Angiopoietin-1 Opposes VEGF-Induced Increase in Endothelial Permeability by Inhibiting TRPC1-Dependent Ca 2 Influx

Abstract

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.