Hemoglobin in ARDS: a novel mediator of aveolar epithelial cell dysfunction

描述（由申请人提供）：ARDS是美国发病和死亡的主要原因。除了保护性机械通气和保守的液体管理外，ARDS没有其他特异性治疗方法，死亡率仍然高达30-50%。因此，迫切需要针对ARDS基本机制的特异性治疗。肺上皮通透性增加是ARDS的病理生理标志，临床表现为肺水肿、气体交换受损和急性呼吸衰竭。我们已经确定细胞游离血红蛋白是ARDS中上皮通透性增加的新介质。我们的初步数据显示，ARDS患者的空腔游离血红蛋白水平较高，与肺通透性增加和临床预后差有关。此外，在ARDS中，血红蛋白的铁酰氧化形式在空气中升高。在一项针对严重脓毒症（ARDS的主要原因）的临床试验中，使用铁酰血红蛋白抑制剂对乙酰氨基酚治疗可降低脂质过氧化和减轻急性肾损伤。在培养的肺上皮细胞中，血红蛋白处理通过细胞和线粒体活性氧的产生诱导上皮屏障通透性。此外，气管内给药小鼠肺部无细胞血红蛋白会导致氧化铁蛋白的产生，并增加肺泡毛细血管屏障的通透性。这些发现支持了本研究的总体假设，即急性呼吸窘迫综合征空域中Ferryl-Hgb的形成导致肺泡上皮细胞氧化应激和上皮通透性增加，参与急性呼吸窘迫综合征的病理生理。为了验证这一假设，我组建了一个跨学科的共同研究人员和顾问团队，他们在临床ARDS （Ware）、Hgb氧化还原化学（Roberts）、初级肺泡上皮细胞分离和培养（Guttentag）、线粒体氧化应激（Dikalov）、ARDS人类模型（Matthay）和先进的体内成像技术（West）方面具有专业知识，他们的专业知识补充了我自己在上皮通透性细胞模型和ARDS动物模型方面的专业知识。我们将利用从急性呼吸窘迫综合征和静压性肺水肿患者收集的肺水肿液临床样本，以及小鼠和上皮细胞培养研究，表明人类急性呼吸窘迫综合征患者空气中铁蛋白水平升高，并导致小鼠和培养的上皮细胞肺上皮通透性增加（目的1）。我们将定义血红蛋白诱导上皮通透性增加以及上皮细胞和线粒体氧化应激的细胞和分子机制（目的2）。最后，我们将在细胞培养、呼吸机诱导的小鼠肺损伤临床相关模型和分离的灌注人肺模型中测试针对铁蛋白（对乙酰氨基酚）的治疗方法（Aim 3）。这一建议极具创新性，解决了一个临床意义重大的问题。这些研究结果将极大地促进我们对急性肺损伤中肺上皮通透性机制的理解，并为未来针对ARDS的新型靶向治疗铺平道路。

DESCRIPTION (provided by applicant): ARDS is a major cause of morbidity and mortality in the United States. There are no specific therapies for ARDS other than protective mechanical ventilation and conservative fluid management and mortality remains high at 30-50%. Thus, there is a critical need for specific therapies that target fundamental mechanisms of ARDS. Increased lung epithelial permeability is a pathophysiologic hallmark of ARDS and is manifest clinically by pulmonary edema, impaired gas exchange and acute respiratory failure. We have identified cell free hemoglobin as a novel mediator of increased epithelial permeability in ARDS. Our preliminary data show that airspace levels of cell-free hemoglobin are high in ARDS and are associated with increased lung permeability and poor clinical outcomes. Furthermore, it is the Ferryl oxidized form of hemoglobin that is elevated in the airspaces in ARDS. In a clinical trial in severe sepsis, the leading cause of ARDS, treatment with an inhibitor of Ferryl hemoglobin, acetaminophen, decreased lipid peroxidation and attenuated acute kidney injury. In cultured lung epithelial cells, hemoglobin treatment induces epithelial barrier permeability through cellular and mitochondrial reactive oxygen species generation. Furthermore, intratracheal administration of cell-free hemoglobin to the lungs of mice leads to generation of oxidized Ferryl hemoglobin and increased lung alveolar capillary barrier permeability. These findings support the overall hypothesis for the proposed studies, that formation of Ferryl-Hgb in the airspace in ARDS causes alveolar epithelial cell oxidative stress and increased epithelial permeability, contributing to the pathophysiology of ARDS. To test this hypothesis I have assembled a cross-disciplinary team of co-Investigators and consultants with expertise in clinical ARDS (Ware), Hgb redox chemistry (Roberts), primary alveolar epithelial cell isolation and culture (Guttentag), mitochondrial oxidative stress (Dikalov), human models of ARDS (Matthay) and advanced in vivo imaging techniques (West) whose expertise complements my own in cellular models of epithelial permeability and animal models of ARDS. We will utilize clinical samples of pulmonary edema fluid already collected from patients with ARDS and hydrostatic pulmonary edema along with mouse and epithelial cell culture studies to show that levels of Ferryl hemoglobin in the airspace are increased in human ARDS and cause increased lung epithelial permeability in mice and cultured epithelial cells (Aim 1). We will define the cellular and molecular mechanism of hemoglobin induced increases in epithelial permeability and cellular and mitochondrial oxidative stress in the epithelium (Aim 2). Finally we will test a therapy targeted at Ferryl hemoglobin (acetaminophen) in cell culture, in a clinically relevant model of ventilator induced lung injury in mice and in an isolated perfused human lung model (Aim 3). This proposal is highly innovative and addresses a clinically significant problem. Results from these studies will greatly advance our understanding of the mechanisms of lung epithelial permeability in acute lung injury and will pave the way for future novel targeted therapeutics in ARDS.