Background: Preserving the integrity of the blood-brain barrier (BBB) is beneficial to avoid further brain damage after acute ischemic stroke (AIS). Astrocytes, an important component of the BBB, promote BBB breakdown in subjects with AIS by secreting inflammatory factors. The glucagon-like peptide-1 receptor (GLP-1R) agonist exendin-4 (Ex-4) protects the BBB and reduces brain inflammation from cerebral ischemia, and GLP-1R is expressed on astrocytes. However, the effect of Ex-4 on astrocytes in subjects with AIS remains unclear. Methods: In the present study, we investigated the effect of Ex-4 on astrocytes cultured under oxygen-glucose deprivation (OGD) plus reoxygenation conditions and determined whether the effect influences bEnd.3 cells. We used various methods, including permeability assays, western blotting, immunofluorescence staining, and gelatin zymography, in vitro and in vivo. Results: Ex-4 reduced OGD-induced astrocyte-derived vascular endothelial growth factor (VEGF-A), matrix metalloproteinase-9 (MMP-9), chemokine monocyte chemoattractant protein-1 (MCP-1), and chemokine C-X-C motif ligand 1 (CXCL-1). The reduction in astrocyte-derived VEGF-A and MMP-9 was related to the increased expression of tight junction proteins (TJPs) in bEnd.3 cells. Ex-4 improved neurologic deficit scores, reduced the infarct area, and ameliorated BBB breakdown as well as decreased astrocyte-derived VEGF-A, MMP-9, CXCL-1, and MCP-1 levels in ischemic brain tissues from rats subjected to middle cerebral artery occlusion. Ex-4 reduced the activation of the JAK2/STAT3 signaling pathway in astrocytes following OGD. Conclusion: Based on these findings, ischemia-induced inflammation and BBB breakdown can be improved by Ex-4 through an astrocyte-dependent manner.

Rationale: Mesenchymal stromal cells (MSCs) are emerging as a novel therapeutic strategy for the acute ischemic stroke (AIS). However, the poor targeted migration and low engraftment in ischemic lesions restrict their treatment efficacy. The ischemic brain lesions express a specific chemokine profile, while cultured MSCs lack the set of corresponding receptors. Thus, we hypothesize that overexpression of certain chemokine receptor might help in MSCs homing and improve therapeutic efficacy. Methods: Using the middle cerebral artery occlusion (MCAO) model of ischemic stroke, we identified that CCL2 is one of the most highly expressed chemokines in the ipsilateral hemisphere. Then, we genetically transduced the corresponding receptor, CCR2 to the MSCs and quantified the cell retention of MSC^CCR2 compared to the MSC^dtomato control. Results: MSC^CCR2 exhibited significantly enhanced migration to the ischemic lesions and improved the neurological outcomes. Brain edema and blood-brain barrier (BBB) leakage levels were also found to be much lower in the MSC^CCR2-treated rats than the MSC^dtomato group. Moreover, this BBB protection led to reduced inflammation infiltration and reactive oxygen species (ROS) generation. Similar results were also confirmed using the in vitro BBB model. Furthermore, genome-wide RNA sequencing (RNA-seq) analysis revealed that peroxiredoxin4 (PRDX4) was highly expressed in MSCs, which mainly contributed to their antioxidant impacts on MCAO rats and oxygen-glucose deprivation (OGD)-treated endothelium. Conclusion: Taken together, this study suggests that overexpression of CCR2 on MSCs enhances their targeted migration to the ischemic hemisphere and improves the therapeutic outcomes, which is attributed to the PRDX4-mediated BBB preservation.

Intestinal microbiota is an important environmental factor in the initiation and progression of autoimmune diseases. However, investigations on the gut microbiome in neuromyelitis optica spectrum disorders (NMOSD) are relatively insufficient, especially for that of the Asia population. To evaluate whether or not the intestinal microbiota of NMOSD patients had specific microbial signatures. Next-generation sequencing and gas chromatography were employed to compare the fecal microbial composition and short-chain fatty acids (SCFAs) spectrum between patients with NMOSD (n = 84) and healthy controls (n = 54). The gut microbial composition of NMOSD distinguished from healthy individuals. Streptococcus, significantly increased in NMOSD, is positively correlated with disease severities (p < 0.05). The use of immunosuppressants results in a decrease of Streptococcus, suggesting that Streptococcus might play a significant role in the pathogenesis of NMOSD. A striking depletion of fecal SCFAs was observed in NMOSD patients (p < 0.0001), with acetate and butyrate showing significantly negative correlation with disease severities (p < 0.05). The fecal organismal structures and SCFAs level of patients with NMOSD were distinctive from healthy individuals. These findings not only could be critical events driving the aberrant immune response responsible for the pathogenesis of these disorders but could also provide suggestions for disease therapy.

Astrocytes mediate the destruction of the blood–brain barrier (BBB) during ischemic stroke (IS). IL‐9 is a pleiotropic cytokine that we previously found to be highly expressed in peripheral blood mononuclear cells from patients with IS, and the presence of IL‐9 receptors on astrocytes has been reported in the literature. Here, we detected the effect of IL‐9 on astrocytes using an anti–IL‐9‐neutralizing antibody to treat rats with experimental stroke. Supernatants from astrocytes treated with or without oxygen–glucose deprivation and/or IL‐9 were incubated with bEnd.3 cell monolayers after blocking the IL‐9 receptor on the endothelium. Immunofluorescence staining and Western blot analyses were conducted to observe the change in tight junction proteins (TJPs) in bEnd.3 cells as well as the level of VEGF‐A and possible signal pathways in astrocytes. We also applied middle cerebral artery occlusion (MCAO) models to determine the effect of anti–IL‐9‐neutralizing antibodies on IS. As a result, astrocyte‐conditioned medium treated with IL‐9 aggravated the disruption of the BBB accomplished by the degradation of TJPs in endothelial cells. In addition, IL‐9 increased the level of VEGF‐A in astrocytes, and blocking the effect of VEGF‐A reversed the breakdown of the BBB. In the MCAO model, anti–IL‐9‐neutralizing antibody reduced the infarct volume and BBB destruction. Mechanistically, the anti–IL‐9‐neutralizing antibody repaired the damaged TJPs (zonula occludens 1, occludin, and claudin‐5) and induced a decrease in VEGF‐A expression in ischemic lateral brain tissue. In contrast, a local injection of recombinant murine IL‐9 to the brain resulted in a marked up‐regulation of VEGF‐A in the striatum. In conclusion, anti–IL‐9‐neutralizing antibody can reduce the severity of IS partially by alleviating the destruction of the BBB via down‐regulation of astrocyte‐derived VEGF‐A. This finding suggests that targeting IL‐9 or VEGF‐A could provide a new direction for the treatment of IS.—Tan, S., Shan, Y., Lin, Y., Liao, S., Zhang, B., Zeng, Q., Wang, Y., Deng, Z., Chen, C, Hu, X., Peng, L., Qiu, W., Lu, Z. Neutralization of IL‐9 ameliorates experimental stroke by repairing the blood–brain barrier via down‐regulation of astrocyte‐derived vascular endothelial growth factor‐A. FASEB J. 33, 4376–4387 (2019). www.fasebj.org