Regulatory microRNAs-mediated cerebrovascular protection and traumatic braininjury

创伤性脑损伤(TBI)是军人和退伍军人的主要医疗问题，目前没有 治疗可以缓解创伤后的神经功能障碍。血脑屏障(BBB)提供了 动态接口，将大脑与循环系统分开，保持大脑稳定 微环境。作为血脑屏障的主要组成部分，脑微血管内皮细胞(BMECs)与 与细胞间紧密连接(TJ)一起，在调节血脑屏障完整性和细胞旁起主导作用 渗透性1.越来越多的证据表明，颅脑损伤后血脑屏障的崩溃会导致毁灭性的 一系列事件，如外周免疫细胞移位，脑部炎症反应，水肿， 和出血性转化，导致神经创伤的继发性损伤。因此，有必要对 确定机制并制定有效的治疗策略，以保护血脑屏障的完整性并防止 颅脑损伤后的永久性脑损伤。 MicroRNAs(MiRs)是一类主要的非编码小RNA，对蛋白质进行负性调节 表情。除了在各种生物过程中发挥关键作用外，miRs还参与了一种 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 脑外伤的发病机制。然而，miR分子在人类免疫系统中的功能意义和分子机制 调节脑血管发病机制，特别是血脑屏障紊乱/功能障碍，以及由此产生的长期 颅脑损伤患者的神经结局知之甚少。 MiR-15a/16-1簇是第一个被发现与人类致癌相关的miR基团。 在脑外伤患者中发现了血浆miR-15a/16-1水平失调，显示出巨大的潜在的 在临床诊断和预后中有价值的生物标志物。有趣的是，miR-15a/16-1水平的分子抑制 已被证明可以预防心肌梗死(MI)和缺血性脑损伤。因此，美国人 欧洲制药公司认为miR-15a/16-1集群是最重要的miR之一 目标是开发基于miR的治疗心肌梗死的药物。 在我们最近的初步研究中，我们已经表明miR-15a/16-1簇的表达是 脑挫伤后小鼠脑血管内皮细胞选择性增加。值得注意的是，内皮细胞(EC)选择性miR- 15A/16-1遗传缺陷导致血脑屏障漏出减少，神经元丢失、白质(WM)损伤， 以及脑创伤后小鼠的神经行为损害。我们还发现miR-15a/16-1簇可以与 主要血脑屏障紧密连接的3‘-UTRs，大脑丰富的Claudins，并抑制它们的翻译，以及基因 MiR-15a/16-1簇的沉默或缺失显著增加血管内皮细胞或脑组织中 克拉丁斯。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-15a/16-1减轻脑损伤后血脑屏障的破坏和随后的病理级联反应， 从而有助于提高血脑屏障的稳定性，减少神经元/WM的损失，并改善长时间。 颅脑损伤患者的长期神经学结局。这项提案将实现三个目标。目标1：确定 血管miR-15a/16-1在创伤后血脑屏障功能障碍和脑外伤预后中的作用；目的2：阐明其机制 MiR-15a/16-1对创伤后血脑屏障功能障碍的调节；目的3：探索miR-1的系统递送 15A/16-1 Anagomir(抑制剂)通过稳定血脑屏障成为治疗脑外伤的潜在药物。阐明分子 MiR-15A/16-1介导的血脑屏障功能障碍的机制可能引导我们发现新的有效的治疗方法 对TBI的攻击。

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.