Role of Nesprin in Flow-Mediated Endothelial Mechanotransduction

Nesprin 在血流介导的内皮力转导中的作用

• 批准号: 7483752
• 负责人: ABDUL I BARAKAT
• 金额: $ 19万
• 依托单位: UNIVERSITY OF CALIFORNIA AT DAVIS
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-08-15 至 2011-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7483752
• 关键词: Actinin; Actins; Affect; Anti-Inflammatory Agents; Anti-inflammatory; Apical; Arterial Fatty Streak; Atherosclerosis; Back; Binding; Blood Vessels; Blood flow; C-terminal; CCL2 gene; Cardiovascular Diseases; Cardiovascular system; Cell Nucleus; Cell membrane; Cell surface; Cells; Cellular Mechanotransduction; Class; Cytoskeleton; Data; Development; Disruption; Dominant-Negative Mutation; Endothelial Cells; Endothelium; Enzyme-Linked Immunosorbent Assay; F-Actin; Family; Fluorescence; Fluorescence Microscopy; Gene Expression; Gene Proteins; Goals; Human; IL8 gene; Image Analysis; Inflammatory; Intercellular adhesion molecule 1; Interleukin-10; Intermediate Filaments; Invasive; Laser Microscopy; Laser Scanning Confocal Microscopy; Life; Link; Liquid substance; Localized; Mechanics; Mediating; Messenger RNA; Methods; Microfilaments; Mutation; N-terminal; Northern Blotting; Nuclear; Nuclear Envelope; Nuclear Outer Membrane; Numbers; Pathway interactions; Phenotype; Physiological; Play; Positioning Attribute; Protein Family; Proteins; Regulation; Reporting; Research; Role; Role playing therapy; Second Messenger Systems; Signal Transduction; Site; Small Interfering RNA; Spectrin; Standards of Weights and Measures; Structure; Transmembrane Domain; Vascular Cell Adhesion Molecule-1; Vascular Endothelial Cell; Western Blotting; Width; atheroprotective; cellular imaging; mRNA Expression; member; novel; protein expression; response; retinal rods; second messenger; shear stress; transcription factor; transmission process

DESCRIPTION (provided by applicant): The long-term objective of our research is to elucidate the mechanisms governing the responsiveness of vascular endothelial cells (ECs) to fluid mechanical shear forces. This information is necessary for understanding why early atherosclerotic lesions localize in arterial regions exposed to low and/or oscillatory shear stress. The specific goal of the present application is to probe the role of Nesprin-1, a member of a novel class of nuclear membrane-anchored spectrin-repeat proteins, in the transmission of shear forces from the EC surface to the nucleus and in the regulation of EC gene and protein expression. Nesprin-1 forms a direct physical bridge between the actin cytoskeleton and the nucleus. We hypothesize that this direct link provides a medium for the transmission of a physical force to the nucleus and hence mediates flow-induced changes in EC gene and protein expression. The specific aims are: 1. Establish the effect of steady and oscillatory shear stress on Nesprin-1 topography and expression in human aortic ECs (HAECs). Topographic changes will be assessed using standard fluorescence microscopy, laser scanning confocal microscopy, and fluorescence- reporting constructs for live-cell imaging. Protein expression will be studied using Western blot analysis. 2. Establish if Nesprin-1 remodeling in response to shear stress occurs through the actin cytoskeleton. HAEC F- actin expression and organization will be altered using siRNA methods, microchannel confinement, or pharmacological agents. The effect of F-actin alterations on shear stress-induced changes in Nesprin-1 topography and expression will be studied. 3. Characterize the role of Nesprin-1 in shear stress transmission to the nucleus and shear-induced changes in gene and protein expression in HAECs. The effect of a Nesprin-1 dominant negative construct on shear stress-induced changes in mRNA and protein expression of pro- and anti-inflammatory markers will be investigated. mRNA expression will be studied using Northern blot analysis while protein expression will be investigated using Western blot analysis and ELISA. Research relevance: Understanding how the cells of the arterial wall respond to blood flow is important for determining why cardiovascular disease develops preferentially in arterial regions of disturbed flow. In vascular cells, fluid mechanical forces alter the expression of genes that are important for the development and progression of cardiovascular complications. The proposed research aims to understand how a fluid mechanical force is transmitted from the cell surface to the nucleus where the genetic changes occur.

描述（由申请人提供）：我们研究的长期目标是阐明管理血管内皮细胞（EC）对流体机械剪力力的反应性的机制。该信息对于理解为什么早期动脉粥样硬化病变定位于暴露于低和/或振荡性剪切应力的动脉区域所必需。本应用的具体目标是探测Nesprin-1的作用，Nesprin-1是新型核膜锚定谱蛋白的成员，在剪切力从EC表面传播到核以及EC基因和蛋白质表达的调节中。 Nesprin-1在肌动蛋白细胞骨架和细胞核之间形成直接的物理桥。我们假设这种直接链接为将物理力传递到核提供了一种介质，因此介导了流动诱导的EC基因和蛋白质表达的变化。具体目的是：1。建立稳定和振荡性剪切应力对人主动脉EC（HAEC）中Nesprin-1地形和表达的影响。将使用标准荧光显微镜，激光扫描共聚焦显微镜以及用于活细胞成像的荧光报告结构来评估地形变化。将使用蛋白质印迹分析研究蛋白质表达。 2。确定NESPRIN-1是否针对剪切应力进行重塑是否通过肌动蛋白细胞骨架发生。 HAEC F-肌动蛋白的表达和组织将使用siRNA方法，微通道限制或药理剂改变。将研究F-肌动蛋白改变对剪切应力诱导的NESPRIN-1地形变化和表达的影响。 3。表征Nesprin-1在剪切应力传播向细胞核中的作用，以及剪切诱导的HAEC基因和蛋白质表达的变化。 Nesprin-1显性负结构对剪切应力诱导的mRNA变化以及促炎和抗炎标记的蛋白质表达的影响。将使用Northern印迹分析研究mRNA表达，同时将使用Western印迹分析和ELISA研究蛋白质表达。研究相关性：了解动脉壁细胞对血流的反应对于确定为什么心血管疾病在干扰流动的动脉区域优先发展。在血管细胞中，流体机械力改变了基因的表达，这对于心血管并发症的发展和进展至关重要。拟议的研究旨在了解如何将流体机械力从细胞表面传播到发生遗传变化的细胞核。

项目成果

Nesprin-3 regulates endothelial cell morphology, perinuclear cytoskeletal architecture, and flow-induced polarization.

• DOI: 10.1091/mbc.e11-04-0287
• 发表时间: 2011-11
• 期刊: Molecular biology of the cell
• 影响因子: 3.3
• 作者: Morgan JT;Pfeiffer ER;Thirkill TL;Kumar P;Peng G;Fridolfsson HN;Douglas GC;Starr DA;Barakat AI
• 通讯作者: Barakat AI