International Review of Cytology, Volume 185: A Survey of Cell Biology

VOLUME 185 SERIES EDITORS Geoffrey H. Bourne James F. Danielli Kwang W. Jeon Martin Friedlander Jonathan Jarvik 1949...

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VOLUME 185

SERIES EDITORS Geoffrey H. Bourne James F. Danielli Kwang W. Jeon Martin Friedlander Jonathan Jarvik

1949-1 988 1949-1 984 19671984-1 992 1993-1 995

EDITORIAL ADVISORY BOARD Aimee Bakken Eve Ida Barak Rosa Beddington Howard A. Bern Robert A. Bloodgood Dean Bok Stanley Cohen Rene Couteaux Marie A. DiBerardino Charles J. Flickinger Hiroo Fukuda Elizabeth D. Hay P. Mark Hogarth Anthony P. Mahowald

M. Melkonian Keith E. Mostov Andreas Oksche Vladimir R. Pantic L. Evans Roth Jozef St. Schell Manfred Schliwa Robert A. Smith Wilfred D. Stein Ralph M. Steinman M. Tazawa Donald P. Weeks Robin Wright Alexander L. Yudin

Edited by

Kwang W. Jeon Department of Biochemistry University of Tennessee Knoxville, Tennessee

VOLUME 185

ACADEMIC PRESS San Diego London Boston New York Sydney Tokyo Toronto

Front cover photograph: Serum-free collagen gel culture of rat aortic and renal venous explants. (For more details, see Chapter 1, Figure 4.)

This book is printed on acid-free paper.

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Copyright 0 1999 by ACADEMIC PRESS All Rights Reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy, recording, or any information storage and retrieval system, without permission in writing from the Publisher. The appearance of the code at the bottom of the first page of a chapter in this book indicates the Publisher’s consent that copies of the chapter may be made for personal or internal use of specific clients. This consent is given on the condition, however, that the copier pay the stated per copy fee through the Copyright Clearance Center, Inc. (222 Rosewood Drive, Danvers, Massachusetts 01923), for copying beyond that permitted by Sections 107 or 108 of the U.S. Copyright Law. This consent does not extend to other kinds of copying, such as copying for general distribution, for advertising or promotional purposes, for creating new collective works, or for resale. Copy fees for pre-1999 chapters are as shown on the title pages. If no fee code appears on the title page, the copy fee is the same as for current chapters. 0074-7696199 $25.00

Academic Press a division of Harcourt Brace & Company 525 B Street, Suite 1900, San Diego, California 92101-4495, USA http:l/www.apnet.com Academic Press 24-28 Oval Road, London NW1 7DX, UK http:/lwww.hbuk.co.uk/apl International Standard Book Number: 0-12-364589-1 PIUNTED IN THE UNlTFD STATES OF Ah4ERICA 9 8 9 9 0 0 0 1 0 2 0 3 E B 9 8 7 6 5 4 3 2 1

CONTENTS

Contributors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

ix

Autoregulation of Angiogenesis by Cells of the Vessel Wall R. F. Nicosia and S. Villaschi I. I1. 111. IV. V.

Introduction ........................................................... Vasculogenesis and Angiogenesis ......................................... Evidence That Blood Vessels Can Autoregulate Angiogenesis: Rat Aorta Model . . . . . Interactions between Endothelial Cells and Fibroblasts or Smooth Muscle Cells . . . . . Growth Factors and Other Soluble Regulators of Angiogenesis Produced by Cells of the Vessel Wall . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VI . Proteolytic Enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VII. Extracellular Matrix and Cell Adhesion Molecules ............................. VIII. Vasoactive Factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IX. Summary and Future Directions ........................................... References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1 2 5 7 9 17 19 23 26 31

Fibroblast Growth Factors as Multifunctional Signaling Factors Gyorgyi Szebenyi and John F. Fallon I. I1. Ill. IV.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Structure. Regulation of Synthesis. Subcellular Localization. and Release of FGFs . . . . FGF-Binding Proteins and Signaling Pathways ................................ The Biological Activities of FGFs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V

45 47 56 72

vi

CONTENTS

V . Concluding Remarks ..................................................... References ............................................................

85 87

Structural and Functional Characteristics of the Centrosome in Gametogenesis and Early Embryogenesis of Animals Marina M. Krioutchkova and Galina E. Onishchenko I . Introduction ............................................................ II . Behavior of the Centrosome during Gametogenesis ............................

107 110

Ill. Behavior of Centrioles and the Centrosome in Cells in Early Development and during Differentiation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. Conclusions ............................................................ References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

122 143 144

Cell and Molecular Biology of the Pars Tuberalis of the Pituitary Werner Wittkowski. Jijrgen Bockmann. Michael R. Kreutz. and Tobias M. Bockers I . Introduction ............................................................ II . Characteristics of Pars Tuberalis ........................................... 111 . Gene Expression of Pars Tuberalis-Specific Cells .............................. IV. Biorhythmic Alterations ................................................... V. Physiological Significance: Pars Tuberalis as the Major "Zeitgeber" of Pars Distalis Activity? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VI . Concluding Remarks ..................................................... ............................................ References . . . . . . . . . . . .

157 158 165 177 182 186 187

Polarization of the Na+.K+-ATPasein Epithelia Derived from the Neuroepithelium Lawrence J . Rizzolo I. Introduction ........................................................... 195 II . Polarity and Transepithelial Transport ....................................... 197 111. Plasticity: Remodeling Cell Polarity ......................................... 202 IV. General Models for Epithelial Polarity ....................................... V . Diversity of Na+.K+-ATPase lsoforms and Tissue Specificity ..................... VI. Mechanisms That Polarize the Distribution of the Na+.K+-ATPase . . . . . . . . . . . . . . . .

205 210 214

CONTENTS

VII. Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

vii 226 228

Desmosomes: Intercellular Adhesive Junctions Specialized for Attachment of Intermediate Filaments Andrew P. Kowalczyk. Elayne A . Bornslaeger. Suzanne M. Norvell. Helena L. Palka. and Kathleen J. Green Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Ultrastructural Properties and Tissue Distribution of Desmosomes . . . . . . . . . . . . . . . . Transmembrane Components of the Desmosome: Desmosomal Cadherins . . . . . . . . . Plaque Components of the Desmosome .................................... Protein-Protein Interactions in the Desmosome: A Model for Desmosome Assembly .......................... VI . Regulation of Desmosome Assembly ....................................... VII . Desmosomal Components in Signal Transduction and Development . . . . . . . . . . . . . . VIII. Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

268 270 278 282 283

Index ............................................................

303

I. I1. 111. IV. V.

237 238 241 248

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CONTRIBUTORS

Numbers in parentheses indicate the pages on which the authors' contributions begin

Tobias M. Bockers (157), lnsfifute of Anafomy, AG Molecular Neuroendocrinology, Wesiralische Wilhelms-Universitat, 0-48149 Miinster, Germany Jurgen Bockrnann (157), Institute of Anatomy, AG Molecular Neuroendocrinology, Wedfalische Wilhelms-Universitat, 0-48149 Munster, Germany Elayne A. Bornslaeger (237), Departmenfs of Pathology and Dermatology and the R.H. Lurie Cancer Center, Northwestern University Medical School, Chicago, Illinois 6061 1-3008 John Fallon (45), Department of Anatomy, University of Wisconsin Medical School, Madison, Wisconsin 53706- 1532 Kathleen J. Green (237),Departments of Pathology and Dermatology and the R.H. Lurie Cancer Center, Northwestern University Medical School, Chicago, Illinois 6061 1-3008 Andrew P. Kowalczyk (237), Department of Dermatology and the R.H. Lurie Cancer Center, Northwestern University Medical School, Chicago, lllinois 606I 1-3008 Michael R. Kreutz (157), lnstitute of Medical Psychology, University of Magdeburg, 39120 Magdeburg, Germany Marina M. Krioutchkova (107), Department of Cytology and Histology, Moscow State University, Moscow- I 19899, Russia Roberto F. Nicosia (1), Department of Pathology and Laboratory Medicine, Al/egheny University of the Health Sciences, Philadebhia, Pennsylvania 19102 Suzanne M. Nowell (237),Departments of Pathology and Dermatology and the R.H. Lurie Cancer Center, Northwestern University Medical School, Chicago, Illinois 60611-3008 ix

X

CONTRIBUTORS

Galina E. Onishchenko (107),Department of Cflology and Histology, Moscow State Universiity, Moscow-119899, Russia Helena L. Palka (237),Departments of Pathology and Dermatology and the R.H. Lurie Cancer Center, Northwestern University Medical School, Chicago, Illinois 60611-3008 LawrenceJ. Rizzolo (195),Departmentof Surgery, Section ofAnatomy, Yale Universify School of Medicine, New Haven, Connecticut 06520 Gyorgyi Szebenyi (45),Department of Cell Biology and Neuroscience, University of Texas Southwestern Medical Center, Dallas, Texas 75235-91I 1 Sergio Villaschi (I), Department of Surgery, Universify of Rome "Tor Vergata," Rome, Italy Werner Wittkowski (157), Institute of Anatomy, AG Molecular Neuroendocrinology, Westfalische Wilhelms-Universitit, 0-48149 Munster, Germany

Autoregulation of Angiogenesis by Cells of the Vessel Wall R. F. Nicosia* and S. Villaschit *Department of Pathology and Laboratory Medicine, Allegheny University of the Health Sciences, Philadelphia, Pennsylvania 19102; and tDepartment of Surgery, University of Rome “Tor Vergata,” Rome, Italy

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The cells of the vessel wall can regulate angiogenesis by producing growth factors, proteolytic enzymes, extracellular matrix components, cell adhesion molecules, and vasoactive factors. This properly enables preexisting blood vessels to generate new vessels in the absence of exogenous angiogenic stimuli. Vascular autoregulation of angiogenesis can be studied by culturing rat aortic or venous explants in collagen gels under serum-free conditions. In this system, the combined effect of injury and exposure of explants to collagen triggers a self-limited angiogenic response. Interactions among endothelial cells, smooth muscle cells, and fibroblasts play a critical role in the regulation of this process. This chapter reviews the literature on angiogenesis, focusing on the vessel wall as a highly specialized and plastic tissue capable of regenerating itself through autocrine, paracrine, and juxtacrine mechanisms. KEY WORDS: Angiogenesis, Endothelium, Smooth muscle cells, Pericytes, Fibroblasts, Growth factors, Matrix metalloproteinases, Plasminogen activators, Extracellular matrix.

1. Introduction Angiogenesis, the process by which blood vessels develop from the endothelium of a preexisting vasculature, plays a fundamental role in the growth, survival, and function of normal and pathologic tissues. The cardiovascular system is the first functioning organ system (Wilting er al., 1995a) and its developmental failure results in the early intrauterine death of the embryo (Shalaby er aZ., 1995). Once blood vessels have formed and remodeled during embryonal development and fetal growth, they become quiescent International Review of Cytology, VoL 185

0074-7696199 $25.00

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Copyright 0 1999 by Academic Press. All rights of reproduction in any form reserved.

