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

• 批准号: 10475228
• 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
• 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/10475228
• 关键词: 5' -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

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作为一种新的氧化还原感受器的作用。 传递血管内皮生长因子衍生的过氧化氢信号，通过调节内皮细胞促进血管生成 (EC)糖酵解。新血管的诱导对于组织修复是至关重要的，因为损伤的反应包括 外周动脉疾病(PAD)，在糖尿病中受损。过氧化氢等活性氧物种(ROS) 来源于NADPH氧化酶(NOX)和线粒体在正常水平上作为信号分子促进 血管内皮生长因子诱导内皮细胞血管生成和修复性新生血管。然而，它仍然 尚不清楚“可扩散的过氧化氢信号如何被特定地传递以促进治疗性血管生成”。 ROS的信号转导功能主要是通过氧化活性半胱氨酸残基生成半胱氨酸亚磺酸 酸(Cys-OH)“(亚磺化)，参与与目标蛋白和氧化还原形成二硫键 发信号。此外，内皮细胞利用糖酵解作为ATP的主要来源来促进血管生成。然而， NOX-线粒体ROS(MitoROS)/氧化还原信号与EC代谢(糖酵解)之间的机制联系 在血管内皮生长因子诱导的血管生成中，我们完全不知道。Drp1 GTP酶是线粒体(Mito)分裂的关键调节因子 通过其翻译后修饰，但其在ROS依赖的血管内皮细胞VEGFR2信号和血管生成中的作用 而在活体内也从未报道过。我们的初步数据与血管内皮细胞生长因子 通过NOX衍生的过氧化氢诱导Drp1的硫苯化，从而驱动mito分裂-mitoROS轴，从而 通过形成二硫键促进关键代谢酶AMPK的氧化激活(早期) 以及PFKFB3在内皮细胞中的表达(晚期)。这反过来又增强了内皮糖酵解，并 在缺血性血管疾病中恢复新生血管所需的血管生成。Aim1将 血管内皮生长因子诱导的DRp1亚磺化及其在ROS依赖的血管生成中的作用 在ECS中的反应。AIM2将确定血管内皮生长因子诱导Drp1亚磺化的分子机制 内皮细胞通过线粒体ROS依赖的方式促进糖酵解。目标3将确定职能角色 内皮细胞DRp1在ROS依赖的修复性新生血管中的表达及其潜在机制 活体采用PAD动物模型(后肢缺血模型)。我们还将解决糖尿病引起的过度行为 ROS通过关注S616上的Drp1磷酸化，而不是Drp1-CysOH，从而损害内皮细胞和体内的血管生成。 我们将使用各种创新的试剂、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-蛋白质原位相互作用；利用氧化还原敏感技术对胞浆和有丝分裂原进行实时成像 生物传感器；新开发的EC特异性Drp1/-小鼠和CRISPR/Cas9产生的“氧化还原死亡”半胱氨酸 氧化缺陷的Drp1或AMPK敲入突变小鼠。我们的提议将提供新的机制 对Cys氧化的线粒体分裂蛋白Drp1的洞察，该蛋白协调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。