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Investigation of the function of PMCA1 in physiological and pathological angiogenesis

Njegic, Alexandra Maria

[Thesis]. Manchester, UK: The University of Manchester; 2019.

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Abstract

Angiogenesis occurs under physiological and pathological conditions and the ability to manipulate vessel growth makes it a promising therapeutic target; however, pro-angiogenic therapies have failed to translate successfully into the clinic therefore more therapeutic options are required. One such novel target are the plasma membrane calcium ATPases (PMCAs), with PMCA4 shown to negatively regulate angiogenesis both in vitro and in vivo. The PMCAs (PMCA1-4) act to extrude Ca2+ from the cytosol into the extracellular space and can also mediate intracellular signalling. Given this role for PMCA4 in angiogenesis we hypothesised that PMCA1, which is also present in endothelial cells, may play a role in angiogenesis. In this study, the contribution of PMCA1 to physiological angiogenesis was assessed using both in vitro and in vivo methods. To determine if PMCA1 plays a role in angiogenesis in vitro, siRNA-mediated ATP2B1 knockdown was performed in human umbilical vein endothelial cells (HUVECs) which were then used to determine changes to endothelial cell biology and angiogenic characteristics. Knockdown of PMCA1 in HUVECs led to an increase in basal intracellular Ca2+, a reduction in cell viability and impaired VEGF-mediated tubule formation. Mechanistically, preliminary work identified changes to proteins involved in regulation of the eukaryotic cell cycle; loss of PMCA1 leads to a reduction in 5 out of 6 core components of the mini chromosome complex. In addition, to determine the effect of PMCA1 on angiogenesis in vivo, a novel mouse line was generated using the pan-endothelial transgene Tie2 Cre (PMCA1Tie2). These mice are viable and display no overt phenotype under basal conditions; however, the extent of PMCA1 knockdown in endothelial cells from PMCA1Tie2 mice was only 30%. Furthermore, when subject to surgical pressure-overload induced hypertrophy, PMCA1Tie2 mice show a similar extent of cardiac remodelling when compared to littermate controls but have increased levels of the pro-angiogenic protein RCAN1.4. Overall, PMCA1 is required for effective VEGF-mediated angiogenesis and endothelial cell viability in vitro and loss of PMCA1 leads to downregulation of cell cycle components. However, the understanding of the contribution of PMCA1 to physiological and pathological angiogenesis in vivo is still not conclusive; the partial endothelial knockout achieved in this study suggests that endothelial cells may require PMCA1 in order to function normally, as such, the complete ablation of endothelial PMCA1 may not be achievable in vivo.