Author's Accepted Manuscript

Oxidative stress and vascular inflammation in

aging
Mariam El Assar, Javier Angulo, Leocadio
Rodríguez-Mañas

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PII: S0891-5849(13)00330-4
DOI: http://dx.doi.org/10.1016/j.freeradbiomed.2013.07.003
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To appear in: Free Radical Biology and Medicine

Received date: 17 January 2013

Revised date: 28 June 2013
Accepted date: 2 July 2013

Cite this article as: Mariam El Assar, Javier Angulo, Leocadio Rodríguez-Mañas,
Oxidative stress and vascular inflammation in aging, Free Radical Biology and
Medicine, http://dx.doi.org/10.1016/j.freeradbiomed.2013.07.003

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Oxidative stress and vascular inflammation in aging

Mariam El Assar1, Javier Angulo2, Leocadio Rodríguez-Mañas1,3

1
Fundación para la Investigación Biomédica, Hospital Universitario de Getafe, Getafe,

Spain.
2
Instituto Ramón y Cajal de Investigación Sanitaria, Hospital Universitario Ramón y

Cajal, Madrid, Spain.

3
Servicio de Geriatría, Hospital Universitario de Getafe, Getafe, Spain.

Corresponding author:

Dr. Leocadio Rodríguez-Mañas

Head of Servicio de Geriatría, Hospital Universitario de Getafe

Ctra de Toledo, km 12,500

28905 – GETAFE

Madrid – Spain

Phone: +34 91 683 93 60 / ext. 6411

E-mail: [email protected]

1
ABSTRACT

Vascular aging, a determinant factor for cardiovascular disease and health status in the

elderly, is now viewed as a modifiable risk factor. Impaired endothelial vasodilation is

an early hallmark of arterial aging that precedes the clinical manifestations of vascular

dysfunction, being the first step to cardiovascular disease and influencing vascular

outcomes in the elderly. Accordingly, the preservation of endothelial function is thought

to be an essential determinant of healthy aging. Paying preferential attention to address

the effect of aging on the endothelial function, this review is focused on the two main

mechanisms of aging-related endothelial dysfunction: oxidative stress and

inflammation. Aging vasculature generates an excess of the reactive oxygen species

(ROS), superoxide and hydrogen peroxide that compromise vasodilatory activity of

nitric oxide (NO) and facilitate the formation of the deleterious radical, peroxynitrite.

Main sources of ROS are mitochondrial respiratory chain and NADPH oxidases

although NOS uncoupling could also account for ROS generation. In addition, reduced

antioxidant response mediated by erythroid-2-related factor-2 (Nrf2) and down-

regulation of mitochondrial manganese superoxide dismutase (SOD2) contributes to the

establishment of chronic oxidative stress in aged vessels. This is accompanied by a

chronic low-grade inflammatory phenotype that participates in defective endothelial

vasodilation. The redox-sensitive transcription factor, nuclear factor-NB (NF-NB), is up-

regulated in vascular cells from old subjects and drives a pro-inflammatory shift that

feedbacks oxidative stress. This chronic NF-NB activation is contributed by increased

angiotensin-II signaling and down-regulated sirtuins and precludes adequate cellular

response to acute ROS generation. Interventions targeted to recover endogenous

antioxidant capacity and cellular stress response rather than exogenous antioxidants

could reverse oxidative stress – inflammation vicious cycle in vascular aging. Lifestyle

2
attitudes such as caloric restriction and exercise training appear as effective ways to

overcome defective antioxidant response and inflammation, favoring successful

vascular aging and decreasing the risk for cardiovascular disease.

Key words. Arterial aging; endothelial dysfunction; nitric oxide; oxidative stress;

reactive oxygen species; inflammation; nuclear factor-B; sirtuins.

3
 Aging is the main risk factor for cardiovascular diseases [1]. This influence is

not only due to a higher prevalence of cardiovascular risk factors in aged people but

also to an independent effect of aging on cardiovascular health by itself [2]. Although

aging is an inevitable process, the increasing knowledge of the mechanisms underlying

the process of aging in the cardiovascular system has led to change the view of the

vascular aging as a non-modifiable risk factor. Thus the concept of vascular aging as a

modifiable cardiovascular risk factor is gaining force [3]. As a consequence the way to

prevent or delay cardiovascular aging is now an issue of debate as prevention of

cardiovascular disease is a critical goal to reduce morbidity, disability and health care

costs. Indeed some authors suggest that prevention is worthy in older patients at any age

[4]. Finally, recent evidence suggests that vascular dysfunction not only has an impact

on classical cardiovascular outcomes but also to other adverse consequences.

Deterioration of vascular function with ageing also leads to cognitive impairment and

dementia in later life [5] that represent a fundamental issue in the overall health of the

elderly subject and an outstanding challenge in the practice of geriatric medicine.

Among the different mechanisms leading to vascular dysfunction the endothelial

damage is one of the earliest and crucial events. Because of that, in this review we will

pay a preferential attention to address the effect of aging on the endothelial function.

Vascular dysfunction associated to the aging process

Vascular aging is characterized by functional and structural changes of the

endothelium and smooth muscle cells, as well as alterations of the communication

routes between these two cell layers that form the vascular wall. Impaired endothelial

vasodilation is an early manifestation of arterial aging that precedes in years the clinical

4
manifestations of vascular dysfunction, being the first step to cardiovascular disease and

influencing vascular outcomes in the elderly [6]. Another hallmark of vascular aging is

an increase in the arterial stiffness secondary to the loss of arterial elasticity,

compromising vascular adaptation to blood flow and pressure changes. This high

arterial stiffness is manifested by an increase in the speed of propagation of

pressure/flow waves that can be measured by different ways, being the determination of

the Pulse Wave Velocity (PWV) the most usual method. Aging-associated arterial

stiffness is related to alteration of morphology and composition of extracellular matrix.

Elastic fibers undergo fragmentation and thinning, transferring mechanical load to

collagen, which is 100-1000 times stiffer than elastin. Fragmentation might be due to

mechanical fatigue or enhanced matrix metalloproteinase (MMP) activity [7]. Increased

expression and activity of MMP-2 has been reported in vessels from aged animals [8-

10] and an aging-related increase of MMP-2 activity has also been demonstrated in

human internal mammary artery [11]. In fact, a single-nucleotide polymorphism in the

gene coding for 1 chain of type 4 collagen, the substrate for MMP-2, is associated with

arterial stiffness, determined by PWV increase, in two unrelated populations [12].

Although MMP-2 up-regulation is more commonly related to vascular aging, plasma

MMP-1 and circulating markers of type 1 collagen (MMP-1 substrate) degradation

positively correlate with PWV and augmentation index as indicators of arterial stiffness

in human subjects [13].

Arterial stiffness is always preceded by an impaired endothelial vasodilation

suggesting that this arterial alteration is also linked to endothelial dysfunction [14]. On

this background of endothelial dysfunction, other functional disturbances associated

with aging and some diseases (including the classical cardiovascular risk factors), will

contribute to the development of structural alterations, which in turn contribute to

5
vascular stiffness and to an additional impairment of the endothelial function. In

addition to the role of endothelium in promoting vascular disease in the great vessels,

endothelial dysfunction plays also a significant role in the age-associated microvascular

dysfunction [15]. It is important to note that large and small arteries are not isolated

systems and a cross-talk between alterations in large and small arteries is evident. In this

sense, preservation of small arteries would result in limitation of blood pressure raise

and arterial stiffness of large arteries, a situation that prevents small artery remodeling

and target organ damage [16]. This cross-talk between large artery alteration and small

cerebral arteries could be a determinant factor for cognitive decline in older individuals

[17].

Functional decline and structural alterations of aged vasculature are also

accompanied by defective capacity of vascular repair that contributes to the increased

severity of vascular diseases observed in older persons [18]. Reduced repair capacity

with aging could be related to the decrease in number and/or the impairment of function

of endothelial progenitor cells (EPCs), which play an integral role in the cellular

mechanisms for endothelial regeneration and maintenance [19]. Human studies have

consistently shown impaired function (colony formation, survival, migration and

proliferation) of EPCs with aging that translate into reduced ability to reendothelialize

injured arteries of nude mice in vivo [20] while the number or circulating EPCs in

elderly subjects has been reported to be reduced [21, 22] or unchanged [23].

Interestingly, the number of circulating EPCs correlated with the elasticity of large and

small arteries in persons with advancing age [21] and migration and proliferation

abilities of EPCs correlate with flow mediated dilation in aged subjects [23], suggesting

the interconnection of functional, structural and repair alterations in the process of

vascular aging.

6
 Taking into account the relevant role of endothelial dysfunction in the

development of macro- and microvascular dysfunction that may explain why

endothelial dysfunction is associated with the major causes of morbidity and mortality,

the maintenance of a correct function of this vascular layer is thought to be an essential

determinant of healthy aging [24, 25].

Aging-related decline in endothelial vasodilation

There is a huge amount of evidence showing the presence of endothelial

dysfunction in the aged vasculature both in animal models and humans. In particular,

the reduction in the endothelium-dependent vasodilatations has been consistently

described both in vitro and in vivo in different vascular beds from old animals [26-28]

and elderly humans [15, 25, 26, 28]. These evidences demonstrated that aging is an

independent factor associated with endothelial dysfunction even in the absence of other

cardiovascular risk factors [15, 29]. The reported scientific evidences strongly suggest

that the pathogenesis of the age-dependent endothelial dysfunction is clearly

multifactorial, with several pathophysiological mechanisms contributing to the

functional deterioration of vascular endothelial cells.

Endothelium-derived hyperpolarizing factor (EDHF). Together with

prostacyclin and nitric oxide (NO), EDHF is one of the main three players in endothelial

vasodilation. Contribution of EDHF to endothelial vasodilation of human arteries has

been demonstrated in different vascular regions such as coronary arteries [30], omental

and subcutaneous microvessels [31, 32] and penile resistance arteries [33]. Impairment

of EDHF-mediated vasodilation in different vascular districts from aged animals is

generally observed [34-36] but preserved responses that partially compensates for the

loss of NO- and prostacyclin-mediated vasodilation have also been reported [37]. In

7
human gastroepiploic arteries, EDHF-induced relaxation inversely correlated with the

age of the patients from whom the arteries were obtained [38].

Age-induced decline in EDHF-mediated vasodilation in rats is prevented by

antioxidant treatment with red wine polyphenols [39] and seems to be related to up-

regulation of renin-angiotensin system (RAS) [40] and reduced expression of small- and

intermediate-conductance calcium-activated potassium channels (SK and IK) [39]. A

decline on the hyperpolarizing responses mediated by large conductance calcium-

activated potassium channels (BK) has also been suggested to play a role in vascular

dysfunction with aging [41]. Defective BK-mediated responses were also observed in

coronary arteries from aged rats and can be recovered by exercise training [42].

