VEGF Stimulates RCAN1.4 Expression in Endothelial Cells via a Pathway Requiring Ca2+/Calcineurin and Protein Kinase C-δ

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.

Plasma Membrane Calcium ATPase Isoform 4 Inhibits Vascular Endothelial Growth Factor–Mediated Angiogenesis Through Interaction With Calcineurin

“…Although our results do not directly demonstrate that reduction of these cellular functions is linked to PMCA4-mediated downregulation of RCAN1.4 and Cox-2, both proteins have been described to promote endothelial cell migration and tube formation, 17,18,31 suggesting that they likely play a significant role in the PMCA4-directed inhibition of angiogenesis. In agreement with this idea, PMCA4-deficient MLECs presented higher expression of Cox-2 ( Figure The results of our gain-or loss-of-PMCA4 function experiments might be particularly relevant in the context of pathophysiological processes associated with changes in the expression of PMCA proteins.…”

“…4 and Cox-2, whose products actively participate in the onset of angiogenesis. [5][6][7][17][18][19] The inhibitory effect produced by PMCA4 on the VEGF-induced activity of the calcineurin/NFAT pathway in endothelial cells prompted us to next examine the role of PMCA4 as a negative regulator of angiogenesis gene expression.…”

Exogenous Recombinant Dimeric Neuropilin-1 Is Sufficient to Drive Angiogenesis

“…Scratch Wound Migration Assay-The assay was performed as described previously (39). Briefly, HDMECs and HUVECs were seeded in a 24-well plate coated with gelatin at 3 ϫ 10 4 and 2 ϫ 10 4 cells per well, respectively.…”

“…Fibroblast-HDMEC Co-culture-The assay was performed as described previously (39). Briefly, human dermal fibroblasts were seeded at 2 ϫ 10 4 cells per well in a 24-well gelatinized plate and grown until confluent for 3-4 days in fibroblast growth medium (Promocell).…”

In vitro 3D angiogenesis assay in egg white matrix: comparison to Matrigel, compatibility to various species, and suitability for drug testing

“…6 Other methods use substitutes of extracellular matrix (ECM) such as fibrin, collagen, or Matrigel to induce capillary-like formation principally from ECs. [7][8][9][10][11] These matrices are commercially available from a variety of providers, enabling a highthroughput screening in multiwell plates. In such matrix, ECs grow and migrate as a network representing a preliminary step of neocapillary formation.…”