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Nck Adaptor Proteins in Atherogenic Endothelial Activation

Nck 接头蛋白在致动脉粥样硬化内皮激活中的作用

基本信息

批准号：
9158158
负责人：
Anthony Wayne Orr
金额：
$ 36.25万
依托单位：
LOUISIANA STATE UNIV HSC SHREVEPORT
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-06-01 至 2020-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9158158
关键词：
Adaptor Signaling Protein Address Adult Affect Animal Model Anti-Inflammatory Agents Anti-inflammatory ApoE knockout mouse Apolipoprotein E Arterial Fatty Streak Arthritis Atherosclerosis Binding Binding Proteins Binding Sites Biological Models Blood Vessels C-terminal CD31 Antigens Cell Adhesion Molecules Cell Line Cell physiology Cells Chronic Clinical Complement Factor B Complex Data Development Diet Disease Endothelial Cells Family Gene Expression Genes In Vitro Individual Inflammatory Intercellular Junctions Intercellular adhesion molecule 1 Knock-out Laboratories Leukocytes Ligation Maps Mediating Modeling Molecular Mus Nuclear Pattern Peptides Permeability Phosphorylation Process Protein Isoforms Proteins Proteomics Publishing Recruitment Activity Research Role SH3 Domains Signal Transduction Site Testing Tyrosine Vascular Cell Adhesion Molecule-1 Vascular Permeabilities Work base domain mapping hemodynamics in vivo inhibitor/antagonist knock-down leukocyte homing member new therapeutic target novel oxidant stress p21 activated kinase prevent protective effect response shear stress src Homology Region 2 Domain

项目摘要

Alterations in hemodynamic shear stress and oxidant stress at atherosclerosis-prone sites promote a low level, chronic endothelial activation that predisposes these sites to plaque development. Work from my laboratory has identified the Nck family of signaling adaptors, including both Nck1 and Nck2, as novel regulators of endothelial activation. Nck1/2 knockdown or addition of a peptide inhibitor blunts shear and oxidant stress- induced p21 activated kinase (PAK) signaling, thereby reducing endothelial permeability, cytoskeletal remodeling, and NF-κB-driven proinflammatory gene expression. Additionally, we provide compelling evidence that PECAM-1 phosphorylation in response to shear and oxidant stress recruits Nck to endothelial cell-cell junctions, promotes Nck-dependent PAK activation, and stimulates Nck-dependent induction of proinflammatory signaling (NF-κB) and gene expression (ICAM-1, VCAM-1). These data suggest a model whereby Nck recruitment to PECAM-1 alters the local signaling milieu thereby switching PECAM-1 signaling from anti-inflammatory to pro-inflammatory. Endothelial deletion of both Nck1 and Nck2, but not individual isoforms, impairs vascular development. However, several studies have found distinct functions for Nck1 and Nck2, and disturbed flow patterns differentially affect Nck1 and Nck2 expression both in vitro and in vivo. While no studies to date have examined endothelial Nck1 or Nck2 function during pathological conditions in adults, treatment with a Nck- blocking peptide significantly blunts leukocyte recruitment and vascular permeability in multiple model systems in vivo. However, these studies cannot determine whether this peptide acts through Nck or affects endothelial function directly. Our preliminary data utilizing endothelial-specific Nck1/2 deletion in atherosclerosis-prone ApoE knockout mice show reduced proinflammatory gene expression in response to disturbed flow and reduced early plaque development. Therefore, we hypothesize that PECAM-1 phosphorylation in response to shear and oxidant stress facilitates the formation of distinct Nck-based signaling complexes that drive permeability and proinflammatory gene expression under atherogenic conditions. In Aim 1, we will characterize PECAM-1/Nck complex formation during endothelial activation by mapping the Nck-binding sites on PECAM-1, verifying the interaction in vivo, and determining the crosstalk with local oxidant stress in this response. In Aim 2, we will characterize the composition and function of Nck-based signaling complexes during endothelial cell activation by assessing the mechanisms of altered Nck1/Nck2 expression, testing their relative roles in endothelial cell activation, and utilizing domain mapping and spatial proteomics to characterize the Nck1/2 interactome during endothelial activation. In Aim 3, we will determine whether endothelial Nck signaling affects endothelial activation in vivo utilizing mice containing conditional deletion of the Nck genes in endothelial cells to assess their relative roles in endothelial activation under atherogenic conditions.

动脉粥样硬化易发部位的血流动力学剪切应力和氧化应激的改变促进了