Vascular endothelial growth factor stimulates a novel calcium-signaling pathway in vascular smooth muscle cells

Background. Vascular endothelial growth factor (VEGF) is a potent vascular milogen that selectively stimulates vascular smooth muscle cell (VSMC) migration through an unknown mechanism while having no effect on VSMC proliferation. It is known that VSMC migration and proliferation are dependent on the second messenger Ca2+ and, in particular, mitogen-stimulated Ca2+ influx. We hypothesized that the selective effect of VEGF on VSMC migration versus proliferation was a result of differential VEGF-stimulated Ca2+ signaling pathways. Methods. Primary cultured human aortic smooth muscle cells (VSMCs) were grown to subconfluency and assigned to the following experimental groups: no stimulation, stimulation with platelet-derived growth factor-BB (PDGF-BB) (20 ng/mL) as positive control, and stimulation with VEGF(165) (40 ng/mL). Total increase in [Ca2+](cyt) and intracellular calcium release was quantified with the use of a fura-2 fluorescence assay. Assays for the following receptors VEGFR-1 (Flt-1), VEGFR-2(KDR/Flk-1) and PDGFR-P were performed by immunoprecipitation, while PLC gamma(1), Akt 1/2, and phospholamban B phosphorylation were assessed with Western immunoblotting. Results. VSMCs stimulated with VEGF(165) exhibited no intracellular Ca2+ release, compared with a 75 +/- 30 nmol/L intracellular calcium release after PDGF-BB stimulation, (P < .02) VEGF(165)-stimulated VSMCs in Ca2+-containing media exhibited 192 +/- 26 nmol/L increase in [Ca2+](cyt), compared with 354 +/- 54 nmol/L increase after PDGF-BB stimulation (P < .02). VEGF(165) did not phosphorylate PLC gamma(1) after 1, 5, or 10 minutes of treatment. VEGF165 treatment did not result in P13-K/Akt activation at 1-, 5-, or 10-minute time points. Calmodulin-dependent kinase II (CaMKII) was activated by both VEGF(165) and PDGF-BB after 1 and 5 minutes of stimulation. The presence of the receptors VEGFR-1, VEGFR-2, and PDGFR-P was confirmed in all experimental groups. Conclusions. VEGF induces extracellular calcium influx but no intracellular calcium release in VSMCs. This lack of intracellular Ca 2, release stems from the inability of VEGF(165) to activate the PLC gamma(1) cascade and IP3 receptor-mediated Ca2+ release. The lack of P13-K/Akt activation at these time points indicates a novel extracellular Ca2+ influx pathway sufficient to activate CaMKII. A paradigm relating extracellular Ca2+ influx to CaMKII activation and migration is suggested and may account for the selective effects of VEGF on VSMC migration.