Angiopoietin-1 opposes VEGF-induced increase in endothelial permeability by inhibiting TRPC1-dependent Ca2 influx.

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.