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(Folkman and Cotran, 1976) but retain their vasoformative properties, which become manifest again in the adult during wound healing (Knighton et al., 1990) and the female menstrual cycle (Jakob et aL, 1977). Angiogenesis is also reactivated during pathologic processes such as cancer, rheumatoid arthritis, psoriasis, complicated atherosclerosis, diabetic retinopathy, and hemangiomas (Folkman, 1995). Because the neovasculature contributes to the progression of these disorders, antiangiogenic drugs have been proposed as possible therapeutic agents and are currently being tested in clinical trials (Folkman, 1996). There is also considerable interest in the pharmacologic stimulation of angiogenesis in patients suffering from delayed wound healing, venous stasis ulcers, and obliterative vascular disorders (Takeshita et al., 1994; Isner et al., 1996). New blood vessels develop as a result of complex interactions between endothelial cells and angiogenic regulators such as soluble growth factors (Moses et al., 1995),insoluble extracellular matrix (ECM) molecules (Ingber and Folkman, 1989), and matrix-degrading proteolytic enzymes (Ray and Stetler-Stevenson, 1994; Cornelius et al., 1995). Growth factors capable of stimulating angiogenesis are secreted by a variety of cell types, including embryonal cells (Drexler et al., 1992), cancer cells (Folkman and Cotran, 1976), epithelial cells (Brown et al., 1992), macrophages (Sunderkotter et al., 1994), and lymphocytes (Lutty et aL, 1983). Angiogenic factors are also produced by the cells of the vessel wall: endothelial cells (Hannan et al., 198Q smooth muscle cells (Brogi et al., 1994), and fibroblasts (Hlatky et al., 1994). Thus, blood vessels that have switched from quiescence to an activated state as a result of injury or other stimulatory conditions have the capacity to generate new vessels without the intervention of nonvascular cells. This autoregulation of the angiogenic process is observed when vascular explants are cultured in three-dimensional biomatrix gels in the absence of serum or exogenous growth factors (Kawasaki et al., 1989; Nicosia and Ottinetti, 1990; Brown et al., 1996). Understanding how blood vessels regulate their own growth may provide new insights into the angiogenic process and its mechanisms. The purpose of this chapter is to reappraise critically the literature on angiogenesis,focusing on the vessel wall as a highly specialized and plastic tissue capable of regenerating itself through a vasoformative process mediated by autocrine, paracrine, and juxtacrine mechanisms.

II. Vasculogenesis and Angiogenesis A. Formation of Blood Vessels during Embryonal Development Blood vessels form in the embryo as a result of two fundamental processes: vasculogenesis and angiogenesis (Risau et al., 1988). Vasculogenesis occurs

AUTOREGULATION OF ANGIOGENESIS

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when blood vessels emerge de n o w from a subpopulation of mesenchymal cells known as angioblasts, which differentiate into endothelial cells. After the first capillary tubes have developed, new vessels form as a result of angiogenesis, i.e., sprouting of new microvessels from preexisting vessels, vasculogenesis, or a combination of the two processes (Wilting et al., 1995a). Embryonal endothelial cells generate sprouts that branch, become canalized, and anastomose with each other giving rise to capillary loops and networks. The sprouts grow as a result of both endothelial cell migration and proliferation as well as recruitment of new angioblasts from the surrounding mesenchyme or from other blood vessels (Wilting et al., 1995b). Capillaries give rise to larger vessels by fusion of adjacent vessels and by lateral intercalation of proliferating endothelial cells resulting in luminal expansion. For example, the paired dorsal aortas of the chick embryo develop out of a close-meshed capillary plexus which is remodeled into two longitudinal tubes (Hirakow and Hiruma, 1983). The two aortas then fuse laterally, giving rise to the definitive aorta which eventually expands by intercalated endothelial proliferation (Wilting et al., 1995b). Vascular remodeling during embryonal development is also characterized by regression of capillaries and disappearance of endothelial cells through cell death, emigration, and transdifferentiation (Wilting et al., 1995b). Retraction of capillaries with incorporation of endothelial cells into the main vessels has also been described (Christ et al., 1990). The primary capillary network of the embryo is composed exclusively of endothelial cells. As arteries and veins develop, the mesenchyme around the endothelium differentiates into smooth muscle cells and fibroblasts. Embryonal differentiation of vascular smooth muscle cells proceeds centrifugally at first in the aorta and then in the main veins (Wilting et aL, 1995b). Differentiation of arteries and veins results in the gradual formation of three anatomic layers: the intima, the media, and the adventitia. Two main types of arteries develop: elastic arteries, which include the aorta and its main branches, and muscular arteries. In the newborn, the arterial intima is composed almost exclusively of endothelial cells which rest on a basement membrane. As they mature and eventually age, blood vessels thicken, acquiring more layers of smooth muscle cells. The intima of arteries and veins becomes populated in the adult by medialderived smooth muscle cells known as myointimal cells which produce collagen, elastic tissue, basement membrane molecules, and proteoglycans (Schwartz et al., 1990). The media of elastic arteries is composed of alternating layers of smooth muscle cells and elastic laminas (Dingemans et al., 1981). In contrast, muscular arteries have less elastic tissue which organizes into laminae only at the boundaries of the intima with the media and the adventitia. Veins develop thinner walls than arteries and have tunica medias composed primarily of smooth muscle cells with relatively low amounts of elastic tissue. The adventitia of both arteries and veins is composed primarily of fibroblasts and interstitial collagen. Microvessels known as vasa va-

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R. F. NICOSIA AND S. VlLLASCHl

sorum develop in the adventitia and penetrate into the tunica media of large vessels to nourish the outer layers of smooth muscle cells (Barger et al., 1984). Even after the vascular system has fully differentiated, the boundaries between the adventitia of arteries or veins and the connective tissue around arterioles, capillaries, and venules remain ill-defined. Thus, the fibroblasts that surround the endothelium of arterioles, capillaries, and venules are essentially an anatomic extension of the adventitial fibroblasts of arteries and veins into the peripheral microvascular bed. The capillaries and postcapillary venules are coated by a single discontinuous layer of pericytes which resemble smooth muscle cells and surround the endothelium with dendritic processes (Sims, 1986). During vascular development, smooth muscle cells and pericytes are recruited by the endothelial cells from the surrounding mesenchyme (Schwartz et al., 1990). Pericytes can only be identified in the late fetal period (Donahue and Pappas, 1961). The basement membrane of capillary endothelial cells during embryonal development is poorly developed (Tonnesen et al., 1985). As the capillaries mature, endothelial cells lay down a continuous basement membrane which they share with the surrounding pericytes (Sims, 1986). They also differentiate into morphologically and functionally distinct endothelial subtypes based on interactions with the local environment. For example, capillary endothelial cells of the choroidal plexus become fenestrated in response to induction by the choroidal epithelial cells (Wilting and Christ, 1989), whereas brain capillary endothelial cells acquire tight junctions and bloodbrain features in response to stimuli by surrounding astrocytes (Stewart and Wiley, 1981).

B. Angiogenesis during Adult Life After the vascular system has developed and matured, blood vessels gradually stop growing and become quiescent. Angiogenesis is reactivated in the Adult at the level of the microcirculation, where physiologic or pathologic stimuli can induce endothelial sprouting from preexisting microvessels (Folkman and Cotran, 1976). New vessels can also originate from the endothelium of large or medium vessels during the neovascularization of atherosclerotic plaques, the recanalization of thrombi, and the development of collateral circulation (Takeshita et al., 1994; Sueishi et al., 1997). Formation of microvessels from a preexisting vessel is a multistep process (Folkman, 1985) characterized by an orderly sequence of events which can be summarized as follows: (i) vascular engorgement and dilatation with activation of endothelial cells; (ii) localized degradation of the subendothelial basement membrane; (iii) migration of endothelial cells into the interstitium through gaps in the basement membrane of the parent microvessels;


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(iv) formation of immature sprouts composed of activated endothelial cells; (v) proliferation of endothelial cells behind the migrating endothelium of the sprouts; (vi) remodeling/digestion of the preexisting ECM around the sprouting endothelium and deposition of a new ECM; (vii) canalization of the neovessels with formation of anastomoses and capillary loops followed by establishment of blood flow; (viii) incorporation of pericytes around the endothelium; (ix) formation of a continuous basement membrane; and (x) vascular regression, attributed to both lack of blood flow (Clark and Clark, 1939) and removal of angiogenic stimuli (Ausprunk et al., 1978).

111. Evidence That Blood Vessels Can Autoregulate Angiogenesis: Rat Aorta Model In 1959 Williams found that microscopic wounds of connective tissue produced without damaging blood vessels healed without angiogenesis and proposed that vascular injury was needed to stimulate neovascularization during wound healing. Subsequently, O’Donaghue and Zarem (1971) observed that mouse skin isografts implanted in a transparent chamber stimulated angiogenesis.They also found that purified collagen showed no angiogenic activity and argued that since the full-thickness skin graft consisted primarily of collagen and blood vessels, the failure of collagen to stimulate angiogenesis implicated the blood vessels of the graft as key factors in the stimulation of neovascularization. In 1982 we reported that explants of rat aorta cultured in plasma clot in the presence of fetal bovine serum gave rise to luxuriant outgrowths of branching microvessels (Nicosia et al., 1982). We later discovered that the angiogenic response of the rat aorta did not require exogenous stimuli and also occurred under serum-free conditions in a chemically defined growth medium without addition of angiogenic factors (Nicosia and Ottinetti, 199O)(Fig.1).On this basis, we hypothesized that the aortic wall was capable of regulating its own angiogenic response trough endogenous mechanisms activated by the injury of the dissection procedure. Similar observations were made concurrently by Kawasaki et al. (1989) and were confirmed later by Nissanov et al. (1995) and Akita et al. (1997). The autoregulation of angiogenesis by the vessel wall is not restricted to the rat aorta since rat venous explants (Fig. 2) and human chorionic vessels (Brown et al., 1996) are similarly capable of generating an angiogenic response under serumfree conditions. The outgrowth in the rat aorta cultures is composed of three main cell types: fibroblasts, endothelial cells, and pericytes (Nicosia and Ottinetti, 1990). Fibroblasts originate from the aortic adventitia and appear in the

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FIG. 1 Serum-free collagen gel culture of rat aorta. Microvessels (arrows) sprouted from the aortic explant (asterisk) are surrounded by fibroblasts (arrowheads). Scale bar = 150 p M .

FIG. 2 Serum-free collagen gel culture of rat renal vein (asterisk). The outgrowth is composed of branching microvessels (arrows) and fibroblasts (arrowheads). Scale bar = 100 pM.


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gel at Day 2 of culture. Endothelial cells migrate from the injured edges of the aortic intima at Days 3 or 4, giving rise to microvascular sprouts. The neovessels elongate, branch, develop lumina, anastomose, and eventually stop growing at Days 9 or 10. During the first days of culture, the neovascular sprouts are composed primarily of endothelial cells which closely interact with the surrounding fibroblasts. As the vascular outgrowth expands and eventually matures, pericytes migrate from the root to the tip of the microvessels, crawling along the endothelium, which they use as a surface for attachment, proliferation, and contact guidance. Pericytes originate at least in part from a subpopulation of smooth muscle cells located in the intima or the subintimal layers of the tunica media (Nicosia and Villaschi, 1995). It is also possible that fibroblasts become incorporated into the microvessels and differentiate into pericytes, as reported by Clark and Clark (1939) and Rhodin and Fujita (1989). The vascular remodeling that follows the growth phase is characterized by retraction of the small endothelial branches into the main stems of the microvessels. Regression and remodeling of the microvasculature is accompanied by an increase in the number of pericytes. As a result of endothelial retraction and periendothelial accumulation of pericytes, microvessels become shorter, thicker, and less branched during the regressionhemodeling phase (Nicosia and Villaschi, 1995). The aortic outgrowths are regulated by autocrine, paracrine, and juxtacrine interactions among endothelial cells, pericytes, and fibroblasts. The nature of these interactions can be studied by coculturing isolated cell strains in collagen gels.

