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：
10666540
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/10666540
关键词：
5&apos -AMP-activated protein kinase AMP-activated protein kinase kinase Address Animal Model Biological Assay Biosensor Biotin Blood Vessels CRISPR/Cas technology Cardiovascular Diseases Cell Separation Cellular Metabolic Process Cytosol Data Diabetes Mellitus 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 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 Mice Vascular Diseases Vascular Endothelial Growth Factors angiogenesis cysteinesulfenic acid diabetic disulfide bond 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 transmission process 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.

该赠款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这通过调节内皮细胞来传输VEGF衍生的H2O2信号传导，以增强血管生成（EC）糖酵解。诱导新血管对于响应损伤的组织修复至关重要周围动脉疾病（PAD），糖尿病受损。活性氧（ROS），例如H2O2 源自正常水平的NADPH氧化酶（NOX）和线粒体作为信号分子的促进 VEGF诱导的内皮细胞（EC）和修复新生血管的血管生成。但是，它仍然是不清楚“如何专门传播可扩散的H2O2信号来促进治疗性血管生成”。 ROS的信号传导函数主要是通过氧化反应性Cys保留以产生“半胱氨酸硫酸盐酸（Cys-OH）”（磺苯基），与靶蛋白和氧化还原参与二硫键形成信号。此外，ECS利用糖酵解作为ATP的主要来源来促进血管生成。但是， NOX-Mitochrial ROS（Moritoros）/氧化还原信号传导与EC代谢（糖酵解）之间的机械联系在VEGF诱导的血管生成中，完全未知。 DRP1 GTPase是线粒体（mito）裂变的关键调节剂通过其后翻译后的修改，但在EC中的ROS依赖性VEGFR2信号传导和血管生成中的作用体内从未有过报道。我们的初步数据与VEGF的假设一致通过NOX衍生的H2O2诱导DRP1的磺苯基化，该H2O2驱动Mito裂变轴轴轴通过二硫键形成（早期）促进关键代谢酶AMPK的氧化激活以及EC中的PFKFB3表达（后期）。这反过来增强了内皮糖酵解和恢复缺血性血管疾病中的新血管形成所需的血管生成。 AIM1将表征VEGF诱导的DRP1磺酰化，并在ROS依赖性血管生成中确定其作用 EC中的响应。 AIM2将确定VEGF诱导的DRP1磺酰化的分子机制通过线粒体ROS依赖性方式促进糖酵解。 AIM 3将决定功能角色内皮drp1在ROS依赖性的修复新血管形成和解决基础机制中使用PAD动物模型（后肢缺血模型）的体内。我们还将解决糖尿病诱导的如何超过 ROS在EC和体内损害了血管生成，通过将DRP1磷酸化在S616时，而不是DRP1-CYSOH。我们将使用各种创新试剂，方法和小鼠，包括生物素标记的Cys-OH捕获探针；基于BIFC的蛋白质 - 蛋白质相互作用原位；使用氧化还原敏感的细胞质和身体成像的实时成像生物传感器；新开发的EC特异性DRP1 - / - 小鼠和CRISPR/CAS9生成的“氧化还原” cys 氧化缺陷的DRP1或AMPK敲入突变小鼠。我们的建议将提供新颖的机械对CYS的洞察力氧化的线粒体裂变蛋白DRP1，该蛋白质drp1调整了NOX/MITO ROS信号和糖酵解作为治疗缺血性心血管疾病的潜在治疗靶点。