Regulation of Angiogenesis Via Vascular Endothelial Growth Factor Receptors1

[CANCER RESEARCH 60, 203–212, January 15, 2000] Review

Regulation of Angiogenesis via Vascular Endothelial Growth Factor Receptors1

Tanja Veikkola, Marika Karkkainen, Lena Claesson-Welsh, and Kari Alitalo2 Molecular/Cancer Biology Laboratory, Haartman Institute, University of Helsinki, SF-00014 Helsinki, Finland [T. V., M. K., K. A.], and Department of Medical Biochemistry and Microbiology, Biomedical Center, S-75123 Uppsala, Sweden [L. C-W.] Introduction

Endothelial cell signal transduction mechanisms involved in angio- with several different isoforms generated by alternative splicing, genesis have come into focus in cancer research when it was realized consisting of polypeptides of 121, 145, 165, 189, or 206 amino acid that solid tumors are dependent on neovascularization (1). Unlike residues (15, 24–26). VEGF binds both VEGFR-1 and VEGFR-2 and normal human endothelial cells, which are quiescent except in the is apparently capable of inducing heterodimers between the two reproductive organs of fertile women, endothelial cells in tumors (27–30). VEGF uses symmetrical binding sites at each pole of the express several target features associated with their angiogenic acti- dimer for receptor binding (31, 32). It has been shown that the second vation. On the other hand, unlike the tumor cells, endothelial cells are immunoglobulin homology domain of VEGFR-1 is critical for ligand readily accessible from the blood circulation, and they are not likely binding, but the first three immunoglobulin domains are required to to develop resistant variants to cytostatic therapy (see Ref. 2). Vas- reconstitute full affinity (31, 33–35). Of the other VEGF family cular targeting may involve either the destruction of existing vessels members, PlGF and VEGF-B bind only VEGFR-1, the Orf virus by exploitation of differences between normal and tumor vessels VEGFs bind only VEGFR-2, and VEGF-C and VEGF-D interact with (3–6) or inhibition of the tumor angiogenesis process per se (7–9). In both VEGFR-2 and VEGFR-3 (16–18, 36–42). Recently, NRP-1, a both scenarios, knowledge of the endothelial cell-specific growth cell surface glycoprotein that acts as a receptor for collapsins/sema- factor receptors and their signal transduction and effector mechanisms phorins and controls axon guidance during embryonic development, is essential and will undoubtedly provide additional points of attack to has been identified as an isoform-specific receptor for VEGF165, human cancers. Here we discuss recent results on one important PlGF-2, VEGF-B, and Orf virus VEGFs (39, 43–45). Heterodimers of family of endothelial growth factor receptors implicated in angiogen- PlGF and VEGF are produced by certain cell lines, and they exert esis. Several excellent reviews have appeared on the same topic and mitogenic activity toward endothelial cells (46, 47). VEGF-B/VEGF related topics (10–14). heterodimers have also been obtained in expression vector cotrans- The VEGF3 family currently includes five members in addition to fection experiments (48). In addition to the high-affinity receptors, the prototype VEGF, namely, PlGF, VEGF-B, VEGF-C, VEGF-D, certain splice isoforms of the VEGFs also bind heparan sulfate pro- and Orf virus VEGFs (also called VEGF-E; Refs. 15–18 and Refs. teoglycans on the cell surface and in the pericellular matrix via a below). The VEGFs mediate angiogenic signals to the vascular en- distinct heparin binding domain (16, 49–51). dothelium via high-affinity RTKs. To date, three receptors for the VEGFR-1 is a Mr 180,000 transmembrane glycoprotein, but its VEGFs have been identified. All three are relatively specific for mRNA can also be spliced to produce a shorter soluble protein endothelial cells and demonstrate structural and functional similarities consisting of only the first six extracellular immunoglobulin homol- to the PDGF receptor family (see Refs. 14 and 19). These receptors ogy domains (20, 27, 52). Such a RNA splice variant, originally are currently