Kohn E C, Alessandro R, Spoonster J, Wersto R P, Liotta L A
Signal Transduction and Prevention Unit, National Cancer Institute, Bethesda, MD 20892.
Proc Natl Acad Sci U S A. 1995 Feb 28;92(5):1307-11. doi: 10.1073/pnas.92.5.1307.
During angiogenesis, endothelial cells react to stimulation with finely tuned signaling responses. The role of calcium-regulated signaling in angiogenesis has not been defined. This study investigated the calcium dependency of endothelial cell proliferation and invasion by using an inhibitor of ligand-stimulated calcium influx, CAI (carboxy-amidotriazole). Incubation with CAI significantly inhibited proliferation of human umbilical vein endothelial cells (HUVECs) in response to serum (IC50 = 1 microM) or basic fibroblast growth factor (FGF2; P2 < 0.005 at 10 microM). Statistically significant inhibition of HUVEC adhesion and motility to basement membrane proteins laminin, fibronectin, and type IV collagen was demonstrated (adhesion, P2 < 0.004-0.01; motility, P2 < 0.009-0.018). Marked inhibition of native and FGF2-induced gelatinase activity was shown by zymogram analysis and was confirmed by Northern blot analysis. CAI inhibited HUVEC tube formation on Matrigel and inhibited in vivo angiogenesis in the chicken chorioallantoic membrane assay, 67% at 20 microM and 56% at 10 microM compared with 16% for an inactive CAI analog or 9% for 0.1% dimethyl sulfoxide control. Incubation of HUVECs with CAI and/or FGF2 followed by immunoprecipitation with anti-phosphotyrosine antibody showed inhibition of FGF2-induced tyrosine phosphorylation of proteins in the range 110-150 kDa. These results suggest that calcium-regulated events are important in native and FGF2-stimulated HUVEC proliferation and invasion, perhaps through regulation of FGF2-induced phosphorylation events, and indicate a role for calcium in the regulation of angiogenesis in vivo.
在血管生成过程中，内皮细胞通过精确调节信号反应来对刺激作出响应。钙调节信号在血管生成中的作用尚未明确。本研究使用配体刺激的钙内流抑制剂CAI（羧基 - 氨三唑）来研究内皮细胞增殖和侵袭对钙的依赖性。用CAI孵育可显著抑制人脐静脉内皮细胞（HUVECs）对血清（IC50 = 1 microM）或碱性成纤维细胞生长因子（FGF2；10 microM时P2 < 0.005）的增殖反应。统计学上显著抑制了HUVEC对基底膜蛋白层粘连蛋白、纤连蛋白和IV型胶原的黏附及迁移能力（黏附，P2 < 0.004 - 0.01；迁移，P2 < 0.009 - 0.018）。酶谱分析显示对天然和FGF2诱导的明胶酶活性有明显抑制作用，Northern印迹分析也证实了这一点。CAI抑制了HUVEC在基质胶上形成管样结构，并在鸡胚绒毛尿囊膜试验中抑制了体内血管生成，20 microM时抑制率为67%，10 microM时为56%，而无活性的CAI类似物抑制率为16%，0.1%二甲亚砜对照抑制率为9%。用CAI和/或FGF2孵育HUVECs，然后用抗磷酸酪氨酸抗体进行免疫沉淀，结果显示抑制了FGF2诱导的110 - 150 kDa范围内蛋白质的酪氨酸磷酸化。这些结果表明，钙调节事件在天然和FGF2刺激的HUVEC增殖和侵袭中很重要，可能是通过调节FGF2诱导的磷酸化事件实现的，并表明钙在体内血管生成的调节中发挥作用。
