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ISBN: 0-8247-0264-6

This book is printed on acid-free paper.

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Copyright  2001 by Marcel Dekker, Inc. All Rights Reserved.

Neither this book nor any part may be reproduced or transmitted in any form or by any
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Series Introduction

Tumor Angiogenesis and Microcirculation is Volume 24 in the Basic and Clinical

Oncology series. Many of the advances in oncology have resulted from close
interaction between the basic scientist and the clinical researcher. The current
volume follows, expands on, and illustrates the success of this relationship as
demonstrated by new therapies and promising areas for scientific research.
As editor of the series, my goal has been to recruit volume editors who
not only have established reputations based on their outstanding contributions to
oncology, but also have an appreciation for the dynamic interface between the
laboratory and the clinic. To date, the series has consisted of monographs on
topics such as chronic lymphocytic leukemia, nucleoside analogs in cancer ther-
apy, therapeutic applications of interleukin-2, retinoids in oncology, gene therapy
of cancer, principles of antineoplastic drug development and pharmacology, and
AIDS-related malignancies. Tumor Angiogenesis and Microcirculation is cer-
tainly a most important addition to the series.
Volumes in progress include works on secondary malignancies, chronic
lymphoid leukemias, and controversies in gynecologic oncology. I anticipate that
these volumes will provide a valuable contribution to the oncology literature.

Bruce D. Cheson, M.D.

iii
Preface

In previous decades, it was recognized that the development of new blood vessels
is crucial to support the growth of tumors and metastases. Starting with the hy-
pothesis of Dr. Judah Folkman that tumor growth is angiogenesis dependent, this
area of research now has a solid scientific foundation. Insight into the mechanisms
through which tumors regulate angiogenesis and gain access to the circulation
has led to the development of treatment strategies targeted against the tumor
vasculature. These strategies are based mostly on the observation that newly
formed blood vessels have specific characteristics that allow discrimination from
mature, resting blood vessels.
To use antiangiogenic therapy effectively requires a significant adjustment
of the conventional line of thinking about treating cancer patients. Whereas con-
ventional chemotherapy, radiotherapy, and immunotherapy are directed against
tumor cells, antiangiogenic therapy is aimed at the vasculature of a tumor and
will either cause total tumor regression or keep tumors in a state of dormancy.
This approach has a significant benefit over other treatment modalities in that it
is applicable to tumor growth in general and is not dependent on specific tumor
characteristics.
Because several of these antiangiogenic and antimetastatic approaches have
now reached a stage where they are being tested in clinical trials, we feel that
there is a need to highlight the current developments in this research field. The
aims of this book are therefore: a) to provide a well-balanced overview of the
current biological principles of angiogenesis and microcirculation, b) to outline
the methods involved in discovering angiogenesis stimulators and inhibitors,

v
vi Preface

c) to review promising preclinical modulators of angiogenesis, and d) to show

the recent clinical achievements and possible future applications.
We have attempted to create a book that will be of benefit not only to
basic scientists working in this field but also to clinicians (pathologists; surgeons;
medical, radiation, and hemato-oncologists; and internists) who will perform fu-
ture clinical studies with angiogenesis inhibitors.

Emile E. Voest
Patricia A. D’Amore
Contents

Series Introduction Bruce D. Cheson iii

Preface v
Contributors xi

I Biological Principles of Angiogenesis

1 Endothelial Cells and Pericytes in Tumor Vasculature 1

Diane C. Darland and Patricia A. D’Amore

2 The Extracellular Matrix and the Regulation of

Angiogenesis 9
Joseph A. Madri

3 Matrix Metalloproteinases (Matrixins) and Their Inhibitors

(TIMPS) in Angiogenesis 29
Teresa A. Bennett and William G. Stetler-Stevenson

4 Regulation of Cell Migration in the Process of Angiogenesis 59

Bela Anand-Apte and Bruce R. Zetter

5 Plasmin, Plasmin Inhibitors, and Angiogenesis 73

Martijn F. B. G. Gebbink

vii
viii Contents

II Assays to Study Angiogenesis

6 Assays to Study Angiogenesis 91

Robert Auerbach and Wanda Auerbach

7 Screening for Angiogenesis Inhibitors with the Chick

Chorioallantoic Membrane and the Mouse Corneal Micropocket
Assays 103
Robert J. D’Amato

8 Capillary Morphogenesis In Vitro: Cytokine Interactions and

Balanced Proteolysis 111
Roberto Montesano and Michael S. Pepper

9 Skin Fold Chamber Models 143

Michael Leunig and Konrad Messmer

10 Protease Assays and Their Use in the Discovery of Novel

Regulators of Angiogenesis 155
Li Yan, Inmin Wu, and Marsha A. Moses

III Angiogenic Factors

11 Vascular Endothelial Growth Factor/Vascular Permeability

Factor: Multiple Biological Activities for Promoting
Angiogenesis 167
Donald R. Senger

12 Tie Receptors, Ang Ligands 185

Yuji Gunji, Arja Kaipainen, Kristiina Iljin, Eola Kukk-Valdre,
Berndt Enholm, and Kari Alitalo

13 Vascular Endothelial Growth Factor Receptors 199

Arja Kaipainen, Eija Korpelainen, and Kari Alitalo

14 Regulation of Vascular Endothelial Growth Factor (VEGF)

Expression 213
Ilan Stein and Eli Keshet

15 Fibroblast Growth Factors 227

David A. Moscatelli and Daniel B. Rifkin
Contents ix

16 Role of Proangiogenic Cytokines and Inhibitors of

Neovascularization in Tumor Angiogenesis 265
Peter J. Polverini and Robert M. Strieter

IV Regulation of Angiogenesis

17 The Link Between Oncogenes, Signal Transduction Therapy,

and Tumor Angiogenesis 285
Robert S. Kerbel, Alicia Viloria-Petit, Futoshi Okada,
and Janusz Rak

18 Genetic Control of Angiogenesis by Tumor Suppressor Genes 307

Maartje Los and Emile E. Voest

19 Regulation of Neoplastic Angiogenesis by the Organ

Microenvironment 321
Rakesh Kumar and Isaiah J. Fidler

20 Role of Macrophages in Tumor Angiogenesis 335

Peter J. Polverini

21 Phenotypic Analysis of Endothelium from the Tumor

Vasculature 349
Gerard Groenewegen and Arjan W. Griffioen

V Angiogenesis Inhibitors: Preclinical and Clinical Drugs

22 Drug Delivery and Angiogenesis Inhibition in the Treatment of

Brain Tumors 361
Laurence D. Rhines, Matthew G. Ewend, and Henry Brem

23 Matrix-Associated Endogenous Inhibitors of Angiogenesis 375

Raghu Kalluri and Vikas P. Sukhatme

24 αvβ3 and Its Antagonists in the Control of Angiogenesis 387

Brian P. Eliceiri and David A. Cheresh

25 The Role of Vascular Endothelial Growth Factor in

Angiogenesis 399
Napoleone Ferrara
x Contents

26 TNP-470: Preclinical and Clinical Development 431

Deborah M. Milkowski and Rachelle A. Weiss

27 Matrix Metalloproteinase Inhibitors 449

Peter D. Brown

28 Tumoral Vascularity: What Does It Tell Us About the Growth

and Spread of Cancer? 465
Noel Weidner

29 The Prognostic and Diagnostic Value of Circulating Angiogenic

Factors in Cancer Patients 487
Olaf A. J. Kerckhaert and Emile E. Voest

VI Future Perspectives

30 Perspectives in Vascular Cancer Therapy: An Introduction 501

Geert H. Blijham

31 The Combination of Antiangiogenic Therapy with Cytotoxic

Therapy: A Systems Approach 507
Beverly A. Teicher

32 Targeting the Vasculature of Solid Tumors 549

Philip E. Thorpe and Sophia Ran

33 Combining Antivascular Approaches with Radiotherapy:

A Perspective 579
Juliana Denekamp

34 Antiangiogenic Gene Therapy 597

Jaap C. Reijneveld and Emile E. Voest

Index 609
Contributors

Kari Alitalo, M.D., Ph.D. Research Professor of the Finnish Academy of Sci-
ences Molecular/Cancer Biology Laboratory, Haartman Institute, University of
Helsinki, Finland

