Abstract TP144: Extracellular Vesicles Derived From Stroke Patient Clots Impair Cerebral Endothelial Cell Function

Abstract

Introduction: Most patients undergoing thrombectomy fail for full reperfusion. The present study tested whether small extracellular vesicles (sEVs) from stroke patient clots impair cerebral endothelial cell (CEC) function, which may contribute to dysfunction of downstream CECs, leading to reduction of cerebral tissue perfusion. Methods: Per IRB-approved protocol, clots were collected from 60 patients (30 males) with acute ischemic stroke subjected to thrombectomy. Red and white clots were determined by immunochemistry. Clot-sEVs were isolated and characterized using ultracentrifugation, size distribution, and Western blot. The effects of clot-sEVs on human CEC (hCEC) permeability and proteins were measured using trans-endothelial assay and trans-endothelial electrical resistance (TEER) and Western blot, respectively. Nystatin, an inhibitor of caveolin-dependent endocytosis, was employed to study hCEC uptake of clot-sEVs. Results: Clot-sEVs had a mean size of 73±2nm. Cellular source analysis of clot-sEVs revealed that 22%, 30%, and 44% of sEVs from red clots and 28%, 28%, and 52% of sEVs from white clots were from neutrophils, endothelial cells, and platelets, respectively. Compared to sEVs derived from donor plasma (plasma-sEVs), treatment of hCECs with clot-sEVs significantly increased cell permeability (227% vs 100% of plasma-sEVs) and reduced TEER (66.6ω.cm 2 vs 87.3ω.cm 2 ). Clot-sEVs significantly increased pro-inflammatory and pro-coagulation proteins in the hCECs including ICAM1, VCAM1, TNF-a, HMBG1, tissue factor, and significantly reduced tight junction proteins ZO-1 and Claudin-5. The effects of white clot-sEVs on hCEC dysfunction were greater than red clot-sEVs. Nystatin significantly reduced hCEC uptake of clot-sEV from 232% to 142% and the clot-sEV-increased permeability. Moreover, Pearson correlation showed that the severity of clot-sEV impaired hCEC function was significantly correlated with final TICI scores of the individual patients (permeability vs TICI, r=-0.3348, p=0.02; TEER vs TICI, r=0,3666, p=0.01). Conclusion: Clot-sEVs contribute to hCEC dysfunction by augmenting pro-inflammatory and pro-coagulation proteins. Blocking uptake of clot-sEVs by recipient cells may improve patient outcomes.