designated VEGFR-1, VEGFR-2, and VEGFR-3 and detected in a HUVEC cDNA library, encodes 31 unique amino acid were originally named flt (fms-like tyrosine kinase), KDR (kinase residues before a stop codon (52). VEGFR-2 is a Mr 230,000 protein, insert domain-containing receptor)/flk-1 (fetal liver kinase-1), and and no splice variants have been reported for this receptor. In human Ј FLT4, respectively (11, 12, 20–23). All have seven immunoglobulin VEGFR-3, alternative 3 polyadenylation signals result in a 4.5-kb homology domains in their extracellular part and an intracellular transcript and a more prevalent 5.8-kb transcript (53). The latter tyrosine kinase signaling domain split by a kinase insert (Fig. 1). In transcript encodes 65 additional amino acid residues and is the major form detected in tissues. After biosynthesis, the glycosylated M adults, VEGFR-1 and VEGFR-2 are expressed mainly in the blood r vascular endothelium, whereas VEGFR-3 is restricted largely to the 195,000 VEGFR-3 is proteolytically cleaved in the fifth immunoglobulin homology domain, but the resulting M 120,000 and M 75,000 lymphatic endothelium. r r chains remain linked by a disulfide bond (41, 54). The VEGF molecule is an antiparallel disulfide-linked homodimer Because hypoxia is a major inducer of VEGF expression in tumors, and the VEGFRs are enhanced in tumor endothelia (see Fig. 2), Received 4/2/99; accepted 11/15/99. The costs of publication of this article were defrayed in part by the payment of page hypoxic regulation of the VEGFR genes has been studied by several charges. This article must therefore be hereby marked advertisement in accordance with groups. In vivo, VEGFR-1 and VEGFR-2 appear to be up-regulated 18 U.S.C. Section 1734 solely to indicate this fact. by hypoxia (55–57). In vitro, VEGFR-1 expression was increased by 1 Supported by the Finnish Academy of Sciences, The University of Helsinki, the Finnish and Swedish Cancer Research Foundation, the Ludwig Institute for Cancer hypoxia, whereas VEGFR-2 was either down-regulated or not af- Research and the Novo Nordisk Foundation. fected (58–60). On the other hand, culture medium from hypoxic 2 To whom requests for reprints should be addressed, at Molecular/Cancer Biology Laboratory, Haartman Institute, University of Helsinki, SF-000014 Helsinki, Finland. cells up-regulated VEGFR-2 protein (59). The latter effect was prob- 3 The abbreviations used are: VEGF, vascular endothelial growth factor; VEGFR, ably mediated by VEGF in the culture medium because VEGF treat- VEGF receptor; PlGF, placenta growth factor; RTK, receptor tyrosine kinase; PDGF, ment has been shown to up-regulate VEGFR-2 expression in endo- platelet-derived growth factor; NRP-1, neuropilin-1; HUVEC, human umbilical vein endothelial cell; HIF, hypoxia-inducible factor; MAPK, mitogen-activated protein kinase; thelial cells and in cerebral slice cultures (61, 62). The promoter for PKC, protein kinase C; PLC, phospholipase C; PI3-K, phosphatidylinositol 3Ј-kinase; VEGFR-1 contains sequences matching the HIF-1␣ consensus bind- STAT, signal transducer and activator of transcription; KS, Kaposi’s sarcoma; VE- ␣ cadherin, vascular endothelial cadherin; ES, embryonic stem; VHL, von Hippel-Lindau; ing site, whereas the closely related HIF-2 may stimulate VEGFR-2 HHV-8, human herpes virus-8/KS herpes virus; Ang, angiopoietin. promoter activity (60, 63, 64). 203

Fig. 1. The currently known VEGFs and their receptors. VEGFR-1 and VEGFR-2 have seven extracellular immunoglobulin homology domains, but in VEGFR-3, the fifth immunoglobulin domain is cleaved on receptor processing into disulfide-linked sub- units. VEGFR-1 and VEGFR-2 mediate angiogenesis, whereas VEGFR-3 is also involved in lymphangiogenesis. NRP-1 binds to specific COOH-terminal sequences present only in certain VEGFs ␣ ␤ that bind to VEGFR-1 and/or VEGFR-2. v 3 integrin and VE- cadherin have been found in complexes with activated VEGFR-2, and the latter also associates with an activated VEGFR-3 complex. sVEGFR-1, soluble VEGFR-1; VEC, VE-Cadherin.

