MicroRNAs in blood brain barrier protection

描述（由申请人提供）：在过去十年中，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、中风、阿尔茨海默病和帕金森病、脑感染和癫痫。炎症在BBB损伤中起重要作用，继发于脑或血液中产生的促炎因子和脑内皮细胞的白细胞参与。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，其表达需要微调调控。microRNAs（miRNAs）是近年来发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研究脑血管内皮细胞中miRNAs的作用，用于BBB的功能研究和神经炎症模型中脑微血管的体内成像。基于我们对神经炎症过程中BBB功能障碍的研究，我们提出，当干预剂具有抗炎特性并且可以稳定紧密连接时，屏障保护最好，正如我们通过抑制糖原合成酶激酶（GSK 3）所显示的那样。为了鉴定一组相关的miRNA，我们用炎症刺激激活了原代人BMVEC，TNF-α鉴定了137个下调的miRNAs和180个上调的miRNAs。在复制的BMVEC中，我们在TNF刺激过程中抑制了GSK 3的BMVEC，并检测到几种表达模式受GSK 3调节的miRNA。从暴露于TNF的小鼠中分离的脑微血管中也改变了相同的miRNA，miRNA过表达阻止了BMVEC中炎症介质的分泌。在我们提出的研究中，我们将测试所选miRNA对BBB紧密性的过表达或抑制以及对单核细胞-内皮细胞接合（粘附/迁移）的影响。使用生物信息学，我们将确定其他miRNAs的靶点。接下来，我们将在体内进行miRNA转染，并监测这将如何改变神经炎症动物模型中的白细胞粘附/迁移。提出的实验将提供脑内皮中miRNA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.