Protective role of let-7 microRNAs in brain endothelial dysfunction during ischemia/reperfusion injury

已经认识到血脑屏障(BBB)功能障碍存在于大多数神经系统疾病中， 包括中风、多发性硬化症、阿尔茨海默氏症和帕金森氏症、脑部感染和癫痫。 炎症在血脑屏障损伤中起重要作用，仅次于大脑中产生的促炎因子 或血液和白细胞与脑内皮细胞的接触。脑微血管内皮细胞(BMVEC) 炎症部位炎症过程的积极参与者和调节者。炎症反应 在大脑中，内皮涉及数百个基因，这些基因的表达需要微调调节。MicroRNAs (MiRNAs)最近成为基因表达的主要调节因子。关于他们的信息非常有限 参与脑内皮细胞缺血/再灌注(I/R)事件引起的炎症反应。 我们建议使用成熟的体外系统研究miRNAs在脑内皮细胞中的作用 在活体神经炎模型中血脑屏障的功能研究和脑微血管成像。 基于我们对神经炎症时血脑屏障功能障碍的研究，我们认为屏障保护是最好的。 当介入剂具有抗炎性能并能稳定紧密连接时实现。 最近，我们发现了高度修饰的miRNA，属于let-7 miRNA家族，它们是重要的 用于内皮细胞的维护。LET-7和miR-98被预测为针对炎症分子CCL2， CCL5、IL8和IP10/CXCL10。LET-7和miR-98在体内外的过表达导致细胞数量减少 白细胞与脑内皮细胞的黏附和迁移与促炎因子表达的降低 细胞因子。在氧糖剥夺(OGD)再灌注(OGD/R)模型中， 这些miRNAs的过表达导致血脑屏障的紧密性增加，从而减弱屏障的“泄漏性”。 这些miRNAs的过表达导致脑梗塞体积缩小和中性粒细胞在脑内的浸润 TMCAO，一种在体I/R卒中动物模型。在我们提议的研究中，我们将测试过度表达或抑制 选定的miRNAs对血脑屏障密封性和白细胞-内皮细胞参与(黏附/迁移)的影响。vbl.使用 生物信息学，我们将确定miRNAs的其他目标。接下来，我们将在体内进行miRNA转基因，并 监测miRNAs的存在如何改变中风动物模型中白细胞的黏附/迁移。 拟议的实验将提供脑内皮细胞miRNAs的鉴定和功能评估， 并为未来预防缺血/再灌注对心脏的有害影响提供了新的治疗方法。 大脑。

It has been recognized that blood brain barrier (BBB) dysfunction exists in most neurological diseases, including stroke, multiple sclerosis, Alzheimer's and Parkinson's diseases, brain infections and epilepsy. Inflammation plays a significant role in BBB injury, secondary to pro-inflammatory factors produced in the brain or blood and leukocyte engagement of brain endothelium. Brain microvascular endothelial cells (BMVEC) are active participants and regulators of inflammatory processes at a site of inflammation. Inflammatory responses in brain endothelium involve hundreds of genes whose expression requires fine-tuned regulation. microRNAs (miRNAs) recently emerged as major regulators of gene expression. Very limited information exists about their participation in inflammatory responses caused by ischemia/reperfusion (I/R) events in brain endothelial cells. We propose to investigate the role of miRNAs in brain endothelium using well-established in vitro systems for functional studies of the BBB and imaging of brain microvasculature in an in vivo model of neuroinflammation. Based on our studies of BBB dysfunction during neuroinflammation, we propose that barrier protection is best achieved when the intervening agents possess anti-inflammatory properties and can stabilize tight junctions. Recently we have identified highly modified miRNAs, belonging to the let-7 miRNA family, which are important for endothelial maintenance. let-7 and miR-98 were predicted to target the inflammatory molecules, CCL2, CCL5, IL8 and IP10/CXCL10. Overexpression of let-7 and miR-98 in vitro and in vivo resulted in reduced leukocyte adhesion to and migration across brain endothelium and diminished expression of pro-inflammatory cytokines. In oxygen glucose deprivation (OGD) followed by reperfusion (OGD/R), an in vitro I/R model, overexpression of these miRNAs led to increased BBB tightness, thereby attenuating barrier `leakiness'. Overexpression of these miRNAs resulted in decreased infarct volume and neutrophil infiltration in the brain in tMCAO, an in vivo I/R stroke animal model. In our proposed study, we will test the overexpression or inhibition of selected miRNAs on BBB tightness and leukocyte-endothelial cell engagement (adhesion/migration). Using bioinformatics, we will identify other targets for miRNAs. Next, we will perform miRNA transfection in vivo and monitor how the presence of miRNAs will change leukocyte adhesion/migration in an animal model of stroke. Proposed experiments will provide identification and functional assessment of miRNAs in brain endothelium, and lead to future therapeutic developments for prevention of deleterious effects of ischemia/reperfusion on the brain.