Role of mitochondrial dysfunction in hyperoxia-induced pulmonary vascular endothelial injury

线粒体功能障碍在高氧诱导的肺血管内皮损伤中的作用

• 批准号: 10455405
• 负责人: ELIZABETH R JACOBS
• 金额: --
• 依托单位: CLEMENT J. ZABLOCKI VA MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-04-01 至 2023-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10455405
• 关键词: Acute Lung Injury; Alcohol abuse; Animals; Blood; Blood Vessels; Breathing; Cells; Cessation of life; Chimeric Proteins; Chronic lung disease; Critical Illness; Cultured Cells; DNA; DNA Damage; Data; Edema; Electrical Resistance; Endothelial Cells; Endothelium; Environment; Exhibits; Experimental Designs; Exposure to; Extravasation; Filtration; Functional disorder; Fusion Protein Expression; Gases; Hydrogen; Hyperoxia; Image; Inhalation; Injury; Intervention; Investigation; Label; Lead; Lentivirus; Link; Liquid substance; Lung; Lung Capacity; Lung diseases; Measures; Mechanical ventilation; Mediating; Microvascular Permeability; Mitochondria; Mitochondrial DNA; Molecular; Morbidity - disease rate; Mus; Nuclear; Obesity; Oxidative Stress; Oxygen; Patients; Permeability; Prevention; Prevention approach; Production; Proteins; Pulmonary Edema; Reactive Oxygen Species; Recombinants; Risk; Rodent; Role; Secondary to; Signal Transduction; Site; Small Interfering RNA; Stress; Structure; Testing; Time; Tissues; Vascular Endothelium; Veterans; Work; animal tissue; base; clinically relevant; genetic manipulation; human tissue; hyperoxia induced lung injury; imaging biomarker; in vivo; in vivo imaging; military veteran; mitochondrial dysfunction; mitochondrial membrane; monolayer; mortality; novel; overexpression; protein expression; repair enzyme; repaired; response; response to injury; single photon emission computed tomography; targeted nucleases; therapeutic target; tool

More than 250,000 veterans are placed on mechanical ventilation annually. These patients often require high fractions of oxygen (hyperoxia) which significantly exacerbates the injury that triggered mechanical ventilation initially. Pulmonary endothelial cells (PECs) are particularly sensitive to hyperoxia, exhibiting increased production rates of mitochondrially-derived reactive oxygen species (mtROS), mitochondrial (mt) dysfunction, pulmonary edema and ultimately increased morbidity/mortality in critically ill patients, but mechanisms are incompletely understood. Cells adapt to stress by increasing both mitochondrial fission and fusion. Our data identify for the first time hyperoxia-enhanced mt-fragmentation in PECs, and decreased expression of mt- fusion and increased expression of mt-fission promoting proteins which underlie the increased mt- fragmentation. In addition, we show that mitochondrial targeted endonuclease repair protein (mt-ENDO-III) protects from hyperoxic PEC loss. Finally, we have demonstrated that inhaled 2% hydrogen gas (H2) can protect against hyperoxia-induced lung injury, and that this protection can be identified by single photon emission computed tomography (SPECT) imaging. The molecular basis of hyperoxia-associated mt- fragmentation and subsequent pulmonary microvascular permeability is the focus of this proposal. Our hypothesis is that hyperoxia-induced pulmonary edema results from mtDNA damage which signals a shift to pro-fission protein expression and mt-fragmentation, leading to increased microvascular permeability and edema. Furthermore, we believe that 2% H2 in atmospheric gases will counteract hyperoxia-evoked pulmonary edema with diminished mtDNA damage and mt-fragmentation. Using novel tools including Dendra- 2 mice, which express a fluorescent protein targeted to the mitochondrial membrane in endothelial cells to quantify mt-fragmentation in intact tissue, recombinant adeno- and lentivirus, siRNA, unique genetically modified rodents, vertical experimental designs from cultured cells to intact animals and human tissue, our work will determine mechanisms linking hyperoxia-induced mtDNA damage, mt-fragmentation and pulmonary edema. Specific Aims: 1) To determine if hyperoxia-induced pulmonary endothelial mtDNA damage modifies expression/activation ratios of specific mt-fission and fusion proteins, thereby enhancing mt-fragmentation, and increasing microvascular permeability. We will use mt-ENDO-III to repair mtDNA damage in cultured PECs and in vivo and measure hyperoxia-induced changes in pro-fission or fusion protein expression, mt-fragmentation, mt-function, monolayer transendothelial electrical resistance (TEER) or filtration coefficient (Kf) as measures of endothelial permeability. 2) To test if pulmonary endothelial mt-fragmentation, independent of mtDNA damage, increases microvascular permeability. Using genetically modified rodents, siRNA and overexpression of pro-fission or fusion protein in cultured PECs and in vivo, we will measure pulmonary endothelial mt-fragmentation, mt-function, and Kf in intact lungs or TEER in cultured PECs. 3) To determine if (i) H2 protects from hyperoxia-induced mtDNA damage, increased mt-fragmentation, or increased microvascular permeability, and (ii) SPECT imaging can identify protection secondary to limited mtDNA damage, diminished mt-fragmentation, or diminished microvascular permeability in vivo. In this translational aim, we will assess the effect of 2% H2 on mtDNA integrity, shifts in pro-fission/fusion proteins, and mt- fragmentation. Genetic manipulations of fission/fusion proteins or ENDO-III will be employed to modify mt- fragmentation, and in vivo SPECT imaging markers of death or oxidoreductive state will identify clinically relevant endpoints associated with changes in pulmonary endothelial mt-fragmentation. Key results will be confirmed in human tissue. This work will provide critical new information about the role of mitochondrial damage in mediating hyperoxia-induced changes in pulmonary microvascular permeability and is expected to lead to mechanism-based approaches to the prevention and treatment of hyperoxia-induced lung disease.