VEGFR Signaling inhibitors of nitric oxide synthase, suggesting that nitric oxide contributes to MAPK activation (75). The role of Ras in the VEGFR- Like other RTKs, the VEGFRs are thought to dimerize and undergo MAPK pathway remains to be elucidated. In primary endothelial trans-autophosphorylation on ligand binding. Both VEGFR-2 and cells, Ras was not activated in response to VEGF, and MAPK acti- VEGFR-3 are tyrosine phosphorylated when stimulated with their vation was mediated mainly via a PKC-dependent pathway (74). PKC respective ligands (40, 65), but VEGFR-1 autophosphorylation is less has also been implicated in VEGF-induced MAPK activation in obvious and has been studied mostly in receptor-overexpressing trans- VEGFR-2-transfected fibroblasts (71). Seetharam et al. (66) have fected cells (37, 65–67). Phosphorylated tyrosine residues may serve to control the kinase activity of the receptor and to create docking sites observed activation of the Ras guanine nucleotide exchange factor Sos for cytoplasmic signaling molecules, which provide substrates for the in VEGF-stimulated endothelial cells, but phosphorylation of the kinase. These molecules, either adapters or enzymes themselves, link adapter protein Shc was barely detectable. On the other hand, Shc VEGFRs to the signaling pathways that are discussed below. phosphorylation in response to VEGF stimulation has been detected in VEGFR-1 and VEGFR-2. Activation of the MAPK pathway in porcine aortic endothelial cells overexpressing VEGFR-2 (69), and response to VEGF has been observed in many types of endothelial Sck, a Shc homologue, has been shown to interact with the VEGFR-2 cells (66–74). Interestingly, MAPK activation was delayed in cytoplasmic domain in the yeast two-hybrid system (76). Several VEGFR-2-transfected fibroblasts, and the mitogenic response was investigators have reported phosphorylation of the Ras GTPase-acti- weaker than in endothelial cells, suggesting the involvement of cell vating protein in endothelial cells after VEGF stimulation (65, 66, 77). type-specific signaling mechanism(s) (71). In postcapillary venular Ras GTPase-activating protein activation has also been observed in endothelium, VEGF-mediated induction of MAPK was blocked by VEGFR-1-transfected fibroblasts and porcine aortic endothelial cells,

Fig. 2. Immunostaining of VEGFRs in the vascular endothelium of a melanoma metastasis. Adjacent sections were stained with VEGFR-1, VEGFR-2, VEGFR-3, and control antibodies. Arrows point to endothelial cells. Anti-VEGFR-1 and anti-VEGFR-2 were kind gifts from Dr. Herbert Weich.

