MicroRNAs in blood brain barrier protection

描述(申请人提供)：在过去的十年中，人们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，包括多发性硬化症、中风、阿尔茨海默氏症和帕金森氏症、脑感染和癫痫。炎症在血脑屏障损伤中起重要作用，其次是脑或血液中产生的促炎因子和白细胞与脑内皮细胞的结合。脑微血管内皮细胞(BMVEC)是炎症部位炎症过程的积极参与者和调节者。脑内皮细胞的炎症反应涉及数百个基因，这些基因的表达需要微调调节。MicroRNAs(MiRNAs)是近年来出现的基因表达的主要调控因子。关于它们参与脑内皮细胞炎症反应的信息非常有限。我们建议使用成熟的体外系统来研究脑内皮细胞中miRNAs在脑内皮细胞中的作用，该系统用于神经炎症模型中血脑屏障的功能研究和脑微血管的活体成像。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，当介入剂具有抗炎特性并能够稳定紧密连接时，屏障保护效果最好，正如我们通过抑制糖原合成酶�(GSK3miRNAs)所表明的那样。为了确定一组相关的miRNA，我们用炎症刺激激活了原代人的BMVEC，肿瘤坏死因子�鉴定了137mRNAs下调和180miRNAs上调。在复制的BMVEC中，我们抑制了肿瘤坏死因子�刺激过程中的GSK3�n BMVEC，并检测到几种受GSK3�调控的miRNA的表达模式。在暴露于肿瘤坏死因子�的小鼠脑微血管中，miRNAs的过表达阻止了BMVEC中炎症介质的分泌。在我们拟议的研究中，我们将测试选定的miRNAs对血脑屏障紧密性的过度表达或抑制，以及对单核细胞-内皮细胞接触(黏附/迁移)的影响。利用生物信息学，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，并监测这将如何改变神经炎症动物模型中的白细胞黏附/迁移。拟议的实验将提供脑内皮细胞中miRNAs的鉴定和功能评估。

DESCRIPTION (provided by applicant): During the last decade it has been recognized that blood brain barrier (BBB) dysfunction exists in most neurological diseases, including multiple sclerosis, stroke, Alzheimer's and Parkinson's diseases, brain infections and epilepsy. Inflammation plays 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 which 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 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 BBB and in vivo imaging of brain microvasculature in the 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 as we have shown via inhibition of glycogen synthase kinase (GSK3� To identify a set of relevant miRNAs, we activated primary human BMVEC with the inflammatory stimulus, TNF�identified 137 down-regulated miRNAs and 180 up-regulated miRNAs. In replicate BMVEC, we inhibited GSK3�n BMVEC during TNF�timulation and detected several miRNAs with expression patterns that were regulated by GSK3�The same miRNAs were changed in brain microvessels isolated from mice exposed to TNF�miRNA overexpression prevented secretion of inflammatory mediators in BMVEC. In our proposed study, we will test the overexpression or inhibition of selected miRNAs on BBB tightness and the effects on monocyte-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 this will change leukocyte adhesion/migration in an animal model of neuroinflammation. Proposed experiments will provide identification and functional assessment of miRNAs in brain endothelium.