Structure function analysis of vascular endothelial growth factor receptors in angiogenesis

Heart attack or stroke can block blood vessels leading to local tissue damage or even death; there is therefore a need to increase blood flow and promote circulation by making new blood vessels. In contrast, inhibiting blood vessel growth prevents cancer and blindness respectively. The formation of blood vessels (angiogenesis) is complex: They are lined with endothelial cells, which divide and re-arrange themselves into new tube-like vessels. Vascular endothelial growth factor is an essential protein for the blood vessel formation and works by binding to receptors on the endothelial cells. This project will determine how these receptors signal to co-ordinate new vessel formation. Discoveries from this study will provide new insight for the development of novel therapeutic approaches to promote or block the growth of new blood vessels.

Nitric oxide (NO) not only regulates blood vessel tone but also promotes angiogenesis. Vascular endothelial growth factor-A (VEGF) is critical for vascular development, angiogenesis and revascularisation after endothelial injury and acts via two receptors, VEGFR-1 and VEGFR-2. The loss of either receptor leads to death in utero. In contrast, the loss of VEGFR-1 catalytic activity does not impair vascular development, but inhibits pathological neovascularisation suggesting that VEGFR-1 is also important in angiogenesis-driven diseases. We hypothesise that VEGF utilises alternative signalling pathways via VEGFR-1 or VEGFR-2 to regulate NO and promote angiogenesis. We will determine the relative roles of these two receptors using chimeric receptors in which the extracellular domain of the epidermal growth factor receptor was substituted for that of VEGFR-1 or VEGFR-2 and transduced into human endothelial cells. To determine the structural-functional relationship between VEGFR-1 and VEGFR-2, NO and angiogenesis, a series of tyrosine-to-phenylalanine mutants will be analysed in NO release and capillary-like tube formation assays. A proteomic approach will be employed to identify the proteins associated with key VEGF receptor tyrosine residues that are required for eNOS activation and/or capillary morphogenesis and their downstream effectors. The downstream effectors of VEGFR-1 and VEGFR-2 and identified tyrosine residues will then be investigated by manipulating the signal transduction pathways of phosphatidylinositol 3-kinase (PI3K/PDK-1/Akt), phospholipase C-gamma1 and protein kinase C isozymes. This will be achieved by selective inhibition using either gene knockdown or over-expression of dominant-negative mutants of the signalling proteins using lentiviral and adenoviral vectors respectively. These studies will identify new sites of modulation in VEGF-mediated vascular morphogenesis and NO release that may highlight new therapeutic targets for management of vascular insufficiencies and cancer.