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Sphingosine-1-phosphate receptor 3 is implicated in BBB injury via the CCL2-CCR2 axis following acute intracerebral hemorrhage.

ABSTRACT:

Background

Intracerebral hemorrhage (ICH) is a catastrophic cerebrovascular disease with high morbidity and mortality. Evidence demonstrated that sphingosine-1-phosphate receptor (S1PR) plays a vital role in inflammatory damage via the upregulation of CCL2 expression. However, whether S1PR3 is involved in blood-brain barrier (BBB) breakdown via CCL2 activation after ICH has not been described.

Methods

We investigated the expression profiles of all S1PRs using high-throughput RNA-seq analysis and RT-PCR. The potential role of S1PR3 and interaction between S1PR3 and CCL2 were evaluated via Western blotting, immunofluorescence, and flow cytometry. BBB disruption was examined via magnetic resonance imaging, transmission electron microscopy, and Evans blue extravasation. Microglial activation, proliferation, and polarization were assessed via histopathological analysis. The expression levels of CCL2, p-p38 MAPK, ICAM-1, and ZO-1 were examined in vitro and in vivo.

Results

The present results showed that the levels of S1PR3 and its ligand, sphingosine 1-phosphate (S1P), were dramatically increased following ICH, which regulated the expression of CCL2 and p38MAPK. Moreover, reductions in brain edema volume, amelioration of BBB integrity, and improvements in behavioral deficits were achieved after the administration of CAY10444, an S1PR3 antagonist, to rats. Remarkably increased CCL2, p-p38MAPK, and ICAM-1 expression and decreased ZO-1 expression were observed in cocultured human astrocytes (HAs) and hCMEC/D3 cells after S1P stimulation. However, the expression levels of CCL2, p-p38 MAPK, and ICAM-1 were decreased and ZO-1 expression was increased after S1PR3 inhibition. In addition, microglial proliferation and M1 polarization were attenuated after CAY10444 administration.

Conclusion

To the best of our knowledge, this is the first demonstration of the neuroprotective role of S1PR3 modulation in maintaining BBB integrity by inhibiting the S1PR3-CCL2 axis after ICH, providing a novel treatment for ICH by targeting S1PR3.

SUBMITTER: Xu D

PROVIDER: S-EPMC8111497 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundBlood 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.MethodsADEVs 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.ResultsOGD-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.ConclusionTaken 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.