Ferrara_Angiogenesis-From Basic Science to Clinical Applications.pdf

AngiogenesisFromBasic Science toClinical Applications

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Edited by

Napoleone Ferrara

AngiogenesisFromBasic Science toClinical Applications

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Library of Congress CataloginginPublication Data

Angiogenesis : from basic science to clinical applications / editor, Napoleone Ferrara.

p. ; cm.Includes bibliographical references and index.ISBN13: 9780849328442 (hardcover : alk. paper)ISBN10: 0849328446 (hardcover : alk. paper)1. BloodvesselsGrowth. I. Ferrara, Napoleone.[DNLM: 1. Vascular Endothelial Growth Factors. 2. Angiogenesis Modulating

Agentstherapeutic use. 3. Clinical Trials. 4. Neoplasmspathology. 5. Neovascularization, Pathologic. 6. Neovascularization, Physiologic. QU 107 A5873 2006]

QP102.A54 2006612.13dc22 2006018322

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and the CRC Press Web site athttp://www.crcpress.com

2004 by CRC Press LLC

Contents

Chapter one Vascular endothelial growth factor: pathophysiology and clinical implications .......................................................... 1Napoleone Ferrara

Chapter two Signal transduction of VEGF receptors towardangiogenesis............................................................................. 37Masabumi Shibuya

Chapter three Growth factors and lymphangiogenesis ............................. 53Paula I. Haiko, Marika J. Karkkainen, Marc G. Achen, Steven A. Stacker, and Kari Alitalo

Chapter four Notch and vascular development ........................................ 75Carrie J. Shawber and Jan Kitajewski

Chapter five Neural guidance molecules in vascular development ..... 89Li Yuan, Pierre Corvol, and Anne Eichmann

Chapter six Neuropilins, receptors for the VEGF and semaphorin families, link angiogenesis and axon guidance ....................................................................... 111Peter Kurschat, Diane Bielenberg, and Michael Klagsbrun

Chapter seven Non-angiogenic functions of VEGF................................... 133Jody J. Haigh, Carmen Ruiz de Almodovar, Martin Schneider, and Peter Carmeliet

Chapter eight Contribution of pro-angiogenic hematopoietic cellsto vascularization of tumor and ischemic tissue.............. 163Shahin Rafii, David Lyden, David Jin, and Andrea Hooper

Chapter nine Antiangiogenic drugs as broadly effectivechemosensitizing agents ...................................................... 181Robert S. Kerbel, Francesco Bertolini, Shan Man, Daniel J. Hicklin, Urban Emmenegger, and Yuval Shaked

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Chapter ten Using an anti-VEGF monoclonal antibody totreat cancer............................................................................ 199Zev A. Wainberg and Fairooz Kabbinavar

Chapter eleven Ocular neovascularization ................................................ 215Peter A. Campochiaro

Chapter twelve Targeting VEGF for neovascular age-related macular degeneration and macular edema ................... 229Philip J. Rosenfeld and Anne E. Fung

Chapter thirteen Therapeutic angiogenesis for cardiovascular disease ....................................................... 253Rohit Khurana and Michael Simons

Index ..................................................................................................................... 269

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Preface

Angiogenesis has long been known to be fundamental for such diversephysiological processes as embryonic and postnatal development, reproduc-tive functions, and wound repair. The blood vessels provide oxygen andnutrients and carry key regulatory signals to the growing tissues. Earlypioneers, including Glenn Algire and Isaac Michaelson, observed severaldecades ago that tumorigenesis and certain eye disorders resulting inimpaired vision are accompanied by increased vascular proliferation, andproposed that the new blood vessels play an important pathogenic role inthese conditions. However, the vast potential therapeutic importance of thefield was not appreciated until 1971, when Judah Folkman proposed thatanti-angiogenesis might represent a therapy for solid tumors. Since then,much effort has gone in the elucidation of the mechanisms of angiogenesis.Indeed, the last two decades have seen an explosive progress in our under-standing of the molecular mechanisms underlying growth and differentia-tion of blood vessels. Key angiogenic inducers and several anti-angiogenicmolecules have been identified and characterized. This broad interest in thefield translates in to almost 27,000 Medline citations under the keywordangiogenesis as of April 2006.

So why a new book on angiogenesis? In spite of the many efforts andthe remarkable progress in basic biology, unequivocal evidence that target-ing angiogenesis results in a clinical benefit in humans is a very recentaccomplishment. After several setbacks that led numerous skeptics to doubtthat such an approach would ever be successful, over the last 2 years thefirst antiangiogenic agents have been approved by the FDA for the treatmentof cancer and age-related macular degeneration. Blocking angiogenesisresulted in a clear benefit and survival advantage in patients affected byadvanced malignancies. Impressive results were obtained in age-relatedmacular degeneration. For the first time, a significant number of patientsmay experience not only a slowing down of vision loss but they may in factenjoy a meaningful and sustained vision gain. Only a few years ago, this wouldhave been only a remote possibility. However, this clinical success has raiseda new set of basic and clinical questions that will need to be addressed beforethe field may advance in a significant manner. It will be important to eluci-date the pathways that mediate resistance to antiangiogenic treatments, aug-ment the efficacy of existing treatments by selecting appropriate combinatorial

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therapies, and determine how to select the patients who are most likely torespond to the treatment.

While this is not a comprehensive overview of the field, the reader willfind in the present volume some of the latest advances in basic science andin the clinical studies with inhibitors of angiogenesis, contributed by severalexperts in their respective fields. The book should be of broad interest tobasic scientists interested in vascular biology and to clinicians engaged inoncology, ophthalmology, and cardiovascular medicine.

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Editor

Napoleone Ferrara, M.D. was born in Catania, Italy, on July 26, 1956. Heearned his M.D. from the University of Catania Medical School in 1981. Hejoined Genentech in 1988 after postdoctoral training at the University ofCalifornia at San Francisco. At present, he is a Genentech Fellow.

His research interests concern the regulation of angiogenesis. In 1989,his laboratory isolated vascular endothelial growth factor (VEGF) and sub-sequently investigated its basic biology and potential clinical applications.In 1993, Dr. Ferrara and colleagues demonstrated that inhibition of VEGFusing a monoclonal antibody suppressed tumor growth in vivo. This workled to the clinical development of bevacizumab (Avastin), a humanized anti-VEGF monoclonal antibody, the first anti-angiogenic agent to be approvedby the U.S. Food & Drug Administration. Dr. Ferraras work on intraocularneovascularization led to the clinical development of ranibizumab (Lucentis)for the treatment of wet age-related macular degeneration.

