Regulatory microRNAs-mediated cerebrovascular protection and traumatic braininjury

创伤性脑损伤（TBI）是服务人员和退伍军人的主要医疗问题， 治疗可用于减轻创伤后神经功能缺损。血脑屏障（BBB）提供了一个 动态接口将大脑与循环系统分离，保持大脑稳定 微环境脑微血管内皮细胞（brain microvascular endothelial cells，BMEC）是血脑屏障的主要组成部分， 与细胞间紧密连接（TJ），在调节BBB的完整性和细胞旁 渗透性1.越来越多的证据表明，TBI后BBB的破坏促进了破坏性的 事件的级联，例如外周免疫细胞的迁移，脑炎症反应，水肿， 和出血性转化，导致神经创伤中的继发性损伤。因此需要 确定保护BBB完整性和预防BBB损伤的机制并开发有效的治疗策略 脑外伤后的永久性脑损伤 microRNAs（miRs）是一类重要的非编码小分子RNA， 表情除了它们在各种生物过程中的关键作用之外，miR还涉及一种新的生物学过程。 各种人类神经系统疾病。我们和其他人已经证明了miR参与了 TBI的发病机制。然而，miR分子在肿瘤中的功能意义和分子机制尚不清楚。 调节脑血管发病机制，特别是BBB破坏/功能障碍， 对TBI的神经学结果了解甚少。 miR-15 a/16-1簇是第一个被鉴定的与人类癌发生相关的miR组。 在TBI个体中发现了血浆miR-15 a/16-1水平失调，显示出作为治疗TBI的巨大潜力。 在临床诊断和预后中有用的生物标志物。有趣的是，miR-15 a/16-1水平的分子抑制 已经显示出对心肌梗塞（MI）和缺血性脑损伤的保护作用。因此，美国 和欧洲制药公司认为miR-15 a/16-1簇是最重要的miR 目标是开发用于治疗MI的基于miR的药物。 在我们最近的初步研究中，我们已经表明miR-15 a/16-1簇的表达是 在TBI后小鼠脑血管中选择性增加。值得注意的是，内皮细胞（EC）选择性miR- 15 a/16-1遗传缺陷导致BBB渗漏减少，神经元损失、白色物质（WM）损伤减少， 和神经行为障碍的影响。我们还发现miR-15 a/16-1簇可以结合到 主要BBB紧密连接的3 '-UTR，脑富集的Claudin，并抑制其翻译，以及遗传 miR-15 a/16-1簇的沉默或缺失显著增加了内皮或脑组织中miR-15 a/16- 1的表达。 claudins这些发现为我们的中心假设提供了基础，即基因缺失 血管miR-15 a/16-1减弱TBI后BBB破坏和随后的病理级联反应， 从而有助于增加BBB稳定性，减少神经元/WM损失，并改善长- TBI中的神经系统结局。本提案将实现三个目标。目标1：确定 血管miR-15 a/16-1在创伤后BBB功能障碍和TBI结局中的作用;目的2：阐明其机制 miR-15 a/16-1对创伤后BBB功能障碍的调节;目的3：探索miR-15 a/16 - 1的系统递送 15 a/16-1曲洛莫（抑制剂）通过稳定血脑屏障作为TBI的潜在治疗。阐明分子 miR-15 a/16-1介导的血脑屏障功能障碍的机制可能会引导我们发现新的有效的治疗方法 针对TBI。

Traumatic brain injury (TBI) is a major medical concern in service personnel and veterans as currently no therapy is available to alleviate post-trauma neurological deficits. The blood-brain barrier (BBB) provides a dynamic interface to separate the brain from the circulatory system, maintaining a stable brain microenvironment. As a major component of the BBB, brain microvascular endothelial cells (BMECs), together with intercellular tight junctions (TJs), play a dominant role in modulating BBB integrity and paracellular permeability1. Accumulating evidence has demonstrated that BBB breakdown after TBI promotes a devastating cascade of events such as transmigration of peripheral immune cells, cerebral inflammatory responses, edema, and hemorrhagic transformation, contributing to secondary injury in neurotrauma. Thus, it is necessary to identify mechanisms and develop effective therapeutic strategies that protect BBB integrity and prevent permanent brain damage after TBI. MicroRNAs (miRs) function as a major class of small non-coding RNAs that negatively modulate protein expression. In addition to their critical role in various biological processes, miRs have also been implicated in a variety of human neurological diseases. We and others have shown the involvement of miRs in the pathogenesis of TBI. However, the functional significance and molecular mechanisms of miR molecules in regulating cerebrovascular pathogenesis, in particular BBB disruption/dysfunction, and resultant long-term neurological outcomes are poorly understood in TBI. The miR-15a/16-1 cluster is the first identified miR group associated with human carcinogenesis. Dysregulated plasma miR-15a/16-1 levels have been found in TBI individuals, showing great potential as useful biomarkers in clinical diagnosis and prognosis. Interestingly, molecular inhibition of miR-15a/16-1 levels has been shown to protect against myocardial infarction (MI) and ischemic brain injury. Therefore, American and European pharmaceutical companies consider the miR-15a/16-1 cluster as one of the most important miR targets to develop miR-based drugs for the treatment of MI. In our recent preliminary studies, we have shown that expression of the miR-15a/16-1 cluster is selectively increased in the mouse cerebral vasculature after TBI. Of note, endothelial cell (EC)-selective miR- 15a/16-1 genetic deficiency leads to reduced BBB leakage, and less neuronal loss, white matter (WM) injury, and neurobehavioral impairments in mice after TBI. We also found that the miR-15a/16-1 cluster can bind to the 3’-UTRs of major BBB tight junctions, brain-enriched Claudins, and inhibit their translation, and genetic silencing or deletion of the miR-15a/16-1 cluster significantly increased endothelial or cerebral expression of claudins. These findings have provided the basis for our Central Hypothesis that genetic deletion of vascular miR-15a/16-1 attenuates BBB disruption and subsequent pathological cascades after TBI, thereby contributing to increased BBB stabilization, reduced neuronal/WM loss, and improved long- term neurological outcomes in TBI. Three aims will be performed in this proposal. Aim 1: Define the role of vascular miR-15a/16-1 in post-traumatic BBB dysfunction and TBI outcomes; Aim 2: Elucidate the mechanisms of miR-15a/16-1 regulation of post-traumatic BBB dysfunction; Aim 3: Explore systematic delivery of miR- 15a/16-1 antagomir (inhibitor) as a potential therapy in TBI through BBB stabilization. Elucidating the molecular mechanisms of miR-15a/16-1-mediated BBB dysfunction may lead us to discover novel and effective treatment against TBI.