Angiogenesis is the sprouting of blood vessels from existing ones. This process is essential during embryonic vascular development, but in the healthy adult only occurs during wound healing and the development of the corpus luteum. However, in many disease states such as cancer, diabetic retinopathy, and rheumatoid arthritis, angiogenesis plays a role in the progression of the disease, thus making it an attractive target for the development of inhibitors. In other diseases such as ischaemic heart and peripheral vascular disease, improvement of blood supply by promoting angiogenesis could improve tissue survival and patient prognosis. Vascular endothelial growth factors are probably the most important angiogenic growth factors.

Five related mammalian growth factors have now been identified (VEGF-A, VEGF-B, VEGF-C, VEGF-D and placental growth factor (PlGF)), which signal through two related receptor tyrosine kinases VEGFR-1/Flt1 and VEGFR-2/KDR in vascular endothelial cells. Inhibition of these receptors has shown promise as a strategy to inhibit tumour growth. Different VEGFs bind different combinations of receptor. PlGF and VEGF-B bind selectively to VEGFR-1, whereas VEGF-A binds both receptors and VEGF-C and -D bind only to VEGFR-2 and to the related receptor VEGFR-3, which is only expressed in lymphatic endothelium. Despite much progress recently, the precise contribution of each of the VEGF receptors to physiological and pathological angiogenesis remains to be defined, and the signalling pathways initiated by each receptor are not completely characterised. The ability of VEGFR-2 to activate the MAP kinase pathway in endothelial cells, but not when over-expressed in other cell types such as fibroblasts and human embryonic kidney cells suggests that an endothelial cell-specific component may be required for effective VEGF-R signalling. In an attempt to identify novel proteins which interact with the VEGF receptors, we have screened yeast two-hybrid libraries with the cytoplasmic domains of each receptor. Several proteins have been identified by this approach including PLC-γ, the adaptor ShcB/Sck and the ubiquitin ligase, Nedd4. The sites of interaction of these proteins with the receptors, and their potential signalling roles, will be discussed.

As well as comparing the proteins that interact with VEGFR-1 and VEGFR-2 by yeast two-hybrid analysis, we also sought to compare the ability of the receptors to activate defined signalling pathways in endothelial cells. Previous attempts to assign individual responses to a particular receptor have used either transfected cell lines, receptor-specific growth factors or antisense oligonucleotides. Such studies have attributed the majority of VEGF-induced responses to activation of VEGFR-2. As a consequence of poor growth factor-induced VEGFR-1 autophosphorylation, however, observations from these studies may instead reflect the relative activation of the two receptors. We have generated novel chimeric VEGF receptors in which the dimerization domain of the B subunit of DNA gyrase is fused to the cytoplasmic domain of VEGFR-1 and -2. When expressed in porcine aortic endothelial cells, both chimeric VEGFR-1 and -2 autophosphorylate in response to addition of the small-molecule dimerizing agent, coumermycin. Once activated, both receptors induce downstream activation of MAPK, PLC-γ and PKB/Akt. Mutational analysis has shown that the Y1175 residue of VEGFR-2 is essential for the activation of PLC-γ mediated by this chimeric receptor. In contrast to previous reports which show a limited ability of VEGFR-1 to mediate signalling cascades, our results suggest that once sufficiently activated, VEGFR-1 signals in a similar manner to VEGFR-2 in endothelial cells. Similar drug-activatable receptors may be of potential use for therapeutic angiogenesis.