THE NEW ANGIOTHERAPY pdf

Stewart 8 Angiogenesis and Vascular Endothelial Growth Factor VEGF in Reproduction .... “Angiogenesis” wasdefined as the formation of new blood vessels by extension of existing blood ves

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The new angiotherapy/edited by Tai-Ping Fan and Elise C Kohn

p.; cm.

Includes bibliographical references and index.

ISBN 0-89603-464-X (alk paper)

1 Neovascularization 2 Neovascularization inhibitors I Fan, Tai-Ping D II Kohn, Elise C [DNLM: 1 Neovascularization, Pathologic 2 Neovascularization, Physiologic WG 500 N5324 2001] QP106.6 N49 2001

612.1'3—dc21

00-050029

D EDICATION

To Dorothy, our two children, Victoria and Patrick, as a token of my love and tion for their immense patience and many sacrifices in making my dreams come true Also

apprecia-to my grandmother, parents, and brothers and sisters for their constant encouragement.-TPF

To my husband, Gary Claxton, for his loving support, to my mentor, Dr Lance Liotta,for his valuable advice and encouragement, and to very dear friends without whom thisbook could not have been finished.-ECK

v

F OREWORD

vii

In this very timely and informative book, Tai-Ping Fan and Elise Kohn have

assembled, in The New Angiotherapy, chapters written by many of the leading scientists

in the field of angiogenesis research The molecular underpinnings of the angiogenicprocess in reproduction, development, repair, and disease are clearly developed Broadcoverage is given to preclinical and clinical applications of angiogenesis inhibitors, andthe book is a valuable source of references to the many aspects of the angiogenic process

It is, however, far more than a reference book It is organized so that the reader begins torecognize the emergence of unifying themes in the study of angiogenesis

The growth of new microvessels from resting vessels is the outcome of a fine balancebetween molecules that are either positive or negative regulators of angiogenesis Some

of these regulators reside in the extracellular matrix, while others circulate Severalangiogenesis regulators are cleavage products of larger proteins that have differentfunctions

An analogy of the angiogenic process to the clotting process, in which approximately

40 proteins determine whether or not a clot will form, is not too farfetched Negativeregulators of angiogenesis—angiogenesis inhibitors—can be thought of as a new class

of drugs They are being tested in clinical trials for neoplastic and non-neoplastic eases Certain of these drugs are fairly specific angiogenesis inhibitors and block mainlythe endothelial cells in the tumor bed Other inhibitors block endothelial cell prolifera-tion, but also have direct antitumor effects (2-methoxy-estradiol, for example) It is evenpossible to shift the effect of certain conventional cytotoxic agents toward an anti-angio-genic function by changes in dose and schedule It may be prudent to distinguish between

dis-“indirect” angiogenesis inhibitors that block a tumor from producing an angiogenic factor(such as small molecules designed to target an oncogene product such as vascularendothelial growth factor) in contrast to“direct” inhibitors that block vascular endothelialcells from responding to a spectrum of angiogenic factors

The need for surrogate markers that could measure efficacy of anti-angiogenic therapy

or could indicate in some way the angiogenic activity of a tumor is thoroughly discussed

in The New Angiotherapy It is now recognized that angiogenesis inhibitors may be most

effective if the dose is titrated against total angiogenic output of a patient’s tumor, inmuch the same way that insulin is titrated against the pulse rate, and coumarin dose isadjusted according to prothombin level In other words, as Sir James Black has empha-sized for drugs like propranolol, clinical trials may be optimally designed with a goal ofequal effect, rather than equal dose

The importance of quantifying the effectiveness of anti-angiogenic therapy is just one

of many new directions in angiogenesis research, both basic and clinical, that are lighted by the authors Future directions are also emphasized in the chapters on genetherapy, and therapeutic angiogenesis for ischemic disease of the heart and limbs, as well

high-as in new thinking about preventive anti-angiogenesis therapy

Judah Folkman, MD

Children’s Hospital, Harvard Medical School,

Boston, MA

P REFACE

ix

Angiogenesis is the development of new blood vessels from an existing vascular bed.Normal vascular proliferation occurs only during embryonic development, the femalereproductive cycle, and wound repair Many pathological conditions are characterized

by persistent, unregulated angiogenesis, such as cancer, atherosclerosis, rheumatoidarthritis, and diabetic neuropathy Conversely, inadequate angiogenesis can often lead tochronic pressure ulcers, duodenal ulcers, and myocardial infarction Control of vasculardevelopment will permit new therapeutic approaches to these disorders, whereas enhance-ment of angiogenesis by exogenous growth factors can prevent or limit the damage inchronic wounds and duodenal ulcers

The New Angiotherapy covers the recent progress in basic and applied research in

angiogenesis Critical reviews contributed by an international team of experts discuss thefundamental concepts in the physiology and pathophysiology of angiogenesis and evalu-ate the potential of angiotherapy in the management of angiogenic disease, highlightingsome of the angiogenics and antiangiogenics both in development and in clinical trials.The future prospects of receptor antagonists, enzyme inhibitors, and vascular targetedapproaches, especially that of gene therapy in the development of angiotherapy, are alsocovered

Over the past five years, angiogenesis research has been expanding rapidly To keepabreast of the most recent developments in the principles and practice of angiotherapy,

we recommend Angiogenesis—the only specialist journal in the field Please visit the

website at http://www.wkap.nl/journalhome.htm/0969-6970

We would like to thank the following colleagues for their generous assistance inreviewing some of the manuscripts: Adriana Albini, Robert Auerbach, Lee Ellis, ArjanGriffieon, Pamela Jones, Pieter Koolwijk, Macro Presta, David Walsh, JohannesWaltenberger, and David West

Tai-Ping D Fan, P h D Elise C Kohn, MD

C ONTENTS

Foreword viiPreface ixList of Contributors xv

1 Vasculogenesis and Angiogenesis 1

Robert Auerbach and Wanda Auerbach

Amy D Bradshaw and E Helene Sage

II Angiogenesis in Health and Disease

5 Integrin Receptors and the Regulation of Angiogenesis 67

Dorothy Rodriguez and Peter C Brooks

6 Vasoactive Peptides in Angiogenesis 81

David Andrew Walsh and Tai-Ping D Fan

7 Angiogenesis in Wound Healing and Surgery 105

Wayne A Morrison, Geraldine M Mitchell, Jane E Barker, Tamara Konopka, and Alastair G Stewart

8 Angiogenesis and Vascular Endothelial Growth Factor (VEGF)

in Reproduction 115

D Stephen Charnock-Jones, Yulong He, and Stephen K Smith

9 Rheumatoid Arthritis: A Target for Anti-Angiogenic Therapy? 129 Ewa M Paleolog and Jadwiga M Miotla

10 Diagnostic and Prognostic Significance of Tumor Angiogenesis 151

Stephen B Fox and Adrian L Harris

III Stimulation of Angiogenesis

[Therapeutic Angiogenesis]

11 Hyaluronan Oligosaccharides Promote Wound Repair:

Its Size-Dependent Regulation of Angiogenesis 177 David C West and Tai-Ping D Fan

12 Role of Thymic Peptides in Wound Healing 189

Hynda K Kleinman, Derrick S Grant, and Katherine M Malinda

xi

13 Angiogenesis and Growth Factors in Ulcer Healing 199

Sandor Szabo, Yuen Shing, M Judah Folkman, Aron Vincze, Zoltan Gombos, Xiaoming Deng, Tetayana Khomenko, and Masashi Yoshida