Cyclooxygenase (COX) pathway alteration. During aging, there is a shift in the

balance in favor of increased COX-derived contractile factors (thromboxane A2, TXA2)

over COX-derived vasodilators (prostacyclin, PGI2), therefore, endothelium-dependent

contractions increase [43]. In humans, the loss of contribution of prostacyclin to

endothelium-dependent vasodilatation in aging has been reported in vivo [44] and in

mesenteric microvessels in vitro [15] while a reduced capacity of prostacyclin to cause

vasodilation in vivo has also been observed in older humans [45]. Conversely, the

existence of COX-derived vasoconstrictor factors associated with aging has been

described in vivo by using plethysmographic studies [46, 47] and in vitro studying

isolated mesenteric arteries [15]. Endothelium-dependent vasoconstrictions in aging

could be mediated by TXA2 and prostaglandin H2 but prostacyclin has also been

proposed to act as a contractile factor [48]. A role for superoxide is also possible, as

these reactive oxygen species have been described as COX activity-derived

endothelium-dependent vasoconstrictors [47].

8
 No consensus has so far been established regarding the COX isoform

responsible for the age-related vasoconstrictions since both COX-1 and COX-2 have

been demonstrated to contribute [49-51]. No differences related to age have been

detected in the expression of mRNA for COX-1 and COX-2 isoforms in human

mesenteric microvessels [15]. However, protein expression may be not the only factor

accounting for COX-mediated effects, as the existence of post-translational changes in

the activity of these enzymes cannot be ruled out. In this sense, there is an important

regulation of COX activity by NO and ONOO- [52]. Furthermore, a physiological

binding interaction between COX-2 and iNOS has been reported, bringing NO or

ONOO- to activate COX-2 in a synergistic molecular interaction between these two

inflammatory pathways [53].

Alteration of NO pathway. Synthesized by NO synthase [54] isoforms, endothelial NOS

(eNOS), neuronal NOS (nNOS), both constitutive, and inducible NOS [54], NO is a

molecule key to cardiovascular health. NO production seems to have a role in longevity,

since triple knockout mice for NOSs display a survival rate at 10 month-old reduced by

80%, approximately, with respect to wild type [55] and survival rate for eNOS knockout

mice is 50% of wild type at 18 months of age [56]. Furthermore, lifespan extension

caused by caloric restriction is strongly attenuated in eNOS knockout mice [57]. There

is no doubt about the existence of a deficit in the vascular NO bioavailability associated

with aging, but the impact of aging on eNOS expression is controversial. eNOS

expression in aged vessels has been shown to be unchanged [58], decreased [59] or

increased [60, 61]. In the mesenteric microvessels from healthy young and old humans,

no age-dependent changes have been detected concerning eNOS mRNA levels [15].

Moreover, human endothelial cells from brachial artery and peripheral veins do not

differ significantly with age in the eNOS expression [62]. In contrast, it is well accepted

9
that eNOS activity is reduced with aging [63]. This could be due to substrate or cofactor

deficiency and post-translational modifications. eNOS presents a significant regulation

at post-translational level involving PI3 kinase/Akt-dependent phosphorylation at Ser

1177, resulting in increased activity of eNOS. Although reduced Akt-dependent

phosphorylation of eNOS in micro- and macro-vasculature of aged animals has been

demonstrated [64, 65], the involvement of reduced eNOS phosphorylation in human

vascular aging needs to be confirmed. Reduced availability of tetrahydrobiopterin (BH4)

in vascular aging leads to eNOS uncoupling that not only decreases NO production but

also causes deleterious effects on vascular aging through superoxide production [66] as

it will be further discussed later. Although reduced eNOS activity possibly accounts for

the decline in endothelial vasodilation with aging, two mechanisms outstand as

responsible for endothelial dysfunction in aging: an excessive chronic oxidative stress

and an increased pro-inflammatory activity both compromising bioavailability of nitric

oxide (NO) [67-70]. These two mechanisms act in a complementary way, reinforcing

each other and represent the main focus of this review.

THE ROLE OF OXIDATIVE STRESS IN THE VASCULAR SYSTEM

Reactive oxygen species (ROS) are physiologically produced at basal rate into the

vascular wall in a controlled regulated manner. Oxidases generate superoxide by

donating an electron to oxygen, starting ROS production. The presence of different

forms of superoxide dismutase (SOD) in intracellular regions lower superoxide

concentrations into the picomolar range [71]. SOD activity as well as spontaneous auto-

reaction of superoxide generates hydrogen peroxide (H2O2). Considering its production

rate and its metabolism by scavenging systems such as glutathione peroxidase (GP) and

10
catalase, peroxidase levels are thought to be in the low nanomolar concentration range

[72].

Although ROS exert many roles in the cellular physiology, one of the most relevant is

its function as cell signaling initiators by their ability to introduce reversible post-

translational protein modifications [73] but also by activation/inhibition of redox

sensitive transcription factors.

Depending upon the balance between ROS generation and antioxidant defenses

concentration or activity the oxidative stress will change. Eventually, different

physiological and pathophysiological stimuli can generate acute increases in oxidative

stress that influence cellular processes and signaling. In this regard, the concentrations

of ROS may increase in pathological situations, as a consequence of either an increased

ROS production and/or a reduction in the antioxidant defenses, leading to accumulation

of damaged/misfolded proteins, increased mutagenesis rate, inflammation, and

endothelial dysfunction [74].

Physiological ROS not only act as a signaling system that modifies cell function but

also may determine cell fate. ROS generation needs to be controlled by the cell to avoid

cellular senescence since ROS induce endothelial senescence by inhibiting telomerase

and by telomere-independent actions [75]. Furthermore, since it has been proposed that

NO inhibits endothelial senescence [76], the interference of ROS on NO signaling

system would influence endothelial aging. ROS-mediated signaling also participates in

other fundamental biological processes of the endothelium such as apoptosis [77, 78]

and repair/regeneration [79, 80].

ROS and vascular damage in aging

11
Since Denham Harman formulated the free radical theory of aging [81], the connection

of oxidative stress with the aging process and aging-related diseases is widely accepted.

Several ROS are involved in the development of age-associated changes in the vascular

wall.

Superoxide reacts with nitric oxide (NO) three to four times faster than it reacts with

SOD but the biological significance of this reaction becomes evident when superoxide

concentration rises or when NO levels are very high [82]. The reaction with superoxide

quenches the biological activity of NO generated by vascular cells, hampering NO-

mediated responses and leading to a decrease in the endothelium-dependent relaxations

[83]. Conversely, reduced vascular NO availability would increase ROS generation

since it has been demonstrated that NO modulates superoxide production in human

vessels [84]. In addition, reaction of NO with superoxide generates peroxynitrite

(ONOO-) a highly reactive molecule that has been associated with vascular aging [85].

Peroxynitrite can easily penetrate into the cell causing oxidative modifications of

macromolecules, especially lipids, DNA, and proteins via direct oxidative reactions

through the nitrosylation of tyrosine and cysteine residues or via indirect radical-

mediated mechanisms [86].

There are plenty of evidences supporting the implication of oxidative stress in the

genesis of vascular damage in aging process. Increased oxidative stress and ROS

production have been detected during the aging process in different vascular beds from

animal models, that includes rat aorta [87-89] and coronary arteries [90] and mouse

aorta [91], inducing a decreased endothelial NO-bioavailability. Moreover, NO-

bioavailability could influence longevity. The concept is supported by the fact that

increased longevity of Peromyscus leucopus, a species closely related to mouse but

having double lifespan potential, is associated with limited endothelial production of

12
ROS and augmented expression of antioxidant enzymes (catalase, glutathione

peroxidase and hemeoxygenase) with respect to mice [92]. This suggests that larger

lifespan potential is associated with decreased vascular ROS generation and increased

oxidative stress resistance.

There also are evidences demonstrating an increased ROS production in vessels from

aged humans. Nitrotyrosine, a substance that reflects the impact of peroxynitrite on

proteins and constitutes a cellular marker of oxidative stress, was twofold increased in

endothelial cells from healthy older subjects (63±1 years) who also manifested blunted

in vivo endothelium-dependent dilation with respect to young subjects (23±1 years)

[67]. Furthermore, in addition to an increase in nitrotyrosine in mesenteric microarteries

from older subjects (> 60 years), enhanced generation of superoxide was detected in

these vessels, accompanied by a reduction of NO-induced relaxation [15].

ROS may ultimately influence structural alterations indirectly through long-term impact

of endothelial dysfunction but could also directly interfere with important players in

age-related vascular remodeling. In this sense, increased vascular smooth muscle cell

expression of MMP-2 and MMP-9 seems to be related to ROS production by

mitochondria and NADPH oxidase, respectively [93, 94], which is consistent with the

reported correlation between NADPH-dependent superoxide production by phagocytes

and plasma MMP-9 levels in humans [95].

The association of increased superoxide production with vascular aging becomes

relevant when the withdrawal of superoxide results in improved endothelial vasodilation

of aged arteries. For instance, preincubation with the synthetic SOD mimetic,

TEMPOL, or the NADPH oxidase inhibitor, apocynin, results in improved

endothelium-dependent dilation in soleus feed arteries from old rats but not from young

rats [96]. In the same way, oral administration of TEMPOL for 3 weeks normalizes

13
aortic superoxide production, restores endothelium-dependent dilation and reverses the

aging-associated increase in aortic PWV (arterial stiffness) in old rats [97]. With respect

to human vasculature, preincubation of mesenteric microvessels from healthy elderly

subjects with SOD or TEMPOL causes a significant improvement of endothelium-, NO-

dependent relaxation [15]. An age-dependent formation of nitrotyrosine was observed in

these vessels, while the endothelial dysfunction associated to aging is partially restored

by scavenging peroxynitrite with uric acid [15]. It is important to note that the

improvement of endothelial vasodilation by scavenging superoxide is not due to the

existence of other cardiovascular risk factors (CVRF) in the older populations since

SOD enhanced endothelium-dependent dilation in arteries from elderly subjects having

or not CVRF while did not modify endothelial responses in adult subjects [29]. All

these evidences suggest that the relevant changes in vascular function that are observed

in aging are driven, at least partially, by these highly reactive molecules.

Sources of oxidative stress

Although many other enzymes can generate ROS, four enzymatic systems predominate

as the sources for ROS production in the human vasculature, namely NADPH oxidases,

xanthine oxidase, uncoupled NO synthase and the mitochondrial respiratory chain [28,

84, 98-100]

Mitochondrial ROS

It is widely assumed that mitochondria are a major source of ROS relevant to the aging

process [101, 102]. Mitochondria are indeed considered the predominant intracellular

site of superoxide production which is generated from electron leaks in the

mitochondrial electron transport system [103]. Superoxide mainly produced by

complexes I and II is rapidly dismutated by manganese superoxide dismutase (SOD2) in

14
mitochondrial matrix to form H2O2 that may leave mitochondria due to its neutral

conformation. Approximately, 0.1% of oxygen consumed by mitochondria is reduced to

superoxide under physiological conditions [104], but when metabolic alterations lead to

inefficient nutrient oxidation, mitochondria increase the production of superoxide and

other ROS [105]. In this sense, superoxide formation increases as a result of the age-

related decline in mitochondrial biogenesis in rat aorta, being the vascular cell content

of cytochrome c oxidase negatively correlated with mitochondrial superoxide

production [106]. Mitochondrial ROS production has been demonstrated to impair

vasodilation [107], an interference that explains the negative correlation of aortic

endothelium-dependent relaxation with mitochondrial superoxide generation in aorta

from aged mice [108]. Elevated production of mitochondrial ROS could not only cause

aging-related diminished endothelial vasodilation but it also might be a determinant

factor for arterial stiffness since PWV is increased in old mice with one single copy of

the mitochondrial SOD, (SOD2+/-), with respect to wild type mice or those with a single

copy of SOD1 [109]. This study also shows that aged SOD2+/- mice have increased

levels of superoxide but decreased levels of H2O2 in smooth muscle cells, suggesting

that, in addition to endothelium, increased mitochondrial ROS production could affect

vascular smooth muscle too.

