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

项目摘要 败血症，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，是一种关键的 公共卫生问题。脓毒症每年影响近5000万人，是全球死亡的主要原因， 并对全球经济产生重大影响。脓毒症的实质性负担的主要原因是 对增强其发病机制的生物学机制认识不足。的标志之一 脓毒症是内皮损伤，其表现为内皮屏障通透性过高，并导致器官损伤。 包括急性呼吸窘迫综合征（ARDS）的功能障碍。一个已知的破坏 脓毒症中的内皮屏障完整性是无细胞血红蛋白（CFH），释放到循环中的血红蛋白 来自溶解的红细胞。CFH在大多数脓毒症患者中升高，并且与较高的 器官功能障碍的发生率，如ARDS和死亡。这项建议旨在确定病理生理作用， CFH在脓毒症内皮细胞通透性增高中的作用。内皮通透性的主要调节因子是 内皮糖萼，一种排列在血管腔中的糖蛋白和蛋白聚糖基质。在败血症中， 这种功能由于乙酰肝素酶活性的增加而受损，乙酰肝素酶是一种降解内皮细胞的酶， 糖萼重要的是，更多的糖萼分解与更差的脓毒症结局相关。鉴于 乙酰肝素酶的表达部分地受到受活性氧刺激的转录因子的调节 CFH在脓毒症的炎症环境中发生氧化，产生活性氧 包括超氧化物，我假设CFH产生的超氧化物触发了糖萼 通过诱导类肝素酶表达进行切割，从而作为内皮细胞的关键介导剂 脓毒症中渗透性过高和随之而来的器官损伤。我将测试CFH对肺部的影响， 在培养的原代人肺微血管中使用机械方法检测内皮糖萼 内皮细胞和鼠多微生物败血症。这两个模型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， 脓毒症相关的肺损伤、严重程度和死亡率。目的2将定义CFH在调节 乙酰肝素酶表达和活性。我还将询问乙酰肝素酶表达的改变和 活性影响内皮屏障通透性和脓毒症结果。最后，我将描述CFH的影响- 产生的超氧化物对乙酰肝素酶表达和活性的影响，完成我的调查，提出了 通路在解决CFH在糖萼降解和内皮功能障碍中的作用时，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 揭示了脓毒症相关肺损伤治疗药物开发的新方法。 此外，这个项目的完成将促进我的技术，批判性思维， 沟通技巧，这将是至关重要的，我作为一个独立的物理学家，科学家的成功。

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.