14 Angiogenesis in Skeletal and Cardiac Muscle:

Role of Mechanical Factors 213 Margaret D Brown and Olga Hudlická

15 Therapeutic Angiogenesis for the Treatment

of Cardiovascular Disease 249

Jeffrey M Isner and Takayuki Asahara

16 Therapeutic Angiogenesis for the Heart 279

Johannes Waltenberger and Vinzenz Hombach

17 Contact-Guided Angiogenesis and Tissue Engineering 295

Robert A Brown, Giorgio Terenghi, and Clive D McFarland

IV Anti-Angiogenesis

18 Inhibitors of Matrix Metalloproteinases 315

Peter D Brown and William G Stetler-Stevenson

19 Control of Angiogenesis by Microbial Products 329

Tsutomu Oikawa

20 Interaction of Angiogenic Growth Factors with Endothelial Cell

Heparan Sulfate Proteoglycans: Implications for the Development of Angiostatic Compounds 357 Marco Rusnati, Giovanni Tulipano, and Marco Presta

21 2-Methoxyestradiol: A Novel Endogenous Chemotherapeutic and Anti-Angiogenic Agent 387 Victor S Pribluda, Theresa M LaVallee, and Shawn J Green

V Novel Targets

22 Receptor Tyrosine Kinases in Angiogenesis 409

Laura K Shawver, Kenneth E Lipson, T Annie T Fong, Gerald McMahon, and Laurie M Strawn

23 Targeting Gene Therapy to the Tumor Vasculature 453

Kelvin K W Lau and Roy Bicknell

24 Antibody Targeting of Tumor Vasculature 475

J Wilson, David C West and Philip E Thorpe

25 Endothelial Monocyte-Activating Polypeptide II:

A Novel Injury Signal? 491 Cliff Murray and Maarten Tas

26 CD105 Antibody for Targeting of Tumor Vascular

Endothelial Cells 499

Ben K Seon and Shant Kumar

VI Angiotherapy in the Clinic

27 Design of Pharmacological and Diagnostic Strategies

for Angiogenesis-Dependent Diseases 517

Lucia Morbidelli and Marina Ziche

28 Design of Clinical Trials for Anti-Angiogenics 527

Elise C Kohn

29 Angiogeneis Therapies: Concepts, Clinical Trials,

and Considerations for New Drug Development 547 William W Li, Vincent W Li, and Dimitris Tsakayannis

30 Angiostatin and Endostatin: Recent Advances in Their Biology, Pharmacokinetics, and Potential Clinical Applications 573 Jesus V Soriano and B Kim Lee Sim

Index 597

Amy D Bradshaw, Department of Biological Structure, University of Washington, Seattle, WA

Peter C Brooks • Department of Biochemistry and Molecular Biology, Norris Cancer Center, USC School of Medicine, Los Angeles, CA

Margaret D Brown • School of Sport and Exercise Sciences, University of Birmingham, Birmingham, UK

Peter D Brown • Department of Clinical Research, British Biotech Pharmaceuticals, Oxford, UK

Robert A Brown • University College London, Plastic and Reconstructive Surgery, Tissue Repair Unit, London, UK

D Stephen Charnock-Jones • Reproductive Molecular Research Group, Department

of Obstetrics and Gynaecology, University of Cambridge, The Rosie Hospital, Cambridge, UK

Xiaoming Deng • Departments of Pathology and Pharmacology, University of California, Irvine, CA; and Pathology and Laboratory Medicine Service, VA Medical Center, Long Beach, CA

Tai-Ping D Fan • Angiogenesis Laboratory, Department of Pharmacology, University

Zoltan Gombos • Departments of Pathology and Pharmacology, University of California, Irvine, CA; and Pathology and Laboratory Medicine Service, VA Medical Center, Long Beach, CA

Derrick S Grant • Jefferson Medical College, Thomas Jefferson University, Philadelphia, PA Shawn J Green • EntreMed, Rockville, MD

Adrian L Harris • Molecular Oncology Laboratory, Imperial Cancer Research Fund, University of Oxford Institute of Molecular Medicine, John Radcliffe Hospital, Oxford, UK

Yulong He • Reproductive Molecular Research Group, Department of Obstetrics and Gynaecology, University of Cambridge, Cambridge, UK

Vinzenz Hombach • Department of Internal Medicine II, Ulm University Medical Center, Ulm, Germany

Olga Hudlická • Department of Physiology, University of Birmingham, Birmingham, UK Jeffrey M Isner • St Elizabeth’s Medical Center, Boston, MA

Pamela F Jones • Molecular Medicine Unit, University of Leeds, Clinical Sciences Building, St James’s University Hospital, Leeds, UK

Tetayana Khomenko • Departments of Pathology and Pharmacology, University

of California, Irvine, CA; and Pathology and Laboratory Medicine Service, VA Medical Center, Long Beach, CA

Hynda K Kleinman • Cell Biology Section, National Institute of Dental Research, NIH, Bethesda, MD

Tamara Konopka • Department of Pharmacology, University of Melbourne,

Theresa M LaVallee • EntreMed, Rockville, MD

William W Li • The Angiogenesis Foundation, Cambridge, MA

Vincent W Li • The Angiogenesis Foundation, Cambridge, MA

Kenneth E Lipson • Preclinical and Pharmaceutical Development, SUGEN, South San Francisco, CA

Katherine M Malinda • Cell Biology Section, National Institute of Dental Research, National Institutes of Health, Bethesda, MD

Stefano J Mandriota • Department of Morphology, University of Geneva Medical Center, Geneva, Switzerland

Clive D McFarland • CSIRO, Division of Biomolecular Engineering, Sydney Laboratory, North Ryde, New South Wales, Australia

Gerald McMahon • Preclinical and Pharmaceutical Development, SUGEN, South San Francisco, CA

Geraldine M Mitchell, Bernard O’Brien Institute of Microsurgery, St Vincent’s Hospital, Fitzroy, Australia

Jadwiga M Miotla, Kennedy Institute of Rheumatology, London, UK

Roberto Montesano, Department of Morphology, University of Geneva Medical Center, Geneva, Switzerland

Lucia Morbidelli • Institute of Pharmacological Sciences, University of Siena, Siena, Italy

Wayne A Morrison • Bernard O’Brien Institute of Microsurgery, St Vincent’s Hospital, Fitzroy, Australia

Cliff Murray • University of Nottingham Lab of Molecular Oncology, CRC Department

of Clinical Oncology, City Hospital, Nottingham, UK

Tsutomu Oikawa • Department of Cancer Therapeutics, The Tokyo Metropolitan Institute

of Medical Science (Rinshoken), Tokyo, Japan

Ewa M Paleolog • Endothelial Cell Biology Group, Kennedy Institute of Rheumatology, London, UK

Michael S Pepper • Department of Morphology, University of Geneva Medical Center, Geneva, Switzerland

Marco Presta • General Pathology and Immunology, University of Brescia School

of Medicine, Brescia, Italy

Victor S Pribluda • EntreMed, Rockville, MD

Dorothy Rodriguez • Department of Biochemistry and Molecular Biology, Norris Cancer Center, University of Southern California School of Medicine,

Yuen Shing • Department of Surgery, Children’s Hospital, Harvard Medical School, Boston, MA

Stephen K Smith • Reproductive Molecular Research Group, Department of Obstetrics and Gynaecology, University of Cambridge, Cambridge, UK

William G Stetler-Stevenson • The Extracellular Matrix Pathology Section, Laboratory

of Pathology, National Cancer Institute, Bethesda, MD

Alastair G Stewart • Department of Pharmacology, University of Melbourne, Parkville, Australia

Laurie M Strawn • Preclinical and Pharmaceutical Development, SUGEN, South San Francisco, CA

Sandor Szabo • Pathology and Laboratory Medicine Service, VA Medical Center, Long Beach, CA; Departments of Pathology and Pharmacology, University

of California, Irvine, CA

Maarten Tas • University of Nottingham Lab of Molecular Oncology, CRC Department

of Clinical Oncology, City Hospital, Nottingham, UK

Giorgio Terenghi • Blond-McIndoe Research Center, Queen Victoria Hospital, East Grinstead, Sussex, UK