IV. Interactions between Endothelial Cells and Fibroblasts or Smooth Muscle Cells Relatively few studies have explored the mechanisms that regulate the paracrine and juxtacrine interactions between the cells of the vessel wall. These studies have been limited by the use of fetal bovine serum (FBS), which is added to growth media in order to propagate and maintain cells in culture. FBS contains many growth and attachment factors which have profound effects on cell behavior and disrupt the paracrine cross-talk between different cell types. Advances in tissue culture have led to the development of serum-free media for endothelial cells (Knedler and Ham, 1987). These optimized media enable us to perform coculture experiments in the absence of FBS. Rat aortic endothelial cells grown in serum-free medium on a gel of interstitial collagen reorganize into a network of microvascular tubes when

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overlaid with a second layer of collagen (Nicosia et aL, 1994~).These microvessels resemble the microvascular outgrowths of the rat aortic cultures except that they are not surrounded by fibroblasts or pericytes and have a limited life span since they disintegrate within 3 to 4 days. Fibroblasts added to the collagen gel stabilize the microvessels which remain viable for at least 21 days (longest time studied). At variance with serum-stimulated cultures in which the endothelium is overgrown by fibroblasts, serum-free cocultures promote the coexistence of the two cell types in what becomes a symbiotic system of reciprocal paracrine stimulation (Villaschi and Nicosia, 1994).The stabilization of the microvessels requires the contiguous presence and juxtacrine stimulation of the fibroblasts. Fibroblasts have been shown by our laboratory and by others to produce soluble factors that stimulate angiogenesis (Sato et aL, 1987; Villaschi and Nicosia, 1994). Factors other than angiogenic molecules, however, are likely to contribute to the juxtacrine effect of fibroblasts on the microvasculature since fibroblastconditioned medium, which has angiogenic activity (Villaschi and Nicosia, 1994), prolongs the survival of isolated endothelial networks by only 4 to 5 days. These factors may include the insoluble components of the basement membrane which accumulate around the microvessels in endothelial/fibroblast cocultures and have stabilizing effects on neovessels (Nicosia et al., 1998). Fibroblasts may cooperate with the endothelium during microvascular sprouting because of their ability to activate proteolytic enzymes which digest the ECM (Gilles et aL, 1997). Endothelial cells and fibroblasts also stimulate each other’s capacity to contract the collagen gel. This phenomenon is mediated, at least in part, by soluble factors since collagen gel contraction by either isolated endothelial cells or fibroblasts is stimulated by growth medium conditioned by the other cell type. Endothelial cells promote the transformation of fibroblasts into myofibroblasts, specialized connective tissue cells with contractile ability (Gabbiani etal., 1971;Villaschi and Nicosia, 1994). Fibroblast-mediated collagen gel contraction is promoted, at least in part, by the endothelial-derived peptide endothelin-1 (ET-1). The fibroblast-derived factors that promote endothelial contraction of the collagen gel are unknown. The combined contraction of collagen in the cocultures is reminiscent of the contraction of granulation tissue which results in wound closure during tissue repair (Gabbiani et aL, 1971). The serum-free collagen gel overlay coculture method can also be used to investigate the juxtacrine and paracrine interactions between endothelial cells and smooth muscle cells. Smooth muscle cells isolated from the intimal aspect of the rat aorta (ISMCs) or the tunica media (MSMCs) and cultured in collagen gel without endothelial cells exhibit a round shape and tend to degenerate. In the coculture experiments, the ISMCs that are close to the endothelium become dendritic and migrate toward the microvessels, which are eventually surrounded by these cells (Nicosia and Villaschi, 1995). In


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contrast, MSMCs stay round, degenerate, and only rarely migrate toward the endothelium. ISMCs stabilize the microvessels, prolonging the life span of the endothelium. The morphology of the microvessels also suggests that the ISMCs promote endothelial cell differentiation. Ultrastructural studies demonstrate a striking similarity between the microvessels of the endothe1iaVISMCs cocultures and those of the primary aortic cultures. The ISMCs that have migrated around the microvessels of the cocultures are virtually indistinguishable from the pericytes of the primary aortic cultures. These experiments demonstrate that endothelial cells chemotactically attract a subpopulation of smooth muscle cells which transform themselves into pericytes establishing contacts and junctional communications with the endothelium. The behavior of ISMCs is similar to that of microvessel-derived pericytes which have been shown by others to adhere to capillaries in collagen gel culture (Minakawa et al., 1991). Pericytes, depending on their functional state, degree of differentiation, and spatial relation to the endothelium, may play different roles during the angiogenic process. The observation in the rat aorta model that the progressive increase in the number of pericytes coincides with the arrest of vascular proliferation is consistent with the idea that pericytes may function as negative regulators of angiogenesis and as inducers of endothelial differentiation and stabilization (Orlidge and D’Amore, 1987; Nicosia and Villaschi, 1995). Pericytes are believed to inhibit endothelial migration and proliferation by juxtacrine mechanisms mediated by physical contacts. On the other hand, it is possible that pericytes may promote microvascular sprouting during early stages of angiogenesis when they lose contact with the endothelium and produce endothelial growth factors in response to angiogenic stimuli (Diaz-Flores et al., 1992; Nehls et al., 1992). In the next sections we review the vascular-derived soluble factors, proteolytic enzymes, ECM molecules, cell adhesion molecules, and vasoactive substances that are likely to participate in the autocrine, paracrine, and juxtacrine mechanisms regulating the interactions among endothelial cells, fibroblasts, and smooth muscle cells/pericytes during the angiogenic response of the vessel wall.

V. Growth Factors and Other Soluble Regulators of Angiogenesis Produced by Cells of the Vessel Wall We describe here the soluble factors implicated in the autoregulation of angiogenesis by the vessel wall. This section discusses vascular-derived regulators of angiogenesis and does not include molecules such as transforming growth factor-a, angiogenin, and epidermal growth factor, which

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contribute primarily to epithelium- and cancer-related angiogenesis, and interleukins, which are mostly involved in inflammation-related and immune-mediated angiogenesis.

A. Growth Factors 1. Fibroblast Growth Factors Fibroblast growth factors (FGFs) are a family of heparin-binding polypeptides with potent angiogenic activity. To date, at least nine FGFs and four types of FGF receptors have been described (Moses et aL, 1995; Gorlin, 1997). Endothelial cells, smooth muscle cells, and fibroblasts produce acidic fibroblast growth factor (aFGF, FGF- 1) and basic fibroblast growth factor (bFGF, FGF-2) and express FGF receptors, which are transmembrane proteins with tyrosine kinase activity (Schweigerer et aL, 1987; Root and Shipley, 1991; Hughes et aL, 1993). The FGF-FGF receptor system represents an important endogenous stimulator of endothelial migration, proliferation, proteolytic activity, and blood vessel formation (Gospodarowicz and Neufeld, 1986; Montesano et al., 1986; Schweigerer et al., 1987; Mignatti et al., 1989). FGFs also promote the migration and proliferation of smooth muscle cells and fibroblasts (Root and Shipley, 1991; Lindner, 1995a). FGFs are stored intracellularly and in the basement membrane, where they are bound to heparan sulfate proteoglycans. The mRNA of bFGF and its receptor FGFR-1 is upregulated in endothelial cells and smooth muscle cells after vascular injury (Lindner and Reidy, 1993). aFGF and bFGF lack signal peptide sequences and are not secreted through the conventional endoplasmic reticulum/Golgi pathway. Injury plays an important role in the release of FGFs since the plasma membrane of transiently injured cells becomes permeable to large molecules, allowing free diffusion of FGFs into the extracellular space (Mutsukrishnan et aL, 1991). It has also been postulated that FGFs may be released through an exocytotic pathway independent of the endoplasmic reticuludGolgi complex (Mignatti and Rifkin, 1991). Basement membrane-bound FGFs are released by heparinases or plasmin (Vlodavsky et aL, 1987; Saskela and Rifkin, 1990). Once they are secreted in the extracellular space, the FGFs are free to interact with their receptors on the cell surface. After receptor binding, aFGF and bFGF are internalized and translocated to the nucleus just a few hours before the onset of DNA synthesis (Hawker and Granger, 1994). Although the mechanism by which nuclear-bound FGFs stimulate DNA synthesis is unknown, the process of FGF nuclear translocation is believed to be a requirement for quiescent cells to reenter the cell cycle.


11

Immunohistochemical stain of the rat aorta reveals that bFGF is stored in the cytoplasm of endothelial and smooth muscle cells (Villaschi and Nicosia, 1993). In the rat aorta model, the injury of the dissection procedure causes a release of bFGF from its storage sites. bFGF levels in aortaconditioned medium are highest during the first days of culture and gradually decrease over time, becoming undetectable when microvessels stop growing. Treatment of rat aortic cultures with anti-bFGF antibodies causes a 40% inhibition of the angiogenic response. Anti-bFGF antibodies also inhibit the angiogenic effect of endogenous bFGF in cultures of human chorionic vessels (Brown et al., 1996) and bovine capillary endothelial cells (Sato et al., 1991).

2. Vascular Endothelid Growth Factor Vascular endothelial growth factor (VEGF), also known as vascular permeability factor because of its capacity to greatly increase vascular permeability (Dvorak et al., 1995),is a heparin-binding angiogenic factor with endothelial target specificity (Ferrara et al., 1992). VEGF belongs to a family of endothelial growth factors which includes the recently discovered VEGF-B and the lymphatic endothelium-specific VEGF-C (Enholm et al., 1997; Kukk et al., 1997). VEGF stimulates endothelial migration, proliferation, and proteolytic activity (Ferrara et al., 1992). At variance with aFGF and bFGF, VEGF has a signal peptide and is secreted through conventional pathways. VEGF is produced by a variety of cells, including endothelial cells (Ladoux and Frelin, 1993), smooth muscle cells (Ferrara et al., 1991), and fibroblasts (Minchenko et al., 1995). Secretion of VEGF is markedly stimulated by hypoxic conditions (Shweiki et al., 1992; Minchenko et al., 1995). Four VEGF isoforms are produced by alternative splicing of the VEGF gene (Ferrara et al., 1992). The shortest form, VEGFlZ1,is secreted and can be recovered in conditioned media. The two longer forms, VEGFlS9and VEGF206,are secreted but bind almost exclusively to the cell membrane or the ECM and are not found in the soluble phase. VEGF165 has an intermediate behavior. As for the FGFs, heparin, heparan sulfate, and heparanase all induce release of substrate-bound VEGF, suggesting that heparan sulfate proteoglycans are extracellular binding sites for VEGF (Houck et al., 1992; Ferrara et al., 1992). VEGF165and VEGFls9 can also be released from their binding sites by plasmin (Houck et al., 1992). VEGF binds to the tyrosinase kinase receptors flk-UKDR and flt-1, which are expressed in the endothelium of blood vessels (Terman et al., 1992;Millauer et al., 1993; Quinn et al., 1993). Knockout of the flk-1 receptor gene causes failure of vasculogenesis in the embryo (Shalaby et al., 1995). Knockout of pr-I results in endothelial disorganization daring vasculogenesis and formation of abnormally dilated vascular structures (Fong et al., 1995).

