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Endothelium-targeted deletion of the miR-15a/16-1 cluster ameliorates blood-brain barrier dysfunction in ischemic stroke.

ABSTRACT: The blood-brain barrier (BBB) maintains a stable brain microenvironment. Breakdown of BBB integrity during cerebral ischemia initiates a devastating cascade of events that eventually leads to neuronal loss. MicroRNAs are small noncoding RNAs that suppress protein expression, and we previously showed that the miR-15a/16-1 cluster is involved in the pathogenesis of ischemic brain injury. Here, we demonstrated that when subjected to experimentally induced stroke, mice with an endothelial cell (EC)-selective deletion of miR-15a/16-1 had smaller brain infarcts, reduced BBB leakage, and decreased infiltration of peripheral immune cells. These mice also showed reduced infiltration of proinflammatory M1-type microglia/macrophage in the peri-infarct area without changes in the number of resolving M2-type cells. Stroke decreases claudin-5 abundance, and we found that EC-selective miR-15a/16-1 deletion enhanced claudin-5 mRNA and protein abundance in ischemic mouse brains. In cultured mouse brain microvascular ECs (mBMECs), the miR-15a/16-1 cluster directly bound to the 3' untranslated region (3'UTR) of Claudin-5, and lentivirus-mediated ablation of miR-15a/16-1 diminished oxygen-glucose deprivation (OGD)-induced down-regulation of claudin-5 mRNA and protein abundance and endothelial barrier dysfunction. These findings suggest that genetic deletion of endothelial miR-15a/16-1 suppresses BBB pathologies after ischemic stroke. Elucidating the molecular mechanisms of miR-15a/16-1-mediated BBB dysfunction may enable the discovery of new therapies for ischemic stroke.

SUBMITTER: Ma F

PROVIDER: S-EPMC7173187 | biostudies-literature | 2020 Apr

REPOSITORIES: biostudies-literature

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Endothelium-targeted deletion of the miR-15a/16-1 cluster ameliorates blood-brain barrier dysfunction in ischemic stroke.

Ma Feifei F Sun Ping P Zhang Xuejing X Hamblin Milton H MH Yin Ke-Jie KJ

Science signaling 20200407 626

The blood-brain barrier (BBB) maintains a stable brain microenvironment. Breakdown of BBB integrity during cerebral ischemia initiates a devastating cascade of events that eventually leads to neuronal loss. MicroRNAs are small noncoding RNAs that suppress protein expression, and we previously showed that the miR-15a/16-1 cluster is involved in the pathogenesis of ischemic brain injury. Here, we demonstrated that when subjected to experimentally induced stroke, mice with an endothelial cell (EC)- ...[more]

PMID: 32265338

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Project description:RationaleAngiogenesis promotes neurological recovery after stroke and is associated with longer survival of stroke patients. Cerebral angiogenesis is tightly controlled by certain microRNAs (miRs), such as the miR-15a/16-1 cluster, among others. However, the function of the miR-15a/16-1 cluster in endothelium on postischemic cerebral angiogenesis is not known.ObjectiveTo investigate the functional significance and molecular mechanism of endothelial miR-15a/16-1 cluster on angiogenesis in the ischemic brain.Methods and resultsEndothelial cell-selective miR-15a/16-1 conditional knockout (EC-miR-15a/16-1 cKO) mice and wild-type littermate controls were subjected to 1 hour middle cerebral artery occlusion followed by 28-day reperfusion. Deletion of miR-15a/16-1 cluster in endothelium attenuates post-stroke brain infarction and atrophy and improves the long-term sensorimotor and cognitive recovery against ischemic stroke. Endothelium-targeted deletion of the miR-15a/16-1 cluster also enhances post-stroke angiogenesis by promoting vascular remodeling and stimulating the generation of newly formed functional vessels, and increases the ipsilateral cerebral blood flow. Endothelial cell-selective deletion of the miR-15a/16-1 cluster up-regulated the protein expression of pro-angiogenic factors VEGFA (vascular endothelial growth factor), FGF2 (fibroblast growth factor 2), and their receptors VEGFR2 (vascular endothelial growth factor receptor 2) and FGFR1 (fibroblast growth factor receptor 1) after ischemic stroke. Consistently, lentiviral knockdown of the miR-15a/16-1 cluster in primary mouse or human brain microvascular endothelial cell cultures enhanced in vitro angiogenesis and up-regulated pro-angiogenic proteins expression after oxygen-glucose deprivation, whereas lentiviral overexpression of the miR-15a/16-1 cluster suppressed in vitro angiogenesis and down-regulated pro-angiogenic proteins expression. Mechanistically, miR-15a/16-1 translationally represses pro-angiogenic factors VEGFA, FGF2, and their receptors VEGFR2 and FGFR1, respectively, by directly binding to the complementary sequences within 3'-untranslated regions of those messenger RNAs.ConclusionsEndothelial miR-15a/16-1 cluster is a negative regulator for postischemic cerebral angiogenesis and long-term neurological recovery. Inhibition of miR-15a/16-1 function in cerebrovascular endothelium may be a legitimate therapeutic approach for stroke recovery.

