Targeting cell-free hemoglobin in sepsis to reduce lung microvascular permeability: mechanistic and translational studies

项目摘要 脓毒症引起的急性呼吸窘迫综合征（ARDS）是急性呼吸衰竭的主要原因， 病危发病率和死亡率很高，没有经过证实的药物治疗， 抗菌剂肺微血管内皮细胞通透性增加是一种明确的致病因素， 在脓毒症相关的ARDS中导致急性肺水肿和肺功能障碍的特征。虽然 调节微血管通透性的机制是一个深入研究的领域， 在脓毒症相关的ARDS中，肺微血管通透性增加的触发因素并不清楚， 明白有必要确定这些微血管通透性增加的早期触发因素， 脓毒症，以提高我们对病理生理学的理解，关键是，以确定新的治疗靶点 预防和早期治疗脓毒症引起的ARDS。我们最近在患者中的转化研究， 离体灌注的人肺和败血症小鼠模型已经将无细胞血红蛋白（CFH）鉴定为 增加微血管渗透性的关键近端介质，（1）在超过 80%的严重脓毒症患者，（2）与严重脓毒症患者的死亡率独立相关 脓毒症，（3）在我们的模型系统中对肺微血管通透性具有强效作用，（4）可以 在临床和实验性脓毒症中氧化为高反应性铁基（4+）血红蛋白，一种有效的氧化剂，和（5） 可以被血红素蛋白还原剂对乙酰氨基酚机械地靶向。此外，初步 研究表明，氧化剂介导的线粒体损伤和内皮细胞凋亡的激活是 CFH介导其对微血管通透性影响的关键机制。这方面的研究 建议将建立在这一初步工作的特点机制，CFH触发增加 脓毒症中的肺微血管通透性。我们的主要目标是将这些发现转化为新的目标 这些疗法将在我们的新型人类肺模型中进行测试，为快速转化为临床试验做准备。 败血症。在目标1中，我们将研究CFH增加的细胞和生理机制， 在离体人肺和临床相关模型中的微血管通透性和急性肺损伤 脓毒症引起的ARDS在目标2中，我们将研究原发性肺微血管内皮细胞沿着与我们的 在体内模型，以确定CFH诱导内皮细胞凋亡的分子机制。在目标3中， 将测试在肺微血管中用对乙酰氨基酚靶向氧化CFH的治疗潜力， 内皮细胞和临床相关的人和鼠脓毒症诱导的ARDS模型。研究 在这些目标中提出的目标有可能产生重大和持续的科学影响。针对早期CFH 预防和治疗脓毒症中的ARDS是一种新的方法，可能对临床产生重大影响。 结果。此外，关注对乙酰氨基酚作为微血管增加的靶向治疗， 由于氧化CFH导致的渗透性的降低可以重新利用廉价且安全的化合物来治疗脓毒症。

Project Summary Sepsis-induced acute respiratory distress syndrome (ARDS) is a leading cause of acute respiratory failure in critical illness. Morbidity and mortality are high and there are no proven pharmacologic therapies other than antimicrobials. Increased permeability of the pulmonary microvascular endothelium is a defining pathogenic feature that leads to acute pulmonary edema and lung dysfunction in sepsis-associated ARDS. Although the mechanisms that regulate microvascular permeability are an area of intensive research effort, the proximal triggers of increased pulmonary microvascular permeability in sepsis-associated ARDS are not well understood. There is a vital need to identify these early triggers of increased microvascular permeability in sepsis, both to enhance our understanding of pathophysiology, and critically, to identify new therapeutic targets for prevention and early treatment of sepsis-induced ARDS. Our recent translational studies in patients, the isolated perfused human lung, and mouse models of sepsis have identified cell-free hemoglobin (CFH) as a key proximal mediator of increased microvascular permeability that (1) is released into the circulation in over 80% of patients with severe sepsis, (2) is independently associated with mortality in patients with severe sepsis, (3) has potent effects on pulmonary microvascular permeability across our model systems, (4) can be oxidized in clinical and experimental sepsis to highly reactive ferryl (4+) hemoglobin, a potent oxidant, and (5) can be mechanistically targeted by the hemoprotein reductant acetaminophen. Furthermore, preliminary studies suggest that oxidant-mediated mitochondrial injury and activation of apoptosis in endothelial cells are key mechanisms through which CFH mediates its effects on microvascular permeability. The studies in this proposal will build on this preliminary work to characterize the mechanisms by which CFH triggers increased pulmonary microvascular permeability in sepsis. Our primary goal is to translate these findings to new targeted therapies that will be tested in our novel human lung model as preparation for rapid translation to clinical trials in sepsis. In Aim 1, we will study the cellular and physiologic mechanisms by which CFH increases microvascular permeability and acute lung injury in the isolated human lung and clinically relevant models of sepsis-induced ARDS. In Aim 2 we will study primary pulmonary microvascular endothelial cells along with our in vivo models to define the molecular mechanisms by which CFH induces endothelial apoptosis. In Aim 3, we will test the therapeutic potential of targeting oxidized CFH with acetaminophen in pulmonary microvascular endothelial cells and clinically relevant models of human and murine sepsis-induced ARDS. The studies proposed in these aims have the potential for major and sustained scientific impact. Targeting CFH for early prevention and treatment of ARDS in sepsis is a new approach that could have a major impact on clinical outcomes. Furthermore, the focus on acetaminophen as a targeted therapy for increased microvascular permeability due to oxidized CFH could repurpose an inexpensive, and safe compound for treatment of sepsis.