Essential role of calcium in vascular endothelial growth factor A-induced signaling: mechanism of the antiangiogenic effect of carboxyamidotriazole

Vascular endothelial growth factor-A (VEGF-A) plays a major role in tumor angiogenesis and raises the concentration of intracellular free calcium ([Ca2+]i). Carboxyamidotriazole (CAI), an inhibitor of calcium influx and of angiogenesis, is under investigation as a tumoristatic agent. We studied the effect of CAI and the role of [Ca2+]i in VEGF-A signaling in human endothelial cells. VEGF-A induced a biphasic [Ca2+]i signal. VEGF-A increased the level of intracellular inositol 1,4,5-trisphosphate (IP3), which suggests that VEGF-A releases Ca2+ from IP3-sensitive stores and induces store-operated calcium influx. Reduction of either extracellular or intracellular free Ca2+ inhibited VEGF-A-induced proliferation. CAI inhibited IP3 formation, both phases of the calcium signal, nitric oxide (NO) release, and proliferation induced by VEGF-A. CAI prevented neither activation of VEGF receptor-2 (VEGFR-2) (VEGFR2/KDR/Flk-1/VEGFR2/KDR/Flk-1), Phospholipase C-g, or mitogen-activated protein kinase (MAP kinase) nor translocation of nuclear factor of activated T cells (NFAT). We conclude that calcium signaling is necessary for VEGF-A-induced proliferation. MAP kinase activation occurs independently of [Ca2+]i but is not sufficient to induce proliferation in the absence of calcium signaling. Inhibition of the VEGF-A-induced [Ca2+]i signal and proliferation by CAI can be explained by inhibition of IP3 formation and may contribute to the antiangiogenic action of CAI. Calcium-dependent NO formation may represent a link between calcium signaling and proliferation.