Nck1 in Ischemia Reperfusion Injury

项目摘要 中风仍然是全世界死亡和残疾的主要原因。血栓清除是目前唯一的治疗方法 以恢复脑血流量。然而，脑灌注的恢复本身就会引发氧化应激 最终导致脑水肿和神经炎症的途径，因此需要寻找新的途径 神经保护策略。 Nck1 基因与缺血性中风的风险相关，一项大型 GWA 研究表明 确定 Nck1 是缺血性心脏病患者的一个新基因。然而，Nck1 如何贡献 缺血性中风的发病机制在很大程度上尚不清楚。在此观察 Nck1 的重要作用。在试点研究中， 我们发现 1) EC 中 Nck1 的敲低会减弱 NF-kB 的激活和粘附分子的表达 对缺氧/复氧损伤的反应。 2）EC中Nck1的消耗显着减少了内源性ROS 水平，表明 Nck1 在内皮细胞诱导 ROS 中发挥直接作用。 3) Nck1显着全局缺失 减弱提睾肌模型中缺血/再灌注引起的白细胞粘附和迁移。 这与通透性显着降低相关，表明 Nck1 在血管中具有直接作用。 IRI 后的炎症和渗透性。该应用测试了内皮细胞 Nck1 的假设 促进内源性 ROS 并关键介导氧化应激诱导的神经炎症和 BBB通透性促进缺血性中风的缺血/再灌注损伤。 在 COBRE 计划的三年期间，我们将确定 Nck1 在缺氧后如何被激活 再氧合损伤。在人 Nck1 KO 和过表达脑内皮细胞中，我们将确定 Nck1 结合介质 通过亲和力下拉和质谱分析。此外，Nck1 在 ROS 产生中的作用将通过 确定 ROS 的线粒体和 Nox 来源。为了确定 Nck1 内皮抑制的影响，我们将 在暂时性大脑中动脉闭塞中测试含有 Nck1 条件性内皮缺失的新型小鼠 （丝，T. MCAO）/再灌注模型。中风的主要特征，包括梗塞体积、 将评估神经缺陷和神经炎症的免疫组织化学分析。此外， 内皮细胞Nck1对BBB的直接影响将通过注射荧光造影剂、缺血后 活体再灌注以评估血管渗漏。 我将继续我的专业和科学发展，为R01申请做好准备 LSUHSC 的 COBRE 项目。该项目将促进我继续接受科学和专业培训， 促进我在学术界建立独立的研究计划。

PROJECT ABSTRACT Stroke remains a leading cause of death and disability worldwide. Thrombus removal is the only current therapy to restore cerebral blood flow. However, the restoration of cerebral perfusion can itself trigger oxidative stress pathways that eventually lead to brain edema and neuro-inflammation, raising the need to find new neuroprotective strategies. Nck1 gene is associated with ischemic stroke’s risk, and a large GWA study has identified Nck1 as a novel gene in ischemic heart disease patients. However, how Nck1 contributes to the pathogenesis of ischemic stroke is largely unknown. Herein observe an important role for Nck1. In pilot studies, we found that 1) knockdown of Nck1 in ECs blunts NF-kB activation and adhesion molecule expression in response to hypoxia/re-oxygenation injury. 2) Nck1 depletion in ECs significantly reduces endogenous ROS levels, suggesting a direct role for Nck1 in endothelial induction of ROS. 3) Global deletion of Nck1 significantly attenuates ischemia/reperfusion-induced leukocyte adhesion and emigration in the cremaster muscle model. This is associated with remarkable reduction in permeability, suggesting a direct role for Nck1 in vascular inflammation and permeability after the IRI. This application tests the hypothesis that endothelial Nck1 promotes endogenous ROS and critically mediates oxidative stress-induced neuro-inflammation and BBB permeability to promote the ischemia/reperfusion injury in ischemic stroke. During the three years of the COBRE program, we will determine how Nck1 is activated following the hypoxia reoxygenation injury. In human Nck1 KO and overexpressing brain endothelial cells, we will determine the Nck1 binding mediators by affinity pulling down and mass spectrometry analyses. Furthermore, the role of Nck1 in ROS production will be assessed by determining the mitochondrial and Nox sources of ROS. In order of determining the effects of endothelial inhibition of Nck1 we will test novel mice containing conditional endothelial deletion of Nck1 in transient middle cerebral artery occlusion (filament, T. MCAO)/reperfusion model. The major characteristics of stroke, including infarct volume, neurological deficits, and immunohistochemical analysis for neuro- inflammation will be assessed. In addition, the direct effects of endothelial Nck1 on BBB will by injecting fluorescent contrast agents, post- ischemic reperfusion intravital to assess vascular leak. I will continue my professional and scientific development in preparation for R01 application taking the advantage of the COBRE program in LSUHSC. This project will foster my continued scientific and professional training and facilitate my establishing an independent research program in academia.