Polarized release of brain microvascular endothelial cell-derived extracellular vesicles supports leukocyte transendothelial migration in vitro

Abstract Background Abundant evidence indicates extracellular vesicles (EVs) (exosomes and microvesicles) are mediators of intercellular communication. Previous reports from this group further showed that EVs from brain microvascular endothelial cells (BMEC) transferred the tight junction protein (TJ) claudin-5 (CLN-5) to leukocytes in vitro and during experimental autoimmune encephalomyelitis, leading us to hypothesize that such interaction might facilitate transendothelial migration (TEM) by a “zipper mechanism” whereby CLN-5 molecules on leukocyte-bound EVs temporarily replace those at interendothelial junctions. Such a mechanism is likely to be under strict spatiotemporal control. A corollary to this is that EVs are released from BMEC in a polar manner, such that only those released from the apical surface interact with leukocytes, while those from the basolateral surface bind adventitial targets. Methods To assess polar EV release from BMEC and subsequent BMEC EV:leukocyte interactions during TEM using transwell assays, flow cytometry, nanoparticle tracking, super-resolution and live-time imaging were used. Results It was shown that EV release was highly polarized, as EVs accumulated predominantly in the chamber facing their membrane of origin. Supportive of polarized exosome release, apical and basolateral membrane material appeared segregated into discrete multivesicular body populations. Consistent with a role in TEM, apical-derived BMEC EVs preferentially bound leukocytes in a manner dependent on leukocyte adhesion, with TEM suppressed when EV release was inhibited. Polarized EV release was maintained under physiological flow, wherein CLN-5 + EVs exhibited near exclusive release at the apical surface, possibly reflecting their predisposition toward interacting with circulating immune cells. Conclusion BMEC EVs are released in a polarized manner and those derived from the apical surface focally interact with adherent leukocytes. That inhibition of EV release further suppressed TEM suggests that BMEC EV:leukocyte binding is functionally coupled to the TEM process.