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

描述（由申请人提供）：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、高海拔脑水肿、急性高山病、心脏骤停和缺血性中风。目前中风治疗的目的是恢复缺血脑的灌注; 然而，当血流/氧供应重新建立时，会发生相当大的脑细胞损伤和BBB功能障碍。因此，迫切需要开发新的治疗策略，其可以在H/R期间“拯救”可挽救的脑组织免受损伤和/或保护BBB完整性。在这项资助中，我们将测试的假设，有机阴离子转运多肽（Oatps）和多药耐药蛋白（Mrps），两个家庭的内源性血脑屏障转运蛋白，可以为治疗H/R的目标。两个具体目标将检验这一假设。目的1：研究Oatps介导的中枢药物释放。在这个目标中，我们将集中在Oatp 1a 4，主要药物运输Oatp在啮齿动物血脑屏障。我们将在体内研究Oatp 1a 4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）（目标1A）。然后，我们将Oatp 1a 4介导的他汀类药物转运的变化与神经保护和抗氧化功效的指数相关联（目的1B）。转化生长因子（TGF-β）信号调节Oatp 1a 4的表达/活性。因此，我们将评估dorsomorphin和SB 431542（两种TGF-β受体抑制剂）对Oatp 1a 4的BBB功能表达的影响，以评估该途径控制CNS药物递送的靶向作用（Aim 1C）。目的2：评价Mrps在BBB的药理学靶向是否保护H/R后的BBB完整性。谷胱甘肽（GSH）是一种重要的中枢神经系统抗氧化剂，是Mrp 1、Mrp 2和Mrp 4的底物。因此，我们将在体内研究H/R诱导的这些Mrps在BBB的表达/活性变化（目的2A）。然后，我们将研究GSH及其氧化形式GSH二硫化物（GSSG）在BBB的GSH转运系统和酶（目的2B）的改变所造成的变化。我们将通过核因子红细胞2相关因子2（Nrf 2）信号传导（Aim 2C）评估Mrps和GSH合成/代谢酶在BBB的调节。由于Nrf 2信号传导被氧化应激激活，我们的研究将在存在和不存在活性氧（ROS）清除剂TEMPOL（即，4-羟基-2，2，6，6-四甲基哌啶-N-氧基）来确定氧化应激的治疗靶向是否可以控制BBB Mrp介导的转运。我们在这项资助中的目标是促进发现新的方法，通过治疗靶向内源性转运蛋白在BBB治疗H/R组分的疾病。

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.