CHARACTERIZATION OF VASCULAR ENDOTHELIAL GROWTH-FACTORS EFFECT ON THEACTIVATION OF PROTEIN-KINASE-C, ITS ISOFORMS, AND ENDOTHELIAL-CELL GROWTH

Vascular endothelial growth factor (VEGF) is a potent endothelial cell mitogen which mediates its effects by binding to tyrosine kinase rece ptors, We have characterized the VEGF-activated intracellular signal t ransduction pathway in bovine aortic endothelial cells and correlated this to its mitogenic effects, VEGF induced concentration- and time-de pendent increases in protein kinase C (PKC) activation with a maximum of 2.2-fold above the basal level at 5 x 10(-10) M within 10 min as me asured both by in situ and translocation assays, Immunoblotting analys is of PKC isoforms in cytosolic and membrane fractions indicated that after VEGF stimulation the content of Ca2+-sensitive PKC isoforms (alp ha and beta II) was increased in the membrane fractions, whereas no ch anges were observed for PKC isoforms delta and epsilon. The stimulatio n of PKC activity by VEGF was preceded by the activation of phospholip ase C gamma (PLC gamma), This was demonstrated by parallel increases i n PLC gamma tyrosine phosphorylation, [H-3]inositol phosphate producti on, and [H-3]arachidonic acid-labeled diacylglycerol formation in bovi ne aortic endothelial cells, In addition, VEGF increased phosphatidyli nositol 3-kinase activity 2.1-fold which was inhibited by wortmannin, a phosphatidylinositol 3-kinase inhibitor, without decreasing the VEGF -induced increase in PKC activity or endothelial cell growth, Interest ingly, genistein, a tyrosine kinase inhibitor, and GFX or H-7, PKC inh ibitors, abolished both VEGF-induced PKC activation and endothelial ce ll proliferation, VEGF's mitogenic effect was inhibited by a PKC isofo rm beta-selective inhibitor, LY33-3531, in a concentration-dependent m anner. In contrast, antisense PKC-alpha oligonucleotides enhanced VEGF -stimulated cell growth with a simultaneous decrease of 70% in PKC-alp ha protein content, Thus, VEGF appears to mediate its mitogenic effect s partly through the activation of the PLC gamma and PKC pathway, invo lving predominately PKC-beta isoform activation in endothelial cells.