MicroRNA-29a-5p attenuates hemorrhagic transformation and improves outcomes after mechanical reperfusion for acute ischemic stroke

Abstract

Background: Hemorrhage transformation (HT) following endovascular reperfusion treatment is associated with worse clinical outcomes in acute ischemic stroke patients. MicroRNA (miR) modulates several aspects of cerebral ischemia-reperfusion injury, including blood-brain barrier (BBB) integrity, inflammation, oxidative stress, and apoptosis, significantly impacting cerebral recovery and function. This study investigated the role of astrocytic miR-29a-5p in HT in the transient middle cerebral artery occlusion (MCAO) model and oxygen-glucose deprivation reoxygenation (OGD/R) model of astrocytes.

Methods: MiR-29a-5p expression in the OGD/R astrocyte model was assessed. The astrocyte injury, the expression of A1 and A2 phenotypes of reactive astrocytes, and the regulation of miR-29a-5p target genes were evaluated after the miR-29a-5p intervention. A mechanical reperfusion-induced HT model was established in hyperglycemic rats using 5-h MCAO following reperfusion at 6 h. MiR-29a-5p agomir was administered intravenously before reperfusion. Infarct volume, HT, BBB damage, neurological score, the expression of miR-29a-5p, and its target genes were evaluated.

Results: MiR-29a-5p expression decreased in OGD/R-treated astrocytes and the peri-infarction tissue and blood of the MCAO model. Elevating miR-29a-5p levels reduced astrocyte injury, suppressed neurotoxic A1 astrocyte markers (C3, Fkbp5, and Serping1), while enhanced neuroprotective A2 astrocyte markers (S100a10 and Emp1) in the OGD/R and MCAO models. Intravenous administration of miR-29a-5p agomir increased the expression of miR-29a-5p and reduced infarct volume, reperfusion-induced HT, and BBB breakdown after ischemia, improving neurological outcomes in the MCAO model. Overexpression of miR-29a-5p effectively suppressed the expression of its direct target genes, glycogen synthase kinase 3 beta and aquaporin 4 in the OGD/R and MCAO models.

Conclusions: MiR-29a-5p alleviates astrocyte injury and regulates A1 and A2 astrocyte markers, glycogen synthase kinase 3 beta, and aquaporin 4 in astrocytes subjected to ischemia-reperfusion injury. Astrocytic miR-29a-5p may be a protective target for reducing HT and improving outcomes following mechanical reperfusion in acute ischemic stroke.

Keywords: Acute ischemic stroke; Astrocyte; Cerebral ischemia-reperfusion injury; Hemorrhagic transformation; MicroRNA; Oxygen-glucose deprivation reoxygenation.

Fig. 1

Elevating miR-29a-5p levels was protective in ischemia-reperfusion injury in the OGD/R astrocyte model. A, miR-29a-5p mimics increased the expression of miR-29a-5p in astrocytes following OGD/R. B, miR-29a-5p mimics alleviated cell damage shown by CCK-8 assay. C, miR-29a-5p mimics inhibited LDH release from astrocytes. D and E, miR-29a-5p mimics decreased the expression of neurotoxic A1 reactive astrocyte markers (C3, Fkbp5, and Serping1) while increasing the expression of neuroprotective A2 reactive astrocyte markers (S100a10, Ptx3, and Emp1). Each sample was tested in triplicate. ∗P < 0.05; ∗∗P < 0.01; #P < 0.001.

Fig. 2

miR-29a-5p regulated the expression of target genes in the OGD/R astrocyte model. A, the top predicted twenty hub genes for miR-29a-5p were identified by bioinformatics analysis. B, miR-29a-5p mimics suppressed the upregulated expression of Gsk3b and increased the downregulated expression of Fzd2 and Dvl3 shown by RT-PCR. C, D, and E, Western blot analysis showed miR-29a-5p mimics significantly suppressed the upregulated expression of GSK3β, FKBP5, and AQP4. F and G, Gsk3b and Aqp4 are the direct target genes for miR-29a-5p by a dual-luciferase reporter assay. Each sample was tested in triplicate. ∗∗P < 0.01; #P < 0.001.

Fig. 3

Elevating miR-29a-5p levels regulated the expression of miR-29a-5p, predicted target genes, and A1/A2 astrocyte markers in the hyperglycemia-associated reperfusion-induced HT model. A, miR-29a-5p expression decreased in the peri-infarction tissue and blood of the MCAO model (n = 6). B, miR-29a-5p agomir treatment before reperfusion reduced the miR-29a-5p expression in the peri-infarction tissue and blood of the MCAO model (n = 6). C, miR-29a-5p agomir treatment suppressed the mRNA expression of A1 markers (C3, Fkbp5, and Serping1), Gsk3b, and Aqp4, while enhanced A2 markers (S100a10 and Emp1) in the peri-infarction tissue (n = 6). ∗P < 0.05; ∗∗P < 0.01; #P < 0.001.

Fig. 4

MiR-29a-5p agomir reduces infarct volume, reperfusion-induced hemorrhage, and BBB disruption and improves neurological outcomes via regulating GSK3β, FKBP5, and AQP4. A, TTC staining of brain slices and quantification for infarct volume (n = 6). B, Hemoglobin content index is determined by spectrophotometric hemoglobin assay (n = 6). C, BBB permeability in the ischemic hemisphere was shown by Evans blue staining (n = 6). D, Western blot shows the expression of GSK3β, FKBP5, AQP4, and occludin in the peri-infarction tissue (n = 6). ∗∗P < 0.01; #P ≤ 0.001.