Since PKC activation is believed to be a crucial signaling event in VEGF-induced endothelial cell proliferation, we examined whether the PKC activator phorbol 12-myristate acetate (PMA) had a similar effect as VEGF on endothelial cell proliferation. Incubation of BRECs with 20 nM PMA for 96 hours increased cell numbers to a similar extent as 4-day treatment with 100 ng/mL VEGF
(Fig. 2A) . Addition of 0.1 to 10 μM riluzole inhibited this PMA-induced cell proliferation
(Fig. 2A) . To confirm PKC activation by VEGF, we measured levels of phospho-PKC by testing cell lysates in immunoblot assays using an anti-phospho-PKC antibody. We found that 24-hour incubation of HUVECs with 20 nM PMA or 100 ng/mL VEGF for 24 hours increased levels of phospho-PKC. Addition of riluzole or GF109203X (5 μM) inhibited the increase in phospho-PKC levels by either stimulant
(Fig. 2B 2C) . We examined which PKC isoforms were activated by VEGF by immunoprecipitating isoforms using specific antibodies and then performing immunoblotting using an anti-phospho-PKC antibody. We found that VEGF increased the level of phospho-PKC βII, but had minimal effect on the phosphorylation of PKC α, PKC βI, or PKC γ
(Fig. 2D) . Addition of riluzole substantially reduced the VEGF effect on phospho-PKC βII levels, whereas addition of GF109203X with VEGF decreased levels of all phospho-PKC isoforms to below those in control cultures
(Fig. 2D 2E) . To further test the possibility that PKC βII inhibition was the main mechanism of the riluzole effect on VEGF-induced endothelial cell proliferation, we turned to siRNA technology. Treatment with siRNA targeting PKC β markedly reduced PKC βII levels in HUVEC cultures, both VEGF-treated and untreated
(Fig. 2F) . As expected, in the presence of the siRNA, VEGF treatment did not induce cell proliferation
(Fig. 2G) . Control RNA used to assess the transfection efficiency of siRNA did not inhibit VEGF-induced proliferation (not shown). In the presence of siRNA, the inhibitory effect of riluzole on HUVEC proliferation disappeared. On the other hand, in sister cultures that were not treated with siRNA, riluzole markedly inhibited VEGF-induced proliferation.