Comparative Analysis of the Impact of Mitochondrial Dysfunction on Endotheliopathy in Pediatric Sepsis and Diabetic Ketoacidosis

Endotheliopathy is associated with adverse patient outcomes in children suffering from critical illnesses, but pathobiological mechanisms are not well understood and no therapies targeting endotheliopathy are currently available. Increasing data indicates that mitochondrial dysfunction contributes to the pathophysiology of critical illness including sepsis and diabetic ketoacidosis (DKA). However, no studies have identified systemic inflammatory mediators present in pediatric critical illness that contribute to endothelial mitochondrial dysfunction. In this proposal, we will test the hypothesis that endotheliopathy is associated with both systemic mitochondrial dysfunction and exacerbated by plasma cytokines and matrix metalloproteinases (MMPs) that impair mitochondrial function within endothelial cells (ECs) in these two pediatric critical illnesses. In Aim 1, we will measure plasma biomarkers of endotheliopathy and circulating plasma mitochondrial DNA (mtDNA) in children hospitalized in the intensive care unit for sepsis and DKA. In Aim 2, we will determine the extent to which plasma from children with sepsis- and DKA-associated endotheliopathy causes mitochondrial dysfunction ex vivo using a human umbilical endothelial cells (HUVEC) reporter assay. Our group previously optimized this assay as part of an undergraduate mentorship experience in the Nemours Critical Care Mitochondrial Unit, and we demonstrated that plasma from children with DKA lowers HEK293 mitochondrial respiration in acute kidney injury. Building on this experience, we expect to find a correlation between high levels of plasma biomarkers of endotheliopathy and mtDNA from Aim 1 with the level of reduction in HUVEC mitochondrial respiration. This correlation would support systemic mediators in plasma contribute to endothelial mitochondrial dysfunction. In Aim 3, we will identify systemic mediators, including cytokines and MMPs, that cause mitochondrial dysfunction in children with sepsis- and DKA-associated endotheliopathy. Elevated mediators associated with both endotheliopathy and plasma mtDNA (from Aim 1) and those that were elevated in plasma decreased mitochondrial respiration (from Aim 2) will be further tested as causes of mitochondrial dysfunction using co-incubation with targeted inhibitors in our reporter assay. By comparing blood cytokine and MMP profiles across children with sepsis and DKA and examining their associations with both endotheliopathy biomarkers and inducible mitochondrial dysfunction in ECs, we will identify candidate proteins most likely to cause mitochondrial dysfunction in pediatric critical illness. With completion of these aims, we will learn if mitochondrial dysfunction is linked to endothelial dysfunction and begin to identify novel therapeutic strategies designed to prevent, mitigate or reverse mitochondrial dysfunction, thereby reducing adverse outcomes associated with sepsis and DKA in pediatric populations.