Protein kinase C promotes angiogenic activity of human endothelial cells via induction of vascular endothelial growth factor

Abstract

Protein kinase C (PKC) plays an important role in the regulation of angiogenesis. However, downstream targets of PKC in endothelial cells are poorly defined. mRNA expression of vascular endothelial growth factor (VEGF) was analysed by quantitative real-time RT-PCR in human umbilical vein endothelial cells (HUVEC) and HUVEC-derived EA.hy 926 cells. siRNA was used to knockdown PKC isoforms and VEGF. Matrigel tube formation assay was used to analyse the angiogenic activity of endothelial cells. Phorbol-12-myristate-13-acetate (PMA) enhanced the ability of HUVEC to organize into tubular networks when plated on Matrigel, a phenomenon that could be prevented by PKC inhibitors. PMA markedly increased the expression of VEGF in HUVEC and EA.hy 926 cells. The enhancement in VEGF expression was prevented by PKC inhibitors and by an inhibitor of the Erk1/2 pathway. PMA-induced tube formation was reduced by inhibition of the VEGF receptor kinase, or by VEGF knockdown. PMA led to an activation of PKC isoforms alpha, delta and epsilon in HUVEC. Knockdown of PKC alpha diminished PMA-induced VEGF expression and angiogenesis. Also endothelial progenitor cells isolated from human peripheral blood showed enhanced VEGF expression and improved angiogenic activity in response to PKC activation. Moreover, incubation of HUVEC with VEGF led to PKC alpha activation and PKC-dependent VEGF upregulation. PKC alpha activation promotes angiogenic activity of human endothelial cells. This is likely to be largely mediated by induction of VEGF. VEGF enhances its own expression via a PKC alpha-dependent positive feedback mechanism.