Dr. Ferrara is author or co-author of over 220 publications and is therecipient of several scientific awards.

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Contributors

Marc G. AchenLudwig Institute for Cancer

ResearchRoyal Melbourne HospitalVictoria, Australia

Kari AlitaloMolecular/Cancer Biology LaboratoryHaartman Institute and

Biomedicum HelsinkiUniversity of HelsinkiHelsinki, Finland

Francesco Bertolini European Institute of OncologyMilan, Italy

Diane BielenbergDepartment of SurgeryChildrens HospitalHarvard Medical SchoolBoston, Massachusetts

Peter A. CampochiaroDepartments of Ophthalmology

and NeuroscienceThe Johns Hopkins University

School of MedicineBaltimore, Maryland

Peter CarmelietCenter for Transgene Technology

and Gene TherapyFlanders Interuniversity Institute

for BiotechnologyLeuven, Belgium

Pierre Corvol Collge de FranceParis, France

Anne Eichmann Collge de FranceParis, France

Urban Emmenegger Sunnybrook & Womens College

Health Sciences CentreToronto, Canada

Napoleone FerraraGenentech, Inc.South San Francisco, California

Anne E. FungBascom Palmer Eye InstituteUniversity of Miami School of

MedicineMiami, Florida

Jody J. HaighDepartment for Molecular

Biomedical ResearchFlanders Interuniversity Institute

for BiotechnologyUniversity of GhentGhent, Belgium

Paula I. Haiko Molecular/Cancer Biology LaboratoryHaartman Institute and


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Daniel J. HicklinImClone Systems, Inc.New York, New York

Andrea Hooper Department of Genetic Medicine

and PediatricsWeill Cornell Medical CollegeNew York, New York

David Jin Department of Genetic Medicine


Fairooz KabbinavarUCLA Medical CenterLos Angeles, California

Marika J. Karkkainen Molecular/Cancer Biology

LaboratoryHaartman Institute and


Robert S. KerbelSunnybrook & Womens College

Health Sciences Centre andDepartment of Medical

BiophysicsUniversity of TorontoToronto, Canada

Rohit Khurana Department of MedicineUniversity College LondonLondon, United Kingdom

Jan KitajewskiDepartments of Obstetrics and

Gynecology and PathologyColumbia University Medical

CenterNew York, New York

Michael KlagsbrunDepartments of Surgery and

PathologyChildrens HospitalHarvard Medical SchoolBoston, Massachusetts

Peter Kurschat Department of SurgeryChildrens HospitalHarvard Medical SchoolBoston, Massachusetts

David LydenDepartment of Genetic Medicine


Shan ManSunnybrook & Womens College


Shahin RafiiDepartment of Genetic Medicine


Philip J. Rosenfeld Bascom Palmer Eye InstituteUniversity of Miami School of MedicineMiami, Florida

Carmen Ruiz de AlmodovarCenter for Transgene Technology



Martin SchneiderCenter for Transgene Technology



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Yuval ShakedSunnybrook & Womens College


Carrie J. ShawberDepartment of Obstetrics and

GynecologyColumbia University Medical

CenterNew York, New York

Masabumi Shibuya Institute of Medical ScienceUniversity of TokyoTokyo, Japan

Michael SimonsSection of CardiologyDartmouth Medical SchoolLebanon, New Hampshire

Steven A. StackerLudwig Institute for Cancer ResearchRoyal Melbourne HospitalVictoria, Australia

Zev A. WainbergUCLA Medical CenterLos Angeles, California

Li YuanSchool of Life SciencesXiamen UniversityXiamen, China

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1

chapter one

Vascular endothelial growth factor: pathophysiology and clinical implications

Napoleone Ferrara

Contents

Introduction .................................................................................................... 2Identification of VEGF .................................................................................. 4Biological activities of VEGF-A ................................................................... 4VEGF isoforms ............................................................................................... 5Regulation of VEGF gene expression......................................................... 6

Oxygen tension.................................................................................. 6Growth factors, hormones, and oncogenes .................................. 7

VEGF receptors .............................................................................................. 7VEGFR-1 (Flt-1) ................................................................................. 8VEGFR-2 (KDR, human; Flk-1, mouse)......................................... 9Neuropilin (NRP)1 and NRP2 ...................................................... 10

Role of VEGF in physiological angiogenesis .......................................... 11Role of VEGF in pathologic conditions ................................................... 12

Tumor angiogenesis........................................................................ 12Preclinical studies............................................................. 12Clinical trials of VEGF inhibitors in cancer patients.. 14

Intraocular neovascular syndromes............................................. 15Perspectives................................................................................................... 16References...................................................................................................... 17

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2 Angiogenesis

IntroductionThe observation that tumor growth can be accompanied by increased vascu-larity was reported more than one century ago (for review, see Reference 1).In 1939, Ide et al. postulated the existence of a tumor-derived blood vesselgrowth stimulating factor on the basis of the observation that tumors trans-planted in transparent chambers induced intense neovascular responses inthe host.2 These authors proposed that such a factor might promote thedevelopment of a neovascular supply needed for delivery of nutrients to thegrowing tumor.2 In 1945, Algire et al. advanced this concept, hypothesizingthat the rapid growth of tumor transplants is crucially dependent on thedevelopment of a neovascular supply.3 In 1971, Folkman proposed thatanti-angiogenesis may be a valid strategy for treating human cancer, and asearch for regulators of angiogenesis that may represent therapeutic targetsbegan.4

Neovascularization is essential also for physiological processes such asembryogenesis, tissue repair, and reproductive functions.5 The developmentof the vascular tree initially occurs by vasculogenesis, the in situ differenti-ation of endothelial cell precursors, the angioblasts, from the hemangio-blasts.6 The juvenile vascular system then evolves from the primary capillaryplexus by subsequent pruning and reorganization of endothelial cells in aprocess called angiogenesis.7 Recent studies suggest that the incorporationof bone marrow-derived endothelial progenitor cells (EPCs) in the growingvessels complements the sprouting of resident endothelial cells.812 Chapter8 by Rafii and colleagues discusses the role of EPCs in neovascularization.Additionally, a subset of perivascular monocytes seems to be particularlyimportant for new vessel growth.13

