Divergence of angiogenic and vascular permeability signaling by VEGF: inhibition of protein kinase C suppresses VEGF-induced angiogenesis, but promotes VEGF-induced, NO-dependent vascular permeability.

ABSTRACT

Vascular endothelial growth factor (VEGF) promotes angiogenesis by a variety of mechanisms including stimulation of endothelial cell proliferation and migration and increasing vascular permeability. Although its mitogenic activity is mediated primarily by the beta(2)-isoforms of protein kinase C (PKC), little is known about the signaling pathways transducing its other physiological properties. Accordingly, we used a novel inhibitor molecule to examine the role of PKC isoforms alpha and beta in mediating VEGF-induced angiogenesis and vascular permeability. Because conventional inhibitors of PKC, such as staurosporine or calphostin C, also inhibit a variety of other protein kinases, we used a novel compound to specifically inhibit PKC. A myristoylated peptide, which mimics the pseudosubstrate motif of PKC-alpha and -beta subtypes, has been shown to be a highly selective and cell-permeable inhibitor of PKC. Blocking led, as expected, to abrogation of VEGF-induced endothelial cell proliferation in vitro. In vivo, VEGF-induced angiogenesis was impaired by myristoylated peptide. Surprisingly, selective inhibition of PKC induced vascular permeability in vivo via a NO-dependent mechanism. Moreover, PKC inhibition led to a 6.4-fold induction of NO synthase (NOS) activity in endothelial cells. Our findings demonstrate that activation of PKC is a major signaling pathway required for VEGF-induced proliferation and angiogenesis, whereas vascular permeability was enhanced by blocking PKC. Inhibition of calcium-dependent PKC by itself led to induction of NOS. Although NOS is a downstream target for VEGF-induced angiogenesis, its induction by PKC inhibition was not sufficient to promote neovascularization. These results reveal that angiogenesis and vascular permeability induced by VEGF are mediated by mechanisms which ultimately diverge.