Some substances seem to play a relevant role in the relationship between the

mitochondria-derived oxidative stress and the vascular aging process. This is the case

for the p66Shc adaptor protein. p66Shc plays an important role as a redox enzyme

implicated in mitochondrial ROS production. After being imported to the mitochondrial

inter-membrane space, it mediates electron transfer from reduced cytochrome c to

molecular oxygen producing H2O2 [110]. Its relevance on aging process is highlighted

by the fact that genetic deletion of p66Shc reduces ROS production and increases

15
lifespan by 30% [111]. Although the influence of p66Shc on longevity could be

controversial in light of the high expression of p66Shc mRNA and protein detected in

skin fibroblasts from human centenarians [112], the involvement of this redox protein in

vascular aging is supported by substantial evidence. In this sense, aortic rings from old

p66Shc-/- knockout mice display preserved endothelial vasodilation and NO-release while

do not exhibit age-related superoxide generation increase in contrast to old wild type

mice [91]. Consistently, overexpression of p66Shc reduces NO production in endothelial

cells [113]. These evidences and the implication of p66Shc in cardiovascular diseases

point to this protein as a key factor in arterial dysfunction and cardiovascular disease

associated with aging [114, 115].

An additional important link between mitochondrial oxidative stress and cardiovascular

aging is the induction of programmed cell death. The available evidence suggests that

age-associated increase in oxidative stress causes an increased rate of endothelial

apoptosis [116]. Furthermore, recent studies show that mitochondria-derived ROS, in

addition to causing oxidative damage, play an important role in activating numerous

redox-sensitive transcription factors, including NF-B and AP-1 [116]. Activation of

another redox sensitive factor, kruppel-like factor 4 (KLF-4) could be responsible for

up-regulation of mitochondrial SOD isoform (MnSOD) [117]. An additional member of

this family, KLF-2, seems to be responsible for angiogenic response in endothelial

progenitor cells but its expression is reduced in senescent HUVEC and senescent

proangiogenic cells [118]. In fact, oxidative stress can modulate activity of zinc fingers-

containing transcription factors that contain cysteine residues with sulfhydryl groups

susceptible to be oxidized [119].

NADPH oxidases

16
Despite the assumed impact of ROS generated in mitochondria on the aging process,

some authors points to an alternative source of ROS, the NADPH oxidases (NOX), as

equally, or even more, important players in free radical production associated with

aging [120]. NOX are transmembrane enzymes that transport electrons from

cytoplasmatic NADPH to molecular oxygen leading to superoxide generation.

Depending of the localization NOX generate superoxide on the outside of plasma

membrane or the lumen of intracellular organelles but the ROS generated by NOX also

reach the cytoplasm through anion channels (superoxide) or diffusing through

membranes after dismutation (H2O2). There are seven NOX family members that differ

in their subcellular localization and the specific ROS generated. NOX1 is expressed in

vascular smooth muscle cells [121] and NOX2 has been detected in endothelial and

smooth muscle cells [122, 123]. Both generate superoxide [124] and are upregulated in

the presence of different cardiovascular risk factors NOX4 is constitutively expressed in

vascular smooth muscle and endothelial cells [121, 125] and it generates hydrogen

peroxide [124, 126]. NOX5 activity is regulated by calcium to produce H2O2 and is

overexpressed in coronary vessels (sequentially in endothelium and vascular smooth

muscle) from patients with coronary disease [127].

Numerous evidences of vascular aging-related upregulation of NADPH oxidase

expression and/or activity have been obtained in aged rats [61, 128, 129]. In fact,

NADPH oxidase-derived H2O2 has been shown to be responsible for the altered NO

production [130] while inhibition of NADPH oxidase improves endothelium-dependent

dilation [96, 131] in arteries from old rats.

Data available in literature have implicated different components of the superoxide

producing enzyme NADPH oxidase in the oxidative stress associated to vascular aging.

Increased NADPH oxidase p67 subunit expression and/ or enzyme activity with age

17
was observed in the aorta of mice [97, 132]. As well, aging-related up-regulation of the

NADPH oxidase subunits NOX-1 and p22phox were detected in mesenteric arteries of

middle-aged rats [39]. Donato et al. reported increased NADPH oxidase-p47phox subunit

in venous endothelial cells from older men in the absence of upregulation of xanthine

oxidase, another key oxidant producing enzyme [67].

In mesenteric microvessels from aged humans without other known cardiovascular risk

factors, it was demonstrated both increased expression of the NOX-4 subunit of

NADPH oxidase as well as improved endothelial vasodilation after NADPH oxidase

inhibition with apocynin [15]. This finding suggests that NADPH oxidase-derived ROS

would be related to the age-associated endothelial dysfunction also in the human

vasculature.

A number of studies have implicated ROS generated by NADPH oxidases in the

activation of Nrf2-dependent pathways [93, 133]. However all of these studies have

relied on ablating NADPH oxidase activity by the use of pharmacological inhibitors,

and direct genetic evidence for the involvement of any specific NOX isoform has so far

been lacking. Recently, it has been revealed by microarray screen a significant increase

in the expression of many antioxidant and detoxifying genes regulated by Nrf2 in

transgenic compared to wild-type mouse hearts in which NOX4 was overexpressed

[134]. A number of transcription factors have been shown to regulate NOX4 promoter

activity including NF-NB [135], HIF1 [136], and Nrf2 [137] and these pathways are

likely dependent on the stimulus and cell type.

Xanthine oxidase

Xanthine oxidase (XO) is another enzymatic source of ROS and plays an essential role

in several oxidative stress-related diseases [138]. Several reports suggested that

18
oxidative stress associated with aging is related to increased expression and activity of

XO in liver, cerebral cortex and plasma of old mice [138] and in the aortic wall [139,

140], but not in coronary arterioles of aged rats [141] when compared to adults.

Inhibition of XO has been shown to reduce oxidative stress and to improve skeletal

muscle function in aged mice [142]. In the same sense, XO seems to contribute to

pressure-induced superoxide formation in mesenteric arteries from old but not young

rats [143]. However, the role of this enzyme as a source of oxidative stress in vascular

aging is controversial in humans. Although a positive correlation between XO activity

and age has been found in human plasma [140], no differences were observed in

vascular endothelial protein expression of XO between young and older subjects [67,

144]. In the same way, mRNA expression of XO was unaltered and oxypurinol failed to

modify bradykinin-evoked relaxations in mesenteric arteries derived from old subjects

[15]. Furthermore, in vivo inhibition of this enzyme with allopurinol did not improve

aged-dependent endothelial dysfunction [144].

NOS uncoupling

Nitric oxide synthase [54] normally produces the potent vasodilator NO by catalyzing

the conversion of L-arginine to L-citruline. This normal function of endothelial NOS

(eNOS) requires dimerization of the enzyme, the substrate L-arginine, and the essential

cofactor tetrahydrobiopterin (BH4) [145]. However, in the absence of either L-arginine

or BH4, eNOS can produce superoxide. This phenomenon has been referred to as NOS

uncoupling [146]. There is a growing body of evidence that eNOS uncoupling plays a

role in increased ROS production associated to the aging process [28].

There are a number of potential mechanisms leading to eNOS uncoupling. The

availability of the cofactor BH4 may be limited due to a decreased synthesis or an

19
increased oxidation. In this situation eNOS becomes uncoupled, and superoxide is

produced [147]. In addition this increase in the level of superoxide or peroxynitrite

induces the oxidation of BH4 to BH2, further lowering the bioavailability of BH4 [148-

150]. This mechanism has been shown by Delp et al. in aging rat skeletal muscle

arterioles [151], by Yang et al. in small arteries of aged mice [66] and by Antoniades et

al. [152] in human vessels. Furthermore, the treatment of aged vessels with sepiapterin

(exogenous BH4), increased endothelium-dependent vasodilation [66, 149, 151].

However, in large conduit arteries, neither the concentration of BH4 nor the ratio of

reduced BH4 to the oxidation products was different between young and aged mice

[153].

In human studies, BH4 supplementation increased forearm blood flow in response to

acetylcholine in healthy elder subjects [69]. In addition, treatment of isolated mesenteric

arteries from healthy aged subjects with BH4 ameliorated dysfunctional endothelium-

dependent relaxations, suggesting that the enhancement of the intracellular levels of

BH4 would prevent NOS uncoupling, in this case likely involving the inflammatory

inducible iNOS isoform [15].

In addition to these BH4-related mechanisms, other sources of NOS uncoupling has

been described in different vascular beds and experimental models. Activation of

mammalian target of rapamycin (mTOR)/S6K1 signalling has been demonstrated to

play a causal role in endothelial aging and endothelial dysfunction related to eNOS

uncoupling in a cell culture model and in a rat aging model [154].

Limited L-arginine availability also promotes eNOS uncoupling [155]. From a

biochemical point of view, a reduction in the availability of the substrate is difficult to

sustain because the endothelial concentration of L-arginine is more than one order of

magnitude higher than the substrate concentration required for the optimal function of

20
the enzyme. However, an improvement in endothelial function in older subjects after

oral administration of L-arginine has been reported [156], raising the concept of the “L-

arginine paradox”. A similar observation was obtained in cutaneous microcirculation of

aged subjects [157] but this was not confirmed in braquial artery flow-mediated dilation

in old subjects after the intra-braquial infusion of L-arginine, despite a 23-fold increase

in its plasmatic concentrations [158]. Although a lower availability could be explained

by a reduction in the transport of L-arginine to endothelial cell, there is evidence

showing that age-related deterioration of endothelial function is not associated to a

change in the transport of L-arginine [159]. Other possible explanation for the lower

availability of L-arginine with aging could be related to an increased expression and/or

activity of arginase, the enzyme that degrades L-arginine, leading to a decrease in

substrate availability for eNOS and the consequent reduction of NO synthesis [160].

Increasing evidence suggests that upregulation of arginase functionally inhibits NOS

activity and contributes to the pathophysiology of age-related vascular dysfunction

[160-162]. Furthermore, pharmacological inhibition and antisense knockdown of

arginase 1 restored endothelial NO production and endothelial function ex vivo [163]

and endothelial vasodilation was recovered after arginase inhibition in elderly subjects

[164]. Recently, it was documented that iNOS induction and arginase activation in the

aged rat aortic endothelium contribute to eNOS uncoupling and therefore to increased

ROS production, which promotes endothelial dysfunction and arterial stiffening [165].

In vivo chronic arginase inhibition restores eNOS coupling and improves endothelial

function and arterial compliance in aging vasculature [165]. Increase in endothelial

concentrations of asymmetric dimethylarginine (ADMA) could represent a compelling

explanation for the “L-arginine paradox”. ADMA inhibits eNOS activity by competing

with L-arginine for binding sites on this enzyme [166]. Dimethylarginine

21
dimethylaminohydrolase II (DDAH II) is the predominant isoform responsible for

ADMA metabolism in endothelial cells, which, interestingly, is inhibited by ROS

leading to ADMA accumulation [167]. A possible role for this compound has been

proposed in the physiological process of aging, since a positive correlation has been

reported in healthy subjects between the plasmatic levels of ADMA and age [168].

