Activated neutrophil-derived exosomes contribute to blood-brain barrier damage and hemorrhagic transformation after cerebral ischemia/reperfusion

Abstract

Background: Hemorrhagic transformation (HT) caused by blood-brain barrier (BBB) damage is closely correlated with the poor prognosis of ischemic stroke. Neutrophils are proven to mediate BBB injury after ischemic stroke, but the mechanism remains to be further investigated. Therefore, the present study aims to investigate the effect of neutrophil-derived exosomes on BBB integrity.

Method: A tMCAO-HT model was constructed to assess neutrophil infiltration and its co-localization with brain microvascular endothelial cells (BMEC). After using quiet (Q-Neu) and activated neutrophil (A-Neu) and their exosomes to treat the BBB model in vitro, TEER and permeability were assayed to assess the BBB integrity. Small RNA sequencing was performed to identify differentially expressed miRNAs (DE-miRNAs) in A-Neu- and Q-Neu-derived exosomes, and the function and pathways of DE-miRNA targets were analyzed by GO and KEGG enrichment.

Result: Different degrees of cerebral hemorrhage were observed in the tMCAO-HT model. The expression of the neutrophil marker Ly6G was significantly increased in tMCAO-HT model compared to the sham group, and co-localized with the BMEC marker CD31. Notably, Ly6G expression was positively correlated with hemoglobin content in brain tissue. A-Neu and its derived exosomes reduced TEER and elevated permeability in the BBB model in vitro. Moreover, BBB-related proteins Claudin 5, Occludin and ZO-1 expression were significantly reduced in BMEC after treatment with A-Neu and its derived exosomes. Nevertheless, Q-Neu and its exosomes had no significant effect on BBB integrity. A total of 84 DE-miRNAs are present in Q-Neu- and A-Neu-derived exosomes, and their target genes are involved in the regulation of "positive regulation of establishment of endothelial barrier", "cell junction", "ECM-receptor interaction" and "VEGF signaling pathway". Moreover, RT-qPCR revealed that the expression trends of miR-409-3p, miR-6909-5p, miR-3473d, miR-370-3p and miR-6904-5p in exosomes were consistent with the sequencing results.

Conclusion: Neutrophils are abnormally recruited in HT after ischemic stroke, and are associated with cerebral hemorrhage. In vitro, A-Neu-derived exosomes facilitate BBB injury, which may be accomplished by exosomal transport of miRNAs.

Keywords: Blood brain barrier; Exosome; Hemorrhagic transformation; Ischemic stroke; MicroRNA; Neutrophil.