A NOVEL SELECTIVE INHIBITOR FOR PLASMA MEMBRANE CALCIUM ATPase 4 IMPROVES VEGF-MEDIATED ANGIOGENESIS

A thesis submitted in fulfilment of the requirement of the University of Wolverhampton for the degree of Doctor of Philosophy.Ischaemic cardiovascular diseases are the leading cause of death worldwide. Therapeutic angiogenesis provides a valuable tool to treat these conditions by stimulating the growth of new blood vessels in the ischaemic tissue. The pro-angiogenic factor VEGF is the most potent inducer of angiogenesis, and exogenous delivery of VEGF has been a key element of therapeutic strategies. Unfortunately, VEGF-based pro-angiogenic procedures have produced only limited patient benefit. Failure to restore efficient VEGF activity remains a major problem. VEGF-mediated activation of the calcineurin/NFAT signalling pathway has been identified as a crucial regulator of angiogenesis. Our laboratory has recently shown a novel role for the plasma membrane calcium ATPase 4 (PMCA4) protein as a negative regulator of VEGF-induced angiogenesis via interaction with calcineurin. The recent identification of aurintricarboxylic acid (ATA) as a selective inhibitor of PMCA4 prompted us to hypothesise that inhibition of PMCA4 with ATA should enhance VEGF-induced angiogenesis. Here, we show that treatment of endothelial cells with nanomolar concentrations of ATA notably enhances calcineurin/NFAT signalling by disrupting the PMCA4/calcineurin interaction. ATA mediated inhibition of PMCA4 results in a significant increase in endothelial cell motility and in vitro and in vivo blood vessel formation. Low concentrations of ATA do not have any deleterious effects on the viability of endothelial cells or zebrafish embryonic development. However, high ATA concentrations impaired endothelial cell viability, and were associated with toxicity in zebrafish embryos. This study highlights the potential of targeting PMCA4 to improve VEGF-based pro-angiogenic therapeutic strategies. This goal will require the development of refined versions of ATA without associated toxicity, or the identification of novel PMCA4 inhibitors