Jho David, Mehta Dolly, Ahmmed Gias, Gao Xiao-Pei, Tiruppathi Chinnaswamy, Broman Michael, Malik Asrar B
Department of Pharmacology and The Center of Lung and Vascular Biology, The University of Illinois, Chicago, IL 60612, USA.
Circ Res. 2005 Jun 24;96(12):1282-90. doi: 10.1161/01.RES.0000171894.03801.03. Epub 2005 May 26.
Angiopoietin-1 (Ang1) exerts a vascular endothelial barrier protective effect by blocking the action of permeability-increasing mediators such as vascular endothelial growth factor (VEGF) through unclear mechanisms. Because VEGF may signal endothelial hyperpermeability through the phospholipase C (PLC)-IP3 pathway that activates extracellular Ca2+ entry via the plasmalemmal store-operated channel transient receptor potential canonical-1 (TRPC1), we addressed the possibility that Ang1 acts by inhibiting this Ca2+ entry mechanism in endothelial cells. Studies in endothelial cell monolayers demonstrated that Ang1 inhibited the VEGF-induced Ca2+ influx and increase in endothelial permeability in a concentration-dependent manner. Inhibitors of the PLC-IP3 Ca2+ signaling pathway prevented the VEGF-induced Ca2+ influx and hyperpermeability similar to the inhibitory effects seen with Ang1. Ang1 had no effect on PLC phosphorylation and IP3 production, thus its permeability-decreasing effect could not be ascribed to inhibition of PLC activation. However, Ang1 interfered with downstream IP3-dependent plasmalemmal Ca2+ entry without affecting the release of intracellular Ca2+ stores. Anti-TRPC1 antibody inhibited the VEGF-induced Ca2+ entry and the increased endothelial permeability. TRPC1 overexpression in endothelial cells augmented the VEGF-induced Ca2+ entry, and application of Ang1 opposed this effect. In immunoprecipitation studies, Ang1 inhibited the association of IP3 receptor (IP3R) and TRPC1, consistent with the coupling hypothesis of Ca2+ entry. These results demonstrate that Ang1 blocks the TRPC1-dependent Ca2+ influx induced by VEGF by interfering with the interaction of IP3R with TRPC1, and thereby abrogates the increase in endothelial permeability.
血管生成素-1（Ang1）通过尚不清楚的机制阻断血管内皮生长因子（VEGF）等增加通透性介质的作用，从而发挥血管内皮屏障保护作用。由于VEGF可能通过磷脂酶C（PLC）-肌醇三磷酸（IP3）途径发出内皮细胞高通透性信号，该途径通过质膜储存-操纵性通道瞬时受体电位香草酸亚型1（TRPC1）激活细胞外Ca2+内流，我们探讨了Ang1通过抑制内皮细胞中这种Ca2+内流机制发挥作用的可能性。在内皮细胞单层中的研究表明，Ang1以浓度依赖的方式抑制VEGF诱导的Ca2+内流和内皮通透性增加。PLC-IP3 Ca2+信号通路抑制剂可防止VEGF诱导的Ca2+内流和高通透性，类似于Ang1的抑制作用。Ang1对PLC磷酸化和IP3产生没有影响，因此其降低通透性的作用不能归因于对PLC激活的抑制作用。然而，Ang1干扰了下游IP3依赖的质膜Ca2+内流，而不影响细胞内Ca2+储存的释放。抗TRPC1抗体抑制VEGF诱导的Ca2+内流和内皮通透性增加。内皮细胞中TRPC1的过表达增强了VEGF诱导的Ca2+内流，而应用Ang1则对抗这种作用。在免疫沉淀研究中，Ang1抑制IP3受体（IP3R）与TRPC1的结合，这与Ca2+内流的偶联假说一致。这些结果表明，Ang1通过干扰IP3R与TRPC1之间的相互作用，阻断了VEGF诱导的TRPC1依赖的Ca2+内流，从而消除了内皮通透性的增加。