Project description:AimsIschemic stroke is a life-threatening disease with limited therapeutic strategies. Blood-brain barrier (BBB) disruption is a critical pathological process that contributes to poor outcomes in ischemic stroke. We previously showed that the microglial inhibition of the inflammasome sensor absent in melanoma 2 (AIM2) suppressed the inflammatory response and protected against ischemic stroke. However, whether AIM2 is involved in BBB disruption during cerebral ischemia is unknown.MethodsMiddle cerebral artery occlusion (MCAO) and oxygen-glucose deprivation/reoxygenation (OGD/R) were used to mimic cerebral ischemia in mice and brain microvascular endothelial cells (HBMECs), respectively. The infarct volume, neurological deficits, and BBB permeability were measured in mice after MCAO. Transendothelial electrical resistance (TEER) and neutrophil adhesion to the HBMEC monolayer were assessed after OGD/R treatment. Western blot and immunofluorescence analyses were conducted to evaluate the expression of related proteins.ResultsAIM2 was shown to be expressed in brain endothelial cells and upregulated after ischemic stroke in the mouse brain. AIM2 deletion reduced the infarct volume, improved neurological and motor functions, and decreased BBB disruption. In vitro, OGD/R significantly increased the protein levels of AIM2 and ICAM-1 and decreased those of the tight junction (TJ) proteins ZO-1 and occludin. AIM2 knockdown effectively protected BBB integrity by promoting the expression of TJ proteins and decreasing ICAM-1 expression and neutrophil adhesion. Mechanistically, AIM2 knockdown reversed the OGD/R-induced increases in ICAM-1 expression and STAT3 phosphorylation in brain endothelial cells. Furthermore, treatment with the p-STAT3 inhibitor AG490 mitigated the effect of AIM2 on BBB breakdown.ConclusionOur findings indicated that inhibiting AIM2 preserved the BBB integrity after ischemic stroke, at least partially by modulating STAT3 activation and that AIM2 may be a promising therapeutic target for cerebral ischemic stroke.