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Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 2000 American Association for Cancer Research. REGULATION OF ANGIOGENESIS VIA VEGFRs but these cells showed no clear MAPK activation or proliferation in VEGF may require the association of VEGFR-2 with cell surface response to VEGF (65, 66). Interestingly, activation of MAPK in adhesive proteins. Activated VEGFR-2 was found in a complex with ␣ ␤ bovine brain capillary endothelial cells by VEGF was selectively integrin v 3, an adhesion molecule specifically expressed on angio- ␣ ␤ inhibited by the Mr 16,000 NH2-terminal fragment of prolactin, an genic endothelium (93). v 3 binds to pericellular matrix proteins antiangiogenic factor that also inhibited proliferation of these cells containing an arginine-glycine-aspartic acid (RGD) peptide motif, ␣ ␤ (68, 78). In addition to MAPK, VEGF has been shown to activate the such as vitronectin and fibronectin. v 3 has been shown to be p38 stress kinase pathway in HUVECs, and this response has been involved in the regulation of the cell cycle and the survival of linked to F-actin reorganization and cell migration (72). endothelial cells (94–96). Various antagonists of this integrin, such as The PLC-␥-PKC pathway has also been implicated in the mitogenic antibodies and cyclic binding peptides, as well as cytokines that action of VEGF. VEGFR-1 interacts with the PLC-␥ SH2 domain in ␣ ␤ interfere with v 3 activity, are capable of inhibiting tumor angio- the yeast two-hybrid assay (79), and several groups have reported that genesis (97). VEGFR-2 tyrosine phosphorylation and VEGF-induced VEGF induces the phosphorylation and activation of PLC-␥ (66–68, mitogenicity were enhanced when endothelial cells were plated on the 70, 71, 73, 77, 80), leading to the hydrolysis of phosphatidylinositol ␣ ␤ v 3 ligand vitronectin, demonstrating that this integrin contributes to 4,5-bisphosphate to diacylglycerols and inositol 1,4,5-trisphosphate. VEGFR-2 signaling (93). Inositol 1,4,5-trisphosphate is likely to be responsible for the increase VE-cadherin, an endothelium-specific cell-cell adhesion protein, 2ϩ in intracellular Ca after VEGF stimulation (27), whereas diacyl- has also been implicated in molecular interactions with the VEGFRs. glycerol, in turn, activates certain PKC isoforms expressed in the Deficiency or truncation of VE-cadherin was lethal in mouse embryos 2ϩ target cells. VEGF selectively activated the Ca -sensitive PKC iso- due to increased endothelial cell apoptosis (98). In normal endothelial ␣ ␤ forms and 2 in bovine aortic endothelial cells, and the mitogenic cells, VEGFR-2 colocalized with VE-cadherin, and VEGF stimulation ␤ effect of VEGF in these cell was inhibited by a PKC- -selective resulted in the formation of a complex between VE-cadherin, ␤-cate- inhibitor (80). nin, PI3-K, and VEGFR-2 and subsequent Akt activation. On the Xia et al. (80) reported activation of PI3-K in response to VEGF other hand, disruption of VE-cadherin function prevented the endo- and demonstrated that PI3-K activity is not required for mitogenesis thelial cells from responding to survival signals induced by VEGF. or activation of PKC (74, 80). However, PI3-K was shown to activate Interestingly, VEGFR-3, but not VEGFR-1, also coimmunoprecipi- Akt, a serine kinase involved in antiapoptotic signaling, and to sub- tated with VE-cadherin. sequently deliver a survival signal in HUVECs treated with a Vascular Permeability May Be Transduced via an Unknown VEGFR-2-selective VEGF mutant (81). VEGFR-2 and the PI3-K/Akt VEGFR. Tumor vessels are leaky in general, and VEGF is one of the pathway may therefore play a role in VEGF-mediated survival of most potent inducers of vascular permeability (99). VEGF-C also has immature vessels (82). Akt has also been reported to directly activate vascular permeability activity, but the response is not transduced via endothelial nitric oxide synthase, suggesting that Akt may regulate the VEGFR-3 (89). On the other hand, PlGF, which only binds to increased production of nitric oxide in response to VEGF stimulation VEGFR-1 (36, 37), is incapable of inducing permeability. These data (83, 84). imply that either VEGFR-2 or some other as yet unidentified receptor STATs are latent cytoplasmic transcription factors. STAT activa- mediates the permeability response. When the third variable domain tion by the VEGFRs has been studied in transient transfection assays in the VEGF polypeptide was substituted with the analogous region of (85). All