Philip E Thorpe • Harold C Simmons Comprehensive Cancer Center, Dallas, TX Dimitris Tsakayannis • The Angiogenesis Foundation, Cambridge, MA

Giovanni Tulipano • Unit of General Pathology and Immunology, Department

of Biomedical Science and Biotechnology, University of Brescia School of Medicine, Brescia, Italy

Aron Vincze • Departments of Pathology and Pharmacology, University of nia, Irvine, CA; and Pathology and Laboratory Medicine Service, VA Medical Center, Long Beach, CA

Califor-David Andrew Walsh • Academic Rheumatology, University of Nottingham Clinical Sciences Building, City Hospital, Nottingham, UK

Johannes Waltenberger • Department of Internal Medicine II (Cardiology), Ulm University Medical Center, Ulm, Germany

David C West • Department of Immunology, Faculty of Medicine, The University

Marina Ziche • Institute of Pharmacological Sciences, University of Siena, Siena, Italy

C OLOR P LATES

Color plate for Chapter 15 appears as an insert following page 262 Color plates forChapters 23 and 26 appear as an insert following page 490

Plate 1 (Fig 2 from Chapter 15, for full caption see page 255)

Plate 2 (Fig 1 from Chapter 23, for full caption see page 457)

Plate 3 (Fig 2 from Chapter 23, for full caption see page 461)

Plate 4 (Fig 3 from Chapter 26, for full caption see page 506)

Plate 5 (Fig 8 from Chapter 26, for full caption see page 512)

xix

I C ONCEPTS

From: The New Angiotherapy

Edited by: T.-P D Fan and E C Kohn © Humana Press Inc., Totowa, NJ

1

INTRODUCTION

The principle that underlies the targeting of blood vessels, “Angiotherapy,” is that thedifferentiation, maintenance, and homeostasis of blood vessels may be targets forintervention in a variety of normal and disease processes Wound healing, placentadevelopment, estrus-associated uterine changes, vascular events accompanying hairgrowth, and normal vessel maintenance are among the many normal processes that maybenefit from angiotherapy Tumor-associated angiogenesis, ocular neovascularization,inflammation-associated vascular manifestations, autoimmune disease-relatedneovascularization, vascular repair following myocardial damage, dermatologicalvascular pathologies associated with scleroderma and psoriasis — there is an almost unend-ing list of “angiogenic diseases” where angiotherapy can be of critical importance

In this context it is of paramount significance that the mechanisms underlying vessel development are by no means fully understood Where conflicting views haveemerged from the many experimental studies of blood-vessel formation, they have led toefforts to define and compartmentalize these views, thereby generating a sense ofconfidence concerning our understanding of vascular development that tends to over-shadow our uncertainties

blood-The origin of blood vessels during embryogenesis has been a subject of debate at leastsince the middle of the 19th century when His proposed that all blood vessels originate from

pre-existing blood vessels (1) This view was widely accepted until the 1920s when, in a

series of experimental studies of avian and mammalian embryos, Sabin, Regan, Clarke, and

others reported on de novo in situ development of blood vessels during the differentiation

of various organ rudiments (2–4) For a detailed discussion of these historically important experiments, see reviews by Wagner (5) and Auerbach and Joseph (6).

Vasculogenesis and Angiogenesis

Robert Auerbach and Wanda Auerbach

CONTENTS

INTRODUCTIONANGIOGENESIS AND VASCULOGENESIS COEXIST DURING EMBRYONICDEVELOPMENT

ANGIOGENESIS AND VASCULOGENESIS IN THE ADULTCONCLUDING COMMENTS

REFERENCES

More recently, two types of experiments have been used to further our understanding

of blood vessel development In the first, organ rudiments from one type of embryohave been grafted into another embryo For example, mouse metanephric rudiments were

transplanted onto the chorio-allantoic membrane (CAM) of a chick embryo (7) Simple

histological preparations using staining of DNA as well as comparison of nuclearsize could readily distinguish mouse from chick nuclei, because mouse nuclei were largerand contained about four times as much DNA as chicken nuclei These experimentsclearly showed that chicken blood vessels invaded the grafted mouse metanephric rudi-ment, thus supporting His’s early concepts of vascular origins

The second type of experiment was superficially similar, and involved the grafting

of quail embryonic tissues into chicken embryos (8) Again nuclear morphology and

DNA staining (Feulgen) could be used to distinguish donor from host cells and tissues.However, by using more primitive sources of donor tissues, donor cells could be found

in blood vessels throughout the embryo, even in regions distant from the original graftsite These results supported the view that blood vessel development occurred bydifferentiation of precursor cells found within a developing organ rudiment rather than

by extension of existing blood vessels

In an effort to resolve these differences, Poole and Coffin (9) and Werner Risau (see ref 10) proposed two definitions: “Vasculogenesis” was defined as the devel-

opment of blood vessels from precursor cells (angioblasts) “Angiogenesis” wasdefined as the formation of new blood vessels by extension of existing blood vessels.Vasculogenesis was seen as a process restricted to early embryonic development,whereas angiogenesis was associated with new blood-vessel formation operativeduring subsequent organogenesis and continuing throughout the life span of theindividual These definitions have found general acceptance By those definitions,angiotherapy may aim to induce angiogenesis, as, for example, in the eliciting ofcollateral blood vessels in the heart, or it may aim to inhibit angiogenesis, as in thecontrol of tumor growth and metastasis Vasculogenesis is seen to be of little or nosignificance in angiotherapy

But we are in danger of oversimplifying in an effort to understand blood-vesseldevelopment and thereby missing important aspects of angiotherapy Foremost is theidea that vasculogenesis and angiogenesis are mutually exclusive This idea is simply

wrong As we have pointed out previously (11) blood-vessel development in the

metanephros involves both angiogenesis and vasculogenesis

Still, these are embryonic rudiments, and therefore do not answer the importantquestion of whether vasculogenesis may still occur in adults That this is in fact the case

is argued best by combining two types of experimental results: tumor transplantationexperiments, and recovery from vascular insult In tumor transplantation experiments

carried out with adult mice, Wang et al (12) were able to show that individual primitive

endothelial cells could participate in angiogenesis, i.e., embryonic endothelial cells orprecursor cells, co-injected subcutaneously with tumor cells gave rise to new blood

vessels associated with the growing tumor Isner and his group (13,14) showed that there

were circulating angioblasts in adults, and that these could assist in (or give rise to)endothelial cells during neovascularization or vascular repair Moreover, it had long beenreported and recently confirmed that there are angioblasts (endothelial-cell precursors)

in the adult bone marrow, and that these can pass through the circulation to contributecells to blood vessels arising during neovascularization

ANGIOGENESIS AND VASCULOGENESIS COEXIST DURING

resulted in all vessels originating from the chick embryo (7,15) When Robert et al (16)

cultured 12-d mouse rudiments in vitro, they were able to find only scattered flk-1+ cells,

(flk-1 marking angioblasts) and no vessels developed in vitro, supporting the Ekblom findings.However, when they then grafted these rudiments into syngeneic adult hosts, numerous flk-1+ endothelial cells developed in the vessels and glomeruli of the graft, and all were of donororigin Thus, it appears that the xenograft experiments may have led to misleading conclusionsconcerning the origin of at least some of the vasculature of the developing kidney.The developing brain rudiment has always been considered the prime example for a

vasculature that is exclusively derived by angiogenesis (10,17) In our own laboratory we

have shown that yolk-sac endothelial cells co-cultured with brain rudiments develop

brain-specific properties (18) However, although this in vitro model demonstrated that

individual endothelial cells could acquire organ specificity when introduced intodeveloping embryonic rudiments such as the brain, the model did not distinguish betweenthe possibility that such organ-specific differentiation would occur if endothelial cellsmigrated as a sheet from pre-existing blood vessels and the alternative that they entered

as individual cells through the circulation Hatzopoulas et al (19) recently injected

cultured endothelial cells originating from isolates of early mouse embryonic mesoderminto the extra-embryonic chick-embryo circulation, and these cells were subsequentlylocalized in several sites, including the brain vasculature These studies suggest, althoughthey do not give conclusive evidence, that vasculogenesis plays a role in brain blood-vessel development, and especially supports the idea that maintenance of a healthyvasculature may depend on a repopulating pool of endothelial cell precursors (see also

the earlier studies of Stewart and Wiley, ref 20).