Many potential angiogenic factors have been described over the pasttwo decades.14,15 Much evidence indicates that vascular endothelial growthfactor (VEGF) is a particularly important regulator of angiogenesis.1 Whilenew vessel growth and maturation are highly complex and coordinatedprocesses requiring the sequential activation of a series of receptors (e.g.,Tie1, Tie2, and PDGFR-) by numerous ligands in endothelial and muralcells,16,17 VEGF signaling often represents a rate-limiting step in angiogenesis.VEGF (referred to also as VEGF-A) belongs to a gene family that includesplacenta growth factor (PlGF),18,19 VEGF-B,20 VEGF-C,21,22 and VEGF-D.23,24

Homologues of VEGF have been identified in the genome of theparapoxvirus Orf virus25 and shown to have VEGF-like activities.26,27 VEGF-Cand VEGF-D regulate lymphangiogenesis28,29 (see Chapter 3, this volume,covering the unique role of this gene family in controlling growth anddifferentiation of multiple anatomic components of the vascular system).Recent data have shown that inhibiting VEGF-A results in clinical benefitsincluding increased survival in patients with advanced malignancies, pro-viding the first clinical validation of the hypothesis that blocking angio-genesis is a strategy for treating cancer.30


Chapter one: Vascular endothelial growth factor 3

Figure 1.1 (See color insert following page 82.) Tumor cells produce VEGF-A andseveral other angiogenic factors such as PlGF, VEGF-C, FGF, angiopoietins, inter-leukin-8, etc. An additional source of angiogenic factors is represented by the stroma,a heterogeneous compartment consisting of fibroblastic, inflammatory, and immunecells. Recent studies indicate that bone marrow-derived cells, either authentic EPC orpro-angiogenic monocytes, participate in tumor angiogenesis. VEGF-A, PlGF, andseveral chemokines (e.g., SDF-1) may recruit BMCs. Tumor cells may also releasestromal cell recruitment factors such as PDGF-A, PDGF-C, and TGF-. Awell-established function of tumor-associated fibroblasts is the production of growthand survival factors for tumor cells such as EGFR ligands, hepatocyte growth factor,heregulin, etc. Endothelial cells produce PDGF-B, which promotes recruitment ofpericytes in the microvasculature following activation of PDGFR-.


4 Angiogenesis

Identification of VEGFIndependent lines of research contributed to the discovery of VEGF andemphasized the biological complexity of this molecule.1 In 1983, Senger etal. described the partial purification from a conditioned medium of a guineapig tumor cell line of a protein able to induce vascular leakage in the skin.The protein was designated tumor vascular permeability factor (VPF).31 Theauthors proposed that VPF could be a mediator of the high permeability oftumor blood vessels. However, these efforts did not result in the isolationand amino acid sequencing of the VPF protein. Therefore, VPF remainedmolecularly undefined for several years.

In 1989, we reported the isolation of a diffusible endothelial mitogenfrom medium conditioned by bovine pituitary follicular cells. It was namedvascular endothelial growth factor (VEGF) to reflect its restricted target cellspecificity.32 NH2-terminal amino acid sequencing of purified VEGF provedthat this protein was distinct from the known endothelial cell mitogens andindeed did not match any known proteins mentioned in available data-bases.32 Connolly et al., following up on the work of Senger, independentlyreported the isolation and sequencing of VPF.33 cDNA cloning of VEGF34and VPF35 revealed that VEGF and VPF were the same molecule. This wassurprising, considering that other endothelial cell mitogens such as FGF donot increase vascular permeability.

Biological activities of VEGF-AVEGF-A promotes the growth of vascular endothelial cells derived fromarteries, veins, and lymphatics (for review, see Reference 36). VEGF inducesangiogenesis in tridimensional in vitro models, inducing confluent microvas-cular endothelial cells to invade collagen gels and form capillary-like struc-tures.37,38 VEGF-A also induces angiogenesis in a variety of in vivo modelsystems.39

VEGF-A is also a survival factor for endothelial cells.4044 In vitro, VEGFprevents endothelial apoptosis induced by serum starvation. Such activityis mediated by the PI-3 kinase/Akt pathway.42,45 Also, VEGF induces expres-sion of the Bcl-2, A1,41 XIAP,46 and survivin47 anti-apoptotic proteins inendothelial cells in vivo. The pro-survival effects of VEGF are developmen-tally regulated. VEGF inhibition results in extensive apoptotic changes inthe vasculatures of neonatal (but not adult) mice.48 Furthermore, VEGFdependence has been demonstrated in endothelial cells of newly formed butnot of established vessels within tumors.43,44 Coverage by pericytes is one ofthe key events resulting in loss of VEGF-A dependence.43

Endothelial cells are the primary targets of VEGF-A, but several studieshave reported mitogenic and non-mitogenic effects of VEGF-A on certainnon-endothelial cell types including retinal pigment epithelial cells,49pancreatic duct cells,50 and Schwann cells.51 Chapter 7 by Haigh and col-leagues discusses the non-mitogenic functions of VEGF.



The earliest evidence that VEGF-A can affect myeloid cells came from areport describing its ability to promote monocyte chemotaxis.52 Subse-quently, VEGF-A was reported to have hematopoietic effects.53 VEGF deliv-ery to adult mice inhibits dendritic cell development.54,55 Also, VEGFincreased production of B cells and the generation of immature myeloidcells.56 Recently, conditional gene knock-out technology has been employedto achieve selective VEGF gene ablation in bone marrow cell isolates andhematopoietic stem cells (HSCs).57 VEGF-deficient HSCs and bone marrowmononuclear cells failed to repopulate lethally irradiated hosts despiteco-administration of a large excess of wild-type cells.57

As previously noted, VEGF is known also as VPF, based on its abilityto induce vascular leakage.31,58 Such permeability-enhancing activity under-lies important roles of this molecule in inflammation and various patholog-ical circumstances including intraocular neovascular syndromes (reviewedby Dvorak et al.59,60).

Several studies have pointed to the role of nitric oxide (NO) inVEGF-induced vascular permeability and angiogenesis.61,62 Fukumura et al.assessed the relative contributions of the NO synthase (NOS) isoforms,inducible (i)NOS, and endothelial (e)NOS to these processes.63 Angiogenesis,vessel diameter, blood flow rate, and vascular permeability were propor-tional to NO levels and were most impaired in eNOS/ mice. VEGF signif-icantly increased permeability in both wild-type and iNOS/ mice, but notin eNOS/ mice. VEGF-induced angiogenesis was markedly reduced ineNOS/ mice, although the mice developed normally and showed no appar-ent defect in the vasculature.