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Knockout of the VEGF gene, which is expressed during vasculogenesis in the mesenchyme surrounding developing blood vessels, causes failure of vasculogenesis not only in the homzygous animal but also in the heterozygous form (Ferrara et al., 1996). Thus, the VEGFNEGF receptor system is an absolute requirement for embryonal vasculogenesis. VEGF is also critical for angiogenesissince anti-VEGF antibodies (Borgstrom et al., 1996) or dominant-negative forms of the fik-1 receptor (Millauer et al., 1994) have potent antiangiogenic activity in vivo. In the rat aorta model, the explants and their outgrowths secrete VEGF in the culture medium. Inhibition of VEGF with a neutralizing antibody causes a 70% reduction of the angiogenic response (Nicosia et al., 1998). Inhibition of endogenous VEGF with a neutralizing antibody also reduces angiogenesis in cultures of human chorionic vessels. However, maximal inhibition of angiogenesis in these cultures is obtained with a combination of anti-bFGF and anti-VEGF antibodies (Brown et al., 1996), suggesting that vascular-derived bFGF and VEGF cooperate in promoting the angiogenic response of injured blood vessels. 3. Platelet-Derived Growth Factor

Platelet-derived growth factor (PDGF) is a potent mitogen and chemotactic factor for smooth muscle cells and fibroblasts (Ross et al., 1974; Antoniades et al., 1975). PDGF is composed of two polypeptides, A and B, which can associate forming homodimeric (AA and BB) or heterodimeric (AB) complexes (Hart et al., 1990). The PDGF receptor is composed of two subunits, CY and p chains. These two chains dimerize forming ma, pp, and Cup receptors (Seifert et al., 1988). PDGF AA binds only to the PDGF act receptor. PDGF BB binds to all three receptors. PDGF AB binds to the (Y(Y and receptors. PDGF is produced by endothelial cells (Kazlauskas and DiCorleto, 1985), smooth muscle cells (Majesky et al., 1988), and fibroblasts (Antoniades et al., 1991).Large vessel endothelial cells do not respond in vitro to PDGF (Kazlauskas and DiCorleto, 1985) but may express the PDGF aa receptor in vivo after injury (Lindner, 1995b). Microvascular endothelial cells apparently express the PDGF p/3 receptor (Smits et al., 1989) and proliferate in response to PDGF (Bar et al., 1989).The angiogenic activity of PDGF may be due to both a direct effect on the microvascular endothelial cells (Bar et al., 1989) and an indirect effect mediated by smooth muscle cells and fibroblasts, which in response to PDGF stimulation secrete endothelial cell growth factors such as bFGF and VEGF (Finkenzeller et al., 1992; Brogi et al., 1994). PDGF, which is upregulated in injured blood vessels (Schwartz et al., 1990), is a potent stimulator of angiogenesis in the rat aorta model (Nicosia et al., 1994b). The outgrowth of PDGF-stimulated aortic cultures contains a markedly increased number of spindly mesenchy-


13

ma1 cells which probably mediate the angiogenic effect of PDGF in the system. The PDGF BIP ligandheceptor system also seems to play an important role in the recruitment of smooth muscle cells by the endothelium. The endothelium of injured rat carotid arteries expresses PDGF B, whereas the underlying smooth muscle cells express the PDGF /3 receptor (Lindner, 1995b). A similar mechanism may regulate the interactions between microvascular endothelial cells and pericytes because pericytes migrate and proliferate in response to PDGF B (D’Amore and Smith, 1993). PDGF B knockout mouse embryos have markedly dilated vessels and lack mesangial cells, which are glomerular capillary-supporting cells closely related to pericytes (Leveen et aZ., 1994). Abnormal development of the cardiovascular system is also observed in patch mice whose PDGF a receptor gene has been deleted. The vessels of patch mice are lined by a normal endothelium but contain a reduced number of smooth muscle cells and exhibit abnormal fragility (Schatteman et aZ., 1995).

4. Insulin-like Growth Factor Insulin-like growth factor-I (IGF-I) is produced by a variety of cell types, including endothelial cells (Bar, 1992), smooth muscle cells (Delafontaine et al., 1991), and fibroblasts (Pietrzkowski et aZ., 1992). IGF-I expression is observed in connective tissue mesenchyme during embryonal development and is upregulated in the vessel wall of the adult animal during vascular injury (Khorsandi et aZ., 1992) and angiogenesis (Hansson et al., 1989). IGFI production in smooth muscle cells is stimulated by PDGF and bFGF (Delafontaine et al., 1991). IGF-11, a peptide shorter than IGF-I, elicits similar growth responses as IGF-I (Baker et aZ., 1993). The activity of IGF-I and IGF-I1 is modulated by IGF-binding proteins which may either potentiate or inhibit the autocrine and paracrine effects of the IGFs (Bar, 1992). IGF-I and IGF-I1 are probably not required for vasculogenesis and angiogenesis since knockout of IGF-I, IGF-11, or the IGF-I receptor (IGFIR) gene, which both growth factors use to transduce their signals, has no effect on the development of the cardiovascular system (Baker et al., 1993; King et al., 1985). The small size of the embryos lacking IGF-IR, IGF-I, and IGF-11, alone or in combination, indicates that IGFs play an important role in maintaining normal growth rate and in maximizing the efficiency of growth in the various organ systems of the embryo including the cardiovascular system (Baker et aZ., 1993). In fact, addition of IGF-I to serumfree cultures of rat aorta causes a potentiation of the angiogenic response of the aortic explants (Nicosia et aZ., 1994b). 5. Scatter Factor Scatter factor (SF), also known as hepatocyte growth factor, is a secreted endothelial mitogen produced by fibroblasts (Coffer et aZ., 1991). SF binds

14


to the c-met receptor, which is coded by a protooncogene (Rosen and Goldberg, 1997). SF, which was first characterized as a potent epithelial mitogen that caused scattering of cultured epithelial cells, was subsequently found to stimulate endothelial cell migration, proliferation, capillary tube formation, and angiogenesis (Rosen et al., 1991; Bussolino et al., 1992). Secretion of SF by fibroblasts is potentiated by bFGF (Roletto et al., 1996). Like other angiogenic factors, SF binds to the ECM (Lamszus et al., 1996).

6. Heparin-Binding Epidermal Growth Factor-like Factor Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a member of the epidermal growth factor family which, unlike EGF, has strong affinity for heparin (Moses et al., 1995). Cell surface heparan sulfates play a critical role for the binding and function of HB-EGF. HB-EGF is produced by a variety of inflammatory and noninflammatory cells, including endothelial cells and smooth muscle cells (Yoshizumi et al., 1992; Dluz et al., 1993). HB-EGF is a potent mitogen for smooth muscle cells and fibroblasts but not for endothelial cells (Moses et al., 1995). HB-EGF binds to the EGF receptor (Higashiyama et al., 1992) but it is not yet clear which of the EGF receptors is involved in the biological function of this growth factor (Moses et al., 1995). HB-EGF mRNA is overexpressed in injured blood vessels (Moses et al., 1995) and can be demonstrated in wound fluid 1 day following skin injury (Powell et al., 1993). HB-EGF is upregulated by bFGF and PDGF (Dluz et al., 1993). 7. Transforming Growth Factor+

Transforming growth factor+ (TGF-P) is a multifunctional peptide capable of regulating the migration, proliferation, and differentiation of a variety of cell types. Three related proteins with high structural similarities have been described in mammalian cells: TGF-Pl, TGF-p2, and TGF-p3 (Massague, 1990).The effects of TGFPs are critically dependent on the differentiation state of the target cells and the presence or absence of other growth factors (Sporn et al., 1987). TGF-Pl stimulates the proliferation and ECM production of fibroblasts (Fine and Goldstein, 1987) and promotes the transformation of these cells into myofibroblasts, which have the capacity to contract collagen (Desmouliere et al., 1993).TGF-Pl can either stimulate or inhibit smooth muscle cell proliferation depending on its concentration and on the density of the cultured cells (Majack et al., 1990). The effect of TGF-P1 on endothelial cells and angiogenesis appears to be contextual because TGF-P1 inhibits the migration and proliferation of endothelial cells in two-dimensional culture (RayChaudury and D’Amore, 1991) but promotes the organization of endothelial cells into capillary tubes in three-


15

dimensional collagen gel culture (Madri et al., 1988). This phenomenon may be related to the capacity of TGF-P1 to stimulate production of ECM molecules that have morphogenetic and stabilizing effects on endothelial cells (Madri et al., 1988;Nicosia et aL, 1993) and to modulate the production of proteolytic enzymes and their inhibitors (RayChaudury and D’Amore, 1991). TGF-P1 can either promote or inhibit the in vitro angiogenic activity of bFGF or VEGF in collagen gel culture depending on its concentration in the culture medium (Pepper et al., 1993b). In vivo injection of TGF-P1 in the subcutaneous tissue induces formation of granulation tissue and fibrosis (Roberts et al., 1986). The in vivo angiogenic activity of TGF-P1 has been attributed to the capacity of this growth factor to recruit at the site of injectionhmplantation macrophages and other inflammatory cells which secrete direct-acting angiogenic factors (Roberts et al., 1986; Wahl et al., 1987; RayChaudury and D’Amore, 1991). TGF-P1 is secreted in an inactive form and is believed to be activated by plasmin or an acid microenvironment (Sporn et aL, 1987; RayChaudury and D’Amore, 1991). Active TGF-P1 in turn stimulates production of plasminogen activator inhibitor-1 (PAI-l), which blocks production of plasmin by inhibiting the activity of plasminogen activators (RayChaudury and D’Amore, 1991). During angiogenesis this self-regulating system may be triggered when endothelial cells and pericytes develop cell-cell contacts which activate TGF-P1 (RayChaudury and D’Amore, 1991).Endogenous TGF-P1 production is upregulated in response to vascular injury (Majesky et al., 1991) and during cutaneous wound healing (Cromack et al., 1987). The role of TGF01 in embryonal vasculogenesis and angiogenesis was initially unclear since mice that survived the knockout of TGF-P1 had a normal cardiovascular system and suffered from a lethal disorder of the immune system (Kulkarni et al., 1993). The lack of cardiovascular abnormalities in these mice was attributed to the replacement of TGF-P1 by other embryonal TGF-P isoforms or by maternal TGF-P1 (Kulkarni et al., 1993). Subsequent studies showed that TGF-P1 null mouse embryos that died in utero had defective yolk sac vasculogenesis due to inadequate endothelial differentiation and capillary tube formation (Dickson et aL, 1995).