Moreover, it has been described an ADMA accelerating effect of endothelial cells

senescence [169]. Although some authors could not find an increase in endothelial

production of ADMA in older subjects with respect to young subjects [158], other

investigators found a significant increase in plasma levels of ADMA in subjects older

than 70 years and confirmed that the recovery of flow-mediated dilation after L-arginine

infusion was associated with the normalization of the L-arginine/ADMA ratio [156].

Interestingly, although the majority of the reports point to eNOS as the isoform that can

be uncoupled producing ROS [28], iNOS isoform can also serve as a source of ROS in

the absence of sufficient amounts of substrate (L-arginine) or cofactor (BH4) [100].

Cross-talk between ROS sources

Conciliation of evidences pointing to different sources of ROS as the predominant

contributor to vascular aging may be possible looking at the cross-talk pathways

existing between the systems responsible for ROS generation. Increased concentrations

of ROS originating in anyone of the systems can lead to stimulation of the production of

mitochondrial ROS, activation of NADPH oxidases, conversion of xanthine

dehydrogenase to xanthine oxidase or uncoupling of NOS [170]. Particularly interesting

and relatively well delineated is the cross-talk between mitochondrial ROS and NADPH

oxidases. In this regard, increase in mitochondrial ROS production induced by

angiotensin II (AngII) is dependent on both NADPH oxidase activity and activation of

22
mitochondrial ATP-sensitive potassium channels (mitoKATP) [171]. These channels are

activated by superoxide and H2O2 [172] and its opening causes mild mitochondrial

uncoupling and ROS production [173]. In addition, mitochondria-derived ROS enhance

endothelial oxidative stress by a feed-forward mechanism involving mitoKATP [171].

In the other way, AngII-induced NADPH oxidase stimulation is abrogated in human

aortic endothelial cells transfected with the mitochondrial SOD2 or treated with the

mitochondria-targeted SOD mimetic, mitoTEMPO. In contrast, depletion of SOD2

enhances AngII-induced NADPH oxidase activation and even increases NADPH

oxidase activity in the absence of stimuli [170, 174]. Additionally, mitochondrial ROS

stimulation by hypoxia causes NADPH activation via PKC [175]. This feed-forward

cycle in which NADPH oxidases increase mitochondrial ROS that further activates

NADPH oxidases and increases superoxide and H2O2 leading to BH4 depletion/eNOS

uncoupling [176, 177] and reduced NO bioavailability could be a reasonable picture of

endothelial dysfunction associated with oxidative stress in vascular aging (Figure 1).

Defence against oxidative stress in vascular aging

As previously stated, oxidative stress may result from increased ROS generation,

defective antioxidant defense system or both. In contrast to the existing consensus

regarding the increase in ROS production as a hallmark of vascular aging, the results

obtained from studies in which antioxidant defense levels were determined have been

controversial.

The presence of superoxide anion definitely impacts biological availability of NO [178].

An efficient defence mechanism against superoxide radicals produced during oxidative

stress is provided by the activity of superoxide dismutase (SOD). The three distinct

isoforms of SOD, the cytosolic copper-zinc SOD (Cu/ZnSOD, SOD-1), mitochondrial

23
manganese (MnSOD, SOD-2), and extracellular SOD (ecSOD, SOD-3), have evolved

as the key enzymatic system for converting oxygen radical to hydrogen peroxide and

molecular oxygen [179]. Although the increase in ROS production in aged vessels is

generally accepted, a consensus with respect to the impact of aging on the vascular

expression of SOD has not been reached. For instance, unaltered expression of

Cu/ZnSOD and MnSOD isoforms and elevated expression of ecSOD has been reported

in conduit arteries from aged rats [132] while microvasculature from aged rats displayed

increased expression of all three SOD isoforms [180]. Unaltered expression of

Cu/ZnSOD and MnSOD isoforms has also been observed in pulmonary [181] or

coronary [141] arteries of aged rats. Furthermore, similar expression of Cu/ZnSOD and

MnSOD was detected in aortas from rodents with disparate longevity [92]. However,

other studies suggest that vascular aging could be associated with reduced

expression/activity of different SOD isoforms [31, 107, 109, 115].

The ecSOD is the major SOD isoform in the vascular extracellular space, thereby it

protects against NO inactivation by free radicals during its diffusion to smooth muscle

[182]. In studies using ecSOD deficient mice it was demonstrated that this isoform

plays an essential role in protection against oxygen free radical mediated endothelial

dysfunction during the aging process. In fact, endothelium-dependent vasodilation

induced by acetylcholine was impaired in both old and adult ecSOD-/- mice when

compared to wild type mice at each age while the infusion of the SOD mimetic

TEMPOL improved endothelial function in these animals [183]. Reduced mRNA levels

of ecSOD, but not that of Cu/ZnSOD or MnSOD, were described in aged when

compared to younger mice [183]. Those findings are consistent with a previous study in

ecSOD-/- mice, where the absence of compensatory up-regulation of the Cu/Zn SOD or

24
MnSOD isoform was reported [184]. Furthermore, reduced ecSOD activity has been

shown in plasma from older healthy subjects [185, 186].

MnSOD is the main antioxidant enzyme that scavenges superoxide anions in the inner

mitochondrial matrix, and acts as a first line of defense against mitochondrial oxidative

stress [187]. Various aging models revealed that MnSOD was impaired in parallel to an

increase in oxygen free radical and protein tyrosine nitration that results from enhanced

peroxynitrite formation [85]. Studies done in MnSOD deficient mice demonstrate the

detrimental effect of superoxide anions and therefore of ROS and Reactive Nitrogen

Species [188] formation in mitochondria. Indeed, MnSOD-/- deficient mice die soon

after birth while MnSOD +/- animals show age-dependent endothelial dysfunction and

enhanced atherosclerosis [189]. Lesions in mitochondrial DNA (mtDNA) increase in an

age dependent fashion and are severely augmented in MnSOD +/-, due to the close

proximity of the mtDNA to the ROS formation which makes mtDNA particularly

sensitive to mutations and to oxidative damage [108]. Several studies reported

decreased expression of MnSOD antioxidant enzyme in aorta of old mice [97, 108]. In

humans, the available results are controversial; while some authors have not observed

an alteration in MnSOD expression in endothelial progenitor cells with aging [190],

others have reported a decrease of this enzyme expression in the endothelial cells of

sedentary old subjects [186].

Cu/ZnSOD is the predominant isoform in vascular vessels [179]. Like the two other

isoforms, its activity is necessary to limit the increase in superoxide and therefore to

maintain normal endothelial vasodilations. Studies from animal models suggested that

normal Cu/ZnSOD expression protects endothelial function and that a deficiency in a

single copy of its gene accelerates endothelial function with aging [191]. In humans, a

25
reduction in Cu/ZnSOD protein expression and total SOD enzymatic activity was

observed in aged mesenteric lymphatic vessels [192].

Definitely, each isoform of SOD expressed in a different location of the vessel wall,

seems to play an important specific role in protecting vascular function during the aging

process.

Response to oxidative stress – ROS signaling

In addition to its above discussed deleterious effects, ROS generation also functions as a

signaling system that is activated in specific circumstances (metabolic alterations,

hypoxia,…) and promotes a response by the cell. Incapacity to overcome acute ROS

generation or inability to generate an adequate response may constitute an important

problem that could account for vascular dysfunction in aging. Among transcription

factors involved in cellular response to ROS generation, nuclear factor erythroid-2-

related factor-2 (Nrf2) is an evolutionarily highly conserved redox sensitive

transcription factor that is activated by ROS production in the vasculature [193, 194].

Activation of Nrf2 up-regulates the expression of numerous genes for proteins that

detoxify ROS or have antioxidant properties. It has been proposed an important role for

Nrf2 in regulating the aging process by orchestrating the cellular response to oxidative

stress [195]. Despite an increase in superoxide production, aortae from aged rats display

downregulation of Nrf2 mRNA and protein as well as nuclear activity of Nrf2 that

results in a decrease of its target genes (NADPH quinone oxidoreductase-1, -

glutamylcysteine synthetase and heme oxygenase-1) which inversely correlate with the

expression of NF-B target genes [88]. Similar results were observed in carotid arteries

from aged non-human primates [196]. These studies also revealed defective Nrf2

activation and increased sensitivity to apoptotic effects in response to H2O2 or high

26
glucose [89, 196]. An impairment of Nrf2 system could contribute to the reduced

angiogenic capacity of aged vasculature and its defective response to ischemic injuries

since disruption of Nrf2 signaling impairs angiogenic processes in human coronary

arterial endothelial cells [197]. Although the analysis of different species with disparate

longevity suggest that lifespan potential is inversely related to ROS production by

mitochondria [198], other evidences grant a key role to stress resistance rather than to

reduced ROS production in obtaining larger healthspan and longevity. This is based in

the fact that extremely long-lived rodents, such as naked mole rat, show ROS

production similar to mice and intracellular pro-oxidant conditions [199]. The

contributing factor to a positive healthspan and extended longevity would rely in

advantageous cytoprotection and stress resistance, resulting in stable proteome and

genome that retard cellular senescence and aging and confers enhanced protection

against inflammation and degenerative processes [188]. Together with upregulation of

p53, cytoprotection and stress resistance in naked mole rats is likely afforded by

enhanced expression/activity of Nrf2 [195], a key role that would be conserved among

phylogenetically distant organisms, as suggested by the fact that genetic activation of

Nrf2 signaling causes significant increases in lifespan of invertebrates [200, 201]. In

this sense, the naked mole rat differs from mice in displaying a significant higher

resistance to arterial apoptosis and DNA fragmentation induced by exposure to H2O2

[202]. Vascular Nrf2 dysfunction associated to aging would exacerbate cellular

oxidative stress and increase the sensitivity of aged vessels to cellular damage.

Furthermore, endothelium-dependent relaxation impairment caused by H2O2 addition in

aorta is exacerbated in old rats despite an upregulation of catalase activity [203]. In fact,

vasodilation of soleus feed arteries from young rats in response to H2O2 is blunted in

aged rats [96]. This idea of an age-related failure of oxidative stress-mediated signalling

27
is clearly exemplified by evidences obtained in skeletal muscle. Muscles contraction

generates a redox-sensitive increase in cytoprotective heat shock proteins and

antioxidant defense enzymes that is abolished in muscles from old rodents [204, 205].

Muscles from old human and mice show chronic constitutive activation of redox-

sensitive transcription factors and an inability to further activate these transcription

factors following muscle contraction [205, 206]. Then, in muscle from young and adults

the increase in ROS following contraction triggers transcription of cytoprotective

proteins. In contrast, an elevated ROS production in old muscles at rest drives

constitutive expression of redox-sensitive transcription factors such as NF-B that

impedes the additional transcription of cytoprotective genes following contraction,

making these muscles vulnerable to oxidative stress [207]. The role of excessive ROS

accumulation in this phenomenon is supported by the fact that is mimicked by deleting

SOD1 in young mice [208]. Further evidences in human mesothelial cells have been

provided. High concentrations of early glycosylated proteins (Amadori adducts) trigger

the expression of NF-B-related pro-inflammatory genes (TNF, IL-6, IL-1, COX-2,

iNOS) through ROS generation in human mesothelial cells [209]. However, mesothelial

cells from old subjects do not display such a response to the exposure to this stimulus,

associated to an elevated basal expression of NF-B-related pro-inflammatory

molecules [210], an increased expression that correlates with age and is sensitive to

antioxidants [211]. Nrf2 and NF-B behave as antagonistic pathways. Then, diverse

Nrf2 activators, such as phenethyl isothiocyanate (PEITC) attenuate lipopolysaccharide

(LPS)-induced NF-B activation [212]. Furthermore, activation of Nrf2 promotes

suppression of NF-B signaling by inhibiting IKK/IB phosphorylation and p65 NF-B

subunit nuclear translocation [213]. Conversely, it has been reported that NF-B

competes against Nrf2 for transcription co-activator CREB binding protein (CBP),

28
directly repressing Nrf2 signaling at the transcription level [21]. This mechanism could

be additive to the recruitment of histone deacetylase 3 (HDAC3) by NF-B causing

local hypoacetylation to hamper Nrf2 signaling [214].

