Mechanisms driving endothelial angiopoietin-2 expression and vascular dysfunction during pediatric sepsis

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This K08 proposal describes a 5-year research training program that will prepare the candidate for a career as an independent, NIH-funded translational scientist focused on mechanisms driving sepsis-mediated vascular disease. The scientific premise for the aims is that vascular endothelial dysfunction is a key driver of organ injury in pediatric sepsis. Morbidity and mortality remain unacceptably high in pediatric sepsis despite advances in modern medicine, principally due to knowledge gaps in the mechanisms driving vascular disease during sepsis. Endothelial cell-derived angiopoietin-2 (Ang-2) has emerged as a critical promoter of vascular injury and organ impairment in sepsis through its antagonism of the endothelial Tie2 receptor. Plasma Ang-2 levels are significantly elevated in children with sepsis and are associated with measures of organ injury and outcomes. Preliminary data suggest that heparan sulfate cleavage from the surface glycocalyx of flow conditioned primary human lung microvascular endothelial cells promotes Ang-2 expression, implicating a novel paradigm by which Ang-2 is upregulated in sepsis. Preliminary data and prior work also suggest that inactivation of liver kinase B1 (LKB1) and downstream adenosine-monophosphate-activated protein kinase (AMPK) may be integral in this process. Further, the candidate discovered that Ang-2 is bound to the surface of exosomes isolated from plasma of septic children, suggesting that exosomal Ang-2 may significantly contribute to vascular disease and organ injury during sepsis. Together, these data support the novel mechanistic hypothesis that enzymatic heparan sulfate erosion from the endothelial glycocalyx during pediatric sepsis upregulates expression of Ang-2 that, when bound to exosomes, has potent vascular destabilizing effects. To test this hypothesis, in vitro, ex vivo, and clinical studies will be performed in the following two aims. Aim 1: Test the hypothesis that glycocalyx heparan sulfate erosion increases Ang-2 expression from flow conditioned human lung microvascular endothelial cells via attenuated LKB1 activity and downstream AMPK pathway signaling. Aim 2: Test the hypothesis that exosomal Ang-2 (a) is biomarker for organ injury and clinical outcomes in pediatric sepsis and (b) promotes endothelial permeability via Tie2 receptor antagonism. Harnessing the combined expertise of the candidate’s mentoring team and utilizing the wealth of resources available at the candidate’s institution, the candidate will train in advanced translational science techniques germane to the completion of these aims, including microvascular flow modeling, nanoparticle tracking analysis and characterization with flow cytometry, and electric cell-substrate impedance sensing. The proposed research program has defined benchmarks that will facilitate the candidate’s career advancement and will culminate in the submission of an R01 to continue uncovering the mechanisms driving vascular disease in pediatric sepsis that will foster the discovery of novel therapeutics.