B. Other Soluble Regulators of Angiogenesis

1. Tissue Factor Tissue factor (TF; thromboplastin) is a procoagulant protein secreted by endothelial cells, smooth muscle cells, and pericytes in response to injury and blood flow-mediated changes in shear stress (Taubman, 1993; Grabowski and Lam, 1995; Bouchard et al., 1997). TF is a chemoattractant for

16


smooth muscle cells with the same potency as that of PDGF or bFGF (Sato et al., 1996). The observation that yolk sac blood vessels of mouse embryos lacking the TF gene have no pericytes and are prone to hemorrhage suggests that TF may also play a developmental role in the differentiation of the perivascular mesenchyme and the recruitment of mural cells by the endothelium (Carmeliet et al., 1996). The possibility that a similar mechanism may operate in the adult is supported by the observation that the endothelium of angiogenic microvessels expresses TF, which is otherwise not detectable in quiescent vessels (Contrino et al., 1996). 2. Tumor Necrosis Factor-a

Tumor necrosis factor-a (TNF-a), also known a cachectin for its ability to induce cachexia, plays a role in inflammation and hematopoiesis. Endothelial effects of TNF-a include the induction of class I major histocompatibility antigens, procoagulant activity, interleukin-1, and leukocyte adhesiveness. TNF-a has been shown to stimulate endothelial cell migration and capillary tube formation in vitro (Leibovich etal., 1987).Like TGF-P, however, TNFa inhibits endothelial cell proliferation in vitro (Frater-Schroder et al., 1987). The in vivo angiogenic activity of TNF-a (Leibovich et al., 1987) may be attributed to the molecule’s ability to attract macrophages and mast cells, which in turn secrete direct-acting angiogenic factors (D’Amore and Smith, 1993; Leibovich et al., 1987). TNF-ais also produced by smooth muscle cells (Warner and Libby, 1989) and may contribute to the mechanisms that regulate vascular-related angiogenesis during wound healing.

3. Angiopoietins The angiopoietins are a newly discovered family of peptides which play a critical role in the development of the vessel wall during embryonal vasculogenesis and angiogenesis.Angiopoietin-1 (Ang-1) and angiopoietin2 (Ang-2) bind to the Tie-2 tyrosine kinase receptor which is expressed in endothelial cells (Sato et al., 1995; Davis et al., 1996; Maisonpierre et al., 1997). Ang-1 and Ang-2 are produced by the perivascular mesenchyme and smooth muscle cells during embryonal development (Davis et al., 1996). Genetic ablation of Ang-1 or the Tie-2 receptor results in abnormal vasculogenesis with failure of the newly formed vasculature to differentiate (Sato et al., 1995; Suri et al., 1996). Vessels formed in the absence of a functioning Ang-l/Tie-2 system do not acquire a properly assembled mural layer of smooth muscle cells or pericytes and become dilated (Puri et al., 1995; Sat0 et al., 1995;Suri et al., 1996).Ang-1 is unable to induce endothelial migration or proliferation even though it induces phosphorylation of the endothelial Tie-2 receptor (Sun et al., 1996). These findings suggest that endothelial


17

cells respond to stimulation by Ang-1 by secreting factors that recruit mural cells and contribute to the harmonious differentiation of the vascular wall (Suri et al., 1996). Interestingly, patients with arteriovenous malformations, which exhibit abnormal layering of smooth muscle cells around the endothehum, have point mutations of the Tie-2 receptor (Vikkula et al., 1996). Ang-2 competes with Ang-1 for the binding to the Tie-2 receptor. The overexpression of Ang-2 in transgenic mice causes abnormal vascular development similar to that observed in Ang-1 and Tie-2 knockout mice, which suggests that Ang-2 is a natural inhibitor of Ang-1 (Maisonpierre et al., 1997). Abnormal vasculogenesis is also observed in mouse embryos with Tie-1 receptor knockout, but the ligand for the Tie-1 receptor has not yet been identified (Sato et al., 1995).

VI. Proteolytic Enzymes During the early stages of angiogenesis, activated endothelial cells create localized gaps in the basement membrane through which they sprout into the surrounding tissue. As they migrate through the interstitial collagen, endothelial cells generate additional defects in the ECM for the sprouting neovessels. These processes are regulated by proteolytic enzymes and enzyme inhibitors which act in concert to ensure a balanced degradation of the perivascular matrix. Two major classes of proteolytic enzymes and respective inhibitors have been implicated in angiogenesis: plasminogen activators (PAS) and matrix metalloproteinases (MMPs).

A. Plasminogen Activators The PAS hydrolyze plasminogen, a zymogen present in plasma, to form plasmin, a broad spectrum serine protease which breaks down a variety of protein substrates (Saskela, 1985). Endothelial cells can directly synthesize tissue-type PA (t-PA) and urokinase-type PA activator (u-PA) (Loskutoff and Edgington, 1979; Pepper et al., 1987). t-PA is a secreted enzyme which plays an important role in the degradation of fibrin by both arterial and venous endothelial cells. Localized production of plasmin, unaffected by inhibitors of fibrinolysis, is mediated by uPA which has been demonstrated at the tip of growing microvessels (Bacharach and Keshet, 1992). The uPA receptor (uPAr) binds and localizes uPA at critical sites of proteolysis during angiogenesis (Loskutoff and Edgington, 1979; Barnathan et al., 1990). Receptor-bound uPA generates plasmin in close proximity of the sprouting endothelial cell. Endothelial cells also produce PAI-1 which inhib-

18

R. F. NICOSIA AND

S. VlLIASCHl

its PA activity and ensures a balanced degradation of the ECM (Bacharach and Keshet, 1992). Angiogenic factors, such as bFGF and VEGF, induce upregulation of both uPA and PAI-1 (Moscatelli and Rifkin, 1988; Pepper et al., 1987,1991). Vascular injury causes upregulation of tPA, uPA, uPAr, and PAI-1 in both endothelial cells and smooth muscle cells (Reidy et aL, 1996). The expression of uPA, uPAr, and PAI-1 is restricted to the site of injury and at the wound edge, implicating these molecules in endothelial and smooth muscle cells migration. Capillary tube formation in vitro and angiogenesis in vivo can be inhibited with uPAr antagonists (Min et aL, 1996).Plasmin functions either directly by digesting noncollagenous components of the ECM (Liotta et al., 1981) or indirectly by activating MMP-1, which in turn digests interstitial collagen (Gross et aL, 1982). Gene knockout experiments indicate that the PA/PAI-1 system is not required for embryonal vasculogenesis and angiogenesis since mice that lack the uPA, tPA, or PAI-1 genes have marked defects in fibrinolysis and coagulation but exhibit a normal cardiovascular system (Carmeliet et aL, 1997a). The PA/PAI-1 system may, however, play a role in reactive and pathologic processes. For example, in vivo intimal migration of smooth muscle cells in response to injury is reduced in mice having a disruption of the plasminogen gene (Carmeliet et al., 1997b).

B. Matrix Metalloproteinases The MMPs are a family of zinc-dependent endopeptidases which are secreted as zymogens and activated extracellularly. MMPs are classified into three groups depending on their substrate specificity: interstitial collagenases (substrate: interstitial collagen), stromelysins (substrates: laminin and fibronectin), and gelatinases (substrate: type IV collagen) (Ray and StetlerStevenson, 1994). There is some functional overlap between the groups since some gelatinases (MMP-2) can digest fibrillar collagen, whereas stromelysins can digest type IV collagen. The function of the MMPs is regulated at multiple levels, including gene activation, transcription, mRNA stability, translation, secretion, binding to ECM components, proenzyme activation, and inactivation by tissue inhibitors of metalloproteinases (TIMPs) (Ray and Stetler-Stevenson, 1994). Endothelial cells, smooth muscle cells, and fibroblasts produce both MMPs and TIMPs (Cornelius et aL, 1995; Forough et aL, 1996; Gilles et aL, 1997). The function of MMPs is modulated by growth factors. For example, expression of interstitial collagenase (MMP1) is induced by both bFGF and VEGF (Unemori et aL, 1992; Kennedy et aL, 1997). These growth factors also stimulate the plasminogen system promoting the formation of plasmin which in turn transforms proMMP-1 into its active form (Unemori et d.,1992). The regulation of MMP synthesis and secretion is cell and tissue specific and the capacity of differ-


19

ent growth factors to induce these enzymes varies with the cell type. Stimuli other than growth factors such as cell shape changes, as well cell-cell and cell-matrix interactions, can influence MMPs production (Woessner, 1994). For example, gelatinase B (MMP-9) is induced by the ECM molecule thrombospondin-1 (Quian et aZ., 1997). Gelatinase A (MMP-2) exhibits only a slight response to growth factors and its synthesis is modulated by the ECM and intracellular calcium influx (Woessner, 1994). A cell-associated enzyme known as membrane-type metalloproteinase (MT-MMP-1) has been shown to activate MMP-2 by proteolytic mechanisms (Sato et aZ., 1994). MT-MMP-1 belongs to a family of cell surface enzymes which are believed to play an important role in the activation of MMPs by cell-cell contact. Recent studies indicate that MT-MMP-1 expression by fibroblasts, which results in the activation of MMP-2, is upregulated by interstitial collagen (Gilles et al., 1997). The activity of the MMPs is neutralized by a group of endogenous inhibitors known as TIMPs (Ray and StetlerStevenson, 1994; Woessner, 1994). TIMP-1 forms a complex with activated MMP-1, stromelysin, or MMP-9, whereas TIMP-2 binds to MMP-2. Based on their capacity to inhibit MMP activity,TIMPs can regulate the angiogenic process by modulating the degradation of the ECM. Thus, while they can inhibit angiogenesis by suppressing MMP activity, TIMPs can also promote angiogenesis by preventing excessive proteolytic degradation of the ECM. Up to a certain level, MMPs promote capillary tube formation in vitro, but they have the opposite effect when their concentration is too high (Schnaper et aZ., 1993; Qian et al., 1997). The promoting or inhibitory effects of MMP2 can be regulated by an adequate concentration of TIMP-2. TIMP-2 also has the capacity to inhibit endothelial cell proliferation independently of its anti-MMP activity (Ray and Stetler-Stevenson, 1994). MMPs, which have been localized by in situ labeling techniques in developing microvessels (Galis et al., 1994; Brooks et al., 1996), are believed to be necessary for angiogenesis since neutralization of MMP activity inhibits capillary tube formation in vitro (Schnaper et aZ., 1993; Qian et al., 1997). MMP-2-deficient mice develop normally without anatomical abnormalities but have a reduced capacity to generate new vessels during pathologic processes such as tumor growth (Itoh et aZ., 1998). The absence of cardiovascular defects in these mice has been attributed to the functional redundancy of matrix metalloproteinases (Itoh et al., 1998).

VII. Extracellular Matrix and Cell Adhesion Molecules A. Extracellular Matrix Molecules The ECM that surrounds the endothelium of microvessels is both a mechanical barrier and a substrate requirement for the angiogenic process. The

20

R. F. NICOSIA AND S. VlLIASCHl

morphogenetic behavior of endothelial cells and the response of these cells to soluble growth factors and proteolytic enzymes is tightly linked to the insoluble molecules of the ECM. Endothelial cells are anchorage-dependent cells which must attach to adhesive substrates in order to survive and grow. This process is mediated by a family of cell surface receptors known as integrins (Hynes, 1992) which transduce ECM cues to the nucleus through intracellular pathways of biochemical and mechanochemical signals (Ingber and Folkman, 1989). The importance of the ECM in angiogenesis is underscored by experimental evidence indicating that the stimulatory effect of growth factors and other soluble regulators of angiogenesis is not sufficient to explain how blood vessels form (Ingber, 1991).

