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，造成全球20%的死亡。缺乏高度 有效的治疗方法使重症患者的全身器官功能障碍， 损伤血管内皮。我们的实验室已经表明，血管的驱动因素之一， 败血症中的功能障碍是循环氧化无细胞血红蛋白（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+）和氧化三价铁（3+，高铁血红蛋白）氧化的循环 forms.来自我们实验室的数据表明，只有氧化的3+形式的CFH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 线粒体功能障碍，如超氧化物产生增加和总 线粒体与脓毒症患者循环CFH增加平行， 循环细胞外线粒体DNA（mtDNA）在败血症。重要的是， 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。此外还 不知道mtDNA是作为自由可溶性分子释放出来的，还是在细胞内。 细胞外囊泡（EV）。了解mtDNA是否包含在EV中可以提供信息 循环mtDNA的潜在影响、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mtDNA从血管中释放的假说 这导致下游大血管屏障完整性的丧失。第一个目标是 该项目的重点是确定CFH诱导的氧化损伤及其 线粒体DNA从肺微血管内皮释放的作用。我们将评估 CFH对线粒体氧化损伤和通透性孔激活影响。此外，本发明还提供了一种方法， 我们将量化mtDNA是否是自由可溶的或包含在EV中，如果是， 抗氧化剂阻止这种分泌。该项目的第二个目标将决定 mtDNA对内皮屏障功能的影响。我们还将使用来自高血小板的贫血小板血浆。 脓毒症患者的特征性前瞻性队列，以定量循环mtDNA并与 死亡率、ARDS发展和内皮损伤标志物的水平。在 本研究的结论，我们将揭示一个新的分子机制CFH诱导 血管功能障碍，并描述了线粒体DNA的影响以及它是如何从血管中释放出来的。 内皮细胞这个拟议的项目将提供多学科的经验和增长， 建立一个成功的职业生涯作为一个机械和翻译科学家的基础。

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.