Interestingly, acute handgrip exercise causes vasodilation of brachial artery that is

blunted in older subjects. This vasodilation is, at least partially, mediated by acute ROS

generation since it is inhibited by administration of an antioxidant cocktail (vitamins C

and E, and -lipoic acid) in young but not in older subjects [215]. This suggests that

acute exercise triggers ROS generation that leads to vasodilation in young arteries but

the elevated basal ROS production hampers ROS-mediated signaling in aged arteries

causing altered vascular function. Supporting this concept, endothelium-dependent

vasodilation in soleus arterioles has been reported to involve ROS signaling and

exercise training-induced enhancement of endothelial function is partially produced by

stimulating ROS signaling [216]. Thus, multiple evidences demonstrated an inadequate

response of aged tissues, including vascular tissue, to the signaling triggered by acute

ROS generation due to the presence of an age-related chronic increase in ROS

production that precludes the appropriate response.

ROS have been demonstrated to be beneficial by triggering hormetic signals that protect

the organism from later insult. In this sense, neural differentiation in N2a neuroblastoma

cells is dependent of superoxide anion production that increases mitochondrial

biogenesis through MAPK activation [217]. In seals, apnea stimulates oxidative stress

and hypoxic hormetic responses that include Cu-ZnSOD, catalase, HIF-1 and Nrf2

upregulation [218]. Finally, oxidative stress-induced hormesis is associated with

reduction of mitochondrial superoxide production by activating antioxidant system in

mutant invertebrates with expanded longevity [219], suggesting that preserved capacity

of ROS to trigger hormetic responses could be important in aging.

29
INFLAMMATION

In addition to increased oxidative stress, inflammation outstands as a determinant

process in the development of vascular aging. It is difficult, in fact, to understand the

impact of these factors without each other, since numerous interplays exist between

inflammation and oxidative stress and vice versa (Figure 2). One of the key changes to

arteries with age is the development of chronic, low-grade inflammation [220] which

has been referred as “ inflammaging” [221]. Molecular inflammation is a possible

converging process linking normal aging and age-related pathological processes [222].

Furthermore, the increase in inflammatory markers with aging is not only independent

of traditional CV risk factors, but it also accelerates arterial thickening and stiffness

independently of traditional CV risk factors levels [223]. Abundant data demonstrate an

increase in systemic inflammatory markers such as Tumor Necrosis Factor alpha (TNF-

D), Interleukin 1beta (IL-1E), members of the super family of Interleukin 6 (IL-6), as

well as higher levels of C-reactive protein (CRP) in plasma of older subjects when

compared with young adults [224]. This increase is positively correlated with age,

independently of other cardiovascular risk factors [225]. Among the above mentioned

cytokines, the pleiotropic pro-inflammatory IL-6 has been importantly implicated in age

associated vascular disease [226]. Indeed, high plasma levels of IL-6 are correlated with

greater disability and mortality in older people [227]. The levels of CRP are also

associated with increased arterial stiffness in middle-aged and elderly subjects [228,

229]. On the other hand, expression of MCP-1 and matrix metalloproteinase are greater

in the thickened arterial intima of vessels obtained in autopsies from older people

compared with those from young adult [230].

30
In experimental models, there is emerging evidence concerning the association between

aging and vascular inflammation [90, 220, 226, 231]. Inflammation involves the

activation of the transcription factor NF-NB viewed as the master regulator of the

inflammatory molecules including TNF-D, interleukins (IL-1E, IL-2, and IL-6),

chemokines (IL-8 and RANTES), adhesion molecules (ICAM, and VCAM), and

enzymes (iNOS and COX-2) [232]. Previous reports demonstrated an increase of the

inducible isoform [54] protein expression associated with aging in rats [165],[141].

Moreover, iNOS mRNA expression is enhanced in several human cell types from aged

subjects, ranging from mesothelial cells [211] to microvessles [15]. Many evidences

have reported that iNOS expression is mainly localized to the endothelium in arteries

from aged animals [60, 141]. On the other hand, it was found that SMCs from the media

of old rats have a much higher capability to produce NO than SMCs of young adult rats,

in part due to their differences in their ability to transcribe iNOS gene [233].

Furthermore, iNOS expression is particularly prominent in the type of SMCs that

populate the arterial intimal lesion at the site of injury, whereas SMCs derived from the

normal arterial media express much lower levels or iNOS [234]. Experiments made in

apoE/ mice revealed that iNOS absence resulted in a reduction of advanced

atherosclerotic lesions while its presence contributed to the oxidation of LDL by

activated smooth muscle cells [235].

An age related up-regulation in TNF-D has been described in coronary arteries,

associated with a gene expression profile suggestive of an inflammatory response [90,

236]. In fact, administration of exogenous TNF-D can induce oxidative stress by

activating NADPH oxidase [237], endothelial dysfunction [238], endothelial apoptosis

[236], and up-regulation of pro-atherogenic inflammatory mediators, such as iNOS and

31
adhesion molecules. In addition, exposure to TNF stimulates mitochondrial superoxide

production in human retinal endothelial cells [239]. Moreover, chronic TNF-D

inhibition improves flow-mediated arterial dilation in resistance arteries of aged

animals, while reduces ICAM-1 and iNOS expression [240, 241]. All these effects

closely mimic aging-induced functional alterations of the vascular endothelium [90].

The proinflammatory phenotype accompanying arterial aging is likely involved in the

impairment of endothelium-dependent dilations. In this sense, selective inhibition of

iNOS partially reversed the endothelial dysfunction associated with aging in human

microvessels [15]. Moreover, COX-derived molecules are also involved in the defective

endothelial responses that are associated to the normal vascular aging. A lost of the

prostacyclin-mediated vasodilator responses together with the release of a contractile

factor (thromboxane A2) is the most outstanding features [15].

Inflammatory cells could also play a role in vascular dysfunction associated to aging. In

this sense, in healthy subjects older than 55 years, neutrophil, eosinophil and monocyte

count (within the normal ranges) as well as myeloperoxidase activity inversely

correlated with forearm blood flow responses, which were improved after BH4

administration [242]. This suggests that moderate increment of inflammatory cells

through myeloperoxidase activity would reduce BH4 availability compromising NO-

mediated vasodilation.

In addition to impact the functional status, inflammation directly participates in

processes leading to alterations in the structure of arterial wall with aging. MMPs are

up-regulated by pro-inflammatory cytokines [243, 244] and local inflammation is

indeed responsible for the up-regulation of MMPs in aged vasculature [245].

Nuclear factor-kappa B (NF-NB) and vascular aging

32
NF-NB is a redox sensitive transcription factor encoded by members of the Rel gene

family. Under non-inflammatory conditions the heterodimeric Rel domain subunits, p50

and p65, are constitutively expressed in the cytoplasm bound to the inhibitory protein

INB that mask the NF-NB nuclear localization signal (NLS) and prevents its activity

[246]. NF-NB activation via the canonical pathway is mediated by the upstream INB

kinase [165], a heterodimer consisting of 2 catalytic subunits, IKKD and IKKE, and a

regulatory subunit termed IKKJ or NF-NB essential modulator (NEMO) [247]. In

response to a variety of factors, including proinflammatory cytokines, pathogens,

oxidative stress, and growth factors, IKK is activated and phosphorylates INB, leading

to its polyubiquitination and subsequent proteasomal degradation. INB degradation

allows p50 and p65 to translocate to the nucleus which in turn activates a myriad of

genes that mediate diverse cellular processes such as inflammation, proliferation,

apoptosis and cellular senescence [248]. Numerous studies report increased NF-NB

activity with aging. Binding of NF-NB to DNA is increased in skin, liver, kidney,

cerebellum, cardiac muscle, gastric mucosa and aorta of old rodents compared with that

in young rodents [249-254]. Furthermore, NF-NB was identified as the transcription

factor most associated with mammalian aging, based on patterns of gene expression

[255].

In human studies, augmented expression and activity of NF-NB has been documented in

peritoneal mesothelial cells accordingly to age [211]. Donato et al [67] found that

endothelial cells obtained from older non-diabetic adults have greater protein expression

of NF-NB than young adults. This NF-NB increase was positively related to oxidative

stress as determined by nitrotyrosine content. Moreover, an in situ age-dependent NF-

NB activation has been demonstrated in the vascular wall of human mesenteric

microvessels from aged subjects by Southwestern histochemistry techniques, which

33
clearly correlates with the development of endothelial dysfunction [15].

There is an emerging view that interactions between inflammation and oxidative stress

occurs, as different redox-sensitive transcriptional factors such as AP-1 and NF-NB are

activated by ROS, increasing the gene expression of cytokines (TNF-D, IL-1, and IL-6),

adhesion molecules (ICAM, VCAM), and pro-inflammatory enzymes (iNOS, COX-2)

[256] (Figure 3). In this regard, Ungvari et al [257] recently presented advances on the

role of mitochondrial-derived ROS in activating NF-NB downstream signalling inducing

a characteristic pro-inflammatory phenotype in aging. They reported that oxygen free

radical overproduced in the mitochondria of both vascular endothelial and smooth

muscle cells from aged rat arteries, is dismutated to H2O2 by means of Mn-SOD. The

increased release of H2O2 was the responsible for NF-NB activation in the cytoplasm of

aged cells. Although NF-B seems to be the main transcriptional factor mediating

H2O2-induced response in vascular cells, senescence elicited by H2O2 have been

proposed to be mediated by other transcriptional factors such as STAT-1 in human

glomerular mesangial cells [258] and p38 MAPK-ATF-2 in human fibroblasts [259].

On the other hand, specific functional inhibition of endothelial NF-B signalling in

mice prevented age-related insulin resistance and vascular senescence, prolonging life

span. This was associated with decreased oxidative stress, increased muscle blood flow,

enhanced locomotor activity and upregulation of mitochondrial sirtuin-related proteins

in the aorta of these mice [260]. Interestingly, the NF-NB inhibitor salsalate efficiently

increased the expression of NF-NB inhibitor INB and reduced NF-NB in endothelial

cells, as well reduced the expression of NADPH oxidase p47phox and the levels of

nitrotyrosine in association to an improved brachial artery flow-mediated dilation in

older humans [261].

