Abstract
BackgroundVascular endothelial growth factor (VEGF) has previously been shown to upregulate the expression of the endogenous calcineurin inhibitor, regulator of calcineurin 1, variant 4 (RCAN1.4). The aim of this study was to determine the role and regulation of VEGF-mediated RCAN1.4 expression, using human dermal microvascular endothelial cells (HDMECs) as a model system.Methodology/Principal FindingsWe show that VEGF is able to induce RCAN1.4 expression during cellular proliferation and differentiation, and that VEGF-mediated expression of RCAN1.4 was inhibited by the use of inhibitors to protein kinase C (PKC) and calcineurin. Further analysis revealed that siRNA silencing of PKC-delta expression partially inhibited VEGF-stimulated RCAN1.4 expression. Knockdown of RCAN1.4 with siRNA resulted in a decrease in cellular migration and disrupted tubular morphogenesis when HDMECs were either stimulated with VEGF in a collagen gel or in an endothelial/fibroblast co-culture model of angiogenesis. Analysis of intracellular signalling revealed that siRNA mediated silencing of RCAN1.4 resulted in increased expression of specific nuclear factor of activated T-cells (NFAT) regulated genes.Conclusions/SignificanceOur data suggests that RCAN1.4 expression is induced by VEGFR-2 activation in a Ca2+ and PKC-delta dependent manner and that RCAN1.4 acts to regulate calcineurin activity and gene expression facilitating endothelial cell migration and tubular morphogenesis.
Highlights
Angiogenesis is defined as the formation of new blood vessels from pre-existing vessels, and is an essential process in embryonic development and normal physiology
In order to establish the role of each receptor in RCAN1.4 upregulation, Vascular endothelial growth factor (VEGF)-B and VEGF-E, which bind VEGF Receptor 1 (VEGFR-1) and VEGFR-2 respectively, were used to stimulate human dermal microvascular endothelial cells (HDMECs)
VEGF-A has previously been shown to upregulate RCAN1.4 expression in endothelial cells, triggering a negative feedback loop which regulates the activity of calcineurin, and results in decreased angiogenesis [10,17,38]
Summary
Angiogenesis is defined as the formation of new blood vessels from pre-existing vessels, and is an essential process in embryonic development and normal physiology. The vascular endothelial growth factor (VEGF) family of homodimeric glycoproteins have been shown to be critical for angiogensis. In addition there are a number of structurally related proteins, including parapoxvirus Orf VEGF (VEGF-E). These ligands bind in an overlapping pattern to 3 receptors; VEGF Receptor 1 (VEGFR-1), VEGFR-2 and VEGFR-3 (reviewed in [2]). Vascular endothelial growth factor (VEGF) has previously been shown to upregulate the expression of the endogenous calcineurin inhibitor, regulator of calcineurin 1, variant 4 (RCAN1.4). The aim of this study was to determine the role and regulation of VEGF-mediated RCAN1.4 expression, using human dermal microvascular endothelial cells (HDMECs) as a model system
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