ml after
a 100 ml red wine intake (Duthie et al., 1998). Most interestingly, the vasorelaxant effect of
polyphenols from red wine was confirmed also in men in which NO and normalized flow-
mediated dilation were measured before and 30, 60, and 120 minutes after red wine
consumption (Boban et al., 2006; Papamichael et al., 2004). Moreover, RWPC are not only
able to improve NO production, for their anti-oxidant and anti-inflammatory properties but
also increase the NO bioavailability in the vascular wall, by decreasing its transformation in
peroxynitrite induced by O
2
-
during oxidative stress.
Altogether, these findings suggest a possible beneficial effect of a diet rich in these
vasoactive polyphenolic compounds to prevent hypertension as the effective concentrations
of these molecules can be reached in human plasma and they might act on the endothelium
in vivo. The RWPC responsible of this effect (resveratrol, delphinidin and tannins) could be
used for hypertension treatment.
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327
4.2 Protective role in cardiac function and ischemic diseases
RWPC, administrated in a preventive purpose way, are able to reduce cardiac or cerebral
ischemic injuries in rat models of myocardial infarct and stroke, respectively. Left ventricular
hypertrophy, myocardial fibrosis and vascular remodeling were investigated in rats during
chronic inhibition of NOS activity by L-NAME. The in vivo treatment of rats with Provinols
TM

(40 mg/kg per day) reduced not only the increase in blood pressure caused by L-NAME
treatment, but also protein synthesis in the heart and aorta caused by the chronic inhibition of
NO synthesis, finally reducing myocardial fibrosis. These effects were associated with an
increase of NOS activity, a moderate enhancement of eNOS expression and a reduction of
oxidative stress in the left ventricule and aorta (Pechanova et al. 2004).
The protective cardiac effect of polyphenols was confirmed by another study, conducted in
rats and observing, ex vivo, the effects of short-term oral administration of RWPC (20 mg/kg
per day for one week) on cardiac responsiveness and ischaemia-reperfusion injury. The
involvement of NO in the cardiac effects of RWPC was evaluated using L-NAME (2 mg/kg
per day for one week), a dose which did not affect blood pressure, in a group of rats
previously treated with polyphenols. Heart reactivity was studied in perfused isolated
hearts by the Langendorff method. The hearts harvested from RWPC-treated rats showed a
lower basal pressure, a greater heart rate and decreased inotropic responses to either
isoprenaline or carbachol, the agonists of beta-adrenoceptors or muscarinic receptors,
respectively. RWPC treatment did not modify cardiac expression of eNOS or Cu/Zn
superoxide dismutase, a protein involved in oxidative stress protection. However, it was
found increased nitrite levels in the coronary effluent from hearts harvested from RWPC-
treated rats, suggesting an increased NO production. Most interestingly, in ischaemia-
reperfusion protocols, RWPC treatment reduced infarct size, oxidative stress, and the
myocardial content of end products resulting from lipid peroxidation, malondialdehyde and
4-hydroxynonenal, without affecting post-ischaemic contractile dysfunction. All these
observed effects were prevented by L-NAME treatment, suggesting the involvement of NO
in this protective role of RWPC on heart. In conclusion, these data showed that short-term
treatment with RWPC could prevent the heart injury caused by cardiac ischemia through
oxidative stress decrease and NO pathway improvement (Ralay-Ranaivo et al., 2004).
The presence of melatonin in red wine was demonstrated in most recent studies. Lamont
and co-workers investigated the cardio-protective role of both melatonin and resveratrol.
These molecules improve heart protection via the activation of the newly discovered
survivor activating factor enhancement (SAFE) pathway. This pro-survival signaling
pathway involves the activation of pro-inflammatory molecules such as tumor necrosis
factor alpha (TNF) and interleukin 6 (IL6) and the signal transducer and activator of
transcription 3 (STAT3). They realized ex vivo studies in isolated perfused hearts from either
wild type or total TNF receptor 2-knockout or cardiomyocyte-specific STAT3-deficient
mice. The protocols of heart injury by ischemia-reperfusion showed that both resveratrol
and melatonin, at concentrations found in red wine, significantly reduced infarct size in
wild-type mice (25% 3% versus 69 3% in the control non treated mice) but failed to
protect hearts in both knockout mice. Perfusion with either melatonin or resveratrol
increased STAT3 phosphorylation prior to ischemia by 79% and 50% versus the control,
respectively. These findings suggest that both melatonin and resveratrol contained in red
wine, protect heart via the SAFE pathway, in an experimental model of myocardial
infarction (Lamont et al., 2011).

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Concerning cerebral ischemia, Ritz and co-workers investigated the beneficial effects of
chronic or acute treatment of RWPC in rats submitted to an experimental model of stroke.
Rats were treated for the chronic treatment with RWPC (30 mg/kg per day) dissolved in
drinking water for one week, before being subjected surgically to a transient middle cerebral
artery occlusion followed by reperfusion. The volume of the ischemic lesions was assessed
24 h after reperfusion and a proteomic analysis of brain tissues was performed, to study the
effects of RWPC on expression of proteins involved in cerebral stroke injury. Treatment with
RWPC partially or completely prevented the increased levels of excitatory amino acids
(aspartate, glutamate and taurine) that characterized the response to ischemia in control
rats, significantly reduced brain infarct volumes, and enhanced residual cerebral blood flow
after brain ischemia. This was associated to lower basal concentrations of energy metabolites
including glucose, lactate, and free radical scavengers such as ascorbate, in the brain
parenchyma, compared with untreated rats. No difference in uric acid levels was found.
These effects resulted in arterial vasodilatation, as the internal diameters of several arteries
were significantly enlarged after RWPC treatment. Proteomic analysis revealed that RWPC
could be able to modulate in vivo the expression of proteins involved in maintenance of
neuronal caliber and axon formation, in protection against oxidative stress, and in energy
metabolism (Ritz et al., 2008a). These data were confirmed in the second work of the same
team, about the protective effects of an acute treatment with RWPC (a bolus of 0.1 mg/kg),
realized by an intracerebral microdialysis started at the beginning of the stroke. In this
study, RWPC induced increased residual blood flow after 10 minutes of the reperfusion
following ischemia and reduced size of the cerebral ischemic infarct in both cortex and
striatum. The acute treatment of rats with RWPC dramatically decreased the extracellular
concentrations of excitatory amino acids and, concomitantly, increased the levels of free
radical scavengers such as uric and ascorbic acids (Ritz et al., 2008b). Altogether these
findings provide an experimental evidence of the advantage to use RWPC for the
prevention, in patients with high risk to developing ischemic events, or in the acute
treatment of patients during stroke.
Angiogenesis is a main process involved in the repair of ischemic injury. The role of RWPC
in angiogenesis was also investigated and several studies evidenced that these molecules are
able to modulate, at the molecular and cellular levels, several actors of the pivotal pathways
involved in vascular cell proliferation and migration. Previous studies had demonstrated an
anti-angiogenic role of polyphenols both in vitro and in vivo (Fotsis et al., 1998; Igura et al.,
2001). In contrast, most recently, Baron-Menguy and co-workers evidenced a dose-
dependent effect on angiogenesis of RWPC, and in particular of delphinidin, in a model of
post-ischaemic neovascularization in rats submitted to femoral artery ligature. Indeed, high
doses of RWPC (i.e. 7 glasses of red wine) reduced arterial, arteriolar, and capillary densities
and blood flow, inhibited the phosphoinositol 3-kinase (PI3-K)/Akt/eNOS pathway,
decreased vascular endothelial growth factor (VEGF) expression, and reduced
metalloproteinase-2 (MMP-2) activation. In contrast, low doses of RWPC (i.e. 1/10
th
glass of
red wine) increased neovascularisation in ischemic legs compared to control level in
association with an increased blood flow. The angiogenic effect was linked to the
overexpression of PI3-K/Akt/eNOS pathway and to increased VEGF production, without
effect on MMP-2 activation. These anti- or pro-angiogenic effects of RWPC were reproduced
when they used delphinidin, administrated alone at low or high doses. This dual dose-
dependent effect of polyphenols in angiogenesis is particular interesting because of its
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329
potential applications both in the therapy of diseases requiring the block of angiogenesis
such as in some cancers, and in the treatment of post-ischemic injuries to improve
angiogenesis and ameliorate reperfusion of tissues, at high and low doses, respectively.
4.3 Role in metabolic diseases
It has been extensively evidenced the strict correlation between metabolic dysfunctions and
the development of cardiovascular diseases. Endothelial dysfunction, an independent
predictor of cardiovascular events, has been consistently associated with obesity and the
metabolic syndrome in a complex interplay with insulin resistance. Deficiency of eNOS is
considered as the primary defect that links insulin resistance and endothelial dysfunction
(Cersosimo and Defronzo, 2006; Defronzo, 2006; Fornoni and Raij, 2005). Furthermore,
several epidemiological studies have shown that patients affected by metabolic diseases are
often also affected by hypertension and other cardio-vascular complications such as
atherosclerotic plaque formation and increased levels of pro-thrombotic factors, associated
to an elevated risk of mortality by vascular thrombotic events (Kopelman, 2000).
More recently, we have suggested a protective role of RWPC in metabolic syndrome
(Agouni et al., 2009). In our study, Zuker fatty (ZF) rats (Fa/Fa), an experimental model of
metabolic syndrome, or their lean littermates, received normal diet or a diet
supplemented with Provinols
TM
for 8 weeks in food. This treatment significantly reduced
the plasmatic levels of metabolic products such as glucose, fructosamine, total and LDL-
cholesterol, and triglycerides, and finally improved cardiac and endothelial vascular
functions. Regarding vascular function, Provinols
TM
corrected endothelial dysfunction in
aortas and mesenteric arteries from ZF rats by improving endothelium-dependent
relaxation in response to acetylcholine. This beneficial effect in endothelium was associated
to an enhanced NO bioavailability due to increased NO production and eNOS activity, and
reduced oxidative stress and O
2
-
release. The effect on eNOS activity was associated to a
decreased expression of caveolin-1, a protein known to inactivate eNOS by cell membrane
sequestration, while the reduction of free radical production was linked to a decrease of
Nox-1 (NADPH oxidase membrane sub-unit) expression (Agouni et al., 2009). In agreement
with our work, this protective effect of RWPC in plasmatic metabolic parameters and
oxidative stress linked to metabolic disorders was confirmed recently in hamsters submitted
to high-fat diet (Suh et al., 2011).
Because of these interesting results, polyphenols might be good candidates for prevention
and treatment of metabolic syndrome and cardiovascular risk reduction. This was
previously suggested by another study of Napoli and coworkers who have shown that red
wine consumption improved insulin resistance in type 2 diabetic patients (Napoli et al.,
2005). Thus, RWPC could represent a new class of medicinal products against obesity-
associated diseases.
5. The oestrogenic receptors in cardiovascular protection
Several epidemiological studies suggested a protective effect of oestrogens in
premenopausal women in vascular and metabolic diseases development. These numerous
studies showed that the incidence of hypertension and other cardiovascular diseases is
significantly lower in premenopausal females compared to males and that, after the onset of

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330
menopause, the incidence increases dramatically, eventually approaching the level observed
in age-matched males (Mendelsohn and Karas, 1999). This effect has been attributed to the
fall in circulating oestrogen levels, contributing to a menopause-related increase in blood
pressure, and thus to a greater predisposition to cardiovascular disease. Consistent with
this, oestrogen replacement therapy has been reported to reduce the risk of cardiovascular
disease, and in particular of hypertension and atherosclerosis, in postmenopausal women to
that observed in premenopausal women (Barton et al., 2007; Mendelsohn and Karas, 1999).
Oestrogens have been shown to have direct vasodilatory and anti-atherosclerotic effects via
the oestrogen receptors expressed on human and rat arteries (Haas et al., 2007; Shaw et al.,
2001). The mechanisms involved in the protective role played by these hormones is
associated to vascular inflammation reduction (Nilsson, 2007), increased endothelial NO
production (Chen et al., 1999) and the prevention of smooth muscle vascular cell
proliferation (Pareet al., 2002). But the ability of oestrogens to elicit effects on autonomic
functions involved in cardiac control appears also to constitute a major part of its beneficial
effects (Spary et al., 2009). Despite wealth of evidences for its central autonomic role, the
sites and mechanisms of oestrogenic action on the neural pathways of cardiovascular
regulation are still poorly understood.
Oestrogens act on specific receptors which are transcription factors, the nuclear oestrogenic
receptors (ERs). Two ERs have been described, ER and ER, with several structurally and
functionally conserved domains, and involved in genomic signaling mechanism or
associated to plasma membrane, influencing cytosolic non-genomic signaling. ER was first
characterized in mid-1980 and the cloning of ER following in late 1995 (Kuiper et al., 1996).
In addition, as a result of alternative splicing of the eight exons encoding rat ER, five
different isoforms of this ER exist (1, 2, 13, 23 and 14) with a not yet completely
determined role (Maruyama et al., 1998; Petersen et al., 1998; Price et al., 2000). It has been
suggested that ER may modulate ER gene transcription, acting in some conditions by
opposite actions to ER (Lindberg et al., 2003; Maruyama et al., 1998; Zhao et al., 2008).
In the absence of oestrogens, the receptors are conserved in an inactive state in a complex
with one of the several chaperone molecules, such as heat shock protein 90 (Beato and Klug,
2000). Following binding to oestrogens, the receptor undergoes a conformational change,
activating an intracellular cascade leading to the ER release from the chaperone. ER can
forms homo- or hetero-dimers that interact with target gene promoters, inducing the up- or
the down-regulation of several genes (Figure 1) (Hall et al, 2001). The ER subtypes have also
been shown to interact differently with a range of other transcription factors, including
activating protein-1 (Paech et al., 1997; Webb et al., 1999; Zhao et al., 2008). This genomic
response usually occurs within hours after oestrogen exposure and is believed to be the
result of a direct action, not involving the second messenger signaling pathways. In contrast,
the non-genomic oestrogenic signaling is also possible but less well understood. It is
associated to the cytosolic pathways with classical second messengers and occurs
considerably faster than the genomic signaling (Kang et al., 2010). It is possible that these
rapid non-genomic events are mediated by cytoplasmic, rather than nuclear ER and ER,
suggesting the involvement of another plasma membrane receptor, a particular G protein-
coupled receptor (GPCR) which is not related to ER or ER. To confirm this hypothesis
more recently, another membrane-bound ER was emerged. This GPCR, the G protein
coupled oestrogen receptor 1 (GPER1), also called GPR30, is able to bind with a high affinity
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331
to 17-estradiol (E2), mediating oestrogenic signals in cardiovascular and metabolic
regulations (Nilsson et al., 2011). GPER1 is expressed in different vascular segments and in
the heart of several species. In rats, the mRNA of this receptor was found both in endothelial
and in smooth muscle cells; but in mice and humans, it seems to be expressed primarily in
endothelial cells of small systemic arteries, suggesting a direct role of GPER1 in endothelial
function regulation, while the effects of its activation in vascular smooth muscle cells and
vascular tone are indirect, via the endothelium (Nilsson et al., 2011). GPER1 is located to the
endoplasmic reticulum of vascular cells mediating the rapid oestrogen signaling (Revankar
et al., 2005).
The role of GPER1 activation by its specific agonist, G-1, on vascular tone was
investigated in rat vessels. Several studies showed the involvement of this receptor in
vascular relaxation by reducing angiotensin II (AngII) and/or endothelin-1 (ET-1)-
induced vascular contractions. This was not influenced by the endogenous oestrogenic
levels and it was gender independent (Haas et al, 2009, Lindsey et al., 2009; Meyer et al.,
2010). This effect was not found in serotonin-dependent vascular contraction, suggesting a
direct effect of GPER1 activation on the renin-angiotensin system, probably independent
of NO production (Nillson et al, 2011). In contrast, another study suggests that GPER1
causes arterial relaxation via an endothelial and a NO-dependent mechanism (Broughton
et al., 2010). Thus, the involvement of endothelial NO in this vascular relaxation cannot be
excluded. Moreover, an hypotensive effect of GPER1 activation was observed in
ovariectomized animals, in agreement with the hypertensive phenotype of GPER1 knock-
out mice (Martensson et al., 2009). Furthermore, GPER1 activation could play a protective
role in atherosclerosis and/or excessive angiogenesis during cancer, reducing vascular
smooth muscle or endothelial cell proliferation, respectively (Haas et al., 2009; Holm et al.,
2011).
If the non-genomic effects of E2 are realized through GPER1, ER is the receptor
implicated in the anti-atherogenic effects of oestrogens. Indeed, the ER, when stimulated
by E2, induces endothelial cell proliferation, vascular re-endothelialization, endothelial
NO production, vascular inflammation attenuation, and reduction of smooth muscle cell
proliferation (Brouchet et al., 2001; Pare et al., 2002; Vegeto et al., 2003). Nevertheless,
studies conducted on vessels harvested from ER or ER knockout mice showed that both
these ERs are responsible for E2-dependent vascular relaxation (Guo et al., 2005). It was
previously evidenced the association of a subpopulation of ER with the endothelial
membrane and the complex structure of caveolae (Chambliss and Shaul, 2002). The
binding of E2 with ER in caveolae leads to the MAPK/Akt pathway activation, resulting
in eNOS phosphorylation and activation, and subsequent increased NO production
(Figure 1) (Chambliss and Shaul, 2002). This beneficial effect on vascular function played
by oestrogens was confirmed by epidemiological studies, in which the presence of
endogenous oestrogens and their effect on cardiovascular homeostasis appear to be
closely related to the degree of atherosclerosis progression throughout a woman's life
(Clarkson 2007). Experimental studies suggest that in the mouse, ER appears to be
largely responsible for the protective effects of oestrogens against atherosclerotic vascular
disease (Hodgin et al., 2001). In turn, according to some studies, the abundance of both ER
subtypes, ER and ER, in human aorta, decreases with the progression of

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332
atherosclerosis, aggravating the endothelial dysfunction of atherosclerotic vessels by the
reduction of oestrogenic-dependent eNOS activation and NO release (Losordo et al., 1994;
Nakamura et al., 2004).
On the light of the effect of E2 via ER in eNOS pathway activation and NO production, a
vascular role of oestrogens, similar to that evidenced for RWPC on endothelium, was
evoked. Some researchers and our studies started to investigate if RWPC or one of the
polyphenolic compounds contained in red wine, resveratrol or delphinidin, could play a
role of phytoestrogens, interacting at high affinity with ERs and inducing their beneficial
vascular effects via these endothelial receptors.
6. Oestrogenic receptor alpha and polyphenols
After the description of these encouraging findings, nobody exactly identified the pivotal
compound responsible of RWPC vascular effects and, most important, how this molecule
was able to interact with the vascular endothelium, thus improving endothelial function.
It was previously described that resveratrol is able to enhance eNOS expression and
activity, but the mechanisms by which this polyphenol induced these effects were still not
well known (Wallerath et al., 2002). In a study conducted in vitro in BAECs, nanomolar
concentrations of resveratrol induced ERK1/2 signaling activation, similar to that of E2,
since this was dependent of ER activity triggering eNOS activation and NO release
(Klinge et al., 2005). The same team, in another study in vitro (in HUVECs), better
determined the mechanisms by which resveratrol was able to improve eNOS activation
pathway. The authors of this work demonstrated for the first time that resveratrol
increased interaction between ER, Caveoline-1 (Cav-1) and proteins involved in eNOS
activation such as Src, by a G-protein-coupled mechanism. A main role for ER in the
NO production induced by resveratrol in endothelial cells was suggested because they
observed attenuated effects of resveratrol in cells in which ER was depleted using a
siRNA. Resveratrol and E2 did not stimulate ER/Cav-1 interaction (Klinge et al., 2008).
Moreover ER is 4.5 times more expressed then ER in HUVECs and no effect of a siRNA
directed versus ER was found on resveratrol action in endothelium. This study implies
that dietary intake of resveratrol might offer possible vascular protective effects via the
activation of ER in vivo.
In contrast, experiments conducted in rats did not evidence a role of oestrogen receptors
in aorta endothelium-dependent relaxation to RWPC (Kane et al., 2009). The authors of
this work showed that RWPC caused redox-sensitive PI3-K/Akt-dependent eNOS
activation and NO-mediated relaxation in rat aortas ex vivo. This vascular effect was more
pronounced in the aorta of female than male rats, but it was due most likely to increased
expression levels of eNOS rather than activation of oestrogen receptors, because the
inhibition of ER by the oestrogen antagonist, ICI 182780, did not modify the ability of
RWPC to induce their vascular effects (Kane et al., 2009). Interestingly, another study
conducted in female SHR rats evidenced that the chronic treatment with RWPC of
ovariectomized rats induced reduction of arterial pressure and vascular dysfunction
characterizing this hypertensive model in a manner independent of the ovarian function
(Lopez-Sepulveda et al., 2008).
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Cardiovascular Protection: The Oestrogenic Receptor- Implication

333

Fig. 1. ER activation by polyphenols or oestrogens induces eNOS increased activity and
NO production. Signaling pathway by which delphinidin or E2 interacting with ER,
activates rapidly eNOS and increases NO production in endothelial cells by PI3-K/Akt or
via Src/ERK1/2 pathways. ER is associated in endothelial cells caveolae with Cav-1 which
links to the membrane the inactive form of eNOS. When ER binds E2 or polyphenols,
eNOS is phosphorylated in its active site, thus improving NO release. The same pathways
implicated in delphinidin-ER activation were proposed by Klinge and coworkers for
endothelial cell stimulation by resveratrol at nanomolar concentrations (Klinge et al., 2008).
NO is able to activate guanilyl-cyclase (GLc) in smooth muscle cells inducing increased levels
of cyclic-GMP (GMPc) with subsequent protein-kinase G activation (PKG), reducing
intracellular calcium and inducing vascular relaxation. On the right of the figure, is
represented the homo-dimer formation and nuclear translocation of E2-activated ER,
inducing the genomic response.
Conversely, we investigated the hypothesis that ER is one of the key targets involved in
vivo in the vasculoprotective effects of RWPC (and in particular of delphinidin) interacting
with the endothelium. Thus, the ER implication in the French Paradox was first tested
using ER-deficient mice (Chalopin et al., 2010). We have shown the necessity of this
oestrogenic receptor in the Provinols
TM
- or delphinidin-induced endothelial-dependent
relaxation, eNOS activation, and NO release. Indeed, no effect of these products on
endothelium were observed in vessels harvested from ER-deficient mice or in wild-type
vessels without endothelium. The activation of ER by RWPC or delphinidin alone induced
the activation of the same pathway, evidenced by the previously described in vitro work of
Klinge and colleagues with resveratrol. Indeed, E2 and the selective agonist of ER, 1,3,5-
tris(4-hydroxyphenyl)-4-propyl-1H-pyrazole (PPT), as well as Provinols and delphinidin,
are able to activate molecular pathways involving Src, ERK1/2, eNOS and caveolin-1
phosphorylations (see Figure 1). The mechanism involved required ER activation because

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334
of the absence of effect in vessels or cells from ER-deficient mice, and after silencing, in
wild-type endothelial cells, ER activity or expression either with a pharmacological
inhibitor (fulvestran) or with a siRNA, respectively. Moreover, using a binding assay and a
docking study, we have shown that delphinidin fits on ER's activation site, exerting 73% of
specific inhibition against E2 on ER, in the binding assay. Most importantly, ER is also
implicated in the in vivo effects observed in mice treated with Provinols administrated in
the food, with respect to the improvement in endothelial function given by the concomitant
increase in NO and decrease in O
2

release in vessels. Indeed, these vascular and anti-
oxidant effects of the in vivo treatment with Provinols
TM
were not found in ER-deficient
mice (Chalopin et al., 2010). Then, we have demonstrated for the first time the physiological
relevance of ER in the in vivo vascular effects of RWPC.
It is important to note that ER, ER, and GPER-1 are all expressed in the arterial wall of
both women and men (Meyer et al., 2006; Haas et al., 2007), and that E2 has potent dilator
effects on vascular tone of human coronary and internal mammary arteries harvested from
patients without gender differences (Haas et al., 2007; Mugge et al., 1993). These findings
suggest a potential function for oestrogen receptor also in male cardiovascular system. Thus,
RWPC could have the same protective vascular properties in both women and men through
ER. In line with the fact that ER mediates atheroprotective effects, in a man with a
disruptive mutation in the ER gene, it was noted an impaired vascular function and a
premature coronary artery disease (Sudhir et al., 1997). Thus, not only the female but also
the male cardiovascular system appears to be an important target for oestrogens affecting
vascular disease development (Haas et al., 2007; Meyer et al., 2008). Nevertheless, studies in
humans comparing oestrogen plasma concentrations and the progression of cardiovascular
diseases have revealed conflicting results (Meyer et al., 2008). Actually, there is doubt about
the interest to treat male patients with oestrogen receptor agonists to interfere with
atherosclerosis progression.
Finally, further works are needed to confirm if ERs are implicated in all the vascular and
metabolic effects of RWPC or if ER activation by RWPC induces only the eNOS pathway
improvement. For instance, the role of ERs activation by RWPC in inhibition of endothelial
cell proliferation and cell cycle progression or in angiogenesis has not been investigated yet.
7. Conclusion
The first epidemiological studies played a main role in the demonstration of a French
Paradox existence, leading to the start of about forty years of scientific findings concerning
the protective properties of polyphenols and, more particularly, those contained in red
wine. Currently, the numerous data obtained in vitro, ex vivo, and in vivo, on their beneficial
effects in heart and vessels, validly suggest a therapeutic potential for RWPC.
The last findings have identified in delphinidin and resveratrol some of the key molecules
involved in the vascular effects of RWPC via ER activation, adding a new piece to the
puzzle explaining the French Paradox (Chalopin et al., 2010; Klinge et al., 2008). Indeed,
despite a previous study (Kane et al., 2008), which evidenced no implication of ERs in
RWPC-dependent vascular relaxation in rats, the last studies clearly showed that the
beneficial endothelial effects of RWPC require ER activation. This is followed by a rapid
response to the polyphenolic stimuli in endothelial cells, involving the pathways associated
French Paradox, Polyphenols and
Cardiovascular Protection: The Oestrogenic Receptor- Implication

335
to eNOS activation and subsequent NO release. Furthermore, the phytoestrogenic role of
RWPC, and especially of delphinidin, was confirmed by binding experiences which found
high affinity of delphinidin against ER compared to its natural agonist E2 (Chalopin et al.,
2010). Similar mechanisms and a phytoestrogenic role on ER activation were suggested
also for resveratrol on endothelial cells by Klinge and coworkers (Klinge et al., 2005, 2008).
In this chapter, we have focalized our attention on the red wine because it contains both,
delphinidin and resveratrol, the main vasoactive compounds contained in non-alcoholic red
wine extract. In particular, we wanted to explain the main mechanisms by which these
compounds are able to induce cardiovascular protection against hypertension, cardiac
ischemia, stroke and atherosclerotic plaque formation as one of the complications linked to
metabolic syndrome. It is important to note that the effects of these substances could be
different according to the concentrations employed as evidenced in experimental models of
angiogenesis (Baron-Menguy et al., 2007). It is also relevant to remember of other beneficial
properties of RWPC, as anti-oxidant, anti-inflammatory, anti-tumor or antithrombotic
agents, that we have not extensively described here. Indeed, RWPC are also able to
modulate the apoptotic, proliferative or migration processes in cells (Martin et al., 2003) by
acting directly on vascular remodeling and angiogenesis (Brownson et al., 2002; Favot et al.,
2003). Here, we have chosen to stress on strong properties of RWPC as vasodilators
inducing endothelial NO production, because this effect implicates ER activation as
demonstrated in the last studies.
Furthermore, despite the favorable effect of some molecules contained in red wine in the
prevention of several cardiovascular pathologies, alcohol is a serious problem of public
health and, actually, it is important to remenber that these beneficial effects are due to the
non-alcoholic fractions of red wine. Interestingly, in multinational studies it was shown an
increased risk of mortality by myocardial infarction, especially in women who take no
alcohol, but compared to moderate drinkers (Yusuf et al., 2004). Moreover, on the light of
other epidemiological data, it seems to be developed the view that modest alcohol but
neither zero nor more than modest intake reduces total mortality and cardiovascular risk by
cardio and neuroprotection (Collins et al., 2009; Opie and Lecour, 2007).
According with the French Paradox, the moderate intake of wine (1 or 2 glasses per day)
could be beneficial for health by reducing the risk of CVD mortality. As evidenced in Table
1, the content of these vasoactive substances is more relevant in red wine compared to other
food and beverages. Finally on the light of all the epidemiological and fundamental studies
analyzed in this chapter, and our works, we can suggest that RWPC, and in particular
delphinidin and resveratrol, could be used for their therapeutic potential in the prevention
and treatment of cardiovascular pathologies. We think that ER activation might be the
main molecular target triggering the beneficial effects of dietary supplementation of RWPC.
Nevertheless, further studies are needed to verify the implication of ER in other
physiological effects of polyphenols and not only in NO release and vascular relaxation.
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17
Importance of Dermatology
in Infective Endocarditis
Servy Amandine, Jones Meriem
and Valeyrie-Allanore Laurence
*

Department of Dermatology,
Hpital Henri Mondor, Crteil,
France
1. Introduction
Infective endocarditis (IE) is a rare affection with an annual incidence of between 15 to 60
cases per million. If untreated, IE is fatal, and the overall mortality is evaluated above 20%.
IE is an endovascular microbial infection of intracardiac structures. The early characteristic
lesion corresponds to variable sized vegetation leading to valvular destruction and abscess
formation.
Epidemiologic profile evolved progressively with decreasing proportion of IE on abnormal
native valve compensated by an increased proportion of prosthetic valve IE and native
valve IE with previously unrecognized predisposing conditions. Among causative
microorganisms, the responsibility of staphylococci is more frequently observed.
Diagnosing IE remains a clinical challenge because evolution is insidious and symptoms are
polymorphous. This diagnosis must be systematically considered in the presence of
purpura, distal necrosis but also in patients who had have chronic dermatosis which
correspond to an underestimated potential source of IE.
2. Pathophysiology
Secondary to damage of endothelium, extracellular matrix proteins are exposed leading to
development of non-bacterial thrombotic endocarditis (NBTE) with fibrin and platelets.
Endothelial damage can occur after mechanical lesions (devices, repeated intravenous
injection of particulate material), turbulent blood flow (congenital heart disease, prosthetic
valves), inflammation (chronic rheumatic fever) or degenerative lesions (European society
of cardiology [ESC], 2009). NBTE facilitates micro-organism adherence and infection of
endothelium (Figure 1).
International specialists (American Heart Association [AHA], 2007; ESC, 2009) no longer
differentiate acute, subacute and chronic IE based on usual progression of untreated disease.
Indeed, although clinical manifestations are more insidious in subacute IE, severe

*
Corresponding Author

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complications can occur and it is currently difficult to determine the onset of the disease.
Presently, IE are classified depending on the type of valve damage (right/left-sided,
native/prosthetic valve).

Fig. 1. Pathophysiology of IE with aseptic and septic phases (A. Servy; August 2011). NBTE:
non- bacterial thrombotic endocarditis

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347
3. Epidemiology
3.1 Incidence
IE is a rare disease with 2 to 6 per 100 000 persons affected per year (Que, 2011). Classically
described in young patients after chronic rheumatic heart disease. Its incidence in
industrialized countries is more elevated in at-risk groups, mainly persons older than 65 years
old (15 per 100 000 per year). At present, the average is 57 years old (Que, 2011; ESC, 2009).
Native valve IE is the most frequent. Left-sided native valve IE represents 70% of disease
incidence and the mortality is evaluated at 15% (25-45% with healthcare-associated). 5-10%
of IE affect right-sided native valve, mainly in intravenous-drug users, and patients with
congenital heart disease or devices. Staphylococcus spp is most frequently involved in right-
sided native valve IE and mortality is less than 10% (Que, 2011).
Prosthetic valve IE is also increasing (10-30% of IE), mechanical and bioprosthetic equally.
The prevalence of valve prostheses IE is above 6% (0.3-1.2% per year). Left-sided prosthetic
valve endocarditis (20% of IE) is the most severe with 20 to 40% mortality. The main germs
involved in early prosthetic valve IE (less than one year after cardiac surgery) include
staphylococci, fungi and Gram-negative bacilli, whereas late IE is associated with
staphylococci, oral streptococci, S. bovis and enteroccoci (ESC, 2009).
3.2 Risk factors
3.2.1 Characteristics of patient
The main risk factor is age (median age above 60) (Murdoch, 2009) due to degenerative
valve, immunosuppressive conditions and multiple comorbidities. However, edentate
people have a lower risk of IE. Digestive portal of entry is frequent in this population,
mainly in S. bovis and enterococcus IE and should be researched.
Many comorbidities increase the risk of IE, leading to heart diseases. At present, in
industrialized countries, chronic rheumatic heart disease has become exceptional and the
proportion of degenerative valve lesion and congenital heart disease is more important as
well as their responsibility for IE (Moreillon, 2004). Chronic immunosuppressive therapy
(chemotherapy, topical corticosteroid) or affections are predisposing conditions, mainly
diabetes mellitus (16% of IE), hemodialysis (8%), cancer (8%) and HIV infection (2%)
(Murdoch, 2009). Physicians should be aware of the risk of EI in cases of acute or chronic
dermatosis. Chronic bacteria carriers, wounds, and percutaneous invasive procedure
increase significantly the risk of bacteremia.
3.2.2 Situations at risk
All iatrogenic invasive procedures are at risk of bacteremia such as catheter, urinary
surgery, and endoscopy (Table 1). Nevertheless, intravenous drug users are more at risk
(10% of IE) due to poor hygiene. Indeed 55% of active heroin, cocaine and
methamphetamine injection drug users report a lifetime history of skin infection mainly in
cases of intramuscular injection or frequent heroin or speedball injection (Phillips, 2010). In
these cases, S. aureus and fungi must be suspected and treated. Dental treatments are too

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348
easily suspected (AHA, 2007; Strom, 1998) whereas most of the time, no procedure or
situation at risk are identified and daily bacteremia is often involved (AHA, 2007). In a
recent French study (Association pour l'Etude et la Prvention de l'Endocardite Infectieuse
[AEPEI], 2002), 63% of IE cases had no situation at risk identified.

