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风。这些患者通常需要很高的 氧的几个部分(高氧)，它显著加剧了引发机械通气的损伤 最初是这样。肺内皮细胞(Pec)对高氧特别敏感，表现为 线粒体衍生的活性氧(MtROS)的生成率，线粒体(Mt)功能障碍， 危重患者的肺水肿和最终增加的发病率/死亡率，但其机制是 不完全理解。细胞通过增加线粒体的分裂和融合来适应压力。我们的数据 首次发现高氧增强的PECs线粒体碎裂，并降低mt-1的表达 Mt-裂解促进蛋白的融合和表达增加是mt-ras增加的基础。 碎片化。此外，我们还发现线粒体靶向核酸内切酶修复蛋白(mt-endo-III) 防止高氧性PEC损失。最后，我们证明了吸入2%的氢气(H2)可以 防止高氧诱导的肺损伤，这种保护可以通过单光子来识别 发射计算机断层扫描(SPECT)成像。高氧相关线粒体DNA损伤的分子基础 碎裂和随后的肺微血管通透性是这项提议的重点。 我们的假设是，高氧引起的肺水肿是线粒体DNA损伤的结果，线粒体DNA损伤是转变的信号。 促进裂解蛋白表达和线粒体碎裂，导致微血管通透性增加和 浮肿。此外，我们相信，大气中2%的氢气将抵消高氧引起的 肺水肿，线粒体DNA损伤和线粒体碎裂减少。使用包括Dendra在内的新工具- 2小鼠，表达针对内皮细胞线粒体膜的荧光蛋白，以 量化完整组织、重组腺病毒和慢病毒、siRNA中独特的遗传基因的mt-片段 改良的啮齿动物，从培养细胞到完整的动物和人体组织的垂直实验设计，我们的 工作将确定高氧诱导的线粒体DNA损伤、线粒体碎裂和肺组织之间的联系机制 浮肿。具体目的：1)确定高氧诱导的肺内皮细胞线粒体DNA损伤是否改变 特定mt-裂解蛋白和融合蛋白的表达/激活比率，从而增强mt-碎裂，以及 增加微血管通透性。我们将使用mt-endo-III修复培养的PECs和 在体内并测量高氧诱导的裂变或融合蛋白表达的变化，mt-片段， MT功能，单层经内皮电阻(TEER)或滤过系数(KF)作为 内皮通透性的测量。2)测试肺内皮细胞mt-碎裂是否独立于 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PECs和活体中过表达裂解或融合蛋白，我们将测量肺组织 完整肺的内皮线粒体碎裂、线粒体功能和KF或培养的PECs的TEER。3)确定是否 (I)氢气保护免受高氧诱导的线粒体DNA损伤、线粒体片段化增加或增加 微血管通透性，以及(Ii)SPECT成像可以识别有限mtDNA的继发性保护 损伤，减少线粒体碎裂，或体内微血管通透性降低。在这个翻译版本中 目的：我们将评估2%氢气对线粒体DNA完整性、分裂/融合蛋白位移和线粒体DNA损伤的影响。 碎片化。分裂/融合蛋白或Endo-III的遗传操作将被用来修饰mt-III。 碎裂和体内死亡或氧化还原状态的SPECT成像标记物将在临床上识别 相关终点与肺内皮细胞mt-碎裂改变相关。主要结果将是 在人体组织中得到证实。这项工作将提供有关线粒体作用的关键新信息 损伤在介导高氧诱导的肺微血管通透性改变中的作用 导致以机制为基础的方法来预防和治疗高氧引起的肺部疾病。

项目成果

Pneumocytes are distinguished by highly elevated expression of the ER stress biomarker GRP78, a co-receptor for SARS-CoV-2, in COVID-19 autopsies.

• 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