Protease-Regulated Blood Brain Barrier Permeability

描述（由申请人提供）：在脑缺血等病理情况下，血管通透性过度增加导致血脑屏障（BBB）开放和血管源性水肿，这是急性卒中患者死亡的主要原因。任何旨在减弱BBB破坏并因此减轻血管源性水肿严重程度的治疗策略将对缺血性卒中患者的死亡率产生显著影响。组织型纤溶酶原激活剂（tPA）主要存在于血液中，其主要功能是作为血栓溶解酶。tPA也在中枢神经系统（CNS）中表达，在中枢神经系统中它被认为具有不同的功能。在缺血性卒中中，缺血半球内内源性tPA活性增加，并且tPA的遗传缺陷或药理学抑制与梗死体积减少相关。初步研究表明，响应于缺血性损伤而从细胞内储存释放的内源性tPA和作为栓塞性中风的溶栓治疗而施用的外源性tPA两者都直接引起BBB的破坏。由于tPA是FDA批准的用于治疗急性卒中患者的唯一药物，因此确定这种蛋白酶在脑缺血期间是否对BBB具有有害作用至关重要。因此，本申请将集中于了解tPA作为脑缺血期间脑血管通透性的调节剂的作用。本研究综合运用生物化学、分子生物学、药理学和遗传学方法，在体外和体内研究tPA和脂蛋白受体相关蛋白（LRP）对脑血管通透性影响的基础上，验证了以下假设：1）脑缺血后，早期tPA释放增加，可能来自胶质细胞; 2）这种tPA与BBB中的LRP的结合诱导特异性细胞信号传导事件，可能通过促分裂原活化蛋白激酶（MAPK）家族成员的磷酸化;和3）MAPK磷酸化导致细胞骨架和紧密连接（TJ）蛋白的变化，随后脑血管通透性增加。

DESCRIPTION (PROVIDED BY APPLICANT): In pathological situations such as cerebral ischemia, excessive increases in vascular permeability lead to opening of the blood brain barrier (BBB) and vasogenic edema, which is a major cause of death in acute stroke patients. Any therapeutic strategy aimed at attenuating the disruption of the BBB and therefore the severity of vasogenic edema will have a significant impact in the mortality of patients with ischemic stroke. Tissue-type plasminogen activator (tPA) is predominantly found in the blood where its primary function is as a thrombolytic enzyme. tPA is also expressed within the central nervous system (CNS) where it is thought to have a different function. In ischemic stroke, endogenous tPA activity increases within the ischemic hemisphere and either genetic deficiency or pharmacological inhibition of tPA have been associated with decrease in infarct volume. Preliminary studies demonstrate that both endogenous tPA released from intracellular stores in response to the ischemic insult and exogenous tPA administered as a thrombolytic therapy for embolic stroke, directly cause disruption of the BBB. Since tPA is the only FDA-approved medication for the treatment of patients with acute stroke, it is of vital importance to establish whether this proteinase has a deleterious effect on the BBB during cerebral ischemia. Accordingly, this application will focus on understanding the role of tPA as a regulator of cerebrovascular permeability during cerebral ischemia. By using a combination of biochemical, molecular, pharmacological and genetic approaches, both in vitro and in vivo, and building on previous studies of the effect of tPA and the Lipoprotein Receptor Related Protein (LRP) on cerebrovascular permeability, we will test the following hypotheses: 1) following the onset of cerebral ischemia there is an early increase in the release of tPA, possibly from the glial cells; 2) the association of this tPA with LRP in the BBB induces a specific cell signaling event, likely through phosphorylation of a member of the Mitogen-Activated Protein Kinase (MAPK) family; and 3) MAPK phosphorylation results in changes in cytoskeletal and tight junction (TJ) proteins with subsequent increases in cerebrovascular permeability.