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Mitochondria Dynamics Protein Drp1 in ROS Signaling, Endothelial Metabolism and Angiogenesis

线粒体动力学蛋白 Drp1 在 ROS 信号传导、内皮代谢和血管生成中的作用

基本信息

批准号：
10317794
负责人：
Masuko Ushio-Fukai
金额：
$ 49.52万
依托单位：
AUGUSTA UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-09-01 至 2025-07-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10317794
关键词：
5&apos -AMP-activated protein kinase AMP-activated protein kinase kinase Address Animal Model Attenuated Biological Assay Biosensor Biotin Blood Vessels CRISPR/Cas technology Cardiovascular Diseases Cellular Metabolic Process Cytosol Data Diabetes Mellitus Diffuse Disease Endothelial Cells Endothelium Enzymes Fractionation Future Gene Transfer Glycolysis Goals Grant Growth Guanosine Triphosphate Phosphohydrolases Hindlimb Human Hydrogen Peroxide Impairment In Situ Ischemia KDR gene Knock-in Knock-in Mouse Label Ligation Link Measures Metabolic Metabolism Methods Mitochondria Modeling Molecular Mus Muscle Mutant Strains Mice Myocardial Ischemia NADPH Oxidase Outer Mitochondrial Membrane Oxidation-Reduction Oxidative Phosphorylation Oxides Patients Peripheral arterial disease Phase Phosphorylation Physiological Play Post-Translational Protein Processing Process Production Protein Dynamics Proteins Reactive Oxygen Species Reagent Regulation Reporting Role Sampling Signal Transduction Signaling Molecule Site Source Transgenic Organisms Vascular Diseases Vascular Endothelial Growth Factors angiogenesis base cysteinesulfenic acid diabetic disulfide bond hypoxia inducible factor 1 in situ imaging in vivo innovation insight limb ischemia mutant neovascularization new therapeutic target novel overexpression oxidation postnatal protein protein interaction real-time images response response to injury sensor therapeutic angiogenesis therapeutic target tissue repair treatment strategy

项目摘要

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.

这项资助的目的是阐明线粒体动力学蛋白Drp1作为一种新型氧化还原传感器的作用