Risk factors of IE
Patient characteristics Situations at risk
Age Invasive procedures:
- percutaneous (drug, catheter)
or
- dental
Daily bacteremia
- Brushing teeth
- Chewing
Comorbidities Heart disease and prosthetic
valve
Diabetes mellitus
Chronic renal failure
Immunosuppressive affection
Treatment Immunosuppressive therapy
Table 1. Procedures and situations at risk of bacteremia.
3.3 Causal microorganisms
Distribution of causative microorganisms of IE is different, depending on the patients
characteristics (Table 2) and portal of entry. Gram-positive bacteria are the most frequent
microorganisms. They are responsible for more than 80% of IE because they have greatest
ability to adhere and colonize damaged valves (Que, 2011).
Microorganisms (%)
Valve affected
Native valve IE Intracardiac device IE
Drug abusers
Others
patients
Prosthetic
valve
Others
Staphylococcus aureus 68 28 23 35
Coagulase-negative
staphylococcus
3 9 17 26
Viridans group streptococci 10 21 12 8
Streptococcus bovis 1 7 5 3
Enterococcus ssp 5 11 12 6
HACEK 0 2 2 1
Fungi 1 1 4 1
Polymicromial 3 1 0.8 0
Negative culture findings 5 9 12 11
Table 2. Microbiologic etiology of IE depend on patients characteristics (Murdoch, 2009).
HACEK: Haemophilus (parainfluenzae, aphrophilus, paraphrophilus and influenza),
Actinobacillus actinomycetemcomitans, Cardiobacterium hominis, Eikenella corrodens,
Kingella (kingae and dentrificans).
Although increasing involvement of oral streptococci, streptococcaceae remain the main
pathogen (nearly 60% of IE). In streptococci group, group D (S. bovis) are found in 25%,
oral streptococci in 17% and pyogenic streptococci (S. agalactiae, S. pyogenes) in 6% of IE.

Importance of Dermatology in Infective Endocarditis

349
Enterococci (mainly E. faecalis) are also frequent (8%), mainly in elderly people and
prosthetic valve carriers (AEPEI, 2002). Urinary and digestive portal of entry (including
colon cancer, and diverticulitis) must be researched with colonoscopy and imaging.
Presence of S. bovis equally implies digestive portal of entry.
The role of Staphylococcaceae is increasing (20-34% of IE) with 23% of IE due to S. aureus
and 6% to coagulase-negative staphylococci (AEPEI, 2002; Miro, 2005). Staphylococcus IE is
more frequent in intravenous drug users, HIV patients, right-sided IE and iatrogenic
infection. The prevalence of IE in patients with Staphylococcus aureus bacteremia is elevated
(22%) and some authors recommend a systematic echocardiography in this situation
(Rasmussen, 2011).
The responsibility of others germs is lesser and unusually several microorganisms are
associated in rare instances (less than 5%). No germ is identified in 5% of cases (AEPEI, 2002).
3.4 Portals of entry
Any site of infection can be responsible of IE. However, some portals of entry are more
frequent and must be investigated. Cutaneous portal of entry is frequent (20%) and often
misdiagnosed by physicians. In these cases, IE mainly developed on traumatic or chronic
wounds, infected or inflammatory dermatosis, intravenous drug use, percutaneous
iatrogenic procedures Dental portal of entry is observed in 9% of cases (poor dental
condition, dental procedure). Genitourinary and digestive portal of entry are observed
respectively in above 2-11% and 5-9% (AEPEI, 2002; Tornos, 2005).
4. Diagnosis
4.1 Clinical manifestations
Clinical diagnosis of IE is often difficult because of various clinical manifestations and
insidious evolution. Moreover, atypical presentation is usual in elderly,
immunocompromised patients (lack of fever) and carriers of prosthetic valve, mainly in
earlier phase (less than one 1 year after surgery). In fact, in this last group, blood cultures are
frequently negative, echocardiography is difficult (ESC, 2009) and inflammatory syndrome
and fever is classical even in absence of IE. So, clinical suspicion of IE should be
systematically discussed in these cases, and complementary investigations performed.
4.1.1 General signs
Fever is the most frequent sign (approximately 90%) and usually temperature normalizes
within 1 week (5-10 days) under adaptive antibiotherapy. An impaired general health
condition can be observed with weight loss, fatigue and anorexia.
4.1.2 Cardiological signs
Cardiological manifestations are nearly constant. Heart murmurs are found in up 85% of IE
(ESC, 2009) but occurrence of new ones (48%) or increasing of an older murmur (20%) are
more evocative (Murdoch, 2009). Clinical manifestations of heart complications can be
added (mainly heart failure).

The Cardiovascular System Physiology, Diagnostics and Clinical Implications

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4.1.3 Extracardiac manifestations
Extracardiac manifestations are also frequent, particularly in right-sided IE (78% versus 52%
in left-sided IE) with 68% of pulmonary embolism (AEPEI, 2002).
If IE is suspected, dermatological manifestations should be systematically searched and
discussed despite rarity (5 to 25% of IE present skin manifestations) not only for diagnosis but
also for prognostic (Table 5). They can easily lead to suspicion of IE. In our recent study
(unpublished data), we demonstrated a link between the presence of cutaneous signs and
embolic events (18.4% of embolic events in lack of cutaneous sign versus 33%) without higher
mortality. Dermatological manifestations (Figures 2 and 3) seem to be also less frequently
observed with enteroccoci infection (14.5% versus 27.1%) (Martnez-Marcos, 2009).

Fig. 2. Vascular purpura on trunk and arms during IE.

Importance of Dermatology in Infective Endocarditis

351

Fig. 3. Necrotic lesions of fingers (same patient): old Janeway lesion or purpura lesion.
- Oslers lesions are specific and described as purple painful nodes on palms, soles,
fingertips, pulp of the toes or sometimes on ears (Farrior, 1976). Unfortunately,
prevalence is low (3-3.6%) (AEPEI, 2002; Murdoch, 2009) and lesions disappear in a few
days without sequelae. In a study including 43 intravenous-drug users IE, Oslers nodes
were observed in 50% of left-sided IE whereas none were noticed in right-sided IE (33
right-sided). Moreover, bacteriological study of nodes revealed the same
microorganisms as in blood (S. aureus).
- Janeway lesions are small non tender erythematous and painless macular (sometimes
nodular!) localized on palms or soles (2-5% of IE) (AEPEI, 2002; Murdoch, 2009). These
lesions are equally specific and their differenciation difference with Osler nodes is often
as difficult, clinically as histologically.

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- Purpura is more frequent (7.3%) (AEPEI, 2002) but not specific. Its pathophysiology is
still unclear including often septic embolism and/or leucocytoclastic cutaneous
vasculitis by complex immune depositions (Lvesque 1999). Vascular purpura is
characterized by red lesions that dont blanch on applying pressure, caused by
erythrocyte extravasation. Lesions are localized on lower parts of the body (legs, back).
In IE, lesions are also described on the neck and near the clavicles. IE mucosal purpura
is often observed on conjunctivae and mouth (Heffner, 1979).
- Splinter haemorrhages are common in many diseases and found in 8 to 14% of IE
(Konstantinou, 2009; Murdoch, 2009).
30% of IE (ESC, 2009) has at least one vascular or immunological phenomenon. Vascular
phenomenon includes systemic arterial embolism (17-33%) (AEPEI, 2002; Murdoch, 2009),
infectious embolism (septic pulmonary infarct, infectious aneurysm) and classically Janeway
lesions. Immunological manifestations are mainly represented by Oslers nodes and Roth
spots (2%) (Murdoch, 2009).
Musculoskeletal symptoms are common with mainly arthralgia (14%) (Murdoch, 2009),
myalgia and back pain. In the presence of, spondylodiscitis (3-15%) (ESC, 2009) mainly
observed in streptococci IE must be systemically discussed. Splenomegaly is less frequently
noticed (11%) (Murdoch, 2009).
4.2 Laboratory studies
4.2.1 Biology findings
- Inflammatory syndrome
In most cases, unspecific inflammatory syndrome is observed, including neutrophils
hyperleucocytosis, elevated erythrocyte sedimentation rate (ESR) and C-reactive protein.
- Microbiological diagnosis
Three sets of blood cultures, including at least one aerobic and one anaerobic samples and
spaced of at least 30 minutes, should be obtained from a peripheral vein before beginning
any antimicrobial therapy. The blood cultures are positive in 85% of cases (ESC, 2009).
However, blood culture can be negative in cases of prior antibiotherapy or specific
microorganisms (Table 3). In this last case, other bacteriological investigations are
performed, such as serologies, specific PCR and culture on surgical material, catheter and
device (pacemaker, defibrillator) or embolus samples.
Negative blood culture
Frequently Constantly: bacteria intracellular
Fastidious Gram-negative bacilli of HACEK
group
Nutritionally variant streptococci
Brucella
Fungi
Coxiella burnetii
Bartonella
Chlamydia
Trophynema whipplei
Table 3. Microorganisms and negative blood culture. HACEK group: Haemophilus
(parainfluenzae, aphrophilus, paraphrophilus and influenza), Actinobacillus
actinomycetemcomitans, Cardiobacterium hominis, Eikenella corrodens, Kingella (kingae
and dentrificans).

Importance of Dermatology in Infective Endocarditis

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- Rheumatoid factor is an immunological phenomenon, not specific but found in 5% of IE
(Murdoch, 2009).
4.2.2 Histologic findings
Valvular histology after cardiac surgery is the gold standard for diagnosis of IE and
observed vegetations, microorganisms and/or valvular inflammation (Greub, 2005).

Fig. 4. Cutaneous leucocytoclastic vasculitis (H&E stain; x200).
Kidney fragments can reveal different unspecific lesions including glomerulonephritis or
interstitial lesions.
Skin biopsies for histological study are often performed. Oslers nodes are classically
explained by immune complex deposition, mainly responsible of leucocytoclastic vasculitis.
Janeway lesions are associated with septic emboli; however, all histological findings can be
observed in both lesions (Cardullo, 1990; Kerr, 1979; Loewe, 2009; Espinosa Parra, 2002).
4.3 Imaging studies
4.3.1 Echocardiography
Echocardiography is the second fundamental examination for IE diagnosis and its heart
complications. In first-line, transthoracic echocardiography (TTE) must be systematically
performed in case of suspicion. Its sensibility only ranges from 40 to 63%. So, in cases with

The Cardiovascular System Physiology, Diagnostics and Clinical Implications

354
negative examination, poor quality of the exam, prosthetic valve transoesophageal
echocardiography (TEE) is recommended if there is high clinical suspicion. In the other
cases, a second echocardiography must be performed 7-10 days later if suspicion remains
(ESC, 2009). Evocative signs of IE are vegetations (mobile echogenic masses implanted in the
endothelium in the trajectory of valvular regurgitation or implanted in prosthetic material),
abscess and new dehiscence of a valvular prosthesis (Evangelista, 2004). However,
echocardiography does not permit differentiation between septic and aseptic vegetations; so
lesions persisting after effective treatment must not be interpreted as a clinical recurrence of
the disease unless supported by clinical features and bacteriological evidence.
Echocardiography is repeated as soon as new complications are suspected or at completion
of antibiotic therapy for evaluation of cardiac and valve function.
4.3.2 Other imaging
Computed tomography can be used in second intention to diagnose (good evaluation of
valvular abnormalities) IE (Feuchtner, 2009) and its systemic complications.
Magnetic resonance imaging is also useful for detection of complications such as cerebral
emboli.
4.4 Duke criteria
Various manifestations of IE exist and diagnosis is often difficult. Therefore, the Duke
criteria combining clinical and biological criteria have been proposed (Table 4) (Li, 1999).
5. Differential diagnoses
IE is an insidious disease associated with a clinical polymorphism. Differential diagnoses are
multiple and it is impossible to give an exhaustive list. Suspicion of IE must be
systematically discussed in cases of unexplained fever until proof of contrary. Note
echocardiographic differential diagnoses: aseptic vegetations in Libman-Sacks endocarditis
(in systemic lupus erythematosus and antiphospholipid syndrome) and marantic
endocarditis associated with gastric and pulmonary adenocarcinoma.
6. Severe complications
6.1 Morbidity
6.1.1 Heart complications
Heart failure is the most frequent complication (50 to 60% of IE) mainly on aortic native
valve IE (29%). It can be explained by valve insufficiency after native valve destruction
causing acute regurgitation (chordal rupture, leaflet rupture or perforation) or prosthesis
dehiscence. Other causes of heart failure include intracardiac fistulae, myocarditis,
pericarditis (in S. aureus infection mainly) or valve obstruction by big vegetations. Surgery is
often indicated (Table 5) in emergency because this complication is the worst predictive
factor of in-hospital and 6-month mortality.

Importance of Dermatology in Infective Endocarditis

355

Definition of term used
Pathologic
criteria
Microorganisms demonstrated by culture or histologic examination of a vegetation, a
vegetation that has embolized, or an intracardiac abscess specimen
Pathologic lesions showing active IE: vegetation or intracardiac abscess confirmed by
histologic examination
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Typical microorganisms consistent
with IE from 2 separate blood culture
Viridans streptocci
Streptococcus bovis
HACEK group
Staphylococcus aureus
Community-acquired enteroccoci in
the absence of a primary focus
Microorganisms consistent with IE
from persistently positive blood
culture
At least 2 positive cultures of blood
samples drawn > 12h apart
All of 3 or a majority of 4 separate
cultures of blood (with first and last sample
drawn at least 1h apart)
Single positive blood culture for Coxiella burnettii or antiphase I IgG antibody titer
>1:800
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Echocardiogram positive for IE
TEE recommended in patients with
prosthetic valves rated at least
possible IE by clinical criteria or
complicated IE (paravalvular abscess)
TTE as first test in others patients
Oscillating intracardiac mass on valve or
supporting structures in the path of
regurgitant jets or on implanted material in
the absence of an alternative anatomic
explanation
Abscess
New partial dehiscence of prosthetic valve
New valvular regurgitation (worsening or changing of pre-existing murmur not
sufficient)
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Predisposition, predisposing heart condition or injection drug use
Fever, temperature >38C
Vascular phenomena, major arterial emboli, septic pulmonary infarcts, mycotic aneurysm,
intracranial hemorrhages, Janeways lesion
Immunologic phenomena: glomerulonephritis, Oslers nodes, Roths spots, rheumatic
factor
Microbiological evidence: positive blood culture but does not meet a major criterion,
serological evidence of active infection with organism consistent with IE
Definition of IE
Definite IE Pathologic criteria 1
Clinical criteria 2 major criteria
1 major + 3 minor criteria
5 minor criteria
Possible IE 1 major + 1 minor criteria
3 minor criteria
Rejected Firm alternative diagnosis explaining evidence of IE
Resolution of IE syndrome with antibiotic therapy for 4 days
No pathologic evidence of IE at surgery or autopsy, with antibiotic therapy for 4
days
Does not meet criteria for possible IE as above
Table 4. Modified Duke criteria (Li, 1999) (TEE: transesophageal echocardiography; TTE:
transthoracic echocardiography)

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Perivalvular complications should be suspected in case of persistent fever, unexplained or
occurrence of atrioventricular block. They included abscess (most common in aortic and
prosthesis IE), pseudoaneurysms, fistulae and signed uncontrolled infection. Staphylococcus
aureus is most often implicated. Despite surgical treatment, 41% of patients die during
hospitalization (ESC, 2009).
6.1.2 Uncontrolled infection
Resistant microorganisms, persisting systemic infection, other sites of infection, septic shock
etc can explain locally uncontrolled infection leading to acute coronary syndrome and
third degree atrioventricular block. Indication of surgery should be discussed in these cases.
Persisting fever, after 7-10 days of antibiotherapy, may discuss uncontrolled infection,
adverse reaction to antibiotic, perivalvular complication, thrombosis, emboli A complete
infectious investigation with blood sample examination and intravenous line replacement
and cultures, should be performed as well as echocardiography.
6.1.3 Systemic embolism
Migration of cardiac vegetations is responsible for systemic embolism (20-50% of IE) mainly
in brain and spleen in left-sided IE and lung in native right-sided and pacemaker lead IE
(ESC, 2009). However, all organs can be affected in case of patent foramen ovale. Embolisms
are not uncommonly silent (20%) and often life-threatening. The incidence of embolic events
increases during the first 2 weeks after the onset of antibiotherapy. Risk factors of embolism
are individualized (Table 5) and prompt antibiotherapy can limit its occurrence (Thuny,
2005). Addition of antithrombotic therapy (thrombolytic drugs, anticoagulant or antiplatelet
therapy) doesnt appear helpful in preventing whereas cardiac surgery during the first week
of antibiotherapy (embolic risk peak) seems beneficial.

Risk factors of embolism
Vegetation
characteristics
Location Mitral valve
Multivalvular IE
Size >10mm
Mobility Increasing or decreasing under
antibiotherapy
Microorganisms Bacteria Staphylococci
Streptococcus bovis
Fungi Candida spp
Past history Previous embolism
Biology Elevated C-reactive protein
Table 5. Risk factors of embolism in IE (Durante Mangoni, 2003; ESC, 2009)
6.1.4 Neurological complications
Neurological damages after vegetation embolism are observed in 20 to 40 % of IE, mainly
due to Staphylococcus aureus infection. These complications include stroke, infectious
aneurysm (or mycotic aneurysm), brain abscess, meningitis, toxic encephalopathy and

Importance of Dermatology in Infective Endocarditis

357
seizure and are associated with poor prognosis (mainly ischaemic or haemorragic strokes)
(ESC, 2009; Thuny, 2007). Cerebral imaging (computed tomography or better magnetic
resonance imaging) should be performed in the presence of neurological signs or headaches
(infectious aneurysm).
Only poor neurological prognostic factors (coma, severe comorbidities and severe brain
damage) can prohibit cardiac surgery (Table 5). In case of haemorragic stroke, cardiac
surgery must be postponed for at least 1 month. In emergency cardiac situation, cooperation
with neurosurgeon is mandatory. The best way to prevent these complications is to quickly
start antibiotherapy (ESC, 2009).
For patients with previous antithrombotic treatment and in the absence of stroke, oral
anticoagulant therapy should be replaced by unfractionned heparin for a period of 2 weeks,
mainly in case of S. aureus IE (higher risk of bleeding). In case of an ischaemic stroke, the
same schema of replacement is proposed. Anticoagulation has to be stopped in case of a
haemorragic stroke and a mechanical valve; unfractionned heparin should be reinitiated as
soon as possible. Previous antiplatelet therapy must be stopped only in the occurrence of
major bleeding (ESC, 2009).
6.1.5 Metastatic infection
Infectious aneurysms (3% of IE) (AEPEI, 2002) are secondary to arterial septic embolism,
mainly in the brain. Most of them are silent but rupture is associated with poor prognosis.
No predicting factor has been individualized, however treatment (neurosurgery or
endovascular surgery) is proposed in case of large, enlarging or already ruptured
aneurysms. After specific antibiotherapy, most of unruptured infectious aneurysms resolve.
Systemic abscesses (other than cerebral) are rare and should be suspected in case of
persistent fever and bacteremia. Clinical criteria and imaging investigation help to find the
site of the infection s (tomography, ultrasound etc). Treatment can be completed by surgery
or percutaneous drainage in case of partial response to antibiotics. All organs can be
affected: spleen, bone (spondylodiscitis 3-15%) etc (ESC, 2009).
6.1.6 Renal complications
Acute renal failure is frequent (30%) but often reversible. Causes are multiple:
glomerulonephritis by immune complex deposition, renal infarction, haemodynamic
impairment and antibiotic or contrast agent toxicity (ESC, 2009).
6.1.7 Recurrences: Relapses and re-infections
Relapse is mainly observed after inadequate antibiotic treatment (insufficient duration,
resistant microorganisms, empirical antibiotherapy in IE with negative blood culture) or
persistent focus of infection. Conversely, re-infection is a new IE with different
microorganism(s) and mainly includes patients with previous IE, intravenous drug abusers,
prosthetic valve carriers and chronic dialysis patients. Re-infection increases risk of death
and of valve surgery (ESC, 2009).

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6.2 Mortality
In-hospital mortality varies from 9.6 to 26%. Prognosis is influenced by many factors (Table
6) but the mortality is higher (79%) in presence of heart failure associated with periannular
complications and Staphylococcus infection (Chu, 2004; ESC, 2009). Operative mortality is
also significant (16%) mainly in patients with prosthetic valves (Fayad, 2011).
Predictors of a poor prognostic
Patient
characteristics
Presence of
complications
Microorganisms Echocardiographic findings
- Older age
- Prosthetic
valve IE
- Previous IE (=
reinfection)
- Insulin-
dependent
diabetes
mellitus
- Comorbidities
- Heart failure
- Renal failure
- Stroke
- Septic chock
- Periannular
complications
- S. aureus
- Fungi
- Gram-
negative
bacilli
- Periannular complications
- Severe left-side valve
regurgitation
- Low left-ventricular ejection
fraction
- Large vegetation
- Severe prosthetic
dysfunction
- Premature mitral valve
closure and other signs of
elevated diastolic pressure
Table 6. Predictors factor of a poor prognosis in IE (ESC, 2009)
7. Treatment: Prolonged antimicrobial therapy and infectious source
eradication
7.1 Medical treatment
Medical treatment should be started quickly after carrying out of bacteriological samples, in
particular blood cultures (3 independent sets at 30 minutes intervals). Antimicrobial therapy
is first empirical (Table 7) and as soon as possible, it is adapted to micro-organism
sensitivity (ESC, 2009). In all the cases, this treatment should be prolongated for several
weeks and toxicity should be followed-up. As soon as possible, portal of entry and
complications should be found and treated. Symptomatic care is usual and classical.
7.2 Surgical treatment
Cardiac surgery is often necessary to treat or prevent complications or eradicate infectious
sites (Table 8). Surgery is more frequently necessary in some types of IE such as native valve
IE (87% of IE operated with 57% in aortic IE and 50% for mitral IE), Staphylococci and
Streptococci IE (respectively 35 and 33% of IE operated) (Fayad, 2011).
With the exception of an emergency, extracardiac infections must be eradicated before
surgery. Coronary angiography is also recommended in patients at risk (men older than 40,
post-menopausal women, patients with at least one cardiovascular risk factor or a history of
coronary disease) excluding emergency or cases with large aortic vegetation (risk of
dislodgment during examination). Repair and replacement of the valve are possible but the
last technique is preferred in complex cases. Intra operative transoesophageal

Importance of Dermatology in Infective Endocarditis

359
echocardiography is precious to guide surgeons. The operative mortality is moderate (16%)
and is more frequent with prosthetic valve carriers (ESC, 2009).
Characteristics of patient
Antibiotherapy suggested for adults patients
Association of
antibiotics
Dosage
Duration
(weeks)
Native
valve or
Prosthetic
valve
since
more
than 12
months
Ampicillin-
sulbactam IV
12g/day (in 4 doses)
4-6
Gentamicin IV or IM 3mg/kg/day (in 2 or 3 doses) 4-6
Allergy
to -
lactams
Vancomycin IV 30mg/kg/day (in 2 doses) 4-6
Gentamicin IV or IM 3mg/kg/day (in 2 or 3 doses) 4-6
Ciprofloxacin 1000 mg/day (in 2 doses) po or
4-6
800mg/day (in 2 doses) IV
Prosthetic valve since
less than 12 months
Vancomycin IV 30mg/kg/day (in 2 doses) 6
Gentamicin IV or IM 3mg/kg/day (in 2 or 3 doses) 2
Rifampicin po 1200mg/day (in 2 doses) 2
Table 7. Proposed antibiotic regimens for initial empirical treatment (po: per os/ IM:
intramuscular/ IV intravenous). Be careful with chronic use of gentamicin and vancomycin.
Serum levels of these antibiotics should be measured once a week for both and additional
renal function testing should be performed for gentamicin.
7.3 Follow-up
Complications are usual and should be searched for regularly. This requires a daily clinical
examination during the first weeks. Electrocardiogram should be performed frequently
(mainly in aortic or prosthesis IE) looking for new atrioventricular block or ischemia signs.
Bacteriological samples should be analyzed until their negativity. Heart failure and death
can occur after several months, so echocardiography is recommended in case of
cardiological signs but also after antibiotic treatment and should be repeated regularly
during the first year (at 1, 3, 6 and 12 months) (ESC, 2009).
Recurrence is frequent. Consequently, patients should be informed about this risk and
prevention rules should be applied closely.
8. Prevention
8.1 Antibiotic prophylaxis
In recent years, antibiotic prophylaxis has become more and more limited. In fact, no
antibiotic permit disappearance of bacteremia after at-risk at-risk procedures. Until now, no
study has proven the benefit of prophylactic treatment in the prevention of IE. At present,
only antibiotic prophylaxis is recommended by ESC (ESC, 2009) for highest risk dental
procedures in patients with highest risk cardiac conditions (Table 9). AHA (AHA, 2007) also
recommends antibiotic prophylaxis for procedures on the respiratory tract or on infected
skin in patients with highest risk of IE. Prophylaxis is associated with a small risk of death
by anaphylaxis but no case has been reported to date and the main risk is microbial
resistance development.

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Indica-
tions
Location of IE
Left-sided native valve IE Prosthetic valve IE (PVE) Right-sided IE
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+
Severe acute regurgitation or
valve obstruction causing
refractory oedema pulmonary or
cardiogenic shock
Severe prosthetic
dysfunction (dehiscence or
obstruction)



Right heart
failure
secondary to
severe tricuspid
regurgitation
with poor
response to
diuretic therapy
Emergency Emergency
Fistula into a cardiac chamber or pericardium causing
refractory pulmonary oedema or shock
Emergency
Severe acute regurgitation or
valve obstruction and persisting
heart failure or
echocardiographic signs of poor
haemodynamic tolerance (early
mitral closure or pulmonary
hypertension)
Severe prosthetic
dysfunction and persisting
heart failure
Urgent Urgent

-
Severe regurgitation and no heart
failure
Severe prosthetic dehiscence
without heart cardiac

Elective Elective
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Locally uncontrolled infection (abscess, false aneurysm, fistula,
enlarging vegetation)

Microorganism
s difficult to
eradicate
(persistent
fungi) or
bacteremia for
> 7 days (S.
aureus, P.
aeruginosa)
Urgent
Persisting fever and positive blood cultures > 7-10 days
Urgent
Fungi or multiresistant organisms
Urgent / Elective
Staphylococci or Gram
negative bacteria (most of
cases of early PVE)
Urgent/Elective
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Large vegetation (>10mm)
following one or more embolic
episodes despite appropriate
antibiotic therapy
Recurrent emboli despite
appropriate antibiotic
treatment
Persistent
tricuspid valve
vegetation >
20mm after
recurrent
pulmonary
emboli with or
without
concomitant
heart failure
Urgent Urgent
Large vegetation (>10mm) and other predictors of complicated
course
(heart failure, persistent infection, abscess)
Urgent
Isolated very large vegetation (>15mm)
Urgent
Table 8. Indications and timing of surgery (ESC, 2009). Emergency: within 24 hours. Urgent:
within a few days. Elective: after 1 -2 weeks of antibiotic treatment.

Importance of Dermatology in Infective Endocarditis

361

Cardiac conditions at highest risk of IE Dental procedures at high risk
Prosthetic cardiac valve or material used for
cardiac valve repair
Previous IE
Some congenital heart disease (CHD)
Cyanotic CHD
- without surgical repair or
- with residual defects, palliative shunts or
conduits
CHD with complete repair with prosthetic
material whether placed by surgery or by
cutaneous technique, up to 6 months after
the procedure
CHD when a residual defect persists at
the site of implantation of a prosthetic
material or device by cardiac surgery or
percutaneous technique
Manipulation of gingival region
Manipulation of periapical region of
the teeth
Perforation of oral mucosa
Antibiotic prophylaxis
Single dose 30-60 minutes before
procedure
Adults
Amoxicill
in 2g po
or IV
If allergy:
Clindamy
cin 600mg
po or IV
Children
Amoxicilli
n
50mg/kg
po or IV
If allergy:
Clindamy
cin
20mg/kg
po or IV

Table 9. Recommendations for antibiotic prophylaxis of IE for patients undergoing dental
procedures (ESC, 2009) (po: per os/ IV: intravenous)
8.2 Hygienic rules
Most IE occurs without history of procedure more at-risk situation of bacteremia (Strom,
1998).
Daily activities like chewing or tooth brushing carry transient but significant bacteremia and
can cause IE (AHA, 2007). Consequently, it is recommended to maintain a good oral hygiene
for all population.
For patients and drug users, disposable intravenous material is mandatory.
8.3 Others rules
In medical practice, percutaneous iatrogenic procedures should be avoided especially on
skin injuries and topical corticosteroid should be used with caution. Regular bacteriological
skin analysis is recommended during the follow-up of erosive dermatosis because it allows
quick adapted antibiotherapy in the case of secondarily advent IE. Of course, all prospective
portals of entry and all comorbidities have to be searched and supported.
9. Conclusion
Infective endocarditis (IE) is a severe disease the diagnosis of which remains difficult due to
clinical polymorphism and frequent insidious evolution over several days or months. Skin
manifestations are very useful for diagnosis but should alert practitioners for presence of
embolic complications. Epidemiologic profile of IE has changed in recent years and so has

The Cardiovascular System Physiology, Diagnostics and Clinical Implications

362
prophylactic and therapeutic recommendations. IE concerns all practitioners and we have to
keep it in mind with any patient.
10. Acknowledgment
Thank you to Pr Olivier Chosidow (Department of dermatology, Hpital Henri Mondor,
Crteil; France) and Dr Nicolas Ortonne (Department of pathology, Hpital Henri Mondor,
Crteil; France) for histological pictures.
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18
Cardiovascular Risk Factors:
Implications in Diabetes, Other
Disease States and Herbal Drugs
Steve Ogbonnia
Department of Pharmacognosy,
University of Lagos, Lagos,
Nigeria
1. Introduction
The danger and the increasing prevalence of heart diseases world wide are now of a great
concern and are attributed to the cardiovascular risk factors. Cardiovascular risk factors
have been identified to be the underlying latent or potent causes of death in all heart
diseases and also in many other disease states such as diabetes. Reduction in the risk factors
with synthetic drugs or drugs of natural products origin in the course of treatment of some
disease states where implicated has been found to improve tremendously the health of the
patient.
Cardiovascular risk factors include triacylglycerols (triglycerides), cholesterol, cholesteryl
esters, very low density lipoproteincholesterol (VLDL-c), low density lipoprotein-
cholesterol (LDL-c), and anti-athrogenic high density lipoproteincholesterol (HDL-c) and
are collectively referred to as plasma lipids. An increase in plasma lipids concentrations
beyond certain level give rise to physiological condition known as Hyperlipidemia.
Hyperlipidemia is, therefore, characterized by abnormal elevation in plasma triglyceride,
cholesterol and low density lipoprotein-cholesterol (LDL-c) and very low lipoprotein -
cholesterol (VLDL-c) and has also been reported to be the most prevalent indicator for
susceptibility to atherosclerotic heart disease (Maruthapan and Shree, 2010). Managing
cardiovascular disease states, therefore, requires drugs that would be capable of lowering
blood plasma lipids in order to reduce mortality and morbidity associated with the
cardiovascular complications (Hasimun et al., 2011). It has been reported in epidemiological
studies that a strong positive correlation exists between increase in the blood cholesterol
level and incidence of cardiovascular heart disease (CHD) (Hamed et al., 2010; Imafidon
2010; Maruthapana and Shree 2010), and also increase in the incidence of
atherosclerosis(Hasimun et al., 2011). A strong relationship between increase in C-reactive
protein (CRP) and cardiovascular risk factors has also been reported as well as increase in
myocardial infection and coronary artery disease among individuals with angina pectoris
(Ghayour Mobarhan et al. 2007).
Atherosclerosis arises from the deposition of fatty substances, cellular waste products,
calcium and fibrin in the arteries, resulting in clotting (Lewis et al., 2002) and is considered

The Cardiovascular System Physiology, Diagnostics and Clinical Implications

366
one of the major causes of coronary heart disease. It is recognized as a common threat to life,
usually seen in individuals consuming high quantities of cholesterol and saturated fats in
their diets. It has also been established in animal studies that raising dietary cholesterol
alone could increase atherosclerosis susceptibility (Madhumathi et al., 2006). Atherosclerosis
is characterized by endothelia dysfunction, muscular inflammation resulting from build up
of plasma lipids which tantamount to vascular remodeling, acute and chronic luminal
obstruction, abnormalities in the blood flow and diminished oxygen supplies to target
organs (Madhumathi et al., 2006). The development of atherosclerosis could also be
attributed to other factors such as oxidative stress which is responsible for the oxidation of
low-density lipoprotein-cholesterol (LDL-c) and is considered as one of the first steps of
atherosclerotic pathogenesis. Local inflammatory processes have also been identified to play
a crucial role in the transition from reversible accumulation of cholesterol in the arterial wall
to irreversible damage of the arteries (Brunner-La Rocca1, et al., 2005). Many factors
contributing to etiology of atherosclerosis in addition to diet include diabetes mellitus,
psychological factors and the presence of glucocorticoids.
Diabetes mellitus (DM) is a major degenerative disease in the world today afflicting many
lives both in the developed and developing countries (Ogbonnia et al., 2011). It has been
succinctly described as the common metabolic disorder of carbohydrate and fat metabolism,
which is due to absolute or relative lack of insulin and is characterized by hyperglycaemia
and hyperlipidemia (Sharon and Marvin, 1975; Walter, 1977). Diabetes is a multiple disease
state and has been defined as a state of premature cardiovascular death that is associated
with chronic hyperglycemia and also associated with blindness and renal failure (Fisher
and Shaw, 2001). This assertion was to draw attention and to encourage multiple clinical
approaches that would altogether help reduce cardiovascular risk factors in diabetic patients
(Ogbonnia et al., 2011). Diabetes especially the type 2 model might be postulated to occur
primarily due to underlying abnormality of insulin resistance - that is resistance of the body
to the biological actions of insulin. The consequences of insulin resistance lead to
hyperinsulinaemia and are associated with CRFs - dyslipidaemia including athrogenic lipid
profile with increase in low and very-low density lipoprotein-cholesterols (LDL-c and
VLDL-c) and reduction in the anti-athrogenic high density lipoprotein-cholesterol (HDL-c).
Cardiovascular risk factors have been implicated and even occur at a frequency much
higher than expected in some other disease states such as benign prostatic hyperplasia
(BPH). Benign prostatic hyperplasia is a neoplastic enlargement of the prostate gland and is
common in elderly men (Ejike and Ezeanyika, 2010). Epidemiological studies have
demonstrated that many of the risk factors associated with cardiovascular diseases are the
same as found in BPH (Dharmananda, 2011), and these risk factors include obesity,
hypertension and diabetes. The diabetes connection may be considered very strong and the
risk centers on the non-insulin dependent diabetes mellitus (NIDDM) which most often
involves excessive insulin levels, a possible direct contributor to the growth of the prostrate
(Hammarten and Hogstedt, 2011). The treatment of BPH became a medical issue mainly in
1970s at the same time that the cardiovascular disease therapy came to fore and the
incidence of the disease has become higher (Dharmananda, 2011). Herbal or phytomedicines
are now being investigated with some recorded successes for the management of
cardiovascular risk factors with the accompanied disease states. Herbal remedies with active
components understood to be sterols, such as beta-sitosterol has been used as a therapeutic
agent for BPH (Bombardelli and Morazzoni, 1997).