1. Developmental Changes in the Extracellular Matrix during Angiogenesis The endothelium of quiescent microvessels is ensheathed by a basement membrane and fibrils of interstitial collagen admixed with proteoglycans. During angiogenesis, the microvascular ECM undergoes complex maturational changes due to the proteolytic degradation of the preexisting matrix and the deposition of a new matrix (Nicosia and Madri, 1987). At the ultrastructural level, developing neovessels are surrounded by a tenuous and discontinuous basement membrane (Schoefl, 1963). Because of the high permeability of the sprouting endothelium, the perivascular space is edematous and may be permeated by plasma-derived large molecules such as fibrinogen, fibronectin, vitronectin, von-Willebrand factor, and plasminogen. The extravascular activation of the coagulation cascade results in the formation of fibrin which provides a permissive ECM scaffold for endothelial cell migration, proliferation, and tube formation. Vitronectin, fibronectin, and von-Willebrand factor promote endothelial migration by acting as substrates for the integrin receptors expressed by sprouting endothelial cells (Hynes, 1992). Plasminogen functions as a zymogen for the formation of plasmin, which endothelial cells use to break down fibrin and other ECM molecules and to generate active MMP-1 (Liotta et al., 1981; Gross et al., 1982). The provisional ECM secreted by endothelial cells contains fibronectin, type V collagen, and small amounts of laminin and type IV collagen (Tonnesen et aL, 1985; Nicosia and Madri, 1987). As the microvessels mature, laminin and type IV collagen accumulate in the subendothelial space forming a basement membrane (Nicosia and Madri, 1987). Additional ECM molecules produced by vascular cells which may modulate angiogenesis include the glycoproteins entactin (Nicosia et al., 1994a), thrombospondin-1 (Koch et aL, 1993), osteopontin (O’Brien et al., 1994), and tenascin (Zagzag et al., 1996) as well as the heparan sulfate proteoglycans perlecan (Aviezer et al., 1994) and syndecan-1 (Kainulainen et aL, 1996). As the basement membrane matures, fibrils of the interstitial colla-


21

gens type I and I11 are deposited by the endothelial cells, pericytes, and surrounding fibroblasts around the newly formed microvessels (Nicosia and Madri, 1987).

2. Role of the Extracellular Matrix in Angiogenesis The ECM plays a fundamental morphogenetic role in angiogenesis because it promotes the organization of endothelial cells into capillary tubes. As endothelial cells migrate into the connective tissue, collagen and the other ECM molecules that surround the endothelium induce capillary tube morphogenesis. This phenomenon can be reproduced in vitro by culturing endothelial cells on basement membrane-like matrices (Kubota et aL, 1988) and in gels of interstitial collagen (Montesano et aL, 1983)or fibrin (Fournier and Doillon, 1992). Destabilization of endothelial cell-ECM contacts causes detachment of endothelial cells from the basement membrane and disruption of microvessels. Detached endothelial cells round up, cease to proliferate, and undergo apoptosis. This effect can be achieved by treating developing microvessels with angiostatic steroids, inhibitors of collagen synthesis, inhibitors of basement membrane synthesis, or fungal-derived antibiotics which destabilize cell-matrix interactions (Ingber and Folkman, 1989). Angiogenesis can also be inhibited by interfering with the binding of integrin receptors to RGD (Arg-Gly-Asp)-containingcomponents of the perivascular matrix (Nicosia and Bonanno, 1991). The RGD-sensitive a& and avosintegrin receptors have been proposed as potential targets for antiangiogenic therapy because they are expressed by the microvascular endothelium during angiogenesis. Expression of the avosor avosreceptors in endothelial cells appears to be regulated by separate growth factor pathways since angiogenesis induced by bFGF apparently depends on aVP3, whereas angiogenesis initiated by VEGF requires avPs(Brooks et aL, 1994; Friedlander et al., 1995). RGD-containing synthetic peptides inhibit angiogenesis by interfering with the binding of RGD-sensitive receptors to RGD-containing ECM molecules (Haymann et aL, 1985;. Nicosia and Bonanno, 1991). Because the perivascular matrix is particularly rich in RGD-containing molecules and the avP3and avPsintegrin receptors can bind to a variety of ECM molecules (Hynes, 1992), it is likely that sprouting endothelial cells utilize a redundant system of RGD-containing substrates for attachment and migration. It is clear, however, that some of these matrix molecules are required for proper vascular morphogenesis. Gene knockout studies have shown that ablation of the fibronectin gene causes malformations of the cardiovascular system (George et al., 1993). Similar results have been observed in mice lacking the as chain of the fibronectin receptor (Yang et aL, 1992).

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Based on their capacity to influence endothelial cell adhesion and shape (Ingber and Folkman, 1989) and to bind angiogenic factors (Moses et al., 1995), ECM molecules can modulate the migratory and proliferative response of endothelial cells. Thus, fibronectin, laminin, and entactin added to a basal gel of interstitial collagen promote the elongation of microvessels sprouting from explants of rat aorta (Nicosia et al., 1993, 1994a). ECM molecules may, however, have opposite effects on angiogenesis depending on their concentration. These effects can be observed in the rat aorta model by varying the amounts of ECM molecules added to the collagen gel. For example, the basement membrane-derived laminin-entactin complex added to a basal gel of interstitial collagen stimulates rat aorta angiogenesis at 30-300 pglml, whereas it has inhibitory effects at 3000 pglml. High concentrations of laminin-entactin also have stabilizing effects and prevent the spontaneous regression of microvessels (Nicosia et al., 1994a). A similar concentration of laminin-entactin is found in native basement membranelike matrices (Kleinmann et al., 1986). This suggests that the mature basement membrane that surrounds the endothelium during the late stages of blood vessel formation may function as a stop signal that turns angiogenesis off and stabilizes the newly formed microvasculature. TGFP-1, which is presumably activated when the endothelial cells of mature microvessels are surrounded by pericytes, may contribute to this late stage of the angiogenic process by stimulating the production and deposition of basement membrane molecules (RayChaudury and D’Amore, 1991). Heparan sulfate proteoglycans, which are present in the basement membranes and on the cell surface, can modulate the response of endothelial cells and smooth muscle cells/pericytesto bFGF, VEGF, and other heparinbinding angiogenic factors by sequestering these molecules in the ECM (Ferrara et al., 1992; Moses et al., 1995). Heparitinases secreted in response to injury or pathologic stimuli may release the growth factors from the ECM making them available for angiogenic stimulation (Vlodavsky et al., 1987; Houck et al., 1992; Ferrara et al., 1992). Heparin, a highly sulfate glycosaminoglycan,is stored in the cytoplasmicgranules of mast cells, which are believed to modulate angiogenesis by releasing this molecule in the extracellular space (Meininger and Zetter, 1992). Heparan sulfate proteoglycans may also act as low-affinity receptors for FGFs, which are more soluble and stable when complexed with the sulfate groups of these molecules (Moses et al., 1995). Conformational changes induced by the heparan proteoglycans may allow the FGFs to better interact with their high-affinity receptors on the endothelial cell surface. Perlecan, a basement membrane proteoglycan, is a potent inducer of bFGF-mediated angiogenesis (Aviezer et al., 1994). Similarly, syndecan-1, a cell surface proteoglycan, is believed to play a role in angiogenesis because it is transiently expressed by endothelial cells during formation of microvessels (Kainulainen et. al., 1996). Be-


23

cause of its ability to bind angiogenic factors and extract them from tissues, heparin may also exhibit antiangiogenic activity if administered at pharmacologic doses together with hydrocortisone or other angiostatic steroids (Moses et al., 1995). The enzymatic digestion of the ECM by vascular cells generates fragments which may have opposite effects of the intact molecule. For example, hyaluronan, a large glycosaminoglycan present in the interstitial ECM, has antiangiogenic activity,whereas its oligosaccharidefragments potentiate the action of angiogenic factors (Rooney et aL, 1995). Conversely, proteolytic digestion of fibronectin (Homandberg et al., 1985), plasminogen (O’Reilly et al., 1996), or collagen type XVIII (O’Reilly et al., 1997) generates fragments with antiangiogenic activity.

6.Cell Adhesion Molecules The organization of endothelial cells into capillary tubes is mediated not only by cell-matrix adhesive events but also by cell-cell interactions. Cytoadhesive proteins such as the selectins and cadherins are believed to play a role in regulating cell-cell interactions that take place during capillary tube morphogenesis (Koch et al., 1995; Dejana, 1996; Dejana et al., 1997). Cell-cell interactions during capillary morphogenesis are also regulated by integrins, which in addition to engaging ECM molecules have the capacity to bind to each other (Dejana et al., 1997), and by molecules of the Ig superfamily such as platelet-endothelial cell adhesion molecule-1 (Lu et al., 1996; DeLissler et al., 1997). E-selectin, a surface molecule that localizes in adherent junctions, contributes to the formation and maintenance of capillary tubes by interacting with sialyl Lewis-X and sialyl Lewis-A carbohydrate moieties on adjacent cells (Nguyen et al., 1993). Similarly, vascular endothelial cadherin, which is localized in endothelial adherent junctions, is required for capillary tube formation and maintenance (Dejana et al., 1997; Matsuma et al., 1997).

VIII. Vasoactive Factors

Vasoactive events may play a role in angiogenesis because vasodilatation and increased blood flow promote capillary growth (Ziada et al., 1984). During angiogenesis in vivo capillaries with higher flow generate more sprouts and change gradually into arterioles and venules (Thoma, 1911; Clark and Clark, 1939). Conversely, neovessels that are not supplied with blood tend to regress (Clark and Clark, 1939). Vascular regression is associ-

