Brain endothelial cells release extracellular vesicles in a polarized way that aids leukocyte crossing in vitro

Brain microvascular endothelial cells, which form the walls of the blood-brain barrier, do not shed their extracellular vesicles randomly. A new in vitro study, published in Fluids and Barriers of the CNS, shows that these cells release vesicles in a strikingly polarized fashion, with vesicles accumulating predominantly in the chamber facing the membrane surface from which they originated. The work also links this polarized release to leukocyte transendothelial migration, the process by which immune cells cross the endothelial barrier.

The research team, led by Dylan Krajewski and Joel S. Pachter, built on earlier findings from their own group showing that vesicles from brain microvascular endothelial cells can transfer the tight junction protein claudin-5 to leukocytes, both in cell culture and during experimental autoimmune encephalomyelitis, an animal model of inflammatory disease. That observation led them to hypothesize a so-called zipper mechanism: claudin-5 molecules carried on vesicles bound to leukocytes might temporarily replace those at the junctions between endothelial cells, smoothing the path for immune cells squeezing through. For such a mechanism to work, the researchers reasoned, vesicle release would need strict spatiotemporal control, with vesicles from the apical, blood-facing surface interacting with leukocytes while vesicles from the basolateral surface bind perivascular or parenchymal targets.

To test this, the team combined transwell assays, flow cytometry, nanoparticle tracking analysis, super-resolution microscopy and live-cell time-lapse imaging. The results showed that vesicle release was highly polarized, with vesicles accumulating predominantly in the chamber facing their membrane of origin. Supporting the idea of polarized exosome release specifically, apical and basolateral membrane material appeared to be segregated into discrete late endosomal populations within the cells, suggesting the cells maintain separate intracellular routing for material destined for each surface.

The functional link to immune cell migration emerged when the researchers examined vesicle-leukocyte interactions during transendothelial migration. Apical-derived vesicles preferentially associated with leukocytes that had adhered to the endothelial layer, and when vesicle release was inhibited, transendothelial migration was suppressed. In other words, blocking the cells’ ability to shed vesicles also blocked, at least in part, the ability of immune cells to cross the barrier in this culture system.

Importantly, the polarization was not an artifact of static culture conditions. When the researchers applied physiological flow, mimicking the shear forces of blood moving through a vessel, polarized vesicle release was maintained. Under flow, vesicles carrying claudin-5 showed predominant release at the apical surface, a pattern the authors suggest may reflect a predisposition of these vesicles toward interacting with circulating immune cells.

The authors conclude that brain microvascular endothelial cell vesicles are released in a polarized manner, and that those derived from the apical surface focally interact with adherent leukocytes. Because inhibiting vesicle release suppressed transendothelial migration, they propose that binding between these vesicles and leukocytes supports the migration process itself.

The findings are limited to an in vitro system, and the authors note that the study was shared early as a peer-reviewed, accepted manuscript subject to further edits before the final version of record. Nevertheless, the work offers a mechanistic picture of how the blood-brain barrier’s own cells may actively participate in immune cell trafficking, by handing vesicles decorated with junctional proteins to the immune cells attempting to cross.

Subject of Research: Polarized release of extracellular vesicles by brain microvascular endothelial cells and its role in leukocyte transendothelial migration in vitro

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

Article References: Krajewski, D., Ge, S., Jellison, E. R., Wu, Y., Galpayage Dona, K. N. U., Andrews, A. M., Ramirez, S. H., McGrath, J. L., Chan, S. J. W., Zhu, J.-Y., Bazan, G. C., Adler, A. J., & Pachter, J. S. (2026). Polarized release of brain microvascular endothelial cell-derived extracellular vesicles supports leukocyte transendothelial migration in vitro. Fluids and Barriers of the CNS. https://doi.org/10.1186/s12987-026-00891-6

Image Credits: AI Generated

DOI: 10.1186/s12987-026-00891-6

Keywords: extracellular vesicles, blood-brain barrier, brain microvascular endothelial cells, leukocyte transendothelial migration, claudin-5, exosomes, polarized release, in vitro, tight junctions, immune cell trafficking, Fluids and Barriers of the CNS, Polarized