Cardiovascular Risk Factors: Implications in Diabetes, Other Disease States and Herbal Drugs

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2. Cardiovascular risk factors
Cardiovascular risk factors consisting mostly of plasma lipids including triacylglycerol
(triglycerides), cholesteryl esters and cholesterol are synthesized by the liver and adipose
tissues and may also be absorbed from the diet (Stryer, 1988). They are also efficiently
synthesized from carbohydrate diets largely in the intestinal epithelia tissues in addition to
the liver (Metzler, 1974), and are transported between various tissues and organs for
utilization and storage. These plasma lipids like other lipids are generally insoluble in water
and pose a transportation problem in aqueous blood plasma. This problem is overcome by
associating the nonpolar lipids comprising triacylglycerol (triglycerides) and cholesteryl
esters with amphipatic lipids such as phospholipids, cholesterol and proteins to produce
water-miscible lipoproteins (Conn and Stumpf, 1976; Stryer, 1988). A lipoprotein is a particle
consisting of core hydrophobic lipids surrounded by a shell of polar lipid and apoprotein
and mediates the cycle by transporting lipids from the intestine as chylomicrons and from
the liver as very low density lipoproteins-cholesterol (VLDL-c) - to most tissues for
oxidation and to adipose tissue for storage. Lipoproteins are grouped according to
increasing densities by centrifugation as follow:
i. Chylomicrons which incorporate intestinal absorbed triacylglycerol from intestinal
absorption of triacylglycerol and other lipids.
ii. Very low density lipoprotein-cholesterol (VLDL-c, or pre - Lipoprotein), are derived
from the combination of newly synthesized triacylglycerol together with small amounts
of phospholipids and cholesterol and apolipoproteins all synthesized in the liver
(Stryer, 1988).
iii. Intermediate density lipoprotein (IDL)
iv. Low density lipoprotein-cholesterol (LDL-c) (LDL-c or -Lipoproteins), representing a
final stage in the catabolism of VLDL-c, and
High density lipoproteins-cholesterol (HDL-c, or -Lipoproteins), involved in cholesterol
transport and also in VLDL-c and chylomicron metabolism.
Major groups of lipoproteins have been identified to be physiologically important and are
used in clinical diagnosis. The primary role of LDL-c appears to be the transport of esterified
cholesterol to tissue while that of the high density lipoproteins-cholesterol (HDL-c) is to
carry excess cholesterol away from most tissues to the liver. The size of the lipoprotein
particles also varies from a 200 to 500 nm diameter for chylomicrons to as little as 5 nm
for the smallest HDL particles (Metzler, 1974).
Lipoprotein is made up of triacylglycerol (16%) which is the predominant lipid in
chylomicrons and VLDL-c, while phosholipids (30%) and cholesterol (14%) are the
predominant lipids of HDL-c and LDL-c respectively and cholesterol esters (36%) (Stryer,
1988). It also contains much smaller fraction of unesterified long chain fatty acids (free fatty
acids) which are metabolically the most active of plasma lipids. These constitute what is
collectively known as Cardiovascular Risk Factors which are implicated in many disease
states as potent or latent causes of death. Lipoproteins may be separated according to their
electrophoretic properties into: -, -, and pre -- Lipoproteins (Holme and Peck, 1998).
The protein moiety of a lipoprotein is known as apolipoprotein or apoprotein constituting
nearly 70% of HDL-c and as little as 1% of chylomicrons. Some apolipoproteins are integral
and can not be removed, whereas others are free to transfer to other lipoprotiens. Seven

The Cardiovascular System Physiology, Diagnostics and Clinical Implications

368
principal apoprotein, A 1, A 2, A 4, B 48, B 100, C and E have been isolated and
characterized. They are synthesized and secreted by the liver and the intestine and generally
have two principal roles: they solubilize highly hydrophobic lipid and also they contain
signals that regulate the movement of particular lipid into and out of specific target cells and
tissues.
Lipoprotein Source/major
core lipid
Diameter
(nm)
Density
(g/mL)
Composition Main Lipid
Components
Mechanism of
lipid delivery
Protein (%) Lipid
(%)
< 0.95
Chylomicrons Dietary
triacylglycerol
Intestine
90-1000 < 0.95 1-2 98-99 < 1.006 Hydrolysis by
lipoprotein
lipase
Chylomicrons
remnants
Dietary
cholesterol
esters
Chylomicrons
45-150 < 1.006 6-8 92-94 0.95-
1.006
Receptor-
mediated
endocytosis
by liver
VLDL Endogenous
triacylglycerols
Liver
(Intestine)
30-90 0.95-
1.006
7-10 90-93 1.006-
1.019
Hydrolysis by
liproprotein
lipase
IDL Endogenous
cholesterol
esters VLDL
25-35 1.006-
1.019
11 89 1.019-
1.063
Receptor-
mediated
endocytosis
by liver and
conversion to
LDL
LDL Endogenous
cholesterol
esters VLDL
20-25 1.019-
1.063
21 79 Receptor-
mediated
endocytosis
by liver and
other tissues
Endogenous
cholesterol
esters
Transfer of
cholesterol
esters to IDL
and LDL
Table 1. Composition of the Lipoproteins in plasma of humans.
Each apolipoproteins carry out one or more distinct roles.
i. The apo B, stabilizes lipoproteins micelles and as the sole protein of LDL-c serves the
function of solubilizing cholesterol within LDL-c complex which in turn increases the
transport capacity of LDL-c for subsequent deposit on arterial wall (Madhumathi et al.,
2006).
ii. They are enzyme cofactors. The apoC-II has specific function of activating the
lipoprotein lipase that hydrolyses triacylglycerols of chylomicrons and VLDL. Lack of
either C-II or the lipase results in a very high level of triacylglycerol in the blood.
iii. They act as ligands for interaction with lipoprotein receptors in tissues, e.g apoB-100
and apo E for the LDL receptors, apo E for the LDL receptor related protein (LRP)
which has been identified as the remnant receptor, and apo A-1 for the HDL-c receptor.
The function of Apo A-IV and apo D, however, are not yet clearly defined, although
apo D, is believed to be an important factor in human neurodegenerative disorders.

Cardiovascular Risk Factors: Implications in Diabetes, Other Disease States and Herbal Drugs

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2.1 Cholesterol
Cholesterol is physiologically very essential for all animal life, and is primarily synthesized
from simpler substances within the body. It is an amphipathic waxy steroid of fat that is
manufactured in the liver or intestines. It constitutes essentially structural component of
membrane required in establishing proper membrane permeability and fluidity and is also a
constituent of the outer layer of plasma lipoproteins. Cholesterol is the principal sterol
synthesized by animals and transported in the blood plasma of all mammals (Leah, 2009). It
is also an important component implicated in the manufacture of bile acids, steroid
hormones, and vitamin D (Jain, 2005; Maxifield and Tabas, 2005; Hasimun, 2011). The
hydroxyl group on cholesterol interacts with the polar head groups of the membrane
phospholipids and sphingolipids, while the bulky steroid and the hydrocarbon chain are
embedded in the membrane, alongside the nonpolar fatty acid chain of the other lipids. In
this structural form, cholesterol reduces the permeability of the plasma membrane to
protons (positive hydrogen ions) and sodium ions.

Fig. 1. Chemical structure of cholesterol
Most cholesterol is carried in the blood by low density lipoprotein (LDL), which delivers it
directly to cells where it is needed. Both a 74-kDa cholesteryl ester transfer protein and a
phospholipid transfer protein are also involved in this process. Cholesterol esterases, which
release free cholesterol, may act both on lipoproteins and on pancreatic secretions. The LDL-
cholesterol complex binds to LDL receptors on the cell surfaces. These receptors are specific
for apolipoprotein B-100 present in the LDL. The occupied LDL-receptor complexes are
taken up by endocytosis through coated pits; the apolipoproteins are degraded in
lysosomes, while the cholesteryl esters are released and cleaved by a specific lysosomal acid
lipase to form free cholesterol.
Animal fats are complex mixtures of triglycerides, with fewer amounts of phospholipids
and cholesterol. As a consequence all food containing animal fats contain cholesterol to
varying extent. Plasma cholesterol concentration elevation is, therefore ,one of the important
CRFs as its transportation within lipoprotein is affected and is strongly associated with
progression of atherosclerosis.
2.2 Triacylglycerols
Triacylglycerols (figure 2b see the figure below) serve as biochemical energy reserves in the
cell and may be oxidized in the liver to provide energy or deposited as depot fat in
characteristic regions of the animal where they act as a long-term food store and insulator
(Plummer, 1998). They are the neutral and saponifiable lipids found in most organisms.
Triacylglycerols (triglycerides) which are chemically fatty acid esters of the trihydroxy

The Cardiovascular System Physiology, Diagnostics and Clinical Implications

370
alcohol, glycerol (Figure 2a), are compounds that usually make up the bulk of ingested
lipids and are transported to the blood via the lymphatic system in the form of
chylomicrons. Triacylglycerols synthesized endogenously as against those obtained from
the diet, are carried by VLDL produced primarily by the liver (Styer, 1988). Studies have
suggested that triacylglycerol (TG)-rich lipoprotein(TRL) plays an important role in the
development of atherosclerosis because both coronary artery disease and myocardial
infarction have been associated with abnormal postprandial lipoprotein pattern (Moreno-
Luna et al., 2007)

(a) (b)
Fig. 2. (a) Chemical structure of cholesterol (b) chemical structure of triacylglycerol
Most of the fatty acids synthesized or ingested by an organism are either transformed into
triacylgylcerols and stored for metabolism to give energy or incorporated into
phospholipids components of the membrane. Triacylgylcerols have as precursor fatty acyl-
CoAs and glycerol--phosphate but many enzymatic steps are involved in their biosynthesis
in animal tissues. Although the triglycerides have been found to be important predictors of
CVD in many studies, no clinical trial data has established that lowering triglycerides in
individuals with or without diabetes independently leads to lowering of CVD occurring
rates even after changes in HDL-cholesterol are adjusted for. From the foregoing, it is
evident that elevated cholesterol, low HDL-c, high TG and high LDL-c are all risk factors for
CVD. The pattern of occurrence of these abnormalities in type 2 DM especially has been
severally reported in both developed and developing economies (Idogun et al., 2007;
Williams et al., 2008).
2.3 Very Low Density Lipoprotein-cholesterol (VLDL-c or pre- lipoproteins)
VLDL-c is synthesized in the liver and contains primarily triglycerides in their lipid cores
for their export and also some cholesterol ester (Botham and Mayes, 2006). As their
triglycerides are cleaved by endothelial lipoprotein lipase and transferred to hepatic tissues,
the VLDL (very-low-density lipoprotein) particles lose most of their apolipoprotein C and
become intermediate-density lipoproteins. VLDL is one of the five major groups of
lipoproteins which functions to enable fats and cholesterol to move within the water-based
solution of the bloodstream. VLDL-c particles have a diameter of 30-80 nm each and
transports endogenous products such as triglycerides, phospholipids, cholesterol, and
cholesteryl esters, whereas chylomicrons transport exogenous (dietary) products. It
functions as the body's internal transport mechanism for lipids.

Cardiovascular Risk Factors: Implications in Diabetes, Other Disease States and Herbal Drugs

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2.4 Low Density Lipoprotein-cholesterol (LDL-c)
The primary role of LDL-c appears to be the transport of esterified cholesterol to tissues
(Guyton and Hall, 2006). Low density lipoprotein results when triacylglycerols are released
from VLDL-c by the action of the same lipase that acts on chylomicrons and the remnants
which are rich in cholesterol esters are called intermediate density lipoprotein (IDL). IDL
particles have two fates as half of them are taken up by the liver and the other half
converted into LDL which is the major carrier of cholesterol in blood (Styer, 1988). LDL-c or
- lipoprotein represent the final stage in the metabolism of VLDL. Originally, LDL-
cholesterol was determined by a lengthy, laborious process called ultracentrifugation of
serum. A much more rapid test became available based on the following Friedwald
equation: Total cholesterol = LDL-cholesterol + HDL-cholesterol + VLDL-cholesterol
(VLDL-cholesterol = triglycerides/5). One can rapidly and easily do a lipid profile by
enzymatically measuring the important lipidstotal cholesterol, HDL- cholesterol, and
triglycerides. Dividing triglycerides by five gives the relatively unimportant, but hard to
measure, VLDL-cholesterol, which is useful in then calculating the very important LDL
cholesterol (Holme and Peck, 1998).
2.5 High Density Lipoprotein-cholesterol (HDL-c or -lipoprotein)
HDL-c is involved in the cholesterol transport and in VLDL and chylomicron metabolism.
Unlike LDL which primary role appears to be the carriage of esterified cholesterol to the
tissues, HDL functions to carry excess cholesterol away from most tissues to the liver. The
apoA-I present in the HDL-c particle binds lipid and also activates lecithin cholesterol
acyltransferase (LCAT), which catalyzes formation of cholesteryl esters which migrate into
the interior of the HDL-c and are carried to the liver (Metzler, 1974). Recent studies on
patients with LCAT deficiency have shown a modest but significant increase in incidence of
cardiovascular disease consistent with a beneficial effect of LCAT on atherosclerosis
(Rousset et al., 2009) HDL particles compared to other lipoproteins, are assembled outside of
cells from lipids and proteins, some of which may be donated from chylomicrons or other
lipoprotein particles. HDL has higher protein content than other lipoproteins and is more
heterogeneous. The major HDL protein is apolipoprotein A-I, but many HDL particles also
contain A-II,

and apolipoproteins A-IV, D, and E may also be present. A low plasma level of
HDL-cholesterol is associated with a high risk of atherosclerosis.
3. Implicated disease states
3.1 Diabetes
Diabetes mellitus is a major global health problem and is now recognized as one of the leading
causes of death in the developing countries, where the high prevalence of the disease could be
attributed to improved nutritional status coupled with a gross lack of modern facilities for the
early diagnosis of the disease (Uebanso et al., 2007; Ogbonnia et al., 2008
a
). Diabetes mellitus
(DM) is a complex disease characterized by abnormal pattern of fuel usage resulting from over
production of glucose and its under utilization by other organs (Stryer, 1988). Diabetes has
been succinctly described as the common metabolic disorder of carbohydrate and fat
metabolism, which is due to absolute or relative lack of insulin and is characterized by
hyperglycaemia (Walter, 1977; Shah et al., 2008 and Sharma et al., 2010; Dinesh et al., 2011).

The Cardiovascular System Physiology, Diagnostics and Clinical Implications

372
Diabetes mellitus is therefore a multifactorial disease associated with hyperglycemia, (Shah et
al., 2008; Sharma et al., 2010); lipoprotein abnormalities, raised basal metabolic rate and high
oxidative stress inducing damage to beta cells. The abnormalities in carbohydrates and lipid
metabolism in diabetes also result in excessive production of reactive oxygen species (ROS)
and defect in ROS scavenging enzymes in addition to oxidative stress. The low level of insulin
associated with diabetes has been found to increase the activity of anti-enzyme, fatty acyl
Coenzyme A oxidase, which initiates the -oxidation of the fatty acids, resulting in lipid
peroxidation (Shah et al., 2008). Increased lipid peroxidation has also been found to impair
membrane function by decreasing membrane fluidity and changing the activity of the
membrane-bound enzyme and receptors. The resulting lipid radicals and lipid peroxides are
harmful to the cell in the body and are associated with atherosclerosis and brain damage.
Chronic hyperglycemia which occurs in diabetes causes glycation of body proteins which in
turn leads to secondary complications affecting eyes, kidneys, nerves and arteries (Mishra
and Garg, 2011). These may be delayed, lessened or prevented by maintaining blood glucose
values close to normal in modern medicine, though no satisfactory effective therapy is
available for total cure of diabetes mellitus.
Diabetes mellitus is also associated with hyperlipidaemia with profound alteration in the
concentrations and compositions of plasma lipid. Changes in the concentration of the lipids
in diabetes contribute to the development of vascular disease. Excessive levels of blood
cholesterol accelerate atherogenesis and lowering high blood cholesterol reduces the
incidence of CHD (Grundy, 1986). One of the risk factors for coronary heart disease is
elevated total cholesterol (TC), low density lipoprotein-cholesterol (LDL-c) and lowered
high density lipoprotein-cholesterol (HDL-c). The development of cardiovascular disease in
DM is often predicted by several factors which include central obesity, hypertriglyceridemia
and hypertension. Hypertriacylglyceridemia and low high-density liopoproteinaemia are
two components of the atherogenic profile seen in DM. Elevated low density lipoprotein-
cholesterol (LDL-c) has also been found to be an independent risk factor for the
development of cardiovascular disease and is often reported to be the commonest lipid
abnormality found in patients with DM (Udawat and Goyal, 2001; Idogun et al., 2007).
3.2 Atherosclerosis
Atherosclerosis or arteriosclerosis is a disease of large and medium size muscular arteries
and is characterized by endothelial dysfunction vascular inflammation and build up of
lipids, cholesterol, calcium and cellular debris within intima of vessel wall. This build up
results in plaque formation, vascular remodeling, acute and chronic luminal obstruction,
abnormalities in the blood flow and diminished oxygen supply to the target organ.
(Madhumathi et al., 2006). Atherosclerotic disease has been found to be the most common
cause of myocardial ischemia. Myocardium is said to be ischaemic when the pumping
capability of the heart is impaired as a result of fall in the coronary blood flow which could
not meet up with the metabolic need of the heart. In artherosclerotic disease, there is a
localised lipid deposits called plaques develop within the arterial walls. In the severe cases
of the disease these plaques become calcified and are so large that they physically narrow
the lumen of the arteries producing stenosis (Mohrman and Heller, 2006). umerous studies
have revealed important risk factors for the development of arthrosclerosis and these

Cardiovascular Risk Factors: Implications in Diabetes, Other Disease States and Herbal Drugs

373
include diseases such as diabetes mellitus, arterial hypertension, and also smoking and
elevated blood cholesterol
Current concepts in atherosclerosis suggest that oxidation of LDL-c is involved in its
pathogenesis. The critical role of oxidized LDL-c in atherogenesis may be due to its rapid
uptake by the foam cells lining the arterial intima, which are thought to have macrophage-
like properties. When LDL-c is oxidized chemotactic effect is exerted on monocytes and this
increase the uptake of LDL-c leading to the formation of arterial plaque. Lipid oxidation can
be inhibited by the use of antioxidants such as vit E which inhibit the formation of lesions in
hypercholesterolemic rabbits (Chein and Frishman, 2003).
Hypercholesterolemia has also been implicated in the process of atherogenesis and a
curvilinear relationship has been documented between increasing cholesterol and increasing
incidence of CVD (Brunzell et al., 2008). The role of LDL-c in the development of CVD
cannot be overemphasized as there is documented evidence that high levels of LDL-c not
only cause atherosclerosis but pharmacological interventions that reduce LDL-c are
associated with stabilization and regression of atherosclerosis in proportion to the
cholesterol lowering achieved (O'Keefe et al., 2004). Low levels of HDL-c have been
consistently reported in cardiovascular diseases (Idogun et al., 2007; Sani-Bello et al., 2007,
Singh et al., 2007). Primary treatment of coronary artery disease (and atherosclerosis in
general) should include attempts to lower blood lipid by dietary and pharmacological
techniques to prevent and possibly reverse further deposit of plaques.
3.3 Benign Prostatic Hyperplasia (BPH)
Benign Prostatic Hyperplasia (BPH) is a neoplastic enlargement of the prostate gland, and is
a common problem among aging men (Ejike and Ezeanyika, 2010; Dharmananda, 2011). The
etiology of this disease is still poorly understood, but it has been proposed to have two
phases:
One of the phases involves no clinical sign but there maybe some microscopic changes while
the other manifests as the disorder of urination caused by the obstruction of the urinary tract
by an enlarged prostate gland (Dharmananda, 2011).
Epidemiological studies have demonstrated that many of the risk factors associated with
cardiovascular diseases apply also as risk factors for BPH. The problems associated with
diabetic may be considered very strong as the risk in non-insulin dependent diabetes
(NIDDM); which most often connected with insulin resistance may be a possible direct
contributor to the growth of BPH (Dharmananda, 2011). NIDDM which arises from either
impairment of insulin utilization or dysfunction on the metabolism of carbohydrates, fats
and protein or both culminates in hyperlipidemia- hence elevation in plasma cardiovascular
risk factor. BPH is therefore associated with metabolic syndrome (Kasturi et al., 2006;
Ozden, 2007).
4. Herbal drugs used to control CRF in the disease states
Herbal medicines may be described as medicines prepared either with a single plant part or
combinations of different plant parts either fresh,dried or as extract are now recognized as

The Cardiovascular System Physiology, Diagnostics and Clinical Implications

374
potent therapeutic agents. Plants derived medicines commonly referred to as
phytomedicines have been effectively employed in the management of variety of
pathological conditions and are associated with fewer side effects (Nirmala et al., 2011;
Ogbonnia et al, 2011). In recent years, they have been found to be effective both as
hypoglycaemic and hypolipidemic agents (Ogbonnia et al., 2008
c
; 2010
b
) and have also been
empirically used by many people from various cultures to lower cholesterol levels (Hamed,
et al., 2010). Herbal medicines owe their therapeutic activities to the presence in them of
secondary organic compounds or natural products constituents called the active
constituents.
4.1 Herbal active constituents
Herbal drugs contain natural products or secondary metabolites as the active constituents
responsible for their physiological and pharmacological activities. The physiological and
pharmacological activities have been found amongst alkaloids, phenolics and flavonoid
compounds, glycosides (steroidal and saponins), and terpenoids, and is brought about
through one or combination of two or more of the mechanisms that are the same as in the
disease state they are being used. The mechanisms of their antidiabetic and antilipidemic
activities which contribute to lowering of plasma lipids are the same mechanisms
responsible for lowering of cardiovascular risk factors. These include the following:
Glycosidase (Glucosidase) inhibition mechanism; alpha-amylase inhibition mechanism;
antioxidant activities mechanism; inhibition of hepatic glucose metabolizing enzymes
mechanism and inhibition of glycosylation of haemoglobin mechanism. These different
mechanisms of activities are briefly discussed below
4.2 Possible mechanism of actions
The different classes of secondary product active constituents present in different herbal
medicines may act through one or different mechanisms to bring about lowering or clearing
of cardiovascular risk factors in a patient which may be probably the same mechanism
through which they act exert their pharmacological action to control the disease state in
question. Notably some of these possible mechanisms of actions may include:
4.2.1 Glycosidase (Glucosidase) inhibitor mechanism
One of the earliest features of type II diabetes and also observed in pre-diabetic phase is the
loss of early phase secretion of insulin. Early phase insulin secretion is seen after a meal or
after oral or intravenous ingestion of glucose and it is responsible for inhibition of hepatic
glucose output and its absence results in postprandial hyperglycemia. (Ogbonnia and
Anyakora 2009
c
). The -glucosidase inhibitors category of drugs have been found to
decrease postprandial glucose level by interfering with carbohydrate digestion and delaying
gastrointestinal absorption of glucose . Slowing down digestion and breakdown of starches
may have beneficial effects on insulin resistance and glycaemic index control on people
suffering from diabetes. In this group some cryptic or water soluble alkaloids especially
polyhydroxy alkaloids, have been identified to be potent glucosidase inhibitor (Kameswara
et al., 2001). This as a whole comprises of relatively simple monocyclic pyrrolidine and

Cardiovascular Risk Factors: Implications in Diabetes, Other Disease States and Herbal Drugs

375
piperidine alkaloids, necines, amino alcohols which are derivatives bicyclic pyrrolizidine,
and are mostly esters of amino alcohols and of aliphatic carboxylic acids.
4.2.2 Inhibition of hepatic glucose metabolizing enzymes mechanism
Synthesis of glucose by the liver and kidney from non carbohydrate precursor such as
lactate, glycerol and amino acid constitutes a process known as gluconeogenesis. The liver
hydrolytic enzymes glucose-6-phosphatase and fructose-1, 6- diphoshatase have been
shown to play a crucial role in gluconeogenesis contributing to hyperglycaemic condition
found in diabetes. Herbal drug products may act by binding with the enzymes. Treatment
with an herbal drug has been observed to decrease the activities of these liver enzymes
significantly with a concomitant decrease in blood sugar level (Lazar, 2006).
4.2.3 Antioxidants effects
Phenolics and polyphenolics are associated with antioxidant properties and have been
reported to categorically reduce the oxidation of the LDL-c (Kar, 2007). Flavonoids in
hawthorn extract have been found to reduce wall tension in normal and sclerotic blood
vessels. These chemicals are also presumed to stimulate beta-2-receptors and thus widen
coronary arteries and blood vessels in skeletal muscle . Flavonoids and other antioxidants
act to destroy free radicals which are particles that can damage cell membranes, interact
with genetic material and possibly develop heart diseases and cancer. They have also been
found to decrease two other markers of cardiovascular disease, homocysteine and C-
reactive protein. C-reactive protein (CRP) has been reported to be associated with increased
risk of cardiovascular disease, myocardial infection (MI) coronary artery disease mortality
among individuals with angina pectoris.
Oxidative stress has been reported to increase in diabetic patients and is regarded as
common pathway by which many classical cardiovascular disease (CVD) risk factors and
postprandial dysmetabolism may initiate and promote atherosclerosis (WHO 1985). Studies
have shown that treatment with antioxidant reduces diabetic complications (Negappa et al.,
2003). Flavonoids have been shown to scavenge reactive oxygen species (ROS) that are
produced under severe stress conditions and protect plant cell and animal cell from
oxidative stress and may have important role in human health.
4.2.4 Inhibition of glycosylation of haemoglobin mechanism
It has now become apparent that both fasting and postprandial hyperglycaemia contributes to
overall glycaemic burden and therefore total glycosylation of haemoglobin, HbA
.,
Many
studies have shown that there is substantial evidence and a very strong correlation between
hyperglycaemia and the risk of developing cardiovascular disease and mortality. Postprandial
hyperglycemia has been found to occur together with postprandial hyperlipidaemia which is
also associated with increased oxidative stress and endothelia dysfunction. However, one
could therefore postulate that herbal drug products that are effective in the reduction of
postprandial hyperglycaemia may not only play a role in managing type II diabetes but could
also offer a tantalizing possibility of reducing cardiovascular risk.

The Cardiovascular System Physiology, Diagnostics and Clinical Implications

376
S/no Plant/Herbal Drugs Work Done Reference
1. Alstonia congensis
Engler (Apocynaceae)
bark and Xylopia
aethiopica (Dunal) A.
Rich (Annonaceae) fruits
Evaluation of acute in mice
and subchronic toxicity
Ogbonnia et al., 2008
a
2 Leone Bitters, a Nigerian
polyherbal formulation
Antimicrobial evaluation,
acute and subchronic toxicity
studies
Ogbonnia et al., 2008
a
,
2010
a
3. Parinari curatellifolia
Planch,
(Chrysobalanaceae)
seeds
Assessing plasma glucose
and lipid levels, body weight
and acute toxicity following
oral administration of an
aqueous ethanolic extract.
Ogbonnia et al., 2008
b
4. poly-herbal formulation on alloxan- induced diabetic
rats
Ogbonnia et al., 2008
b
,
2010
b
5. Treculia africana Decne
and Bryophyllum
pinnatum Lam
Evaluation of Hypoglycaemic
and Hypolipidaemic Effects
of Aqueous Ethanolic
Extracts
Ogbonnia et al., 2008
c
6. Stachytarpheta
angustifolia
Evaluation of acute and
subchronic toxicity in animals
and phytochemical profile
Ogbonnia et al., 2009a

7. Parinari curatellifolia
Planch
(Chrysobalanaceae)
seeds
Evaluation of acute in mice
and subchronic toxicity
Ogbonnia et al., 2009
b
8. Parinari curatellifolia
and Anthoclista vogelli
Diabetes and cardiovascular
factors
Ogbonnia et al., 2011
9. Azadirachta indica Diabetes Mellitus and
hypolipidemic effects
Dinesh et al., 2011
10. Holarrhena
antidysenterica
Diabetes Ali et al., 2009
11.

Annona muricata Linn
Centratherum
anthelmintica
Diabetes
Diabetes
Shah et al., 2008
12. Grape seed extract cholesterol http://www.nativeremed
ies.com/article/cholestero
l-education-heart-
disease.html
13.

Red yeast rice contains a
natural form of
lovastatin
cholesterol
14. Vilis vinifera extract and
Oroxylum indicum
cholesterol DMello et al., 2011
15. Garlic Cholesterol, antithrombic
Cardiovascular diseases

Hoareau and DaSilva,1999
16. Hawthorn leaf and
flower



Cardiovascular Risk Factors: Implications in Diabetes, Other Disease States and Herbal Drugs

377
17. Cinnamomic camphoric
aetherolleum
Coronary artery disease
(CAD)







18. Rosmarini folium
(Rosemary leaf)
19. Pini aetheroleum(pine
neddle)
20. Eucalypti folium
(Eucalyptus leaf)
21. Menthae aetheroleum
(menthol)
22. Bacopa monnieri Linn Diabetes Ghosh et al., 2006
23. Feronia elephantum Corr Diabetes Mishra and Garg, 2011
24. Achilleamellifolium
(yarrow) Convallaria
majalis (lilly of the
valley)Crategeus
laevigata (hawthorn)
Cynarascolymus
(globeantichoke)
Gingko biloba (gingko)
Vibumum opulus
Cardiovascular diseases Hoareau and DaSilva,
1999
Table 2. Some researched plants and plant medicines found to have lowering effects on
cardiovascular risk factors.
5. Summary
The danger and the increasing prevalence of heart diseases world over are now of a
great concern
These diseases could be attributed to the cardiovascular risk factors which also have
been identified to be the underlying latent or potent causes of death in heart diseases in
particular and also in many other disease states
Cardiovascular risk factors include triacylglycerols (triglycerides), cholesterol,
cholesteryl esters, very low density lipoprotein cholesterol (VLDL-c), low density
lipoprotein-cholesterol (LDL-c), and anti-athrogenic HDL which are collectively
referred to as plasma lipids
Cholesterol is a fat-like substance that is present in cell membranes and is a precursor to
steroid hormones and bile acids.
Coronary atherosclerosis is the deposition of cholesterol and fibrin complexes within
the lumen of a coronary artery that narrows the lumen, thereby limiting blood flow.
Coronary heart disease (CHD) is atherosclerosis of one or more coronary arteries that
has resulted in symptomatic disease such as angina pectoris, myocardial infarction, or
congestive heart failure, or has required coronary artery surgery or coronary
angioplasty.
Lipoproteins are lipid-containing proteins in the blood that transport cholesterol
throughout the body.
Disease states with underlying cardiovascular risk factors include diabetes,
atherosclerosis and benign prostatic hyperplasia.

