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

Abstract

Vascular endothelial growth factor‐α (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‐α signaling in human endothelial cells. VEGF‐α induced a biphasic [Ca²⁺]_i signal. VEGF‐α 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) (KDR/Flk‐1), phospholipase C‐γ, 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‐Ainduced [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.