three receptors were shown to be strong activators of STAT3 PlGF, creating a chimeric molecule with significantly reduced binding and STAT5, whereas STAT1 was not activated by the VEGFRs. to VEGFR-2, the mutant still induced vascular permeability with an However, the role of this pathway in endothelial cell biology is unknown. activity similar to that of wild-type VEGF (100). Therefore, it may be VEGFR-3. Whereas both VEGFR-3 isoforms bind to and phos- that vascular permeability is not mediated by any of the known phorylate adapter protein Shc, phosphorylation is stronger in cells VEGFRs. Interestingly, the c-Src protein seems to be a necessary expressing the long isoform (54, 86, 87). The Shc PTB domain is mediator of the permeability response because adenoviral vectors required for ligand-induced Shc tyrosine phosphorylation by expressing VEGF do not induce vascular permeability in mice defi- VEGFR-3 (88), and mutations in Shc phosphorylation sites increased cient in c-Src (101). VEGFR-3 transforming activity in the soft agar assay, indicating that Importance of the Cellular Background in VEGF Signaling Shc has a negative role in VEGFR-3 signaling. Studies. Because most VEGFR signal transduction studies have been Both VEGFR-3 isoforms bind Grb2 via its SH2 domain in an carried out using endothelial cells that express more than one type of inducible manner (54, 87). Stimulation of VEGFR-3 also activates VEGFR, it has not been possible to attribute the results to a particular MAPK, at least in transfected cells (89). Similarly, both isoforms bind receptor. Attempts to study signal transduction by individual receptors to a glutathione S-transferase-SH2 (PLC-␥) fusion protein on stimu- using transfected cells have been compromised by the lack of a proper lation (54, 86). In contrast, no direct interaction has been observed cellular background, which may be an important factor in VEGFR with PI3-K (86). In a human erythroleukemia cell line and in KS cells, signaling, depending on the substrate under study. Use of receptor- VEGF-C induced tyrosine phosphorylation of the cytoskeletal protein specific ligands or mutants of the VEGF family may offer an alter- paxillin by related adhesion focal tyrosine kinase, a recently identified native approach to these questions (89, 102). Another variable in the member of the focal adhesion kinase family (90, 91). signal transduction studies is introduced by the heterogeneity of Although very little is known about the specific signal transduction endothelial cells and their tendency to lose differentiated properties in of VEGFR-3 in the lymphatic endothelium, mutations in VEGFR-3 culture. Keeping these aspects in mind, the signaling mechanisms have recently been linked with hereditary lymphedema, an autosomal discovered thus far are of interest and may offer possible targets for dominant disorder of the lymphatic system that leads to disabling therapeutic intervention (103). For example, global assessment of swelling of the extremities and, in rare cases, to lymphangiosarcomas endothelial cell gene expression by RNA microarray analysis may (see Fig. 1; Ref. 92). This indicates that disturbed VEGFR-3 signaling help to better define the activated state of the endothelial cells on may play key a role in the development of this disease. angiogenesis and drug treatment. However, for candidate drugs, ad- VEGFRs Associate with Cell Adhesion Receptors. According to ditional validation has to be provided by experiments in transgenic new data, endothelial cell proliferation and survival in response to and gene targeted mice. 205

The VEGFRs in Vascular Development limb buds, and expressed more widely in the mesenchyme (43, 120). Targeted disruption of the mouse NRP-1 gene caused severe abnor- The various VEGFRs are first expressed in the mouse embryonic malities in the peripheral nervous system (121). Interestingly, VEGF mesoderm between days 7.5–9.5 of development and distinct expres- has recently been reported to act also as a neurotrophic survival factor sion patterns are also found in 17 week human fetuses (104–106). and as a mitogenic agent for Schwann cells (122). Mice overexpress- Disruption of any of the three VEGFR genes leads to embryonic ing NRP-1 formed excess capillaries and abnormal blood vessels and lethality. In embryos with targeted null mutations of VEGFR-2, died in utero (120). It is possible that aberrant angiogenesis in NRP-1 differentiation of both endothelial and hematopoietic cells is blocked, transgenic embryos resulted from inappropriate VEGF