Nck1 in Ischemia Reperfusion Injury

项目摘要 中风仍然是全球死亡和残疾的主要原因。血栓清除是目前唯一的治疗方法 来恢复脑血流量然而，脑灌注的恢复本身可以触发氧化应激 最终导致脑水肿和神经炎症的通路，增加了寻找新的 神经保护策略Nck1基因与缺血性卒中风险相关，一项大型GWA研究显示， 在缺血性心脏病患者中发现了Nck 1作为一个新基因。然而，Nck1如何贡献于 缺血性中风的发病机制在很大程度上是未知的。在此观察Nck 1的重要作用。在试点研究中， 我们发现：1）在内皮细胞中敲低Nck1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)内皮细胞中Nck1的缺失显著降低了内源性ROS 水平，表明Nck 1在ROS的内皮诱导中的直接作用。3)Nck1的整体缺失显著 减弱提睾肌模型中缺血/再灌注诱导的白细胞粘附和迁移。 这与渗透性的显著降低有关，表明Nck 1在血管内皮细胞中的直接作用。 IRI后的炎症和渗透性。该应用测试了内皮Nck 1 促进内源性ROS和关键介导氧化应激诱导的神经炎症， 血脑屏障通透性促进缺血性脑卒中缺血/再灌注损伤。 在COBRE计划的三年中，我们将确定Nck1在缺氧后如何被激活 复氧损伤在人Nck 1 KO和过表达脑内皮细胞中，我们将确定Nck 1结合介质 通过亲和下拉和质谱分析。此外，Nck1在ROS产生中的作用将通过以下方法进行评估： 确定ROS的线粒体和Nox来源。为了确定Nck 1的内皮抑制作用，我们将 在短暂的大脑中动脉闭塞中测试含有Nck 1的条件性内皮缺失的新小鼠 （filament，T. MCAO）/再灌注模型。卒中的主要特征，包括梗死体积， 将评估神经缺陷和神经炎症的免疫组织化学分析。此外，本发明还提供了一种方法， 内皮细胞Nck 1对血脑屏障的直接作用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应用做准备， 在LSUHSC的COBRE计划。这个项目将促进我继续科学和专业培训， 帮助我在学术界建立一个独立的研究项目。

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.