The vascular endothelial growth factors (VEGFs) have central roles in initiating blood vessel formation by both stimulating angioblast differentiation and activating angio-genesis from existing vessels. There are six members of the VEGF ligand family: VEGF-A to VEGF-E and placental growth factor (PlGF). These ligands signal via three VEGF-RTKs: VEGF-receptors-1 to 3. VEGF-Ais the predominant form of the ligand and can bind all three receptors, whereas PlGF and VEGF-B recognize only VEGFR-1, and VEGF-E is specific for VEGFR-2. VEGF-C and D bind VEGFR3 and to some extent VEGFR2. All three RTKs have a similar overall structure, with a ligand binding extracellular domain containing seven immunoglobulin-like repeats and intracel-lular domain comprising a juxtamembrane sequence, tyro-sine kinase domain with kinase insert, and carboxy-terminal tail. VEGFR-3 is mainly confined to lymphatic endothelia, where it has important roles in lymphangiogenesis. The principal VEGF-RTKs of the microvasculature are VEGFR-1 and VEGFR-2, which are the focus of this section. Because most endothelial cells express both VEGFR-1 and -2, defining the specific roles and signaling events mediated by each receptor in response to VEGF has been challenging. One way to address this issue has been by overexpression of chimeric receptors, allowing specific activation of each receptor, or the expression of each receptor individually in cells that do not normally express VEGFRs. Such approaches are useful, but care is required in extrapolating to the situation in primary endothelial cells expressing physiological levels of receptors. Some of the discrepancies reported in signaling by VEGFRs may be a result of these limitations.
Of the two microvessel VEGF-RTKs, VEGF-A binds with highest affinity to VEGFR-1. However, this receptor appears to have relatively low kinase activity, and VEGF-A-activated increase in kinase activity or autophosphorylation of VEGFR-1 is difficult to detect in endothelial cells. One function of the receptor may therefore be to act as a negative regulator of VEGF-A activity by sequestering the ligand and preventing it from activating VEGFR-2.
In addition to any role in regulating VEGF-A availability, VEGFR-1 does have signaling activity. Transgenic mice expressing a truncated form of VEGFR-1, lacking the intra-cellular domain, exhibit normal developmental angiogenesis but have compromised pathological angiogenesis, indicating involvement of the intracellular VEGFR-1 signaling domain. VEGFR-1-specific ligands also activate distinct patterns of gene expression, again consistent with the receptor having discrete signaling activity. The signaling pathways utilized by VEGFR-1, however, are poorly understood. Phosphorylation has been observed in bac-ulovirus expressed intracellular domains of the receptor and in cells overexpressing VEGFR-1 in response to VEGF-A. The principal phosphorylation sites identified in these studies were Y1169, Y1213, and Y1333. VEGF-A activation of nonoverexpressing endothelial cells increases Y1213 phos-phorylation. PLCg Nck, and Crk bind to phosphopeptides containing the Y1333 site, and SHP2, phospholipase Cg (PLCg), and Grb2 bind to Y1213 phosphopeptides. Increased tyrosine phosphorylation of PLCg Crk, and SHP2 has also been observed in response to VEGF-A stimulation in cells overexpressing VEGFR-1. These data suggest that the receptor could modulate Ca++/DAG (via PLCg) and Ras/Raf pathways (via SHP2/Grb2), although more work is necessary to establish interaction of the receptor with these intermediates in endothelial cells expressing physiological levels of receptor in their normal cellular background.
VEGF-A and the VEGFR-1-specific ligand PlGF appear to have some distinct effects on VEGFR-1 in endothelial cells expressing normal levels of the receptor. The two ligands induce different patterns of gene expression and activate phosphorylation of different tyrosine residues in the receptor. In contrast to the effects of VEGF-A on Y1213, PlGF activates phosphorylation of Y1309. The signaling and functional consequences of Y1309 phosphorylation have yet to be defined. Such differential effects of ligands on the receptor provide a means for distinct activities of the ligands on endothelial cells and raise the important question of the mechanism for ligand-specific phosphorylations in VEGFR-1.
There is cross-talk between VEGFR-1 and VEGFR-2. Specific activation of VEGFR-1 by PlGF enhances VEGF-A-activation of VEGFR-2 by a mechanism involving transphosphorylation of VEGFR-2 by VEGFR-1. The two receptors exist as homomeric complexes and preformed het-eromeric complexes in endothelial cells.