The Cardiovascular System Physiology, Diagnostics and Clinical Implications

378
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19
Morphology and Functional Changes
of Intestine, Trophology Status and
Systemic Inflammation in Patients
with Chronic Heart Failure
G.P. Arutyunov and N.A. Bylova
The Russian State Medical University (RSMU),
Russia
1. Introduction
1.1 Morphological and functional changes of the small intestine in patients with
different classes of chronic heart failure
Current understanding prompts to view chronic heart failure (CHF) as a systemic condition.
Traditionally, the following organs are considered the target organs of CHF: heart, kidneys,
brain. However, low cardiac output and increased activity of the renin-angiotensin-
aldosterone system (RAAS), which lead to vasospasm and ischemia, are bound to have
effect on functions of other organs, including small intestine, large intestine, and adipose
tissue. The increased activity of RAAS is likely to have effect on morphological restructuring
of the intestine as well. It can be assumed that accumulation of collagen in the intestinal
wall, as well as development of edema and ischemia, decrease the functional activity of the
intestinal wall and are a major factor of the malabsorption syndrome, which, in its turn,
leads to progressive loss of body mass. This results in progression of certain clinical
manifestations in patients with CHF, such as: weakness, fatigue, and progressive decrease of
exercise tolerance, which cannot be explained by changes in peripheral circulation alone.
The incidence of these complaints is known to increase as the NYHA class of the CHF
grows, and it reaches its peak in patients with class IV (Harrington & Anker, 1997).
Loss of body mass means a significantly worse prognosis for the patients with CHF.
According to SOLVD study, 6 % decrease of body mass in patients with CHF is a potent
predictor of negative impact on survival along with other factors such as age, gender, LV
ejection fraction, NYHA class (Anker et al., 2003).
Therefore, decrease of body mass should be considered an important sign, equal in
significance to such symptoms as dyspnea and edema.
Obviously, a search for new methods to correct the nutritional status in patients with CHF is
necessary. The method of nutritional support may be one of these promising options to treat
and stop development the malabsorption syndrome, as this method constitutes a system
with pathogenesis-based rationale that implies prescription of balanced nutritive mixtures

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characterized by maximum degree of absorption even in the setting of morphological
changes in the intestine.
1.2 Materials and methods
This was an open-label prospective study approved by the Ethics Committee of the Russian
State Medical University. All of the patients participating in this study had signed the
informed consent.
The study included 110 patients with New York Heart Association (NYHA) class I-IV
ischemic CHF, with history of CHF for more than 24 months. Age of the patients was 45 to
65 years, mean age was 58.75.3 years. All of the patients were allocated to different groups
based on the severity of their CHF. The control group included 38 patients (24 male and 14
female), 45 to 65 years old (mean age 60.62.6), in which no signs of CHF were found after
testing. The patient populations had comparable basic parameters at baseline.
All of the patients with the signs of CHF received standard basic therapy, which included
ACE inhibitors, -blockers, loop diuretics or thiazide diuretics, cardiac glycosides, nitrates,
aspirin, and aldosterone antagonists. Patients in the control group received therapy for their
main condition.
Patients were investigated using the following sets of tests:
- Endoscopy with biopsy of the small intestine (the samples were taken 10 cm below the
plica duodenojejunalis). Preparation and analysis of microscopic specimens were
performed in the Pathology Department of the Bakulev Scientific Center of
Cardiovascular Surgery.
- Assessment of functional activity of the small intestine (measurement of excretion of fat
in feces, biochemical measurement of total protein and protein fractions in feces via
nitrogen content, measurement of carbohydrate absorption in the small intestine using
the D-xylose test).
Statistical analyses of the results were performed using standard statistical formulas with
Microsoft Excel 7.0 and BIOSTAT software. Arithmetic means of values in the sample
population (M) and standard deviations () were determined. The significance of differences
between groups was determined using Student test at p < 0.05. Relationship between
parameters was assessed by calculating the correlation coefficient (r) with the level of
errorless prognosis at 95 % (p<0.05).
1.3 Results
1.3.1 Morphological changes of the small intestine in patients with NYHA class I-IV
CHF
The photographs of microscopic specimens (Fig. 1) demonstrate the pattern of the mucosa of
the small intestine typical for patients with NYHA class IV CHF (a) and healthy individuals
(b). Collagen fibers are stained pink.
These photographs demonstrate that in patients with NYHA class III-IV CHF the collagen
fibers stained pink take up a significant area of the small intestine mucosa, while in patients
without signs of CHF only solitary collagen fibers are present.
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Fig. 1. The microscopic sample of the mucosa of the small intestine from a patient with
NYHA class IV CHF (a) and a patient without signs of CHF (b). Van Giesons stain,
magnification 400.
a
b

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Collagen deposition level in patients with NYHA classes III and IV was significantly different
from values measured in patients with NYHA classes I or II and in patients without signs of
CHF. Comparison of collagen relative density between patients without CHF and with NYHA
class I or II CHF did not reveal significant differences. However, a clear trend towards increase
of collagen level in the latter was noted. Also, the difference in the amount of collagen between
CHF patients with NYHA classes III and IV was not significant.
When assessing the microscopic specimens of the small intestine, high amount of collagen
was observed to mechanically push enterocytes away from a capillary vessel. This increases
the intestine-blood barrier and may have an impact on nutrient absorption.
The measured data are presented in Table 1.
Groups
Distance between
EBM and
a capillary vessel,
m
Significance of difference between groups
1. No CHF 8.40.7 p1-2 >0.05; p1-3>0.05; p1-4<0.05; p1-5<0.05
2. NYHA class I 10.11.2 p2-3>0.05; p2-4>0.05; p2-5>0.05
3. NYHA class II 11.31.1 p3-4<0.05; p3-5<0.05
4. NYHA class III 18.61.4 p4-5>0.05
5. NYHA class IV 19.11.2
Table 1. Distance between EBM (enterocyte basal membrane) and the capillary wall
Even primary assessment of microscopic photographs revealed atrophy of villi of the small
intestine mucosa in patients with NYHA class III-IV CHF. Fig. 2 shows pattern of the mucosa
in a patient with NYHA class IV CHF (a) compared to a patient from the control group (b).

a
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Fig. 2. Villi of the small intestine mucosa in a patient with NYHA class IV CHF (a) and a
patient without CHF (b).Magnification x100. Hematoxylin-eosin stain.
Quantitative values describing changes in the villi of the small intestine in patients with
different classes of CHF are shown in the Table 2.
Groups
Mean length
of a villus, m
Mean width
of a villus, m
Significance of difference between
groups
No CHF 3729.9 984.4
p1-2>0.05; p1-3>0.05; p1-4<0.05;
p1-5<0.05
Class I CHF 3697.4 943.5 p2-3>0.05; p2-4>0.05; p2-5>0.05
Class II CHF 3746.9 916.1 p3-4<0.05; p3-5<0.05
Class III CHF 2545.5 653.1 p4-5>0.05
Class IV CHF 2236.1 593.0
Table 2. Length and width of mucosal villi in the small intestine of patients with CHF.
To conclude, analysis of the small intestine mucosa biopsy samples demonstrated significant
morphological changes, which worsen with the severity of CHF. Increase of intestine-blood
barrier and decrease of the absorbing area of the villi determined further study of nutrient
absorption, which, supposedly, should have decreased significantly in patients with CHF.
1.3.2 Functional changes of the small intestine in patients with NYHA class I-IV CHF
Analysis of protein absorption parameters revealed the following pattern: in patients with
NYHA class I CHF, losses of total nitrogen were virtually below the upper limit of normal
range, reaching 7.10.2 % of daily consumption. No significant differences were found
b

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between the patients of the control group (5.90.21 %) and classes I or II (p>0.05). With
NYHA class IV CHF, the loss of protein was significantly higher 18.61.3 %; this was on
average 3.1 times higher than in the group of patients without CHF (p<0.05). In the group of
patients with NYHA class III, the loss of protein was 16.71.8 %. Comparison of the protein
loss levels in CHF patients with NYHA class III and IV vs. the protein loss levels in patients
with NYHA class I and II showed a significant difference (p>0.05).
Levels of total fat loss were most pronounced in CHF patients with NYHA class III and IV
(22.42.1 % and 24.12.12 % of the daily consumption, respectively) and exceeded the levels
in patients without CHF (5.50.86 %) 4-fold on average. For NYHA classes I and II, fat loss
levels were at the upper limit of normal (6.11.1 and 7.20.9 % of daily consumption,
respectively) and were not significantly different from the values in the group of patients
without CHF (p>0.05).
Analysis of D-xylose test results demonstrated a dependency similar to the one observed for
absorption of proteins and fats. In patients with NYHA class IV CHF, the 5-hour excretion of
D-xylose was 0.890.05 g. This was 1.4 times lower than the values for the control group. For
NYHA class III CHF, D-xylose excretion was 0.960.03 g. This was also significantly
different from normal values. Patients with NYHA class I and II did not show significant
differences compared to the control group.
To conclude, patients with CHF experience deterioration of absorption for all basic
nutrients, and the absorption reduction demonstrates dependence upon the severity of the
CHF.
1.4 Attempts for correction
Taking into consideration the pronounced changes of the small intestine in patients with
severe CHF that lead to development of malabsorption syndrome and protein-energy
insufficiency, such patients demand specific correction of their nutritional status. Naturally,
raw nutrients will hardly be utilized in the intestine that underwent these changes. A
possibility to use specifically treated nutrients in the form of standard mixes for oral feeding
was the objective that we assessed in the second phase of this study.
This part was an open-label, randomized, prospective, 24-week study.
Patients with NYHA class III and IV CHF were screened for this study. Total number of
subjects was 74 (46 males, 28 females).
Randomization was performed using a random number method, with even numbers
corresponding to the standard-of-care group (Group 1), and odd numbers corresponding to
the nutritional support group (Group 2). The number of patients assigned to Group 1 was
36. Group 2 included 38 patients.
Figure 3 shows the study design.
Subjects in Group 1 (n=36) received standard basic therapy and their usual nutrition within
the standard diet for cardiovascular patients.
Subjects in Group 2 (n=38), in addition to the basic therapy and standard diet, received a
balanced nutritive mix, comprising 25 % of daily calories.
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Fig. 3. Study Design.
The absolute majority of the patients were male and had history of ischemic origin of their
disease. Their mean age was over 60 years. Mean NYHA class within these groups was 3.5
and 3.4, respectively. The resulting groups were identical in structure by gender, age and
other clinical characteristics, warranting their comparability. The following tests were
performed during this study: 6-minute test, echocardiography (LVEF), diet review,
measurement of body mass and BMI, estimation of body fat mass and lean body mass, total
protein, albumin, absolute lymphocyte count, hand dynamometry, assessment of absorption
for proteins, fats, and carbohydrates, morphometric study of small intestine mucosa biopsy
specimens, count of hospitalizations due to CHF progression.
Estimations of energy and nutrient demands were performed for all subjects before
therapeutic diet and nutritional support were prescribed.
Subjects in Group 2 had 25 % of their energy demands (daily energy consumption) covered
using balanced nutritive mixes, and the remaining 75 % were covered using the standard
diet. All patients maintained dietary diaries, which allowed us to control the amount of
energy they have received with their usual diet.
The balanced nutritive mixes used for nutritive support were Peptamen (Nestle,
Switzerland), Berlamin Modular (Berlin-Chemie, Germany), Unipit and Nutrien-standard
(Nutritek, Russia).
Assessment of nutritional support efficacy included change of 6-minute test parameters over
time, change of LBM over time, and number of hospitalizations due to CHF progression
compared between the experimental group and the control group.
During 24-week observation, a total of 11 patients died in 2 groups: 4 males and 2 females in
Group 1 (standard-of-care), and 3 males and 2 females in Group 2 (oral nutritional support).
Total number of hospitalization events throughout 24 weeks was 54 and 42 in Group 1 and
Group 2, respectively. Table 3 shows causes of death and hospitalization.

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Cause Hospitalization Death
Group 1 Group 2 Group 1 Group 2
CHF progression 36 (66,7%) 28 (66,7%) 4 (66,7%) 3 (60%)
Recurrent MI 6 (11,1%) 7 (16,7%) 1 (16,7%) 2 (40%)
Pneumonias 10 (18,5%) 6 (14,3%) 1 (16,7%) 0
Other 2 (0,4%) 3 (0,7%) 0 0
Total 54 42 6 5
Table 3. Causes of death and hospitalization for Groups 1 and 2.
Three patients chose to withdraw from study: 2 of them were from the standard-of-care
group, and another one from the nutritional support group (the reason was moving to
another city). Compliance was assessed using patient diaries, where their adherence to basic
therapy and to nutritional support was recorded. Compliance below 80 % was reported for
2 subjects in Group 1 and 2 subjects in Group 2 (these subjects were excluded from the final
analysis). As a result, in the standard-of-care group 26 patients completed the study, and in
the nutritional support group 30 patients completed the study.
1.4.1 Changes of 6-minute test parameters over time
The trend for growth of the parameters in Group 2 was noticeable starting from Week 2. The
curves of 6-minute test for the 2 groups diverged significantly starting from Week 8. After
24 weeks, the exercise tolerance in patients receiving standard nutrition decreased
significantly (p=0.025). The baseline values for 6-minute test in this group were 85 to 243 m,
mean 203.441.6 m. After 24 weeks, the mean distance walked in 6 minutes decreased by
19 % (164.748.1 m).
Six patients with NYHA class III CHF experienced substantial deterioration of their health
worsening of dyspnea, weakness, edema, i. e. their condition progressed to NYHA class IV.
In the group of patients receiving the nutritional support, the baseline for the 6-minute
test was 182.245.6 m (range 34 m to 221 m), and Week 24 mean was 231.341.1 m (75 m
to 295 m), i. e. a statistically significant (p=0.015) increase in exercise tolerance was
observed. In this group, progression of NYHA class III to class IV was recorded for
2 patients only.
To conclude, in patients receiving the standard diet, Week 24 exercise tolerance decreased
significantly, whereas in patients receiving nutritional support, significant increase of the
exercise tolerance was observed. Statistical significance for the difference between the
groups was p=0.021.
1.4.2 Change of hand dynamometry measurements over time
After 24 weeks of monitoring, no significant differences were found for dynamometry
variables when comparing pre-treatment and post-treatment values; however, there was a
trend towards increase for these variables in the nutritional support group (mean increase
0.2 kg, p=0.084). In the standard-of-care group, the variables decreased, with mean change
of 0.4 kg; this change was not statistically significant (p=0.09).
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1.4.3 Change of LBM over time
For most of the patients receiving nutritional support, a significant increase of LBM was
shown (mean change 5.81.2 kg or 8.9 %, p=0.038). For 2 patients, a progressive decrease of
LBM was recorded on treatment (the LBM decreased by 2.1 kg and 3.4 kg). In the standard
nutrition group, 23 patients experienced statistically significant decrease of their LBM (mean
decrease 3.60.7 kg or 4.9 %, p=0.036). The LBM did not change significantly in 2 patients,
and an increase of LBM (+1.7 kg) was reported for 1 patient.
To conclude, long-term nutritional support leads to statistically significant increase of LBM,
while in the standard nutrition group the LBM continues to decrease progressively. The
differences between groups were statistically significant (p=0.04).
1.4.4 Changes of laboratory variables over time (absolute lymphocyte count and
serum albumin)
In the nutritional support group, a statistically significant increase of nutritional status was
demonstrated: the absolute lymphocyte count increased from 1590 to 1710 x109 (+12.5 %,
p=0.04), and the albumin level increased from 25.1 to 29.4 g/L (+17.1 %, p=0.045). In the
standard-of-care group, no significant changes were observed after 24 weeks of monitoring
for the lymphocyte count and albumin. The differences between groups were statistically
significant (p=0.04).
To conclude, the nutritional support demonstrated a favorable effect for all basic nutritional
status variables.
1.4.5 Other variables
For the effect of nutritional support on protein, fat and carbohydrate absorption variables
see Table 4.
Absorption variables
Group 1 Group 2
Baseline Week 24 Baseline Week 24
Total protein loss,
% of daily consumption
18.10.3 17.90.2 17.90.2 15.480.9
Total fat loss,
% of daily consumption
22.50.6 21.60.4 23.40.4
20.71.3

D-xylose excretion with urine,
g/5 h
0.810.05 0.850.04 0.820.04 0.830.04
Table 4. Absorption of nutrients in patients with NYHA class III-IV CHF, pre-treatment and
post-treatment.
No statistically significant changes were demonstrated in either group for morphometric
variables during the treatment period of 24 weeks.
1.5 Conclusion
The study of the small intestine condition revealed marked changes of structure and
functional activity of the intestine in all patients with CHF. The degree of the small intestine

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impairment directly depends on the severity of the CHF. This suggests direct impact of
heart failure on gastrointestinal restructuring.
We consider the oral nutrition system involving prescription of balanced nutritional mixes
to be one of the promising options to treat cardiac cachexia, as it has a pathogenesis-based
rationale. This study was an attempt to evaluate the efficacy of the nutritional support
during CHF progression.
2. Morphological and functional changes of the large intestine in patients
with different classes of chronic heart failure
2.1 Introduction
Current understanding acknowledges the role of the following factors in the pathogenesis of
CHF: neuroendocrine imbalance with excessive production of neurohormones, impairment
of various target organs (cardiac muscle, kidneys, skeletal muscles, small intestine).
However, many authors observed increased level of pro-inflammatory cytokines in CHF
patients that cannot be explained by neuroendocrine activation. A number of authors
reported a correlation between cytokine levels and blood plasma concentration of the
endotoxin (lipopolysaccharide of gram-negative bacteria).
In an attempt to find the origin of the endotoxin, various bacteria were considered,
particularly the bacteria of upper and lower respiratory tract, H. pylori, microorganisms of
urinary tract and intestine. The strongest changes were found in the flora of the large
intestine, where increase of the total number of microorganisms was observed,
predominantly gram-negative. However, there were no reports of detailed analysis of the
intestinal flora composition, and the parietal mucin layer flora was not taken into account.
The data on flora changes suggested potential methods of correction, particularly the
selective decontamination method. However, according to literature reports, a course of
selective decontamination reduces the number of microorganisms in the large intestine in
the phase of antibacterial treatment only, while as early as 6 weeks after their
discontinuation characteristics of the flora return to baseline. This shows lack of efficacy of
the selective decontamination alone in CHF patients with high NYHA classes.
From our perspective, there are two possible approaches to correct the endotoxemia that
leads to systemic inflammation: (1) development of methods that act on the intestinal wall
by decreasing its permeability to the endotoxin, and (2) treatment that has direct effect on
the intestinal flora towards normalization of both numbers and the composition of the flora.
2.2 Materials and methods
Laboratory tests.
Quantitative assay of endotoxin level using Kinetic-QCL test 50-650 U "Bioscience
Cambrex Wallkersville", USA.
Quantitative assay of IL-6 (IL-6 Human ELISA Kit (1 x 96 Well Plate), Cytokine company,
Russia), TNF-alpha (TNF alpha Human ELISA Kit (1 x 96 Well Plate), Cytokine company,
Russia), CRP (C Reactive Protein Human ELISA Kit - 1 x 96 Well Plate, Abcam, USA)
plasma levels using solid phase ELISA.
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2.3 Assessment of the microbial landscape, large intestine wall structure, endotoxin
levels, and pro-inflammatory cytokines in CHF patients with different NYHA classes
To study these variables, three consecutive groups were enrolled: Group 1: 65 patients with
NYHA class III-IV CHF; Group 2: 60 patients with NYHA class I-II CHF; Group 3: 56
patients, control group (patients with ischemic heart disease and arterial hypertension
without signs of CHF).
2.3.1 Changes in lumen flora of the large intestine in CHF patients with different NYHA
classes and in the control group
Comparison of the first and the second study groups revealed statistically significant
differences (p<0.05) for the following variables: total number of enterobacteria was 10
9
colony-
forming units (CFU)/g in Group 1 vs. 10
7
CFU/g in Group 2. Enterobacteria pool growth was
predominantly formed by E. coli (10
7
CFU/g in NYHA I-II group vs. 10
9
CFU/g in NYHA III-
IV group, p<0.0001), various Klebsiella sp. (10
5
CFU/g in NYHA I-II group vs. 10
7
CFU/g in
NYHA III-IV group, p<0.005), and citrate-assimilating enterobacteria (10
6
CFU/g in NYHA I-II
group vs. 10
8
CFU/g in NYHA III-IV group, p<0.005). Differences in concentrations of
Citrobacters, Enterococci and Candida yeasts were also statistically significant.
Comparison of the results for Group 1 (NYHA class III-IV) and control group subjects
showed differences similar to the comparison of Group 1 versus Group 2, with the exception
of differences in Clostridia populations (lecithinase- and hydrogen sulfide-positive strains):
10
7
CFU/g in CHF patients with NYHA III-IV vs. 10
5
CFU/g in the control group (p<0.05).
Comparison of Group 2 (NYHA I-II CHF) versus control group demonstrated minimal
changes in the gut microbiome. Statistically significant differences were shown for
Bacteroides only (10
9
CFU/g in NYHA class I-II patients vs. 10
10
CFU/g in the control group,
p<0.05). In CHF patients with NYHA class III-IV the levels of Bacteroides were not
significantly different from results reported for the control group; therefore, from our
perspective, these data can be ignored in practice.
Statistically significant differences were demonstrated for Clostridia (hydrogen sulfide-
positive, lecithinase-positive): 10
5
CFU/g in the control group vs. 10
7
CFU/g in CHF
patients with NYHA class III-IV, as well as for Enterococci and Candida yeasts (p<0.05). No
statistically significant differences between groups were demonstrated for other
microorganisms.
Conclusion: the higher is NYHA class of CHF, the stronger are the changes in the large
intestine flora due to growth of gram-negative species.
2.4 Changes of the parietal mucin layer flora in CHF patients with NYHA class I-IV
The microorganisms located in the parietal mucin layer have the most significant impact on
the host. Therefore, our objective was to study the changes in parietal mucin layer flora in
CHF patients with NYHA class I-IV.Taking into account the minimal changes in lumen flora
between NYHA class I-II patients and the control group, and considering technical
difficulties associated with parietal flora studies, we decided to skip investigation of parietal
mucin layer flora in the large intestine for the control group.We decided to approximate the
results from biopsies of CHF patients with NYHA class I-II as normal.

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Biopsy studies showed changes similar to those reported for feces. A statistically significant
changes of the enterobacteria population in CHF patients with NYHA class III-IV was
demonstrated (10
8
CFU/g vs. 10
5
CFU/g in patients with NYHA classes III-IV and I-II,
respectively; p<0.0001).These changes were due to growth of E. coli (10
8
CFU/g vs.
10
5
CFU/g in NYHA III-IV vs. NYHA I-II, respectively; p<0.0001), Klebsiella (10
8
CFU/g vs.
10
5
CFU/g in NYHA III-IV vs. NYHA I-II, respectively; p<0.005), and citrate-assimilating
enterobacteria (10
8
CFU/g vs. 10
5
CFU/g in NYHA III-IV vs. NYHA I-II, respectively;
p<0.005).No statistically significant differences were found in the biopsy specimens for
Clostridia, Enterococci, as well as for Candida yeasts. However, statistically significant
differences were demonstrated for the population of Bifidobacteria: 10
3
CFU/g vs. 10
6
CFU/g
in NYHA III-IV vs. NYHA I-II, respectively (p<0.05).
Conclusion: the parietal mucin layer flora changes corresponded to the changes of the
lumen flora: the higher is NYHA class, the greater is the population of gram-negative
microorganisms. No changes in the population levels of gram-positive flora was
demonstrated for lumen flora, while in the parietal mucin layer a decrease of Bifidobacteria
population was reported.
2.5 Changes in the structure of the intestinal wall in CHF patients with NYHA class I-IV
Changes found in the microbial landscape of both lumen flora and parietal mucin layer flora
prompted us to study the large intestine wall structure in CHF patients with NYHA class I-
IV.
In Group 2, no changes were observed in the biopsy specimens of large intestine mucosa. In
Group 1, hyperemic vessels and focal dense lymphoid-cellular infiltrates were reported in
the lamina propria of the large intestine mucosa (Fig. 4). This pattern is consistent with
marked chronic inflammation.
To confirm the lymphoid nature of the infiltrates, OLA reaction (common lymphocytic
antigen) was performed (Fig. 5).
A strong positive infiltrate (derivates of white blood cell line) was observed in the lamina
propria of the large intestine mucosa. Particularly, intraepithelial lymphocytes were seen
clearly. CD8+ staining (Fig. 6) revealed intraepithelial CD8+-positive T-lymphocytes in the
superficial layer of the large intestine mucosa.
Ki67 reaction revealed positive cells of gland epithelium (Fig. 7) in the proliferation phase.
At the same time, there were solitary cells positive for Muc5 reaction (i. e. containing mucin
5) in the superficial epithelium of the large intestine mucosa.
Histopathology also revealed a large number of siderophages (Fig. 8), suggesting chronic
congestion in the large intestine vessels. We considered these changes to be a sign of the
chronic heart failure.
Conclusion: the CHF patients with NYHA class III-IV showed signs of marked chronic
inflammation in the large intestine mucosa, along with tissue edema and venous congestion.
The severity of these changes increased with higher NYHA class and the severity of CHF
decompensation symptoms.
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Fig. 4. Biopsy sample of the large intestine mucosa. Hematoxylin-eosin stain.

Fig. 5. Biopsy sample of the large intestine mucosa. OLA reaction.

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Fig. 6. Biopsy sample of the large intestine mucosa, CD8+ lymphocyte stain. 1 CD8+
lymphocytes.
2.6 Endotoxin level assessment
Plasma levels of the endotoxin were 1.20.03 EU/L in NYHA class III-IV subjects and
0.460.01 EU/L in NYHA class I-II subjects (EU endotoxin units). The level of the
endotoxin in the control group subjects was 0.350.02 EU/L. Notably, plasma endotoxin
levels directly correlated with the changes in population numbers of the large intestine
gram-negative flora.
The CHF patients with NYHA class III-IV had levels of IL-6 at 11.50.3 U/L, TNF-alpha at
6.60.4 U/L, and CRP at 80.65 mg/ml. The CHF patients with NYHA class I-II had levels
of IL-6 at 4.60.3 U/L, TNF-alpha at 3.70.4 U/L, and CRP at 5.50.29 mg/ml. The levels of
these pro-inflammatory cytokines in the control group were within normal limits: : IL-6 was
2 U/L, TNF-alpha was 1.5 U/L, and CRP was 2.9 mg/ml. The identified changes prompted
us to suggest two approaches to correction of these conditions:
1. Targeting the large intestine wall using different diuretic regimens, including agents
with tissue activity.
2. Selective decontamination in combination with probiotics.
2.7 Use of different diuretic regimens
Figure 9 shows the study design.
1
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Fig. 7. Biopsy sample of the large intestine mucosa, Ki67 reaction.
1 proliferating epithelial cells of a gland.
The study drugs were prescribed for the first 5 days after the screening visit. After that, the
study drug was discontinued and patients remained on the standard-of-care therapy for the
30 days of follow-up (until compensation of their clinical status).
The following tests were performed in this study:
body mass and the volume of excreted fluid,
results of 6-minute test,
Clinical Status Assessment Scale score (points),
plasma levels of the endotoxin,
feces flora composition and enzyme activity of the microorganisms,
results of colonoscopy with cecum biopsy and further histopathology and
histochemistry of the obtained samples.
These tests were performed on Day 1, Day 6 and Day 30 of the study.
After the study treatment period, all patients were switched to the supportive care regimen
and received the standard-of-care therapy; this phase lasted for 30 days.
One patient died while on study (CHF decompensation was the cause of death).
1

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Fig. 8. Biopsy sample of the large intestine mucosa. Hematoxylin-eosin stain. 1
siderophage.

Fig. 9. Study design for the assessment of efficacy of Diacarb (acetazolamide) and
Hypothiazide (hydrochlorothiazide) in comprehensive therapy of CHF patients with NYHA
III-IV.
Data from 79 subjects who completed the study was therefore used for the analysis of study
results.
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During the study, 15 adverse reactions were reported, but none of these caused
discontinuation of the study treatment.
No statistically significant differences between groups were demonstrated for the main
variables.
2.8 Changes of body mass and volume of excreted urine over time
To determine efficacy of each study diuretic regimen, changes of the body mass and the
volume of excreted urine were evaluated throughout the study. After the first five days, a
decrease of body mass to 830.5 kg and 83.10.36 kg was demonstrated in both Group 1 and
Group 2, respectively. The decrease was statistically significant against the baseline body
mass of 87 kg, but the difference between groups was not statistically significant (p=0.872).
The volume of excreted urine in the study groups was the highest on the first day of
treatment, comprising 2.510.1 L and 2.50.25 L for Group 1 and Group 2, respectively. This
effect decreased proportionally during the following five days in both groups. Notably, no
statistically significant difference was detected between groups both in terms of body mass
change (p=0.99) and in terms of the volume of excreted urine.
2.9 Changes of endotoxin levels over time
Substantial decrease of endotoxin levels on the follow-up Day 21 (1.20.02 EU/L to
0.20.01 EU/L) was demonstrated in all groups. However, in the Diacarb group, this
process was substantially faster: as early as on Day 5, the endotoxin levels were at
0.40.02 EU/L, whereas in the thiazide diuretic group the levels were at 0.780.01 EU/L
(p=0.012). Notably, while the diuretic effects in the Hypothiazide and Diacarb groups were
almost identical, the latter group demonstrated faster decrease of the endotoxin level.
This is probably due to tissue pH change caused by Diacarb, which facilitates fast
dehydration of the large intestine wall and decreases its permeability for the endotoxin. To
support this hypothesis, we performed histopathology and histochemistry studies using the
biopsy samples from the large intestine at Day 1 and Day 5. To exclude the role of the large
intestine flora, we also monitored its composition throughout the study.
2.10 Assessment of diuretic regimen effects on structural changes in the large
intestine wall over time
Assessment of the histological and histochemical patterns in the large intestine mucosa
during Diacarb or Hypothiazide treatment demonstrated the following changes.In the
thiazide group (Fig. 10 and Fig. 11), there were signs of reduced mucosal edema on
treatment (Day 6); however, a rise of local inflammatory reaction was also evident
(lymphoplasmocytic infiltration, increased number of segmented WBCs, predominantly
eosinophils).This may be a relative effect caused by reduction of edema and shortening of
intercellular distances rather than an absolute growth of the inflammatory infiltration.
A decrease of edema on Day 6 was also seen in the Diacarb group (Fig. 12, Fig. 13), but, in
addition to that, the level of local inflammation decreased (the infiltration by lymphoid cells
is consistent with low-intensity chronic inflammation).

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Most probably, the difference between Diacarb and Hypothiazide in their effects on large
intestine wall edema causes different permeability of the wall for the endotoxin, which
results in different kinetics of blood endotoxin level reduction in the CHF patients with
NYHA class III-IV. These findings may result from changes in pH of the intestinal wall,
better microcirculation, and consequent decrease of infiltration. Another possibility is that
Diacarb, a carbonic anhydrase inhibitor, blocks alpha-carbonic anhydrase of gram-negative
bacteria, depressing their pathogenic effect on the intestinal wall.
2.11 Assessment of effects of diuretic regimens on changes of flora over time
No statistically significant changes in concentration of gram-negative bacteria both in feces
and in biopsy material were demonstrated, regardless of the treatment regimen.

Fig. 10. Magnification x 400 (good), hematoxylin and eosin stain.Before treatment, fuzzy
lymphoid-cellular infiltration and solitary eosinophils were noted in the lamina propria of the
large intestine mucosa.
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Fig. 11. Magnification x 400 (good), hematoxylin and eosin stain.Day 6 of the treatment
period.Lymphoplasmocytic infiltration increases. The population of segmented WBCs,
particularly eosinophils, increases significantly.
These results demonstrate that plasma endotoxin levels in CHF patients with NYHA
class III-IV are affected not only by the gram-negative flora population in the intestine, but
also by the severity of edema, and therefore by the degree of decompensation of patients
clinical status.The endotoxin levels in this case are probably affected by the increase of the
intestinal wall permeability for the endotoxin caused by the edema.
As patients status improves towards compensation, the endotoxin levels decrease to normal
values. This process is faster with the use of Diacarb compared to the use of Hypothiazide.
However, this is not accountable to their diuretic effects, because there were no statistically
significant differences in the changes of body mass and the volume of excreted urine
between Diacarb and Hypothiazide.It is likely that Diacarb improves microcirculation by
changing tissue pH, and causes not only improvement of renal urine filtration, but also
faster shrinking of tissues, particularly shrinking of the intestinal wall, which decreases its
permeability for the endotoxin.
Differences in the inflammatory infiltrate intensity between Hypothiazide and Diacarb
groups were discovered (in addition to decreased edema, Diacarb reduces the inflammation
in the large intestine mucosa). This effect is probably accountable to improved
microcirculation in the intestinal wall in the Diacarb group, as well as carbonic anhydrase
inhibition produced by Diacarb leading to block of alpha-carbonic anhydrase of gram-
negative bacteria, which reduces their pathogenic effect on the intestinal wall.

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Fig. 12. Magnification x 400 (good), hematoxylin and eosin stain.Before treatment, there
were hyperemic vessels and focal dense lymphoid-cellular infiltration in the lamina propria
of the large intestine. This pattern is typical for strong chronic inflammation.

Fig. 13. Magnification x 400 (good), hematoxylin and eosin stain. Day 6 of the treatment
period.Fuzzy lymphoid-cellular infiltration is seen in the lamina propria of the large
intestine mucosa, which is consistent with the pattern of weak chronic inflammation.
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Conclusion: administration of Diacarb facilitates faster decrease of plasma endotoxin levels,
which allows for faster compensation of patients clinical status.
2.12 Use of selective decontamination alone vs. selective decontamination in
combination with probiotics in comprehensive therapy of NYHA class III-IV CHF
All patients included in this phase of the study received standard-of-care therapy,
including:
ACE inhibitors/ angiotensin receptor blockers (mean daily dose of 10 mg/160 mg);
beta-blockers (prescribed from Day 5 after the start of the therapy, dose titration from
the minimum therapeutic dose, mean daily dose was 50 mg of metoprolol per day);
digoxin 0.00025 g per day (in case of atrial fibrillation with tachycardia or LVEF below
25 %);
aspirin 125 mg/day (secondary prophylaxis method);
Cordarone 200 mg/day (in case of ventricular disturbances with risk of high Lown
grades);
loop diuretics (Lasix) with mean daily dose of 70 mg/day.
The following drugs were chosen for the study:
antibacterial fluoroquinolone: ciprofloxacin with daily dose of 1000 mg;
probiotic: Primadophilus Bifidus, 1 capsule per day.
At the study start, all patients received a 5-day selective decontamination with oral
ciprofloxacin 1000 mg/day.After that, patients were randomized into two groups:
Group 1 (n=45) received standard-of-care;
Group 2 (n=45) received the probiotic Primadophilus Bifidus, 1 capsule per day for
14 days.
After the completion of probiotic treatment, both groups received standard-of-care.
The following study variables were evaluated on Day 1, Day 5, and Day 21:
results of 6-minute test,
Clinical Status Assessment Scale score (points),
plasma levels of the endotoxin,
feces flora composition and enzyme activity of the microorganisms.
In this phase, 2 deaths occurred in the group receiving selective decontamination alone.In
the Primadophilus Bifidus group, one patient experienced adverse effects leading to
patients decision to discontinue the drug.
Data from 87 subjects who completed the study was therefore used for the analysis of study
results.
Ten cases of adverse reactions were reported during the study. Only one of these cases
caused the patient to stop the drug.
The resulting groups had comparable basic characteristics.

