Regulation of angiogenesis via vascular endothelial growth factor receptors.

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