The inner lining of the vasculature is a monolayer of endothelial cells that performs a multitude of functions with importance for cardiovascular health.
Many of the functions are regulated by or are dependent on the calcium ion (Ca2+), a major intracellular signalling factor.
The overall aim of this thesis was to investigate molecular mechanisms and pharmacology of Ca2+ entry mechanisms and their downstream signalling in endothelial cells. Molecular, biochemical and electrophysiology techniques and
cell functional assays were used to address two main objectives. The first objective was to investigate putative Ca2+ channel regulator proteins, CRACR2A and golli-MBP. Based on previous reported studies, it was hypothesised that both of these proteins would have a role in regulating
endothelial cell store-operated Ca2+ entry. However, unexpectedly these proteins had Ca2+ channel-independent functions, leading to alternative hypotheses about their roles in endothelial cells. The second objective was to
generate new information about pharmacology targeted to a Ca2+ channel subunit, TRPC6, which has been suggested to be important in endothelial cell biology. Specifically, it was hypothesised that the anti-cancer agent carboxyamidotriazole (CAI) and the flavanol galangin and its derivatives, which have previously been shown to inhibit intracellular Ca2+, were TRPC6 channel
inhibitors.
Endothelial cells did not express CRACR2A as reported for T cells (Srikanth et al., 2010) but did express a longer splice variant arising from the same gene, EFCAB4B isoform a (EFCAB4B-a). CRACR2A and EFCAB4B-a have the same
putative Ca2+ binding domains at their N termini but EFCAB4B-a, unlike CRACR2A, had no role in the regulation of Ca2+ release-activated Ca2+ (CRAC) channels. Instead, EFCAB4B-a contained a predicted Rab domain in its C
terminus and was therefore a putative monomeric G protein. It localised to endothelial cell specific Weibel-Palade bodies and influenced the abundance of the pro-thrombotic agent von Willebrand factor. Golli-MBP was found expressed in endothelial cells but again there was little effect on CRAC channel activity. Instead golli-MBP was identified as a positive regulator of the important endothelial growth factor receptor, VEGFR2. The small-molecule CAI, was identified as an inhibitor of TRPC6 channels
without effect on TRPC5 or TRPV4 channels. A range of flavonol compounds was also identified as TRPC6 inhibitors.
In summary, this research has generated new information and hypotheses about a putative Ca2+-regulated Rab protein of Weibel-Palade bodies, a novel regulator of a key endothelial growth factor receptor, and a molecular target for CAI. It has, therefore, led to understanding of molecular mechanisms and pharmacology in endothelial cells which may be useful for devising strategies to treat life-threatening conditions such as cardiovascular disease and cancer.
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