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 treat...