activity. and no blood vessels are formed (107). However, at least in vitro, endothelial/hematopoietic precursor cells can be derived from VEGF and VEGFRs in Tumors VEGFR-2-deficient ES cells, demonstrating that VEGFR-2 is not required for the formation of the common hematopoietic/endothelial Expression of VEGF and its receptors correlates with the degree of progenitor cell, the so-called hemangioblast (108, 109). Rather, vascularization of many experimental and clinical tumors as detected VEGFR-2 signaling seems to be necessary for endothelial commit- by in situ hybridization and immunohistochemistry (123–132), and ment because endothelial development is terminated at an early stage both have been used as prognostic indicators of an increased meta- in VEGFR-2-negative ES cell cultures (109). Hematopoietic require- static risk (see Ref. 133). Although the detailed molecular mechanism ment for VEGFR-2, on the other hand, is conditional and depends on of the “angiogenic switch” by which quiescent endothelium becomes the cell culture conditions (108, 109). Although VEGFR-2 is not activated is unknown, VEGF seems to be the main inducer of tumor required for hemangioblast formation, it appears to be essential for the angiogenesis. Tumor hypoxia and oncogenes up-regulate VEGF lev- subsequent VEGF-directed hemangioblast migration to appropriate els in the neoplastic cells, and hypoxia, in combination with the environments in the developing embryo (108–110). In the absence of locally increased VEGF concentrations, up-regulates VEGFR-1 and VEGFR-2 signaling, such cells may rapidly disappear, explaining the VEGFR-2 on tumor endothelial cells (62, 127, 131). VEGF-negative lack of endothelial and hematoipoietic precursors in VEGFR-2 null ES cells or Ras-transformed fibroblasts grow poorly as tumors in embryos. syngeneic mice (134, 135). Interestingly, ES cells lacking HIF-1␣ In human postnatal hematopoietic tissues, VEGFR-2 apparently (deficient in hypoxic induction of VEGF) are tumorigenic. The tumors persists as a specific marker of hematopoietic stem cells, differenti- formed by HIF-1␣-deficient ES cells are poorly vascularized, but they ating them from the lineage-committed hematopoietic precursor cells are apparently more resistant to tumor cell apoptosis under hypoxic (111). Endothelial progenitors or angioblasts have also been isolated conditions (136). A recent report has suggested that in some cases, from peripheral blood and claimed to be incorporated into sites of tumor vessels can also be formed without endothelial cells (137). active angiogenesis (112, 113). Apparently, tissue ischemia or sys- Autocrine VEGF and VEGFR-1 expression has been shown to temic VEGF treatment can mobilize such bone marrow-derived occur in angiosarcomas (138). Of the nonendothelial tumors studied, mononuclear progenitor cells (114, 115). only a few melanomas and leukemia cell lines aberrantly expressed Mouse embryos homozygous for a targeted mutation of the VEGFRs, whereas several tumor cell lines expressed NRP-1 (22, VEGFR-1 locus develop an excess of endothelial cells in both em- 139–141).4 In tumors associated with the VHL disease, VEGF and bryonic and extraembryonic locations, but the endothelial cells fail to VEGFRs are constitutively up-regulated in the absence of hypoxia organize into normal vascular channels (116). The increase in endo- (see Ref. 142). Whereas the normal VHL protein functions as a thelial cell numbers is due to an alteration in cell fate determination component of a ubiquitin-protein ligase complex that targets selected among mesenchymal cells, leading to increased hemangioblast com- proteins such as HIF-1␣ for ubiquitin-mediated proteolysis (143), the mitment (117). However, when only the tyrosine kinase domain of cells containing a defective VHL protein fail to degrade HIF-1␣, VEGFR-1 was deleted, leaving the ligand binding extracellular part leading to constitutive up-regulation of many hypoxia-regulated and the transmembrane domain intact, the gene-targeted mice devel- genes, including VEGF (144). oped normal vessels and survived (118). One possible explanation for VEGFR-3 is up-regulated in tumor angiogenesis in general, for these and other findings is that during embryogenesis, VEGFR-1 acts example, in breast carcinomas (145, 146). In addition, VEGFR-3 has as a VEGF sink, regulating the amount of free VEGF available for been shown to be increased in the endothelium of lymphatic vessels in vascular development. In the absence of VEGFR-1, there would be an metastatic lymph nodes