The developing limb is yet another organ whose vascularization was always believed to

be entirely through the process of angiogenesis, but has now also been shown to involve

both angiogenesis and vasculogenesis (21) These investigators use the terms “angiotrophic”

and “angioblastic” to describe blood-vessel formation, a terminology that, although notadopted generally, is clearer, because vessel formation (literally, vasculogenesis) involvesboth formation of blood vessels by extension of existing vessels and by de novo formationfrom angioblasts

Even the early embryonic neural tube, long thought to be comprised entirely ofneurogenic cells, has been shown to become vascularized both by sprouting of pre-existing endothelial cells and by angioblasts that have entered the neural tube prior to

establishment of patent blood vessels (22).

ANGIOGENESIS AND VASCULOGENESIS IN THE ADULT

The fact that hematopoietic and endothelial stem cells can both develop from a singleprecursor cell, the hemangioblast, and that endothelial cells and hematopoietic cellsoriginate in close apposition during development of the embryonic yolk sac and the fetal

liver suggested that a similar close relationship between endothelial and hematopoietic

precursor cells could exist in adult life as well (23) That this was so was clearly reported

by Shi et al (13) In vitro experiments demonstrated that human CD34+ hematopoietic

precursor cells generated adherent endothelial cells with high proliferation potential whencultured in the presence of vascular endothelial growth factor (VEGF) Moreover, usingmolecular markers to distinguish host from donor cells in a canine model of bone marrowtransplantation, these investigators demonstrated that 12 wk after placing a Dacron graft indog recipients, bone marrow-derived donor cells were the only source of the CD34+ endot-helial cells in the newly vascularized graft The authors conclude that “vasculogenesis is notonly restricted to early embryogenesis, but may play a physiological role as demonstrated

in this study, or may contribute to the pathology of vascular diseases in adults” (13) The results were not, in fact, unexpected In the 1960s, Stump et al (24) and Gonzales

et al (25) had shown that endothelial cells, apparently derived from the circulation,

could seed Dacron grafts in vivo There was, however, no direct evidence that thesecells were originally of bone marrow origin On the other hand, the in vitro and in vivo

results of Shi et al (13) extend the earlier finding that bone marrow CFU-A (in vitro)

and CFU-F (in vivo) colonies obtained from adult bone marrow can exhibit

endothe-lial-cell phenotypes (26).

The results of Asahara et al., demonstrating that circulating CD34+ cells contribute

to new blood vessel formation in adults are most readily interpreted as the result ofrelease of bone marrow-derived progenitor cells In this connection it is interesting thatthese investigators have also reported unpublished observations that indicate angioblast

contributions to VEGF-induced corneal neovascularization (27) This finding is

particularly important, because the corneal model has universally been accepted as amodel for angiogenesis and not for vasculogenesis It is particularly intriguing thatendothelial-cell progenitors may express tie-2, the receptor for angiopoietin 1 and 2

(28), growth factors whose action previously were considered only as affecting

angio-genesis and not vasculoangio-genesis

If one accepts the concept that angioblasts in the circulation can give rise to newblood vessels, then vasculogenesis as well as angiogenesis should become a target forangiotherapy Targetting vasculogenesis may require different strategies than thoseused when targetting angiogenesis: for example, angioblasts express many cell-sur-face receptors shared with hematopoietic stem cells, receptors that to a large extentare no longer expressed on mature endothelial cells Critical among these are homing(adhesion) molecules, for in order to participate in local blood-vessel formation theymust recognize a target-site receptor, attach, and extravasate from the circulation.Differentiated endothelial cells resident within blood vessels need no such homingdevices, but, on the other hand, must be able to traverse the basement membranes/extracellular matrix that stabilize mature blood vessels

Targeting only angiogenesis or only vasculogenesis, moreover, may not be sufficient toprevent neovascularization One may draw an analogy from hematopoietic repair followingirradiation There are short-term and long-term aspects to hematopoiesis, and distinctionsare best seen when comparing low-dose and high-dose irradiation Short-term restitutionmay be achieved by transplanting bone marrow cells, most of which disappear within a fewweeks after transplantation Long-term restitution is also achieved with bone marrow trans-plantation, but here the source of cells is a minority CD34+ stem-cell population whosedifferentiation, with time, leads to complete restitution

CONCLUDING COMMENTS

This review has focused on the potential cooperation between two processes,angiogenesis and vasculogenesis This does not mean that each of these cannot occurindependently of the other There is, for example, no compelling evidence thatvasculogenesis, i.e., the differentiation of angioblasts during blood-vessel formation, is

an obligatory component of tumor-induced angiogenesis However, because circulatingprecursor cells can enter new blood vessels, and because these cells have been clearlydemonstrated to exist, it would seem reasonable to assume that they do, in fact, participate

in neovascularization Similarly, although there is no doubt that vasculogenesis is themode of origin of the initial vascular plexus of the yolk sac, three-dimensional structuresthat form even before the circulation is established may well provide a platform fromwhich sprouting, i.e., angiogenesis, can become a major element during even the primitiveblood-vessel development of the early embryo

At present, most, if not all, angiotherapeutic protocols target blood vessels formingduring the process of angiogenesis As we learn more about the intricate interplay betweenangioblasts and the expanding sprouting vasculature, we may be able to improve long-term efficacy of treatment by including the angioblast and the process of vasculogenesis

in angiotherapeutic protocols

REFERENCES

1 His, W (1868) Untersuchungen über die erste Anlage des Wirbelthierleibes Leipzig, F C W Vogel, Germany.

2 Sabin, F R (1917) Origin and development of the primitive vessels of the chick and of the pig Contrib.

Embryo Carnegie Inst Publ (Washington) 6, 61–124.

3 Reagan, P R (1917) Experimental studies on the origin of vascular endothelium and of erythrocytes.

Amer J Anat 21, 39–176.

4 Clark, E R and Clark, E L (1935) Observations on changes in blood vascular endothelium in the living

animal Am J Anat 57, 385–438.

5 Wagner, R C (1980) Endothelial cell embryology and growth Adv Microcirc 9, 45–75.

6 Auerbach, R and Joseph, J (1984) Cell surface markers on endothelial cells: a developmental

perspective, in Biology of Endothelial Cells (Jaffe, E A., ed.), Martinus Nijhoff, Boston, pp 393–400.

7 Ekblom, P., Sariola, H., Karkinen, M., and Saxen L The origin of the glomerular endothelium Cell Diff.

11, 35–39.

8 Noden, D M (1991) Development of craniofacial blood vessels, in The Development of the Vascular

System [Issues in Biomedicine 14] (Feinberg, R., Shearer, G., and Auerbach, R., eds.), Karger, pp 1–24.

9 Poole, T J and Coffin, J D (1989) Vasculogenesis and angiogenesis: Two distinct morphogenetic

mechanisms establish embryonic vascular pattern J Exp Zool 251, 224–231.

10 Risau, W (1997) Mechanisms of angiogenesis Nature 386, 671–674.

11 Auerbach, R and Auerbach, W (1997) Profound effects on vascular development caused by

perturbations during organogenesis Am J Pathol 151, 1183–1186.

