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

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Abstract
Intracellular signaling mechanisms by the angiogenesis inhibitors endostatin and angiostatin remain poorly understood. We have found that endostatin (2 μg/ml) and angiostatin (5 μg/ml) elicited transient, approximately threefold increases in intracellular Ca2+ concentration ([Ca2+]i). Acute exposure to angiostatin or endostatin nearly abolished subsequent endothelial [Ca2+]i responses to carbachol or to thapsigargin; conversely, thapsigargin attenuated the Ca2+ signal elicited by endostatin. The phospholipase C inhibitor U-73122 and the inositol trisphosphate (IP3) receptor inhibitor xestospongin C both inhibited endostatin-induced elevation in [Ca2+]i, and endostatin rapidly elevated endothelial cell IP3 levels. Pertussis toxin and SB-220025 modestly inhibited the endostatin-induced Ca2+signal. Removal of extracellular Ca2+ inhibited the endostatin-induced rise in [Ca2+]i, as did a subset of Ca2+-entry inhibitors. Peak Ca2+responses to endostatin and angiostatin in endothelial cells exceeded those in epithelial cells and were minimal in NIH/3T3 cells. Overnight pretreatment of endothelial cells with endostatin reduced the subsequent acute elevation in [Ca2+]i in response to vascular endothelial growth factor or to fibroblast growth factor by ∼70%. Intracellular Ca2+ signaling may initiate or mediate some of the cellular actions of endostatin and angiostatin.