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，通过调节内皮细胞来增强血管生成 (EC) 糖酵解对于响应损伤（例如，损伤）的组织修复至关重要。外周动脉疾病 (PAD)，在糖尿病中会受到活性氧 (ROS) 的影响，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，促进然而，VEGF 诱导的内皮细胞 (EC) 血管生成和修复性新血管形成仍然存在。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此外，EC 利用糖酵解作为 ATP 的主要来源来促进血管生成。 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 Drp1 GTPase 在 VEGF 诱导的血管生成中的作用尚不清楚。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 Aim1 恢复缺血性血管疾病中新血管形成所需的血管生成。表征 VEGF 诱导的 Drp1 磺酰化并确定其在 ROS 依赖性血管生成中的作用 ECs 中的反应将决定 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 将决定其功能作用。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；使用氧化还原敏感的基于 BiFC 的蛋白质-蛋白质相互作用原位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 氧化缺陷的 Drp1 或 AMPK 敲入突变小鼠我们的建议将提供新的机制。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。

项目成果

期刊论文数量（1）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Embracing Diversity, Equity, and Inclusion in the Scientific Community-Viewpoints of the Diversity, Equity, and Inclusion Committee of the North American Vascular Biology Organization.

在科学界拥抱多样性、公平性和包容性——北美血管生物学组织多样性、公平性和包容性委员会的观点。

DOI：
发表时间：
2022
期刊：
影响因子：
0
作者：
Garelnabi, Mahdi;Cowdin, Mitzy;Fang, Yun;Shrestha, Bandana;Ushio;Aikawa, Elena;Graham, Garth;Molema, Grietje;Yanagisawa, Hiromi;Aikawa, Masanori
通讯作者：
Aikawa, Masanori