Nck1 in Ischemia Reperfusion Injury

项目摘要

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.