每年有超过25万名退伍军人接受机械通气。这些患者往往需要高 氧分率（高氧），显著加剧了触发机械通气的损伤 一开始肺内皮细胞（佩奇）对高氧特别敏感，表现出增加的氧浓度。 脑源性活性氧（mtROS）的产生率，线粒体（mt）功能障碍， 肺水肿，并最终增加危重患者的发病率/死亡率，但机制是 不完全理解。细胞通过增加线粒体分裂和融合来适应压力。我们的数据 首次发现佩奇中高氧增强的mt片段化，以及mt表达降低， 融合和增加的MT-分裂促进蛋白的表达，这是MT-分裂促进蛋白增加的基础。 碎片化此外，我们发现线粒体靶向核酸内切酶修复蛋白（mt-ENDO-III） 防止高氧PEC损失。最后，我们已经证明，吸入2%的氢气（H2）可以 保护免受高氧诱导的肺损伤，并且这种保护可以通过单光子识别 发射计算机断层扫描（SPECT）成像。高氧相关mt-的分子基础 碎片化和随后的肺微血管渗透性是该提议的焦点。 我们的假设是高氧引起的肺水肿是由线粒体DNA损伤引起的， 促分裂蛋白表达和线粒体断裂，导致微血管通透性增加， 水肿此外，我们认为，大气中2%的H2将抵消高氧诱发的 肺水肿，线粒体DNA损伤和线粒体碎片减少。使用包括Dendra在内的新工具- 2只小鼠，表达靶向内皮细胞线粒体膜的荧光蛋白， 定量完整组织中的mt片段化，重组腺病毒和慢病毒，siRNA，独特的遗传学 改良啮齿动物，从培养细胞到完整动物和人体组织的垂直实验设计， 这项工作将确定高氧诱导的线粒体DNA损伤、线粒体断裂和肺损伤之间的联系机制。 水肿具体目的：1）确定高氧诱导的肺内皮细胞mtDNA损伤是否改变了 特异性mt-分裂和融合蛋白的表达/活化比率，从而增强mt-片段化，和 增加微血管渗透性。我们将使用mt-ENDO-III来修复培养的佩奇中的mtDNA损伤， 并测量在促分裂或融合蛋白表达、MT-片段化 mt函数、单层跨内皮电阻（TEER）或滤过系数（Kf）， 内皮渗透性的测量。2)检测肺内皮细胞线粒体碎片是否独立于 线粒体DNA损伤，增加微血管通透性。使用转基因啮齿动物，siRNA和 在培养的佩奇和体内，我们将测量肺组织中促分裂或融合蛋白的过度表达， 完整肺中的内皮mt-断裂、mt-功能和Kf或培养佩奇中的TEER。3)以确定是否 (i)H2保护免受高氧诱导的mtDNA损伤，增加线粒体碎片化，或增加 微血管通透性，以及（ii）SPECT成像可以识别继发于有限mtDNA的保护作用 损伤、减少的MT碎裂或减少的体内微血管通透性。在这个翻译 目的，我们将评估2%H2对mtDNA完整性、促分裂/融合蛋白的变化以及线粒体- 碎片化将采用裂变/融合蛋白或ENDO-III的遗传操作来修饰mt-1。 碎片，并且死亡或氧化还原状态的体内SPECT成像标记物将在临床上识别 与肺内皮MT片段化变化相关的相关终点。主要成果将是 在人体组织中得到证实这项工作将提供关于线粒体的作用的重要新信息， 在介导高氧诱导的肺微血管通透性变化中的损伤， 导致以机制为基础的方法来预防和治疗高氧诱导的肺部疾病。

项目成果

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• DOI: 10.1007/s12192-021-01230-4
• 发表时间: 2021-09
• 期刊: Cell stress & chaperones
• 影响因子: 3.8
• 作者: Puzyrenko A;Jacobs ER;Sun Y;Felix JC;Sheinin Y;Ge L;Lai S;Dai Q;Gantner BN;Nanchal R;North PE;Simpson PM;Rui H;Benjamin IJ
• 通讯作者: Benjamin IJ

Polypharmacy to Mitigate Acute and Delayed Radiation Syndromes.

• DOI: 10.3389/fphar.2021.634477
• 发表时间: 2021
• 期刊: Frontiers in pharmacology
• 影响因子: 5.6
• 作者: Gasperetti T;Miller T;Gao F;Narayanan J;Jacobs ER;Szabo A;Cox GN;Orschell CM;Fish BL;Medhora M
• 通讯作者: Medhora M