12 Wang, S J., Greer, P., and Auerbach, R (1996) Isolation and propagation of yolk sac-derived endothelial

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Vitro Cell Dev Biol 32, 292–299.

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from vascular repair to therapeutics Cardiovasc Res 38, 54–68.

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cells expressing flk-1 are intrinsic, vasculogenic angioblasts Am J Physiol 271, F744–F753.

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18 Yu, D and Auerbach, R (1999) Brain-specific differentiation of mouse yolk sac endothelial cells Brain

Res Dev Brain Res 117(2), 159–169 [cf.Yu, D (1999) Mouse yolk sac endothelial cells and their organ

specific differentiation PhD dissertation, University of Wisconsin.]

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characterization of endothelial progenitor cells from mouse embryos Development 125, 1457–1468.

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26 Eckmann, L., Freshney, M., Wright, E G., Sprout, A., Wilkie, N., and Pragnell, I B (1988) A novel in

vitro assay for murine haematopoietic stem cells Br J Cancer Suppl 9, 36–40.

27 Asahara, T., Chen, D., Takahashi, T., Fujikawa, K., Kearney, M., Magner, M., et al (1998) Tie2 receptor

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progenitor endothelial cells for angiogenesis Science 275, 965–967.

From: The New Angiotherapy

Edited by: T.-P D Fan and E C Kohn © Humana Press Inc., Totowa, NJ

2

INTRODUCTION

The establishment and maintenance of a vascular supply is an absolute requirement forthe growth of normal and neoplastic tissues, and as might be predicted, the cardiovascularsystem is the first organ system to develop and to become functional during embryogen-esis Both during development and in postnatal life, all new blood vessels begin as simpleendothelial-lined tubes Some become capillaries after developing an intimate associa-tion with pericytes, whereas others develop into vessels of larger diameter (arteries andveins) and acquire a variable number of concentrically disposed smooth-muscle celllayers Traditionally, the formation of new blood vessels has been ascribed to two inter-related but separable processes, vasculogenesis, and angiogenesis (Fig 1)

Definitions

Vasculogenesis is a series of differentiation and morphogenetic events which result inthe formation of a primary capillary plexus, and is comprised of at least three stages: 1)

the in situ differentiation of mesodermal cells into angioblasts or hemangioblasts; 2) the

differentiation of angioblasts and hemangioblasts into endothelial cells (and etic cells in the case of the hemangioblast); and 3) the organization of newly formedendothelial cells into a primary capillary plexus The existence of the angioblast, whichdifferentiates exclusively into endothelial cells, has been well-established However,definitive proof for the existence of the hemangioblast, which is purported to havethe dual capacity to differentiate into either endothelial or hematopoietic cells, is still

hematopoi-lacking (reviewed in refs 1–4).

The term angiogenesis, derived from the Greek words “angeion” and “genesis,”

mean-ing vessel and production respectively, was coined by Hertig in 1935 (5) to describe the

formation of new blood vessels in the placenta As will become apparent however,angiogenesis is not limited to this setting, and a more contemporary definition would be

“the formation of new capillary blood vessels by a process of sprouting from pre-existingvessels in a variety of developmental, physiological, and pathological settings.” A similaralthough far less well-studied process also occurs in the lymphatic system, and is some-times referred to as lymphangiogenesis

Evidence has recently been provided for the existence, in the peripheral circulation, of

an endothelial-cell precursor that contributes to the formation of new blood vessels in

postnatal life (6) These findings are likely to have a major impact on the current

defini-tion of angiogenesis Recall that although the formadefini-tion of capillary-like tubes is implicit

in the definitions of both vasculogenesis and angiogenesis, primary differentiation ofmesoderm into angioblasts is a process that is limited exclusively to vasculogenesis.Because mesoderm does not persist into postnatal life, the definition of vasculogenesis

is unlikely to be affected by this new finding However, the existence of a circulatingendothelial precursor does mean that the source of new endothelium during angiogenesiscan no longer be ascribed exclusively to sprouting from pre-existing vessels Thus thedefinition of angiogenesis will have to be extended to include the incorporation of

Fig 1 Vasculogenesis and angiogenesis Blood vessels are formed by two processes:

vasculogenesis, the in situ differentiation of mesodermal precursors into endothelial cells, which

subsequently organize into tube-like capillaries to form a primary capillary plexus, and esis, in which new capillaries are formed by a process of sprouting from pre-existing capillaries

angiogen-or post capillary venules Adapted from ref 21, with copyright permission from Springer-Verlag

GmbH & Co KG.

endothelial progenitors and/or their progeny into newly forming vessels Important questionsconcerning the origin of circulating precursors (possibly from the bone marrow) as well astheir precise relationship to angioblasts (and hemangioblasts) remain to be answered.

As indicated earlier, the immature endothelial-lined tubes that arise during vasculogenesisand angiogenesis subsequently differentiate into capillaries (after association with pericytes)

or into larger vessels such as arteries and veins (after forming a media composed essentially

of smooth-muscle cells) Furthermore, capillaries in many organs undergo further tiation and develop organ-specific functions Examples include formation of the blood-brainbarrier (BBB) in the central nervous system (CNS), formation of fenestrated endothelium inendocrine and other organs, formation of sinusoids in the liver and spleen, and formation ofhigh endothelial venules in lymph nodes These processes, which occur in primitive vesselsresulting either from vasculogenesis or angiogenesis, should be referred to as secondarydifferentiation, in order to distinguish them clearly from primary angioblast and endothelialcell differentiation

differen-The multiple cell functions that occur during angiogenesis belong either to a phase ofactivation or to a phase of resolution The activation phase encompasses initiation andprogression, and includes: 1) increased vascular permeability and extravascular fibrindeposition; 2) basement-membrane degradation; 3) cell migration and extracellularmatrix invasion; 4) endothelial-cell proliferation; and 5) capillary lumen formation Thephase of resolution encompasses termination and vessel maturation, and includes: 1)inhibition of endothelial cell proliferation; 2) cessation of cell migration; 3) basement-membrane reconstitution; and 4) junctional complex maturation As indicated earlier, thedefinition of vasculogenesis includes both primary endothelial-cell differentiation, aswell as the organization of these endothelial cells into capillary-like tubes With respect

to the phases of activation and resolution, many components are equally as applicable tovasculogenesis as they are to angiogenesis Although a great deal is known about thosefactors that induce the activation phase, very little is known about the factors involved

in the phase of resolution, in which the dominant activity of negative regulators is calledinto play Furthermore, it is at present unclear as to whether the resolution phase is anactive phase, or whether it is the consequence of exhaustion of positive regulators thatpredominated during the phase of activation If the latter hypothesis is correct, thisassumes that endothelial cells have the inherent capacity to synthesize their own base-ment membrane and to organize into capillary-like tubes, and that this is mediated in part

by the autocrine activity of endogenous regulators

Angiogenesis in Pre- and Postnatal Life

Although, by definition, vasculogenesis must precede angiogenesis, the two processescontinue in parallel during early development However, unlike vasculogenesis, whichappears to be restricted to early development, angiogenesis is also required for the main-tenance of functional and structural integrity of the organism in postnatal life Thus itoccurs during wound healing, in inflammation, in situations of ischemia, and in femalereproductive organs (in the ovary during ovulation and corpus luteum formation; in theplacenta and mammary gland during pregnancy) Angiogenesis in these situations istightly regulated, and is limited by the metabolic demands of the tissues concerned.Angiogenesis also occurs in pathological situations such as proliferative retinopathy,

rheumatoid arthritis (RA) and juvenile hemangioma (reviewed in refs 7–9).