24

R. F. NICOSIA AND

S. VlLLASCHl

ated with contraction of the neovessels and endothelial retraction (Clark and Clark, 1939; Nicosia and Villaschi, 1995). Vasodilating agents have been reported to have angiogenic activity (Ziche et al., 1982; Ziada et al., 1984; Masferrer et aZ., 1989). Vasoactive events may regulate the angioformative behavior of endothelial cells by activating through mechanochemical mechanisms endothelial genes involved in angiogenesis (Ingber, 1991;Gimbrone et al., 1997). Angiogenic factors may modulate vascular tone and blood flow. For example, aFGF, bFGF, and VEGF cause vasodilation (Ku et al., 1993; Wu et al., 1996b), whereas PDGF is a potent vasoconstrictor (Berk et aL, 1986). Vascular cells produce a variety of vasoactive molecules, including nitric oxide, prostaglandins, and endothelins, all of which may contribute to the mechanisms by which the vessel wall regulates angiogenesis. Flow-dependent vasodilation is mediated by intracellular influx of calcium, calcium-mediated activation of nitric oxide (NO) synthase, and synthesis of NO, also known as endothelial-derived relaxing factor (Moncada, 1997).Endothelial cells express constitutively a form of nitric oxide synthase which generates NO from L-arginine. Endothelial cell-derived NO maintains a vasodilator tone that is essential for the regulation of normal blood flow and pressure. Smooth muscle cells, like many other cell types, can express an inducible form of nitric oxide synthase (iNOS) in response to cytokines such as IL-1 or TNF-a. Small arteries and arterioles are a major site of NO-mediated vasodilation in response to increase blood flow and shear stress. NO mediates the vasodilator effect of ADP, histamine, serotonine (Moncada, 1997), aFGF, bFGF, and VEGF (Ku et aZ., 1993; Wu et aZ., 1996b). NO apparently also regulates the increased vascular permeability caused by VEGF at the level of postcapillary venules via a signaling cascade involving guanylate cyclase stimulation and guanosine 3'3'-cyclic monophosphate-dependent protein kinase (Wu et al., 1996b). It has also been proposed that NO mediates the endothelial-specific mitogenic effect of VEGF (Morbidelli et aZ., 1996). NO promotes the proliferation of and PA production by endothelial cells through upregulation of endogenous bFGF (Ziche et aZ., 1997). Expression of iNOS by nonendothelial cells has been implicated in angiogenesis since the angiogenic activity of monocytes requires a functioning NOS system (Leibovich et aZ., 1994). Interestingly, NO inhibits the migration and proliferation of smooth muscle cells (Garg and Hassid, 1989). Thus, increased levels of NO during the early stages of angiogenesis may explain not only the vasodilation and increased blood flow but also the finding of immature endothelial sprouts without pericytes or smooth muscle cells. The role of NO in angiogenesis,however, is debated since inhibitors of NO synthase have antiangiogenic activity in the rabbit cornea model (Ziche et aZ., 1994) but increase vascular density in the chorioallantoic membrane of the chick embryo (Pipili-Synetos et aZ., 1993).


25

The ETs are a family of small peptides secreted by both vascular endothelial and smooth muscle cells. Three different isoforms have been described (ET-1, ET-2, and ET-3). Endothelial cells produce exclusively ET-1, whereas other tissues produce ET-2 and ET-3. ET-1 production is induced by a variety of factors, including TGFP-1 and hypoxia (Luscher and Wenzel, 1995). ET-1 causes transient vasodilation followed by profound and sustained vasoconstriction. ET-1 also stimulates smooth muscle cell migration and proliferation (Bobik et aZ., 1990) and potentiates the effect of PDGF BB on these cells (Weissberg et aZ., 1990). The mitogenic effect of ET-1 is mediated by the ETA and ETB receptors (Luscher and Wenzel, 1995). The ETA receptor is responsible for vasoconstriction, whereas the ETB receptor, which is also expressed in endothelial cells, is linked to NO and prostacyclin release (Luscher and Wenzel, 1995). During embryonal development, ET-1 is expressed intensely in the endocardium of the outflow tract and in the endothelium of arch arteries and dorsal aorta (Kurihara et al., 1997). Neural crest-derived ectomesenchymal cells migrate from the pharyngeal arches to the heart outflow tract and arch arteries where they contribute to the formation of great vessels. Ectomesenchymal cells, which provide support to the endothelial cells and give rise to smooth muscle cells, may be recruited to their final destination sites by ET-1. In fact, ET-1 knockout mouse embryos exhibit a variety of cardiovascular malformations, including interrupted aortic arch, hypoplasia of the aortic arch, aberrant subclavian artery, and ventricular septa1 defects with abnormalities of the outflow tract (Kurihara et aZ., 1997). The frequency of these abnormalities increases by treatment with neutralizing anti-ET-1 monoclonal antibodies or selective ETA receptor antagonists (Kurihara et al., 1997). Prostaglandin El(PGEJ and, to a lesser extent PGE2 and PGFh, stimulate angiogenesis in vivo (Ben-Ezra, 1978). The angiogenic activity of prostaglandins is probably mediated by cytokines and growth factors secreted by inflammatory cells which are chemotactically attracted to the site of angiogenesis (Ziche et al., 1982; Odedra and Weiss, 1991) and by fibroblasts which secrete VEGF in response to prostaglandin stimulation (Ben-Av et al., 1995). Prostaglandins such as PGEl may also facilitate angiogenesis by causing vasodilation and increased blood flow. Adenosine, a vasodilator metabolite which accumulates in tissues during hypoxia and anaerobic metabolism, has been shown to stimulate endothelial cell migration and proliferation in vitro as well as angiogenesis in vivo (Ziada et al., 1984).Although the relatively high doses of adenosine required to stimulate angiogenesis have raised doubts about its physiologic role, these observations suggest that small molecules of nonproteic nature released during wound healing or other processes associated with hypoxia may influence the angiogenicresponse of blood vessels. For example, inosine-a metabolic derivative of adenosine-and nicotinamide, both of which accu-

26

R.


mulate in tissues during anaerobic metabolism, have been shown to have angiogenic activity (Odedra and Weiss, 1991).

IX. Summary and Future Directions Our current knowledge of the endogenous angiogenic properties of blood vessels can be summarized as follows: (i) Preexisting blood vessels have the capacity to generate new vessels in the absence of exogenous angiogenic stimuli; (ii) vascular autoregulation of angiogenesiscan be studied by culturing arterial or venous explants in vitro; (iii) angiogenesis in vascular organ culture is induced by the combined effect of injury and exposure of the explants to collagen; (iv) the angiogenic response of the vessel wall is mediated by autocrine, paracrine, and juxtacrine interactions among endothelial cells, smooth muscle cells/pericytes, and fibroblasts; (v) the cells of the vessel wall regulate angiogenesis by producing growth factors, proteolytic enzymes, components of the ECM, cell adhesion molecules, and vasoactive factors; and (vi) molecules involved in the regulation of angiogenesis are expressed by blood vessels during embryonal development, the female menstrual cycle, wound healing, hypoxic conditions, and pathologic processes. The cascade of cellular events occurring during the angiogenic response of the vessel wall to injury and the putative endogenous regulators of this process are schematically represented in Fig. 3. Observations made with models of vascular organ culture pose intriguing questions for future research. For example, can formation of blood vessels be inhibited by targeting a single angiogenic factor? Gene knockout experiments indicate that this is possible when blood vessels develop from the undifferentiated mesenchyme during vasculogenesis in the embryo (Ferrara et al., 1996). Vascular organ culture experiments, however, suggest that formation of neovessels from preexisting blood vessels is only partially inhibited with antibodies against individual growth factors (Villaschi and Nicosia, 1993; Brown et al., 1996; Nicosia et al., 1998). This observation, which may also apply to pathologic angiogenesis, raises the possibility that antiangiogenic factor therapy in the adult may not succeed until the combinations of growth factors regulating angiogenesis in different pathologic conditions have been characterized. Alternatively, drugs capable of blocking signal transduction pathways possibly shared by different angiogenic factors may overcome this limitation. What molecular events initiate angiogenesis? In vivo and in vitro experiments suggest that endothelial growth factors, such as bFGF and VEGF, can directly stimulate angiogenesiswithout the involvement of other molecules or cell types. During in vivo experiments, however, the local delivery

27

AUTOREGULATION OF ANGIOGENESIS INJURY

ACTIVATION OF ENDOTHELIAL CELLS, SMOOTH MUSCLE CELLSPERICYTESAND FIBROBLASTS

LOCALIZED PROTEOLYSIS OF TEEBASEMENTMEMBRANJ?

GROWTH FACTORS NITRIC OXIDE

INTERSTITIALCOLLAGEN

1

b ENDOTEELIALMIGRATION AND PROLIFERATION

CAPILLARY TUBE MORPHOGENESIS

I

CELL ADHESION MOLECULES

.c BALANCED DEGRADATION OF THEINTERSTITIALECM

CHEMOTACTICFACTORS (?) VASOACTIVE FACTORS

FORMATION OF ANASTOMOSES AND CAPILLARY LOOPS AND ESTABLISHMENTOF BLOOD FLOW

1

RECRUITMENTOF PERICYTFS SMOOTH MUSCLE CELLS

TGFbeta-1

I

+

b ENDOTEELIAL DIFFERENTIATION/ QUIESCENCE

1 T

STABILIZATION AND SURWAL OF NEOVESSELS

FIG. 3 Autoregulation of angiogenesis by the vessel wall: putative cascade of cellular and molecular events regulating formation of new vessels in response to injury.

28

R. F. NICOSIA AND S. VlUASCHl

of growth factors causes connective tissue injury which may activate fibroblasts or inflammatory cells. During in vitro experiments, fetal bovine serum may influence the response of isolated endothelial cells to bFGF and VEGF because it contains a variety of platelet-derived factors capable of activating the endothelium (Pepper et al., 1993a;Montesano et al., 1996). In the serumfree environment of the rat aorta model, bFGF functions as an angiogenic factor during the vasoproliferative response of the explants, but it behaves as a trophic factor after the angiogenic growth phase (Villaschi and Nicosia, 1997). Once they have stopped growing, neovessels survive but no longer proliferate in response to bFGF. This suggests that the angiogenic activity of bFGF manifests itself only when the aortic endothelium has been injured. The observation that bFGF and VEGF may be expressed by the vessel wall in the absence of angiogenesis (Cordon-Cardo et al., 1990; Couffinhal et al., 1997) further supports the idea that the mere presence of angiogenic factors is not sufficient to initiate angiogenesis. If this hypothesis is correct, what molecular events in addition to secretion of angiogenic factors are required for the induction of angiogenesis? Conditions that promote the angiogenic activity of growth factors may vary depending on the context in which angiogenesis takes place. In the rat aorta model, injury and exposure of the endothelium to collagen create a permissive environment for angiogenesis (Nicosia and Ottinetti, 1990; Nicosia et al., 1998). Similar conditions may occur in vivo during wound healing or the recanalization of thrombi when the apical surface of injury-activated endothelial cells is exposed to fibrin, collagen, and other ECM molecules (Sueishi et al., 1997). Injury of vascular cells may also release lysosomal enzymes, such the cathepsins B and L, which are capable of degrading the main components of the basement membrane (Guinec et al., 1993). In tumor-related angiogenesis, cancer cells may stimulate fibroblasts to produce collagen, which has been shown to indirectly activate MMP-2 by upregulating fibroblast MT-MMP1 (Gilles et al., 1997). Fibroblast-derived MMP-2 may in turn digest the microvascular basement membrane and bind to the a& receptor of endothelial cells, thereby promoting endothelial sprouting (Brooks et al., 1996). In inflammation-related angiogenesis, TNF-a produced by macrophages (Leibovich et al., 1987) may contribute to the induction of angiogenesis by activating endothelial proteolytic enzymes (Ray and Stetler-Stevenson, 1994). In fact, human capillary endothelial cells are unable to penetrate fibrin and form capillary tubes in response to bFGF or VEGF unless TNFa is added to the culture medium (van Hinsbergh et al., 1997). What mechanisms regulate the formation of vascular anastomoses? In the rat aorta model, the neovessels grow toward each other forming loops as seen during angiogenesis in vivo. The tendency of neovessels to form anastomotic connections becomes apparent when two or more vascular


29

explants are embedded in the same collagen gel (Figs. 4 and 5). A possible explanation is that the endothelial tips of the sprouting neovessels secrete endothelial-specificchemoattractants. In addition, endothelial cells, by contracting collagen fibrils, create traction forces which may guide opposing sprouts toward each other (Vernon et al., 1995). How do endothelial cells recruit smooth muscle cells/pericytes? The rat aorta model and the endothelial-smooth muscle cells coculture experiments strongly indicate that these cells interact by paracrine mechanisms. Gene knockout experiments suggest that Ang-1 produced by the perivascular mesenchyme signals the endothelium to secrete pericyte-chemotactic factors (Sun et al., 1996). Does Ang-1 promote the expression of PDGF B, HB-EGF, TF, ET-1 (D’Amore and Smith, 1993; Lindner, 1995a; Luscher and Wenzel, 1995;Moses et al., 1995; Contrino et al., 1996), or other factors capable of attracting pericytes? Does TGF-P1, which has been implicated in the differentiation of microvessels (D’Amore and Smith, 1993),modulate the production of Ang-l? Is TGF-Pl the signal that turns angiogenesis off? After they have established contact with the microvessels, pericytes form junctions with the endothelial cells (Nicosia and Villaschi, 1995). What cell adhesion molecules regulate the interaction between endothelial cells and pericytes?

