BackgroundPreserving 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.MethodsIn 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.ResultsEx-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.ConclusionBased 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 MSCCCR2 compared to the MSCdtomato control. Results: MSCCCR2 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 MSCCCR2-treated rats than the MSCdtomato 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.

β-Amyloid protein (Aβ) is thought to cause neuronal loss in Alzheimer’s disease (AD). Aβ treatment promotes the re-activation of a mitotic cycle and induces rapid apoptotic death of neurons. However, the signaling pathways mediating cell-cycle activation during neuron apoptosis have not been determined. We find that Wnt5a acts as a mediator of cortical neuron survival, and Aβ42 promotes cortical neuron apoptosis by downregulating the expression of Wnt5a. Cell-cycle activation is mediated by the reduced inhibitory effect of Wnt5a in Aβ42 treated cortical neurons. Furthermore, Wnt5a signals through the non-canonical Wnt/Ca2+ pathway to suppress cyclin D1 expression and negatively regulate neuronal cell-cycle activation in a cell-autonomous manner. Together, aberrant downregulation of Wnt5a signaling is a crucial step during Aβ42 induced cortical neuron apoptosis and might contribute to AD-related neurodegeneration.

Salt (sodium chloride, NaCl) accumulation in the brain is associated with various diseases of central nervous system (CNS). Activation of astrocytes is an important manifestation of pathophysiological processes in the CNS. However, the direct impact of high salt (HS) environment on astrocytes is unclear. In the current study, we found that high salt treatment can induce activation of astrocytes both in vivo and in vitro, manifested as morphological alteration coupled with increased expression of glial fibrillary acidic protein (GFAP). Additionally, HS upregulated the expression of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), interleukin-1β (IL-1β) and vascular endothelial growth factor (VEGF); however, its effects on transforming growth factor-β (TGF-β) expression were not evident. Furthermore, HS treatment induced increased phosphorylation of signal transducer and activator transcription 3 (STAT 3). Inhibition of Janus kinase 2 (JAK 2) by specific pharmacological antagonists, AG490, attenuated the activation of JAK2/STAT3 pathway and induction of GFAP and other pro-inflammatory factors, respectively. The results suggest that the aforementioned multiple inflammatory cytokines and mediators that may be linked to the HS induced pathogenesis of CNS via the JAK2/STAT3 signaling pathways.

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). http://www.fasebj.org