Characterization of Extracellular Vesicles from Ex Vivo Perfused Human Lungs and Their Relationship with Allograft Injury

Purpose Primary graft dysfunction (PGD) is an important cause of morbidity and mortality following lung transplantation and results in part from injuries sustained in the donor. Extracellular vesicles (EVs), membrane-bound sacs released by cells, are increasingly being recognized as key modulators of inflammation. EVs have been implicated in the anti-donor response to the lung allograft and may play a role in the pathogenesis of PGD. Perfusate from ex-vivo lung perfusion (EVLP) allows interrogation of donor EVs which may serve as predictive biomarkers for lung injury. In this study, we aim to identify and characterize EVs derived from EVLP perfusates of marginal donor lungs. Methods Hourly perfusate samples of lungs undergoing EVLP were collected, precleared and ultracentrifuged at 100,000xg for 90mins to isolate small EVs. The presence of EVs was validated by Nanoparticle tracking analysis (NTA), Western blot (WB), and flow cytometry (FACS). Next, EV protein arrays were used to investigate differences in protein content between lungs accepted for transplant (n=20), and injured lungs declined for transplant (n=20). Results Our analyses show the presence of EV-sized particles (≤300nm) positive for canonical EV markers. CD9 and CD81 were identified by both WB(Fig1) and FACS, while CD63 was only detectable by FACS(Fig2). Using EV arrays, CD63 and CD31, an endothelial adhesion marker, were found to be elevated in EVs from damaged lungs declined for transplantation (p=0.0183; p=0.0167). There was no difference observed in levels of EV-associated CD9(Fig3). Conclusion Our findings show that EVLP perfusates contain donor EVs. CD63+ and CD31+ EVs may reflect damage in lungs declined for transplantation. Next, we aim to characterize and compare the RNA cargo of EVs acquired from the same cohort of donor samples, to determine whether differences can be observed at the transcript level. Together, these findings will provide an insight to the potential role of EVs as biomarkers for lung injury.