34
OTHER FACTORS INVOLVED IN VASCULAR AGING IN CONNEXION TO

OXIDATIVE STRESS AND INFLAMMATION

Sirtuins

Sirtuins are members of the Sir2 (silent information regulator 2) family, a group of class

III deacetylases [262]. Sirtuins comprise seven members in human and other mammals

and are found in different subcellular locations including the nucleus (Sirt1, Sirt6,

Sirt7), cytosol (Sirt2), and mitochondria (Sirt3, Sirt4, Sirt5). Their enzymatic activity is

nicotinamide adenine dinucleotide (NAD+)- dependent, so that they are directly linked

to the metabolic and redox state of the cell through multiple signalling pathways [263].

As described above, the aging process is associated with a decline in NO- dependent

endothelial vasodilation as a result of impaired eNOS activity and decreased NO

bioavailability caused by increased in superoxide anions. Sirt1 plays a fundamental role

in regulating eNOS activity by deacetylating it to increase NO production [264]. In this

sense, NO-mediated relaxations, assessed using pressure myography in isolated middle

cerebral resistance arteries under physiological levels of shear stress, were significantly

attenuated in the presence of the Sirt1 blockers, sirtinol, and nicotinamide [265].

Previous studies in aorta and femoral arteries have shown that Sirt1 inhibition, either by

viral [264] or pharmacological strategies [266], attenuates endothelium-dependent

dilations.

At present, endothelial dysfunction in arteries from aged mice and humans is associated

with a reduction of vascular expression of Sirt1 [266]. This reduction occurs in the

absence of changes in total eNOS and is associated with an increase in acetylated

eNOS. Treatment with sirtinol eliminated the age-related difference in NO-dependent

endothelium-dependent dilation, without affecting vascular smooth muscle sensitivity to

NO (endothelium-independent dilatation) [266]. Previous studies suggest that Sirt1

35
activation confers vasoprotection in aged rodents. In aorta form older mice, both protein

expression of Sirt1 and eNOS phosphorylation at serine 1177 were lower and acetylated

eNOS were higher when compared to younger animals [267]. Most available work has

focused on the ability of Sirt1 to deacetylate eNOS, while there is much less work in the

vascular smooth muscle cell (SMC). Senescent SMC accumulate in the arteries of aged

animals [3]. SMC exhibit increased expression of genes that contribute to lesion

instability, including adhesion molecules, PAI-1, and matrix metalloproteinases [268].

Recently, it has been shown in cultured human vascular SMC that overexpression of

nicotinamide phosphoribosyltransferase (Nampt), the rate-limiting enzyme for NAD+

salvage from nicotinamide, conferred resistance to acute oxidative stress, delayed

senescence, and increased replicative lifespan [269]. These effects were mediated

through increased activation of Sirt1, resulting in enhanced Sirt1-mediated degradation

of p53. Furthermore, it has been observed that overexpression of Sirt1 reduces

expression of the angiotensin II type 1 receptor (AT1R) [270]. Interestingly, inhibition

of AT1R has been shown to prevent endothelial dysfunction of cerebral arterioles [271].

Further studies are required to address the role of smooth muscle-derived Sirt1 on

vascular function associated to aging.

Sirt1 has recently emerged as a potent inhibitor of the NF-NB system, providing a

mechanistic link between inflammation and aging [272]. Sirt1 binds and deacetylates

RelA/p65, inhibiting the transcriptional activity of NF-NB [273]. Furthermore, with age

the decline in Sirt1 activity and expression in skeletal muscle are accompanied by

increased inflammation, oxidative stress, and reduction in ability to rebuild muscle after

injury or in response to exercise [274]. Like Sirt1, Sirt6 is able to repress NF-NB activity.

Kawahara et al, showed that Sirt6 is physically present at promoters of genes activated

by NF-NB [275].

36
Interestingly, the role of Sirt1 during aging seems to involve the orchestration of

different stress response pathways. This involves targeting of multiple transcriptional

regulators, besides NF-NB such as p53, FOXO, and HSF1 [276]. For example, Sirt1 is

involved in stress responses, cellular metabolism, and aging through deacetylation of

p53 [277]. Activated p53 then enhance ROS production through mitochondrial

dysfunction and/or increased expression of genes that are involved in redox modulation,

such as the p53-upregulated modulator of apoptosis (PUMA), NADPH oxidase

activator A (NOXA), and p53-induced gene 3 (PIG3) [278, 279]. Furthermore, it was

described that p53 via p21 indirectly activates Nrf2, a central transcription factor in the

antioxidant response. Nrf2 counteracts oxidative environment by binding to the

antioxidant-response element 2, and, via increased antioxidant gene expression [280].

Several other sirtuins have been shown to interfere with HIF signalling. For example,

Sirt6 acts as a co-repressor of HIF-1D by deacetylating histones at HIF-1D responsive

promoters [54]. SIRT3 attenuates HIF-1D activity indirectly by controlling intracellular

ROS levels [281]. These results suggest a central regulatory function of sirtuins in the

cellular response to hypoxia [282].

On the other hand, an interaction between sirtuins and oxidative stress has been

documented. Strong evidence supports a role for Sirt1 mediating an oxidative stress

response by directly deacetylating several transcription factors that regulate antioxidant

genes. Notably, Sirt1 activates several members of the FOXO family of transcription

factors that promote the expression of stress response genes including SOD2 [283].

More even, it was described that mitochondrial sirtuin, Sirt3 controls the flow of

mitochondrial oxidative pathways and, consequently, the rate of production of reactive

oxygen species. Sirt3-mediated deacetylation activates enzymes responsible for

37
quenching reactive oxygen species, and thereby exerts a profound protective action

against oxidative stress-dependent pathologies [284, 285].

The beneficial effects described in the literature of low caloric intake are mediated by

members of the sirtuin family. Precisely how Sirt1 functions during CR remains an

open question, but emerging evidence suggests that p53 plays an important role in

modulating Sirt1 during CR [286]. Given that Sirt1 directly deacetylates HSF1 and

therefore regulates the heat shock response, it is possible that the positive effect of

sirtuin on lifespan might be mediated through a dynamic preservation of proteostasis

[276].

Taken together, all available data point at sirtuins as a promising therapeutic target for

drug development to act on age-related cardiovascular diseases. (Reviewed extensively

in reference [287]).

Hypoxia-inducible factor

Vascular system responds to ischemic insults by promoting angiogenesis and

vasculogenesis to recover blood perfusion and avoid tissue damage. Advanced age is

associated with a decrease in the ability to drive angiogenesis in response to ischemia

[18] that results in higher rates of cardiovascular complications and decreased capacity

for tissue preservation/regeneration [27]. Hypoxia-inducible factor-1 (HIF-1) is the

master regulator of cellular adaptation to hypoxia and orchestrates neoangiogenic

response to tissue ischemia [288, 289]. Under normoxic conditions, key prolines in the

oxygen-dependent degradation domain of HIF-1 are hydroxylated by prolyl

hydroxylases (PHD). This hydroxylation leads to rapid polyubiquitination and

degradation by proteasome. When oxygen concentration falls down, PHDs cannot

hydroxylate HIF-1 prolines, allowing dimerization with HIF-1ß subunit to bind DNA

38
at hypoxia response elements (HRE) of target genes. Growing evidence suggests that

impaired angiogenic response to ischemia in aged vasculature relies on a defective

activity of HIF-1 system in vascular aging. Supporting this concept, old mice had

impaired tissular capacity to form HIF-1/HRE complex in response to hypoxia [290]

and reduced transcriptional activity of HIF-1 was also detected in aortic smooth

muscle cells from old rabbits [291]. Similarly, defective upregulation of HIF-1 in

hypoxic brains of old rats has been observed [292]. In mouse and human hearts,

increasing age is associated to decreased expression of HIF-1 while expression of

PHD3 increases [293], an increase that can be counteracted by caloric restriction in rats

[294]. Elevated levels of PHD activity, due to impaired downregulation of PHD1 in

ischemia, could also be responsible for the defective upregulation of HIF-target genes in

ischemic brain of aged mice [295]. In fact, adenoviral delivery of constitutively active

HIF-1 reverses the inability of aged mice to recover perfusion and motor function in

ischemic hindlimbs [288, 296]. Similar results have been obtained by stabilization of

HIF-1 with deferoxamine [297]. Upregulation of HIF-1 also restores the blunted

inotropic response of hearts from old rats [298]. EPCs play a key role in angiogenic

processes. As discussed above, functionality of EPCs accounts for the impaired

reparative capacity of aged vasculature. HIF-1 is a crucial regulator of EPC function and

homing through activation of its target gene coding for stromal cell-derived factor-1

(SDF-1) [299]. In this sense, defective HIF-1 stabilization in ischemic tissues of aged

mice impairs EPC recruitment for angiogenic response [300].

The involvement of HIF-1 in longevity deserves separate mention. Data on this issue

has been mostly obtained by using C. elegans model. However, disparate effects of

HIF-1 on longevity of these worms seems to be produced. Loss of function of HIF-1

was shown to extend lifespan in C. elegans [301] but increased function of HIF-1 was

39
also demonstrated to promote longevity and to increase resistance to oxidative stress in

these animals [302]. In this direction, Lee et al. reported that inhibition of respiration

promoted longevity in C. elegans by activating HIF-1 [303]. Some light on the role of

HIF-1 in C. elegans longevity was brought by the studies showing that HIF-1

knockdown increases in longevity at 25ºC but impairs healthspan at 15ºC [304]. This

suggests that HIF-1 activity is beneficial for longevity when the organism is exposed to

stressing conditions. HIF-1 activity also increases lifespan of human fibroblasts [305].

This ability could be related to the stimulating effects of HIF-1 on hTERT expression

[306]. In this sense, an increased expression of HIF-1 has been detected in long-lived

Mlck (+/-) mice [307]. Lifespan extension caused by inhibition of respiration in both C

elegans and human cells involves mitochondrial ROS generation that triggers HIF-1

activity [303, 305]. It is conceivable that a chronic excessive production of ROS

associated with vascular aging would hinder an adequate response to hypoxia-induced

ROS signalling to activate HIF-1 system in response to ischemia.

In summary, there is strong evidence proposing that the angiogenic response required

for vascular repair and blood perfusion recovery after ischemic insults is impaired in

aged cardiovascular system due to defective upregulation of HIF-1 in response to

ischemia. The role of HIF-1 in longevity needs further clarification although its

potential extending effects on lifespan seems to be restricted to stressing conditions.

Angiotensin II

Angiotensin (Ang II) is the major biologically active component of the renin

angiotensin system. It acts through distinct G protein-coupled receptors, angiotensin

type 1 and type 2 (AT1 and AT2). AT1 and AT2 receptors have counter regulatory

actions in the cardiovascular system [308]. Whereas AT1 activation results in

40
vasoconstriction, proliferation, pro-inflammatory and pro-fibrotic activities [309], Ang

II binding to AT2 induces vasodilation both in conduit and resistance arteries and

improves arterial remodelling in humans and mice [308]. Ang II signalling has been

widely linked to the aging process. Ang II, angiotensin converting enzyme (ACE), and

AT1 receptors are markedly increased with age within the thickened intima in several

species, including humans [230, 310, 311].

A large number of experimental studies have shown that Ang II mediates several

important events of the inflammatory process [312]. Ang II has been widely linked to an

age-associated increase in the migratory capacity of smooth muscle cell and to the pro-

inflammatory features of the arterial aging [311]. It was suggested that Ang II, acting

via AT1 receptor, induces endothelial cell activation and increases the expression of

VCAM-1, via NF-B pathway [313, 314]. Ang II promotes endothelial dysfunction

through COX-2 activation, which in turn generates vasoactive prostaglandins and ROS

[315].

