Angiogenesis, the de novo growth of blood vessels from pre-existing vessels, is an imperative, tightly-regulated process that underpins the expansion and refinement of the developing vascular network. However, in some pathological conditions, such as after a myocardial infarction, the vascular network can be destroyed necessitating the need for angiogenesis. Therefore stimulating angiogenesis could be therapeutically advantageous. Recently, plasma membrane calcium ATPase 4 (PMCA) has been established as a novel mediator of angiogenesis through its role in endothelial cell migration and tubule formation. In addition to PMCA4, both PMCA1 and PMCA2 are also expressed in human endothelial cells but their contribution to angiogenesis remains unknown. Therefore, we hypothesise that PMCA1 also modulates angiogenesis by altering endothelial cell behaviours.
Transient knockdown of PMCA1 was achieved in human umbilical vein endothelial cells (HUVECs) using siRNA (si-PMCA1) and confirmed with qPCR and western blot. Assays to asses HUVEC viability, proliferation and rate of apoptosis were performed over a period of 6 days post-transfection using Alamar Blue, Ki-67 immunofluorescence and Caspase-Glo 3/7 respectively. In addition, live-cell imaging was performed over a period of 21 hours to further evaluate migration of cells in a wound, cell necrotic cytotoxicity and cell apoptotic death. Furthermore, HUVECs were added to Matrigel to evaluate in-vitro tubule formation and DAPI-stained cells were subject to fluorescent activated cell sorting to determine cell-cycle distribution. Molecular mechanisms were investigated using Western blot.
Transient knockdown of PMCA1 in HUVECs resulted in an 85% reduction of PMCA1 at both mRNA and protein level but did not alter the levels of PMCA4 mRNA up to 6 days post transfection. Phenotypically, 3 and 6 days post-transfection, loss of PMCA1 significantly reduced HUVEC viability without a concomitant increase in HUVEC apoptosis or cytotoxicity. Immunofluorescent staining for Ki-67 showed no statistical difference between si-PMCA1 and control HUVECs for the percentage of Ki-67 positive cells. However, FACs-mediated cell cycle analysis revealed si-PMCA1 HUVECs had a significantly larger proportion of cells in G0/G1 phase with fewer cells identified in G2/M-phase compared to control HUVECs after serum starvation followed by 8 hours of full serum media. Additionally, live-cell imaging of the wound healing assay revealed si-PMCA1 HUVECs displayed significantly reduced HUVEC wound density and wound confluence, resulting in an increased wound width over time. Furthermore, si-PMCA1 also show reduced ability to form tubules in-vitro characterised by a reduction in the number of tubules and tubule junctions formed, despite si-PMCA1 HUVECs having higher protein levels of the pro-angiogenic gene RCAN1.4.
Overall, transient knockdown of PMCA1 has adverse effects on HUVEC viability, migration and tubule formation suggesting loss of PMCA1 is detrimental for in-vitro angiogenesis.
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