Much of our interest in angiogenesis comes from the notion that for tumors to growbeyond a critical size, they must recruit endothelial cells from the surrounding stroma to

form their own endogenous microcirculation (reviewed in ref 10) Thus during tumor

progression, two phases can be recognized: a prevascular phase and a vascular phase Thetransition from the prevascular to the vascular phase is referred to as the “angiogenicswitch.” The prevascular phase is characterized by an initial increase in tumor growthfollowed by a plateau in which the rate of tumor-cell proliferation is balanced by anequivalent rate of cell death (apoptosis) This phase may persist for many years, and can

be recognized clinically as carcinoma in situ, which is characterized by few or no

metastases During the vascular phase, which is characterized by exponential growth,tissue invasion, and the hematogenous spread of tumor cells, the rapid increase in tumor

growth is largely because of a decrease in the rate of tumor-cell apoptosis (11,12) An

inverse relationship thus exists between tumor dormancy/tumor-cell apoptosis and tumorangiogenesis In a sense, tumor angiogenesis might almost be considered as “appropri-ate,” in that newly formed vessels serve to meet the metabolic demands of the rapidlygrowing tumor Although this may be beneficial to the tumor itself, it is clearly detrimen-tal to the organism, because it is permissive for tumor growth, for the dissemination oftumor cells, and for the formation of metastasis

Regulation of Angiogenesis: Balance and Context

It is usually stated that with the exception of angiogenesis that occurs in response totissue injury or in female reproductive organs, endothelial-cell turnover in the healthyadult organism is very low The maintenance of endothelial quiescence is thought to becaused by the presence of endogenous negative regulators, because positive regulatorsare frequently detected in adult tissues in which there is apparently no angiogenesis Theconverse is also true, namely that positive and negative regulators often co-exist in tissues

in which endothelial-cell turnover is increased This has led to the notion of the genic switch,” in which endothelial activation status is determined by a balance betweenpositive and negative regulators: in activated (angiogenic) endothelium, positiveregulators predominate, whereas endothelial quiescence is achieved and maintained by

“angio-the dominance of negative regulators (Fig 2) (reviewed in ref 13) Used initially in “angio-the

context of tumor progression to describe the passage from the prevascular to the vascularphase, the notion of the “switch” can also be applied in the context of developmental,physiological, and pathological angiogenesis Although it still remains to be definitivelydemonstrated in vivo, the current working hypothesis is that the “switch” involves eitherthe induction of a positive regulator and/or the loss of a negative regulator With respect

to activated endothelium, an important distinction needs to be made between cal and pathological settings: although many of the same positive and negative regulatorsare operative in both, endothelial-cell proliferation in the former is tightly controlled,whereas in the latter, uncontrolled angiogenesis implies the continuous dominance ofpositive regulators, which results in unchecked endothelial-cell growth

physiologi-Among the factors that affect endothelial-cell activation status, either positively ornegatively, are cytokines and chemokines (chemotactic cytokines) produced by normaland tumor cells Cytokines are polypeptide regulatory factors involved in the control ofcellular proliferation and differentiation Released by living cells or from extracellularmatrix, cytokines act at picomolar to nanomolar concentrations to affect cellular function.Based on the observation that a given tissue can profoundly influence the way in whichits cellular components respond to a given cytokine, it has been suggested that cytokines

should be seen as “specialized symbols in a language of intercellular communication,

whose meaning is controlled by context” (14) Context is determined by (at least) three

parameters: first, by the presence and concentration of other cytokines in the pericellularenvironment of the responding cell; second, by interactions between cells, cytokines, andthe extracellular matrix; and third, by the geometric configuration of the cells (and thustheir cytoskeleton)

With respect to angiogenesis, the molecular mechanisms underlying the notions ofboth the “angiogenic switch” as well as “context,” are likely to be central to the regulation

of this process With respect to vasculogenesis, although the notions of the “switch” and

“context” are not at present widely used, both are also likely to be important

Fig 2 Potential endogenous positive and negative regulators of angiogenesis A large number of

factors, listed here alphabetically, have been shown to regulate angiogenesis in the experimental setting For many of these factors, definitive studies are still required to demonstrate their role in the endogenous regulation of angiogenesis It is nonetheless generally assumed that the switch to the angiogenic state may involve either the loss of a negative regulator or the induction of a positive regulator, or both, although definitive proof for this notion is also still awaited Adapted from

ref 8, with copyright permission from Arnold Publishers.

IN VIVO AND IN VITRO MODELS FOR THE STUDY OF ANGIOGENESIS

The two most widely used in vivo angiogenesis assays are the chick-embryo chorioallantoicmembrane (CAM) and the rabbit corneal micropocket Direct subcutaneous injection or infu-sion of substances of interest has also been used to assess their pro- or anti-angiogenic effects.Quantitative in vivo assays involve subcutaneous implantation of various three-dimensionalsubstrates to which putative angiogenesis-regulating factors can be added These includepolyester sponges, expanded polyfluorotetraethylene (ePTFE) tubes filled with collagen, poly-vinyl-alcohol foam discs covered on both sides by millipore filters (the disc angiogenesissystem), and Matrigel, a basement membrane-rich extracellular matrix These assays are essential

to establish whether a given molecule stimulates blood-vessel formation in the intact organism;however, their interpretation is frequently complicated by the fact that the experimental condi-tions may inadvertently favor inflammation, and that under these conditions the angiogenicresponse is elicited indirectly, at least in part, through the activation of inflammatory or othernonendothelial cells Although this may be relevant to some settings in which angiogenesisoccurs in vivo, it does not allow one to study the consequences of the direct interaction ofangiogenesis regulators with endothelial cells To circumvent this drawback, in vitro assaysusing populations of cultured endothelial cells have been developed for several of the cellularcomponents of the angiogenic process, and based on the geometry of the assay, these can beclassified as either two-dimensional or three-dimensional Conventional two-dimensional assaysinclude measurement of endothelial cell proliferation, migration, and production of proteolyticenzymes such as matrix metalloproteinases (MMPs) and plasminogen activators (PAs) Three-dimensional assays have as their end-point the formation of capillary-like cords or tubes byendothelial-cells cultured either on the surface of (planar models) or within simplified extracel-lular matrices These assays include: 1) long-term culture of endothelial cells in dishes coatedwith a thin layer of matrix proteins; 2) short-term culture of endothelial cells on a thick gel ofbasement membrane-like matrix; 3) suspension of endothelial cells within three-dimensionalgels composed of collagen or fibrin; 4) radial growth of branching tubules from rings of rat aorta

or from fragments of either rat adipose-tissue microvessels or human placental blood vesselsembedded in collagen or fibrin gels; 5) radial growth of tubular sprouts from endothelial cells

grown on microcarrier beads embedded in a fibrin gel (reviewed in ref 15).

In our own studies, we have employed an in vitro model of angiogenesis that assays bothfor the invasive capacity of stimulated endothelial cells as well as their capacity for histotypicmorphogenesis, i.e., the formation of capillary-like tubes The model consists of cultivating

endothelial cells on the surface of three-dimensional collagen (16) or fibrin (17) gels; under

these conditions, the cells form a monolayer on the surface of the gel and do not invade theunderlying matrix (Fig 3A) When the monolayer is treated with an angiogenic factor such

as basic fibroblast growth factor (bFGF) (18) or vascular endothelial growth factor (VEGF) (19), the cells are induced to invade the underlying gel, and by adjusting the plane of focus

beneath the surface monolayer, branching and anastomosing cell cords can be seen within thegel (Fig 3B) In cross-section, the presence of tube-like structures resembling capillaries can

be observed beneath the surface monolayer (Fig 3C) Invasion can be quantitated by ing the total additive length of all cells that have penetrated into the underlying gel to form cell

measur-cords (19) Unlike planar models of in vitro angiogenesis, the model we have developed has

the advantage of accurately recapitulating the invasive nature of the angiogenic process, and

by virtue of its three-dimensional nature, is also permissive for histotypic morphogenesis, i.e.,for the formation of patent capillary-like tubes whose abluminal surfaces are in direct contactwith the extracellular matrix

Fig 3 Collagen gel invasion model for the study of angiogenesis in vitro (A) When viewed from

above by phase-contrast microscopy, endothelial cells grown on the surface of a three-dimensional

collagen gel form a confluent monolayer without invading the underlying matrix (B) Addition of

angiogenic cytokines such as bFGF or VEGF induces the cells to invade the underlying gel and

to form a network of branching cords, which can be viewed by focussing beneath the surface

monolayer (C) When the invading cell cords are viewed in cross-section by electron microscopy,

their tubular nature, morphologically similar to capillaries seen in vivo, can be appreciated (“cg”

= collagen gel, and “m” = surface monolayer.) Bar in (A,B) = 150 µm and in (C) = 10 µm (A,B)

adapted from ref 18; (C) adapted from ref 21, with copyright permission from Springer-Verlag

GmbH & Co KG.

