Nck1 in Ischemia Reperfusion Injury

Nck1在缺血再灌注损伤中的作用

• 批准号: 10715406
• 负责人: Mabruka Alfaidi
• 金额: $ 21.9万
• 依托单位: LOUISIANA STATE UNIV HSC SHREVEPORT
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-08-01 至 2028-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10715406
• 关键词: Academia; Adaptor Signaling Protein; Adhesions; Adult; Adverse event; Adverse reactions; Affinity; Antioxidants; Applications Grants; Attenuated; Binding; Biological Assay; Biology; Blood - brain barrier anatomy; Blood Vessels; Blood flow; Brain Edema; Brain Infarction; Brain Injuries; Cardiovascular Diseases; Cause of Death; Cell Adhesion Molecules; Centers of Research Excellence; Cerebrovascular Circulation; Cerebrum; Characteristics; Clinic; Clinical; Contrast Media; Data; Development; Edema; Education; Emigrations; Endothelial Cells; Endothelium; Enzymes; Excision; Filament; Fostering; Generations; Genes; Human; Hypoxia; Infarction; Inflammation; Injury; Ischemia; Ischemic Stroke; Knock-out; Knockout Mice; Knowledge; Leucocytic infiltrate; Leukocytes; Link; Mass Spectrum Analysis; Mediating; Mediator; Mentors; Methods; Middle Cerebral Artery Occlusion; Mitochondria; Modeling; Molecular; Mus; Myocardial Ischemia; NADPH Oxidase; Nature; Neurologic Deficit; Nuclear; Oxidation-Reduction; Oxidative Stress; Oxidative Stress Induction; Oxidative Stress Pathway; Pathogenesis; Pathway interactions; Patients; Perfusion; Permeability; Pilot Projects; Play; Predisposition; Preparation; Production; Reactive Inhibition; Reactive Oxygen Species; Reperfusion Injury; Reperfusion Therapy; Research; Role; Source; Stroke; Testing; Therapeutic; Thrombectomy; Thrombolytic Therapy; Thrombus; Training; Translating; United States National Institutes of Health; Vascular Permeabilities; blood-brain barrier disruption; blood-brain barrier permeabilization; brain cell; brain dysfunction; brain endothelial cell; brain tissue; cerebral artery; cremaster muscle; disability; genome wide association study; in vivo; knock-down; neuroinflammation; neuroprotection; novel; overexpression; pharmacologic; post stroke; pre-clinical; programs; response; restoration; stroke patient; stroke risk; therapeutic target; transcription factor; vascular inflammation

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.

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 恢复脑血流。但是，脑灌注的恢复本身会引发氧化应激 有时会导致脑水肿和神经炎症的途径，增加了寻找新的需求 神经保护策略。 NCK1基因与缺血性中风的风险相关，并且一项大型GWA研究具有 在缺血性心脏病患者中鉴定为NCK1是一种新基因。但是，NCK1如何贡献 缺血性中风的发病机理在很大程度上未知。在此观察到NCK1的重要作用。在试点研究中， 我们发现1）ECS中NCK1的敲低钝化NF-KB激活和粘合分子的表达 对缺氧/重新氧损伤的反应。 2）EC中的NCK1耗竭大大减少了内源性ROS 水平，表明NCK1在ROS内皮诱导中的直接作用。 3）NCK1的全局删除 衰减在Cremaster肌肉模型中，缺血/再灌注引起的白细胞粘合剂和移民。 这与渗透性的显着降低有关，这表明NCK1在血管中的直接作用 IRI后的炎症和渗透性。该申请检验了内皮NCK1的假设 促进内源性ROS，并严重介导氧化应激诱导的神经炎症和 BBB渗透性促进缺血性中风中缺血/再灌注损伤。 在毛bre计划的三年中，我们将确定如何在缺氧之后激活NCK1 重氧损伤。在人NCK1 KO和过表达的脑内皮细胞中，我们将确定NCK1结合介质 通过亲和力拉下和质谱分析。此外，NCK1在ROS产生中的作用将由 确定ROS的线粒体和NOX来源。为了确定NCK1内皮抑制的影响 测试新的小鼠，其中含有NCK1的条件内皮缺失在瞬时脑中动脉闭塞 （细丝，T。MCAO）/再灌注模型。中风的主要特征，包括梗塞量， 将评估神经系统缺陷和免疫组织化学分析神经炎症。此外， 内皮NCK1对BBB的直接影响将通过注射荧光对比剂，缺血后 再灌注评估血管泄漏的浸润性。 我将继续我的专业和科学发展，为R01应用做准备，利用了优势 LSUHSC中的毛绒计划。这个项目将促进我持续的科学和专业培训以及 促进我在学术界建立独立的研究计划。