and in lymphangiomas, vascular skin tumors, excess of free VEGF available to its major signal transducing recep- and KS spindle cells (105, 147, 148). tor, VEGFR-2. Therefore, coordinated expression of both VEGFR-1 VEGF and VEGFRs in the Development of AIDS-linked KS. and VEGFR-2 would be essential for controlled early vascular devel- KS, which is the major neoplastic manifestation of HIV-induced opment. AIDS, is an angiogenic tumor composed of endothelial and spindle Disruption of VEGFR-3 led to a defective remodeling of the cells (for a review, see Ref. 149). AIDS-linked KS is always accom- primary vascular plexus and cardiovascular failure after embryonic panied by infection with HHV-8. In KS lesions, HHV-8 is present in day 9.5, but differentiation of endothelial cells, formation of primitive endothelial cells and in spindle cells with endothelial characteristics. vascular networks, and vascular sprouting occurred normally (119). HHV-8 encodes for a G-protein-coupled receptor that has been shown This phenotype suggests that VEGFR-3 is required for maturation of to induce VEGF expression in transfected cells (150), and cell lines the primary vascular plexus into a hierarchy of large and small derived from AIDS-linked KS also express high levels of the vessels. Therefore, VEGFR-3 seems to have a general blood vascular VEGFRs (151). On the other hand, HIV-1 encodes a transcriptional function during early development and only later becomes restricted regulator protein, Tat, which also possesses angiogenic properties. Tat mostly to the lymphatic vascular system. Conditional gene targeting is has been shown to bind to and activate VEGFR-2 (152, 153), to needed to better define the lymphatic functions of VEGFR-3. stimulate KS spindle cell growth (154), and to induce neovascular- Other VEGF-binding proteins, such as NRP-1, also seem to be ization in vivo (155, 156) and in transgenic mice (157). VEGFR-3 is involved in the regulation of angiogenesis during development. In also increased in KS (148), and its ligand, VEGF-C, stimulates the normal mouse embryos, NRP-1 is expressed in a temporally restricted manner in endothelial cells of capillaries and blood vessels and in 4 Michael Klagsbrun, personal communication. 206

Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 2000 American Association for Cancer Research. REGULATION OF ANGIOGENESIS VIA VEGFRs proliferation of KS cells in vitro (158). It thus seems probable that an autocrine VEGF/VEGFR activation loop plays a part in the development of AIDS-linked KS. Role of VEGF-induced Vessel Permeability in Tumor Angio- genesis. In contrast to the well-organized normal blood vessels, tumor vessels are characteristically poorly functioning, leaky endothelial channels with incomplete arteriovenous and perivascular differentiation (159). Tumor blood flow is temporally and spatially heterogenous, and the interstitial pressure in solid tumors is generally much higher than that in normal tissues. Lack of functional lymphatic vessels is another factor leading to interstitial hypertension (159). The microvascular hyperpermeability in tumors causes plasma proteins to leak into the extravascular space, leading to clotting of extravasated fibrinogen and introduction of a provisional plasma-derived matrix. Matrix formation precedes and accompanies the onset of endothelial cell migration and vessel sprouting (160). VEGF derived from hypoxic areas of tumors is likely to mediate increased permeability. VEGF therefore contributes to tumor angiogenesis by both direct and indirect mechanisms: on one hand, VEGF stimulates endothelial cell proliferation and migration; on the other, it renders vessels hyperper- meable, leading to formation of a matrix that supports blood vessel growth. Interestingly, new studies have shown that both VEGF- Fig. 3. Schematic structures of the Tie and Tie-2 receptors. Both receptors contain one complete and one incomplete immunoglobulin domain (circles) plus three epidermal induced permeability and pathological angiogenesis are attenuated in growth factor homology domains (ovals) and fibronectin type III homology domains 5 PlGF-deficient mice. (yellow), followed by a transmembrane domain and a split tyrosine kinase catalytic domain (blue). The Tie-2 receptor binds four ligands; of these, Ang-3 and Ang-4 may be Angiogenesis Inhibitors and VEGFR Function isogenic in mice and humans, although they behave as an inhibitor and a stimulator of the receptor, respectively. The Tie-2 receptor has been implicated in tumor angiogenesis (166, Studies