Bela Anand-Apte, M.B.B.S., Ph.D. Staff Scientist, Ophthalmic Research and

Cell Biology, Cole Eye Institute, Cleveland Clinic Foundation, Cleveland, Ohio

Robert Auerbach, Ph.D. Harold R. Wolfe Professor, Department of Zoology,

University of Wisconsin, Madison, Wisconsin

Wanda Auerbach, M.S. Reference Librarian, Department of Zoology, Univer-

sity of Wisconsin, Madison, Wisconsin

Teresa A. Bennett, Ph.D. Laboratory of Pathology, National Cancer Institute,

National Institutes of Health, Bethesda, Maryland

Geert H. Blijham, M.D., Ph.D. Professor of Medicine and Chairman, Univer-

sity Medical Center Utrecht, Utrecht, The Netherlands

Henry Brem, M.D. Harvey Cushing Professor of Neurosurgery, Oncology, and

Ophthalmology, and Chairman, Department of Neurosurgery, Johns Hopkins
University School of Medicine, Baltimore, Maryland

Peter D. Brown, D. Phil. Project Director, Development, British Biotech Phar-

maceuticals Ltd., Oxford, England
xi
xii Contributors

David A. Cheresh, Ph.D. Professor, Department of Immunology and Vascular

Biology, The Scripps Research Institute, La Jolla, California

Robert J. D’Amato, M.D., Ph.D. Assistant Professor, Department of Ophthal-

mology, Harvard Medical School, Boston, Massachusetts

Patricia A. D’Amore, Ph.D. Senior Scientist, Department of Ophthalmology,

Schepens Eye Research Institute, and Professor of Ophthalmology/Pathology,
Harvard Medical School, Boston, Massachusetts

Diane C. Darland, Ph.D. Research Fellow, Schepens Eye Research Institute,

Harvard Medical School, Boston, Massachusetts

Juliana Denekamp, B.Sc., Ph.D., D.Sc. Professor, Translation Research Unit,

Department of Radiation Sciences, Umeå University, Umeå, Sweden

Brian P. Eliceiri, Ph.D. Research Associate, Departments of Immunology and

Vascular Biology, The Scripps Research Institute, La Jolla, California

Berndt Enholm, B.M. Molecular/Cancer Biology Laboratory, Haartman Insti-

tute, University of Helsinki, Helsinki, Finland

Matthew G. Ewend, M.D. Assistant Professor of Neurosurgery, University of

North Carolina, Chapel Hill, North Carolina

Napoleone Ferrara, M.D. Staff Scientist, Department of Cardiovascular Re-

search, Genentech, Inc., South San Francisco, California

Isaiah J. Fidler, D.V.M., Ph.D. Professor and Chairman, Department of Can-

cer Biology, The University of Texas M.D. Anderson Cancer Center, Houston,
Texas

Martijn F. B. G. Gebbink, Ph.D. Laboratory of Medical Oncology, Depart-

ment of Internal Medicine, University Medical Center Utrecht, Utrecht, The
Netherlands

Arjan W. Griffioen Laboratory of Angiogenesis Research, Department of In-

ternal Medicine, University Hospital Maastricht, Maastricht, The Netherlands

Gerard Groenewegen, M.D., Ph.D. Department of Internal Medicine and On-

cology, University Hospital Utrecht, Utrecht, The Netherlands
Contributors xiii

Yuji Gunji, M.D., Ph.D. Molecular/Cancer Biology Laboratory, Haartman In-

stitute, University of Helsinki, Helsinki, Finland*

Kristiina Iljin, M.Sc. Molecular/Cancer Biology Laboratory, Haartman Insti-

tute, University of Helsinki, Helsinki, Finland

Raghu Kalluri, Ph.D. Assistant Professor, Department of Medicine, Harvard

Medical School, and Nephrology Division, Department of Medicine, Beth Israel
Deaconess Medical Center, Boston, Massachusetts

Arja Kapainen, M.D., Ph.D. Molecular/Cancer Biology Laboratory, Haart-

man Institute, University of Helsinki, Helsinki, Finland

Robert S. Kerbel, Ph.D. Director, Biological Sciences Program and Division

of Cancer Biology Research, Sunnybrook and Women’s College Health Science
Centre, University of Toronto, Toronto, Ontario, Canada

Olaf A. J. Kerckhaert Department of Internal Medicine, Laboratory of Medi-

cal Oncology, University Medical Center Utrecht, Utrecht, The Netherlands

Eli Keshet, Ph.D. Professor, Department of Molecular Biology, The Hebrew

University–Hadassah Medical School, Jerusalem, Israel

Eija Korpelainen Molecular/Cancer Biology Laboratory, Haartman Institute,

University of Helsinki, Helsinki, Finland

Eola Kukk-Valdre, M.D. Molecular/Cancer Biology Laboratory, Haartman

Institute, University of Helsinki, Helsinki, Finland

Rakesh Kumar Department of Cell Biology, The University of Texas M.D.

Anderson Cancer Center, Houston, Texas

Michael Leunig, M.D. Attending Physician, Department of Orthopedic Sur-

gery, University of Berne, Berne, Switzerland

Maartje Los, M.D. Department of Internal Medicine, Laboratory of Medical

Oncology, University Medical Center Utrecht, Utrecht, The Netherlands

*Current affiliation: Assistant Professor, Department of Pediatrics, Jichi Medical School, Tochigi,
Japan.
xiv Contributors

Joseph A. Madri, Ph.D., M.D. Professor, Department of Pathology, Yale Uni-

versity School of Medicine, New Haven, Connecticut

Konrad Messmer, M.D. Professor of Experimental Surgery, Institute for Sur-

gical Research, Ludwig-Maximilians-University of Munich, Munich, Germany

Deborah M. Milkowski, Ph.D. Senior Research Investigator, Metabolism and

Pharmacology, TAP Pharmaceutical Products Inc., Lake Forest, Illinois

Roberto Montesano, M.D. Professor, Department of Morphology, University

of Geneva Medical Center, Geneva, Switzerland

David A. Moscatelli, Ph.D. Research Professor, Department of Cell Biology,

New York University School of Medicine, New York, New York

Marsha A. Moses, Ph.D. Associate Professor, Department of Surgery, Chil-

dren’s Hospital and Harvard Medical School, Boston, Massachusetts

Futosha Okada, Ph.D. Instructor, Laboratory of Pathology, Cancer Institute,

Hokkaido University School of Medicine, Sapporo, Hokkaido, Japan

Michael S. Pepper, M.D., Ph.D. Department of Morphology, University of

Geneva Medical Center, Geneva, Switzerland

Peter J. Polverini, D.D.S., D.M.Sc. Professor and Dean, Department of Pathol-

ogy, University of Minnesota School of Dentistry, Minneapolis, Minnesota

Janusz Rak, M.D., Ph.D. Assistant Professor, Department of Medicine, Ham-

ilton Civil Hospitals Research Centre, McMaster University, Hamilton, Ontario,
Canada

Sophia Ran, Ph.D. Research Assistant Professor of Pharmacology, Simmons

Comprehensive Cancer Center, The University of Texas Southwestern Medical
Center, Dallas, Texas

Jaap C. Reijneveld, M.D. Department of Neurology, University Medical Cen-

ter Utrecht, Utrecht, The Netherlands

Laurence D. Rhines, M.D. Assistant Professor, Department of Neurosurgery,

The University of Texas M.D. Anderson Cancer Center, Houston, Texas
Contributors xv

