Targeting Blood-Brain Barrier Transporters to Treat Ischemic Stroke

项目总结 在美国，缺血性中风是一个重要的公共卫生问题。目前的治疗方法 卒中的治疗方法包括用重组组织型纤溶酶原激活剂(r-tPA)或 血管内治疗；然而，许多患者仍然经历残疾。改善中风后症状的目标 结果需要新的神经保护性药物来治疗中风。虽然许多这样的化合物已经被 在临床前卒中研究中确定，这些都没有成功地转化到临床。相比之下， 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)常规用于 中风患者由于天生有能力服用这些药物来改善中风后的功能结局。 在这项资助中，我们将检验他汀类药物对神经保护的机械性假设。 由关键摄取转运体介导的血脑屏障(BBB)转运 有机阴离子转运多肽1A4(Oatp1a4)。这一假设将通过两个目标进行检验。 目的1：探讨Oatp1a4介导的他汀类药物在缺血性卒中的中枢神经系统递送作用。评估 Oatp1a4在卒中期间中枢药物转运中的作用，我们将研究Oatp1a4介导的他汀类药物转运 采用一过性大脑中动脉闭塞(TMCAO)模型。在这些研究中，年龄匹配的 雄性和雌性大鼠将接受tMCAO 90min(目标1A)。然后我们将证明Oatp1a4- 介导性他汀类药物的释放改善了神经保护和血脑屏障完整性的生物标志物(目标1B)。我们还将 执行神经认知、感觉运动和运动表现研究(即，功能神经测试) 给予他汀类药物并接受tMCAO的动物，再灌注时间长达21天(目标1C)。总而言之， 研究表明，他汀类药物将在再灌注时或再灌注2小时后静脉注射，以 表明早期使用神经保护性药物可以改善中风后的预后。 目的2：转化生长因子-b信号通路可靶向调控Oatp1a4- 缺血性卒中患者中枢神经系统他汀类药物的传递。在这些实验中，我们将进行剂量反应 转化生长因子-α对年龄匹配的雌雄SD大鼠的研究及多次给药实验 B/ALK1激动剂BMP-9和转化生长因子-b/ALK5拮抗剂SB431542。我们会研究 脑微血管内皮细胞中控制转化生长因子-b信号转导的特异性Smad蛋白的激活 2a)。我们还将确定Oatp1a4在BMP-9或 SB431542治疗及其对他汀类药物中枢给药的影响(目标2B)。此外，我们还将衡量指数 对大脑中动脉阻塞大鼠的神经保护、血脑屏障保护标志物和神经结局的影响 给予BMP-9或SB431542，并静脉注射他汀类药物(目标2C)。 总体而言，这些研究具有临床意义，因为它们将证明有效的血脑屏障 转运机制是提高神经保护药物在中风中的有效性所必需的。

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.