Cell-Free Hemoglobin Induces Endothelial Mitochondrial Oxidative Damage Causing Extracellular DNA Release

项目摘要 脓毒症是世界各地的一个严重问题，导致全球20%的死亡。缺乏高度的 有效的治疗方法使危重病患者经常出现全身器官功能障碍 损伤血管内皮细胞。我们的实验室已经证明，血管的其中一个驱动因素 脓毒症的功能障碍是循环中氧化的无细胞血红蛋白(CFH)。在败血症的情况下， 红细胞变得越来越脆弱，导致CFH溶解并释放到血管中 允许亚铁(2+)和氧化铁(3+，高铁血红蛋白)氧化的循环 表格。我们实验室的数据表明，只有氧化的CFH的3+形式才能诱导 微血管屏障功能障碍。然而，支持这一点的细胞内机制 功能障碍还没有得到很好的理解。我的初步研究表明，氧化的CFH导致 线粒体功能障碍，如超氧化物生成增加和总 线粒体。脓毒症患者循环CFH增加的同时， 脓毒症时循环细胞外线粒体DNA(MtDNA)重要的是，这种机制 脓毒症期间线粒体DNA的潜在释放仍然是一个关键的知识缺口。此外，它是 尚不清楚线粒体DNA是以可溶分子的形式释放，还是在体内 细胞外小泡(EVS)。了解电动汽车内部是否含有线粒体DNA可以提供信息 循环线粒体DNA的潜在作用、分布和稳定性。在这个项目中，我将测试 CFH诱导的氧化损伤导致血管释放线粒体DNA的假说 内皮导致下游大血管屏障完整性丧失。这样做的第一个目的是 该项目的重点是确定CFH诱导的氧化损伤背后的机制及其 肺微血管内皮细胞线粒体DNA释放的作用。我们将评估 CFH对线粒体氧化损伤和通透性孔激活的影响。此外, 我们将量化mtDNA是可自由溶解的还是包含在电动汽车中，以及 抗氧化剂会阻止这种分泌。该项目的第二个目标将决定 线粒体DNA对内皮细胞屏障功能的影响我们还将使用高度缺乏血小板的血浆 描述脓毒症患者的前瞻性队列以量化循环mtDNA并进行相关性 与死亡率、ARDS发展和内皮损伤标志物相关的水平。在 本研究的结论是，我们将揭示一种新的CFH诱导分子机制 血管功能障碍，并表征了线粒体DNA的影响和它是如何从 内皮细胞。这一拟议的项目将提供多学科经验和增长，以 作为一名机械和翻译科学家，为成功的职业生涯奠定基础。

PROJECT ABSTRACT Sepsis is a critical problem around the world causing 20% of all global deaths. The lack of highly effective therapeutics leaves critically ill patients with systemic organ dysfunction often caused damage the vascular endothelium. Our lab has shown that one of the drivers of vascular dysfunction in sepsis is circulating oxidized cell-free hemoglobin (CFH). During septic conditions, red blood cells become increasingly fragile leading to lysis and release of CFH into the vascular circulation allowing for oxidation from ferrous (2+) and oxidized ferric (3+, methemoglobin) forms. Data from our lab demonstrates that only the oxidized 3+ form of CFH induces microvascular barrier dysfunction. However, the intracellular mechanisms underpinning this dysfunction are not well understood. My preliminary studies suggest that oxidized CFH causes mitochondrial dysfunction such as increased superoxide production and loss of total mitochondria. In parallel to circulating CFH being increased in septic patients, there is increased circulating extracellular mitochondrial DNA (mtDNA) during sepsis. Importantly, the mechanism underlying release of mtDNA during sepsis remains a key knowledge gap. In addition, it is unknown whether the mtDNA is released as freely soluble molecules or if it is inside extracellular vesicles (EVs). Understanding if mtDNA is contained inside EVs could inform potential effects, distribution, and stability of the circulating mtDNA. In this project, I will test the hypothesis that CFH-induced oxidative damage causes mtDNA release from the vascular endothelium leading to downstream loss of macrovascular barrier integrity. The first aim of this project focuses on identifying the mechanisms behind CFH-induced oxidative damage and its role in mtDNA release from the pulmonary microvascular endothelium. We will evaluate the impact of CFH on mitochondrial oxidative damage and permeability pore activation. In addition, we will quantify whether the mtDNA is freely soluble or contained inside EVs, and if antioxidants block this secretion. The second aim of the project will determine the effect of mtDNA on endothelial barrier function. We will also use platelet poor plasma from a highly characterized prospective cohort of sepsis patients to quantify circulating mtDNA and correlate levels with mortality, ARDS development, and markers of endothelial damage. At the conclusion of this study, we will have uncovered a novel molecular mechanism of CFH induced vascular dysfunction, and characterized both the effects of mtDNA and how it is released from endothelial cells. This proposed project will provide multidisciplinary experience and growth to establish the foundation for a successful career as a mechanistic and translational scientist.