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2.13 Changes of the large intestine flora over time with the use of selective
decontamination alone or selective decontamination combined with the probiotic
By Day 5 of the study, a statistically significant decrease was demonstrated in Group 1 for
total population of both gram-negative microorganisms (baseline value was 10
120.1
CFU/g,
the value after the selective decontamination was 10
60.4
CFU/g; p=0.000) and gram-positive
microorganisms (baseline value was 10
60.56
CFU/g, the value after the selective
decontamination was 10
40.32
CFU/g; p=0.000)However, on Day 21 study visit, gram-
negative and gram-positive populations returned to their baseline levels (10
11.9
CFU/g and
10
6.1
CFU/g, respectively; p=0.000). These results support literature reports of low efficacy
of the selective decontamination used alone.
In the group where probiotics were prescribed after the course of selective decontamination,
Day 5 populations decreased similarly to Group 1, both for gram-negative (baseline
10
12.050.6
CFU/g, post-decontamination 10
6.30.4
CFU/g; p=0.000) and gram-positive
(baseline 10
5.20.5
CFU/g, post-decontamination 10
4.20.2
CFU/g; p=0.000)
microorganisms.However, after Primadophilus Bifidus administration for 14 days, gram-
positive flora population grew to 10
8.020.1
CFU/g, and an insignificant growth of gram-
negative flora to 10
7.270.1
CFU/g was demonstrated, which is consistent with normal values
for gram-negative population in the large intestine.
Conclusion: administration of probiotics after a course of selective decontamination
normalizes large intestine flora levels, whereas decontamination alone leads to reduction of
microbial populations for a short term only.
2.14 Changes of the endotoxin level over time
In Group 1, Day 5 endotoxin levels decreased from baseline significantly (baseline:
1.20.9 EU/L, Day 5: 0.550.06 EU/L; p=0.000), which corresponded to the reduction of
gram-negative population in the large intestine. However, at Day 21, as the gram-negative
population in the large intestine grew, the plasma endotoxin levels returned to their baseline
values (1.180.05 EU/L); on Day 30, the endotoxin concentration remained high
(1.210.045 EU/L).
In Group 2, after the selective decontamination was completed and the gram-negative
population in the large intestine reduced, the plasma endotoxin levels also declined
(baseline: 1.240.01 EU/L, Day 5: 0.670.03 EU/L, p=0.000). A trend towards decline of
plasma endotoxin levels and achievement of normal values was demonstrated subsequently
(Day 21: 0.560.02 EU/L, Day 30: 0.260.08 EU/L). These results can be explained by the
reduction of the gram-negative populations in the large intestine due to administration of
the selective decontamination followed by probiotic.
2.15 Assessment of changes in plasma pro-inflammatory cytokine levels over time
With the use of the selective decontamination alone, the decrease of the following variables
on Day 5 was demonstrated: IL-6 to 4.10.03 U/L (baseline 4.90.01 U/L), TNF-alpha to
50.09 U/L (baseline 5.70.04 U/L), and CRP to 4 mg/ml (baseline 8.6 mg/ml).However, as
early as on Day 12, these cytokine variables were shown to return to their baseline levels,
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which persisted till Day 30. This demonstrates poor efficacy of the selective decontamination
alone for the system inflammation marker endpoints.
When the selective decontamination was used in combination with the probiotic, decrease in
the population of enterobacteria in the large intestine and decrease of plasma endotoxin
levels were reported.However, while levels of the pro-inflammatory cytokines decreased by
Day 5 (CRP: 4.20.1 mg/ml on Day 5 vs. baseline 7.820.05 mg/L; IL-6: 3.90.05 U/L on
Day 5 vs. baseline 50.01 U/L; TNF-alpha: 5.010.02 U/L on Day 5 vs. baseline
5.80.02 U/L), but on Day 21 they already rebounded above the baseline levels, and on
Day 30 there was a trend towards further growth of their levels.
This is probably accountable to the decrease of gram-negative flora population in the large
intestine due to the antibacterial treatment, and consequent decline of plasma levels of the
endotoxin.Normal population numbers of the gram-negative flora are further maintained by
the administration of the probiotic. However, the probiotic contains gram-positive
microorganisms, which bind to the Toll-like receptors, initiating the synthesis of pro-
inflammatory cytokines in the large intestine enterocytes, leading to further exacerbation of
the systemic inflammation.
These results demonstrate lack of efficacy for the selective decontamination used
alone.When the selective decontamination was combined with the probiotic, normalization
of the intestinal flora and plasma endotoxin levels was reported. However, a significant
growth of the pro-inflammatory cytokine levels occurs with this regimen, which affects
patients status and is likely to require additional correction.
These data demonstrated that comprehensive therapy for CHF combined with the selective
decontamination alone (i. e. without probiotic) caused to a short-term decline of gram-
negative flora population, while the population numbers of gram-positive flora remained
almost unaffected. A short-term decline in plasma levels of the endotoxin and pro-
inflammatory cytokines was also reported. However, as early as one week after the
discontinuation of the antibacterial treatment, gram-negative flora population numbers
returned to their baseline levels, accompanied by the increase in plasma levels of the
endotoxin and the pro-inflammatory cytokines. A potential explanation for this pattern is
that CHF patients with NYHA class III-IV develop significant restructuring of their large
intestine walls, providing favorable conditions for domination of gram-negative
flora.Isolated use of the selective decontamination, neither supported by any agents that
repair large intestine wall structure, nor combined by any probiotics, fails to provide stable,
long-term changes in the large intestine flora.
However, administration of probiotics added to the antibacterial treatment demonstrated
persistent effect: normalization of gram-negative flora levels and plasma endotoxin levels.
Notably, with the use of the probiotic, blood CRP levels increased, which is probably
accountable to the presence of gram-positive flora in the probiotic, prompting the host to
produce more antibodies. Unfortunately, the CRP levels were not followed for a longer
period of time, and the time needed for the CRP to reach normal levels remained
unknown.It can only be assumed that this period should not take too much time, because
the subjects were exposed to the probiotic for 2 weeks only. However, if a long-term,
persistent growth of the CRP levels in blood do occur, decompensated CHF patients with
NYHA class III-IV might benefit from administration of statins.

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3. Clinical significance of adipose tissue changes over time in patients with
chronic heart failure of ischemic origin. Treatment options
3.1 Introduction
Syndrome of cardiac cachexia is one of the most severe complications of chronic heart
failure. Among the latest advancements in the field of immunology is the concept of
cytokine activation system and its role in the pathogenesis of chronic heart failure and
development of cardiac cachexia. Currently, two main classes of cytokines are known to
participate in the development of heart failure:vasoconstrictive cytokines (endothelin-1 and
big endothelin) and vasodepressive cytokines (TNF-, IL-1, IL-6, IL-8). Patients with signs of
cardiac cachexia are known to have higher levels of inflammation markers than patients
with normal body mass (Francis, 1998; Monteiro, 2007). Notably, adipose tissue is one of the
sources of cytokines.In addition to leptin and adiponectin, adipose tissue was demonstrated
to participate in production of TNF- and IL-6 (Moses, 2004; Nagaya, 2001; Springer, 2010).
We assumed that one of the methods to decrease the activities of pro-inflammatory
cytokines could be the increase of dry body mass (body muscle mass, body fat mass)
(Dostalova et al., 2003). Therefore, one of the options to correct the levels of inflammatory
markers in this category of patients could be nutritional support.
From our perspective, there are two possible approaches to correct the systemic
inflammation:development of methods that can increase the mass of the adipose tissue and
methods that have direct effect on the synthesis of pro-inflammatory cytokines.
3.2 Assessment of body composition, levels of leptin, adiponectin, and pro-
inflammatory cytokines in patients with different NYHA classes of CHF
To study these variables, three consecutive groups were enrolled in the first part of the
study: Group 1 included chronic heart failure patients with NYHA class I-II, Group 2
included chronic heart failure patients with NYHA class III-IV (subgroup A: without
cachexia; subgroup B: with cachexia), Group 3 was a control group.Patient screening was
performed in the population of patients with history of CHF of ischemic origin with
NYHA class I-IV for more than 6 months, older than 40 years of age, admitted to a general
internal medicine department or a cardiology department (n=197).The control group
included outpatients of the Consultation and Diagnostics Polyclinic (n=52).
Clinical characteristics of the patients:
Age:these three groups were comparable in terms of patients age.
From the results of analysis of associated clinical conditions, diabetes mellitus was reported
in 57.1 % of CHF patients with NYHA class III-IV with cachexia and in 48.6 % without
cachexia, as well as in 14 % of patients with NYHA class I-II.
Analysis of concomitant medications: CHF patients with NYHA class III-IV with/without
cachexia were on ACE inhibitors or ARB in 64.2 %/63.8 %, on beta-adrenoblockers in
82.1 %/80 %, on digoxin in 75 %/76.1 %, on diuretics in 35.7 %/34.3 %, and on Cordarone in
28.6 %/19.5 % of cases, respectively.For NYHA class I-II patients, the corresponding
variables were: on ACE inhibitors/ARB 51.6 %, on beta-adrenoblockers 59.4 %, on digoxin
9.3 %, on diuretics 45.3 %, on Cordarone 3.1 %.
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3.3 Comparison of methods used to evaluate body composition in patients with CHF
of different NYHA class and the control group patients
Two methods were used to study body composition: caliper measurement and
bioimpedance analysis.Comparison of these methods in all three groups did not reveal any
statistically significant differences between the values obtained using caliper measurements
and bioimpedance analyzer.However, the method of caliper measurements is subjective and
is not suitable for patients with decompensated heart failure (i. e. with severe edema
syndrome).Moreover, unlike caliper measurements, bioimpedance analyzer of body
composition allows to estimate not only the body fat mass, but also total fluid content,
which is important for the assessment of the body composition in patients with
decompensated chronic heart failure.
Comparison of Group 1 vs. Group 2 revealed statistically significant differences in the
adipose tissue mass, which was 29.72.2 kg in Group 1 and 22.22.1 kg in Group 2 (p<0.001),
as well as in the lean body mass, which was 54.86.9 kg in Group 1 and 59.76.1 kg
(p<0.001) in Group 2. This was associated with changes in total fluid content, which was
38.23.1 kg in Group 1 vs. 48.83.3 kg in patients with NYHA class III-IV (p<0.001).BMI did
not differ significantly between groups.
Comparison of the study results between Group 2 (NYHA III-IV) and the control group
revealed changes similar to those between Group 1 and Group 2.
Comparison of Group 1 (NYHA I-II) vs. the control group revealed statistically significant
differences in the body fat mass, which was 29.72.2 kg in Group 1 vs. 32.62.4 kg in
Group 3 (p<0.001), and statistically significant differences in total fluid content, which was
38.23.1 kg in Group 1 vs. 25.62.9 kg in Group 3 (p<0.001).
In patients with cachexia, a significantly lower LBM of 55.24.9 kg and body fat mass of
15.651.8 kg were reported. Notably, the total fluid content levels in these patients was
greater than levels of this variable in patients without cachexia, but the differences of this
variable were not statistically significant.
Conclusion: the higher is NYHA class of CHF, the stronger are changes of body
composition; with higher NYHA class, adipose tissue mass declines, total fluid content
grows.These changes were stronger in patients with cachexia.
3.4 Levels of leptin, adiponectin, and pro-inflammatory cytokines in patients with
different NYHA classes and in the control group
The patients with NYHA III-IV were found to have adiponectin levels at 18.64.9 g/mL,
leptin levels at 43.88.3 ng/mL, IL-6 levels at 11.70.3 U/L, TNF- levels at 6.60.2 U/L,
CRP levels at 8.80.4 mg/ml. These values were significantly higher than values reported
for patients with NYHA class I-II and for patients in the control group, who had their levels
of adipokines and pro-inflammatory cytokines within normal range.
Comparison of patients with NYHA class III-IV with and without cachexia demonstrated
the following differences: leptin levels were significantly higher in patients without cachexia
(47.94.2 ng/mL), while levels of adiponectin, IL-6, TNF-, and CRP were significantly
higher in patients with cachexia.

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Conclusion: the higher is NYHA class of CHF, the stronger is the intensity of chronic
inflammation. While this may be an effect of growing intoxication in patients with advanced
stages of CHF, this could also be associated with the role of the adipose tissue in production
of pro-inflammatory cytokines and biologic agents that stimulate cytokine production.
Higher classes of NYHA are associated with lower adipose tissue mass, which leads to
increase of adiponectin plasma levels.However, the levels of leptin in patients with NYHA
class III-IV are also high, which may be accountable to big dimensions of adipocytes.
Patients with cachexia have significantly lower levels of leptin when compared to patients
without cachexia; this is probably associated with shrinking of the lipid droplet in the
adipocyte.
3.5 Evaluation of visceral and subcutaneous tissue structure in patients with different
CHF classes
To study this variable, autopsies of deceased patients (with NYHA class I-IV chronic heart
failure diagnosed before their death) were performed. Patients were allocated into two
groups: Group 1: before death, patients were diagnosed with NYHA I-II chronic heart
failure; Group 2: before death, patients were diagnosed with NYHA III-IV chronic heart
failure. Patients of Group 2 were divided in two subgroups: patients with cachexia and
patients without cachexia. All patients were admitted to GKB no. 4 (City Clinical Hospital
no. 4) before death. For NYHA I-II patients, the main reasons for hospitalization were:
unstable angina, acute myocardial infarction, hypertensive crisis, heart rhythm disorder,
cerebrovascular accident; for NYHA III-IV patients, the main reasons for hospitalization
were: decompensation of CHF, acute/recurrent myocardial infarction, heart rhythm
disorder, cerebrovascular accident.The postmortem assessment included measurement of
the subcutaneous fat, measurement of the omentum mass, morphometric study of the
subcutaneous fat, omental fat and pericardial fat.
Of 118 subjects total, 50 subjects were in the first group, 56 subjects were in the second
group, and 12 subjects belonged to the third group. These three groups were comparable in
terms of patients age. The main cause of death for NYHA I-II patients was the acute
myocardial infarction (46.0 %), and for NYHA II-III patients (both with cachexia and
without cachexia) the main cause of death was post-infarction cardiosclerosis (58.3 %,
48.2 %, respectively). The most frequent complication leading to death in patients with
NYHA class I-II CHF was acute heart failure; in patients with NYHA class III-IV the most
frequent complication leading to death was CHF decompensation. Pneumonia incidence in
patients with NYHA class III-IV was higher (22 %) than in NYHA I-II patients (4 %).
Multiple complications were reported for 22.0 % patients with NYHA I-II, 46.4 % patients
with NYHA III-IV without cachexia, and 100 % of patients with NYHA III-IV with cachexia.
During autopsy, the following investigations were performed: measurement of
subcutaneous fat thickness 2 cm below the navel, measurement of omentum weight;
autopsy samples of subcutaneous fat, omental fat, and pericardial fat at the apex of the heart
were collected.
There were no statistically significant differences between patients with NYHA I-II and
NYHA III-IV without cachexia in the thickness of subcutaneous fat: it was 5.31.7 cm and
5.12.2, respectively (p=0.6).In CHF patients with NYHA III-IV with cachexia, the difference
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in thickness of subcutaneous fat (2.41.1 cm) was statistically different from NYHA III-IV
patients without cachexia (p<0.001).
In CHF patients with NYHA class III-IV, omentum weight was significantly lower than in
patients with NYHA I-II (387134 g vs. 521142 g, respectively; p<0.001).In CHF patients
with NYHA classes III and IV, the omentum weight was 16487 g, which is significantly
lower than in patients without cachexia (p<0.001).
The morphometric analysis of the samples showed the following changes.In the
subcutaneous fat, lymphocytic infiltration was the strongest in patients with NYHA class III-
IV with cachexia (average 12.44.7 %); in patients with NYHA class I-II this variable was
3.82.2 % (comparison: p<0.001), and in NYHA III-IV patients without cachexia it was
4.32.4 % (comparison: p<0.001).No significant differences in the proportion of fibrous
tissue in the subcutaneous fat was found between the groups.The percentage of fibrous
tissue was 3.81.9 % in NYHA class I-II patients, 4.12.3 % in NYHA class III-IV patients
without cachexia, and 4.62.6 % in NYHA class III-IV patients with cachexia (p1-2=0.469, p1-
3=0.229, p2-3=0.506). See Fig. 14, 15, 16.

Fig. 14. Subcutaneous fat in a CHF patient with NYHA class I. Romanowsky-Giemsa stain.

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Fig. 15. Subcutaneous fat in a CHF patient with NYHA class III.

Fig. 16. Subcutaneous fat in a CHF patient with NYHA class IV with cachexia.
Romanowsky-Giemsa stain.
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Analysis of the visceral fat (omentum, pericardial fat) showed the strongest lymphocytic
infiltration in subjects with NYHA class III-IV, both with cachexia and without cachexia.
This variable was 53.47.8 % in the omentum and 49.78.4 % in the pericardium in patients
with cachexia, and 42.16.7 % in the omentum and 42.68.8 % in the pericardium in patients
without cachexia (compare to patients with NYHA I-II, who had the lymphocytic infiltration
of 5.12.3 % in the omentum (p1<0.001, p2<0.001) and 4.92.6 % in the pericardium
(p1<0.001, p2<0.001)).The amount of fibrous tissue in NYHA class III-IV patients without
cachexia was higher than in the patients with NYHA class I-II: 15.24.9 % vs. 3.11.2 % in
the omental adipose tissue (p<0.001), 14.85.4 % vs. 3.20.9 % in the adipose tissue of the
pericardium (p<0.001), respectively.In NYHA III-IV patients with cachexia, the amount of
fibrous tissue was significantly higher than in NYHA III-IV patients without cachexia:
24.83.7 % in the omentum (p<0.001) and 24.33.2 % in the pericardium (p<0.001).See
figures 17-22.
Mean diameter of adipocytes was measured, with the following results:In CHF patients
with NYHA class I-II, the diameter of adipocytes was 38.612.2 m in the subcutaneous fat
samples, 44.216.1 m in the omentum, 42.311.4 m in the pericardial fat. In CHF patients
with NYHA class III-IV without cachexia, the diameter of adipocytes was 42.714.2 m in
the subcutaneous fat samples (p=0.116), 56.413.9 m in the omentum (p<0.001), 52.211.3
m in the pericardial fat (p<0.001). In NYHA class III-IV patients with cachexia, the
diameter of adipocytes was 28.211.5 m in the subcutaneous fat samples (p=0.01),
32.814.3 m in the omentum (p=0.028), 30.312.4 m in the pericardial fat (p<0.001).

Fig. 17. Visceral adipose tissue (pericardial fat) in a CHF patient with NYHA class I.
Romanowsky-Giemsa stain.

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Fig. 18. Visceral adipose tissue (omentum) in a CHF patient with NYHA class I.
Romanowsky-Giemsa stain.

Fig. 19. Visceral adipose tissue (pericardial fat) in a CHF patient with
NYHA class III.Romanowsky-Giemsa stain.
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Fig. 20. Visceral adipose tissue (omentum) in a NYHA class III patient without cachexia. Van
Giesons stain.

Fig. 21. Visceral adipose tissue (pericardial fat) in a CHF patient with NYHA class IV with
cachexia.Romanowsky-Giemsa stain.

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Fig. 22. Visceral adipose tissue (omentum) in a NYHA class IV patient with cachexia. Van
Giesons stain.
Therefore, the higher is NYHA class, the lower are the subcutaneous fat and the omentum
weight. The higher is NYHA class, the more intensive is the chronic inflammation,
manifested by the lymphocytic infiltration and the content of fibrous tissue, especially in the
visceral fat.These changes are stronger in NYHA III-IV patients with cachexia.
In patients with NYHA III-IV and cachexia, a decrease in the content of adipose tissue was
demonstrated to be the result of adipocyte shrinking, as well as substitution of the adipose
tissue by fibrous tissue.
Currently, there is no solution for the problem of how to increase the body muscle mass and
body fat mass in patients with cachexia, particularly with cardiac cachexia. Unfortunately,
all attempts to use anabolic steroids in this patient population have been unsuccessful.We
assumed that using nutritional support in this patient population would allow to change the
nutritional status in addition to reducing the malabsorption syndrome. Moreover,
nutritional mixes that have both local and systemic anti-inflammatory effect are currently
available. Administration of these mixes might help fighting the inflammation observed in
the large intestine of the CHF patients with higher classes of NYHA, as well as reducing
their blood levels of pro-inflammatory cytokines.
3.6 Comparison of efficacy of Modulen vs. Peptamen added to standard-of-care
therapy in CHF patients with NYHA III-IV
Screening was performed in a consecutive population of patients hospitalized to general
internal medicine or cardiology departments (n=144).
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415
Inclusion and exclusion criteria are shown in Table 5.
Inclusion criteria Exclusion criteria
CHF of ischemic origin
CHF history for more than 12 months
Age > 40 years old
Patients consent to participation in the study
Acute or chronic infectious diseases
Cancer within last 5 years
Severe impairment of liver or kidneys
(AST, ALT > 3 x upper limit of normal,
creatinine > 250 mol/L)
Mental disorders
Primary or secondary
immunodeficiency conditions
Alcohol or substance dependence
Any conditions that can cause
cachexia (at investigators discretion)
Lack of tolerance to enteral nutrition
regimen
Unable to sign the informed consent
Unable to follow the study procedures
Table 5. Inclusion and exclusion criteria.
Chronic heart failure patients with NYHA class III-IV were randomized into three groups,
40 subjects in each:
- Patients in Group 1 received Modulen (balanced nutritional mix for enteral tube feeding
or oral feeding) in addition to standard-of-care therapy.
- Patients in Group 2 received Peptamen (balanced nutritional mix for enteral tube
feeding or oral feeding) in addition to standard-of-care therapy.
- Patients in Group 3 received the standard-of-care therapy only, as well as the necessary
amount of nutrients in a standard diet designed for cardiology patients.
All patients received standard-of-care therapy, which included:
ACE inhibitors/ angiotensin receptor antagonists (mean daily dose of 10 mg/160 mg);
beta-blockers (prescribed from Day 5 after the start of the therapy, dose titration from
the minimum therapeutic dose, mean daily dose was 12.5 mg of carvedilol per day);
digoxin 0.00025 g per day (in case of atrial fibrillation with tachycardia or sinus rhythm
with LVEF below 25 %);
aspirin 125 mg/day (secondary prophylaxis method);
loop diuretics (Lasix) with mean daily dose of 60 mg/day.
The following procedures were performed for every patient:medical history; physical
examination, including measurement of weight, height, waist circumference, hip
circumference, wrist circumference, arm circumference; casual BP; heart rate; fasting
chemistry lab blood samples; 6-minute test; echocardiography for the measurement of the
ejection fraction; caliper measurements; bioimpedance analysis of body composition to
measure the lean body mass (LBM), body fat mass (BFM), total water content (TW); Clinical
Status Assessment Score.
Group 1 patients received nutritional mix Modulen (100-130 g of dry mix), which accounted
for 25 % of their daily energy demands, in addition to their basic diet.Group 2 patients

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received nutritional mix Peptamen (100-130 g of dry mix), which accounted for 25 % of their
daily energy demands, in addition to their basic diet.Group 3 patients received their
standard therapy only, as well as the necessary amount of nutrients within a standard diet,
based on pre-calculated energy demands.All patients maintained their dietary diaries,
which were used to adjust the diet on an individual basis.
The energy demands were calculated using Harris-Benedict formula with adjustment for
body mass deficit, taking into account body temperature and activity of a patient.
Energy demands:
AEC = EOO*AF*TF*BMD (1)
where:AEC actual energy consumption (kcal/day);
EOO basal metabolic rate, calculated using Harris-Benedict equations:
EOO (men) = 66 + (13.7*body mass, kg) + (5*height, cm) (6.8*age)
EOO (women) = 655 + (9.6*body mass, kg) + (1.8*height, cm) (4.7*age)
AF activity factor (bed rest: 1.1, movement within room: 1.2, no limitations: 1.3), TF
temperature factor (3637.0C: 1.0, 37.138.0C: 1.1, 38.139.0C: 1.2), BMD body mass
deficit (1020 %: 1.1, 2030 %: 1.2, >30 %: 1.3).
This study enrolled 120 patients.Males: 18 subjects (45 %) in Group 1, 19 subjects (47.5 %) in
Group 2, and 21 subjects (52.5 %) in Group 3.Females: 22 subjects (55 %) in Group 1, 21
subjects (52.5 %) in Group 2, and 19 subjects (47.5 %) in Group 3.
Total duration of CHF history was 15.34.3 months, 15.74.1 months, and 15.64.4 months
for Group 1, Group 2, and Group 3, respectively.These characteristics were not significantly
different between study groups.
Compensated type 2 diabetes mellitus was reported in 52.5 %, 55 %, and 42.5 % of subjects
in Group 1, Group 2, and Group 3, respectively.
Charlson index was > 5 in all groups.
As demonstrated above, there were no statistically significant differences between groups in
gender, age, and co-morbidity rates.
In all three groups, decreases of weight and LBM were demonstrated on Day 21 of the
treatment period:in Group 1, Day 21 body weight was 56.42.6 kg (baseline 66.93.5 kg); in
Group 2, Day 21 body weight was 56.42.1 kg (baseline 66.22.9 kg); in Group 3, Day 21 body
weight was 55.41.7 kg (baseline 67.02.9 kg); in Group 1, Day 21 LBM was 40.91.7 kg
(baseline LBM 47.21.9 kg); in Group 2, Day 21 LBM was 42.02.0 kg (baseline LBM 47.32.0
kg); in Group 3, Day 21 LBM was 41.21.9 kg (baseline LBM 46.91.8 kg).However, on Day
224, the body weight in Group 1 increased to 62.62.7 kg, but was significantly lower than in
Group 2 (66.82.4 kg) and Group 3 (68.71.9 kg).The LBM also increased on Day 224, but it
was lower than baseline LBM in Group 1 (44.41.6 kg), whereas it reached the baseline in
Group 2 (47.42.2 kg) and significantly exceeded the baseline in Group 3 (48.11.9 kg).
In Group 2 and Group 3, a statistically significant decrease of total fluid content on Day 21
was demonstrated: 34.61.8 kg in Group 2 (baseline 47.32.1 kg) and 34.41.7 in Group 3
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(baseline 47.11.7 kg).On Day 224, the total fluid content increased in both of these groups:
47.22.0 kg and 48.31.9, respectively.In Group 1, the total fluid content also decreased by
Day 21 (34.31.9 kg) compared to baseline (46.82.0 kg), but there was no significant
increase of the total fluid content on Day 224 (39.31.9 kg), unlike in other two groups.
On Day 224, the body fat mass increased significantly in Group 1 to 18.61.11 kg (baseline
16,81.13 kg), while no significant change in the body fat mass was reported for Group 2
and Group 3:16.81.13 kg (baseline 16.51.16 kg) in Group 2 and 16.61.33 kg (baseline
16.51.19 kg) in Group 3.
On Day 21, the BMI decreased from baseline in all groups:21.20.60 kg/m
2
in Group 1
(baseline 24.10.93 kg/m
2
); 21.40.59 kg/m
2
in Group 2 (baseline 24.10.86 kg/m
2
); and
21.50.64 kg/m
2
in Group 3 (baseline 24.31.01 kg/m
2
).On Day 224, the BMI returned to
baseline and was 23.70.62 kg/m
2
in Group 1, 23.70.58 kg/m
2
in Group 2, and 24.50.60
kg/m
2
in Group 3.
The body mass increased in all three study groups, but in patients on Modulen this was
accountable to increase of muscle and fat mass, and not to increase of total fluid content.This
suggests that administration of Modulen improves the nutritional status profile.
Assessment of NT-proBNP, adiponectin, leptin, CRP, IL-6, TNF- during administration of
Modulen and Peptamen added to standard-of-care therapy in CHF patients with NYHA
class III-IV.
The level of NT-proBNP in all groups was over 3000 pg/mL.
Reduction of chronic inflammation intensity was demonstrated in patients receiving
Modulen:on Day 224, there was a decrease in levels of CRP (4.70.4 mg/ml vs. baseline
8.90.7 mg/mL), IL-6 (5.20.4 U/L vs. baseline 11.80.8 U/L), TNF- (3.40.2 U/L vs.
baseline 6.80.4 U/L).Levels of adiponectin also declined in patients on Modulen: 15.81.5
g/mL vs. baseline 24.41.5 g/mL. No significant changes were demonstrated in Group 2
and Group 3.
There were no significant changes of leptin levels in any of the groups.
Assessment of hospitalization events showed the following results.In Group 1, during
treatment with Modulen, 32 hospitalization events were reported per year: 22
hospitalizations were due to CHF decompensations, in 12 of these cases congestive
pneumonia was also present; 2 events were due to myocardial infarctions; 5 events were for
hypertensive crisis, with 2 of them progressing to CVA; 3 events were due to fibrillation
paroxysms. Per-patient hospitalization rate was 0.550.01 event/person.There were 5 deaths
over one year of observation in Group 1:2 deaths were caused by CHF decompensation, 2
deaths were caused by CVA, 1 death was caused by AMI.
In Group 1, during treatment with Peptamen, 38 hospitalization events were reported over
one year of observation:of these, 27 hospitalization events were due to CHF
decompensations, with 15 cases accompanied by congestive pneumonia; 3 events were due
to acute myocardial infarctions; 7 events were due to hypertensive crisis, 1 of them
progressed to CVA; 4 events were due to fibrillation paroxysms. Per-patient hospitalization
rate was 0.950.02 event/person.There were 8 deaths over one year of observation in Group
2:5 deaths were caused by CHF decompensation, 2 deaths were caused by AMI, 1 death was
caused by CVA.

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In Group 3, 48 hospitalizations were reported over one year of observation: 41 events were
due to CHF decompensations, in 22 of which congestive pneumonia was also present; 2
events were due to acute myocardial infarctions; 4 events were for hypertensive crisis, with
2 cases progressing to CVA; 1 event was due to a fibrillation paroxysm.Per-patient
hospitalization rate was 1.20.03 event/person.There were 12 deaths over one year of
observation in Group 3:8 deaths were caused by CHF decompensation, 2 deaths were
caused by AMI, 2 deaths were caused by CVA.
4. Conclusion
Rates of hospitalization events and deaths over one year were lower in subjects receiving
Modulen compared to Peptamen and standard-of-care therapy.
5. References
Anker S.D., Negassa A, Coats AJ et al. (2003). Prognostic importance of weight loss in
chronic heart failure and the effect of treatment with angiotensin-converting-
enzyme inhibitors: an observational study. Lancet,Vol.361, No.9363, (March 2003),
pp.1077-1083, ISSN0140-6736
Dostalova I., Kavalkova P., Papezova H., Domluvilova D., Zikan V., Haluzik M.
(2010).Association of macrophage inhibitory cytokine-1 with nutritional status,
body composition and bone mineral density in patients with anorexia nervosa: the
influence of partial realimentation.Nutrition & Metabolism,Vol.7, (April 2010), pp.34,
ISSN 1743-7075
Francis G.S. (1998). Changing the remodeling process in heart failure: basic mechanism and
laboratory results. Current opinion in cardiology, Vol.13, No.3, (May 1998), pp.156-
161, ISSN 0268-4705
Harrington D., Anker S.D. (1997).Skeletal muscle function and its relation to exercise
tolerance in CHF.Journal of the American College of Cardiology, Vol.30, No.7,
(December 1997), pp.1758-1764, ISSN 0735-1097
Monteiro M.P., Ribeiro A.H., Nunes A.F. (2007).Increase in grelin levels after weight loss in
obese zucker rats is prevented by gastric banding. Obesity Surgery,Vol.17, No.12,
(November 2007), pp.1599-1607, ISSN 0960-8923
Moses A.W.G., Slater C., Preston T., Barber M.D., Fearon K.C.H. (2004). Reduced total
energy expenditure and physical activity in cachectic patients with pancreatic
cancer can be modulated by an energy and protein dense oral supplement enriched
with n-3 fatty acids. British Journal of Cancer,Vol.90, No.5, (March 2004), pp.996-
1002, ISSN 0007-0920
Nagaya N., Uematsu M., Kojima M. (2001). Elevated Circulating Level of Ghrelin in
Cachexia Associated With Chronic Heart Failure. Circulation,Vol.104, No.17,
(October 2001), pp. 2034-2038, ISSN 0009-7322
Springer J., Adams V., Anker S. D. (2010).Myostatin Regulator of Muscle Wasting in Heart
Failure and Treatment Target for Cardiac Cachexia.Circulation,Vol.121, No.3,
(January 2010), pp. 354-356, ISSN 0009-7322
20
Evaluation and Treatment
of Hypotension in Premature Infants
Shoichi Ezaki and Masanori Tamura
Division of Neonatal Medicine, Center for Maternal,
Fetal and Neonatal Medicine, Saitama Medical Center,
Saitama Medical University,
Japan
1. Introduction
Sixteen to 98% of extremely preterm infants are treated for hypotension within the first
week of life. The enormous variation in this estimate is due to a lack of reliable evidence.
While selecting a vasoactive agent, it is necessary to consider the goals of the therapy. To
achieve those goals, the clinician must assess the mechanisms of action of the potential
therapies. This chapter details the unique characteristics of the neonatal cardiovascular
system and defines hypotension in preterm infants. It provides indications for treatment and
appropriate therapies for individual cases.
2. Characteristic pathophysiology of hypotension in preterm infants
Blood pressure increases with advancing gestational and postnatal age, which is a
developmentally regulated phenomenon (Noori and Seli, 2005). Since cardiac output (CO)
and systemic vascular resistance (SVR) both contribute to blood pressure, elevation in blood
pressure during development may be the result of increased CO, increased SVR, or both
(Fig.1)
2.1 Hypovolemia
In preterm infants, absolute hypovolemia is the most frequent cause of hypotension.
Peripheral vasodilation with or without myocardial failure is the most frequent primary
etiological factor (Seli and Evans J, 2001). Absolute hypovolemia is defined as a loss of
volume from the intravascular compartment; alternatively, relative hypovolemia is defined
as vasodilatation with an inadequate volume to fill the expanded intravascular
compartment. In both situations, the result is inadequate filling pressure (also known as
preload) in the heart. If severe enough, hypovolemia can reduce CO, resulting in inadequate
tissue perfusion and oxygenation (Fig 1).
In cases of absolute hypovolemia, the body releases corticosteroids, adrenaline, and
noradrenaline, which cause vascular contraction in order to maintain blood pressure and
filling pressure and cause increased heart rate and contractility to maintain systemic blood

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flow (SBF). However, in sick or immature infants, this response may be limited (Ng et al.,
2001; Evans N, 2003). In addition, volume administration for the treatment of hypotension in
sick infants has been reported to have a dopaminergic effect (Seli and Evans J, 2001).
In preterm infants with acute blood loss (e.g., intraventricular hemorrhage [IVH]) or
excessive transepidermal water losses (e.g., gestational age 25 weeks), absolute
hypovolemia should be considered the primary cause of hypotension.