Project description:BackgroundMicroglia and infiltrated macrophages (M/M) are integral components of the innate immune system that play a critical role in facilitating brain repair after ischemic stroke (IS) by clearing cell debris. Novel therapeutic strategies for IS therapy involve modulating M/M phenotype shifting. This study aims to elucidate the pivotal role of S100A9 in M/M and its downstream STAT6/PPARγ signaling pathway in neuroinflammation and phagocytosis after IS.MethodsIn the clinical study, we initially detected the expression pattern of S100A9 in monocytes from patients with acute IS and investigated its association with the long-term prognosis. In the in vivo study, we generated the S100A9 conditional knockout (CKO) mice and compared the stroke outcomes with the control group. We further tested the S100A9-specific inhibitor paqunimod (PQD), for its pharmaceutical effects on stroke outcomes. Transcriptomics and in vitro studies were adopted to explore the mechanism of S100A9 in modulating the M/M phenotype, which involves the regulation of the STAT6/PPARγ signaling pathway.ResultsS100A9 was predominantly expressed in classical monocytes and was correlated with unfavorable outcomes in patients of IS. S100A9 CKO mitigated infarction volume and white matter injury, enhanced cerebral blood flow and functional recovery, and prompted anti-inflammation phenotype and efferocytosis after tMCAO. The STAT6/PPARγ pathway, an essential signaling cascade involved in immune response and inflammation, might be the downstream target mediated by S100A9 deletion, as evidenced by the STAT6 phosphorylation inhibitor AS1517499 abolishing the beneficial effect of S100A9 inhibition in tMCAO mice and cell lines. Moreover, S100A9 inhibition by PQD treatment protected against neuronal death in vitro and brain injuries in vivo.ConclusionThis study provides evidence for the first time that S100A9 in classical monocytes could potentially be a biomarker for predicting IS prognosis and reveals a novel therapeutic strategy for IS. By demonstrating that S100A9-mediated M/M polarization and phagocytosis can be reversed by S100A9 inhibition in a STAT6/PPARγ pathway-dependent manner, this study opens up new avenues for drug development in the field.

Project description:P-glycoprotein (P-gp) is expressed on brain microvessel endothelial cells of blood-brain barrier (BBB) and elevated after cerebral ischemia. In this study, we explored the influence and potential mechanisms of P-gp on BBB function in experimental ischemic stroke in vivo and in vitro. Middle cerebral artery occlusion/reperfusion (MCAO/R) was created in mice. Oxygen-glucose deprivation/reoxygenation (OGD/R) was performed in brain microvascular vessel-derived endothelial cells (bEnd.3) to mimic ischemia/reperfusion injury in vitro. P-gp-specific siRNA and pharmacological inhibitor cyclosporine A were used to inhibit P-gp, whereas pcDNA3.1 was utilized to overexpress P-gp. Twenty-four hours after reperfusion, acute ischemic stroke outcome, BBB integrity and permeability, autophagic proteins and relative signaling pathways were evaluated. P-gp levels were markedly elevated in mouse brain and endothelial cells following MCAO/R and OGD/R, respectively. P-gp siRNA silencing or pharmacologically inhibiting (cyclosporine A) reduced infarct volume and brain edema, attenuated brain pathology, and improved neurological behavior in association with attenuated accumulation of neutrophils and macrophages, reduced expression levels of inflammatory cytokines (TNF-α and IL-1β), matrix metalloproteinases (MMP-2 and MMP-9) and adhesion molecules (ICAM-1 and VCAM-1). P-gp silence also counteracted BBB leakage, restored the expressions of tight junction proteins (Claudin-5, Occludin and ZO-1), activated autophagic proteins (upregulated LC3-II/LC3-I and Beclin 1, and downregulated P62), and diminished Akt/mTOR signal activity in mice following MCAO/R. In the endothelial cell OGD/R assay, P-gp silence downregulated the expressions of inflammatory cytokines and adhesion molecules, inhibited leukocytes adhesion and migration, increased tight junction protein levels, and activated autophagy, all were reversible by forceful P-gp expression. Additionally, treatment with an autophagy inhibitor (3-methyladenine) abolished protections against ischemic stroke and tight junction proteins reduction followed by P-gp silence. In conclusion, increased P-gp expression after ischemic injury resulted in BBB dysfunction and hyperpermeability by suppressing Akt/mTOR-induced endothelial autophagy.

Dataset Information

Endothelium-targeted deletion of the miR-15a/16-1 cluster ameliorates blood-brain barrier dysfunction in ischemic stroke.

Publications

Endothelium-targeted deletion of the miR-15a/16-1 cluster ameliorates blood-brain barrier dysfunction in ischemic stroke.

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