Controlling blood-brain barrier integrity in the ischemic brain by modulating gatekeepers at the abluminal endothelial membrane

载脂蛋白-E(ApoE)是缺血性卒中的主要危险因素，近年来发现它控制着缺血血脑屏障(BBB)紧密连接的完整性和ATP结合盒(ABC)转运体的丰度。载脂蛋白E通过低密度脂蛋白(LDL)受体相关蛋白(LRP)发挥作用，LRP存在于内皮细胞和周细胞上，两者的确切作用仍存在争议。在这个方案中，我们将评估不同的LRP(LRP1、LRP5/6、LRP8)及其相关蛋白(即FrizzledTM)和下游信号(例如JNK1/2/c-jun、GSK3β/β-catenin、FAK、RhoA)在控制(A)紧密连接的表达和相互作用中的作用，(B)ABC转运体在脑毛细血管上的表达和细胞定位，以及(C)大脑中动脉闭塞后药物在血和脑之间的分布。将评估现有的唯一治疗中风的重组组织型纤溶酶原激活剂(TPA)对神经血管信号的影响，并将评估治疗阿尔茨海默病的潜在药物--伽马分泌酶抑制剂LY450139的作用，以寻找可能允许规避tPA对缺血血管有害影响的策略。通过将周细胞缺陷小鼠暴露于大脑中动脉阻塞，我们将剖析内皮和周细胞对载脂蛋白E的反应，研究JNK1/2/c-jun和亲环素-A/核因子-kappaB/基质-金属蛋白酶-9通路在维持缺血后血脑屏障完整性中的作用。通过我们的研究，我们希望更深入地了解血管内皮细胞膜作为信号接口的作用，目的是确定可以通过这些靶点来改善神经血管的完整性和卒中治疗的有效性。

Apolipoprotein-E (ApoE), a major risk factor of ischemic stroke, has recently been shown to control tight junction integrity and ATP-binding cassette (ABC) transporter abundance at the ischemic blood-brain barrier (BBB). ApoE acts via low density lipoprotein (LDL) receptor-related proteins (LRP), which are found on endothelial cells and pericytes, the precise contribution of both being still debated. In this proposal, we would like to evaluate the role of different LRPs (LRP1, LRP5/6, LRP8), their associated proteins (i.e., Frizzled) and downstream signals (e.g., JNK1/2/ c-Jun, GSK3beta/ beta-catenin, FAK, RhoA) in controlling (a) the expression and interaction of tight junctions, (b) the expression and cellular localization of ABC transporters on brain capillaries and (c) the distribution of drugs between the blood and brain after middle cerebral artery occlusion. Effects of the only existing stroke therapeutic, recombinant tissue-plasminogen activator (tPA), on neurovascular signaling will be evaluated and the action of a potential therapeutic in Alzheimer's disease, the gamma-secretase inhibitor LY450139, will be assessed, searching for strategies that might allow to circumvent deleterious effects of tPA on ischemic blood vessels. By exposing pericyte deficient mice to middle cerebral artery occlusion, we will dissect endothelial and pericytic responses to ApoE, examining the role of the JNK1/2/ c-Jun and cyclophilin-A/ nuclear factor-kappaB/ matrix-metalloproteinase-9 pathways in maintaining post-ischemic BBB integrity. With our studies we would like to develop a more thorough understanding of the abluminal endothelial membrane as a signal interface, aiming to identify targets via which neurovascular integrity and the efficacy of stroke therapeutics might be improved.