Daniel B. Rifkin, Ph.D. Professor, Department of Cell Biology, New York

University School of Medicine, New York, New York

Donald R. Senger, Ph.D. Department of Pathology, Beth Israel Deaconess

Medical Center, Boston, Massachusetts

Ilan Stein Department of Molecular Biology, The Hebrew University–Hadas-

sah Medical School, Jerusalem, Israel

William G. Stetler-Stevenson, M.D., Ph.D. Senior Investigator, Section

Chief, Extracellular Matrix Pathology Section, Laboratory of Pathology, National
Cancer Institute, National Institutes of Health, Bethesda, Maryland

Robert M. Strieter University of Michigan Medical School, Ann Arbor, Mich-

igan

Vikas P. Sukhatme, M.D., Ph.D. Professor, Department of Medicine, Harvard

Medical School, and Chief, Nephrology Division, Department of Medicine, Beth
Israel Deaconess Medical Center, Boston, Massachusetts

Beverly A. Teicher, Ph.D. Research Advisor, Cancer Drug Discovery, Lilly

Research Laboratories, Indianapolis, Indiana

Philip E. Thorpe, Ph.D. Professor of Pharmacology, Simmons Comprehensive

Cancer Center, The University of Texas Southwestern Medical Center, Dallas,
Texas

Alicia Viloria-Petit, M.Sc. Graduate student (Ph.D.), Department of Medical

Biophysics, Sunnybrook and Women’s College Health Science Centre, Univer-
sity of Toronto, Toronto, Ontario, Canada

Emile E. Voest, M.D., Ph.D. Professor of Medical Oncology, Department of

Internal Medicine, University Medical Center Utrecht, Utrecht, The Netherlands

Noel Weidner, M.D. Professor and Director of Anatomic Pathology, Univer-

sity of California, San Diego, San Diego, California

Rachelle Weiss, Ph.D. Assistant Director, Clinical Development, TAP Phar-

maceutical Products Inc., Lake Forest, Illinois

Inmin Wu, Ph.D. Research Fellow, Department of Surgery, Children’s Hospi-

tal and Harvard Medical School, Boston, Massachusetts
xvi Contributors

Li Yan, M.D., Ph.D. Research Fellow, Department of Surgery, Children’s Hos-

pital and Harvard Medical School, Boston, Massachusetts

Bruce R. Zetter, Ph.D. Professor of Surgery, Children’s Hospital and Harvard

Medical School, Boston, Massachusetts
1
Endothelial Cells and Pericytes in
Tumor Vasculature
Diane C. Darland and Patricia A. D’Amore
Schepens Eye Research Institute, Harvard Medical School,
Boston, Massachusetts

I. ANGIOGENESIS IS A REQUIREMENT FOR TUMOR

GROWTH

In the course of examining tumor establishment, growth, and metastasis, it is

important to consider one vital component in this process—the vasculature. It is
now well accepted that tumor growth and metastasis require vascularization to
provide both nourishment and a route for tumor cell extravasation (1). The basic
premise is that tumors less than 1 mm3 have their metabolic needs met by diffu-
sion. Beyond that size, a more elaborate system for distributing nutrients and
removing toxins and metabolites is needed to support tumor growth.
It is not entirely clear how tumor and vessel growth are temporally intercon-
nected. Does tumor growth in the absence of sufficient vascular support create
relative local hypoxia and trigger hypoxia-dependent neovascularization? Or does
the presence of the tumor lead to changes in the microenvironment, including
locally elevated levels of growth factor(s) that, in turn, promote neovasculariza-
tion to allow continued growth of the tumor (2)? In addition, can the tumor cells
‘‘co-opt’’ adjacent vessels to obtain support for tumor growth (3, 4)? Are tumors
and their vasculatures connected by mechanisms that vary according to tumor
type and location?
Regardless of which mechanism or combination of mechanisms is at play,
it is readily apparent that the vascularization of tumors is an integral component
of tumor growth. This fact makes angiogenesis a viable target for antitumor ther-
apy (5). Angiogenesis promotion or inhibition in a tumor can be likened to a
rheostat, a device that regulates current flow with a series of resistors (Fig. 1A).
1
2 Darland and D’Amore

Figure 1 Rheostat model of tumor angiogenesis. When the microenvironment of the

tumor vasculature is in the angiogenesis phase, vessel production is favored and the tumor
volume is increased (A). When angiogenesis is inhibited, vessel regression predominates
and the tumor volume is reduced (C ). When there is a balance between the cycles of
angiogenesis and vessel regression, the tumor volume remains relatively static (B). Tumor
vasculature can be targeted with agents that promote vessel regression by treating with
inhibitors of angiogenesis, thereby shifting the direction of the ‘‘angiogenesis rheostat’’
and decreasing tumor volume as indicated (C ).

When growth factor production, cell-cell and cell-matrix interactions are aligned
to promote vessel formation, angiogenesis proceeds. When conditions, microen-
vironmental and otherwise, prohibit new vessel growth, there is a net regression
in tumor vasculature and subsequently in tumor volume (Fig. 1C). When both
angiogenesis and vessel regression occur, there is no net increase in the tumor
vasculature, and the tumor is static in size (Fig. 1B). To examine tumor angiogen-
esis and its impact on tumor growth, it is imperative to understand the roles of,
and the factors that control, the cell types that form the vessels.

II. ENDOTHELIAL CELLS IN TUMOR VASCULATURE

The endothelial cell (EC) is the primary cell type involved in angiogenesis. Endo-
thelial cells form the lumens of blood vessels, function in a variety of local roles
Endothelial Cells and Pericytes 3

including the immune response and the maintenance of a non thrombogenic sur-
face, and participate in nutrient and gas exchange. Endothelial cells of mature,
quiescent vessels have characteristically low proliferative rates with estimated
turnover times measured in years. The rate of EC proliferation increases dramati-
cally during neovascularization as occurs in wound healing or in the rapidly grow-
ing tumor vasculature. For instance, assessment of endothelial proliferation in a
Lewis lung carcinoma model of tumor growth revealed a labeling index of 40%,
which reflects a rapid cell turnover (3). Similarly, a survey of turnover times
calculated from labeling indices among a variety of experimental tumors revealed
turnover times ranging from 1 day to about 10 days. This is in contrast to EC
of vessels in normal tissues in which turnover ranged from about 80 days in the
lung to about 8000 days in the brain (6). Once the nascent vessel structure has
become stabilized, EC proliferation is reduced and the EC transition to a quies-
cent state occurs.
Thus, EC of tumor vessels are distinct from those of the normal vasculature,
particularly with regard to their proliferation rate. Endothelial cells in some re-
gions of the tumor vasculature are characterized by a constant high rate of prolif-
eration, reflecting the neovascularization that accompanies increases in tumor
volume, whereas EC in other regions of the tumor are undergoing apoptosis in
parallel with tumor necrosis and vessel regression (4). Tumor vessel EC can thus
be viewed as a heterogeneous population, with the common trait of instability
to unite them. The cellular and molecular basis of tumor vessel instability is not
entirely clear. The absence or paucity of perivascular cells and the dramatically
altered microenvironment with altered levels of proteases, growth factors, and
matrix components all appear to contribute to this unstable phenotype. We use
the term ‘‘pseudostable’’ to characterize the tumor vasculature. Tumor vessels
are functional at the level of tumor support but inherently unstable, and therefore
are distinct from normal, established vessels.
Endothelial cells of tumor vessels also differ from those of normal vessels
in other ways, including the profile and level of cell adhesion molecules that
they express. During angiogenesis, EC go through a phase of proliferation and
migration during which attachment of cells to one another and to the extracellular
matrix is greatly reduced. A number of adhesion molecules have been identified
as candidates to mediate EC-EC interactions, as well as EC interactions with the
microenvironment (7). As these topics will be covered in greater detail in later
chapters, only a few specific proteins will be mentioned here.
One example of a protein likely to be involved in interendothelial cell inter-
actions is vascular endothelial-cadherin (VE-cadherin/cadherin 5). Vascular en-
dothelial cadherin is expressed at high levels on stable vessels where it is local-
ized at cell-cell junctions. Vascular endothelial cadherin is poorly expressed or
is absent in nonjunctional locations as well as in vessels of hemangiomas and
4 Darland and D’Amore