ANGIOGENESIS-REGULATING CYTOKINES

The ultimate target for both positive and negative regulators of angiogenesis is the helial cell This has led to the notion that angiogenesis regulators may either act directly onendothelial cells, or indirectly by inducing the production of direct-acting regulators byinflammatory and other nonendothelial cells The most extensively studied cytokines involved

endot-in the positive regulation of angiogenesis are VEGF and acidic and basic FGFs (aFGF, bFGF).However, although a regulatory role for VEGF in developmental, physiological, andpathological angiogenesis has been well defined, much controversy still exists as to whetherthe FGFs are relevant to the endogenous control of angiogenesis in vivo The finding that invitro, VEGF and FGF positively regulate many endothelial-cell functions including prolifera-tion, migration, and extracellular proteolytic activity, has led to the notion that these factorsare direct-acting positive regulators In contrast, transforming growth factor-`1 (TGF-`1) andtumor necrosis factor-_ (TNF-_1) inhibit endothelial-cell growth in vitro, and have thereforebeen considered as direct-acting negative regulators However, both TGF-`1 and TNF-_ areangiogenic in vivo, and it has been demonstrated that these cytokines induce angiogenesisindirectly by stimulating the production of direct-acting positive regulators from stromal andchemoattracted inflammatory cells In this context then, TGF-`1 and TNF-_ are considered

to be indirect positive regulators In view of TGF-`’s capacity to directly inhibit cell proliferation and migration, to reduce extracellular proteolysis, and to promote matrixdeposition in vitro, as well as to promote the organization of single endothelial cells embedded

endothelial-in three-dimensional collagen gels endothelial-into tube-like structures, TGF-` has also been proposed

to be a potential mediator of the phase of resolution (reviewed in refs 20–22).

Other cytokines which have been reported to regulate angiogenesis in vivo includehepatocyte growth factor (HGF), epidermal growth factor/transforming growth factor-_(EGF/TGF-_), platelet-derived growth factor-BB (PDGF-BB), interleukins (IL-1, IL-6,and IL-12), interferons, granulocyte colony stimulating factor (G-CSF), placental growthfactor (PlGF), proliferin and proliferin-related protein Chemokines that regulate an-

giogenesis in vivo have to date only been identified in the -C-X-C- family, and include

IL-8, platelet factor 4, and gro-` Angiogenesis can also be regulated by a variety ofnoncytokine factors including enzymes (angiogenin, platelet-derived endothelial-cellgrowth factor/thymidine phosphorylase [PD-ECGF/TP], inhibitors of matrix-degradingproteolytic enzymes (tissue inhibitors of metalloproteinases [TIMPs] and plasminogen-activator inhibitors [PAIs]), extracellular matrix components/coagulation factors orfragments thereof (thrombospondin, angiostatin, hyaluronan and its oligosaccharides,endostatin), soluble cytokine receptors, prostaglandins, adipocyte lipids, and copper ions

(Fig 2) (reviewed in refs 7,20,21,23).

It is crucial to bear in mind that although a large number of factors have been shown to beactive in the experimental setting, it does not necessarily follow that these factors are relevant

to the endogenous regulation of new blood-vessel formation, i.e., that they are relevant to thecontrol of vasculogenesis or angiogenesis in the intact organism In the case of molecules thatare active during the phase of activation, only one, namely VEGF, meets most of the criteria

required for the definition of a vasculogenic or angiogenic factor (21,24).

Vascular Endothelial Growth Factor

Vascular endothelial growth factor (VEGF) is a highly conserved multifunctionalglycoprotein that exerts several possibly independent functions on vascular endothelium

VEGF was initially described as a tumor-secreted protein that increases the permeability

of microvessels, hence its alternate (and possibly more appropriate) name, vascularpermeability factor This extremely potent function of VPF/VEGF is 50,000 times greaterthan that of histamine In vivo, VEGF is also a potent positive regulator of angiogenesis,and in vitro, VEGF induces endothelial-cell migration and proliferation and altersendothelial cell gene expression (including the production of matrix-degradingproteolytic enzymes) Although the mitogenic properties of VEGF appear to be endothe-lial cell-specific, in vitro this effect is relatively weak when compared to other posi-tive regulating cytokines such as bFGF VEGF contains a signal peptide and is thereforesecreted from producer cells At least three VEGF isoforms, which vary in theirrelative proportions in different tissues, are generated through alternative splicing of asingle mRNA that arises from a gene containing 8 coding exons The most abundant andmost extensively studied 165 amino acid isoform (VEGF165; 164 amino acids in rodents),arising from exons 1–5, 7, and 8, has been detected in both soluble and cell/matrix-boundforms (This isoform will be referred to simply as VEGF throughout this chapter.) A 121amino acid form (VEGF121; 120 amino acids in rodents), arising from exons 1-5 and 8, hasbeen detected only in soluble form, whereas a 189 amino acid isoform (VEGF189; 188amino acids in rodents), arising from exons 1-8 appears to be localized exclusively to thecell surface and extracellular matrix A polymerase chain reaction (PCR) product inferringthe presence of a fourth isoform, VEGF206, has also been described in humans, although itsbiological significance remains to be determined Although VEGF was initially purified onthe basis of its affinity for heparin, this is substantially lower than that of other heparin-binding growth factors such as bFGF VEGF121does not bind to heparin VEGF isoformsexist as disulfide-bonded homodimers, and have a significant degree of similarity to placentagrowth factor (PlGF, approx 50% identity) and platelet-derived growth factor (PDGF,approx 20% identity) The PlGF gene contains 7 coding exons from which two alternativelyspliced forms can be generated PlGF can form biologically active heterodimers withVEGF VEGF expression is regulated by hypoxia, glucose deprivation, prostaglandins and

estrogens, as well as by a number of cytokines (reviewed in refs 25–28).

