Mechanisms of Cell-Free Hemoglobin-Mediated Injury to the Pulmonary Endothelial Glycocalyx in Sepsis

项目总结 脓毒症，或由于宿主对感染的反应失调而导致的危及生命的器官功能障碍，是一种严重的 公共卫生问题。败血症每年影响近5000万人，是全球主要的死亡原因， 并对全球经济产生重大影响。脓毒症负担沉重的一个主要原因是 对增强其发病机制的生物学机制认识不足。标志之一就是 脓毒症是一种血管内皮损伤，表现为内皮屏障通透性增高，导致脏器损伤。 功能障碍包括急性呼吸窘迫综合征(ARDS)。一位已知的扰乱 脓毒症中内皮屏障的完整性是无细胞血红蛋白(CFH)，即释放到循环中的血红蛋白 来自溶解的红血球。大多数脓毒症患者CFH升高，并与较高的 器官功能障碍，如急性呼吸窘迫综合征和死亡率。这项建议试图定义病理生理学作用 CFH在脓毒症内皮细胞高通透性中的表达。血管内皮细胞通透性的主要调节因子是 内皮糖萼，一种排列在血管管腔内的糖蛋白和蛋白多糖的基质。在脓毒症中， 这一功能因肝素酶活性增加而受损，肝素酶是一种降解血管内皮细胞的酶 糖萼。重要的是，更大的糖萼分解与更糟糕的脓毒症结局相关。考虑到 乙酰肝素酶的表达在一定程度上受受活性氧刺激的转录因子的调节。 物种(ROS)，CFH在脓毒症的炎症环境中经历氧化，产生ROS 将超氧化物包括在这个过程中，我假设CFH产生的超氧化物触发了糖基化 通过诱导乙酰肝素酶的表达进行切割，从而成为内皮细胞的关键介质 脓毒症时的高通透性和继发的器官损伤。我要测试CFH对肺组织的影响 机械方法在两种原代培养人肺微血管内皮细胞中的应用 血管内皮细胞和小鼠多菌败血症。这两种模式都将用于实现每个目标。在目标1中， 我将确定超氧化物和CFH对糖萼降解、内皮屏障功能和 败血症相关的肺损伤、严重程度和死亡率。目标2将定义CFH在调节 乙酰肝素酶的表达和活性。我还会询问乙酰肝素酶表达的变化和 活性影响内皮屏障通透性和脓毒症结局。最后，我将描述CFH的影响- 生成的超氧化物对肝素酶的表达和活性的影响完成了本人对此的研究建议 路径。在解决CFH在糖萼降解和内皮功能障碍中的作用方面，我将提供 对脓毒症期间循环CFH升高的后果的前所未有的见解，有可能 为脓毒症相关性肺损伤的治疗提供新的治疗方法。 此外，这个项目的完成将促进我的技术、批判性思维和 作为一名独立的内科科学家，沟通能力对我的成功至关重要。

PROJECT SUMMARY Sepsis, or life-threatening organ dysfunction due to a dysregulated host response to infection, is a critical public health issue. Affecting nearly 50 million people annually, sepsis is a leading cause of death worldwide, and significantly impacts the global economy. A major reason for the substantial burden of sepsis is an insufficient understanding of the biologic mechanisms that potentiate its pathogenesis. One of the hallmarks of sepsis is endothelial injury, which manifests as endothelial barrier hyperpermeability and results in organ dysfunction including acute respiratory distress syndrome (ARDS). A known contributor to the disruption of endothelial barrier integrity in sepsis is cell-free hemoglobin (CFH), hemoglobin released into the circulation from lysed red blood cells. CFH is elevated in the majority of patients with sepsis and is associated with higher rates of organ dysfunction, such as ARDS, and death. This proposal seeks to define the pathophysiologic role of CFH in endothelial hyperpermeability in sepsis. A primary regulator of endothelial permeability is the endothelial glycocalyx, a matrix of glycoproteins and proteoglycans that lines the vascular lumen. In sepsis, this function is impaired due to increased activity of heparanase, an enzyme that degrades the endothelial glycocalyx. Importantly, greater glycocalyx breakdown correlates with worse sepsis outcomes. Given that heparanase expression is, in part, modulated by transcription factors that are stimulated by reactive oxygen species (ROS), and that CFH undergoes oxidation in the inflammatory environment of sepsis, producing ROS including superoxide in the process, I hypothesize that CFH-generated superoxide triggers glycocalyx cleavage via induction of heparanase expression, thereby serving as a critical mediator of endothelial hyperpermeability and consequent organ injury in sepsis. I will test the effect of CFH on the pulmonary endothelial glycocalyx using mechanistic approaches in both cultured primary human lung microvascular endothelial cells and murine polymicrobial sepsis. Both models will be used to accomplish each Aim. In Aim 1, I will determine the impact of superoxide and CFH on glycocalyx degradation, endothelial barrier function, and sepsis-associated lung injury, severity, and mortality. Aim 2 will define the role of CFH in the modulation of heparanase expression and activity. I will also interrogate whether alterations in heparanase expression and activity affect endothelial barrier permeability and sepsis outcomes. Finally, I will delineate the impact of CFH- generated superoxide on heparanase expression and activity to complete my investigation of this proposed pathway. In resolving the role of CFH in glycocalyx degradation and endothelial dysfunction, I will deliver unprecedented insights into the consequences of elevated circulating CFH during sepsis, with potential to unveil new approaches to the development of therapeutics for the treatment of sepsis-associated lung injury. Furthermore, the completion of this project will facilitate the development of my technical, critical thinking, and communication skills that will be crucial to my success as an independent physician-scientist.