angiosarcomas (8). These observations suggest that loss (or absence) of VE-cad-
herin results in vessel destabilization and may lead to abnormal remodeling.
There is also considerable evidence for an involvement of the integrin αvβ3 in
EC-extracellular matrix interactions as well as EC-platelet interactions. αvβ3, a
cell surface receptor that can bind to fibronectin, fibrinogen, and von Willebrand
factor, is highly expressed in new vessels in wounds (9) in comparison with
quiescent vessels. Tumors treated with antagonists of αvβ3 exhibit increased
apoptosis and vessel regression (9). These results indicate that αvβ3 is an integral
component of the angiogenic process. At least one aspect of αvβ3’s role in the
tumor vasculature is that it mediates tumor EC adhesion to ECM molecules. In
fact, cell anchoring has been clearly shown to be required for EC survival (for
review see [10]).
In contrast to the EC of quiescent vessels, EC of tumor vessels show a
marked dependence on growth factors for survival. One of the earliest experi-
mental demonstrations of this fact dates back to early observations of vessels
induced in the rabbit corneal pocket assay by tumor cells (11). Implantation of
a tumor fragment induced new vessels that arose from the limbal vessels. Re-
moval of the tumor, however, led to rapid vessel regression. Although the cell-
ular and molecular basis for the instability of these vessels was unknown, it
was clear that the new vessels were somehow dependent on the presence of the
tumor.
Insight into the mechanism underlying this phenomenon became possi-
ble once factors involved in tumor vessel growth were identified. A large body
of data has indicated a central role for vascular endothelial growth factor
(VEGF) in the induction of host vessels into the growing tumor (12). Depen-
dence on the continued presence of VEGF was demonstrated in an experimental
model using a C6 glioma with tetracycline-regulated VEGF expression in which
turning off VEGF expression led to vascular collapse and tumor necrosis (13).
Furthermore, not all of the vessels in this glioma regressed. Instead, variable
stability among the tumor vessels was correlated with the extent of pericyte asso-
ciation with the vessels (14) (See below for a discussion of pericytes in tumor
vessels.)
Another area in which tumor vessels differ from normal vessels is in the
degree of vessel integrity, which depends on the association between the EC.
Tumor vessels are distinguished from nontumor vessels in their degree of perme-
ability. At least some of the ‘‘leakiness’’ of tumor vessels is due to the generally
high levels of VEGF in tumors. Vascular endothelial growth factor causes tran-
sient increases in permeability of normal vessels (15). The mechanisms underly-
ing the increased leakiness are not known but have been suggested to include
decreased interendothelial connections (16), stimulation of transcellular transport
(17), and formation of fenestrations (18, 19).
Endothelial Cells and Pericytes 5

III. PERICYTES IN TUMOR VASCULATURE

Interestingly, the greatest area of exchange across the EC in normal vessels is

at the level of the microvasculature that has the lowest level of perivascular cells
or pericytes. It is also the microvasculature that is the initiation site for angiogen-
esis. Undoubtedly, this is not a coincidence and leads to consideration of the
other major cell type in the vasculature, the pericyte. Pericytes are perivascular
cells that are thought to surround and stabilize new vessels. They are smooth
musclelike cells that are associated with the microvasculature and are sparsely
distributed along the length of the vessel (20). Pericytes extend thin processes
around and along the length of the microvascular tubes and have areas of direct
contact with EC. This is in contrast to the vascular smooth muscle cells that
associate with larger vessels and completely surround the vessel. There is a layer
of continuous basement membrane between the smooth muscle cells and the EC,
with points of contact in regions where smooth muscle (SM) cell processes have
breached the membrane to establish direct contact with the EC (21, 22). There
is a positive correlation with vessel size and the complexity of the pericyte/SM
cell layer that surrounds the vessel (22), although some of the functional differ-
ences remain unclear.
Limited information is available as to the role of pericytes in tumor vascula-
ture. Although it is generally commented that tumor vessels have a paucity of
vessel wall cells (pericytes and SM cells), few studies have examined this issue
systematically. Only recently have investigators begun to recognize the possible
role of the pericyte in tumor vessel stabilization (23). In the tetracycline-regulated
VEGF tumor model described above, vessel injury and regression were evident
only in vessels devoid of α-smooth muscle actin (SM-actin)-positive cells (e.g.,
pericytes) (14). Similar analyses were conducted in archival sections of human
prostate tumors in which it was observed that only 40% of vessels larger than
capillaries had associated SM-actin-containing cells. This was in comparison to
human glioblastomas in which only 25% of the vessels larger than capillaries
had SM-positive cells. This difference was of interest, in that prostate tumors are
considered to be slow-growing tumors, whereas glioblastomas grow rapidly.
One recent study has assessed pericyte association with vessels of renal
cell, colon, mammary, lung, and prostate carcinomas, as well as with glioblasto-
mas (24). Assessment of pericyte association with vessels was accomplished by
immunohistochemical staining for SM-actin to identify the pericytes and CD34
to localize the EC. These analyses revealed a large degree of heterogeneity among
the vasculatures of these tumors. For example, 12.7 ⫾ 7.9% of microvessels in
glioblastoma had both pericytes and EC, whereas 67.3 ⫾ 4.2% of vessels in
mammary tumors had vessels with associated pericytes. On the other hand, some
tumors are reported to have significant numbers of pericytes (25).
6 Darland and D’Amore

Most of the information on pericytes that has been accrued to date is derived
from analysis of pericytes in normal vasculature, in wound healing, and in dia-
betic retinopathy (26). In normal angiogenesis, EC recruit and induce the prolifer-
ation of surrounding mesenchymal cells (23). Upon contact with the EC, the
mesenchymal cells are induced toward a pericyte lineage (27). Association of
the pericyte with the nascent EC tube is thought to initiate a transition toward
vessel stability. There appears to be a window of sensitivity during angiogenesis
in which vessels are sensitive to regression. A recent study in glioma and prostate
tumors showed that vessels lacking periendothelial cells were vulnerable to
growth factor withdrawal and underwent apoptosis, but those with associated
pericytes were resistant (14). The role of perivascular cells in vessel stabilization
in vitro and in vivo reflect the importance of pericyte association with vessels
in promoting normal vessel pruning and stabilization.

IV. PERICYTES AND TUMOR VESSEL STABILITY

We propose that the presence of pericytes is one component of vessel heterogene-

ity, with the association of pericytes with tumor vessels leading to vessel stabili-
zation and absence of pericytes leading to vessel destabilization. Regions of the
tumor vasculature that have associated pericytes can be called pseudostable. The
term ‘‘pseudostabilization’’ refers to vessels that are not currently undergoing
neovascularization, but by their nature as tumor vessels do not achieve stability
characteristic of normal vessels. Areas of the vasculature lacking pericytes repre-
sent areas of potential flux. Either vessels can regress because instability or be
invested with pericytes and subsequently stabilized. Where vessels are sparse,
there is resultant relative hypoxia. Angiogenesis is initiated in these areas with
the early phase of EC proliferation from a pre-existing vessel. The growth of the
tumor depends in part on the balance of the vasculature overall, as suggested by
the rheostat model (Fig. 1). A greater percentage of vessels undergoing angiogen-
esis relative to the pseudostable and regressing areas will result in increased tumor
vasculature. This hypothesis is a new perspective on tumor vessel stability for
which there is increasing supporting data (14, 24). However, the hypothesis of
pericyte investment regulating tumor vasculature stability has yet to be thor-
oughly tested as a mechanism for controlling tumor angiogenesis.
Cumulatively, these and other data indicate that there are distinct parallels
between angiogenesis in tumor vessels and angiogenesis in an injury response.
The similarities are close enough to suggest that tumor vasculature represents a
chronic wound situation, unable to cycle through to stasis—an idea that has been
proposed by Dvorak and coworkers in their examination of tumor angiogenesis
(2, 28). This perspective indicates a theoretical avenue for inhibiting tumor angio-
genesis through stabilizing the tumor vasculature and preventing further tumor
Endothelial Cells and Pericytes 7

expansion, although whether the vessel can remain stable in the face of elevated
levels of growth factors is not clear. In addition, it suggests that targeting cell
adhesion molecules, cell-matrix interactions, EC-pericyte interactions, and
growth factors involved in angiogenesis may allow both inhibition and eventual
regression of the tumor vasculature.