in animal models have illustrated the dependence of tumor 184, 185). Thus far, ligand(s) for Tie is unknown, on the other hand, Tie might also interact weakly with the Tie-2-ligand complex (question marks), similar to the mode of vascularization and progression on VEGF signaling. A heterodimeric action of some epidermal growth factors and receptors. VEGF variant in which the receptor binding sites at one pole of the ligand were mutated while the other pole was left intact was shown to act as an inhibitor of VEGF function.6 A four-fold excess of this apparently because VEGF-mediated blood vessel invasion is essential mutant VEGF, which bound to its receptors but failed to mediate for coupling the resorption of cartilage with bone formation (172). receptor dimerization and subsequent signaling, was sufficient for Interestingly, VEGF also supports osteoclast recruitment and bone almost complete inhibition of vascular permeability and tissue factor resorption, and this effect has been shown to be mediated via expression induced by wild-type VEGF. A dominant negative mutant VEGFR-1 expressed in osteoclasts (173). Given the role of VEGF in of VEGFR-2 has been expressed from a retroviral construct and was bone development, anti-VEGF therapy could be useful in treating found to prevent glioblastoma vascularization and growth in nude bone-associated pathologies, for example, bone metastases and osteo- mice (161). The study was later extended to other types of tumors, and sarcomas. in most cases, tumor growth was inhibited (162). Antibodies directed against either VEGFR-2 or the VEGFR-2/VEGF complex have also Cross-Talk between Different Receptor Families Required for been effective in inhibiting VEGF-mediated signaling and endothelial Angiogenesis cell proliferation in vitro (163–165). Because the dominant negative VEGFR-2 inhibits angiogenesis in vivo, compounds that block the Recent reports on the association of activated VEGFR-2 with other tyrosine kinase activity of VEGFR-2 should also prevent neovascu- cell surface receptors raise questions on the possible roles these larization. Indeed, specific VEGFR-2 tyrosine kinase inhibitors have coreceptors play during angiogenesis in vivo. Both NRP-1 and inte- ␣ ␤ been shown to abolish VEGF-induced mitogenesis of human endo- grin v 3 have been shown to increase the mitogenic effects of VEGF thelial cells in vitro and to have an antitumor effect in mice (103). (43, 93). Because NRP-1 binds only to certain VEGFs (39, 43–45), it Several such compounds (see Ref. 166) are currently under evaluation could function to specifically potentiate the effects of these family in clinical trials for the treatment of human cancers. members. In addition, NRP-1 may be able to induce biological re- Because soluble extracellular domains of the VEGFRs compete sponses in cells that express NRP-1 but no VEGFRs. VEGFR-2 ␣ ␤ with endothelial cell surface receptors for VEGF binding and inhibit association with VE-cadherin and v 3 may serve to create active VEGF-induced mitogenicity (52, 167, 168), recombinant VEGFRs signaling complexes by clustering proteins involved in VEGF-medi- ␣ ␤ retaining high ligand binding affinity also provide the potential for ated survival signaling (81, 98). v 3 is required for the full activation inhibition of tumor angiogenesis. Tumor growth in mice has been of VEGFR-2, suggesting that the complex might regulate tyrosine shown to be suppressed by the soluble VEGFR-1 and VEGFR-2 phosphatase activity or ensure correct juxtaposition of the receptor receptor “bodies” (168–170). Adult mice seem to tolerate such ther- and putative cytoskeletal substrates. apy well, whereas newborn mice that receive the inhibitor during their In addition to the VEGF/VEGFR system, Tie and Eph families of first two postnatal weeks fail to thrive and die (171). In young endothelial tyrosine kinases have important functions in the formation animals, systemic administration of soluble VEGFR-1 leads to inhi- and maintenance of the vascular system (174–180). Tie-1 and Tie-2 bition of growth plate differentiation and growth of long bones, are restricted to the vascular endothelium and certain hematopoietic progenitor cells. Four known ligands, the Angs, bind to Tie-2, 5 Peter Carmeliet and M. Graziella Persico, personal communication. whereas there are currently no ligands for Tie-1 (Fig. 3; Refs. 181– 6 Robert de Waal, personal communication. 185). Interestingly, recent findings demonstrate that overexpression of 207

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