Oxygen glucose deprivation-pretreated astrocyte-derived exosomes attenuates intracerebral hemorrhage (ICH)-induced BBB disruption through miR-27a-3p /ARHGAP25/Wnt/β-catenin axis

Abstract

Background: Blood brain barrier (BBB) breakdown is one of the key mechanisms of secondary brain injury following intracerebral hemorrhage (ICH). Astrocytes interact with endothelial and regulate BBB integrity via paracrine signaling factors. More and more studies reveal astrocyte-derived extracellular vesicles (ADEVs) as an important way of intercellular communication. However, the role of ADEV in BBB integrity after ICH remains unclear.

Methods: ADEVs were obtained from astrocytes with or without oxygen and glucose deprivation (OGD) pre-stimulation and the role of ADEVs in ICH was investigated using ICH mice model and ICH cell model. The potential regulatory effect of ADEVs on endothelial barrier integrity was identified by TEER, western blot and immunofluorescence in vitro. In vivo, functional evaluation, Evans-blue leakage and tight junction proteins (TJPs) expression were analyzed. MiRNA sequencing revealed that microRNA-27a-3p (miR-27a-3p) was differentially expressed miRNA in the EVs from OGD-pretreated astrocytes compared with normal control. The regulatory mechanism of miR-27a-3p was assessed using Luciferase assay, RT-PCR, western blot and immunofluorescence.

Results: OGD-activated astrocytes reduced hemin-induced endothelial hyper-permeability through secreting EVs. OGD-activated ADEVs alleviated BBB dysfunction after ICH in vivo and in vitro. MicroRNA microarray analysis indicated that miR-27a-3p is a major component that was highly expressed miRNA in OGD pretreated-ADEVs. OGD-ADEVs mitigated BBB injury through transferring miR-27a-3p into bEnd.3 cells and regulating ARHGAP25/Wnt/β-catenin pathway.

Conclusion: Taken together, these findings firstly revealed that miR-27a-3p, as one of the main components of OGD-pretreated ADEVs, attenuated BBB destruction and improved neurological deficits following ICH by regulating endothelial ARHGAP25/Wnt/β-catenin axis. OGD-ADEVs might be a novel strategy for the treatment of ICH. this study implicates that EVs from OGD pre-stimulated astrocytes.

Keywords: Blood brain barrier; Intracerebral hemorrhage; OGD pretreated astrocyte-derived exosome; miR-27a-3p.

Fig. 1

Astrocyte protected against hemin-induced BBB disruption through secreting exosomes A. TEER of monolayer bEnd.3 cells. B. Immunoblot images showing the expression of occludin, claudin-5 and ZO-1 in bEnd.3 cell monolayers. C. Morphological characterization of ADEVs observed under a TEM. D. The concentration and size distribution of ADEVs analyzed by NTA. E. Western blot analysis of EV surface maker proteins TSG101, CD81, CD63, and CD9. F. Fluorescence microscope was used to observe the uptake of PKH67-labeled ADEVs (green) by bEnd. 3 cells. Scale bar: 20 μm. **p < 0.01 versus indicated group; *p < 0.05 versus indicated group. All data were expressed as mean ± SEM of at least 3 independent experiments

Fig. 2

ADEVs protected against barrier hyperpermeability and TJPs disruption in bEnd.3 cells after hemin treatment A. Determination of TEER to evaluate the barrier integrity of monolayer bEnd.3 cells. B. Western blot analysis of tight junction protein markers (occludin, Claudin-5, and ZO-1) in bEnd.3 cells. C. Immunofluorescence staining for ZO-1 in bEnd.3 cells D–E. Quantification of the relative protein expression levels of occludin, Claudin-5, and ZO-1. **p < 0.01 versus indicated group; *p < 0.05 versus indicated group. All data were expressed as mean ± SEM of at least 3 independent experiments

Fig. 3

ADEVs mitigated ICH-induced BBB injury A. Schematic illustration presented that EVs from OGD-stimulated astrocytes was injected into mice from tail vein. B. Representative images showing EB extravasation 3 days after ICH induction. C. Quantitative analysis of EB extravasation. D. Representative western blot image of occludin, claudin‐5 and ZO-1 in peri-hematoma region of mice brain were measured by western blotting. E. Immunofluorescence staining for ZO-1/CD31 in the peri-hematoma region. Scale bar: 20 μm. **p < 0.01 versus indicated group; *p < 0.05 versus indicated group. All data were expressed as mean ± SEM of at least 3 independent experiments

Fig. 4

miRNA expression signature profiling in OGD-ADEVs A. miRNA volcano plot. Red point in the plot represents differentially upregulated miRNAs, and green point represents downregulated miRNAs, with statistical significance (fold change > 2.0 or < − 2.0; P < 0.05) C–D. The bubble map of GO enrichment and KEGG pathway analysis of miRNA target genes. D. A heat map of the first 20 significantly differentially expressed miRNAs. E. Expression of miR-27a-3p in N-ADEVs and OGD-ADEVs evaluated by qRT-PCR

Fig. 5

ADVEs abrogated hemin-induced barrier injury in bEnd.3 cells through transferring miR-27a-3p A. RT-qPCR analysis of miR-27a-3p expression in ADEVs from different groups. B. RT-qPCR analysis of miR-27a-3p expression in BMECs from different groups. C. Schematic illustration presented that OGD-stimulated astrocyte was transfected with Cy3 labeled miR-27a-3p mimic, ADEVs extracted was labeled with PKH67 and incubated with bEnd. 3 cells. D. Confocal images of PKH67- labeled ADEVs, including Cy3-miR-27a-3p, internalized by bEnd.3 cells. E. TEER of monolayer bEnd. 3 cells. Scale bar: 20 μm F. The protein levels of occludin, claudin-5 and claudin-5 in bEnd.3 cells determined by western blot. **p < 0.01 versus indicated group; *p < 0.05 versus indicated group. All data were expressed as mean ± SEM of at least 3 independent experiments

Fig. 6

ARHGAP25 was a target gene of miR-27a-3p A. Venn diagram analysis of the predicted downstream target genes of miR-27a-3p by miRanda, PITA and RNAhybrid databases. B. The putative miR-27a-3p binding sites in ARHGAP25 C-D. Western blot and quantitative analysis of ARHGAP25. E. Luciferase reporter assay to evaluate the interaction ability between miR-27a-3p 3ʹ—UTR and ARHGAP25. **p < 0.01 versus indicated group; *p < 0.05 versus indicated group. All data were expressed as mean ± SEM of at least 3 independent experiments

Fig. 7

miR-27a-3p suppressed TJPs degradation via ARHGAP25/Wnt/β-catenin pathway A. Western blot analysis of ARHGAP25 B. Protein expression of occludin, claudin‐5, ZO-1, Wnt3a and β-catenin by western-blot. C–G. Quantitation of the bands in B. **p < 0.01 versus indicated group; *p < 0.05 versus indicated group. All data were expressed as mean ± SEM of at least 3 independent experiments