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blood vessels in tumors follows vascular endothelial growth factor withdrawal. J
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2369–2379.
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neity of angiogenesis and blood vessel maturation in human tumors:implications for
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2
The Extracellular Matrix and the
Regulation of Angiogenesis
Joseph A. Madri
Yale University School of Medicine, New Haven, Connecticut

I. INTRODUCTION

Angiogenesis, the formation of new blood vessels from preexisting vessels, occurs
during development of the vasculature, in selected physiological processes (e.g.,
ovulation), during the wound healing process, and during the vascularization of
tumors. It is a tightly controlled process involving endothelial cell activation, migra-
tion, proliferation, multicellular organization and differentiation, stabilization and,
in some cases, involution (1–4). As noted in the development and maintenance
of all other organ systems, angiogenesis occurs in a complex extracellular matrix
environment. The importance of extracellular matrix organization and composition
in modulating endothelial and vascular cell behaviors in the process of angiogenesis
has been demonstrated in various in vitro and in vivo studies over the past several
years (5–13). In this chapter the roles of extracellular matrix composition and
organization in modulating vascular cell behavior during in vivo and in vitro vascu-
logenesis and angiogenesis will be discussed using selected models as examples.

II. EXTRACELLULAR MATRIX COMPONENTS AS

MODULATORS OF ENDOTHELIAL CELL BEHAVIOR

The importance of extracellular matrix components in modulating endothelial

cell behavior has been recognized since the first successful culture of vascular

Supported, in part, by USPHS Grants RO1-HL28373, PO1-DK38979, RO1-HL51018 and PO1-

NS35476.

9
10 Madri

endothelial cells (14). Then, collagenous substrata (gelatin) were shown to confer
beneficial growth and culture characteristics to microvascular endothelial cells
in culture, including in vitro angiogenesis or ‘‘tube formation’’ (14). In the years
after these studies, several investigators began using a variety of extracellular
matrix components as substrata for the culture microvascular endothelial cells.
Such studies documented modulation in a variety of endothelial cell behaviors,
including attachment, spreading, proliferation, extracellular matrix synthetic pro-
files, multicellular organization, and tube formation (7, 8, 15–27). Concurrent
and subsequent in vivo angiogenesis studies showed a good correlation between
matrix-mediated effects noted in vitro and the presence of matrix components
and endothelial cell behavior observed during angiogenesis induced in animal
models (26). In vivo, organ culture and tissue culture studies of angiogenesis
revealed a complex, spatiotemporal deposition and organization of extracellular
matrix components along the angiogenic sprout with eventual formation of a
mature basal lamina (28, 29). In vitro, microvascular endothelial cells have inter-
acted with a wide variety of matrix components, including the interstitial collagens,
collagenous and noncollagenous components of the basement membrane, and many
other noncollagenous extracellular matrix components (4, 6, 26, 27, 30).
Microvascular endothelial cells exhibit varying proliferation rates de-
pending on the underlying extracellular matrix component with which they inter-
act. For example, rat epididymal fat pad-derived microvascular endothelial cells
(RFC) exhibit a high proliferative rate when plated on laminin 1, intermediate
proliferative rates when plated on collagen types I or V, and low proliferative
rates when plated on collagen type IV and fibronectin (26). In addition to effects
on proliferation, extracellular matrix components also affect other aspects of en-
dothelial cell behavior. Extracellular matrix composition and concentration mod-
ulation of endothelial cell shape and spreading has been appreciated by several
investigators and found to affect growth factor responsiveness through integrin
engagement-mediated signaling cascades (15–19, 31–33). Culture on different
extracellular components has also been observed to modulate the extracellular
matrix synthetic profiles of microvascular endothelial cells as well as their ability
and propensity to form multicellular complexes, tubelike structures, and fenestrae
(3, 7, 20, 34–36). Purified basement membrane components (laminin and type IV
collagen) elicit tube formation in cultures of microvascular endothelial cells; Matri-
gel, a complex mixture of basement membrane components (laminin 1, type IV
collagen, entactin, and heparin sulfate proteoglycan) and growth factors (trans-
forming growth factor beta 1 [TGFβ1], platelet-derived growth factor [PDGF],
basic fibroblast growth factor [bFGF], etc.), elicits rapid multicellular organization
of endothelial cells leading to meshlike networks of cells in culture (13, 35).
Extracellular matrix-mediated modulation of endothelial cell behavior af-
fects the process of in vivo angiogenesis, as evidenced by the distinct endothelial
phenotypes noted among the endothelial cells along an angiogenic sprout that
The Extracellular Matrix 11

express similar behaviors to endothelial cells cultured on different extracellular

matrix components. The migratory cells at the tip of a sprout are invested in
cablelike arrays of type V collagen and fibronectin, whereas the proliferating
cells distal to these lead cells express and deposit laminin 1 in a diffuse abluminal
pattern. Further back along the sprout, the mitotically quiescent differentiating
cells rest on a forming basal lamina composed of diffuse fibronectin, laminin,
and type IV collagen (5, 26).

III. EXTRACELLULAR MATRIX ORGANIZATION AS A

MODULATOR OF ENDOTHELIAL CELL BEHAVIOR

Although useful in clarifying the roles of specific extracellular matrix components

in modulating particular aspects of endothelial cell behavior, typical two-dimen-
sional culture systems do not adequately mimic the three-dimensional environ-
ment in which most angiogenesis occurs. During the past several years, investiga-
tors have used three-dimensional culture systems in their studies of angiogenesis.
As noted for a variety of mesenchymal and epithelial cells, endothelial cells cul-
tured in three-dimensional environments (including gels of purified interstitial
collagens, basement membrane components, fibrin, and basal lamina and interstitial
surfaces of amnion membranes) exhibit significantly different phenotypes com-
pared to their culture in typical two-dimensional culture systems (5–7, 37, 38).
Human umbilical vein endothelial cells (HUVEC) have been shown to form
tubelike structures when grown on trypsin-digested fibronectin substrata and de-
prived of endothelial cell growth factor (12). When HUVEC were cultured on
three-dimensional collagen gels and stimulated with phorbol ester (PMA) the
cells invaded and migrated into the gels and formed branching, tubelike structures
with form lumina and junctional complexes between cells (39–41). In other stud-
ies, when HUVEC were dispersed in a type I collagen gel and PMA and angio-
genic factors were added, tube formation was observed. Further, the addition
of specific β1 integrin antibodies, that is, anti-α2β1, enhanced tube formation,
consistent with the concept that matrix-driven, integrin-mediated signaling events
are involved in the angiogenic process (38). More recent studies of PMA and
angiogenic factor-stimulated HUVEC tube formation in type I collagen gels dem-
onstrated an inhibition of vacuole and lumen formation in the presence of anti-
α2β1 (25). This apparent discrepancy is likely due to different culture conditions,
passage numbers, and growth factor additions used in these two studies, as well
as to differences in the anti-α2β1 reagents used in the two studies. These two
studies (25, 38) also described differences in the effects of anti-αvβ3 reagents
on tube formation that are not consistent with several recent in vivo angiogenesis
studies (42–44). In the study by Gamble et al., anti-αvβ3 was noted to enhance
tube formation in fibrin gels but had no appreciable effects in type I collagen
12 Madri