FIG. 4 Serum-free collagen gel culture of rat aortic (A) and renal venous (V) explants. The microvessels of adjacent outgrowths have migrated toward each other forming anastomotic connections (arrows). Scale bar = 450 p M .

30


FIG. 5 Anastomotic connections (arrows) between microvascular outgrowths of adjacent rat aortic explants in serum-free collagen gel culture. Scale bar = 300 p M .

What are the mechanisms responsible for the regression of neovessels? In the rat aorta model of angiogenesis,the neovasculature regresses through a process that resembles vascular regression during angiogenesis in vivo. Endothelial cells retract, becoming reabsorbed into the main stems of the neovessels which become thicker and less branched. Changes in cell-matrix interactions may be involved in this process. Do endothelial cells downregulate critical integrin receptors such as a,& or a,&? What is the role of proteolytic enzymes during vascular regression? Does the lack of blood flow affect the survival of the neovessels through the action (or lack of action) of vasoactive factors? What factors mediate the contraction of the endothelium responsible for the retraction of the neovessels? Finally, do blood vessels vary in their capacity to autoregulate angiogenesis? The observation that rat veins and human chorionic vessels are capable of generating new vessels indicates that autoregulation of angiogenesis is not restricted to the rat aorta. As more experiments with vascular organ models are carried out, we will learn whether there is heterogeneity of angiogenic regulation among blood vessels isolated from different vascular beds and animal species. These studies may ultimately lead to the development of novel therapeutic strategies for the pharmacologic stimulation or inhibition of the angiogenic process.


31

Acknowledgments This work was supported by NIH Grant HL52585.

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Fibroblast Growth Factors as Multifunctional Signaling Factors Gyorgyi Szebenyi and John F. Fallon Anatomy Department, University of Wisconsin, Madison, Wisconsin 53706

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The fibroblast growth factor (FGF) family consists of at least 15 structurally related polypeptide growth factors. Their expression is controlled at the levels of transcription, mRNA stability, and translation. The bioavailability of FGFs is further modulated by posttranslational processing and regulated protein trafficking. FGFs bind to receptor tyrosine kinases (FGFRs), heparan sulfate proteoglycans (HSPG), and a cysteine-rich FGF receptor (CFR). FGFRs are required for most biological activities of FGFs. HSPGs alter FGF-FGFR interactions and CFR participates in FGF intracellular transport. FGF signaling pathways are intricate and are intertwined with insulin-like growth factor, transforming growth factor+, bone morphogenetic protein, and vertebrate homologs of Drosopbih wingless activated pathways. FGFs are major regulators of embryonic development: They influence the formation of the primary body axis, neural axis, limbs, and other structures. The activities of FGFs depend on their coordination of fundamental cellular functions, such as survival, replication, differentiation, adhesion, and motility, through effects on gene expression and the cytoskeleton. KEY WORDS: FGF, FGF receptor, Gene expression, Signaling, Patterning, Development, Cytoskeleton.

1. Introduction Fibroblast growth factors (FGFs) were first isolated in the 1970s from bovine brain extracts based on their mitogenic and angiogenic activities. Subsequent research established that FGFs form a family of structurally related polypeptide growth factors, have diverse activities, and are produced at some point during the development of each of the four histological tissue types (epithelia, muscle, connective, and nervous tissues). Even though Internntional Review of Cyrology, Vol. 185 0074-76%/99 $25.00

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Copyright 0 1999 by Academic Press. All rights of reproduction in any form reserved.

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GYORGYI SZEBENYI AND JOHN F. FALLON

FGFs and their target cells are widely distributed throughout the developing and adult body, each FGF and FGF receptor shows a restricted, albeit overlapping, spatial and temporal expression pattern. The regulation of synthesis and activity of FGFs and their receptors is complex and occurs at all levels of processing, including activation of transcription, posttranscriptional modifications (splicing, polyadenylation, and mRNA stability), translation initiation, posttranslational modifications (glycosylation, phosphorylation, and ribosylation), intracellular trafficking, secretion, bioavailability, and ligand-receptor interactions (see Sections I1 and III,A,3). In addition, postreceptor FGF signaling pathways are intricate: Several biochemical cascades interact and are integrated into a unique response to the multiple, and sometimes antagonistic, signals constantly bombarding cells (see Section 111,D). The effects of FGFs on cellular functions depend on the biochemical state and environment of their target cells. In addition to their initially observed effects on cell replication and angiogenesis, FGFs regulate cell survival and apoptosis, adhesion, motility, and differentiation (see Section IV,B). FGF modulation of complex biological events, such as tumor formation, the remodeling of blood vessels, and effects on morphogenesis, is likely the result of FGF regulation of several cellular functions. Progress in understanding the roles of FGFs in embryonic development has greatly accelerated since clones of fgfs, large amounts of pure recombinant proteins, and novel methods to test these reagents in vivo have become widely available. There is now a massive amount of data that demonstrate FGFs’ involvement in gastrulation, neurulation, the anteroposterior specification of body segments, and organ morphogenesis in both invertebrates and vertebrates (see Section IV,A). This review is organized as follows: first, we discuss how FGFs are synthesized and released, with an emphasis on the formidable variety in their structure; next, we give a description of FGF-binding proteins and the signaling pathways that mediate the actions of FGFs; then, we discuss the biological activities of FGFs in embryonic development and relate these activities to their effects on fundamental cellular activities, such as cell survival,apoptosis, replication rates, cell-matrix adhesion, cell-cell interactions, cell motility, and differentiation. The aim is to summarize the major findings in FGF biology, to indicate the complexity of FGF signaling pathways and ways they integrate with other cellular activities, and to point out areas where research is lacking. We attempt to explore most of the major issues in FGF biology but can cite only a limited number of specific experiments to illustrate each point. Medline currently contains about 3000 references on the subject, over 75% of which were published in the 1990s. It is impossible and, given the accessibility of electronic databases, unnecessary to provide an exhaustive

FGFs AS MULTIFUNCTIONAL SIGNALING FACTORS

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bibliography. We emphasize primary research results reported in 1995-1997 and only cite earlier work that has not been reviewed by others. The list of reviews in the Appendix will help to locate additional primary resources in special fields of interest and provides a variety of perspectives. We hope that our survey of the FGF field will provide a framework to evaluate the mass of information available on this topic.

II. Structure, Regulation of Synthesis, Subcellular Localization, and Release of FGFs FGFs belong to a family of structurally related polypeptide growth factors, designated by the acronym FGF and a number (FGF-n). See Table I for a list of other names for FGFs still encountered in the literature. FGFs also belong to the larger heparin binding growth factor family (HBGF). The fgfihave been cloned from many species, including mammals, birds, fish, amphibians, fruit flies, and worms. The most conserved sequence within the predicted protein sequence of FGFs is within a core of 120 amino acids (Fig. 1). Within this region, fgf orthologs (divergence resulting from speciation) are 71-100% identical in aa composition, whereas the protein sequences of fgf paralogs (divergence due to gene duplication resulting in several isotypes within a single species) are 22-66% identical. Phylogenetic analyses indicate that there were at least two phases of gene duplications that gave rise to the present diversity in fgfs: The first series of duplications resulted in separate genes forfgf-3, -5, -7, -8, and -9 and three ancestor genes for fgf-l/fgf-2, f g f 4 f g f - 6 , and fgf-ll/fgf--12; the second series of duplications gave rise to separate fgf-1, -2,-4, -6, -11,and -12 genes. The first phase of duplications probably occurred at the time of emergence of the vertebrates and the second phase at the time of fin-to-limb transition (Coulier et al., 1997), consistent with the observations that FGFs contribute to the patterning of the body and the development of the limbs (see Section IV,A). It is expected that more homologous sequences will be found as the interest in FGFs grows and the molecular databases become larger. A. Structure and Regulation of fgfGenes

Most fgf genes have a similar exonhtron organization (Table I). There are three coding exons in fgf-1 to -6 and in fgf-1.5 and in the invertebrate fgf genes, egl-17and bnl (Burdine et al., 1997; Sutherland et al., 1996). Each of the exons encode parallel p strands which fold into a distinct structural domain, called the p trefoil, that has the geometry of a trigonal pyramid

TABLE I Nomenclature of FGF and Some Features of FGF Genes

Name

Alternative names

Chromosomal location of genes (humany

Number of coding exons

FGF-1

Acidic FGF (aFGF)

5q31.3-33.2

3

FGF-2 FGF-3 FGF-4 FGF-5 FGF-6 FGF-7 FGF-8 FGF-9 FGF-10 FGF-11 FGF-12 FGF-13 FGF-14 FGF-15 EGL-17 BNL

Basic FGF (bFGF) INT-2 HST-1, k-FGF (Kaposi)

4q26-27 llq13 llq13.3 4q21 12~13 15 10q24 13q12-ql3 5~12-13 17~12 3q28 xq21 13

3 3 3 3 3

HST-2 KGF (keratinocyte GF) AIGF (androgen induced) GGF (glial) FHF-3 FHF-1 FHF-2 FHF-4

Branchless

Based on Emoto et al. (1997). Based on searches of Genbank through September 1997.

6 (1A-D,2,3)

5 3 3 3

Genes cloned fromb Human, hamster, bovine, rat, pig, chick, mouse Human, opossum, bovine, rat, chick, mouse, sheep, Xenopus, newt Human, chick, fish, mouse, Xenopus Human, chick, bovine, mouse, Xenopus Human, mouse, rat Human, mouse Human, mouse, rat, sheep, dog Human, mouse, chick, Xenopus Human, rat, mouse, Xenopus Human, rat, chick, mouse Human, mouse Human, mouse, chick Human, mouse, chick Mouse

Mouse C. elegans Drosophila

FGFs AS MULTIFUNCTIONAL SIGNALING FACTORS

49

cuc GIR repeats nuclear localization signals

AUG sigm sequencl

lation site C

0

a, a,

1PKC phosphorylatioin

site

elycosylation site ADP-ribosylation site

C

CUG: AUG: signal sequence: FGFR 8 HEPARIN

binding regions: glycosylation sites: ADP-ribosylation site:

osphorylation site $::$:$$:

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