Intracellular Ca2+ signaling in endothelial cells by the angiogenesis inhibitors endostatin and angiostatin

Intracellular signaling mechanisms by the angiogenesis inhibitors endostati n and angiostatin remain poorly understood. We have found that endostatin ( 2 mg/ml) and angiostatin (5 mg/ml) elicited transient, approximately threef old increases in intracellular Ca2+ concentration ([Ca2+](i)). Acute exposu re to angiostatin or endostatin nearly abolished subsequent endothelial [Ca 2+](i) responses to carbachol or to thapsigargin; conversely, thapsigargin attenuated the Ca2+ signal elicited by endostatin. The phospholipase C inhi bitor U-73122 and the inositol trisphosphate (IP3) receptor inhibitor xesto spongin C both inhibited endostatin-induced elevation in [Ca2+](i), and end ostatin rapidly elevated endothelial cell IP3 levels. Pertussis toxin and S B-220025 modestly inhibited the endostatin-induced Ca2+ signal. Removal of extracellular Ca2+ inhibited the endostatin-induced rise in [Ca2+](i), as d id a subset of Ca2+-entry inhibitors. Peak Ca2+ responses to endostatin and angiostatin in endothelial cells exceeded those in epithelial cells and we re minimal in NIH/3T3 cells. Overnight pretreatment of endothelial cells wi th endostatin reduced the subsequent acute elevation in [Ca2+](i) in respon se to vascular endothelial growth factor or to fibroblast growth factor by similar to 70%. Intracellular Ca2+ signaling may initiate or mediate some o f the cellular actions of endostatin and angiostatin.