Figure 1 Schematic representation of extracellular matrix compositional and organiza-

tional changes during angiogenesis. A. Cells at the distal tip of the angiogenic sprout
deposit and interact with an extracellular matrix having very different composition and
organization compared to cells nearer the parent vessel. Cells at the distal tip express
several features of undifferentiated EC, having high migratory, proteolytic, and prolifera-
tive rates, PDGF receptors, α smooth muscle actin, and a high TGFβ receptor type II to
type I ratio. In contrast, cells nearer the parent vessel deposit and interact with a mature
basement membrane and express several features of differentiated EC, having low migra-
tory, proteolytic, and proliferative rates, tight junction formation, loss of both PDGFα and
The Extracellular Matrix 13

gels. In the Davis and Camarillo study, anti-αvβ3 reagents had no effect on
HUVEC lumen formation in type I collagen gels (25). In contrast, Brooks et al.
have demonstrated significant inhibition of growth factor-stimulated (vascular
endothelial growth factor [VEGF] and bFGF) in vivo angiogenesis by anti-αvβ3
and αvβ5 reagents (44). These apparent discrepancies might reflect differences
between in vitro and in vivo models and the use of PMA in the in vitro models
described. Thus, in spite of the incompleteness of our understanding of extracellu-
lar matrix modulation of angiogenesis, there is accruing evidence that extracellu-
lar matrix-mediated integrin engagement that initiates specific signaling pathways
is an important modulator of angiogenesis.
The influences of extracellular matrix on the process of angiogenesis have
been demonstrated using endothelial cells derived from several vascular beds.
When RFC were cultured on the interstitial surface of amnion membranes, the
cells invaded and migrated into the interstitial collagen matrix and formed tube-
like structures with lumina, junctional complexes between cells, and abluminal
deposition of morphologically identifiable basal lamina (45). In later studies these
cells, dispersed in three-dimensional type I collagen gels, demonstrated multicel-
lular aggregation followed by tube formation with lumina and tight junction for-
mation at a baseline level in the absence of angiogenic factors, which was en-
hanced by the addition of angiogenic factors including bFGF, TGFβ1, TGFβ2
and endothelial cell growth factor (ECGF) (8, 46–48).
Bovine aortic endothelial cells (BAEC) have also been shown to form tubes
in specific culture conditions. In these studies clonal populations of ‘‘sprouting’’
BAEC were observed to migrate beneath the monolayer of cells and to form
tubelike structures, depositing type I collagen and displaying lumina (20, 49).
In both cases, (RFC and BAEC), the presence of cells in the three-dimen-
sional environment has been associated with the morphological end point of tube
formation and with dramatic changes in extracellular matrix synthetic profiles,
protease/protease inhibitor profiles, and growth factor receptor expression and
responsiveness (29, 34, 50, 51). Rat epididymal fat pad cell growth on type I
collagen coatings in two-dimensional culture expresses PDGF receptors, exhibits
PDGF responsiveness and exhibits an inhibition of proliferation and an extracel-
lular matrix inductive effect in response to TGFβ1. In contrast, RFC cultured in
three-dimensional type I collagen gels lose their PDGF receptor expression and

β receptors and α smooth muscle actin, and a low TGFβ receptor type II to type I ratio.
B. Schematic representation of specific culture conditions which mimic the modulation
of microvascular EC phenotypes during angiogenesis. Specific culture conditions mimic
either EC at the distal tip of an angiogenic sprout (two-dimensional culture) or differenti-
ated EC nearer the parent vessel (three-dimensional culture). Abbreviations: EC, endothe-
lial cells; PDGF, platelet-derived growth factor; TGF, transforming growth factor; V, type
V collagen; Fn, fibronectin; Ln, laminin; IV, type IV collagen.
14 Madri

responsiveness and exhibit a dramatic reduction in their expression of type II

TGFβ receptor. This reduction is associated with a loss of TGFβ1-mediated inhi-
bition of proliferation (34, 50). Placement of RFC in collagen gels also affects
protease expression. Culture of RFC on collagen coatings resulted in no changes
in expression of urokinase plasminogen activator (uPA) or plasminogen activator
inhibitor (PAI)-1 after TGFβ1 treatment, while culture in collagen gels resulted
in significant transient uPA and (PAI)-1 induction after TGFβ1 treatment, thought
to be required for extracellular matrix remodeling during the early phases of
angiogenesis (34). Changes in growth factor responsiveness have been docu-
mented in ‘‘sprouting’’ BAEC. In contrast to cobblestone BAEC, the tube-form-
ing ‘‘sprouting’’ BAEC express PDGF receptors and exhibit both PDGF respon-
siveness and a proliferative response to TGFβ1 (49, 51). These studies illustrate
the importance of extracellular matrix organization in modulating several aspects
of the angiogenic response (Fig. 1).

IV. PECAM-1 PHOSPHORYLATION/DEPHOSPHORYLATION:

A MODEL FOR EXTRACELLULAR MATRIX-
DRIVEN, INTEGRIN-MEDIATED SIGNALING
AFFECTING ENDOTHELIAL CELL
BEHAVIOR

The roles and control of cell adhesion molecules during angiogenesis are an as-
pect of endothelial cell behavior that has received attention recently. Two mem-
bers of this functional family appear early in vasculogenesis: vascular endothelial
cadherin (VE cadherin) and platelet endothelial cell adhesion molecule-1
(PECAM-1/CD31) (52–57). These molecules are thought to be involved in the
early phases of vessel formation, modulating the adhesive interactions of adjacent
endothelial cell processes during tube formation (54, 58–60). Of these two mole-
cules, PECAM-1’s tyrosine phosphorylation state, cellular localization, and func-
tion are modulated by extracellular matrix-mediated integrin engagement (61).
These recent studies have demonstrated that the tyrosine phosphorylation state
of PECAM-1 is decreased transiently during in vitro endothelial cell spreading
and migration on ECM components and during in vivo vasculogenesis in the
murine conceptus (60, 61). In these studies phosphoamino acid analyses and
Western blotting using antiphosphotyrosine antibodies showed that endothelial
PECAM-1 is tyrosine phosphorylated and that the tyrosine phosphorylation was
decreased after endothelial cell spreading and migration on extracellular matrix
components but not on plastic. In addition, cell adhesion to anti-integrin antibod-
ies similarly induced PECAM-1 dephosphorylation while concurrently inducing
pp125FAK phosphorylation. The tyrosine phosphorylation state of PECAM-1
was found to be mediated by both kinase and phosphatase. The PECAM-1 cyto-
The Extracellular Matrix 15

