Protein Disulfide Isomerase as a Novel Redox Sensor in VEGF Signaling

Redox signaling plays an important role in angiogenesis which is required for treatment of ischemic heart/limb disease. Our lab and others demonstrated that reactive oxygen species (ROS) derived from NADPH oxidase (NOX) are involved in VEGF-induced angiogenesis in endothelial cells (ECs) and post-ischemic neovascularization. However, fundamental question remains 'how ROS signal is efficiently transmitted to promote therapeutic angiogenesis'. Signaling function of ROS is mediated through oxidation of reactive Cys residues which generate 'Cysteine sulfenic acid (Cys-OH)', a key intermediate involved in disulfide bond formation and redox signaling. 'Protein disulfide isomerase (PDI)', a key redox sensor, functions as a reductase, oxidase, and isomerase. PDIA1 is a major PDI isoform with reactive Cys residues in active domains. 'Oxidized' PDI acts as an oxidase to promote disulfide bond formation with specific substrates to regulate their activity. However, role of PDIA1 in VEGF signaling and post-ischemic neovascularization has never been investigated. Our preliminary data show that PDIA1 is upregulated in angiogenic ECs in mouse hindlimb ischemia model and that PDIA1+/- mice show impaired post-ischemic neovascularization. In ECs, PDIA1 knockdown inhibits VEGF-induced angiogenesis without affecting ROS production. Mechanistically, VEGF increases Cys-OH formation of PDIA1, which promotes Cys oxidation/activation of AMPK, a key redox sensor and regulator of VEGF-induced EC metabolism and angiogenesis. Thus, we hypothesize that PDIA1 functions as a key redox sensor to transduce VEGF-induced H2O2 signal to promote oxidative activation of AMPK via disulfide bond formation, which is required for enhancing angiogenesis in ECs and restoring neovascularization in ischemic vascular diseases. Aim 1 will examine whether VEGF-induced Cys oxidation of PDIA1 by NOX-derived H2O2 promotes Cys oxidation and activation of AMPK via their disulfide bond formation, which enhances angiogenesis in ECs. Aim 2 will examine whether endothelial PDIA1 promotes reparative neovascularization through Cys oxidation of AMPK using hindlimb ischemia model. We will use EC-specific PDIA1 deficient mice; biotin-labelled Cys-OH trapping probe, BiFC-based molecular interaction imaging. Our study will provide novel insight into Cys oxidized PDIA1 and AMPK and/or their redox-sensitive molecular interaction, as potential therapeutic targets for treatment of ischemic cardiovascular disease. (AHA Program: Grant-in-Aid)