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

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.

Grant Number: 5F30HL170483-03
NIH Institute/Center: NIH
Principal Investigator: Avery Bogart