plasmic sequences flanking tyrosine residues 663 (Y663 TEV) and 686 (Y686 SEV)
are selected for in phosphopeptides that bind to src-homology 2 domains of src
family kinases and abl proto-oncogene (62), suggesting that both tyrosine 663
and 686 are potential phosphorylation sites. Furthermore both Y663 and Y686 were
identified as phosphorylation sites by site-directed mutagenesis to phenylalanine.
When expressed in 3T3 cells, both mutants (Y to F663 and Y to F686) exhibited
reduced PECAM-1 phosphorylation compared to 3T3 cells expressing wild-type
PECAM. Additionally, F686 mutants showed a reversal of PECAM-1 mediated
inhibition of 3T3 cell migration and did not localize PECAM-1 to cell borders.
These data suggest that β1 integrin engagement signals PECAM-1 dephosphory-
lation and that this signaling pathway plays a role during endothelial cell migra-
tion (Fig. 2) (61, 63).
In our studies we have found that PECAM-1 was more highly phosphory-
lated in endothelial cells overexpressing c-src and, in in vitro kinase assays, puri-
fied c-src phosphorylated a glutathione-s-transferase (GST)-PECAM cytoplasmic
tail fusion protein. We also found that the phosphorylated fusion protein did
associate with agarose bead-bound c-src. This association appeared to be medi-
ated by c-src-SH2 domain, as tyrosine phosphorylated PECAM-1 could be pre-
cipitated by a GST-src-SH2 affinity matrix. The binding to the GST-src-SH2
affinity matrix correlated directly with the level of PECAM-1 phosphorylation.
Specifically, more PECAM-1 derived from c-src overexpressing endothelial cells
(which is highly tyrosine phosphorylated) was bound to the SH2 affinity matrix
than PECAM-1 derived from vector-only transfectants or kinase-negative c-src
overexpressing cells (which are less tyrosine phosphorylated) (61, 64). Further,
several as yet unidentified phosphoproteins could also be coimmunoprecipitated
with wild-type but not with Y to F663 or F686 mutant PECAM-1 (65).
These data suggest that differential tyrosine phosphorylation of the
PECAM-1 cytoplasmic domain could function as a binding site for adaptor pro-
teins, kinases, and phosphatases in signaling cascades. Indeed, further analysis
of the PECAM-1 cytoplasmic tail revealed that it resembles an immunoregulatory
tyrosine-based activation motif (ITAM) domain, which has been identified in
subunits of the T-cell receptor (TCR), B-cell receptor (BCR), Fc receptors, brain
immunoglobulin TAM-like molecule (BIT), and natural killer-associated tran-
scripts (NKATs) that function in signal transduction (66–69).

Consensus: DXXX-XXXXDXXYXXLXXXXXXXXXXXXXXXXXXXXYXXL
E E I I
PECAM-1: DNKE-PLNSDVQYTEVQVSSAEWSHKDLGKKDTETVYSEV
663 686
ITAM Consensus and PECAM-1 Sequences

The PECAM-1 ITAM domain may function in a similar manner to the

other members of the ITAM family, becoming phosphorylated by a member of
16 Madri

Figure 2 Schematic representation of the dynamic tyrosine phosphorylation/dephos-

phorylation of PECAM-1 in endothelial cells and 3T3 cells expressing PECAM-1 or a
PECAM-1 Y to F686 mutation and correlations with migratory state of the cells.
Confluent cultures of endothelial cells exhibit a high level of PECAM-1 tyrosine
phosphorylation, with PECAM-1 localized to areas of cell-cell contact along the cell pe-
riphery. In contrast, after a stimulus to migrate, endothelial cells exhibit a low level of
PECAM-1 tyrosine phosphorylation, with PECAM-1 diffusely localized over the cell sur-
face. Levels of PECAM-1 tyrosine phosphorylation are determined by modulation of ki-
nase and phosphatase activities.
The Extracellular Matrix 17

the src kinase family and subsequently bound by a member of the ZAP70/syk
family or by other proteins containing single or tandem SH2 domains (61, 65).
Thus, the PECAM-1 ITAM domain could serve as a functional branch point of
an ‘‘outside to inside’’ signaling cascade, initiated extracellularly by the recogni-
tion of a particular extracellular matrix component by an integrin, which would
then alter the PECAM-1 tyrosine phosphorylation state by modulating kinase/
phosphatase ratios (65, 66, 69, 70). Conversely, the PECAM-1 ITAM domain
may also function in an ‘‘outside to inside’’ signaling cascade generated extracel-
lularly by PECAM-1–PECAM-1 engagement, affecting the PECAM-1 ITAM
phosphorylation state, initiating a signaling cascade that results in changes in
integrin affinity. Experiments using anti-PECAM-1 to ligate PECAM-1 have
shown that integrin function can be affected (57, 69, 71, 72). In addition, using
migration as a readout, we demonstrated that expression of a Y686 to F PECAM-1
construct in BAEC resulted in an increase in migratory rate. This finding is con-
sistent with the notion that the Y686 to F PECAM-1 competes with endogenous
PECAM-1 in adhesive interactions, thus decreasing ‘‘outside-in’’ signaling by
blunting phosphorylation at Y686 and subsequent interactions with SH2-containing
adaptor and signaling molecules, such as the phosphatase SHP-2 (73).
We have also demonstrated differential tyrosine phosphorylation of
PECAM-1 during the formation of blood islands/vessels from clusters of extra-
embryonic and embryonic angioblasts in the murine conceptus. We identified
differential phosphorylation of tyrosine residue, Y686, in the PECAM-1 cyto-
plasmic domain (60). In recent studies we demonstrated a correlation between a
failure of this transient, dynamic PECAM-1 tyrosine dephosphorylation and ar-
rest of the yolk sac vasculature at the primary capillary plexus stage (74). These
findings suggest a pivotal role for PECAM-1 during vasculogenesis that is sup-
ported by data demonstrating a role for PECAM-1 in modulating in vitro and in
vivo angiogenesis (58). These findings, taken together with knockout mouse stud-
ies illustrating critical roles for selected extracellular matrix components (fibro-
nectin), integrin chains (α5), and TGFβ1 in embryonic vascular development,
suggest the possibility that extracellular matrix-induced integrin-mediated modu-

3T3 cells stably expressing wild type PECAM-1 exhibit an inhibition in their migra-
tion rate compared to sham-transfected cells. In these cells, PECAM-1 was localized to
areas of cell-cell contact along the cell periphery. In contrast, 3T3 cells stably expressing
a mutated form of PECAM-1 having a Y to F686 mutation that results in a loss of tyrosine
phosphorylation, exhibit a high migration rate similar to sham-transfected cells. Further-
more, the mutated PECAM-1 expressed on the surface of these cells is localized diffusely
over the cell surface in a pattern similar to that noted for PECAM-1 in migrating endothe-
lial cells. Abbreviation: PECAM, platelet endothelial cell adhesion molecule; ECM, extra-
cellular matrix.
18 Madri

lation of PECAM-1 tyrosine phosphorylation may be a mechanism by which

these embryonic lethal knockouts cause maldevelopment and loss of integrity of
the developing vascular system (9–11, 60). Additional studies investigating the
cascades and specific proteins involved in extracellular matrix-driven, integrin-
mediated PECAM-1 signaling during embryonic vascular development and dur-
ing large vessel repair and angiogenesis during wound healing will lead to a better
understanding of the roles of this molecule in vasculogenesis and angiogenesis.

V. CONTROLLED INVOLUTION AND STABILIZATION OF

THE MICROVASCULATURE OF THE GERMINAL
MATRIX: THE ROLES OF EXTRACELLULAR MATRIX
SYNTHESIS AND ORGANIZATION

The roles of the extracellular matrix in the dynamic regulation of vasculogenesis

and angiogenesis are complex. This is well illustrated during brain development
(75, 76). Microvascular beds in the brain (as well as other organs and tissues)
undergo selective angiogenesis, stabilization, and involution in a tightly regulated
fashion (77–79). One particular microvascular bed in which this process is noted
is the germinal matrix, a periventricular area from which there is controlled neu-
ronal and glial emigration during development (80–83). During development,
the microvascular density is reduced over an extended period, coinciding with
the neuronal and glial emigrations and involution of the germinal matrix (84,
85). This controlled vascular involution occurs rapidly after premature delivery
and is thought to be a cause of perinatal and early postnatal intraventricular and
parenchymal hemorrhage observed in the premature newborn population (81, 86,
87). Animal models of perinatal and early postnatal intraventricular and paren-
chymal hemorrhage that mimic the human condition have been used to study the
roles of extracellular matrix in this process (88–91). Using a beagle pup model,
we have found that the period of the highest incidence of intraventricular and
parenchymal hemorrhage correlated with a morphologically indistinct and imma-
ture vascular basement membrane surrounding the germinal matrix microvessels,
few tight junctions, and incomplete astrocytic investiture of the microvessels (81,
86). In the surviving animals, the incidence of intraventricular and parenchymal
hemorrhage was noted to decrease over postnatal days 4 to 10. This decrease
in hemorrhages correlated with an increased abluminal deposition of basement
membrane components, which was followed by increased formation of tight junc-
tions and complete investiture of the surviving vessels by astrocytic endfeet (92)
(Fig. 3). These data suggest that the deposition and organization of extracellular
matrix components is a prerequisite for, and stimulates the formation of, endothe-
lial cell tight junctions and astrocytic endfeet investiture of the microvessels,
forming a competent blood-brain barrier (Fig. 4). The inductive mechanisms and
The Extracellular Matrix 19

