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

Abstract

Sepsis is a critical public health issue with high morbidity and mortality. 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 glycocalyx degradation, which occurs due to the activation of heparanase, a glycocalyx-cleaving enzyme. This manifests as endothelial barrier hyperpermeability that leads to organ dysfunction including acute respiratory distress syndrome (ARDS). However, much remains unknown regarding the molecular pathways that underlie sepsis-induced heparanase upregulation. This dissertation presents findings that address this key knowledge gap, centering on the concept that increased heparanase activity during sepsis is in part driven by reduction-oxidation (redox) cycling of hemoglobin that is released into the circulation (i.e., cell-free hemoglobin, CFH). Utilizing plasma from sepsis patients, I demonstrate that concentrations of circulating heparanase and heparan sulfate fragments correlate with worse clinical prognosis. My findings also reveal a direct association between plasma CFH, heparanase, and heparan sulfate degradation products in human sepsis. Further, in experimental sepsis models, I show that CFH stimulates heparanase transcription and activation, and that this is mitigated by acetaminophen, an inhibitor of CFH redox cycling. Moreover, in wildtype septic mice, CFH treatment enhances endothelial glycocalyx shedding, inflammation, and illness severity – effects that are not observed in mice lacking endothelial heparanase. Together, these data suggest CFH upregulates active heparanase, exacerbating endothelial glycocalyx breakdown and worsening outcomes. This work thus uncovers a previously unrecognized pathway of endothelial injury, providing novel insight into sepsis pathogenesis.