The importance of VEGF in experimental primary and metastatic tumor growth invivo has been clearly demonstrated using a variety of approaches including anti-VEGFantibodies, soluble VEGF receptors (VEGFRs), antisense VEGF, a VEGF-toxinconjugate, as well as a dominant negative approach using a truncated form of VEGFR-2.The inhibitory effect of soluble VEGFR chimeric proteins and antisense VEGFoligonucleotides in a murine model of proliferative retinopathy has also been described

(reviewed in ref 9) However, the most dramatic demonstration of the requirement for

VEGF in the development of the vascular tree comes from genetic studies involvingtargeted gene disruption in mice In a manner that is unprecedented for a gene that doesnot undergo imprinting, heterozygosity for VEGF inactivation was embryonic lethal

(29,30) The observation that the phenotype of VEGF –/– mice was more severe than that

of VEGF+/– mice, demonstrates the existence of a dose-dependent requirement for VEGFduring embryogenesis, and implies that minimal amounts of VEGF are required in atightly regulated manner for normal vascular development Essentially, although endot-helial-cell development was delayed in VEGF-deficient mice, resulting in the formation

of abnormal vascular structures and massive tissue necrosis, it was not entirely aborted.This is in contrast to VEGF receptor-2 (Flk-1)-deficient mice, in which endothelial-cell

development was completely absent (see below) These findings point to the existence

of VEGFR-2 ligands other than VEGF Because of embryonic lethality, the homozygousphenotype was inaccessible by standard (germ-line) breeding; this required the use ofembryonic stem (ES) cell aggregates combined with tetraploid mouse embryos Underthese conditions, the resulting fetuses contain a mutant ES-derived embryonic compart-ment (in which VEGF is inactivated) and wild-type, tetraploid-derived extraembryonicmembranes This approach allows for rapid examination of mutant phenotypes derived

from genetically altered ES cells without the need for germ-line transmission (31) With

the exception of conditional VEGF knockouts, which are likely to add significantly to ourunderstanding of the role of this cytokine, the ES-tetraploid system is currently the onlymethod available to assess the effect of homozygous deficiency of a gene that is embry-onic lethal in the heterozygous state Finally, when a nude mouse model was used todetermine the role of VEGF in ES-cell tumorigenesis (i.e., formation of teratomas),VEGF–/– ES cell-induced tumor and associated blood-vessel growth were strikingly

reduced when compared to wild-type ES cells (30).

Two proteins with structural homology to VEGF have recently been described The

first has been called VEGF-B (32) or, alternatively, VEGF-related factor (VRF) (33,34).

VEGF-B transcripts are alternatively spliced, and the overall genomic organization ofVEGF-B is conserved between other members of the VEGF gene family (VEGF, PlGF,PDGF) VEGF-B is primarily cell-associated, and is capable of forming heterodimerswith VEGF VEGF-B increases [3H]thymidine incorporation in human and bovineendothelial cells Whether VEGF-B/VRF binds to the same receptors as other members

of the VEGF family (see below) remains to be elucidated VEGF-B is co-expressed with

VEGF in some tissues, and is particularly abundant in embryonic and adult striated(cardiac and skeletal) muscle; however, unlike VEGF, elevated levels of VEGF-B mRNA

were not found in glioblastomas, breast, or renal carcinomas (32,33,35).

The second protein with structural homology to VEGF has been called VEGF-C (36),

or alternatively, VEGF-related protein (VRP) (37) VEGF-C was isolated during a search

for a ligand for VEGFR-3 (Flt-4) VEGF-C displays a high degree of similarity withVEGF, including conservation of the eight cysteine residues involved in intra- and inter-molecular disulfide bonding It appears that the VEGF-C mRNA is first translated into

a precursor from which the mature ligand is derived by cell-associated proteolyticprocessing The cysteine-rich C-terminal half, which increases the length of theVEGF-C polypeptide relative to other ligands of this family, shows a pattern of spacing

of cysteine residues reminiscent of the Balbiani ring 3 protein repeat Like VEGF andVEGF-B, VEGF-C/VRP transcripts are alternatively spliced to give a number ofmajor isoforms VEGF-C binds to the extracellular domain of VEGFR-3 and inducesVEGFR-3 tyrosine phosphorylation In addition to VEGFR-3, VEGF-C appears to bind

to and induce phosphorylation of VEGFR-2 (Flk-1/KDR) VEGF-C/VRP transcripts aredetectable in many adult and fetal human tissues and in a number of cell lines Patterns

of VEGF-C expression during development suggest that this cytokine plays an important

role in lymphangiogenesis (38).

Alterations in endothelial-cell function induced by members of the VEGF family aremediated via transmembrane tyrosine kinase receptors that at present include VEGFR-1(Flt-1), VEGFR-2, and VEGFR-3 Ligands for VEGFR-1 include VEGF and PlGF;ligands for VEGFR-2 include VEGF and VEGF-C; whereas the only ligand reported so

far for VEGFR-3 is VEGF-C (27,39; for references on VEGF-B/VRF and VEGF-C/VRP, see above) Whether VEGF-B binds to the same receptors as other members of the

VEGF family remains to be described VEGFRs are expressed in many adult tissues, despitethe apparent lack of constitutive angiogenesis VEGFRs are however clearly upregulated inendothelial cells during development and in certain angiogenesis-associated/dependent patho-

logical situations including tumor growth (reviewed in refs 25,28) The phenotypes of both

VEGFR-1- and VEGFR-2-deficient mice have been described VEGFR-1-deficient mice die

in utero at mid-somite stages, and although homozygous deficient mice are capable of formingendothelial cells in both intra- and extra-embryonic regions, assembly of these cells intovessels is perturbed, resulting in the formation of abnormal vascular channels The authorsconclude that VEGFR-1 signaling pathways may regulate normal endothelial cell-cell or cell-

matrix interactions during vascular development (40) VEGFR-2-deficient mice also die in

utero between 8.5 and 9.5 d postcoitum, although in contrast to VEGFR-1, this appears to beowing to abortive development of endothelial-cell precursors Yolk-sac blood islands andorganized embryonic blood vessels were not detectable at any stage of development The

development of hematopoietic precursors was also severely reduced (41) By using a

domi-nant-negative approach, the requirement for VEGFR-2 has also been clearly demonstrated in

tumor angiogenesis (42,43) Gene targeting and dominant negative approaches have therefore

clearly defined an essential role for VEGFRs in developmental and tumor angiogenesis.Because VEGFR-expressing endothelial cells are located adjacent to regions of tumorischemia and necrosis, it is possible that the increase in VEGFR expression is mediated by

hypoxia (44; reviewed in ref 45) In this context, it has been demonstrated in some in vitro studies

that hypoxia increases high-affinity VEGF binding to endothelial cells and induces an increase

in VEGFR-2 number (without alterations in receptor affinity), and that this is associated withincreased VEGF-induced mitogenicity and VEGFR-2 tyrosine phosphorylation in endothelial

cells (46,47) It has also been reported that hypoxia increases expression of VEGFR -1 and -2 mRNA in endothelial cells both in vivo and ex vivo in rat lungs (48) Of particular interest is the

observation that conditioned medium from hypoxic skeletal myoblasts or smooth muscle cellscontains a factor that markedly increases VEGFR-2 number (without alterations in receptor

affinity) in endothelial cells in vitro (49) On the basis of neutralizing antibody studies, it was

concluded that this factor is neither VEGF, bFGF, TNF-_, or TGF-`1 Taken together, thesefindings suggest that hypoxia can increase the effect of VEGF via the paracrine induction ofVEGFRs in metabolically deprived tissues With the exception of hypoxia, other fac-tors that increase VEGFR expression have not been published However, we have observedthat high and low glucose concentrations increase VEGFR-2 mRNA levels in bovinemicrovascular but not large vessel-derived endothelial cells in vitro (S.J Mandriota andM.S Pepper, unpublished observations) We have also studied the effect of bFGF onVEGFR-2 expression in bovine endothelial cells, and have found that although bFGF increaseslevels of VEGFR-2 mRNA and total protein, cell surface protein is diminished This may

be owing to the fact that bFGF concomitantly increases expression of VEGF andVEGF-C, which upon secretion may interact with and promote internalization ofVEGFR-2 (S J Mandriota and M S Pepper, unpublished observations) Downregulation

of VEGFR expression in endothelial cells in vitro has been seen with nitric oxide (NO) or an NO-related metabolite in rat lungs ex vivo (48), with TGF-` (50), and with TNF-_ (51).

Basic Fibroblast Growth Factor

Basic fibroblast growth factor (bFGF), also known as FGF-2 (or heparin-bindinggrowth factor-2), is a member of the FGF superfamily that comprises more than 14distinct gene products bFGF is a cationic polypeptide (pI 9.6) with potent angiogenesis-

inducing properties in vivo (reviewed in refs 52–56).