Targeting Blood-Brain Barrier Transporters to Treat Ischemic Stroke

项目摘要 缺血性中风是美国一个重要的公共卫生问题。当前治疗 治疗中风的方法包括用重组组织纤溶酶原激活剂（r-tPA）或 血管内治疗;然而，许多患者仍然经历残疾。改善中风后的目标 结果需要新的神经保护药物用于中风治疗。虽然许多这样的化合物已经被 在临床前中风研究中发现，这些都没有成功地转化为临床。与此相反， 3-羟基-3-甲基戊二酰辅酶A（HMG-CoA）还原酶抑制剂（即，他汀类药物）常规给予 这是由于这些药物具有改善卒中后功能结局的固有能力。 在这项资助中，我们将测试他汀类药物的神经保护作用的机制假说， 通过关键摄取转运蛋白介导的血脑屏障转运 有机阴离子转运多肽1a 4（Oatp 1a 4）。这一假设将通过两个目标进行检验。 目的1：研究Oatp 1a 4介导的他汀类药物在缺血性脑卒中中的中枢传递。评价 Oatp 1a 4在脑卒中期间CNS药物递送中的作用，我们将研究Oatp 1a 4介导的他汀转运， 使用短暂性大脑中动脉闭塞（tMCAO）模型的BBB。在这些研究中， 雄性和雌性大鼠将经受tMCAO 90分钟（目标1A）。然后我们将证明Oatp 1a 4- 介导的他汀类药物递送改善了神经保护和BBB完整性的生物标志物（目的1B）。我们还将 进行神经认知、感觉运动和运动性能研究（即，功能性神经测试） 给予他汀类药物并进行tMCAO的动物，再灌注时间长达21天（Aim 1C）。在所有 研究中，他汀类药物将在再灌注时或再灌注2小时后静脉内给药， 表明早期给予神经保护药物可以改善中风后的结果。 目的2：转化生长因子-b（TGF-b）信号转导可以靶向控制Oatp 1a 4- 在缺血性卒中中介导的CNS他汀类药物递送。在这些实验中，我们将进行剂量反应 在年龄匹配的雄性和雌性Sprague-Dawley大鼠中使用TGF-β 1进行的研究和多次给药实验 B/ALK 1激动剂骨形态发生蛋白（BMP）-9和TGF-B/ALK 5拮抗剂SB 431542。我们将研究 脑微血管内皮细胞中控制TGF-β信号传导的特异性Smad蛋白的激活（Aim 2A）。我们还将确定BMP-9或BMP-10诱导后Oatp 1a 4表达和活性变化的时程。 SB 431542治疗及其对他汀类药物CNS递送的影响（目的2B）。此外，我们还将测量指数 的神经保护，BBB保护的标志物，和神经系统的结果， 施用BMP-9或SB 431542，并静脉内注射他汀类药物（Aim 2C）。 总体而言，这些研究具有临床相关性，因为它们将证明有效的BBB 需要转运机制来赋予中风中神经保护药物的有效性。

PROJECT SUMMARY Ischemic stroke is a significant public health concern in the United States. Current therapeutic approaches for stroke involve thrombolytic therapy with recombinant tissue plasminogen activator (r-tPA) or endovascular treatments; however, many patients still experience disability. The goal of improving post-stroke outcomes requires novel neuroprotective drugs for stroke treatment. While many such compounds have been identified in preclinical stroke studies, none of these have been successfully translated to the clinic. In contrast, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (i.e., statins) are routinely given to stroke patients due to an inherent ability of these drugs to improve post-stroke functional outcomes. In this grant, we will test the mechanistic hypothesis that neuroprotection from statins results from transport across the blood-brain barrier (BBB) that is mediated by the critical uptake transporter organic anion transporting polypeptide 1a4 (Oatp1a4). This hypothesis will be tested by two aims. Aim 1: To investigate CNS delivery of statins mediated by Oatp1a4 in ischemic stroke. To evaluate the role of Oatp1a4 in CNS drug delivery during stroke, we will investigate Oatp1a4-mediated statin transport at the BBB using the transient middle cerebral artery occlusion (tMCAO) model. In these studies, age-matched male and female rats will be subjected to tMCAO for 90 min (Aim 1A). We will then demonstrate that Oatp1a4- mediated statin delivery improves both biomarkers of neuroprotection and BBB integrity (Aim 1B). We will also perform neurocognitive, sensorimotor, and motor performance studies (i.e., functional neurological tests) in animals administered statins and subjected to tMCAO with reperfusion times of up to 21 days (Aim 1C). In all studies, statins will be administered intravenously either at the time of reperfusion or after 2 h of reperfusion to show that early administration of neuroprotective drugs can improve post-stroke outcomes. Aim 2: Transforming Growth Factor-b (TGF-b) signaling can be targeted to control Oatp1a4- mediated CNS statin delivery in ischemic stroke. In these experiments, we will perform dose-response studies and multiple-dosing experiments in age-matched male and female Sprague-Dawley rats using the TGF- b/ALK1 agonist bone morphogenetic protein (BMP)-9 and the TGF-b/ALK5 antagonist SB431542. We will study the activation of specific Smad proteins that control TGF-b signaling in brain microvascular endothelial cells (Aim 2A). We will also determine the time course of Oatp1a4 expression and activity changes following BMP-9 or SB431542 treatment and their effects on CNS delivery of statins (Aim 2B). Additionally, we will measure indices of neuroprotection, markers of BBB protection, and neurological outcomes in rats subjected to tMCAO, administered BMP-9 or SB431542, and injected intravenously with a statin (Aim 2C). Overall, these studies are clinically relevant because they will demonstrate the effective BBB transport mechanisms are required to confer effectiveness of neuroprotective drugs in stroke.