Metabolically aberrant extracellular mitochondria and oxidative endotheliopathy after traumatic brain injury

The National Institutes of Health National Heart, Lung, and Blood Institute awarded Bloodworks $724,351 under the Cardiovascular Diseases Research program (CFDA 93.837) on July 1, 2026, to investigate the mechanisms by which extracellular mitochondria released from traumatic brain injury sites cause endothelial cell injury and contribute to hemostatic dysfunction and coagulopathy. The research addresses a gap in understanding subcellular changes in endothelial cell injury during traumatic brain injury, which drives vascular leakage, tissue edema, and inflammation alongside dysregulated hemostasis.

The award funds in vitro experimental work to define how metabolically active extracellular mitochondria are endocytosed by endothelial cells through dynamin- and CD36-mediated pathways, triggering endoplasmic reticulum oxidative stress, unfolded protein response activation, and mitophagy. The research will evaluate pharmaceutical agents designed to block extracellular mitochondria-induced endotheliopathy and test whether failure of these cellular repair mechanisms leads to endothelial cell apoptosis, increased permeability, and procoagulant activity.

The period of performance runs through April 30, 2030, with performance taking place in Seattle, Washington. This is a Project Grant assistance type. Bloodworks, a tax-exempt nonprofit blood center and laboratory services organization, serves as the recipient and principal investigator institution.

Abstract

Hemostasis is an equilibrium state that stops bleeding at the site of vascular injury without causing spontaneous and systemic intravascular coagulation and thrombosis. IT consists of five distinct but interconnected components: platelets and adhesive ligands, coagulation, fibrinolysis, vascular tone, and the endothelium. All five components are altered during traumatic brain injury (TBI), leading to local and systemic endotheliopathy and resultant coagulopathy. Endothelial injuries have been consistently detected during TBI and serve as a key conduit to not only hemostatic dysregulation but also vascular leakage, tissue edema, and inflammation. However, past studies provide limited insights into the subcellular changes of EC injury, which forms the foundation of TBI-induced endotheliopathy. Here we propose to investigate the mechanism of how extracellular mitochondria (exmt) cause EC injury during acute TBI. Our published and preliminary observations led US to hypothesize that (1) metabolically active exmt released from injured brains are endocytosed by ECS through the gtpase dynamin- and CD36-mediated pathways, (2) endocytosed exmt cause the cytoplasmic reticulum to undergo oxidative stress (ER stress) and trigger unfolded protein response (UPR) while simultaneously facilite mitochondrial autophagy (mitophagy) to remove exmt, and (3) when UPR and mitophagy fail to restore endothelial homeostasis, ECS undergo apoptosis to increase permeability and procoagulant activity. The former causes cerebral edema, high intracranial pressure, and acute lung injury, whereas the latter contributes to systemic consumptive coagulation, thus worsening the outcome of TBI. We will test these hypotheses by conducting in vitro experiments to define exmt-induced subcellular changes, evaluating the efficacy of agents that block this exmt-induced endotheliopathy in mouse models, and analyzing exmt levels and oxidative activity in plasma samples of TBI patients to identify predictive and risk assessment markers for TBI-induced endotheliopathy. We have three specific aims. The specific aim 1 is to define the pathway of exmt-induced endothelial injury in vitro with specific focus on communications between the endoplasmic reticulum and mitochondria. The aim 2 is to evaluate the efficacy of novel agents for specifically blocking exmt-induced endotheliopathy in mouse models. We propose to investigate whether blocking exmt endocytosis or accelerating their clearance can mitigate the risk and reduce the severity of endotheliopathy and coagulopathy in mice subjected to severe TBI and in non-injured mice infused with exmt. The specific aim 3 is to quantify levels and activity of exmt and their derivatives in plasma of TBI patients and explore the possibility to use exmt related markers for clinical risk assessment of TBI-induced endothelial injury. This study will identify new pathways, predictive markers, and therapeutic targets of TBI-induced endotheliopathy and coagulopathy.