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:
- 金额:$ 49.52万
- 依托单位:
- 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:
- 关键词:5&apos-AMP-activated protein kinaseAMP-activated protein kinase kinaseAddressAnimal ModelAttenuatedBiological AssayBiosensorBiotinBlood VesselsCRISPR/Cas technologyCardiovascular DiseasesCellular Metabolic ProcessCytosolDataDiabetes MellitusDiffuseDiseaseEndothelial CellsEndotheliumEnzymesFractionationFutureGene TransferGlycolysisGoalsGrantGrowthGuanosine Triphosphate PhosphohydrolasesHindlimbHumanHydrogen PeroxideImpairmentIn SituIschemiaKDR geneKnock-inKnock-in MouseLabelLigationLinkMeasuresMetabolicMetabolismMethodsMitochondriaModelingMolecularMusMuscleMutant Strains MiceMyocardial IschemiaNADPH OxidaseOuter Mitochondrial MembraneOxidation-ReductionOxidative PhosphorylationOxidesPatientsPeripheral arterial diseasePhasePhosphorylationPhysiologicalPlayPost-Translational Protein ProcessingProcessProductionProtein DynamicsProteinsReactive Oxygen SpeciesReagentRegulationReportingRoleSamplingSignal TransductionSignaling MoleculeSiteSourceTransgenic OrganismsVascular DiseasesVascular Endothelial Growth Factorsangiogenesisbasecysteinesulfenic aciddiabeticdisulfide bondhypoxia inducible factor 1in situ imagingin vivoinnovationinsightlimb ischemiamutantneovascularizationnew therapeutic targetnoveloverexpressionoxidationpostnatalprotein protein interactionreal-time imagesresponseresponse to injurysensortherapeutic angiogenesistherapeutic targettissue repairtreatment 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.
该基金的目的是阐明线粒体动力学蛋白Drp 1作为一种新型氧化还原传感器的作用
通过调节血管内皮细胞,传递VEGF衍生的H2 O2信号,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,
外周动脉疾病(PAD),其在糖尿病中受损。活性氧(ROS),如H2 O2
来源于NADPH氧化酶(NOX)和线粒体的正常水平的信号分子,
VEGF诱导的内皮细胞(EC)血管生成和修复性新血管形成。但委员会仍
目前尚不清楚“可扩散的H2 O2信号如何特异性地传递以促进治疗性血管生成”。
活性氧的信号转导功能主要是通过氧化活性半胱氨酸残基生成“半胱氨酸次磺酸
酸(Cys-OH)”(亚磺酰化),其参与与靶蛋白的二硫键形成和氧化还原
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。但
NOX-线粒体ROS(mitoROS)/氧化还原信号传导和EC代谢(糖酵解)之间的机制联系
VEGF诱导的血管生成是完全未知的。Drp 1 GTdR是线粒体(mito)分裂的关键调节因子
但它在ROS依赖性VEGFR 2信号传导和内皮细胞血管生成中的作用
并且在体内从未报道过。我们的初步数据与以下假设一致:
通过NOX衍生的H2 O2诱导Drp 1的亚磺酰化,其驱动线粒体分裂-线粒体ROS轴,
通过二硫键形成促进关键代谢酶AMPK的氧化活化(早期)
以及PFKFB 3在EC中的表达(晚期)。这反过来又增强了内皮糖酵解,
在缺血性血管疾病中恢复新血管形成所需的血管生成。aim 1将
表征VEGF诱导的Drp 1亚磺酰化,并确定其在ROS依赖性血管生成中的作用
EC中的响应。Aim 2将决定VEGF诱导的Drp 1亚磺酰化的分子机制
通过线粒体ROS依赖的方式促进EC中的糖酵解。目标3将确定职能作用
内皮细胞Drp 1在ROS依赖性修复性新生血管形成中的作用,并阐明
体内使用PAD动物模型(后肢缺血模型)。我们还将讨论糖尿病引起的过量
ROS通过关注S616处的Drp 1磷酸化而不是Drp 1-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
生物传感器;新开发的EC特异性Drp 1-/-小鼠和CRISPR/Cas9产生的“氧化还原死”Cys
氧化缺陷型Drp 1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氧化线粒体分裂蛋白Drp 1,其协调NOX/mito ROS信号传导,
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(0)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
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Masuko Ushio-Fukai其他文献
Masuko Ushio-Fukai的其他文献
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{{ truncateString('Masuko Ushio-Fukai', 18)}}的其他基金
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 - 财政年份:2021
- 资助金额:
$ 49.52万 - 项目类别:
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:
10666540 - 财政年份:2021
- 资助金额:
$ 49.52万 - 项目类别:
Protein Disulfide Isomerase as Novel Redox Sensor in VEGF Signaling
蛋白质二硫键异构酶作为 VEGF 信号转导中的新型氧化还原传感器
- 批准号:
9479934 - 财政年份:2016
- 资助金额:
$ 49.52万 - 项目类别:
Role of Cysteine Sulfenic Acid Formation in Compartmentalization of VEGF Signalin
半胱氨酸磺酸形成在 VEGF 信号蛋白区室化中的作用
- 批准号:
8445715 - 财政年份:2013
- 资助金额:
$ 49.52万 - 项目类别:
Role of Cysteine Sulfenic Acid Formation in Compartmentalization of VEGF Signalin
半胱氨酸磺酸形成在 VEGF 信号蛋白区室化中的作用
- 批准号:
8620710 - 财政年份:2013
- 资助金额:
$ 49.52万 - 项目类别:
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