| Summary: | Regulator of calcineurin 1 (RCAN1) has been shown to act as a negative regulator of vascular endothelial growth factor (VEGF)-signalling in endothelial cells. Two isoforms are detectable in cells, RCAN1.1 and RCAN1.4, produced by alternative splicing of the RCAN1 mRNA. In this study it was demonstrated that only RCAN1.4 is induced in human dermal microvascular endothelial cells (HDMECs) in response to VEGF. Using siRNA-mediated gene silencing this work shows that RCAN1 depletion leads to a reduction in VEGFR-2 internalisation following VEGF-A stimulation. RCAN1 depletion also leads to a reduction in cell polarity and cytoskeletal reorganisation in response to VEGF. siRNA-mediated silencing of RCAN1 led to an inhibition of VEGF-A mediated migration of HDMECs. These effects of RCAN1 knockdown appear to be specific to VEGFR-2, as no apparent effect on hepatocyte growth factor receptor (HGFR) internalisation and cytoskeletal reorganisation and migration in response to HGF was observed in HDMECs. Over- expression of RCAN1.4 isoform using adenoviral mediated gene delivery resulted in increased migration of HDMECs in the absence of ligand. This effect was insensitive to the calcineurin inhibitor cyclosporine, indicating that RCAN1.4 was not operating through the classical calcineurin/NFAT pathway to regulate cell migration. Instead, the RCAN1.4 effect was sensitive to a small molecule VEGFR-2 kinase inhibitor, which blocked cell migration. This study also examined the role of phospholipase D (PLD) in endothelial cell function. By utilising siRNA for PLD1 and PLD2 it was shown that both PLD1 and PLD2 are required for VEGF-A mediated proliferation, migration and tubular morphogenesis in HDMECs. Overall, the data presented in this thesis defines a novel role for both RCAN1 and PLD in regulating in endothelial cell function. Both proteins could be potential therapeutic targets to regulate vascular function.
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