On the other hand, it is clear that both increased ROS generation and activation of

redox-signalling cascades are critical events involved in Ang II-mediated actions [316].

Ang II is a potent inducer of vascular oxidative stress that triggers mitochondrial

dysfunction and cell injury [317]. The increase in mitochondrial ROS induced by Ang II

is associated with decreased NO formation in endothelial cells [171], reduced aortic

endothelium-dependent relaxation and is associated to the development of

cardiovascular alterations and hypertension [318]. Recently, it was suggested that a

possible link between Ang II and mitochondrial dysfunction is the mitochondrial

adaptor protein p66Shc[319]. However, Ang II not only stimulates mitochondrial ROS

production, but also trigers intracellular superoxide production by NAD(P)H oxidase

41
activation and uncoupling of eNOS, an effect mediated by AT1 receptors [320].

Moreover, Ang II increases NO production [316]. By this double action of increasing

both NO and oxygen free radicals Ang II leads to peroxynitrite generation, reducing NO

availability [321].

Recently, it was shown that disruption of AT1 gene promotes longevity in mice,

possibly through the attenuation of oxidative stress and overexpression of pro-survival

genes (nicotinamide phosphoribosyltransferase (NAMPT) and sirtuin 3), suggesting that

the Ang II/AT1 pathway may be targeted to influence life span in mammals [322].

In fact, in animal models the treatment with either AT1 receptor antagonist or with ACE

inhibitor ameliorates the endothelial dysfunction associated with aging in blood vessels

due, in part, to vascular oxidative stress inhibition [323, 324]. Further studies

demonstrated that genetic deficiency in AT1 receptors attenuated increases in

peroxynitrite during aging [322]. However data from humans are inconclusive.

Losartan, an AT1 receptor antagonist, had no effect on brachial flow-mediated dilation

in older adults, despite reducing blood pressure and circulating inflammatory markers

[325]. Another AT1 receptor antagonist, valsartan, improved vascular compliance in

healthy normotensive elderly individuals but once again did not affect flow-mediated

dilation [326].

THERAPEUTIC IMPLICATIONS

Improvements in our knowledge about the mechanisms involved in producing

endothelial dysfunction and vascular disease during aging have led to provocative

hypothesis raising the possibility to intervene on a process that, until very recently, was

seen as immutable: the vascular aging [327].

42
Lifestyle influence on vascular aging

In addition to pharmacological interventions, lifestyle may influence vascular oxidative

stress and regulate vascular function. Avoidance of sedentary attitudes and performing

regular exercise will likely reward with improved vascular health in older persons.

Exercise can partially reverse the effects of aging process on physiological functions

and preserve functional reserve in the elderly. Maintaining a minimum quantity and

quality of exercise decreases the risk of death and increases longevity [328]. In contrast,

sedentary habits could be extremely deleterious to health in the elderly as time spent in

sedentary behaviors is positively associated with mortality. Even more disturbing is the

fact that health risks caused by prolonged time watching television cannot be

completely overcome by exercise [329]. It is assumed that regular exercise improves

endothelial vasodilation, reduces arterial stiffness and decreases cardiovascular risk in

old people [330]. In this same regard, impaired endothelium-dependent, NO-mediated

dilation of carotid arteries from old mice was restored when the animals had access to

voluntary wheel running for 10-14 weeks [132]. This improvement of endothelial

function was associated with an increase in eNOS expression and activation but also

with a reduction of vascular oxidative stress as demonstrated by the reduction in

nitrotyrosine content and NADPH oxidase expression, and an increase in SOD

expression in the aorta from these exercising mice.

Sedentary older men display poorer endothelium-dependent flow-mediated dilation

(FMD) than older athletes, which show similar vascular responses to those observed in

younger men. The improvement in endothelial function in older athletes is accompanied

by the prevention of increased serum malondialdehyde [253] levels (a marker of lipid

peroxidation) and reduced plasma antioxidant capacity that are observed in sedentary

older men [331]. A potential explanation for these findings comes from studies

43
supporting the relationship between regular exercise and a decrease oxidative stress.

Endothelial cells from exercising older subjects showed reduced nitrotyrosine content

and decreased expression of both NADPH oxidase (p42phox subunit) and the redox-

sensitive pro-inflammatory transcription factor NF-B [186]. In addition, knee-extensor

exercise training recovers the previously blunted vasodilation of brachial artery induced

by forearm handgrip acute exercise in older subjects [215]. The reduced angiogenic

response to ischemic insults occurring in aged vasculature could also be benefited from

exercise, which stimulates ischemia-induced neovascularization in hindlimbs from aged

mice by reactivating HIF-1 signaling [332]. These evidences suggest that maintenance

of habitual physical activity in the elderly reduces vascular oxidative stress and

positively influences vascular function.

Vascular health in advanced age seems to be favoured by moderation of food intake

since caloric restriction is an interventional strategy that has been proposed to promote

anti-aging effects and could reduce morbidity and mortality from cardiovascular events

[333]. Although the mechanisms responsible for the beneficial effects of caloric

restriction on vascular function are not completely elucidated, reduction of oxidative

stress and inflammation are likely determinant factors. Improvement of endothelial

dysfunction caused by lifelong caloric restriction in old rats is accompanied by reduced

vascular ROS production, inhibition of NF-B activity and down-regulation of

inflammatory genes [334]. Even when this intervention starts in latter life,

improvements in vascular function are achieved. Old mice (28-30 months) that were

calorie restricted for 8 weeks recovered endothelial vasodilation by increasing eNOS

expression and reducing oxidative stress, as demonstrated by reduced nitrotyrosine

content, superoxide production and NADPH expression and restored SOD2 and sirtuin-

1 expression [267]. Similar results were obtained after 3 months of caloric restriction in

44
old rats [335]. Nrf2 seems to be a required factor for several of the health benefits

afforded by caloric restriction. Increased resistance against oxidative stress achieved by

means of caloric restriction can also be attained by pharmacological stimulation of Nrf2

pathway [336]. The homologue of Nrf2 in worms, SKN-1, is required for lifespan

extending effect of caloric restriction in C. elegans [337]. Deletion of Nrf2 gene

prevents the expression of antioxidant enzymes as well as the cancer protective effects

induced by caloric restriction in mice [338]. In contrast, Nrf2 seems not to be required

for caloric restriction-induced lifespan extension and increase in insulin sensitivity in

these animals [338]. However, insulin signaling influences the activity of the Nrf2

pathway [201]. In fact, the stimulation of AMP-activated kinase (AMPK) with

metformin, a hypoglycemiant drug that potentiates insulin action, induces a state similar

to caloric restriction, augments oxidative stress response and extends healthspan in C.

elegans through a SKN-1(Nrf2)-dependent mechanism [339].

The role of a decrease inflammation in the beneficial effects of caloric restriction is an

issue of controversy [267] that could be explained by the duration of the restriction.

However the majority of studies support a role for it. This is the case for the works

showing a decrease in vascular expression and activity of iNOS, the isoform of NOS

associated with local inflammatory processes [335], or a reduction of inflammatory

mediators such as prostanoids [340] and soluble adhesion molecules [341] after caloric

restriction. Overall, it is proposed that caloric restriction increases NO bioavailability,

reduces ROS generation, activates Nrf2/antioxidant response pathway, induces ROS

detoxification systems and exerts anti-inflammatory effects that suppresses

initiation/progression of vascular disease that accompany to aging [342].

Antioxidants and vascular aging

45
Prospective epidemiologic studies analyzing the effects of antioxidant consumption and

cardiovascular diseases (CVD) provided encouraging results suggesting a protective

effect of antioxidant intake. Some of these studies analyzed aged populations. The

Massachusetts Health Care Panel Study found a reduction of the risk for CVD mortality

(RR 0.54) in subjects older than 66 years consuming high amounts of carotenoids in

fruits and vegetables. This effect was indeed more pronounced in regard to coronary

disease (RR 0.25) [343]. Reduced risk of myocardial infarction (MI) (RR 0.55) was also

associated with high intake of -carotene in another study done in elderly subjects (55-

95 years) while no association was found for vitamin C or vitamin E intake [344]. In

opposite to this last finding showing the lack of influence of vitamin C, another study

including 1,214 participants aged 75-84 years showed that low blood concentrations of

vitamin C were predictive of increased cardiovascular and all-cause mortality in this

elderly population [345]. However, interventional randomized controlled trials have not

confirmed the observational findings. Despite a study demonstrating that elderly

subjects (67-105 years) taking vitamin E supplements from a sample of 11,178

individuals were relatively protected from coronary disease mortality [346], overall, the

positive findings have been few, yielding largely negative results, making necessary

further and more focused clinical research to understand these conflicting data [347].

In this regard a recent clinical trial has shown an acute reversal of endothelial

dysfunction in older people shortly after oral administration of an antioxidant cocktail

(vitamin C+vitamin E+-lipoic acid) [348], confirming previous results showing the

recovery of exercise induced vasodilation of brachial artery in elderly men after

administration of the same antioxidant cocktail [215].

Administration of high dose of ascorbic acid has been reported to increase vascular

conductance and improve resting leg blood flow in healthy older men [349].

46
Accordingly acute acid ascorbic supplementation augmented reflex cutaneous

vasodilation in aged subjects [157]. These clinical evidences demonstrate that oxidative

stress may be an important target for recovering endothelial function in aging.

Other therapeutic interventions combining antioxidant and anti-inflammatory activities

would be definitely interesting in the prevention/reversion of vascular dysfunction

associated with aging. The plant-derived polyphenol, resveratrol (3,5,4'-

trihydroxystilbene) has been shown to possess these characteristics and to have a role in

preventing vascular dysfunction in the elderly [350]. Mice treated with resveratrol after

the first age of life showed a marked reduction in signs of aging including reduced

endothelial apoptosis, increased aortic elasticity and greater motor coordination which

were associated with decreased inflammation [351]. Resveratrol prevents pro-

inflammatory phenotype in vascular smooth muscle cells from old non-human primates

[352] but also increases resistance to oxidative stress in vascular cells [257], probably

through the activation of Nrf2-mediated antioxidant response [116] and sirtuin-1 [353].

In addition, moderate consumption of red wine or the equivalent dose of resveratrol

improves endothelium-dependent dilation in aged rats [354]. Thus, therapeutic

approaches using natural or synthetic compounds with antioxidant and anti-

inflammatory capacities could be of benefit in preventing/reversing vascular

dysfunction in aging. However, it should be noted that these positive studies

demonstrate benefits in terms of surrogate markers of vascular health but there no exists

strong clinical evidence supporting the benefit of antioxidant supplementation in terms

of reduction of mortality or prevention of death from CVD. Although some secondary

prevention trials in men at risk for CVD showed lower RR for CVD death and nonfatal

myocardial infarct in patients supplemented with vitamin E for 18 months [355], the

main bulk of evidence currently available strongly support the absence of any

47
significant effect of antioxidant supplementation on the prevention of CVD. For

instance, a meta-analysis of 15 clinical trials (most including more than 1,000

participants) evaluating antioxidant supplementation (-carotene or vitamin E) on CVD

outcomes failed to show any benefit [356]. More concerning, a systematic review and

meta-analysis of the use of antioxidant supplementation on all-cause mortality

suggested increased harm from supplemental vitamin E, vitamin A, and -carotene

[357]. A possible explanation for detrimental effects by exogenous antioxidant

supplementation could rely on the interference of these compounds with the

physiological and hormetic signaling pathways mentioned above. In this sense, exercise

training improves endothelial vasodilation in rat soleus muscle arterioles from aged rats

by enhancing ROS signaling through upregulation of endogenous antioxidant systems,

while acute treatment with ROS scavengers blunts endothelial vasodilation [216].

