The aim of this grant is to elucidate the role of mitochondrial dynamics protein Drp1 as a novel redox sensor that transmits VEGF-derived H2O2 signaling to enhance angiogenesis via regulation of endothelial cell (EC) glycolysis. The induction of new blood vessels is critical for tissue repair in response to injury such as peripheral arterial disease (PAD), which is impaired in diabetes. Reactive oxygen species (ROS) such as H2O2 derived from NADPH oxidase (NOX) and mitochondria at normal level act as signaling molecules to promote VEGF-induced angiogenesis in endothelial cells (ECs) and reparative neovascularization. However, it remains unclear ?how diffusible H2O2 signal can be specifically transmitted to promote therapeutic angiogenesis?. Signaling function of ROS is mainly through oxidation of reactive Cys residues to generate ?Cysteine sulfenic acid (Cys-OH)? (sulfenylation) which is involved in disulfide bond formation with target protein and redox signaling. In addition, ECs utilize glycolysis as a major source of ATP to promote angiogenesis. However, the mechanistic link between NOX-mitochondrial ROS (mitoROS)/redox signaling and EC metabolism (glycolysis) in VEGF-induced angiogenesis is entirely unknown. Drp1 GTPase is key regulator of mitochondrial (mito) fission via its post translational modification, but its role in ROS dependent VEGFR2 signaling and angiogenesis in ECs and in vivo has never been reported. Our preliminary data are consistent with the hypothesis that VEGF induces sulfenylation of Drp1 via NOX-derived H2O2, which drives mito fission-mitoROS axis that promotes oxidative activation of key metabolic enzyme AMPK via disulfide bond formation (early phase) as well as PFKFB3 expression (late phase) in ECs. This in turn enhances endothelial glycolysis and angiogenesis required for restoring neovascularization in ischemic vascular disease. Aim1 will characterize the VEGF-induced Drp1 sulfenylation and establish its role in ROS-dependent angiogenic responses in ECs. Aim2 will determine the molecular mechanism by which VEGF-induced Drp1 sulfenylation promotes glycolysis via mitochondrial ROS-dependent manner in ECs. Aim 3 will determine the functional role of endothelial Drp1 in ROS-dependent reparative neovascularization and address underlying mechanisms in vivo using animal model of PAD (hindlimb ischemia model). We will also address how diabetes -induced excess ROS impair angiogenesis in ECs and in vivo by focusing on Drp1 phosphorylation at S616, but not Drp1-CysOH. We will use various innovative reagents, methods and mice including biotin-labelled Cys-OH trapping probe; BiFC-based protein-protein interaction in situ; real-time imaging of cytosol- and mitoROS using redox-sensitive biosensors; newly developed EC-specific Drp1-/- mice and CRISPR/Cas9-generated ?redox dead? Cys oxidation-defective Drp1 or AMPK knock-in mutant mice. Our proposal will provide novel mechanistic insights into Cys oxidized mitochondrial fission protein Drp1 that orchestrates NOX/mito ROS signaling and glycolysis as a potential therapeutic target for treatment of ischemic cardiovascular diseases.
- National Heart, Lung, and Blood Institute: $495,249.00
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