VEGF isoformsThe human VEGF-A gene is organized into eight exons separated by sevenintrons64,65 and is localized in chromosome 6p21.3.66 Alternative exon splicingresults in the generation of four different isoforms, having 121, 165, 189, and206 amino acids, respectively, following signal sequence cleavage (VEGF121,VEGF165, VEGF189, VEGF206).64,65 VEGF165, the predominant isoform, lacks theresidues encoded by exon 6, while VEGF121 lacks the residues encoded byexons 6 and 7. Less frequent splice variants have been also reported, includ-ing VEGF145,67 VEGF183,68 VEGF162,69 and VEGF165b, a variant reported to havean inhibitory effect on VEGF-induced mitogenesis.70

Native VEGF is a heparin-binding homodimeric glycoprotein of 45 kDa.32Its properties closely correspond to those of VEGF165, which is now recognizedas the major VEGF isoform.71 VEGF121 is an acidic polypeptide that fails tobind to heparin.71 VEGF189 and VEGF206 are highly basic and bind to heparinwith high affinity.71 VEGF121 is a freely diffusible protein. In contrast, VEGF189and VEGF206 are almost completely sequestered in the extracellular matrix(ECM). VEGF165 has intermediate properties because it is secreted, but asignificant fraction remains bound to the cell surface and ECM.72


6 Angiogenesis

The ECM-bound isoforms may be released in a diffusible form by hep-arin or heparinase, which displaces them from their binding to heparin-likemoieties, or by plasmin cleavage at the COOH terminus that generates abioactive fragment consisting of the first 110 NH2-terminal amino acids.71Based on the importance of plasminogen activation during physiologicaland pathological angiogenesis processes,73 this proteolytic mechanism canbe particularly important in regulating locally the activity and bioavailabilityof VEGF. More recent studies have shown that matrix metalloproteinase(MMP)-3 can also cleave VEGF165 to generate diffusible, non-heparin-binding,bioactive proteolytic fragments.74 Plouet et al. proposed a role for urokinasein the generation of bioactive VEGF.75

Loss of the heparin-binding domain results in a reduction in the mito-genic activity of VEGF.76 These findings suggest that VEGF165 has optimalcharacteristics of bioavailability and biological potency. The importance ofthe heparin-binding VEGF isoforms is also emphasized by the finding that50% of the mice expressing exclusively VEGF120 (VEGF120/120) died shortlyafter delivery, while the rest died within 2 weeks.77 Recent studies have alsodemonstrated a deficit in the distribution of endothelial cells and impairedfilopodia extension in VEGF120/120 mice, suggesting that the heparin-bindingVEGF isoforms provide essential stimulatory cues to initiate vascular branchformation.78

Regulation of VEGF gene expressionOxygen tension

Oxygen tension plays a key role in regulating the expression of a variety ofgenes.79 VEGF mRNA expression is induced by exposure to low pO2 in avariety of pathophysiological circumstances.80,81 A 28-base sequence has beenidentified in the 5 promoter of the rat and human VEGF gene that mediateshypoxia-induced transcription.82,83 Such sequence reveals a high degree ofhomology and similar protein binding characteristics as the hypoxia-induc-ible factor 1 (HIF-1) binding site in the erythropoietin gene.84

HIF-1 is a basic, heterodimeric, helixloophelix protein consisting of twosubunits, HIF-1 and aryl hydrocarbon receptor nuclear translocator (ARNT),known also as HIF-1.85 In response to hypoxia, HIF-1 binds to specificenhancer elements, resulting in increased gene transcription. A gene highlyhomologous to HIF-1, HIF-2, also forms heterodimers with ARNT and reg-ulates VEGF expression.86 Recent studies have demonstrated the critical roleof the product of the von HippelLindau (VHL) tumor suppressor gene inHIF-1-dependent hypoxic responses (for review, see Mole et al.87). The VHLgene is inactivated in patients with von HippelLindau disease, an autosomaldominant neoplasia syndrome characterized by capillary hemangioblastomasin retina and cerebellum, and in most sporadic clear cell renal carcinomas.88

Earlier studies indicated that one function of the VHL protein is toprovide negative regulation of VEGF and other hypoxia-inducible genes.89



The VHL protein is known to interact with a series of proteins includingelongins B and C and CUL2, a member of the Cullin family.90 HIF-1 wasshown to be constitutively activated in VHL-deficient renal cell carcinomacell lines.91 Later studies demonstrated that indeed one function of VHL isto be part of a ubiquitin ligase complex that targets HIF subunits for pro-teasomal degradation following covalent attachment of a polyubiquitinchain.92,93 Oxygen promotes the hydroxylation of HIF at a proline residue, arequirement for the association with VHL.92,93 A family of prolyl hydroxy-lases related to the Egl-9 C. elegans gene product have been identified as HIFprolyl hydroxylases.79,94,95

Growth factors, hormones, and oncogenes

Several growth factors, including EGF, TGF-, TGF-, KGF, IGF-1, FGF, andPDGF up-regulate VEGF mRNA expression,9698 suggesting that paracrineor autocrine release of such factors cooperates with local hypoxia in regu-lating VEGF release in the microenvironment. Also, inflammatory cytokinessuch as IL-1- and IL-6 induce expression of VEGF in several cell typesincluding synovial fibroblasts.99,100

Hormones are also important regulators of VEGF gene expression. Thy-roid stimulating hormone has been shown to induce VEGF expression inseveral thyroid carcinoma cell lines.101 Shifren et al. have also shown thatACTH is able to induce VEGF expression in cultured human fetal adrenalcortical cells, suggesting that VEGF may be a local regulator of adrenalcortical angiogenesis and a mediator of the tropic action of ACTH.102Gonadotropins have been shown to be inducers of VEGF transcription inthe ovary, both in vivo103,104 and in vitro.105 Mueller et al. reported that estra-diol is a direct transcriptional activator of VEGF, mediated by a variantestrogen response element.106 Progestins have also been reported to induceVEGF gene transcription in endometrial carcinoma cells.107

A variety of transforming events also result in induction of VEGFgene expression. Oncogenic mutations or amplifications of ras lead toVEGF up-regulation.108,109 Mutations in the wnt-signaling pathway that arefrequently associated with pre-malignant colonic adenomas result in upreg-ulation of VEGF.110 Interestingly, VEGF is upregulated in polyps of Apcknockout [Apc(Delta716)] mice. Such mice are models for human familialadenomatous polyposis.111 In both benign and malignant mouse intestinaltumors, stromal expression of COX-2 results in elevated PGE2 levels that inturn stimulate cell surface receptor EP2, followed by induction of VEGF andangiogenesis.111113

VEGF receptorsVEGF binding sites were originally identified on the surfaces of vascularendothelial cells in vitro114,115 and in vivo.116,117 Subsequently, VEGF receptorswere shown to exist also in bone marrow-derived cells such as monocytes


8 Angiogenesis

and macrophages.118 VEGF binds two highly related receptor tyrosinekinases (RTK), VEGFR-1 and VEGFR-2. Both VEGFR-1 and VEGFR-2 haveseven immunoglobulin (Ig)-like domains in the extracellular domain, a singletransmembrane region, and a consensus tyrosine kinase sequence inter-rupted by a kinase-insert domain.119121 The signal transduction propertiesof VEGFR-1 and VEGFR-2 are discussed in detail in Chapter 2.

