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