Targeting blood-brain barrier transporters to treat hypoxia/reoxygenation stress

描述(申请人提供)：缺氧/复氧(H/R)是几种脑部疾病的“组成部分”，如创伤性脑损伤、急性呼吸综合征、阻塞性睡眠呼吸暂停、高原脑水肿、急性高原病、心脏骤停和缺血性中风。目前卒中治疗的目标是恢复脑缺血再灌注； 然而，当血流/氧气供应重新建立时，会发生相当大的脑细胞损伤和血脑屏障功能障碍。因此，迫切需要开发新的治疗策略，在H/R期间“拯救”可挽救的脑组织和/或保护BBB的完整性。在这项研究中，我们将检验有机阴离子转运多肽(OATPs)和多药耐药蛋白(MRPs)这两个内源性BBB转运体家族可以靶向治疗H/R的假设。目的1：研究缺氧复氧时OATPs介导的中枢神经系统药物传递，我们将重点研究Oatp1a4，Oatp1a4，Oatp在啮齿动物血脑屏障中的主要转运药物。我们将在体内研究Oatp1a4介导的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)的转运(目标1A)。然后，我们将把Oatp1a4介导的他汀类药物转运的变化与神经保护和抗氧化效果的指数联系起来(目标1B)。转化生长因子-�信号调节Oatp1a4的表达/活性。因此，我们将评估两种转化生长因子-�受体抑制剂多索吗啡和SB431542对Oatp1a4血脑屏障功能表达的影响，以评估这一途径的靶向性以控制中枢神经系统的药物传递(AIM 1C)。目的：评价以血脑屏障为靶点的MRPs在药物作用下是否能保护H/R后血脑屏障的完整性。谷胱甘肽(GSH)是中枢神经系统的重要抗氧化剂，是MRp1、MRp2和MRp4的底物。因此，我们将在体内研究H/R诱导的这些MRP在血脑屏障的表达/活性的变化(目标2A)。然后，我们将检查由于GSH运输系统和酶的改变而引起的血脑屏障中GSH及其氧化形式GSH二硫化物(GSSG)的变化(目标2B)。我们将评估核因子红系2相关因子2(Nrf2)信号对血脑屏障中MRPs和GSH合成/代谢酶的调节(目标2C)。由于NRF2信号是由氧化应激激活的，我们的研究将在存在和不存在活性氧物种(ROS)清除剂Tempoll(即4-hydroxy-2，2，6，6-tetramethylpiperidine-N-oxyl))的情况下进行，以确定氧化应激的治疗靶向是否可以控制BBB MRP介导的转运。我们在这笔赠款中的目标是通过治疗靶向BBB的内源性转运蛋白来促进发现治疗具有H/R成分的疾病的新方法。

DESCRIPTION (provided by applicant): Hypoxia/reoxygenation (H/R) is a "component" of several brain diseases such as traumatic brain injury, acute respiratory syndrome, obstructive sleep apnea, high altitude cerebral edema, acute mountain sickness, cardiac arrest and ischemic stroke. The objective of current stroke therapy is to restore perfusion to ischemic brain; however, considerable brain cellular damage and BBB dysfunction occurs when blood flow/oxygen supply is re-established. Therefore, there is a critical need for development of novel treatment strategies that can "rescue" salvageable brain tissue from damage and/or protect BBB integrity during H/R. In this grant, we will test the hypothesis that organic anion transporting polypeptides (Oatps) and multidrug resistance proteins (Mrps), two families of endogenous BBB transporters, can be targeted for treatment of H/R. Two specific aims will test this hypothesis. Aim 1: To investigate CNS drug delivery mediated by Oatps during H/R. In this aim, we will focus on Oatp1a4, the primary drug transporting Oatp at the rodent BBB. We will investigate, in vivo, Oatp1a4 mediated transport of 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase inhibitors (i.e., statins) (Aim 1A). We will then correlate changes in Oatp1a4-mediated statin transport with indices of neuroprotective and antioxidant efficacy (Aim 1B). Transforming growth factor (TGF-�) signaling regulates Oatp1a4 expression/activity. Therefore, we will evaluate effects of dorsomorphin and SB431542, two TGF-� receptor inhibitors, on BBB functional expression of Oatp1a4 to evaluate targeting of this pathway for control of CNS drug delivery (Aim 1C). Aim 2: To evaluate if pharmacological targeting of Mrps at the BBB protects BBB integrity following H/R. Glutathione (GSH), a critical CNS antioxidant, is a substrate for Mrp1, Mrp2, and Mrp4. Therefore, we will study in vivo H/R-induced changes in expression/activity of these Mrps at the BBB (Aim 2A). We will then examine changes in GSH and its oxidized form GSH disulfide (GSSG) at the BBB resulting from alterations in GSH transport systems and enzymes (Aim 2B). We will evaluate regulation of Mrps and GSH synthetic/metabolic enzymes at the BBB by nuclear factor erythroid 2-related factor 2 (Nrf2) signaling (Aim 2C). Since Nrf2 signaling is activated by oxidative stress, our studies will be conducted in the presence and absence of the reactive oxygen species (ROS) scavenger TEMPOL (i.e., 4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl) to determine if therapeutic targeting of oxidative stress can control BBB Mrp-mediated transport. Our goal in this grant is to facilitate discovery of novel approaches for treating diseases with an H/R component by therapeutic targeting of endogenous transporters at the BBB.