Negative results in interventional studies together with positive results in epidemiologic

studies suggest that healthy diet habits involving higher natural antioxidant intake

and/or enhancing endogenous antioxidants would be more beneficial on cardiovascular

health than administration of antioxidant preparations. To this same direction points the

fact that the adoption of a Mediterranean diet supplemented with nuts or olive oil (both

foods supplying natural antioxidants) decreased incidence for major cardiovascular

events in 7,447 persons with CVD risk in a 4.8 years follow-up period [358]. Moreover,

a healthy Mediterranean type diet for 6 weeks resulted in improved forearm vasodilation

in healthy older subjects while supplementation of vitamin C with oral tablets failed to

cause such effect [359] suggesting that facilitation of an adequate endogenous

antioxidant status is more effective that exogenous addition of one antioxidant. In the

same direction points the fact that individualized exercise training in sedentary middle

aged subjects exerted greater improvement in endothelium-dependent vasodilation when

48
combined with adoption of a Mediterranean diet than in subjects not taking this diet

[360].

In summary we have provocative evidence supporting the potential role of antioxidants

in the prevention, delay or recovery of vascular dysfunction associated to aging. But this

evidence comes from animal studies or from small clinical trials focused on surrogates

(endothelial function). Thus we must wait for new RCTs with outcomes of clinical

relevance (cardiovascular morbidity and mortality, functional status, frailty, etc) to

adopt appropriate recommendations on the use of antioxidants to face the problems

associated to vascular aging. Furthermore, interventions aimed to enhance the

endogenous antioxidant and stress response capacities could probably be more

efficacious in preserving vascular function with aging than the administration of

exogenous antioxidants.

CONCLUSION

The better understanding of the molecular and cellular mechanisms underlying vascular

aging, as well as their potential interactions, provides a growing list of factors that can

be targets for specific interventions aimed to prevent or delay vascular dysfunction

associated to aging. Oxidative stress and inflammation figure among the principal

mechanisms likely involved in the vascular aging process that leads to cardiovascular

disease (Figure 4). Chronic ROS excess in aging prevents vascular response driving by

physiologic ROS signaling which leads to vascular malfunctions and avoids an adequate

response to stress.

Lifestyle attitudes such as caloric restriction and particularly aerobic exercise in the

elderly may help to prevent or delay the onset of endothelial dysfunction through

49
decreasing oxidative stress and inflammation.. In addition, antioxidant/anti-

inflammatory interventions, especially those enhancing endogenous stress response,

could also be important tools to be considered throughout the aging process favoring a

successful vascular aging and therefore, decreasing the risk for cardiovascular disease.

ACKNOWLEDGEMENTS

This work was supported by grants from Ministerio de Economía y Competitividad

[Instituto de Salud Carlos III, PI10/02781, PI11/01068, RETICEF RD06/0013,

RD12/0043], Spanish Government.

50
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FOOT NOTES

Figure 1. Oxidative stress and endothelial dysfunction associated with vascular

aging.

Physiological reactive oxygen species (ROS) generation in young vessels is limited

under basal conditions and is controlled by ROS detoxifying systems. In vascular aging,

mitochondrial respiratory chain leaks higher amounts of superoxide (O2.-) while

manganese superoxide dismutase activity (SOD2) is reduced. Superoxide and hydrogen

peroxide (H2O2) accumulate in mitochondria where they not only impact mitochondrial

DNA and cause mitochondrial dysfunction but also reach cytoplasm. Increased NADPH

oxidase (NOX) activity and uncoupling of endothelial nitric oxide synthase (eNOS) also

contribute to additional production of ROS that react to NO, reducing its availability,

and generate peroxynitrite (ONOO-) which causes cellular alterations including eNOS

uncoupling and reduced NO production. ROS can also be produced at the

extracytoplasmic space or can reach this place coming from the cytoplasm, further

reducing NO availability and causing defective endothelial vasodilation. This

endothelial dysfunction is a hallmark of unsuccessful vascular aging and facilitates the

development of cardiovascular diseases. SOD1: copper-zinc superoxide dismutase.

Figure 2. Proposed scheme for the different mechanisms of action of inflammatory

factors associated to the aging process and to the aging-related vascular diseases.

Increased inflammatory markers such as Tumour Necrosis Factor-alpha (TNF-D),

among others, stimulate superoxide (O2.-) production by the electron transport chain of

the mitochondria and by the NADPH oxidase. Increased Reactive Oxygen Species

(ROS) contributes to the activation and the nuclear translocation of nuclear factor-

kappaB (NF-B), which results in a low-grade chronic pro-inflammatory shift in the

92
gene expression profile. The transcriptional activity of NF- B is regulated by sirtuin-1

(SIRT-1), and this pathway exhibits age-related alteration. Increased superoxide

production and/or down-regulation of constitutive endothelial nitric oxide synthase

(eNOS) are responsible for decreased NO bioavailability and endothelial dysfunction.

Endothelial dysfunction leads to the development of vascular damage associated to the

aging process and to the aging-related vascular disease.

Figure 3. Chronic activation of nuclear factor-kappaB (NF-B) in vascular aging.

Under basal conditions, nuclear factor NF-B is bound to the NF-B inhibitor (IB) in

the cytoplasm that impedes its binding to DNA for triggering gene transcription.

Activation of IB kinase (IK) in response to some stimuli causes phosphorylation of

IB that results in separation of NF-B and IB allowing for transcription of NF-B-

regulated genes. Chronic oxidative stress leads to the presence of high amounts of

reactive oxygen species (ROS) such as superoxide (O2.-) and hydrogen peroxide (H2O2)

in aged vessels, resulting in chronic activation of the redox sensitive nuclear factor NF-

B under basal conditions. This transcriptional activation results in expression of

cellular adhesion molecules such as intercellular adhesion molecule-1 (ICAM-1) and

vascular adhesion molecule-1 (VCAM-1), local mediators of inflammation such as

inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) and pro-

inflammatory cytokines such as Tumor Necrosis Factor- (TNF), interleukin-6 (IL-6)

and interleukin-1 (IL-1) that conform an inflammatory phenotype that contributes to

endothelial dysfunction and promotes further ROS generation, feeding the vicious

cycle. Furthermore, reduced NO availability caused by ROS decreases expression of

sirtuin-1 (SIRT-1) that is a negative modulator of NF-B activation. In addition,

response against oxidative stress through erythroid-2-related factor-2 (Nrf2)-mediated

93
transcription of antioxidant defense enzymes such as NADPH quinone oxidoreductase-1

(NQO-1), -glutamylcysteine synthetase (GCS) and heme oxygenase-1 (HO-1) is

impaired in aging vasculature permitting sustained activation of NF-B. Lifestyle

changes such as exercise training and caloric restriction, as well as the adequate

administration of antioxidants, are interventions that potentially cause disruption of the

vicious cycle: oxidative stress – NF-B – inflammation – oxidative stress in vascular

aging.

Figure 4. General perspective of the chain of events linking aging to cardiovascular

disease.

Aging is associated with increased production of reactive oxygen species (ROS),

reduced antioxidant defense, increased pro-inflammatory factors and enhanced

angiotensin II-mediated signal. This translates into an endothelial phenotype

characterized by chronic oxidative stress and inflammation, two processes which

reinforce each other through activation of redox sensitive, pro-inflammatory nuclear

factor kappaB (NF-B). Excessive ROS generated by defective mitochondrial

respiratory chain, NADPH oxidase (NOX), hyperactivity and nitric oxide synthase

(NOS) uncoupling together with reduced antioxidant defense results in high

accumulation of hydrogen peroxide (H2O2) and superoxide (O2.-) that reduce NO

bioavailability and promote endothelial senescence and apoptosis while hamper

endothelial regeneration/repair. Pro-inflammatory phenotype contributes to endothelial

dysfunction by increasing peroxynitrite (ONOO-) levels and producing contractile

factors. Defective endothelial vasodilation is later accompanied by structural vascular

alterations such as arterial stiffness that in combination with the low-grade

inflammation of vascular wall and the concomitant presence of cardiovascular risk

94
Graphical Abstract (for review)
fig. 1

vasodilation defective vasodilation

• - ONOO-
0 2• - 02
NO NO

NOX
NOX
02• - 02• - 02• -
iNOS
eNOS NO
H202 NO eNOS
NO
BH4 H202 BH4
SOD1
H202 02• - H202
02 •- ONOO-
SOD1
02• -

NOX
SOD2 SOD2
NOX

02• - H202 02• - H202

youth – successful ageing unsuccessful ageing

 fig. 2

Inflammatory factors
(TNF-D)

Cytoplasm Mitochondrial
dysfunction NADPH eNOS
oxidase

ROS O2.- NO

Endothelial dysfunction Vascular damage

Inactive NF-k active NF-k

Age-related diseases

Interleukins (IL-1E, IL-2, IL-6)

SIRT1
TNF D
TNF-D
Low grade
Adhesion molecules (ICAM, VCAM)
inflammation
Nucleus Enzymes (iNOS, COX-2)
Chemokines ((IL-8, RANTES))
 fig.4

AGIN
NG

Increrased oxidants / Decreased antiox. def.. / Angiotensin II / Pro-inflammatory factors

Endothelium OXIDATIVE STRESS INFLAMMATION

senescence NOX TNF
apoptosis mitochondria
NFF-B
regeneration eNOS uncoupling MnSOD SIRT COX-2
iNOS COX 2
repair Nrf2

H2O2 O2·- ONOO- TXA2/PGH2

N
NO ENDOTHELIAL
DYSFUNCTION

NFF-B
Smooth
ARTERIAL STIFFNESS + IN
NFLAMMATION + CVRF
muscle
l

CARDIOVASC
CULAR DISEASE
fig. 3

Age related chronic

Age-related
oxidative stress
NO

O2·- / H2O2 Antioxidants

NF-B SIRT1
IB
I K
IK
IB
P TNF, IL-6, ICAM, NQO-1, GCS,
NF-B VCAM, iNOS, COX-2
COX 2 Nrf2 HO-1
HO 1

Exercise training
Caloric restriction
factors (CVRF) place the older subject in a situation prone to cardiovascular disease.

COX-2: cyclooxygenase-2; iNOS: inducible nitric oxide synthase; Nrf2: erythroid-2-

related factor-2; PGH2: prostaglandin H2; SIRT: sirtuins; SOD2: manganese-superoxide

dismutase; TNF: Tumor Necrosis Factor-; TXA2: thromboxane.

Highlights

x Vascular aging is now viewed as a modifiable cardiovascular risk factor

x Aging-related endothelial dysfunction is the first step to cardiovascular disease

x Oxidative stress and inflammation are main determinants in vascular aging

x Recovery of endogenous antioxidant capacity in aging benefits endothelial

function

x Good food habits and physical activity may improve vascular health in the

elderly

95