In summary, VEGFR-1 has apparently low kinase activity and can act to sequester VEGF-A and regulate signaling by VEGFR-2. Specific ligands for VEGFR-1 can modulate signaling via VEGFR-1 by displacing sequestered VEGF-A and enhancing VEGFR-2 activity by transphosphorylation. Such activities are likely to be involved in the enhancement of VEGF-induced angiogenesis by PlGF. In addition, VEGFR-1 does have distinct signaling capability responsible for regulating specific gene expression profiles, and probably other effects in endothelial cells.
The limited ability to detect VEGFR-1 activation has led to the suggestion that the principal receptor mediating the effects of VEGF-A in endothelial cells is VEGFR-2. The receptor has been implicated in VEGF-induced endothelial migration, proliferation, and differentiation, as well as the pro-inflammatory and permeability effects of the ligand. Reported sites of tyrosine phosphorylation in VEGFR-2 include Y951, Y996, Y1054, Y1059, Y1175, and Y1214. Of these, Y1175 and Y1214 appear to be primary sites of phosphorylation, and Y1175 has been confirmed in endothelial cells expressing physiological levels of receptor.
Specific activation of VEGFR-2 in nonoverexpressing endothelial cells provides an antiapoptotic signal requiring phosphatidylinositol-3-kinase (PI-3K) and Akt. The activity of Akt is stimulated by VEGF in endothelial cells in response to VEGFR-2-specific ligands, and PI-3K and Akt are both activated via VEGFR-2 in cells overexpressing the receptor. The precise way in which PI-3K is activated by VEGFR-2 is not clear, but in some studies the receptor was found to associate directly with the p85 subunit of PI-3K (pY801, pY1175), whereas in others p85 was bound to focal adhesion kinase (FAK) recruited to pY1214 of VEGFR-2, or VEGFR-associated protein (VRAP), which is recruited to pY951. Intriguingly, the antiapoptotic activity of VEGF appears to involve the interendothelial adhesion molecule VE-cadherin. In cells from mice lacking the intracellular domain of VE-cadherin, coupling between VEGFR-2 and Akt is impaired and interaction between VEGFR-2 and p85 is decreased. Further work will be required to define the mechanism by which VE-cadherin modulates VEGFR-2 association with p85.
The signaling pathway by which VEGF induces endothelial migration via VEGFR-2 is unclear. The pathway is likely to involve FAK and PLCg, both of which interact with VEGFR-2 and have been implicated in VEGF-stimulated endothelial migration in studies using mutant receptors and inhibitors. VEGF activation of endothelial proliferation occurs via a signaling pathway involving VEGFR-2 activation of PLCg following its recruitment to pY1175. The resulting generation of diacylglycerol (DAG) activates protein kinase C (pKC), which in turn stimulates Raf and the Erk pathway.
VEGF markedly increases vascular permeability, and use of receptor-specific ligands indicates this is mediated via VEGFR-2. Again, however, the signaling mechanisms involved are not yet understood. There is good evidence that PLCg-mediated DAG generation and Ca++ mobilization contribute to increased nitric oxide (NO) generation, leading to elevation of cGMp and increased vascular permeability. In addition, Akt, activated via VEGFR-2 as described previously, has also been implicated. One action of the Akt is to phosphorylate endothelial nitric oxide synthase (eNOS) on serine1177 and increase NO production. The PLCg pathway appears to predominate in the first few minutes of VEGF-induced vessel permeability, after which the Akt pathway becomes more important. The downstream events mediating the effects of cGMp on permeability have yet to be clearly defined. The lack of effects of VEGF on vascular permeability in transgenic mice lacking p60Src and p62Yes indicate that these members of the Src family of intracellular tyrosine kinases also participate in the permeability signaling pathway, although exactly how is not yet known. Acute increased permeability of microvessels in response to VEGF can occur through formation of fenestrae, which is stimulated by VEGF, and increased transcellular flux. Clear definition of the mechanisms by which fenestrae form and transcellular flux increases will allow delineation of the missing links between the VEGFR-2-NO signaling pathway and increased permeability. Figure 1 summarizes in schematic form some of the best elucidated VEGFR-2 signaling pathways.
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