Regulation of VEGF-activated signalling by the plasma membrane calcium ATPase 4 in endothelial cells

A thesis submitted in partial fulfilment of the requirements of the University of Wolverhampton for the degree of Master of Philosophy.INTRODUCTION: Angiogenesis, the formation of new blood vessels from pre-existing ones. It is a tightly regulated processes involving pro- and anti-angiogenic molecules. Deregulation of this process is associated with aberrant blood vessel formation (excessive or insufficient) in several human pathologies. Among the many pro-angiogenic factors promoting angiogenesis, the vascular endothelial growth factor (VEGF) has been characterised as a major regulator of both physiological and pathological angiogenesis. Therefore, the characterisation of the molecular mechanisms that regulate VEGF-induced angiogenesis is essential to develop therapeutic strategies that correct abnormal angiogenesis. In this sense, our group has previously reported a negative role for the Plasma Membrane Calcium ATPase 4 (PMCA4) protein in endothelial cells, acting via inhibition of the pro-angiogenic calcineurin/NFAT signalling pathway. However, we hypothesise that other intracellular pathways might be regulated by PMCA4 in endothelial cells during VEGF stimulation of angiogenesis. METHODS: To identify PMCA4 regulated pro-angiogenic signalling pathways, we have screened gene arrays related to Notch signalling or extracellular matrix-Cell Adhesion Molecule (ECM-CAM) pathway using RNA isolated from PMCA4-silenced (or control) HUVEC. Changes in gene expression after PMCA4 knockdown have been further validated by TaqMan-based qPCR in HUVEC or HDMEC. RNA levels of PMCA4 in aging HUVEC were analysed by TaqMan qPCR using RNA isolated from HUVEC cultured from different passages (from 3 to 15 passages). RESULTS: siRNA-mediated PMCA4 knockdown led to increased expression of Notch ligand DLL1 and Notch target gene Hey1 in VEGF-stimulated HUVEC. Expression of the transcription factor c-Fos was also elevated after PMCA4 knockdown in HUVEC stimulated with VEGF for 1h. Analysis of a gene array containing genes encoding extracellular matrix and cell adhesion molecules revealed that PMCA4 silencing alters the basal expression of P-Selectin and L-Selectin in HUVEC. The expression of other genes in the array like, ADAMTS-1, E-Selectin, and VCAM-1, was affected by lack of PMCA4, but only when cells were stimulated with VEGF. Examination of changes in the expression of these genes in PMCA4-silenced HUVEC or HDMEC showed differences indicating that PMCA4 might differentially regulate these genes in different sub-types of endothelial cells. In conclusion, our results suggest that PMCA4 negatively regulates Notch signaling pathway, and it is required for proper synthesis of ECM-CAM molecules. A first step to investigate the expression of PMCA4 in endothelial cells during aging has shown that PMCA4 mRNA levels increase along cell culture passage in HUVEC. However, this initial result requires further verification of changes in PMCA4 protein levels and/or in other cellular types to conclude that PMCA4 expression increases with aging