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

Abstract

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) (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.

Arachidonic Acid–Induced Ca2+ Entry Is Involved in Early Steps of Tumor Angiogenesis

“…Different angiogenic factors (including basic FGF and VEGF) able to induce the release of AA exert their biological effect by increasing [Ca 2+ ] i in different cell types, including endothelial cells (5,11,40). To test the hypothesis of a role for Ca 2+ i in AA-induced tubulogenesis in B-TECs, we imaged the entire capillary-like structures to show that Ca 2+ i signals are activated by AA and ETYA in early stages of B-TEC tubule formation in vitro, whereas such signals are down-regulated during organization of the capillary-like structures in defined networks.…”

“…The dose of antiangiogenic agents is peculiar in determining the efficacy of combination therapies. CAI has been shown to exert its blocking effects starting from 1 Amol/L in normal endothelial cells (5,17,20). Higher doses of CAI have antiangiogenic activity in different systems such as mouse presenting ischemic retinopathy, rat aortic ring culture, or chorioallantoic membrane (18,20,62).…”

“…[2][3][4][5]. Therefore, regulation of changes in intracellular concentrations ([Ca 2+ ] i ) is a potent putative molecular target for angiogenesis interference.…”

“…A number of compounds inhibit calcium entry, including carboxyamidotriazole (CAI), an anti-invasive and antiangiogenic agent that inhibits calcium-mediated signal transduction in tumor cells and endothelial cells by blocking agonistactivated calcium entry (5,18). The blockage by CAI results in inhibition of endothelial cell attachment to extracellular matrix substrate, migration, and proliferation in vivo and in vitro (17,(19)(20)(21).…”

Interleukin-1β induces the upregulation of caveolin-1 expression in a rat brain tumor model

“…VEGF has an extremely important role in the proliferation, migration and formation of blood vessels (14)(15)(16)(17)(18). Our experimental results show that IL-1β could induce the expression of VEGF in tumor capillaries; the peak appears at 60 min after infusion and subsequently decreased.…”