Fig. 1. Mechanism of preterm hypotension
2.2 Myocardial dysfunction
Myocardial contraction and relaxation depend on the regulation of cytosolic calcium
concentration and the responsivity of myofilaments to changes in calcium content. Preterm
infants, term infants, and adults all have the membrane systems that control cell calcium
flux and the sarcomeres that make up the myofibrils. However, the components of each
system undergo qualitative and quantitative changes during development. During in the
prenatal and newborn periods, myocytes change in size and shape. There are also changes
in the number of contractile elements and the nuclear-to-cellular volume ratio.
Cardiac contraction is an energy-dependent process that requires ATP, calcium, and an
ATPase located at the myosin head. The processes of contraction and relaxation in immature
myocardium as well as calcium homeostasis are different from those in mature

Evaluation and Treatment of Hypotension in Premature Infants

421
myocardium. Specifically, immature myocytes do not rely as heavily on the release and re-
uptake of calcium from the sarcoplasmic reticulum; instead, they depend more on
extracellular calcium concentration. As such, the immature myocardium of the fetus and
newborn depends on L-type calcium channels as a calcium source for contraction.
Furthermore, immature myocytes have greater cell surface area-to-volume ratios, which
may compensate for their underdeveloped T-tubule systems. The alterations in myocardial
structure and function with maturation and the developmental changes in cardiovascular
function provide the cellular and molecular bases for differences in myocardial contractility
among preterm newborns, term newborns, and older infants (Rowland and Gutgesell, 1995;
Noori and Seli, 2005).
Therefore, preterm infants with hypotension have a limited ability to increase CO in
response to inotropes or changes in volume (Teitel and Sidi, 1985). Furthermore, they have
an elevated sensitivity to increased afterload (Van Hare et al., 1990), which commonly leads
to decreased CO (Belik and Light, 1989).
2.3 Abnormal peripheral vasoregulation
Immediately after birth, there is a sudden increase in SVR. This can have a deleterious effect
on CO and potentially compromise organ blood flow. After the initial transition period,
vasodilation predominates rather than vasoconstriction. Indeed, the complex regulation of
vascular smooth muscle tone involves a delicate balance between vasodilators and
vasoconstrictors (Fig. 1 and Fig. 2).
hANP: human atrial natriuretic peptide, NO: Nitric oxide, GTP: guanosine triphosphate,
cGMP: cyclic guanosine monophosphate
The endogenous vasodilating factors include NO, eicosanoids, hAMP, and endothelin. The
endogenous vasoconstrictive factors include catecholamines, vasopressin, and angiotensin
II. The balance of these factors determines the blood vessel equilibrium and the tendency
toward vasodilation or vasoconstriction (Fig.2). In Figure 2, the vasoconstriction pathway is
shown in red, and the vasodilatation pathway is shown in blue. Phosphorylation of myosin
is the critical step in vascular smooth muscle contraction. Vasoconstrictors, such as
angiotensin II, vasopressin and norepinephrine, activate second messengers to increase
cytosolic calcium concentration, which in turn activates myosin light chain kinase.
Vasodilators, such as human atrial natriuretic peptide (hANP) and nitric oxide (NO),
activate myosin phosphatase, which dephosphorylates myosin to cause vasorelaxation. The
plasma membrane is shown at its resting potential (plus signs). cGMP denotes cyclic
guanosine monophosphate (Landry and Oliver, 2001). In addition, potassium channels in
the smooth muscle cell membrane have recently been implicated in the pathogenesis of
vasodilatory shock (Liedel et al., 2002).
Under normal physiologic conditions, CO remains essentially unchanged throughout
infancy. Therefore, the increased blood pressure with advancing gestational and postnatal
age is primarily the result of increased SVR. Maturation of vascular smooth muscle, changes
in the expression of vascular angiotensin II receptor subtypes, and maturation of the central
autonomic and peripheral nervous systems play significant roles in increasing vascular tone
and SVR. There are 2 major subtypes of angiotensin II receptors. AT1R, which is expressed in

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422
mature tissues and the umbilical artery, mediates smooth muscle contraction and regulates
fluid and electrolyte balance. AT2R, which is expressed in fetal and newborn tissues, has an
unknown function. The developmentally regulated transition from expression of AT2R to
AT1R begins following the first 2 weeks of life and is complete by month 3 (Noori and Seli,
2005; Engle, 2001). The vasodilating factor NO increases under conditions of oxidative stress
and sepsis. Because preterm infants are prone to these conditions (Ezaki et al., 2009a), their NO
levels can easily increase. Together, these physiological characteristics of preterm infants make
them susceptible to vasoregulatory dysfunction.

Fig. 2. Regulation of vascular smooth muscle tone
3. The significance of hypotension requiring treatment in preterm infants
3.1 Clinical outcomes
Hypotension is a common complication among preterm infants. Importantly, there is an
association between systemic hypotension and neonatal morbidities, including IVH and
neurodevelopmental disorders (Watkins et al., 1989; Goldstein et al., 1995). Unfortunately,

Evaluation and Treatment of Hypotension in Premature Infants

423
common conditions among preterm infants, such as sepsis, renal failure, and neonatal
asphyxia, can lead to the development of clinical hypotension and confer a poor prognosis.
3.2 Relationship between systemic blood flow and blood pressure
The most important goal in treating hypotension is to prevent cellular and tissue damage
resulting from hypovolemia. Seli et al. and Greisen et al. have reported important
considerations for the treatment of hypotension (Seli, 2006; Greisen, 2005). If effective
treatment is not promptly initiated, the blood pressure may decrease further to the
ischemic threshold, which is said to be about 30 mmHg, resulting in tissue ischemia and
permanent organ damage. For example, a loss of cerebral blood flow (CBF) triggers
abnormal cerebral function and, finally, tissue ischemia (Fig. 3). Furthermore, high blood
pressure is also deleterious. Although, exact value is not noted in existing reports, when
blood pressure exceeds intraventricular hemorrhage (IVH) threshold, risk of IVH
increases (Fig. 3).
Loss of vascular autoregulation has not been formally proven as a cause of increased
morbidity and mortality in preterm infants (McLean et al., 2008). However, it has
consequences that negatively affect prognosis. For instance, loss of autoregulation often
triggers IVH. In addition, the amount of blood shunted through the patent ductus arteriosus
(PDA), present in preterm infants, can become unstable. Furthermore, the patient is likely to
develop necrotizing enterocolitis (NEC) due to compromised blood flow to gastrointestinal
tract. Finally, once the patient enters the ischemic stage, there is an increased incidence of
periventricular leukomalacia (PVL) and severe renal failure. As such, although the exact
reference blood pressure values that cause failure of autoregulation and CBF remain
unclear, it is important that hypotension be treated properly.

Fig. 3. Proposed relationship between blood flow and mean blood pressure in the cerebral
circulation of the preterm infant

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4. Definitions of normotension and hypotension in the preterm infant
Most preterm infants admitted to the neonatal intensive care unit (NICU) have medical
conditions, such as respiratory disorders, electrolyte abnormalities, or neonatal asphyxia. In
addition, because there is a wide range of ages and body weights, it is difficult to define
hypotension as a single value in preterm infants. A neonate is considered to be hypotensive
if the mean blood pressure is below the fifth or tenth percentile of the normative data
according to gestational and postnatal age and weight (Cunningham et al., 1999). Another
definition of hypotension is a mean blood pressure less than or equal to the patients
gestational age in weeks. Although this definition is a useful tool, it is only valid during the
first 48 hours of life (Nuntnarumit et al., 1999). However, due to its simplicity, this value is a
good indicator for neonatologists to suspect hypotension.
4.1.1 Definition of preterm hypotension and its relationship to low systemic perfusion
Figure 3 indicates blood pressure values that are thought result from a failure of
autoregulation. Although they would be an ideal definition of hypotension, no consensus has
yet been reached. Of preterm infants with a gestational age of 2326 weeks, >90% have a mean
blood pressure >30 mmHg (Nuntnarumit et al., 1999). Recent studies suggest that it may be as
high as 2830 mmHg, even among extremely low birth-weight infants (Munro et al., 2004).
4.1.2 Permissive hypotension
A recent study in very preterm neonates suggested that blood pressure below the clinically-
accepted lower limit during the rst postnatal days may not require intervention, as long as
adequate tissue perfusion is maintained (Dempsey et al., 2009). This study suggested that
although treatment must be initiated promptly, overzealous treatment may worsen the
prognosis. Therefore, a diagnosis of hypotension must be based on clinical and laboratory
findings.
4.2 Clinical signs
Many conditions may trigger hypotension in preterm infants (Table 1). The need for tests
and treatments to prevent decreased tissue perfusion is examined.
Vasoregulation imbalance
Hemorrhage: Placental hemorrhage, abruption placenta prevail, feto-maternal hemorrhage,
birth trauma-subaponeurotic bleed, massive pulmonary hemorrhage.
Other: Twin-to-twin transfusion, third-space losses, asphyxia, sepsis and septic shock,
disseminated intravascular coagulopathy, NEC
Cardiogenic shock
Asphyxia, electrolyte abnormality, cardiac disease: arrhythmias, congenital heart disease,
PDA, cardiomyopathy, myocarditis, air leak syndromes
Endocrine
Adrenal hemorrhage, adrenal insufficiency
Drug induced
Anesthetic drugs, sedative drugs
Table 1. Causes of hypotension in preterm infants

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4.3 Hemodynamic monitoring in preterm infants
An ideal method for monitoring blood pressure would be simple, reliable, non-invasive, and
painless and would provide continuous measurement. However, such an ideal method has
not yet been developed. As such, the only reasonable approach to obtaining meaningful
hemodynamic data in preterm infants is the use of complex, multi-channel, real-time
monitoring towers combined with streamlined data-acquisition systems and observation of
clinical symptoms.
4.3.1 Conventional assessment
Direct invasive measurements (via umbilical or peripheral artery catheterization) allow for
constant monitoring of blood pressure in hypotensive preterm infants. Although this
method is controversial, in our experience, blood pressure values obtained through intra-
arterial catheterization are more accurate than non-intermittent blood pressure
measurements taken during times of vasoconstriction. In addition, once intermittent blood
pressure measurements become necessary, the patients condition is often already severe,
making the insertion of an arterial catheter impossible. It is important to note the risks of an
indwelling catheter, including thrombus formation, hemorrhage, and infection.
In neonates admitted to the NICU, heart rate is continuously, accurately, and routinely
monitored. However, factors such as anemia, drugs affecting the cardiovascular system, and
infection can also affect heart rate. Therefore, heart rate monitoring has a limited role in the
diagnosis of circulatory compromise.
Similarly, SpO2 measurements are performed routinely on neonates admitted to the NICU.
This measures arterial oxygenation as an indicator of the arterial circulation. However, in
contrast to adults, neonates have unique clinical complications. Clinical oximeters cannot
detect carbon monoxide hemoglobin, methemoglobin, fetal hemoglobin, or other hemoglobin
variations. Therefore, blood tests are needed for the accurate assessment a neonates
oxygenation status (Shiao and Ou, 2007). Nevertheless, SpO2 monitors are also useful for
estimating the extent of the peripheral circulation on the basis of oxygenation waveforms.
Conventional monitoring of neonatal hemodynamics was restricted to intermittent
evaluation of indirect clinical and laboratory indices of perfusion, such as peripheral-to-core
temperature difference, skin color, urine output, capillary refill time, acid-base balance, and
serum lactate levels. There are limited data available on capillary refill time in preterm
infants. In the first 24 hours, the use of a capillary refill time of 3 seconds had a 55%
sensitivity and 81% specificity for detecting low superior vena cava (SVC) flow (Osborn et
al., 2004). In addition, abnormalities in skin color, urine output, base excess, and serum
lactate often arise in other conditions of poor tissue oxygenation. For example, anemia can
cause skin color abnormalities; kidney disease can cause abnormal urine output;
dehydration and late metabolic acidosis can exacerbate BE and cause abnormal lactate
levels. Hence, these measurements are not specific to hypotension and must be assessed in
combination with other test findings.
4.3.2 Echocardiography
Echocardiographic examination may provide useful information regarding CO, contractility,
pulmonary hemodynamics, and PDA shunting in hypotensive preterm infants. Recently,

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functional echocardiography has been increasingly used to assess CO, myocardial function,
and organ blood flow in neonates requiring intensive care (Kluckow et al., 2007).
4.3.2.1 Systolic performance
Left ventricular systolic performance can be assessed by measuring the shortening factor
(SF) and ejection fraction. Normal neonatal values for the SF are 2840% (El-Khuffash and
McNamara, 2011). A normal neonatal value for the ejection fraction is approximately 55%
(Evans N and Kluckow, 1996).
4.3.2.2 Cardiac output
Normal left and right ventricular output ranges from 170 to 320 mL kg-1 min-1. Low left and
right ventricular output is defined as < 150 mL kg-1 min-1 (normal values range from 170 to
320 mL kg-1 min-1) (Evans N and Kluckow, 1996). Superior Vena Cava Flow (SVC flow) in
preterm infants is 50110 mL kg-1 min-1. Low SVC flow is defined as below 30 mL kg-1
min-1 at the first 5 hours post-natally or below 46 mL kg-1 min-1 at the first 48 hours
postnatally (Kluckow, 2005). Approximately 35% of preterm infants of < 30 weeks gestational
age encounter a period of SVC flow below 40 mL kg-1 min-1 during the first 12 hours
postnatally. After this point, SVC flow typically improves (Kluckow and Evans N, 2000).
4.3.2.3 Assessment of hypovolemia
The left ventricular end-diastolic diameter (LVEDD) is used to assess hypovolemia. LVEDD
is measured at the point of maximal ventricular filling. Normally, the mean LVEDD
increases from 11 mm at 2325 weeks, 12 mm at 2628 weeks, and 13 mm at 2931 weeks to
14 mm at 3233 weeks (Skelton et al., 1998). However, the utility of LVEDD as an indicator
of hypovolemia in infants has not been systematically examined. In addition to LVEDD,
other factors can affect left ventricular load in the transitional circulation (Evans N, 2003).
However, once a preterm infant has been diagnosed with hypovolemia, LVEDD is a useful
measurement for evaluation.
Thus, echocardiography is the most suitable test for evaluating cardiac activity and systemic
perfusion in hypotensive preterm infants. Its drawback is that it does not allow for
continuous observation. Additionally, there is no evidence that its use is associated with
better outcomes. Alternatively, ultrasound Doppler, which continuously monitors CO, has
also been used in neonates (Meyer et al., 2009).
4.3.3 Assessment of systemic and organ blood flow
Near-infrared spectroscopy (NIRS) measures hemoglobin flow and venous saturation in the
forearm to calculate oxygen delivery and consumption and fractional oxygen extraction. In a
previous study, Nagdyman et al. used NIRS to measure the cerebral tissue oxygenation
index (TOI), regional cerebral oxygenation index (rSO2), venous oxygen saturation SjO2,
and central SvO2 from the SVC. They found an association between cerebral TOI and SjO2,
between cerebral TOI and SvO2, between cerebral rSO2 and SjO2, and between rSO2 and
SvO2 (Nagdyman et al., 2008).
Peripheral and mucosal blood flow can be monitored using laser Doppler (Stark et al., 2009;
Ishiguro et al., 2011), side-stream dark field imaging (Hiedl et al., 2010), and visible light T-

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Sta (Van Bel et al., 2008) technologies. However, these devices have only been used in
neonates for research purposes.
4.3.4 Further assessment of hypotension in preterm infants
As previously described, the diagnosis, treatment determination, and outcome evaluation of
hypotension must be based on a combination of findings rather than a single marker. If
possible, a time-course observation can improve the prognosis of hypotensive neonates.
Soleymani et al. designed a system for hemodynamic monitoring and data collection in
neonates (Soleymani et al., 2010; Cavabvab et al., 2009). The system integrated conventional
technologies (i.e., continuous monitoring of heart rate, blood pressure, SpO2, and
transcutaneous CO2) with novel technologies, including impedance IEC for continuous
assessment of CO and stroke volume and NIRS to monitor blood flow distribution to the
brain, kidney, intestine, and/or muscle.
5. Treatment/ assessment of neonatal hemodynamics during postnatal
transition
The first priority in treating hypotensive preterm infants is to maintain hemodynamics
while the primary etiology is identied and its pathogenesis is addressed. Hemodynamic
therapy consists of 3 broad categories: uid resuscitation, vasopressor therapy, and
inotropic therapy.
5.1 Fluid bolus
There is no evidence from randomized trials to support the routine use of early volume
expansion in very preterm infants with hypotension. Fluid boli are useful in treating
hypovolemia caused by twin-to-twin transfusion, third-space losses, or hemorrhage.
However, circulating blood volumes are normal in most hypotensive infants, and there is
little to no response to volume administration (Bauer et al., 1993). Moreover, preterm infants
have immature cardiac contractile systems and vascular regulation; as such, volume
management through fluid boli is not always effective.
Goldberg et al. observed an increased incidence of IVH among preterm infants receiving
rapid volume expansion (Goldberg et al., 1980). Additionally, adverse neurological
outcomes have been reported in preterm infants receiving colloid infusions (Greenough et
al., 2002). The use of multiple uid boli is also associated with an increased mortality in
preterm infants (Ewer et al., 2003). Moreover, the administration of fluid boli has been
reported be ineffective for cardiopulmonary resuscitation in cases other than at birth
(Wyckoff et al., 2005).
There is insufficient evidence to determine the ideal type of volume expansion for preterm
infants or for early red cell transfusions. Normal saline is equally effective as albumin in
restoring blood pressure in hypotensive preterm infants. Normal saline is efficacious, safe,
readily available, and inexpensive; therefore, it has become the fluid of choice for volume
expansion (Oca et al., 2003). Furthermore, other crystalloids are costly and increase the risk
of infection and neurodevelopmental deficits (Greenough et al., 2002).

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5.2 Vasopressors and inotropes
5.2.1 Catecholamines
5.2.1.1 Mechanisms of action of catecholamines
The term catecholamines encompasses dopamine (DOA), NE (norepinephrine), and
epinephrine (E). Catecholamines are produced by adrenal medullary cells and by neurons,
specifically sympathetic postganglionic neurons. Indeed, adrenal medullary cells can be
considered a subtype of postganglionic sympathetic neurons. Secretion of catecholamines by
the adrenal medulla is regulated mainly by acetylcholine released from sympathetic nerve
endings.
5.2.1.2 Biosynthesis of catecholamines (Fig.4)
First, tyrosine is hydroxylated to form dihydroxyphenylalanine (DOPA) in the rate-limiting
step. DOPA is then converted into DOA through decarboxylation. DOA is packaged into
secretory granules (chromaffin granules). Dopamine--hydroxylase inside the granules
processes DOA to produce NE. In nerve cells, biosynthesis ends at this stage. In adrenal


Fig. 4. Catecholamine biosynthesis
Epinephrine
Phenyl
ethanolamine N-
methyltransferase
Norepinephrine
L-DOPA
L-tyrosine
Cell membrane
Dopamine
Tyrosine
hydroxylase
Aromatic-L-amino acid
decarboxylase
Glucocorticoid
Chromaffin
Granule
Dopamine--hydroxylase
Norepinephrine
Storage form
Dopamine

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medullary cells, NE continues to be processed into E. Once NE is released from the secretory
granules into the cytoplasm, it is processed by phenylethanolamine-N-methyltransferase
(PNMT) to form E. E binds to a protein known as chromogranin and is recaptured into
secretory granules, where it is stored (Goldstein et al., 2003).
The enzyme PNMT, which catalyzes the transformation of NE into E, is induced by
glucocorticoids. The direction of blood flow in the adrenal gland travels from the cortex
toward the medulla; as a result, medullary cells are in contact with the highest levels of
cortisol. Therefore, E production may be regulated by adrenocortical cells.
Acetylcholine is secreted from preganglionic neurons upon stimulation of a sympathetic
nerve. Acetylcholine acts at nicotinic receptors to depolarize chromaffin cells. This opens
voltage-gated Ca2+ channels, increasing intracellular Ca2+ concentration.This is believed to
result in the exocytosis of chromaffin granules.
5.2.1.3 Metabolism of catecholamines
E and NE secreted from the adrenal medulla are incorporated into various tissues and are
metabolized by the kidneys. Their half-life in the blood is approximately 2 minutes. They are
metabolized by 2 enzymes, catecholamine-O-methyltransferase (COMT) and monoamine
oxidase (MAO), which convert them into metanephrine, normetanephrine, and
vanillylmandelic acid. In addition to their actions on the heart and blood vessels,
catecholamines act on the respiratory tract, gastrointestinal tract, urinary tract, sensory
organs, skeletal muscles, adipose tissues, and pancreatic islets. With glucocorticoids,
catecholamines also inhibit the proliferation of Th1 cells and promotes their differentiation
into Th2 cells.
5.2.1.4 Adrenergic receptors
The physiological effects of catecholamines are elicited through receptors. The basic
structure of adrenergic receptors is a seven-transmembrane protein that binds to GTP-
binding proteins. There are 2 major types of adrenergic receptors, and , which are further
classified into subtypes.
There are 2 major -adrenergic receptor subtypes, 1 and 2, which are subdivided into
several pharmacological subtypes. 1 receptors are present at postsynaptic membranes;
their activation causes contraction of vascular smooth muscles. 2 receptors are present at
presynaptic membranes and inhibit the release of NE caused by sympathetic stimulation. 2
receptors are also present in other various cells, such as blood platelets, pancreatic -cells,
and adipocytes. 1 receptors activate phospholipase C by conjugating with Gq protein. 2
receptors act by inhibiting the production of cAMP through inhibitory GTP-binding
proteins (Gi).
-adrenergic receptors are divided into 3 subtypes: 1, 2, and 3. 1 receptors are mainly
distributed in the heart; 2 receptors are mainly distributed in blood vessels, bronchi, and
glomerulus, and 3 receptors are mainly distributed in adipocytes. Therefore, 1 receptors
promote cardiac stimulation; 2 receptors promote bronchodilation, vasodilation, and
glycogenolysis in muscles, and 3 receptors promote lipolysis. -adrenergic receptors
increase the production of cAMP through stimulatory GTP-binding proteins, Gs. This
activates cAMP-dependent protein kinase A.

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5.2.1.5 Action of catecholamines
Catecholamines act through and receptors. The catecholamines differ in their action at
versus receptors. For instance, while E acts on and receptors, NE acts mainly on
receptors (Fig. 5)
5.2.1.6 Cardiovascular effects of catecholamines
Through their actions at 1 receptors, catecholamines increase heart rate and cardiac
contractile force. In coronary arteries, when the -adrenergic effects of catecholamines
trigger vasoconstriction, there is a compensatory 2-receptor-mediated vasodilation. In
general, the vasodilatory effect predominates. Catecholamines also have vasoconstrictive -
adrenergic effects in arteries of the mucosa, kidney, spleen, and skeletal muscles and in
venous vasculature. The -adrenergic vasodilating effects of catecholamines include arterial
vasodilation due to 2-adrenergic receptors in skeletal muscle. Because of their differential
effects on adrenergic receptors, each catecholamine differently affects blood pressure and
blood ow (Fig.6).


NE: Nor epinephrine, E: Epinephrine, DOA :Dopamine, DOB:Dobtamine
Fig. 5. -and -Adrenergic receptors effects of vasoactive inotropes

NE: Nor epinephrine, E: Epinephrine, DOA :Dopamine, DOB:Dobutamine
Fig. 6. Effects of catecholamines on blood pressure and blood flow (partly modified from
Vincent, 2009)

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5.2.1.7 Downregulation of adrenergic receptors
Recently, it has been proposed that exogenous catecholamine administration downregulates
adrenergic receptors and their associated second-messenger systems (Hausdorff et al., 1999;
Collins et al., 1991). During receptor downregulation, adrenergic receptors undergo
lysosomal destruction; therefore, reversal of this process requires new protein synthesis.
5.2.1.8 Levels of catecholamines in hypotensive preterm infants
In extremely low birth-weight infants with hypotension, those in need of high doses of
dopamine (DOA>10g/kg/min) already had high levels of endogenous dopamine compared
to those needing low doses of dopamine (DOA <10g/kg/min) (p<0.05) (Ezaki et al., 2009b).
The ratio of conversion from NE to E before the use of dopamine and 24 hours after
administration were correlated in both infants who needed high doses of dopamine and in
those who did not. This suggested that there was successful conversion of NE to E. In infants
who did not need high doses of dopamine, there was a similar correlation between conversion
of DOA to NE before and 24 hours after administration of dopamine. However, no correlation
was found in infants who needed high doses of dopamine, suggesting that the conversion
from DOA to NE was limited (Ezaki et al., 2009b) (Fig.7). Therefore, an understanding of the
underlying pathological condition is important when administering catecholamines.

Fig. 7. Correlations between plasma levels of dopamine and norepinephrine at administration
(a) and 24 h later (b) and between norepinephrine and epinephrine at administration (c) and 24
h later (d). The severe hypotension (SH, DOA>10g/kg/min) group (n = 9) is represented by
open circles with dotted regression lines, and the mild hypotension (MH,
DOA<10g/kg/min) group (n = 13) is represented by closed circles with solid regression lines
Correlation coefficients and p-values are shown in the respective graphs.

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5.2.2 Dopamine
5.2.2.1 Treatment of dopamine in preterm hypotension
Dopamine is the most commonly used vasopressor/inotrope for the treatment of systemic
hypotension in preterm infants (Seli,1996). DOA stimulates -adrenergic receptors, -
adrenergic receptors, and dopaminergic receptors (See 5.2.1.4). DOA stimulates dopamine
receptors at low doses (0.5 g kg-1 min-1), mainly triggering effects in renal, mesenteric,
and coronary blood vessels. At doses of 24 g kg-1 min-1, DOA acts at -adrenergic
receptors, and at doses of 48 g kg-1 min-1, DOA acts at -adrenergic receptors (Seli,
2006).
With the exception of E administration, DOA administration is the most effective treatment
for elevating blood pressure in preterm infants. The increase in CBF following DOA
administration was found to be greater in hypotensive preterm infants compared to
normotensive preterm infants, suggesting the presence of pressure-passive CBF in
hypotensive neonates (Sassano et al., 2011). Therefore, we recommend the use of DOA as a
first-line inotrope for the treatment of hypotension in preterm infants.
DOA is also an important neurotransmitter that affects both cerebral vasculature and
neuronal activity. This is exemplified by pathological conditions caused by dopaminergic
dysfunction, including abnormalities in CBF and neuronal metabolism (Edvinsson and
Krause, 2002). In the mature brain, CBF is coupled to oxygen consumption (CMRO2). In
contrast, CBF coupling to metabolism is strikingly different in the brains of very preterm
infants, in which cerebral oxygen extraction, not CBF, sustains CMRO2. However, preterm
infants receiving DOA treatment exhibit flow-metabolism coupling similar to that of the
mature brain. This suggest a role for DOA in promoting flow-metabolism coupling in the
preterm brain (Wong et al., 2009). In addition, we previously reported that high-dose
administration of DOA can limit the conversion of NE to DOA (Ezaki et al., 2009b).
Therefore, extreme caution must be taken when administering high doses of DOA.
5.2.2.2 Adverse effects of dopamine treatment
2-adrenergic receptors are important in endocrine regulation; as such, even low doses of
systemically administered DOA have profound endocrine effects. For instance, DOA
infusion reduces thyroid stimulating hormone and thyroxine levels in very low birth-weight
infants (Filippi et al., 2004).
Doses of DOA should rarely exceed 20 g kg-1 min-1, because there is a risk of excessive
-adrenergic-receptor-mediated peripheral vasoconstriction and a subsequent reduction in
CO (Roz et al., 1993). DOA failed to raise blood pressure in more than 30% of preterm
infants with systemic hypotension (Pellicer et al., 2005).
5.2.3 Norepinephrine
5.2.3.1 The use of norepinephrine in the treatment of preterm hypotension
NE is a potent vasopressor with -and, to a lesser extent, -1 receptor agonist activity
(Hollenberg et al., 2004). In the adult, NE is primarily used as a vasopressor in states of
hyperdynamic shock, in which SVR is decreased and mean arterial blood pressure is low
(Corley, 2004). Experimental studies in fetal lambs have shown that NE may decrease basal

Evaluation and Treatment of Hypotension in Premature Infants

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pulmonary vascular tone (Houfflin-Debarge et al., 2001) and elevate pulmonary blood flow
through activating 2-adrenergic receptors and NO release (Magnenant et al., 2003).
NE can reduce damage incurred by neuroinflammatory and neurodegenerative conditions.
It induces the expression of the chemokine CCL2 in astrocytes, which is neuroprotective
against excitotoxic damage (Madrigal et al., 2009). Indeed, early associative somatosensory
conditioning requires NE (Landers and Sulliyan, 1999).
Thus, NE plays an important role not only in the cardiovascular system, but also in neonatal
development. However, there are few studies on the use of NE in the treatment of
hypotension in preterm infants. While no studies have compared NE to other drugs, its
therapeutic effects in neonates have recently been reported (Paradisis and Osborn, 2004).
The use of NE (0.5-0.75 g kg-1 min-1) is effective in the treatment of term and near-term
infants with septic shock that are resistant to DOA and dobutamine (Tourneux et al., 2008a).
In neonates with persistent pulmonary hypertension-induced cardiac dysfunction, NE can
reduce O2 requirements and normalize the systemic artery pressure (Tourneux et al., 2008b).
5.2.3.2 Adverse effects of norepinephrine treatment
In all previous reports describing the use of NE in neonates, NE was administered after
other inotropes, making it impossible to describe the side effects solely attributable to NE. In
addition, there are no reports on the long-term consequences of the use of NE in preterm
infants. In general, excessive peripheral vasoconstriction causes a decrease in the contractile
forces of the immature heart. This may result in tachycardia or decreased tissue perfusion.
Therefore, capillary refill time, lactate levels, and peripheral and organ blood flow should be
monitored.
5.2.4 Epinephrine
5.2.4.1 The use of epinephrine for the treatment of preterm hypotension
Low and moderate doses of E (0.1250.5 g kg-1 min-1) have found to be as effective as
low and moderate doses of DOA (2.510 g kg-1 min-1) for the treatment of hypotension
in preterm infants (Valverde et al., 2006). In addition, the infusion of E increases mean
arterial blood pressure and heart rate without decreasing urine output in very low birth-
weight infants with hypotension that do not respond to dopamine infusion up to 15 g kg-
1 min-1 (Heckmann et al., 2002).
5.2.4.2 Adverse effects of epinephrine
Compared DOA, E use cases temporary dysfunction of carbohydrate and lactate metabolism
(Valverde et al., 2006) and increased metabolic acidosis (Heckmann et al., 2002). E directly
affects lactate metabolism by increasing lactate production and decreasing lactate
metabolism, thus increasing serum lactate concentrations (Cheung et al., 1997). At very high
doses, E induces vasoconstriction sufficient to counteract its inotropic benets, and CO may
fall (Barrington et al., 1995).
Pellicer et al. recently reported that the long-term prognosis of E use was the same as DOA
use, and that both were safe (Pellicer et al., 2009). This important study provided an
additional treatment option for preterm hypotension.

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5.3 Non-catecholamine inotropic/pressor agents
5.3.1 Dobutamine
5.3.1.1 Physiology of dobutamine in preterm hypotension
Dobutamine is a racemic mixture of 2 isomers, the D-isomer with 1- and 2-adrenergic
effects and the L-isomer with 1- and 1- adrenergic effects. Dobutamine is predominantly
inotropic via stimulation of 1 receptors and has a variable effect on blood pressure
(Hollenberg, 2011). Dobutamine administration results in a variable decrease in total SVR.
Unlike DOA, dobutamine increases myocardial contractility exclusively through direct
stimulation of myocardial adrenergic receptors (Noori et al., 2004).
5.3.1.2 The use of dobutamine for the treatment of preterm hypotension
At a dose of 215 g kg-1 min-1, dobutamine increases CO mainly through augmenting
stroke volume (Noori et al., 2004; Roze et al., 1993; Bhatt-Mehta and Nahata, 1989).
5.3.1.3 Adverse effects of dobutamine treatment
Adverse effects of dobutamine occur at high doses and include increased heart rate. At very
high doses, dobutamine may increase blood pressure and SVR (Cheung et al., 1999), likely
due to stimulation of -receptors (Fig.5 and 6). One study suggested that dobutamines
potential benet of increased oxygen delivery to the tissues was offset by increased tissue
metabolic rate (Penny et al., 2001).
5.3.2 Vasopressin
5.3.2.1 Physiology of vasopressin in preterm infants
Vasopressin induces its physiological responses through 4 receptors, V1, V2, V3, and
oxytocin receptors (OTR) (Holmes et al., 2001). When vasopressin binds to V1 receptors in
vascular smooth muscle (Va1 receptors), it activates phospholipase C, triggering calcium
release from intracellular calcium stores (Fig. 2). This results in vasoconstriction and a
subsequent increase in blood pressure. Activation of V2 receptors in the stomach increases
intracellular cyclic AMP levels through the mediation of adenylate cyclase and have an anti-
diuretic effect. V3 receptors (also known as V1b receptors) are involved in vasopressins
adrenocorticotropic hormone (ACTH)-stimulating effects. Finally, OTR receptors mediate
vasopressings oxytocic effects on uterine contractility.
V2 receptors and OTR receptors also have vasodilating effects that are antagonistic to the
effects of V1 receptors. In addition, V1 receptors and OTR receptors have diuretic effects,
which are antagonistic to the anti-diuretic effects of V2 receptors. Vasopressins effects are
most adapted to disease-induced changes.
Previous reports have indicated that blood levels of endogenous vasopressin show a two-
phased response in adults with shock (Holmes et al., 2001; Landry et al., 1997; Morales et al.,
1999). During the initial phase of shock, endogenous vasopressin is released in large
amounts and reaches high blood levels in order to maintain tissue perfusion. However, its
concentration in the blood decreases over time. As such, vasopressin may be depleted due to
its initial release in large amounts. The release of vasopressin from the pituitary gland may
also be inhibited by NO produced by the vascular endothelium or due to autonomic
nervous system disorders (Holmes et al., 2001; Landry et.al, 1997; Morales et al., 1999).

