Abstract 12127: Protein Disulfide Isomerase A1 as Novel Redox Sensor in VEGFR2 Signaling and Angiogenesis

Abstract

Background: VEGFR2 signaling in endothelial cells (ECs) is regulated by reactive oxygen species (ROS) derived from NADPH oxidases (NOXs) and mitochondria, which plays an important role in postnatal angiogenesis. However, it remains unclear how highly diffusible ROS signal enhances VEGFR2 signaling and reparative angiogenesis. Protein disulfide isomerase A1 (PDIA1) functions as an oxidoreductase depending on the redox environment. However, role of PDIA1 in ROS-dependent VEGFR2 signaling and angiogenesis is entirely unknown. Results: Here we showed that PDIA1 co-immunoprecipitated with VEGFR2 or colocalized with either VEGFR2 or early endosome marker Rab5, but not late endosome marker Rab7, at the perinuclear region in human ECs in response to VEGF. PDIA1 silencing significantly reduced VEGF-induced EC migration (43.3%), proliferation (52.8%) and spheroid sprouting via inhibiting VEGFR2 signaling. Mechanistically, VEGF stimulation rapidly increased Cys-OH formation of PDIA1 via the NOX4-mitochondrial ROS axis. Experiments using “redox-dead” mutant PDIA1 with replacement of the active four Cys residues with Ser revealed that VEGF-induced PDIA1 CysOH formation promoted angiogenic responses by increasing phosphorylation of VEGFR2 at Y1175 via oxidative inactivation of PTP1B. In vivo, Pdia1+/- mice showed impaired angiogenesis in developmental retina and Matrigel plug models as well as ex vivo aortic ring sprouting model. Hindlimb ischemia model revealed that PDIA1 expression was markedly increased in angiogenic CD31 positive ECs of ischemic muscles, and that limb perfusion recovery and neovascularization (48%) in response to ischemic injury were significantly impaired in EC-specific Pdia1 conditional knockout mice. Conclusion: PDIA1 can sense VEGF-induced H2O2 signal via CysOH formation to promote VEGFR2 signaling and angiogenesis in ECs via oxidative inactivation of PTP1B, thereby enhancing postnatal angiogenesis. The oxidized PDIA1 is a potential therapeutic target for treatment of ischemic vascular diseases