背景:血脑屏障(BBB)破坏是缺血性卒中(IS)后的主要不良事件。小窝-1(Cav-1),一种支架蛋白,在IS后的BBB渗透率中起着多重作用,而Cav-1对血脑屏障通透性的利弊仍存在争议。大量研究表明,细胞外囊泡(EV),尤其是干细胞衍生的电动汽车,对IS发挥治疗功效;然而,需要清楚阐明BBB通透性的机制。在这里,我们比较了急性IS后骨髓间充质干细胞来源的细胞外囊泡(BMSC-EVs)和来自脑内皮细胞的EVs(BEC-EVs)对BBB完整性的保护效果,并研究了该机制是否与EVs拮抗Cav-1依赖性紧密连接蛋白内吞相关.
方法:分离BMSC-EV和BEC-EV,并通过纳米粒子跟踪分析进行表征,西方印迹,和透射电子显微镜。氧和葡萄糖剥夺(OGD)处理b.End3细胞用于评估脑内皮细胞渗漏。CCK-8和TRITC-葡聚糖渗漏测定用于测量细胞活力和transwell单层通透性。建立永久性大脑中动脉闭塞(pMCAo)模型,和EV在大鼠中静脉内给药。采用动物神经功能试验,从缺血皮质中分离出微血管。通过伊文思蓝(EB)染色和蛋白质印迹分析BBB渗漏和紧密连接蛋白,分别。采用Co-IP测定和Cav-1siRNA/pcDNA3.1载体转染来验证Cav-1对紧密连接蛋白的内吞功效。
结果:两种电动汽车在大鼠24hpMCAo后,在减少脑梗死体积和BBB渗漏以及增强ZO-1和Claudin-5的表达方面均具有相似的功效。同时,BMSC-EV在改善神经功能方面表现突出。同时,两种EV处理都抑制了OGD暴露的b中的高表达的Cav-1。End3细胞和缺血性脑微血管,BMSC-EV给药后,这种疗效更为突出。Cav-1敲低降低了OGD处理的b.End3细胞单层通透性,恢复了ZO-1和Claudin-5的表达,而Cav-1的过表达会加剧渗透性,并增强Cav-1与ZO-1和Claudin-5的共定位。此外,Cav-1过表达在OGD处理的b中通过BMSC-EV和BEC-EV施用部分逆转了较低的细胞渗漏。End3细胞。
结论:我们的结果表明,Cav-1加重了急性缺血性卒中的BBB通透性,和BMSC-EV对Cav-1依赖性ZO-1和Claudin-5内吞作用与BEC-EV具有相似的拮抗功效。BMSC-EVs治疗在Cav-1抑制和神经功能改善方面优于对照组。
BACKGROUND: Blood-brain barrier (BBB) disruption is a major adverse event after ischemic stroke (IS). Caveolin-1 (Cav-1), a scaffolding protein, played multiple roles in BBB permeability after IS, while the pros and cons of Cav-1 on BBB permeability remain controversial. Numerous studies revealed that extracellular vesicles (EVs), especially stem cells derived EVs, exerted therapeutic efficacy on IS; however, the mechanisms of BBB permeability needed to be clearly illustrated. Herein, we compared the protective efficacy on BBB integrity between bone marrow mesenchymal stem cells derived extracellular vesicles (BMSC-EVs) and EVs from brain endothelial cells (BEC-EVs) after acute IS and investigated whether the mechanism was associated with EVs antagonizing Cav-1-dependent tight junction proteins
endocytosis.
METHODS: BMSC-EVs and BEC-EVs were isolated and characterized by nanoparticle tracking analysis, western blotting, and transmission electron microscope. Oxygen and glucose deprivation (OGD) treated b. End3 cells were utilized to evaluate brain endothelial cell leakage. CCK-8 and TRITC-dextran leakage assays were used to measure cell viability and transwell monolayer permeability. Permanent middle cerebral artery occlusion (pMCAo) model was established, and EVs were intravenously administered in rats. Animal neurological function tests were applied, and microvessels were isolated from the ischemic cortex. BBB leakage and tight junction proteins were analyzed by Evans Blue (EB) staining and western blotting, respectively. Co-IP assay and Cav-1 siRNA/pcDNA 3.1 vector transfection were employed to verify the
endocytosis efficacy of Cav-1 on tight junction proteins.
RESULTS: Both kinds of EVs exerted similar efficacies in reducing the cerebral infarction volume and BBB leakage and enhancing the expressions of ZO-1 and Claudin-5 after 24 h pMCAo in rats. At the same time, BMSC-EVs were outstanding in ameliorating neurological function. Simultaneously, both EVs treatments suppressed the highly expressed Cav-1 in OGD-exposed b. End3 cells and ischemic cerebral microvessels, and this efficacy was more prominent after BMSC-EVs administration. Cav-1 knockdown reduced OGD-treated b. End3 cells monolayer permeability and recovered ZO-1 and Claudin-5 expressions, whereas Cav-1 overexpression aggravated permeability and enhanced the colocalization of Cav-1 with ZO-1 and Claudin-5. Furthermore, Cav-1 overexpression partly reversed the lower cell leakage by BMSC-EVs and BEC-EVs administrations in OGD-treated b. End3 cells.
CONCLUSIONS: Our results demonstrated that Cav-1 aggravated BBB permeability in acute ischemic stroke, and BMSC-EVs exerted similar antagonistic efficacy to BEC-EVs on Cav-1-dependent ZO-1 and Claudin-5
endocytosis. BMSC-EVs treatment was superior in Cav-1 suppression and neurological function amelioration.