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The effects of the small amounts of exogenous vasopressin may be a result of enhancing the
effects of catecholamines, inhibiting inducible NO synthase (iNOS), inhibiting increased
cGMP induced by NO and ANP, or inactivating KATP channels in vascular smooth muscles
(Fig.2) (Landry et al., 2001; Hamu et al.,1999).
In preterm infants, the levels of vasopressin were high during the first 24 hours following
birth (Ezaki et al., 2009b). The effects of these high levels of endogenous vasopressin on the
cardiovascular system are not fully understood.
5.3.2.2 The use of vasopressin for the treatment of hypotension in preterm infants
Meyer et al. reported that vasopressin (0.0350.36 U kg-1 hr-1) may be a promising rescue
therapy for catecholamine-resistant shock in extremely-low-birth-weight infants with acute
renal injury (Meyer et al., 2006). Similarly, Ikegami et al. found that administration of
vasopressin (0.0010.01 U kg-1 hr-1) was effective in extremely-low-birth-weight infants
resistant to treatment with catecholamines and steroids (Ikegami et al., 2010).
5.3 Adverse effects of vasopressin treatment
The side effects of vasporessin include severe cutaneous ischemia, hepatic necrosis,
neurological deficits, and dysmetria (Meyer et al., 2006; Rodrguez-Nunez et al., 2006;
Zeballos et al., 2006). Unlike DOA, E, and dobutamine, there are few reports on the side
effects of vasopressin. Moreover, it is unclear whether its side effects are dose-dependent
and what the long-term prognoses are. However, vasopressin is a pharmacological agent
that can be considered for use in patients in whom other drugs are ineffective.
5.4 Lusitropes
5.4.1 Physiology of phosphodiesterase-III inhibitors in preterm hypotension
Phosphodiesterase inhibitors increase intracellular cyclic AMP and thus have inotropic
effects independent of -adrenergic receptors. As such, they result in fewer chronotropic
and arrhythmogenic effects than catecholamines. However, increased cyclic AMP in
vascular smooth muscle cells can cause vasodilation, thus reducing SVR, which can
exacerbate hypotension. In addition, this can reduce pulmonary artery pressure. (Chen et
al.,1997,1998; Kato et al.,1998). Milrinone, a cyclic nucleotide phosphodiesterase-III inhibitor,
improves contractility and reduces afterload in adults and newborns with cardiac
dysfunction.
5.4.2 The use of phosphodiesterase-III inhibitors for the treatment of preterm
hypotension
McNamara et al. reported that intravenous Milrinone (0.330.99 g kg-1 min-1)
administration produced early improvements in oxygenation without compromising
systemic blood pressure in patients with severe persistent pulmonary hypertension
(McNamara et al., 2006). One randomized clinical trial did not support the use of Milrinone
(0.75 g kg-1 min-1 for 3 hrs, then 0.2 g kg-1 min-1 until 18 hours after birth) in the
prevention of low SVC in the early transitional circulation of preterm infants (Paradisis et
al., 2009).

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5.4.3 Adverse effects of Milrinone treatment
Milrinone can cause hypotension and tachycardia (Chang et al., 1995). The long-term effects
of Milrinone in preterm infants have not been reported.
5.5 Corticosteroids
5.5.1 Physiology of corticosteroids
The adrenal glands are involved in the growth and maturation of fetal organs during
intrauterine life. In most mammals, a cortisol surge occurs as the full gestational term
approaches; this triggers increased synthesis of pulmonary surfactant, reduced sensitivity of
the arteries to prostaglandins, and increased conversion of pancreatic -cells from T4 to T3
in the mature liver. These changes allow the fetus to survive in the extrauterine
environment. There is also a surge in catecholamines produced by the adrenal medulla
during delivery. This surge also allows adaptation to the extrauterine environment by
influencing the cardiovascular system, including elevating the blood pressure and
increasing the heart function, and by influencing glucose metabolism, fat metabolism, and
water absorption in the lungs (Fisher, 2002).
The hypothalamic-pituitary-adrenal system in fetuses and neonates has been implicated in
late-onset circulatory collapse (Masumoto et al., 2008) and in the fetal programming of the
cardiovascular system. Preterm infants have low adrenal function due to their low levels of
3-hydroxysteroid dehydrogenase (HSD) (Mesiano and Jaffe, 1997) and weak 11b-HSD2
activity (Donaldson et al.,1991).
Corticosteroids reverse neonatal hypotension by improving capillary-leak syndrome
(Briegel et al., 1994), potentiating transmembrane calcium currents, increasing -receptor
sensitivity to catecholamines, reversing the downregulation of -receptors, increasing the
density of -receptors, and inhibiting NO synthase expression (Prigent et al., 2004).
5.5.2 The use of corticosteroids in the treatment of preterm hypotension
Hydrocortisone administration is effective in the treatment of hypotension and vasopressor
dependence in hypotensive preterm infants. Its clinical benets include increasing blood
pressure and decreasing the requirement for vasopressor administration (Higgins et al., 2010).
Fernandez et al. have reviewed the use of hydrocortisone in the treatment of premature infants
(Fernandez and Watterberg, 2009). Before initiating therapy with hydrocortisone in extremely
preterm infants with refractory hypotension, a blood specimen should be analyzed for cortisol
concentration. Pending that result, an initial dose of 1 mg/kg can be administered. If the blood
pressure improves within 2 to 6 h, 0.5 mg/kg can be administered every 12 h (approximately
810 mg/m
2
per day). This long dosing interval is used, because hydrocortisone has a longer
half-life in immature infants (Watterberg et al., 2005). This dosing strategy increases serum
values by an average of 5 g/100 ml; higher doses are associated with very high serum
concentrations. If the initial cortisol concentration is high (>1520 g/100 ml), drug
administration may be discontinued, especially in the absence of a clinical response.
5.5.3 Adverse effects of corticosteroid treatment
Although corticosteroid therapy improves blood pressure and circulation, there are many
potential complications, including spontaneous gut perforation, hyperglycemia, and

Evaluation and Treatment of Hypotension in Premature Infants

437
hypertension and long-term consequences, including cerebral palsy and intellectual
impairment. These complications necessitate the judicious use of corticosteroids to support
blood pressure in preterm infants (Yeh et al., 2004).
Hydrocortisone therapy administered simultaneously with indomethacin or ibuprofen has
been associated with acute spontaneous gastrointestinal perforation in extremely preterm
infants. Therefore, care should be taken to avoid concurrent therapy (Watterberg et al., 2004;
Peltoniemi et al., 2005). Infants who develop spontaneous perforation often have high
endogenous cortisol concentrations (Watterberg et al., 2004; Peltoniemi et al., 2005).
Watterberg et al. reported that early, low-dose hydrocortisone treatment was not associated
with an increased risk of cerebral palsy. In fact, infants treated with hydrocortisone
displayed improved developmental outcomes. Together with the short-term benefits, these
data support the use of hydrocortisone for the treatment of adrenal insufficiency in
extremely premature infants (Watterberg et al., 2007).
6. Conclusion
The major findings of the present chapter summarized in the following figure (Fig.8).

Fig. 8. Evaluation and treatment of hypotension in premature infants

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7. Acknowledgment
We express gratitude to the efforts of the authors whose research is cited in this article. First
author (FA)s personal research on vasoactive factors in neonates was inspired by my wife, a
physician who introduced me to the use of vasopressin for the management of shock.
Therefore, FA offer my wife, Yuko Ezaki, my most sincere gratitude. Finally, FA dedicate
this chapter to my famiiy: Munenori, Tomi, Yuko, Yoshiko, Yukiko, and Saeka Ezaki.
FA hope that this work will promote future advances in neonatal care.
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21
Role of Echocardiography in Research into
Neglected Cardiovascular Diseases
in Sub-Saharan Africa
Ana Olga Mocumbi
National Health Institute & University Eduardo Mondlane,
Mozambique
1. Introduction
Echocardiography is a non-invasive imaging technique that has been important in
improving the quality and reliability of cardiovascular diagnosis, but access to it remains
limited in most developing countries in Africa due to the costs of the technique and the lack
of highly specialized personnel to perform it. Training in echocardiography is part of the
postgraduate residency training requirements in cardiology in most African countries,
despite the absence of an accreditation process such as that designed in Europe and United
States of America (Ogah et al., 2006). While the use of transthoracic echocardiography has
been spreading slowly around the continent, transesophageal echocardiography is still
limited to few centers.
Barriers to obtaining ultrasound services in Sub-Saharan Africa include distance, time, cost
of transfers and ultrasound charges (Shah et al., 2008). However, compared to other
diagnostic imaging modalities echocardiography is safe, portable and inexpensive, uses
simple power supply, and requires minimal maintenance. These characteristics make it the
most suitable imaging technique for low-resource areas of Sub-Saharan Africa, where the
introduction of smaller and battery-powered ultrasound machines is being used to reach out
for people living in remote areas that traditionally did not have access to specialized
cardiovascular diagnosis and care.
While witnessing an increasing awareness of the epidemic of cardiovascular disease,
encompassing conditions such as hypertension, acute coronary syndrome, stroke and
chronic heart failure, Sub-Saharan Africa still has a high burden of several infectious-related
cardiovascular diseases and specific conditions such as cardiomyopathies. These neglected
cardiovascular diseases include amongst others rheumatic heart disease (RHD) and
endomyocardial fibrosis (EMF), both representing a considerable source of burden to the
communities and playing a major role in determining premature mortality around the
continent. Having recognized the potential of echocardiography as a research tool, African
scientists have been using this technique to describe the epidemiology and profile of
neglected cardiovascular conditions, as well as to bring new insights into the main causes of
heart failure in both pediatric and adult populations (Mocumbi et al., 2008; Sani et al., 2007;
Jaiyesimi & Antia, 1981a, b; Marijon et al., 2007; Adesanya 1979).

The Cardiovascular System Physiology, Diagnostics and Clinical Implications 446
RHD and EMF have been the subject of community- and hospital-based research using
echocardiography. This has resulted in an increase in the number of publications from
Africa in indexed medical journals during the last decade. However, the increase is far from
the desirable as the number of epidemiological and clinical studies using echocardiography
augmented from 6 to 15 for RHD and from 2 to 4 for EMF (Figure 1).
In this chapter we review the recent use of transthoracic echocardiography worldwide for
advancing knowledge about the pathogenesis and natural history of RHD and EMF,
focusing on the modalities most readily available in low-resource settings, namely
bidimensional, M-mode, pulsed and continuous Doppler. Finally, we discuss the specific
role of echocardiography in fostering research into these two endemic diseases in Africa,
and present the current challenges and opportunities of the use of this technique in Sub-
Saharan Africa.

Fig. 1. Number of publications in indexed journals that reported hospital or community-
based epidemiological and clinical studies using echocardiographic diagnosis for Rheumatic
Heart Disease (RHD) and Endomyocardial Fibrosis (EMF) in the last two decades in Africa.
2. Endomyocardial fibrosis
EMF is a restrictive cardiomyopathy of unknown etiology characterised by progressive
fibrous thickening of the ventricular endocardium, leading to restrictive physiology
associated with atrioventricular valve dysfunction. Most of our knowledge of this condition
comes from hospital-based studies in endemic areas of Uganda, Cote dIvoire, Nigeria, India
and Brazil. Data on its exact epidemiology are scarce, but variations in geographical and
ethnic distribution have been reported, stimulating the search for both environmental
factors and genetic factors.
EMF is thought to be the commonest restrictive cardiomyopathy worldwide (Somers, 1990),
affecting mainly children and adolescents of low-income communities from tropical regions
of Africa, Asia and South America. Established and advanced disease can be easily
diagnosed by clinical examination in endemic areas, but the finding of relatively
asymptomatic individuals who present important echocardiographic abnormalities is not
rare (Mocumbi et al., 2008; Salemi et al., 2005). The characterisation of early stages of the
0
2
4
6
8
10
12
14
16
1991-2000 2001-2010
RHD
EMF
Role of Echocardiography in Research into
Neglected Cardiovascular Diseases in Sub-Saharan Africa 447
disease has not been systematically done, leading to major gaps in our knowledge of its
pathogenesis and natural history.
The use of echocardiography for diagnosis of EMF, started almost half a century ago, has
contributed to characterization of the disease and better understanding of its
pathophysiology, resulting in improvements in management and prognosis. Several authors
from different parts of the world have described the clinical and echocardiographic findings
in EMF (Acquatella et al., 1979; Gonzalez-Lavin et al., 1982; Vijayaraghavan et al., 1983;
Okereke et al., 1991; Rashwan et al.,1995), and more recently there have been attempts to use
this technique for understanding its epidemiology (Mocumbi et al., 2008) as well define
prognostic criteria prior to surgery (Mady et al., 2004).
2.1 Echocardiographic features
The hallmark of established EMF is the presence of thickened endocardium, ventricular
obliteration and dilated atria. The typical image of restrictive cardiomyopathy is that of
inversion of the size of heart cavities with small obliterated ventricles and dilated atria
(Hassan et al., 2005; Berensztein et al., 2000). The wide spectrum of distribution and severity
of the fibrotic lesions, as well as the changes in heart shape and distortion mandate a careful
and comprehensive echocardiographic evaluation of each patient, using the usual and less
conventional views.
The most characteristic echocardiographic features of EMF are large endocardial plaques,
patchy endocardial thickening, obliteration of ventricular apices or valve recesses,
ventricular and atrial thrombi, ventricular cavity volume reduction, enlarged atrium,
restricted mobility of the atrioventricular valve leaflets, fusion of the papillary muscles to
the wall and abnormalities of the ventricular regional wall motion (Okereke et al., 1991;
Mady et al., 2005; Hassan et al., 2005; Berensztein et al., 2000). Less specific
echocardiographic abnormalities include diffuse atrioventricular valve leaflet thickening,
enhanced echodensity of the moderator band or trabeculae, abnormal movement of the
interventricular septum and/or posterior LV wall, and presence of thickened left ventricular
false tendon (Mocumbi et al., 2008). Moderate to massive pericardial effusion is a frequent
finding in both left and right forms of EMF (George et al., 1982; Lowenthal & Teeger, 2000).
Occasionally, endocardial calcification may be seen in the ventricles (Lowenthal & Teeger,
2000; Morrone et al., 1996; Trigo et al., 2010).
The pattern of distribution of the morphological and hemodynamic abnormalities allows the
classification of EMF in different forms according to exclusive or predominant distribution
of structural lesions in one or both sides of the heart. Hence the description of right, left and
bilateral EMF.
2.1.1 Endocardial thickening
Thickening of the endocardium is the most characteristic feature of established EMF
(Ojereke et al., 1991; Connor et al., 1967). It may consist of large plaques affecting one or
both ventricles, as well as patchy endocardial thickening evenly distributed in the
ventricular walls or affecting exclusively the interventricular septum. These abnormalities
can be assessed by both bidimensional and M-mode. The most striking and constant

The Cardiovascular System Physiology, Diagnostics and Clinical Implications 448
features are increased amplitude echos at the right ventricular trabecular region, left
ventricular apex and the region of the posterior mitral valve leaflet (Vijayaraghavan et al.,
1983).
2.1.2 Ventricular thrombosis
Spontaneous contrast and ventricular thrombi are frequently seen in normally contracting
ventricles in early stages of EMF, as part of the initial process that leads to endocardial
fibrosis (Berensztein et al., 2000). The presence of ventricular thrombi, calcified or not, is a
major determinant of management and prognosis.
2.1.3 Ventricular obliteration
This characteristic abnormality of EMF consists in partial or complete exclusion of a portion
of the ventricle from the circulation (Figure 2). In right EMF the trabecular portion of the
ventricle is separated from the remaining cavity by a large fibrotic endocardial plaque,
underneath which there is myocardium of apparently normal texture (Trigo et al., 2010).
Left ventricular obliteration affects both the apex and the recesses of the posterior mitral
valve leaflet excluding these parts from the ventricular cavity (Berensztein et al., 2000). It is
thought that obliteration by thrombi and subsequent scarring fibrosis are the mechanisms
involved (Connor et al., 1967), both leading to reduction of the diastolic properties of the
ventricles. Also, thrombi may involve the sub-valvar apparatus, leading to scarring and
fusion of leaflets to the ventricular wall, therefore resulting in leaflet movement restriction
and severe atrioventricular valve dysfunction.

Fig. 2. Left-sided EMF with obliteration and endocardial thickening at the ventricular apex.
The atypical mitral regurgitation jet is frequent in moderate disease.
With progression of the disease to more advanced stages cavity retraction occurs with
further reduction of the effective ventricular cavity volume, seemingly due to progressive
organization and fibrosis of the mural thrombi and adjacent endocardium. Particularly in
the right ventricle, this process is associated with pulling of the wall by the retracted
tricuspid valve apparatus, resulting in the distinctive finding of advanced right-sided EMF
called apical notch (Figure 3). The apical notch gives the heart a shape that resembles the
map of Africa, hence the designation Heart of Africa (Davies, 1960). On the left side the
Role of Echocardiography in Research into
Neglected Cardiovascular Diseases in Sub-Saharan Africa 449
ventricular apex is never retracted; it becomes thicker leading to considerable reduction of
the longitudinal diameter of the ventricle, resulting in a spherical ventricular shape.

Fig. 3. Transthoracic image obtained during field research using a portable ultrasound
machine showing retraction of the trabecular portion of the right ventricle with reduction of
cavity size and aneurysmal right atrium in which a thrombus can be seen.
2.1.4 Diffuse leaflet thickening
Diffuse thickening of the atrioventricular valve leaflets occurs in some patients with EMF.
This pattern helps differentiating left-sided EMF with predominant valvular lesion from
chronic rheumatic disease of the mitral valve in endemic areas for both diseases. In chronic
rheumatic mitral regurgitation leaflet thickening is usually restricted to or exaggerated at
the tip of the valve, extends to the chordae, and is never associated to obliteration of the
contralateral ventricle (Saraiva et al., 1999; Metras et al., 1983).
2.1.5 Septal motion abnormalities
The restricted movement of the fibrotic left ventricular apex and its obliteration are
accompanied by compensatory contractile mechanism that results in exaggerated and
distinctive motion of the basal portion of the left ventricle, the so-called Merlon sign
(Vijayaraghavan et al., 1983; Berensztein et al., 2000). On M-mode the interventricular
septum has a rapid anterior movement in early diastole (Acquatella et al., 1979) assuming an
M-shaped movement. In some patients the septal motion may be reversed (paradoxical
septal movement).
2.1.6 Restrictive filling pattern
A tall E wave with E/A ratio greater than 2, deceleration time less than 120ms and
isovolumic relaxation time inferior to 160ms are the criteria used to define the presence of
ventricular restrictive filling pattern. This evaluation is usually compromised by the
presence of severe mitral regurgitation. The brisk early diastolic filling with poor filling in
the remainder of diastole, the absence of respiratory changes, the presence of normal

The Cardiovascular System Physiology, Diagnostics and Clinical Implications 450
pericardium and the usual association to pericardial effusion, enable distinction from
constrictive pericarditis.
2.1.7 Atrioventricular valve regurgitation
Mild mitral regurgitation is found in initial stages of left EMF. The jet is atypical and seems
to start inside the ventricular cavity (figure 2). In severe left EMF thickening and scarring of
the valve leaflets and the mitral valve apparatus lead to severe mitral regurgitation that is
usually eccentric, due mainly to restricted movement of the posterior leaflet. The
regurgitation has a high velocity jet directed to the posterior wall of the left atrium, reaching
the pulmonary veins in most cases (Figure 4).
The tricuspid valve apparatus is distorted in EMF with restricted movement of the leaflets in
early phases of the disease. In severe right EMF there is massive tricuspid annulus dilatation
and non-turbulent low velocity regurgitant jet, witnessing the absence of pressure gradient
between the two right cavities. In these cases the right filling pressures are very high,
leading to severe dilatation of the cava system and reflux from the right atrium towards the
supra-hepatic veins, a phenomenon easily accessed using pulsed and color Doppler.
2.1.8 Atrial dilatation
Both the restriction to ventricular filling and the atrioventricular valve regurgitation result
in increase in atrial pressure, leading to progressive atrial dilatation. The consequence is
further increase in atrioventricular valve annulus dilatation perpetuating the cycle and
being responsible for the frequent finding of aneurysmal atria (Hassan et al., 2005;
Berensztein et al., 2000). Annular dilatation, leaflet retraction and fibrosis of the sub-valvar
apparatus lead to non-coaptation and free tricuspid valve regurgitation (Okereke et al.,
1991), this later seen as a non-turbulent low velocity jet on color Doppler.

Fig. 4. Mitral regurgitation and left atrial dilatation on a patient with left EMF evaluated
using portable ultrasound machine during a community-based study.
2.1.9 Semilunar valve abnormalities
The pulmonary valve is usually spared from structural abnormalities but there is often
pulmonary regurgitation that allows estimation of the mean and diastolic pulmonary
Role of Echocardiography in Research into
Neglected Cardiovascular Diseases in Sub-Saharan Africa 451
pressures. In severe cases due to the lack of pressure gradient between the atrium, the
ventricle and the pulmonary artery, there is often diastolic opening of the pulmonary valve.
The aortic valve is almost always normal, but in few cases there may be thickening of the
cusps.
2.1.10 Abnormalities of the left side of the heart
Early left-sided EMF is characterized by thickening of the mitral leaflets, presence of apical
thrombus, and/or obliteration of the apex or the recess between the posterior leaflet and the
posterior wall. Thrombi may be found in the sub-valvar apparatus involving the free edges
of both papillary muscles or in the apex. There is moderate left atrial dilatation but the valve
remains non-regurgitant. The flow across the mitral valve reveals early diastolic filling
followed by restriction pattern.
In the established left-sided EMF endocardial thickening is prominent in interventricular
septum, the apex and posterior wall behind the recess of the posterior mitral leaflet, the
ventricular cavity assumes a spherical shape and there is increased contractility at its basal
portion. The left ventricular ejection fraction is usually not calculated due to the presence of
mitral regurgitation and left ventricular distortion. The heart distortion and change in the
position of the heart in the chest explains the fact that contractility is often graded using a
visual scale. In patients without severe distortion of the left ventricular shape and no mitral
regurgitation, the LV end-systolic and end-diastolic volumes and ejection fraction can be
determined from the apical 4-chamber view according to the modified Simpsons rule or the
Teicholz method (Feigenbaum, 1994).
Although in rare patients the mitral valve may be stenotic, most patients present an
eccentric mitral regurgitation with signs of passive pulmonary hypertension. The left atrium
maximal linear dimensions at the end of left ventricular systole are increased in all plans
and, in severe cases the cavity may be aneurysmal. However, there is rarely left atrial
thrombus.
Regarding the mitral valve there is leaflet thickening and shortage, leading to non-
coaptation and severe mitral regurgitation. The posterior mitral valve leaflet appears to be
tethered down to the left ventricular posterior wall, with reduced mobility during diastole.
In severe cases the leaflet, its chordae and papillary muscle are completely adherent to the
wall leading to massive regurgitation.
2.1.11 Abnormalities of the right side of the heart
The initial lesions on the right side consist of thickening of the moderator band. In the
longitudinal view of the right ventricle and short axis of the left ventricle at the level of the
aorta a stretched moderator band is seen, while in 4 chambers-view the ventricular cavity is
separated into two cameras. There may be thickening of the tricuspid leaflets and the
analysis of the tricuspid inflow by pulsed Doppler reveals abnormal compliance.
Right ventricular trabecular cavity obliteration is thought to start by separation of the
trabecular chamber of the right ventricle from the rest of the cavity, as seen in 4-chambers
view (Figure 5). It is usually accompanied by mild to moderate tricuspid regurgitation
caused by restriction to the movement of the anterior and septal leaflets of the tricuspid

The Cardiovascular System Physiology, Diagnostics and Clinical Implications 452
valve. The leaflets may present attachments to the wall leading to an echocardiographic
picture that may mimic Ebstein Malformation (Vaidyanathan et al., 2009), namely with
dilatation of the tricuspid annulus, tricuspid regurgitation with jet originating from the level
of non-cooptation of the leaflets, which is dislocated to the trabecular portion of the
ventricle. The right ventricular systolic function, evaluated through a visual semi-
quantitative scale using two-dimensional guided M-mode in several incidences (four-
chambers, parasternal long axis, parasternal short axis and sub-costal views), is globally
normal, but may be reduced when there are large endocardial plaques and cavity retraction.
Advanced right EMF is defined by retraction of the ventricular cavity due to elimination of
the trabecular portion of the cavity, resulting in the pathognomonic finding of an apical
notch. The right ventricular outflow tract is dilated and hyperdynamic to compensate the
loss of the trabecular portion, and the interventricular septal motion may be reversed.

Fig. 5. Right EMF seen in 4-chambers view showing separation of the cavity in two portions,
a feature that is characteristic prior to complete obliteration of the trabecular cavity.
Severe tricuspid regurgitation with no turbulence is characteristically associated to
restriction of leaflet movements caused by involvement of the papillary muscles in the
fibrotic process and to dilatation of the annulus that results from severe right atrial
dilatation. At this stage most patients have spontaneous contrast inside the right atrium
extending to the inflow tract of RV and also to the inferior vena cava and dilated supra-
hepatic veins. Multiple thrombi may be found some moving freely and others attached to
the atrial wall. The dilated inferior vena cava and supra-hepatic veins, usually with dynamic
echos indicating stasis, do not show the normal respiratory changes, indicating increased
systemic venous pressure. Pericardial, pleural and peritoneal effusions are also frequently
present in patients in heart failure, best seen in subcostal view.
The colour Doppler is used for semi-quantitative estimation of tricuspid regurgitation
severity, taking into account the width and depth of regurgitant jet inside the atrium seen
from different views (four-chambers, short-axis and sub-costal). One criteria used to define
severe tricuspid regurgitation is the lack of aliasing of the jet and its large width at origin,
especially when there is non-coaptation of the tricuspid valve leaflets. The aneurismal right
atrium results in heart distortion and compression of the left cavities making it difficult to
Role of Echocardiography in Research into
Neglected Cardiovascular Diseases in Sub-Saharan Africa 453
evaluate the presence of mitral dysfunction. Abundant pericardial effusion and compression
of left cavities compromise an adequate evaluation of the left ventricular function.
The lateral and supero-inferior dimensions of the right atrium are always increased and an
aneurysmal atrium is usually found. The high pressure inside the atrial cavity pushes the
interatrial septum towards the left side opening the foramen ovale in many occasions, and
allowing a certain degree of right to-left shunt that causes mild cyanosis. Compression of
the left cavities by the severely dilated atrium and tense right ventricle at the level of the
admission chamber may impede adequate ventricular filling as well as mask mitral
regurgitation. On M-mode these findings are associated with interventricular paradoxical
septal motion and small left ventricular cavity.
2.2 Pathological correlation
Surgery can be used to assess the accuracy of transthoracic echocardiography in
determining the severity of EMF. This has been achieved by performing standardized
transthoracic echocardiography on EMF patients prior to surgery, followed by detailed
intra-operative examination of the abnormalities and histopathological evaluation of tissue
obtained from excised biopsies (Mocumbi et al., 2010). In this series of patients from
Mozambique the echocardiographic description coincided with the intraoperative findings
in more than 80% of patients, the concordance being absolute for the most important
pathological lesions of EMF, namely fusion of the posterior papillary muscle and leaflet to
the wall, left ventricular apical fibrosis, thickening of the atrioventricular leaflets, right
ventricular obliteration, right ventricular retraction and ventricular thrombi. This suggested
that transthoracic echocardiography can be used in isolation for diagnosis and surgical
management of chronic EMF in low-resource endemic areas.
2.3 Challenges and opportunities
Echocardiography can make a confident non-invasive diagnosis of EMF (Vijayaraghavan et
al., 1983; Mocumbi et al., 2010), has been useful in determining patients who can benefit
from surgery and allows evaluation of the response to treatment. Access to hand-carried
echocardiography battery-operated systems has allowed for the first time the design and
implementation of epidemiological research in a remote area in Mozambique. In this
community, known to have a high attack rate of the disease from previous hospital-based
data (Ferreira et al., 2002), 1063 individuals of all ages were randomly selected and
submitted to transthoracic echocardiography using a standardized protocol (Mocumbi et al.,
2008). A prevalence of 19.8% was found, with the majority of the individuals being
asymptomatic and having mild or moderate disease.
For such disease with so many gaps in knowledge there is need to build regional or
continental registries starting with phenotypic characterization of individuals in early stages
of EMF through echocardiography, using standardized criteria that can be validated on
follow-up studies in several endemic areas. This may contribute to uncover aspects related
to its natural history, and constitute cohorts to test differences in genetic and to biological
profile between healthy individuals and those affected by the disease in endemic areas.
Follow-up of individuals with well-established echocardiographic phenotype may also be
important to identify predictors of outcome using different disease management strategies.

The Cardiovascular System Physiology, Diagnostics and Clinical Implications 454
3. Rheumatic heart disease
RHD is the most important form of acquired cardiovascular disease in children and
adolescents in Africa. It is the only chronic sequelae of rheumatic fever (RF), a systemic
disease that results from group A streptococcal infections.
Rheumatic Heart Disease (RHD) is still a major concern in Africa (World Health
Organization, 2007) despite the dramatic declines in the incidence and prevalence of this
condition that have occurred over the last 150 years in the developed world (Gordis, 1985).
It is a disease traditionally associated with poverty and overcrowding, and this decline was
achieved through improvement in living conditions and widespread use of penicillin for the
treatment of streptococcal pharyngitis. The unacceptably high rates of RF/RHD in Sub-
Saharan Africa lead to considerable use of health-care resources and a major impact on the
patients, their families and the society as a whole.
Although RHD is still a neglected disease, there has been a new surge on research on this
condition. This has been centered in developing countries and those populations within
middle- and high-income countries where high burdens of disease still exist.
Echocardiography is considered the adequate tool for identifying early stages of heart valve
disease (Carapetis & Zuhlke, 2011).
3.1 Echocardiographic diagnosis
Echocardiography is an essential tool in diagnosis and management of RF and RHD. Several
structural and hemodynamic abnormalities are important for classifying valve lesions, both
in the acute and chronic phases of the disease. Even before the advent of colour Doppler
flow imaging several studies had already highlighted the utility of echocardiography for the
diagnosis of rheumatic carditis, and emphasized its value in defining the mechanisms of
valve disease and heart failure associated with severe attacks of carditis (Vansan et al., 1996;
Narula et al., 1999). Colour flow Doppler imaging was then considered a useful method of
identifying subclinical mitral and aortic valvar disease at all stages of rheumatic fever when
carditis cannot be otherwise detected (Folger et al., 1992). Regarding chronic rheumatic heart
disease, echocardiography may be used to track the progression of valve abnormalities and
to help determine the time for surgical intervention.
3.1.1 Acute carditis
In acute rheumatic disease Doppler-echocardiography identifies and quantifies valve
abnormalities, ventricular dysfunction and pericardial effusion (Narula et al., 1999; Folger et
al., 1992). The valve most commonly affected is the mitral, followed by the aortic valve
(Folger et al., 1992). In the African context, severe pure rheumatic mitral regurgitation is as
prevalent as pure stenosis but has an entirely different time course, surgical anatomy, and
relation to disease activity, suggesting a separate pathophysiologic mechanism (Marcus et
al., 1994).
The usual features of acute rheumatic valvulitis are annular dilatation, elongation of the
chordae to the anterior leaflet, and postero-laterally directed mitral regurgitation jet (Vansan
et al., 1996; Narula et al., 1999; Folger et al., 1992). Nodular thickening of valve leaflets also
occurs (Vansan et al., 1996), and may represent echocardiographic equivalents of rheumatic
Role of Echocardiography in Research into
Neglected Cardiovascular Diseases in Sub-Saharan Africa 455
verrucae seen universally at autopsy in patients who died of acute rheumatic fever
(Baggenstoos & Titus., 1968) and noted macroscopically at surgery in a substantial
proportion of patients subjected to valve surgery during the acute phase (Kinsley et al.,
1981). When acute carditis courses with chordal thickening (Vijayalakshmi et al., 2008), it
suggests acute rheumatic fever recurrence in patients with established rheumatic heart valve
disease. Mild mitral regurgitation present during the acute phase usually resolves weeks to
months after. In contrast, patients with moderate-to-severe carditis have persistent mitral
and/or aortic regurgitation.
Valve insufficiency due to endocarditis, rather than myocardial dysfunction caused by
myocarditis, is the dominant cause of heart failure in acute rheumatic fever, related to
ventricular dilatation and/or restriction of leaflet mobility (Vansan et al., 1996). This has
been supported by demonstration of the absence of cTnI elevations during rheumatic fever
(Kamblock et al., 2003; Essop et al., 1993). The left ventricle is dilated with preserved or
increased fractional shortening in most cases, but variable degree of ventricular dysfunction
is not rare in the African setting probably due to the high prevalence of predisposing factors
such as anemia.
3.1.2 Chronic rheumatic heart disease
Isolated mitral regurgitation or combined mitral and aortic regurgitation are the most
common abnormalities found in chronic RHD (Vansan et al., 1996; Folger et al., 1992;
Marcus et al., 1994). Several morphological abnormalities have been considered features of
chronic mitral RHD namely (a) valve and/or chordal thickening; (b) restrictive leaflet
motion due to chordal thickening, shortening or fusion, commissural fusion and leaflet
calcification or thickening; and (c) chordal elongation, rupture or prolapse (Marijon et al.,
2007; Paar et al., 2010; Namboodiri et al., 2009; Wilkins et al., 1988). In mitral regurgitation
the posterior mitral leaflet is shortened and immobile because its submitral complex is also
thickened, fused and shortened, resulting in a gap or non-coaptation of the two leaflets in
many patients (Okubo et al., 1984). Mitral stenosis occurs when the leaflets of the affected
valves become diffusely thickened, with fusion of the commissures and chordae tendineae,
as well as increased echodensity of the mitral valve that may signify calcification. However,
valvular calcification is rare in juvenile rheumatic heart disease, frequently seen in Africa
(Yuko-Jowi et al., 2005). Left atrial thrombus is a common finding in mitral stenosis.
There are few studies of characterization of aortic valve abnormalities in rheumatic heart
disease. Rheumatic aortic valve disease is usually diagnosed in combination with mitral
disease, and after exclusion of congenital disease, mainly bicuspid aortic valve.
Echocardiographic diagnosis has been based on morphological changes such as the presence
of thickened leaflets, rolled leaflet edges, coaptation defect, deformed leaflets, commissural
fusion, leaflet retraction, abnormal leaflet mobility, systolic doming of leaflet,
hyperechogenicity of leaflet edges and prolapse are used (Marijon et al., 2007; Paar et al.,
2010). For community studies a more accurate case-definition and assessment of severity is
needed since follow up of patients with RHD shows that those with no or mild aortic valve
disease at the time of mitral valve intervention rarely develop severe aortic valve disease,
and seldom require aortic valve surgery over the long-term follow up, while the presence of
mild aortic stenosis at baseline is predictive of relatively more rapid progression in the
minority of cases (Namboodiri et al., 2009).