A member of the same family of RTKs is VEGFR-3 (Flt-4)122 which,however, is not a receptor for VEGF-A, but instead binds the lymphangio-genic factors VEGF-C and VEGF-D28 (see Chapter 3). In addition to theseRTKs, VEGF interacts with a family of co-receptors, the neuropilins, asdiscussed by Eichmann (Chapter 5) and Klagsbrun (Chapter 6).

VEGFR-1 (Flt-1)

Although Flt-1 (fms-like tyrosine kinase) was the first RTK to be identifiedas a VEGF receptor over a decade ago,123 the precise function of this moleculeis still subject to debate. Recent evidence indicates that the conflicting reportsmay be due, at least in part, to the fact that VEGFR-1 functions and signalingproperties can differ, depending on the developmental stage and cell type,e.g., endothelial versus non-endothelial cells. VEGFR-1 expression is up-regulated by hypoxia via an HIF-1-dependent mechanism.124 VEGFR-1 bindsVEGF-A and also PlGF125 and VEGF-B126 which fail to bind VEGFR-2. Analternatively spliced soluble form of VEGFR-1 (sFlt-1) has been shown to bean inhibitor of VEGF activity.127 The binding site for VEGF (and PlGF) hasbeen mapped primarily to the second Ig-like domain.128130 Flt-1 reveals aweak tyrosine autophosphorylation in response to VEGF.123,131

Park et al. initially proposed that VEGFR-1 may not be primarily areceptor transmitting a mitogenic signal, but may serve as a decoy recep-tor, able to regulate in a negative fashion the activity of VEGF on the vascularendothelium by sequestering and rendering this factor less available toVEGFR-2.125 Thus, the observed potentiation of the action of VEGF by PlGFcould be explained, at least in part, by displacement of VEGF from VEGFR-1binding.125 Recent studies have shown that indeed a synergism existsbetween VEGF and PlGF in vivo, especially during pathological situationsas evidenced by impaired tumorigenesis and vascular leakage in Plgf/mice.132 Gille et al. identified a repressor motif in the juxtamembrane regionof VEGFR-1 that impairs PI-3 kinase activation and endothelial cell migrationin response to VEGF.133

Regardless of the conflicting evidence about the role of VEGFR-1 as asignaling receptor, gene-targeting studies have demonstrated the essentialrole of this molecule during embryogenesis. VEGFR-1/ mice died in uterobetween day 8.5 and day 9.5.134,135 Endothelial cells developed but failed toorganize in vascular channels. Excessive proliferation of angioblasts has beenreported to be responsible for such disorganization and lethality,135 indicat-ing that, at least during early development, VEGFR-1 is a negative regulatorof VEGF action.



More compelling evidence in support of this view derives from thefinding that a VEGFR-1 lacking the tyrosine kinase (TK) domain but able tobind VEGF does not result in lethality or any overt defect in vascular devel-opment.136 Nevertheless, one specific biological response, the migration ofmonocytes in response to VEGF-A (or PlGF), has been shown to require theTK domain of VEGFR-1.136,137 Furthermore, Lewis lung carcinoma cells over-expressing PlGF grow in wild-type mice faster than in VEGFR-1 TK-deficientmice, suggesting that VEGFR-1 may be a positive regulator under patholog-ical conditions when a VEGFR-1-specific ligand is highly expressed.138 Thesefindings suggest that VEGFR-1 has a dual function in angiogenesis, actingin a positive or negative manner in different circumstances. Recently,VEGFR-1 signaling has been linked to the induction of MMP-9 in lungendothelial cells and to the facilitation of lung metastases.139

Recent studies have emphasized the effects of VEGFR-1 in hematopoiesisand recruitment of bone marrow-derived angiogenic cells (for more detail,see Chapter 8). VEGFR-1 activation by PlGF reconstitutes hematopoiesis byrecruiting VEGFR-1+ hematopoietic stem cells (HSCs).140 In addition,VEGFR-1 activation by enforced expression of PlGF rescues survival andability to repopulate in VEGF/ HSCs.57 LeCouter et al. recently providedevidence for a novel function of VEGFR-1 in liver sinusoidal endothelial cells(LSECs). VEGFR-1 activation achieved with a receptor-selective VEGFmutant or PlGF resulted in the paracrine release of HGF, IL-6, and otherhepatotrophic molecules by LSECs, to the extent that hepatocytes were stim-ulated to proliferate when co-cultured with LSECs.141

In some cases VEGFR-1 is expressed by tumor cells and may mediatea chemotactic signal, thus potentially extending the role of this receptor.142In this context, it is noteworthy that Bates et al. reported that the epithelialmesenchymal transformation of colonic organoids resulted in the increasedexpression of both VEGF and VEGFR-1, and that the survival of these cellsdepended on a VEGFVEGFR-1 autocrine pathway.143

VEGFR-2 (KDR, human; Flk-1, mouse)

VEGFR-2 binds VEGF-A with lower affinity than VEGFR-1 (Kd = 75 to 250pM versus 25 pM).144146 The key role of this receptor in developmentalangiogenesis and hematopoiesis is evidenced by lack of vasculogenesis andfailure to develop blood islands and organized blood vessels in Flk-1 nullmice, resulting in death in utero between day 8.5 and day 9.5.147 There is nowgeneral agreement that VEGFR-2 is the major mediator of the mitogenic,angiogenic, and permeability-enhancing effects of VEGF.

The binding site for VEGF-A has been mapped to the second and thirdIg-like domains.148 VEGFR-2 undergoes dimerization and strongligand-dependent tyrosine phosphorylation in intact cells and results in amitogenic, chemotactic, and pro-survival signal. Several tyrosine residueshave been shown to be phosphorylated (for review, see Matsumoto andClaesson-Welsh149).


