Inflammation‐induced Extracellular Vesicles Cause Endothelial Barrier Injury

Abstract

Extracellular vesicles (EVs) are a heterogenous population of sub‐micron sized vesicles released by cells during physiological or pathological stimulation. Circulating vesicles not only serve as important diagnostic/prognostic biomarkers, but also interact with blood or vascular cells and regulate multiple cellular processes by transferring bioactive cargo. In this study, we characterized EV production and cargo signature in the blood of healthy human subjects and burn patients, as well as in cultured human endothelial cells (ECs) after inflammatory stimulation. We also examined the effects of EVs on endothelial barrier structure and function. The results showed that the plasma level of EVs was increased during sterile inflammation in burn patients, with a significant portion of them derived from leukocytes and the endothelium. Interestingly, the EVs from burn patients expressed higher levels of c‐Src, protein kinase C and focal adhesion kinase, compared to EVs from healthy blood donors. In cultured ECs treated with tumor necrosis factor alpha, there was an increased production of EVs into the culture media. Furthermore, treating endothelial cells with EVs induced myosin light chain and VE‐cadherin phosphorylation and actin stress fiber formation in concomitance with adherens junction discontinuity. Functionally, EV treatment caused an increase in albumin flux across endothelial monolayers in a concentration‐dependent manner. EVs derived from c‐Src knockdown parent ECs displayed an attenuated effect on barrier protein phosphorylation and permeability. Likewise, pharmacological inhibition of Src activity reduced EV‐induced protein phosphorylation and barrier dysfunction in endothelial cells. Together, the data suggest that EVs produced during inflammation carry protein kinases capable of damaging the endothelial barrier.Support or Funding InformationNIH grants HL126646, HL070752 (to SYY), and GM097270. The human blood study was approved by the University of South Florida IRB and supported by the institutional research funds.