The Cardiovascular System Physiology, Diagnostics and Clinical Implications 456
Two-dimensional echocardiographic criteria of organic rheumatic tricuspid valve disease
include thickened leaflets with restriction in motion, diastolic doming, and encroachment of
the leaflet tips on the wall of the ventricular inlet (Guyer et al., 1984; Meira et al., 2006). Since
pulmonary hypertension is predominant in mitral valve disease, there is commonly annulus
dilatation that results from right cavities dilatation and leads to tricuspid regurgitation. This
must be differentiated from organic valve disease, which has usually morphological changes
similar to that described above for the mitral valve.
3.1.3 Major valvular abnormalities
3.1.3.1 Restrictive or excessive leaflet motion
Restrictive leaflet motion is evident in most patients with established RHD requiring
surgery MR (Chavaud et al., 2001), nearly one third of patients with acute RF (Vijayalakshmi
et al., 2008; Marcus et al., 1989) and all those with rheumatic mitral stenosis (Wilkins et al.,
1988; Naito et al., 1980; Prasad & Radhakrishnan, 1992; Van der Bel-Kahn & Becker, 1986). It
is caused by chordal shortening, thickening and fusion, commissural fusion, and leaflet
calcification and thickening (Chavaud et al., 2001; Van der Bel-Kahn & Becker, 1986;
Carpentier, 1983). The terms used to characterize the abnormal and restricted mobility of the
mitral leaflets include elbow, dog-leg and hockey-stick deformity (Paar et al., 2010; Webb et
al., 2009; Steer et al., 2009; Reeves et al., 2011; Carapetis et al., 2008) (Figure 6).
Chordal elongation and rupture of the primary chords are the mechanisms responsible for
mitral valve prolapse in RHD. These changes must be carefully looked for as they influence
the surgical management (Chavaud et al., 2001; Marcus et al., 1989; Carpentier, 1983).

Fig. 6. Long axis parasternal view of a patient with mitral stenosis due to RHD showing
thickening of the mitral and aortic valves, as well as restricted motion of the mitral leaflets.
Notice a large left atrial thrombus.
3.1.3.2 Valve thickening
The rheumatic valve is fibrotic and firm, with thickening and fusion of leaflets and
commissures (Van der Bel-Kahn & Becker, 1986), mostly seen in stenotic valves. Thickening
Role of Echocardiography in Research into
Neglected Cardiovascular Diseases in Sub-Saharan Africa 457
of the mitral valve, especially the anterior mitral leaflet, appears to be a consistent feature of
RHD (Figure 6), which can be adequately assessed in the parasternal long axial view where
the anterior mitral valve because the ultrasound beam is perpendicular to the leaflet.
Regarding the aortic valve both the parasternal and subcostal views allow adequate
evaluation. Valve thickness of both mitral and aortic valves increases with age, based on an
autopsy study (Sahsakul et al., 1988). However, in populations where RHD is prevalent
there are very few additional conditions that are associated with increased thickness of the
mitral valve in the age groups affected by RHVD, except for endemic areas for both RHD
and EMF (Mocumbi et al., 2008).
3.2 Recent advances and research needs
The knowledge gap regarding epidemiology, pathogenesis and natural history of RF/RHD
in Africa is related to several factors. First, group A streptococcal infections that precede
RHD are subclinical, and most of the clinical cases are of a minor nature compared with
other diseases afflicting children in this setting. Secondly, RF/RHD is not notifiable in most
African countries and its impact is underestimated. Thirdly, many children are not brought
to medical care when they complain of sore throat or a skin lesion. Finally, the diagnosis of
rheumatic fever/carditis, requires clinical sophistication that exceeds the expertise available
at many local hospitals that are manned by nurses or trained health care workers.
The echocardiographic diagnosis of RHD is not standardized and there are few studies
looking systematically at criteria for diagnosing valve disease using modern
echocardiographic tools. However, due to the persisting burden of the disease in some areas
of the world echocardiography has been used in community studies in Mozambique and
Cambodia (Marijon et al., 2007), Tonga (Carapetis et al., 2008), Nicaragua (Paar et al., 2010),
Fiji (Steer et al., 2009; Reeves et al., 2011), Kenya (Anabwani et al., 1996), India (Thakur et al.,
1996; Bhaya et al., 2010), Pakistan (Sadiq et al., 2009; Rizvi et al., 2004) and China (Zhimin
2006). These studies applied different inclusion and diagnostic criteria, raising the issue
about the need for standardization of the definition of rheumatic heart valve disease by
echocardiography.
Patients from African series present severe abnormalities at early ages ( Sliwa et al., 2010;
Marijon et al., 2008). Because rheumatic heart valve disease has an initial latent stage that
can be detected by appropriate tests (among which echocardiography), has adequate
affordable therapy, and may have its prognosis improved by interventions at an early stage,
it should be the target of screening as a tool of preventive medicine.
3.2.1 Developing guidelines for echocardiographic screening
Early detection of subclinical rheumatic valve disease by echocardiography is vital, as it
presents an opportunity for case detection at a time when prophylactic penicillin to
prevent recurrent episodes can stop progression to important valve disease. This is very
important in Africa, where most new patients admitted to hospitals have already advanced
and complicated rheumatic valvular lesions (Sliwa et al., 2010), often resulting in heart
failure and/or arrhythmia that cannot be adequately managed due to unavailability of open
heart surgery. A current challenge for African scientists is therefore to make
echocardiographic screening reliable, affordable and feasible in low-resource settings, using

The Cardiovascular System Physiology, Diagnostics and Clinical Implications 458
diagnostic criteria that are clear, simple, robust and reproducible. This would allow their
incorporation in protocols for performing, reading and interpreting echocardiograms, in
order to avoid over- and under-diagnosis.
Researchers from Africa have been involved in continental efforts to assess the
epidemiology of RHD using echocardiography. This has started with the Awareness,
Surveillance, Advocacy and Prevention Strategy lounged by the Pan African Society of
Cardiology in 2005, which aims at reducing the burden of RF/RHD in the continent (Mayosi
et al., 2006). More recently African researchers have been taking part in a global initiative
aiming at standardization of echocardiographic screening that is led by the World Heart
Federation (World Heart Federation, 2011).
3.2.2 Disseminating echocardiographic screening
The use of highly trained specialists for large scale echocardiographic screening of RHD in
endemic areas of Africa is not practical (Figure 7) but the diffusion of ultrasound technology
to nontraditional users has been rapid and far-reaching in the last years (Shah et al., 2008).
Experiences for dissemination of echocardiography to non-traditional users in Rwanda and
Tanzania have been designed aiming at the evaluation of pericardial effusion, rheumatic
heart disease, congestive heart failure and estimation of global left ventricular function
(Shah et al., 2008; Adler et al., 2008). The impact of this technology diffusion is being
quantified, but early results show that ultrasound is a teachable skill, leads to accuracy of
diagnosis, helps in management of common cardiovascular conditions, and improves
professional satisfaction of local health providers (Shah et al., 2009). The role of task-shifting
inside the health systems to allow non-cardiologists to perform echocardiographic screening
for RHD must therefore be studied. However, there is need to carefully choose the health
providers to be trained and implement measures of quality assessment and sustainability.

Fig. 7. Photograph of a researcher performing echocardiography in an Africa rural setting.
Role of Echocardiography in Research into
Neglected Cardiovascular Diseases in Sub-Saharan Africa 459
3.2.3 Definition of curricula and selection of ultrasound machines
Considering the unique pattern of cardiovascular disease in Africa, there is need for
designing curricula and training materials tailored to the local needs, taking into
consideration the differential diagnosis with conditions such as cardiomyopathy, which are
also highly prevalent in the continent. In the particular conditions of health care provision in
Africa the choice of the ultrasound machines is also of paramount importance. Machine
specificities that are suitable for the African environment include durability, portability,
battery-operated machines and high two-dimensional image quality. In portable machines a
storage bag with room for gel, towels, probe covers and cleaning supplies is recommended
(Shah et al., 2008).
4. Conclusions
There has been an increase in scientific publications from African researchers and
institutions with the dissemination of echocardiography. Echocardiographic-driven research
into neglected diseases such as endomyocardial fibrosis and rheumatic heart disease have
contributed to uncover epidemiology and clinical profile of these conditions in the
continent, confirming the role for this imaging technique in fostering research and
improving quality of care in cardiovascular diseases in resource-deprived areas of Africa.
Echocardiography may also help to quantify the health impact of certain neglected
cardiovascular diseases in Africa, as well as assist in design and implementation of
programs for surveillance, prevention and control of such conditions.
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22
Psychophysiological Cardiovascular
Functioning in Hostile Defensive Women
Francisco Palmero and Cristina Guerrero
Universitat Jaume I
Spain
1. Introduction
Cardiovascular diseases are the leading causes of death and disability in the world, in both
developed and developing countries, and also in both sexes. In fact one third of annual
deaths worldwide are due to cardiovascular problems, according to the WHO (World
Health Organization) estimated 17.3 million people died from CVDs in 2008, over 80% of
CVD deaths take place in low- and middle-income countries, and by 2030, almost 23.6
million people will die from CVDs (in http://www.who.int/cardiovascular_diseases).
Therefore, is a serious problem, and not only in industrialized countries, indeed, is an
epidemic that not only continues but it is precisely in the developing countries, where it
currently is increasing dramatically. On the other hand, prevalence and mortality from these
diseases among women has increased in an exaggerated way. This for several reasons: first,
as mentioned, in women the death rate from CVDs has increased significantly, equaling or
exceeding that of the male population, so we think it is of great importance to focus on
studies considering this sector only a few risk factors have also increased; second, the
sample with which we had consisted mostly of women, given the characteristics of it
(psychology undergraduates), which were removed the few men who participated in the
study. Thus, from these data and indications, and since most studies have focused on people
of both sexes or only male, we considered appropriate to carry out research with a sample of
only women.
Moreover the etiology of CVD is multidimensional, that is, factors involving genetic,
physiological, chemical, nutritional, environmental and psychosocial, and moreover, is not
fully known. It is known that a number of cardiovascular risk factors that may contribute to
the development, progression or maintenance of CVD, called "classic risk factors. Among
the most important are: age, sex, cholesterol, hypertension, smoking, physical inactivity and
obesity. However, surprisingly, these factors fail to explain more than 50% of the variance in
predicting cardiovascular risk, whether considered independently or when considered
together (Chesney, 1996; Gump & Matthews, 1999). Nevertheless a large proportion of
CVDs are preventable, but they continue to increase mainly because preventive measures
are inadequate.
Therefore, research about this topic has been long and extensive, especially directed to seek
and discover other factors, beyond the "classics", are also contributing to the development

The Cardiovascular System Physiology, Diagnostics and Clinical Implications

466
CVD (Brydon et al., 2010; Chida & Steptoe, 2009; Everson-Rose & Lewis, 2005; Jorgensen &
Kolodziej, 2007; Vella & Friedman, 2009). We refer to psychosocial factors. Thus, the
relationship between different psychosocial variables and the risk of CVD has been studied
extensively since the mid past century, mainly for these two reasons.
2. Psychosocial risk factors and cardiovascular disease
In the search and exploration for other risk factors that may explain the etiology of CVD,
psychosocial factors have gained importance to such an extent that research has been able
to explain the mechanisms of action of these variables on CVD. The results obtained in
various research studies have confirmed the relationship between psychosocial factors
and the atheromatous plaque, which constitutes the basic injury occurring in CVD
(Kaplan, et al, 1983; Kaplan, et al, 1987; Manuck, et al, 1983; Manuck, Kaplan & Matthews,
1986; Manuck, et al, 1989; Jennings, et al, 2004; Vale, 2005). The mechanisms involved in
its formation, which are mechanical and chemical factors are seriously affected by
psychosocial processes, and especially by the stress response. In these processes, emotions
cause a faster heart rate and higher blood pressure, leading to increased blood flow and
turbulence. In addition, there is a mobilization of lipids which exceeds the body's
metabolic requirements, and which facilitates aggregation to artery walls and heart tissue.
This relationship between psychosocial factors and CVD has received the generic name of
"Hypothesis of the cardiovascular reactivity", and has been supported by various
prospective studies (Keys & Taylor, 1971; Schiffer, et al, 1976; Manuck, et al, 1992; Steptoe,
et al, 2000).
To date, research has shown that individuals who tend to display strong responses and
reactivity are at increased risk of CVD (Manuck et al., 1992; Palmero et al., 2006; Treiber, et
al, 2003; Matthews, et al, 2006). The argument that defends this refers to the stereotype
response: if the cardiovascular reactivity is a characteristic of an individual and is
physiological stable and consistent, then the same response patterns will be seen every time
the individual is faced with a situation of stress. Evidently with certain limitations,
laboratory situations can be regarded as a procedure that provides information on an
individuals physiological functioning in real life (Allen, et al, 1987; Allen & Matthews, 1997;
Palmero, et al, 2002; Moseley & Linden, 2006; Palmero, et al, 2007). Thus individuals, whose
pattern of cardiovascular functioning is characterized by the expression of exaggerated
responses, are those who, with time, are likely to experience some cardiovascular
dysfunction (Everson, et al, 1996; Markovitz, et al, 1998; Strike, et al, 2003). Given this
relationship between excessive cardiovascular response and CVD, research efforts have
focused on finding any variable causing an increase in such responses as this will mean an
increase in the likelihood of suffering from one of these diseases.
From the study on the classic Type A behavior pattern through the anger-hostility complex
and hostility were conducted, in recent years, research has focused on the defensive hostility
(high hostility and high defensiveness) as a risk factor in CVD (Guerrero & Palmero, 2010;
Helmers & Krantz, 1996; Jamner, et al, 1991; Larson & Langer, 1997; Shapiro, et al, 1995;
Palmero, et al, 2007; Vella & Friedman, 2007). The trait of defensive hostility reflects an
approachavoid conflict between the desire for social approval and distrust of those who

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467
can provide such support, and currently can be considered as one of the psychosocial factors
with more weight and empirical support in its relationship with CVD.
Several studies of CVD patients demonstrate that subjects with high scores in defensive
hostility show higher rates of ischemia during a mental stress situation, greater damage by
infusion and a longer duration of ischemia during daily activities (Helmers, et al, 1995). As
well, a field study conducted with paramedics showed a higher cardiac response by people
with a high hostility defensive when they deal with stress situations (Jamner, et al, 1991).
These results, usually obtained from real situations, appear to be supported by laboratory
studies (Jorgensen, et al, 1995; Shapiro, et al, 1995; Helmers & Krantz, 1996; Larson &
Langer, 1997; Palmero et al., 2002; Palmero et al., 2007), which indicates the existence of a
subgroup of people who are characterized by high "Defensive Hostility" (DH), as well as a
greater cardiovascular response. In general, DH individuals show greater cardiovascular
response during the task phase that other groups can be formed when combining hostility
and defensiveness variables (Larson & Langer, 1997). And, unlike this group, find another
subgroup which is characterized by low hostility and low defensiveness and show a lower
cardiovascular response -low risk group-.
However as these studies show, various inconsistencies were also found, which originated,
at least in part, from the different tasks used to measure the cardiovascular variables. These
results suggest the relevance of broadening the research spectrum whose aim it is to
strengthen the association between psychological variables and cardiovascular response
from the understanding that the exaggerated response would be the link between these and
CVD. In other words, it seems appropriate to establish whether defensive hostility can be
seen as the toxic component in relation to CVD.
Therefore, the general objective pursued with this study refers to the delimitation of the
effects of defensive hostility on cardiovascular response in women, in a real stress situation.
Specifically, exploring the relationship between defensive hostility, which is a better
predictor than hostility alone, and cardiovascular responses (HR, SBP and DBP) in this tonic
dimension. That is, considering all three phases of the experiment (adaptation: A, task: T
and recovery: R) to establish the significance of the functional psychophysiological profiles
in the four experimental groups (DH: high hostility-high defensiveness, HH: high hostility-
low defensiveness, Def: low hostility-high defensiveness, LH: low hostility-low
defensiveness).
From these premises and the proposed main objective has been carried out this research
with the ultimate aim to contribute both to the development of the theoretical basis on
psychosocial variables that can be considered as cardiovascular risk factors and its
subsequent application in clinical practice as well as contribute to methodological
development in the field of psychophysiological research.
The general hypothesis suggests that individuals with high scores in defensive hostility
display the highest values with the psychophysiological variables. Specifically, we expect
that these individuals show the greatest average values recorded in the variables (HR, SBP
and DBP), and in all the phases of the experiment (A, T and R), compared to those shown by
the individuals of the other three groups. In addition, DH group will be characterized by
less adaptive psychophysiological profile.

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3. Empirical study
3.1 Study design
In this section we show data from a recent study published by the authors (Guerrero and
Palmero, 2010). One hundred and thirty female students from a Universitat Jaume I
participated in this research. The mean age of the participants was 20.34 years (SD=2.06).
The criteria to form the groups were the scores obtained with both the Ho Inventory (Cook
& Medley, 1954) and the Social Desirability Questionnaire (Crowne & Marlowe, 1960). We
used the median as the cut-off point to classify participants as "high" or "low" in each
variable, thus the sample was composed as follows: 30 Defensive Hostility (DH), 40 High
Hostility (HH), 42 Defensive (Def), and 18 Low Hostility (LH).
3.2 Instrumentation
Cardiovascular responses were measured with the registration system Biopac MP150 with
NIBP module 100A, both were connected to a personal computer to monitor and store all
the responses. Specifically, this registration system was used to measure the physiological
responses: heart rate, systolic blood pressure and diastolic blood pressure. Also, this system
recorded these cardiovascular parameters continuously and noninvasively.
Hostility was measured with the Hostility Inventory of Cook and Medley (1954), specifically
the Composite Hostility Score (Chost) consisting in three subscales: cynicism, hostile
feelings and aggressive responses. In previous studies, this information led to a scale being
provided with a greater ability to predict the response and cardiovascular reactivity in
comparison with the Ho scale provided as a whole (Barefoot et al., 1989; Christensen &
Smith, 1993; Guerrero, 2008).
Defensiveness was measured with the Spanish version (vila & Tom, 1989) of the Social
Desirability Questionnaire of Crowne and Marlowe (1960). It consists of 33 items of choice
alternatives (true or false) that reflect socially desirable behaviors and cognitions.
Also, reports and self-reports were also used to collect some data on behavioral habits
related to health issues, and various personal and socio-demographic data.
3.3 Study procedure
A real academic exam was used as a situation of stress. More specifically, we used an exam
of the degree of Psychology; this situation represents a real mental stress task for students.
Data were collected individually in one session. Following informed consent, each subject
completed a questionnaire of demographics, previous medical history and noted any
medication. Then, they went into the experimental cabin where they were asked to sit
comfortably in an armchair and a sensor was connected to their non dominant wrist. From this
time onward, both instructions and exam questions were submitted through the projector.
Following this registration session, it was necessary to remove the sensors and to go to
another room to complete the corresponding scales. Finally, they were thanked for their
collaboration and left.

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The recording session consisted in three phases: adaptation (A), task (T) and recovery (R),
with duration of 10, 20 and 10 minutes, respectively.
a. Adaptation phase (10 min): there was no stimulus. The purpose of this period was for
participants to become familiar with the environment. The psychophysiological
variables were recorded in their tonic dimension to establish baseline levels with the
aim of obtaining the participants usual levels under rest conditions.
b. Task phase (20 min): the 20 stimuli that formed the experimental task were presented: an
objective test of 20 questions with four alternative answers. The stimuli were separated
by a one-minute period, and the duration of this phase was therefore 20 minutes. This
phase was considered an overall stressful period, and its variables were recorded in
their phasic dimension.
c. Recovery phase (10 min): no stimulus was presented. The variables were considered in their
tonic dimension to see how these variables recovered their usual levels after the stress
situation. These data allow us to ascertain how long the organism needs to achieve its
usual values following a situation of stress. This is extremely relevant when considering
the consequences caused by the stress situation from a neuroendocrinological viewpoint
because the greater the time needed by the organism to recover its baseline levels, the
greater the exposure to the effects of substances released by the organism because of this
situation of stress (catecolamnines and cortisol).
3.4 Statistical analysis
The first approaches were the descriptive analysis and correlations, and an analysis of
variance (ANOVA) was carried out for a more detailed analysis of the results. Then,
according to the main objective, that of analyzing the relationship between defensive
hostility and the cardiovascular responses to study the functional significance of each
groups profiles during all three phases, namely in their tonic dimension, an ANOVA was
carried out whose design was 4 groups (DH, HH, LH and Def) x 3 phases (adaptation, task
and recovery) with repeated measures for the phase variable. Subsequently, a univariate
analysis of variance has been conducted for each phase to obtain a more accurate
description of the potential differences encountered should such differences be given from
the post hoc Tukey test with which the groups involved in them will be determined.
3.5 Study results
Data will be presented in the following figures, which reflect the psychophysiological
profiles obtained from analysis of data for each physiological variable separately.
Although, before that, we like to refer to the profiles described by Kelsey (1993) on which
we relied. There are different ways to respond physiologically to stressful events, which
depends on external factors (situational) and internal factors (personality variables). In this
regard, and from the classic proposal Kelsey, we noted three response patterns, which
reflect corresponding profiles associated with different forms of reaction to stressful events:
habituation, sensitization and support or constant.
- Habituation.
When you perceive a situation as potentially threatening or novel, occurs an increase in
cardiovascular reactivity. After an exposure time in such a situation, and after that initial

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increase, there is a phenomenon of habituation, during which we see a progressive decrease
of the initial values. This phenomenon is considered essential in the process of adaptation
and regulation of humans and lower animals, demonstrating the ability to self-regulatory
organism, that is, it can be activate to deal with a potentially dangerous or threatening
situation and, in turn, is able to return to baseline (BL) once the situation has gone or is
under control.
- Sensitization.
In this case, the individual responds to an agent or stressor stimulus with a high
cardiovascular reactivity (the phenomenon of sensitization), similar to the previous pattern
but without habituation occur. Instead, it produces a progressive increase in reactivity over
the situation. It shows a organism's inability to return to baseline, which is highly
detrimental to the heart muscle and overall health.
- Constant.
In this third pattern occurs an initial increase, similar to that of the other two patterns, but
no habituation or sensitization occurs. This increase remains constant throughout the stress.
Thus, this pattern is also maladaptive; since there is no preparation behavior is essential in
the adaptation to the environment (Cannon, 1932) or has the ability to return to baseline
levels.
From these profiles is observed that only the first is adaptive, decreasing when the
individual cardiovascular reactivity to stressful events faced long periods of time. On the
one hand, the initial increase in activation allows for better coping with the situation and the
subsequent decline after a period of time, it is necessary to avoid damaging the body and
maintain homeostasis (Palmero, Breva, et al., 1994; Palmero, Espinosa et al., 1995).
Concerning heart rate in Figure 1 shows the obtained profiles by different groups.

Fig. 1. Heart rate and defensive hostility during three phases.

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471
As for the functional significance of the various profiles, as seen in Figure 1, all groups show
the habituation trend, so profiles are adaptive. Although, LH group shows the more
adaptive pattern, as it presents a greater and faster recovery to their baseline levels.
Concerning to systolic blood pressure as seen in Figure 2, all groups show the habituation
trend. Again, LH group presents a more adaptive pattern, with lower values in the recovery
phase.

Fig. 2. Systolic blood pressure and defensive hostility during three phases.
Here we also find that DH is the group that obtained the greatest values in systolic pressure
in all three phases.
An additional interesting fact is the effect of variable defensiveness, although the difference
was not statistically significant, we see that the two groups with high defensiveness, DH
and Def, presented the higher SBP values in all three phases (A, T and R)
Concerning diastolic blood pressure as seen in Figure 3, all groups show the habituation
trend, and again LH group presents a more adaptive pattern.
Again, we see that DH is the group that shows the highest values in all three phases. In this
case also notes the effect of variable defensiveness, although neither are statistically
significant differences. Two groups with high defensiveness, DH and Def, presented the
higher DBP values, but only in adaptation and task phases.
Figure 2 and 3 demonstrate clear differences between SBP and DBP between the
psychophysiological patters of extreme groups: DH presents higher values than LH group.
And the other two groups are in an intermediate position, with values very similar among
them and throughout the three phases.

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Fig. 3. Diastolic blood pressure and defensive hostility during three phases.
4. Conclusion
In general, the DH group presented higher values for the cardiovascular variables during
the three phases, as well as a slow recovery. Thus, we believe that defensive hostility has
proved to be a more appropriate criterion than hostility alone when determining the
possible risk of cardiovascular dysfunction.
So, consistent with our main objective, and in relation to establishing the functional
significance of the general profiles in the four groups through considering the three
experiment phases, our results are in the expected direction. Specifically, the DH group
obtains the highest values in the three phases and variables, particularly in the blood
pressure index. Additionally, extending the findings available to therefore include the
recovery phase, we have found that this group of individuals takes longer to recover after a
stress situation, that is, they score the highest values encountered during the recovery phase.
About the cardiovascular parameters, the three most commonly used in these studies were HR,
SBP and DBP (Swain & Suls, 1996), to combine the criteria and to enhance the comparison
between different results from various research studies. All the parameters have been
captured, recorded, stored and analyzed in a highly reliable, non invasive and continuous
manner, which is especially relevant in the case of blood pressure. They are easily accessible
and provide us with highly reliable information on cardiac and vascular functioning over
different time periods, that is, to reflect the changing psychophysiology profile of
individuals at all times.
Among these parameters, we note blood pressure reflects the greatest differences between
the two extreme groups (DH and LH): introducing a greater response, activation and
recovery for the DH group. On the other hand, HR reveals a clear difference in the lowest
values submitted by the low-risk group (LH) compared to a more homogeneous response
from the other three groups in all the phases. So, we suggest that the cardiovascular

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473
functioning of hostile defensive individuals in such situations is best reflected through
blood pressure, specifically through diastolic pressure, an index which seems to appear
truer for the recovery phase, as reflected by the significant differences found between the
two extreme groups.
More specifically, showing each cardiovascular physiological variable separately, the
following was observed.
Regarding the HR, although there is no effect of interaction between hostility and
defensiveness, since the group HD presents values very similar to those groups HH and
Def, HR shows how the low-risk group, LH , presents the lowest values along the three
phases compared to the other groups.
We must also emphasize for the three groups with higher levels in HR, which is the group
Def -although differences are not statistically significant- which presents the highest levels
in all three phases, reflecting a major effect of the variable defensiveness, specifically in the
adaptation and task phases where groups with high defensiveness (DH and Def) show the
highest levels.
Our data also specifies that it is in the recovery phase where there are statistically significant
differences between the Def group and the LH group.
Regarding the SBP, beside show the effect of interaction between hostility and
defensiveness, a fact that reflects the important values of the DH group, the main effect of
the defensiveness variable also exist in three phases, which means that subjects with high
scores in defensiveness obtain higher values than subjects with low scores in
defensiveness.
In addition, our data indicate that statistically significant differences that exist between DH
and LH groups are specifically at the task and the recovery phases, in turn, indicates that
there are no differences between groups with respect to their baseline levels.
The fact that the DH group presents higher scores for these values, leads us to suggest that
defensive hostility rather than hostility alone better predicts the cardiovascular function in
situations of stress.
Regarding the DBP, it seems that the real meaning of this variable can be seen only with the
consideration of defensive hostility, such as denoting the effects of interaction in all three
phases. Like the previous case, our data indicate that statistically significant differences also
exist between DH and Def groups, but in this case appear in the recovery phase. There was
also a main effect of the defensiveness variable, but is almost imperceptible.
Regarding the functionality of the profiles all four groups in three variables were adaptive. But
in blood pressure case, it seems interesting to note that while the four groups that tend to
indicate profiles of habituation, the fact that the individuals defensive hostile obtains these
statistically significant values during the recovery phase means that this group of
individuals takes significantly longer to return to the baseline values of situations without
stress. Thus, the profile of hostile defensive subjects would be less adaptive, because in this
group the recovery is more slow and gradual. The profiles of the three remaining groups are
adaptive, with a faster recovery.

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Specifically, in our view, while DBP continues the pattern of other variables during the task,
and displays an interaction effect between hostility and defensiveness, it is the most
important variable to detect the cardiovascular functioning of hostile defensive individuals
in the recovery phase. Once again, these arguments lead us to suggest that it is desirable to
consider this variable, along with the inclusion of the recovery phase.
In short, along with the results corresponding to the tonic dimension of the cardiovascular
variables being studied, it appears that defensive hostility is more appropriate than hostility
alone to understand cardiovascular functioning in stressful situations. So, defensive
hostility identifies a dimension of personality that, ultimately, would be a better predictor
of the cardiovascular response in particular and of cardiovascular disease in general. It
would be more fitting than hostility alone to explain and understand cardiovascular
functioning in stressful situations.
The DH group, which shows that those individuals with high scores in hostility and high
scores in defensiveness, are those who reflect the highest values in activation and
cardiovascular response, but only when faced with the demands of a challenging task. For
this reason, and as we have pointed out, it seems appropriate to use a real situation of stress
as an experimental task because this specific environment is the best scenario to see the
psychophysiological response style that characterizes the individuals being studied. In
addition, if the situation of stress is sufficiently long, there is also the possibility of locating
the adjustment mechanism of individuals to the sustained demands of this stressful activity.
Those hostile individuals seeking to perform an action that is not disagreeable to others
display the greatest difficulty in controlling their hostile experiences. The result of this
inability to properly monitor hostility experiences produces a sustained increase in the
activation of the sympathetic system which, in turn, gives rise to significant increases in the
cardiovascular variables studied: HR, SBP and DBP. One particular sensitivity of the SBP is
to capture this sympathetic activation which suggests its suitability differential in such
studies.
Thus, these findings show the relationship between defensive hostility and cardiovascular
functioning in situations of stress by the various cardiovascular register indexes (HR, SBP
and DBP) and by considering the various parameters analyzed, namely response, activation
and cardiovascular recovery, have been demonstrated. As mentioned above, we believe that
defensive hostility has proved to be a more appropriate criterion than hostility alone when
determining the possible risk of cardiovascular dysfunction.
Regarding the three experimental phases, we can state the following. About the recovery phase,
we think that its inclusion in the experimental research laboratory is especially important
since it provides vital information on restoring physiological parameters. This phase is a
basic and essential element in the detection of the possible risks of future dysfunctions
(Guerrero & Palmero, 2006). As seen, the recovery phase profile in the DH group has shown
a slower recovery, especially in terms of blood pressure, and more specifically in DBP. Thus,
the inclusion of the recovery phase in this type of experimental laboratory, it constitutes a
basic and essential element in the detection of possible risks of future dysfunctions. From a
neuroendocrinological viewpoint, it is important to consider the consequences caused by the
situation since the more time the organism needs to recover the baseline levels, the greater
the exposure to the effects of the substances released (catecholamines and cortisol).

Psychophysiological Cardiovascular Functioning in Hostile Defensive Women

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About the task phase, by considering the duration parameter, which is scarcely taken into
account in such research, we believe it appropriate to propose an experimental task that is
long enough to establish a genuine cardiovascular functioning, that is more likely to be
correct, by appreciating how the adjustment to a stressful situation is produced, or not. The
fact that short or moderately long tasks have been systematically used may have masked the
dysfunctional connotations of the response profiles in the different groups.
Regarding the tasks used as a stressful situation in the laboratory, the importance of creating
and using a type of task that involves a real stress situation must be highlighted, that is, one
as close as possible to everyday situations. Thus, one can understand some of the
inconsistencies found which, in turn, provide the ecological laboratory experiments in this
field with more validity. In this respect, we believe that the task used in this research, a real
exam, is a task with connotations of personal and social threats, which also requires
investing considerable effort to be able to deal with it actively and successfully, and this has
been reflected by the significant differences obtained in the three experiment phases.
Concluding, in our modest opinion, with this and other research we have conducted we
provide more empirical support about the great relevance within the theoretical framework
on DH as a possible psychosocial cardiovascular risk factor also in women. However, as a
future research direction, probable variability among females as compared to males
necessitates concentrated research in this area, and we recognize the need for separate data
analysis for males. Also, the study should be replicate with other samples to see if there are
similar results and generalize these obtained data.
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