10 Angiogenesis

Takahashi et al. have shown that Y1175 and Y1214 are the two majorVEGF-A-dependent autophosphorylation sites in VEGFR-2. However, onlyautophosphorylation of Y1175 is crucial for VEGF-dependent endothelialcell proliferation.150 VEGF enhanced cell adhesion, migration, solubleligand binding, and adenovirus gene transfer mediated by v3 and alsoactivated other integrins known to be involved in angiogenesis, v5, 51,and 21.151 VEGFR-2 activation induces endothelial cell growth by acti-vating the RafMekErk pathway. An unusual feature of VEGFR-2 activa-tion of this pathway is the requirement for protein kinase C but not ras.152,153VEGF mutants that bind selectively to VEGFR-2 are fully active endothelialcell mitogens, chemoattractants, and permeability-enhancing agents,whereas mutants specific for VEGFR-1 are devoid of all three activities.154Also, VEGF-E, a homologue of VEGF identified in the genome of theparapoxvirus Orf virus25 that shows VEGF-like mitogenic and permeabil-ity-enhancing effects, binds and activates VEGFR-2 but fails to bindVEGFR-1.26,27

Neuropilin (NRP)1 and NRP2

Certain tumor and endothelial cells express cell surface VEGF binding sitesdistinct from the two known VEGF RTKs.155 VEGF121 fails to bind these sites,indicating that exon 7-encoded basic sequences are required for binding tothis putative receptor.155 Soker et al.156 identified the isoform-specific VEGFreceptor as NRP1, a molecule previously shown to bind the collapsin/sema-phorin family and implicated in neuronal guidance (for review see Neufeldet al.157).

When co-expressed in cells with VEGFR-2, NRP1 enhanced the bindingof VEGF165 to VEGFR-2 and VEGF165-mediated chemotaxis.156 NRP1 appearsto present VEGF165 to the VEGFR-2 in a manner that enhances the effective-ness of VEGFR-2-mediated signal transduction.156 Binding to NRP1 may helpexplain the greater mitogenic potency of VEGF165 relative to VEGF121. Thereis no clear evidence that NRP1 or the related NRP2 signals following VEGFbinding.157 In contrast, in response to semaphorin binding, NRP1 and NRP2signal axon repulsion. The formation of complexes with plexins is a require-ment for NRP signaling in neurons.158,159

The role of NRP1 in the development of the vascular system has beendemonstrated by gene-targeting studies showing embryonic lethality in nullmice.160 Lee et al. have shown that NP1 is required for vascular developmentand mediates VEGF-dependent angiogenesis in zebrafish.161 Other studieshave linked NRP2 to lymphatic vessel development.162 More recent studiesshow that NRP1 and NRP2 are expressed on the cell surfaces of severaltumor cell lines that bind VEGF165 and display chemotactic responses to thisligand, suggesting pro-tumor activities of NRPs with or without the involve-ment of VEGF RTK signaling.163



Role of VEGF in physiological angiogenesisTwo studies demonstrated an essential role of VEGF-A in embryonic vascu-logenesis and angiogenesis in mice.164,165 Inactivation of a single VEGF alleleresulted in embryonic lethality between day 11 and day 12. The vegf +/embryos exhibited a number of developmental anomalies, defective vascu-larization in several organs, and reduced numbers of nucleated red bloodcells within the blood islands in the yolk sacs, indicating that VEGF regulatesboth vasculogenesis and early hematopoiesis. Interestingly, the converseappeared also to be true, as even modest increases in VEGF gene expressionachieved by the insertion of a LacZ cassette in the 3 UTR of the VEGF generesulted in severe abnormalities in heart development and embryonic lethal-ity at E12.5 through E14.166

These findings indicate a critical VEGF-A gene dosage dependence dur-ing development. In contrast, inactivation of PlGF132 or VEGF-B167 genesdid not result in any major development abnormalities. Among the othermembers of the VEGF gene family, only VEGF-C plays an essential role indevelopment, since its inactivation results in embryonic lethality followingdefective lymphatic development and fluid accumulation in tissues.168 It isnoteworthy that aploinsufficiency of the delta-like 4 Notch ligands alsoresults in embryonic lethality due to defective vascular development (seeChapter 4).

VEGF-A plays an important role in early postnatal life. Administrationof a soluble VEGFR-1 chimeric protein48 or anti-VEGF-A monoclonalantibodies169 results in growth arrest when the treatment is initiated at day1 or day 8 postnatally. Such treatment is also accompanied by lethality,primarily due to inhibition of glomerular development and kidney failure.48

The pivotal role of VEGF in kidney development was also demonstratedby a study showing that selective VEGF deletion in podocytes, using aNephin promoter-driven Cre recombinase, leads to glomerular disease in agene dosage-dependent fashion.170 However, VEGF neutralization in fullydeveloped normal mice48 and rats171 had no marked effects on glomerularfunction. In contrast, VEGF inhibition in adult rats with mesangioprolifera-tive nephritis led to a reduction of glomerular endothelial regeneration andan increase in endothelial cell death, indicating that VEGF may be importantfor glomerular endothelial cell repair following injury, but not for endothelialsurvival in healthy animals.171

Endochondral bone formation is a fundamental mechanism for longitu-dinal bone growth. Cartilage, an avascular tissue, is replaced by bone in aprocess named endochondral ossification.172 VEGF-A mRNA is expressedby hypertrophic chondrocytes in the epiphyseal growth plate, suggestingthat a VEGF gradient is needed for directional growth and cartilage invasionby metaphyseal blood vessels.173,174 Following VEGF blockade, bloodvessel invasion is almost completely suppressed, concomitant withimpaired trabecular bone formation, in developing mice and primates.173,175


12 Angiogenesis

Although proliferation, differentiation, and maturation of chondrocytes wereapparently normal, resorption of hypertrophic chondrocytes was inhibited,resulting in a marked expansion of the hypertrophic chondrocyte zone.Importantly, cessation of the anti-VEGF treatment is followed by capillaryinvasion, restoration of bone growth, and normalization of the growth platearchitecture.

Angiogenesis is a key aspect of normal cyclical ovarian function. Folli-cular growth and the development of the corpus luteum (CL) are dependenton the proliferation of new capillary vessels.176 The process of selection of adominant follicle in monovular species has been also associated with angio-genesis, as there is evidence that selected follicles possess more elaboratemicrovascular networks than other follicles.177 After blood vessel growth,the blood vessels regress, suggesting the coordinated action of inducers aswell as inhibitors of angiogenesis in the course of the ovarian cycle.178,179

Previous studies have shown that VEGF-A mRNA expression is tempo-rally and spatially related to the proliferation of blood vessels in the ova-ries.180,181 Administration of VEGF inhibitors delays follicular development182and suppresses luteal angiogenesis in rodents104,183 and in primates.175,184186These studies have established that VEGF is indeed the principal regulatorof ovarian angiogenesis and that blockade of the VEGF pathway is sufficientto disrupt angiogenesis.

