Mitochondrial dynamics protein Drp1 regulation of endothelial metabolism, ROS, and ischemic vascular disease

Project Summary: The goal of this Project 3 is to demonstrate the novel linkage between mitochondrial ROS (redox) signaling and endothelial cell (EC) glycolysis orchestrated by mitochondrial dynamics protein Drp1 sulfenylation that promotes reparative angiogenesis, which is impaired in diabetic peripheral arterial disease (PAD). Reactive oxygen species (ROS) such as H2O2 derived from NADPH oxidase (NOX) and mitochondria at physiological level act as signaling molecules to promote VEGF/VEGFR2-induced angiogenesis in ECs and neovascularization. By contrast, excess ROS induced in diabetes impairs reparative angiogenesis. Little is known “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 a key initial event in redox signaling. In addition, ECs utilize aerobic glycolysis via PFKFB3 as a major source of ATP to promote angiogenesis. However, the mechanistic link between NOX-mitochondrial ROS (mitoROS)/redox signaling and EC metabolism (glycolysis), which drives angiogenesis is poorly understood. Drp1 GTPase is a key regulator of mitochondrial (mito) fission and its activity is regulated by post-translational modification. However, the role of endothelial Drp1 in ROS-dependent VEGFR2 signaling and angiogenesis in ECs and reparative neovascularization in vivo is entirely unknown. Here we hypothesize that VEGF/NOX- derived H2O2 stimulates Drp1 sulfenylation to promote physiological mito fission and mito ROS production, which drives glycolysis and angiogenesis by oxidative activation of AMPK as well as increased PFKFB3 expression in ECs. This is impaired in T1D due to excess PFKFB3/glycolysis-ROS axis that induces oxidative inactivation of VEGFR2 as well as hyperactivation of Drp1-excess mitoROS axis in basal ECs, which limits VEGF-induced VEGFR2 signaling and angiogenesis as well as reparative neovascularization in PAD. Aim1 will determine the role of endothelial Drp1 sulfenylation in ROS-dependent VEGF-induced angiogenesis in ECs as well as reparative neovascularization in vivo and address the underlying mechanisms. Aim 2 will determine the role of excess glycolysis-ROS axis as a mechanism limiting VEGFR2 signaling and angiogenesis as well as reparative neovascularization in diabetes and address the underlying mechanisms. We will use innovative Cys-OH trapping probe; BiFC-based protein-protein interaction; compartment-specific redox-sensitive biosensors; iEC-iDrp1-/- or iPfkfb3-/- mice, Mito-Timer transgenic mice and newly developed cutting-edge CRISPR/Cas9-generated “redox dead” Cys oxidation-defective Drp1 or AMPK knock-in (KI) mutant mice or Glyco-Lo KI mutant mice that reduces glycolysis in the endothelium in an inducible manner. Our study will provide novel insights into the restoring imbalance between EC metabolism (glycolysis) and ROS signaling via targeting Drp1 as a potential therapeutic strategy for treatment of ischemic vascular diseases. Project 3 is highly dependent on the expertise provided by other Projects and Cores of this PPG.