Figure 3 Schematic representation of the temporal relationships of the incidence of

intraventricular and parenchymal hemorrhage (speckled line), the appearance of basement
membrane (solid line), and tight junctional complexes and coverage of germinal matrix
microvessel abluminal areas by supporting astrocytic cell processes (hatched line) from
postnatal days 1 to 10.

signal transduction cascades involved in this process are currently unknown;

however, treatment of the beagle pups with indomethicin induced a more rapid
appearance of basement membrane components that correlated with a reduction
in the incidence of intraventricular and parenchymal hemorrhage (87). These data
suggest that extracellular matrix deposition and organization elicits endothelial
cell tight junction formation and astrocytic endfoot investiture, leading to a stabi-
lized vascular bed resistant to rupture.
Although necessary and useful, animal models are difficult to manipulate
and by necessity result in the sacrifice of many animals. Thus, development of
in vitro models mimicking the in vivo situation are desirable. To this end we
have developed an in vitro culture model using microvascular endothelial cells
isolated from beagle pup germinal matrix cultured in three-dimensional type I
collagen gels (93, 94). These cells exhibited increased basement membrane com-
ponent synthesis, decreased proteolytic activity, and robust tube formation com-
pared to standard two-dimensional cultures. Coculture of these microvascular
endothelial cells and newborn rat type II astrocytes resulted in endothelial tube
formation mimicking in vivo tube formation and stabilization with abluminal
basement membrane deposition and astrocytic process investiture of the tubes,
mimicking the in vivo process of blood-brain barrier formation (Fig. 5). Further,
Figure 4 Schematic representation of the development of vascular integrity of the neu-
ronal microvasculature illustrating the initiation of tight junction formation and astrocytic
endfoot investiture by deposition and organization of the basement membrane and poten-
tial paracrine interactions between the endothelial and supporting astrocytic cells. Abbrevi-
ations: VEGF, vascular endothelial growth factor; bFGF, basic fibroblast growth factor;
TGF, transforming growth factor.

Figure 5 Double-label immunofluorescence micrographs of a three-dimensional cocul-

ture of astrocytes with brain microvascular endothelial cells. Scale bar ⫽ 50 µm. Original
magnification ⫽ 400x. A. Representative field of the coculture stained with fluorescein con-
jugated Bandeiraea lectin illustrating diffusely stained branching, tubelike structures. B.
The same representative field of the coculture stained with antibodies directed against glial
fibrillated acid protein and detected with a rhodamine- conjugated secondary antibody illus-
trating the investiture of segments of the tubelike structures by astrocyte processes. The
astrocyte processes are in intimate contact with endothelial cells that have formed tubes.
The Extracellular Matrix 21

the endothelial and astrocytic cells comprising these cocultures exhibit mutual
paracrine modulation as evidenced by the modulation of both endothelial and
astrocytic uPA, mediated by the close presence and interaction of the two cell
types in coculture.

VI. EXTRACELLULAR MATRIX AS A MODULATOR OF

APOPTOSIS

During brain development the germinal matrix microvascular bed undergoes sig-
nificant involution (86). There is selective concurrent stabilization and involution
of individual vessels in this vascular bed. As discussed above, the synthesis and
deposition of vascular basement membrane plays an important role in the stabili-
zation process, driving increased tight junction formation and astrocytic endfoot
investiture. Just as important is the process of selective vessel involution. Mor-
phological studies in the beagle pup revealed that the immature germinal matrix
vessels (and possibly those vessels destined to undergo involution) exhibit an
incomplete investiture by basement membrane and astrocytic endfeet, suggesting
a relationship between the presence of astrocytic endfeet, protease levels and
basement membrane, and vessel stabilization (81). In vitro studies in which ger-
minal matrix microvascular endothelial cells and astrocytes were cocultured in
three-dimensional type I collagen gels are consistent with this notion, as uPA
levels were decreased compared to monocultures of endothelial cells (93). These
data, and the findings of Cheresh et al. (42–44), suggest that the presence, compo-
sition, and physical state of the surrounding extracellular matrix determine endo-
thelial cell adhesive properties, which, in turn, determine apoptotic state of the
endothelial cells (Fig. 6).
Data accrued from in vivo and in vitro models showed the importance of
soluble factors—VEGF in particular—as modulators of cell survival and the
importance of interactions between the extracellular matrix and soluble factors
in determining endothelial cell behavior (Fig. 4). Using in vivo models, investiga-
tors found that VEGF expression is detectable around stable, quiescent microves-
sel endothelial cells (93); VEGF expression was found to be decreased during
physiological blood vessel regression (95); and VEGF withdrawal resulted in
vessel regression (96–98). Using an in vitro model of microvessel formation and
stabilization, we have demonstrated that even in the presence of a permissive
three-dimensional matrix of native type I collagen and a subendothelial extracel-
lular matrix composed of type IV collagen and laminin, endothelial cells that
have been stimulated to form capillary structures require the continued presence
of VEGF for survival (99). These studies underscore the importance of the com-
plex interrelationships between the signaling cascades initiated by specific recep-
tors whose engagement is modulated by the composition and organization of the
22 Madri

Figure 6 A. Schematic representation of the specific, controlled stabilization and

involution/apoptosis of the germinal matrix vasculature during development, resulting in
significant reduction in vessel density with the surviving vessels becoming the lenticulo-
striate vessels. Involution/apoptosis: changes or loss of astrocytic or endothelial cell fac-
tors or receptors lead to loss of basement membrane structure and integrity (decreased
synthesis/increased proteolysis), which leads to loss of endothelial cell adhesion and
apoptosis. Stabilization: formation and stabilization of the basement membrane maintains
endothelial cell adhesion, tight junction formation, and astrocytic investiture leading to
and maintaining formation of the blood-brain barrier. B. Immunofluorescence micrograph
(60 µ section) of postnatal day 10 beagle pup germinal matrix illustrating a mature, stabi-
lized vessel segment stained positively for laminin 1 and two smaller, atretic, involuting
vessel segments also exhibiting residual laminin staining. L, lumen; Lm, abluminal laminin
staining; arrows, involuting vessel segments; arrowheads, atretic, involuting vessel seg-
ments.

extracellular matrix and the composition and concentrations of soluble factors in

blood vessel formation, survival, and involution (99–101) (Fig. 6).
These ongoing studies illustrate the importance, complexity, and interrela-
tionships of cell-matrix, cell-cell, and cell-soluble factor interactions in the regu-
lation of angiogenesis.

VII. CONCLUSIONS

As discussed throughout this chapter, the extracellular matrix has many in vivo
and in vitro functions. The use of extracellular matrix preparations has enhanced
our ability to culture microvascular endothelial cells from a variety of vascular
beds. Extracellular matrix preparations are useful in modulating endothelial cell
phenotype and allowing the mimicking of in vivo behavior in defined culture
conditions. Extracellular matrix components and mixtures also have aided in the
The Extracellular Matrix 23

investigation and elucidation of a variety of signaling pathways operational in

regulating endothelial cell proliferation, adhesion, spreading, migration, and acti-
vation. Further, natural and engineered matrices are playing an increasingly im-
portant role in the continued development of artificial blood vessels and endothe-
lial cell-based gene delivery systems. The central importance of the extracellular
matrix in the process of angiogenesis also is evident in several of the following
chapters in this volume.

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