Role of VEGF in pathologic conditionsTumor angiogenesis

Preclinical studies Many tumor cell lines secrete VEGF-A in vitro.187In situ hybridization studieshave demonstrated that VEGF mRNA is expressed in the majority of humantumors, including lung,188,189 breast,190,191 gastrointestinal tract,192195 kid-ney,196198 bladder,106 ovary,199201 and endometrium202 carcinomas and severalintracranial tumors including glioblastoma multiforme203205 and sporadicand VHL syndrome-associated, capillary hemangioblastoma.206,207

In 1993, we reported that monoclonal antibodies targeting VEGF-Ainhibited the growth of several tumor cell lines in nude mice, while theantibody had no effect on the proliferation of tumor cells in vitro.208 Subse-quently, many other tumor cell lines were found to be inhibited in vivo byanti-VEGF monoclonal antibodies.209215 For a recent review, see Gerber andFerrara.216 Tumor growth inhibition was demonstrated also with otheranti-VEGF treatments including a retrovirus-delivered dominant negativeVEGFR-2 mutant,217 small molecule inhibitors of VEGFR-2 signaling,218220antisense oligonucleotides,221,222 anti-VEGFR-2 antibodies,223 and solubleVEGF receptors.224228

While tumor cells usually represent major sources of VEGF, tumor-asso-ciated stroma is also an important site of VEGF production.226,229,230



Recent studies have shown that tumor-derived PDGF-A may be espe-cially important for the recruitment of an angiogenic stroma.231 The growthof a variety of human tumor cell lines transplanted in nude mice is substan-tially reduced but not completely suppressed by anti-human VEGF-A mon-oclonal antibodies.208

Administration of mFlt(1-3)-IgG, a chimeric receptor containing the firstthree Ig-like domains of VEGFR-1 that binds both human and mouseVEGF,226 or recently described cross-reactive anti-VEGF-A monoclonal anti-bodies resulted in nearly complete suppression of growth in several tumorcell lines in immunodeficient mice.232 Similar results were obtained using achimeric soluble receptor consisting of domain 2 of VEGFR-1 fused withdomain 3 of VEGFR-2 (referred to as VEGF-trap).228 The use of VEGF inhib-itors that target only human VEGF in human xenograft models frequentlyresults in underestimating the contribution of VEGF to the process of tumorangiogenesis.

Cre-LoxP-mediated gene targeting has been used to show that VEGFinactivation suppresses tumor angiogenesis in the RipTag model, awell-established genetic model of insulinoma.233 Furthermore, at least in theRipTag model, MMP-9-mediated proteolytic events determined an angio-genic switch mediated by enhancement of low constitutive levels of VEGFthat become available to bind VEGFR-2.234,235

Several studies have shown that combining anti-VEGF treatment withchemotherapy236 or radiation therapy237,238 results in greater anti-tumoreffects than either treatment alone. An issue now under debate is the mech-anism of such potentiation, and various hypotheses have been proposed (seeChapter 9). Klement et al. proposed that chemotherapy, especially whendelivered at close regular intervals using relatively low doses, with no pro-longed drug-free break periods (metronomic therapy), preferentially dam-ages endothelial cells in tumor blood vessels and that the simultaneousblockade of VEGF-A blunts a key survival signal for endothelial cells, thusselectively amplifying the endothelial cell-targeting effects of chemother-apy.236 A similar process in principle may occur when more conventionalmaximum tolerated dose chemotherapy regimens are combined.

Jain239 proposed an alternative hypothesis. Anti-angiogenic agents wouldnormalize the abnormal vasculature characteristic of many vessels intumors, resulting in pruning of excessive endothelial and perivascular cells,a drop in the normally high interstitial pressures detected in solid tumors,and temporarily improved oxygenation and delivery of chemotherapy totumor cells.239 However, according to recent studies, the tumor vasculaturecan be normalized only transiently and eliciting synergistic effects throughthis mechanism requires chemotherapy or radiation therapy over a definedtime window after administration of the angiogenesis inhibitor.240

Considering also that in most clinical protocols no such sequentialadministration is performed, it is unclear whether such a mechanism mayaccount for the long-term beneficial effects of combination treatmentsobserved in some clinical trials. On the other hand, acute administration of


14 Angiogenesis

angiogenic inhibitors has been shown to induce vascular changes consistentwith normalization in humans. Willett et al. reported that a single infusionof bevacizumab to patients with rectal carcinoma rapidly decreased tumorperfusion, vascular volume, microvascular density, interstitial fluid pres-sure, and the number of viable, circulating endothelial cells in six colorectalcancer (CRC) patients.241

Clinical trials of VEGF inhibitors in cancer patientsClinical trials of several VEGF inhibitors including a humanized anti-VEGFmonoclonal antibody (rhuMab VEGF; bevacizumab; AvastinTM),242 ananti-VEGFR-2 antibody,223 small molecules inhibiting VEGFR-2 signal trans-duction,219,220 and a VEGF receptor chimeric protein228 are ongoing in cancerpatients. For recent reviews, see Gasparini et al.243 and Ferrara and Kerbel.244Chapter 10 by Wainberg and Kabbinavar focuses on the clinical developmentof bevacizumab.

The pivotal trial with bevacizumab that led to FDA approval was a largerandomized placebo-controlled Phase III study in which bevacizumab wastested in combination with chemotherapy as first-line therapy for previouslyuntreated metastatic colorectal cancer.30 Patients were randomized to receiveweekly boluses of (1) irinotecan, 5-fluorouracil, and leucovorin (IFL) plusbevacizumab (5 mg/kg every two weeks) or (2) IFL plus bevacizumabplacebo. Survival was significantly increased in the IFL/Avastin arm com-pared to the IFL/placebo arm. Hypertension was more common in the IFL/Avastin-treated group, but was readily managed in all cases with oralanti-hypertensive agents.30

Bevacizumab was approved by the U.S. Food & Drug Administration(FDA) on February 26, 2004 as a first-line treatment for metastatic CRC incombination with 5-fluorouracil-based chemotherapy regimens.

