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 中风仍然是世界范围内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。然而，脑血流的恢复本身会引发氧化应激。 最终导致脑水肿和神经炎症的途径，增加了寻找新的 神经保护策略。NCK1基因与缺血性中风的风险有关，一项大规模的GWA研究已经 Nock1被鉴定为缺血性心脏病患者中的一个新基因。然而，Nock1如何对 缺血性中风的发病机制很大程度上是未知的。在此，我们可以观察到Nock1的一个重要作用。在试点研究中， 我们发现：1)内皮细胞中Nck 1基因的敲除抑制了核因子-kB的活化和黏附分子的表达。 对缺氧/复氧损伤的反应。2)内皮细胞中NKK1的缺失显著降低内源性ROS 水平，提示Nock1在内皮细胞诱导ROS中起直接作用。3)Nock1的全局删除显著 减轻提睾肌模型中缺血/再灌流引起的白细胞黏附和迁移。 这与通透性的显著降低有关，表明Nck 1在血管中起直接作用 IRI后的炎症和通透性。这个应用程序测试了内皮细胞Nock1的假设 促进内源性ROS并关键地介导氧化应激诱导的神经炎症和 血脑屏障通透性促进缺血性卒中缺血再灌注损伤 在Cobre计划的三年中，我们将确定Nck 1在缺氧后是如何激活的 复氧损伤。在人类Nck-1 KO和过度表达的脑内皮细胞中，我们将确定Nck-1结合介质 通过亲和下拉和质谱分析。此外，Nck 1在ROS产生中的作用将通过以下方式进行评估 确定ROS的线粒体和NOX来源。为了确定NKK1抑制内皮细胞的效果，我们将 短暂性大脑中动脉闭塞中含有条件性内皮细胞缺失Nock1基因的新型小鼠试验 (细丝，T.MCAO)/再灌流模型。中风的主要特征，包括脑梗塞体积， 神经功能缺陷和神经炎症的免疫组织化学分析将被评估。此外, 缺血后注射荧光造影剂对内皮细胞Nck-1对血脑屏障的直接影响 再灌注术中评估血管渗漏。 我将继续我的专业和科学发展，为R01的应用做准备，利用这一优势 在LSUHSC的科布雷计划。这个项目将促进我继续进行科学和专业培训，并 促进我在学术界建立一个独立的研究项目。