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.

该资助的目的是阐明线粒体动力学蛋白 Drp1 作为新型氧化还原传感器的作用 传递 VEGF 衍生的 H2O2 信号，通过调节内皮细胞增强血管生成 （EC）糖酵解。新血管的诱导对于响应损伤的组织修复至关重要，例如 外周动脉疾病（PAD）在糖尿病中受损。活性氧 (ROS)，例如 H2O2 源自 NADPH 氧化酶 (NOX) 和正常水平的线粒体作为信号分子，促进 VEGF 诱导内皮细胞 (EC) 血管生成和修复性新血管形成。然而，它仍然 尚不清楚“如何特异性传递可扩散的 H2O2 信号以促进治疗性血管生成”。 ROS的信号传导功能主要是通过氧化反应性Cys残基生成“半胱氨酸磺酸” 酸（Cys-OH）”（磺酰化），参与与目标蛋白形成二硫键和氧化还原 发信号。此外，EC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)/氧化还原信号传导与 EC 代谢（糖酵解）之间的机制联系 VEGF 诱导的血管生成的作用尚不清楚。 Drp1 GTPase 是线粒体 (mito) 裂变的关键调节因子 通过其翻译后修饰，但其在 EC 中 ROS 依赖性 VEGFR2 信号传导和血管生成中的作用 且在体内尚未见报道。我们的初步数据与 VEGF 的假设一致 通过 NOX 衍生的 H2O2 诱导 Drp1 的磺酰化，从而驱动 mito 裂变 -mitoROS 轴 通过二硫键形成促进关键代谢酶 AMPK 的氧化激活（早期） 以及 EC 中的 PFKFB3 表达（晚期）。这反过来又增强内皮糖酵解和 恢复缺血性血管疾病中的新血管形成所需的血管生成。目标1将 表征 VEGF 诱导的 Drp1 磺酰化并确定其在 RO​​S 依赖性血管生成中的作用 EC 中的反应。 Aim2 将确定 VEGF 诱导 Drp1 磺酰化的分子机制 在 EC 中通过线粒体 ROS 依赖性方式促进糖酵解。目标 3 将确定职能角色 内皮 Drp1 在 ROS 依赖性修复性新血管形成中的作用并解决其潜在机制 体内使用PAD动物模型（后肢缺血模型）。我们还将讨论糖尿病如何引起过量 ROS 通过关注 Drp1 S616 的磷酸化而不是 Drp1-CysOH 来损害 EC 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；使用氧化还原敏感的细胞质和线粒体ROS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“氧化还原死亡”Cys 氧化缺陷型 Drp1 或 AMPK 敲入突变小鼠。我们的建议将提供新颖的机制 深入了解 Cys 氧化线粒体裂变蛋白 Drp1，该蛋白协调 NOX/mito ROS 信号传导和 糖酵解作为治疗缺血性心血管疾病的潜在治疗靶点。