The role of bevacizumab in other tumor types and settings is currentlyunder investigation, and Phase III clinical trials in non-small cell lung, renalcell, and metastatic breast cancers are ongoing. Recently, an interim analysisof a Phase III study of women with previously untreated metastatic breastcancer treated with bevacizumab in combination with weekly paclitaxelchemotherapy showed that the study met its primary efficacy endpoint ofimproving progression-free survival, compared to paclitaxel alone (Miller,K. Proc. ASCO, May 2005).

The administration of bevacizumab in combination with paclitaxel andcarboplatin to patients with non-small cell lung carcinoma (NSCLC) resultedin increased response rate and time to progression relative to chemotherapyalone in a randomized Phase II trial.245 The most significant adverse eventwas serious hemoptysis. This was primarily associated with centrally locatedtumors with squamous histology, cavitation, central necrosis, and proximityof disease to large vessels.245 Several other VEGF inhibitors in addition tobevacizumab are being clinically pursued.243,244

Among these, a variety of small-molecule RTK inhibitors targeting VEGFreceptors are at different stages of clinical development. The most advanced



are SU11248 and Bay 43-9006. SU11248 inhibits VEGFRs, PDGFR, c-kit, andFlt-3246 and has been reported to have efficacy in imatinib-resistant gas-trointestinal stromal tumors (Maki, RG. et al. Abst. 9011, Proc. ASCO, May2005). An interim analysis of Phase III data indicates that Bay 43-9006 mono-therapy results in a significant increase in progression-free survival inpatients with advanced renal cell carcinoma (Escudier, B. et al, Abst.LBA4510, Proc. ASCO, May 2005). AG-013736, which shows a similar spec-trum of kinase inhibition as SU11248, has also shown promise in metastaticrenal cell carcinoma in a Phase II monotherapy study (Rini, B. et al. Abst.4509, Proc. ASCO, May 2005).

PTK787 is also a VEGF RTK inhibitor in late-stage clinical trials.219 Thismolecule is in Phase III studies in CRC patients and interim findings havebeen presented (Hecht, J.R. et al. Abst. LBA3, Proc. ASCO, May 2005).

According to investigator-based assessments, there was a statisticallysignificant increase in progression-free survival in patients treated withPTK787 in combination with FOLFOX4 chemotherapy compared to chemo-therapy treatment alone. However, a central review failed to document anysignificant difference. In 2006, SU11248 and Bay 43-9006 have been approvedby the FDA for the treatment of metastatic venal cell carcinoma.

Intraocular neovascular syndromes

Diabetes mellitus, occlusion of the central retinal vein, and prematurity withsubsequent exposure to oxygen can all be associated with retinal ischemiaand intraocular neovascularization, which may result in vitreous hemor-rhages, retinal detachment, neovascular glaucoma, and blindness.247,248Chapter 11 by Campochiaro discusses in detail the mechanisms of ocularneovascularization.

Expression of VEGF mRNA spatially and temporally correlates withneovascularization in several animal models of retinal ischemia.40,249,250 Ele-vations of VEGF levels in the aqueous and vitreous humors of human eyeswith proliferative retinopathies secondary to diabetes and other conditionshave been described.251,252 As in the animal models, these studies demon-strated a temporal correlation between VEGF elevations and active prolifer-ative retinopathies.251 Subsequently, animal studies using various VEGFinhibitors including soluble VEGF receptor chimeric proteins,253 monoclonalantibodies,254 antisense oligonucleotides,255 and small-molecule VEGFR-2kinase inhibitors256 have directly demonstrated the role of VEGF as a keymediator of ischemia-induced intraocular neovascularization.

Neovascularization and vascular leakage are also major causes of visualloss in the wet form of age-related macular degeneration (AMD), the overallleading cause of blindness.248 Earlier studies demonstrated the immunohis-tochemical localization of VEGF in surgically resected choroidal neovascularmembranes from AMD patients,257 suggesting a role for VEGF in theprogression of AMD-related choroidal neovascularization. It is unclearwhether such VEGF up-regulation is hypoxia-related.258 Currently,


16 Angiogenesis

anti-VEGF strategies are being explored in clinical trials in AMD patients(see Chapter 12).

The most clinically advanced VEGF inhibitors are an aptamer that selec-tively binds VEGF165 (pegaptanib, Macugen)259 and a recombinant human-ized anti-VEGF Fab that neutralizes all VEGF-A isoforms and proteolyticfragments, which is derived from bevacizumab (ranibizumab, Lucentis).260Pegaptanib was approved by the FDA in December 2004 for the treatmentof AMD, following Phase III studies showing that intraocular administra-tions of the drug reduced visual loss relative to placebo.261 Very recently, acontrolled Phase III study showed that administration of ranibizumab notonly maintained but also improved vision in patients with wet AMD.262

PerspectivesResearch conducted over the past 15 years has clearly established that theVEGF family plays an essential role in the regulation of embryonic andpostnatal physiologic angiogenesis processes such as normal growth48,173 andcyclical ovarian function.104 Furthermore, VEGF inhibition has been shownto suppress pathological angiogenesis in a wide variety of preclinical modelsincluding genetic models of cancer, leading to the clinical development of avariety of VEGF inhibitors. Definitive clinical studies have provided proofthat VEGF inhibition, using bevacizumab in combination with chemother-apy, may provide significant benefits including increased survival in patientswith previously untreated metastatic CRC.30 Ongoing clinical studies aretesting the hypothesis that bevacizumab may have efficacy in treating othertumor types as well.

It would be of great importance to have reliable markers to monitor theactivities of anti-angiogenic drugs. The absence of such biomarkers to datemay have impaired clinical development of various anti-angiogenic drugs.Two potential candidates are circulating endothelial cells and their progen-itor subsets and MRI dynamic measurement of vascular permeability andflow in response to angiogenesis inhibitors, although their long-term pre-dictive values remain to be established.241,263,264 Emphasizing such difficultiesin identifying predictive markers, a recent study found that VEGF andthrombospondin expression or microvessel density in tumor sections didnot correlate with clinical responses to bevacizumab in patients with meta-static CRC; patients showed survival benefits from the treatment irrespectiveof these parameters.265

Finally, therapeutic angiogenesis constitutes a promising approach forthe treatment of ischemic disorders such as coronary or limb ischemia.However, in spite of extensive preclinical and clinical studies with severalangiogenesis factors (VEGF, aFGF, bFGF, FGF4), we still have no proof ofclinical efficacy from any pro-angiogenic treatment. Therefore, it appearsthat numerous issues must be resolved before this field may advance in asignificant manner (see Chapter 13 by Khurana and Simons).



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