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Brain-derived neurotrophic factor (BDNF) is a neurotrophin best characterized for its survival and differentiative effects on neurons. Recent studies demonstrated that BDNF and its receptors are also expressed in the peripheral vasculature, where it stimulates angiogenesis and promotes the survival of endothelial cells. This study was designed to investigate the angiogenic effects of BDNF and its expressional regulation by tumor necrosis factor (TNF-α) and protein kinase C (PKC) in endothelial cells. In the Matrigel angiogenesis assay, BDNF-stimulated vascular tube formation of human umbilical vein endothelial cells (HUVEC) was completely blocked by an inhibition of the TrkB receptor, but only partially inhibited by the inhibition of the p75NTR signaling. Treatment of HUVEC and HUVEC-derived EA.hy 926 cells with TNF-α resulted in a downregulation of BDNF expression, which could be prevented by the TNFR1 antagonist WP9QY. BDNF downregulation by TNF-α was associated with decreased angiogenic activity of HUVEC. The effect of TNF-α on BDNF expression could not be abolished by the inhibition of PKC. Treatment of HUVEC and EA.hy 926 cells with PKC-activating phorbol esters (phorbol-12-myristate-13-acetate, PMA or phorbol-12,13-dibutyrate) resulted in a downregulation of BDNF expression, whereas the inactive 4α-phorbol-12,13-didecanoate was without effect. PMA had no significant effect on BDNF mRNA stability and the downregulation of BDNF mRNA expression by PKC activation was likely a transcriptional event. BDNF downregulation by PMA could be prevented by PKC inhibitors Gö 6983 and rottlerin, but not by Gö 6976. Thus, a Gö 6983/rottlerin-sensitive PKC isoform is likely to be responsible for PMA-induced BDNF downregulation.

Aims Protein kinase C (PKC) plays an important role in the regulation of angiogenesis. However, downstream targets of PKC in endothelial cells are poorly defined. Methods and results 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 α, δ and e in HUVEC. Knockdown of PKC α 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 α activation and PKC-dependent VEGF upregulation. Conclusions PKC α 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 α-dependent positive feedback mechanism.