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）打开和血管源性水肿，这是急性脑卒中患者死亡的主要原因。任何旨在减轻血脑屏障破坏从而减轻血管源性水肿严重程度的治疗策略都将对缺血性卒中患者的死亡率产生重大影响。组织型纤溶酶原激活剂（tPA）主要存在于血液中，其主要功能是溶栓酶。tPA也在中枢神经系统（CNS）中表达，在那里它被认为具有不同的功能。在缺血性卒中中，内源性tPA活性在缺血半球内增加，tPA的遗传缺陷或药理抑制与梗死体积的减少有关。初步研究表明，细胞内储存的内源性tPA在应对缺血性损伤时释放，以及外源性tPA作为栓塞性卒中的溶栓治疗，都直接导致血脑屏障的破坏。由于tPA是fda唯一批准的用于治疗急性脑卒中患者的药物，因此确定这种蛋白酶在脑缺血时是否对血脑屏障有有害作用至关重要。因此，本应用将集中于了解tPA在脑缺血时作为脑血管通透性调节剂的作用。通过体外和体内生物化学、分子、药理学和遗传学方法的结合，并在先前关于tPA和脂蛋白受体相关蛋白（LRP）对脑血管通透性影响的研究的基础上，我们将验证以下假设：1)在脑缺血发作后，tPA的释放早期增加，可能来自胶质细胞；2)这种tPA与血脑卒中中LRP的关联诱导了一个特定的细胞信号事件，可能是通过丝裂原活化蛋白激酶（MAPK）家族成员的磷酸化；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.