Mechanosensing and Mechanotransduction in the Endothelial Nucleus

内皮细胞核中的机械传感和机械转导

基本信息

批准号：
10814132
负责人：
Jocelynda Salvador
金额：
$ 4.3万
依托单位：
NORTHWESTERN UNIVERSITY AT CHICAGO
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-09-08 至 2025-09-07
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10814132
关键词：
ATAC-seq Actins Affect Age Aging Aorta Area Atomic Force Microscopy Binding Biophysics Blood Vessels Blood flow CRISPR/Cas technology Cell Line Cell Nucleus Cell surface Cells Chromatin Chromatin Structure Chromosome Structures Complex Cytoplasm Cytoskeleton Cytosol DNA Double Strand Break Data Endothelial Cells Endothelium Epigenetic Process Evaluation Exhibits Exposure to Fibroblasts Gamma-H2AX Gene Expression Profile Genetic Transcription Geometry Heart Histones Homeostasis In Vitro Individual Institution Intermediate Filaments Invaded Kinetics Knockout Mice Lamins Link Maintenance Mechanical Stress Molecular Nuclear Nuclear Envelope Nuclear Pore Nuclear Structure Pathology Periodicity Phenotype Post-Translational Protein Processing Premature aging syndrome Process Protein Array Proteins Proteomics Reporter Research Role Shapes Signal Transduction Stress Techniques Testing Training Trees Validation Vascular Diseases Vimentin Work age related atheroprotective career experience experimental study fluid flow hemodynamics in vivo interest large datasets live cell imaging mechanotransduction migration plectin prevent resilience shear stress single-cell RNA sequencing skills transmission process viscoelasticity

ATAC-seq Actins Affect Age Aging Aorta Area Atomic Force Microscopy Binding Biophysics Blood Vessels Blood flow CRISPR/Cas technology Cell Line Cell Nucleus Cell surface Cells Chromatin Chromatin Structure Chromosome Structures Complex Cytoplasm Cytoskeleton Cytosol DNA Double Strand Break Data Endothelial Cells Endothelium Epigenetic Process Evaluation Exhibits Exposure to Fibroblasts Gamma-H2AX Gene Expression Profile Genetic Transcription Geometry Heart Histones Homeostasis In Vitro Individual Institution Intermediate Filaments Invaded Kinetics Knockout Mice Lamins Link Maintenance Mechanical Stress Molecular Nuclear Nuclear Envelope Nuclear Pore Nuclear Structure Pathology Periodicity Phenotype Post-Translational Protein Processing Premature aging syndrome Process Protein Array Proteins Proteomics Reporter Research Role Shapes Signal Transduction Stress Techniques Testing Training Trees Validation Vascular Diseases Vimentin Work age related atheroprotective career experience experimental study fluid flow hemodynamics in vivo interest large datasets live cell imaging mechanotransduction migration plectin prevent resilience shear stress single-cell RNA sequencing skills transmission process viscoelasticity

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项目摘要

Project Summary/Abstract Vascular cells are constantly subjected to physical forces associated with the rhythmic activities of the heart, which combined with the individual geometry of vessels further imposes oscillatory, disturbed, or laminar shear stresses on endothelial cells. These hemodynamic forces dictate the phenotype and gene expression profile of endothelial cells in different regions of the arterial tree making them athero-protective or athero-prone. Due to the impact of distinct types of flow in the onset of vascular pathology, significant effort has been placed in identifying mechanosensing proteins and transcriptional profiles linked to different types of shear stress. In contrast, less emphasis has been given to how forces at the cell surface are transmitted to the nucleus, impacting nuclear shape, nuclear pore function, and chromatin organization and integrity in the vasculature, which is the focus of this application. I found that oscillatory and disturbed flow have a progressive deleterious effect in nuclear shape, which is exacerbated by loss of vimentin. Based on this and other preliminary data, I hypothesize that Vimentin is critical for maintaining nuclear shape and chromatin integrity in regions of oscillatory and disturbed flow in the endothelium. Using endothelial cells in vitro for mechanistic studies and vimentin null mice for in vivo validation, I will complete an in-depth characterization of changes in nuclear integrity and function in dysmorphic nuclei associated with aging and loss of vimentin. I will begin with a detailed characterization of nuclear shape, nuclear membrane integrity, and function (via analysis of nuclear-cytoplasmic transport) in ECs under non-laminar flow both in vivo, in the aortic endothelium, and in vitro using live-cell imaging, and atomic force microscopy. I also will characterize vimentin kinetics, including its post-translational modifications, under non-laminar flow (Aim 1). To assess shifts in transcriptional and signaling networks, I will use scRNA sequencing and proteomics to identify and validate the vimentin interactome. I will examine the consequence of vimentin loss on proteins of the Linker of Nucleoskeleton to Cytoskeleton (LINC) complex which directly or indirectly interact with vimentin and are involved in direct force transmission to the nucleus (Aim 2). Finally, I will evaluate how loss of vimentin alters endothelial chromatin integrity and epigenetic states using ATAC-sequencing to provide evidence of vimentin’s critical function in maintaining chromatin homeostasis in the vasculature (Aim 3). Through the completion of this proposed work, I will significantly expand my toolkit of techniques, skills, and concepts while actively contributing to clarify the impact of physical forces on the endothelial nucleus. I am also looking forward to broadening my skills in working with large datasets including analysis, distillation of information and deriving new questions. As part of this process, I will pursue several interactions and scientific connections with centers and collaborators within and outside my institution that will contribute to the advancement and completion of this work, my scientific training, and prepare me for the next stage of my career.

项目摘要/摘要血管细胞不断受到与心脏的节奏活性相关的物理力，结合血管的单个几何形状进一步不可能振荡，干扰或层状剪切内皮细胞的应力。这些血液动力学决定了表型和基因表达谱动脉树不同区域的内皮细胞使它们具有动脉粥样硬化或易动脉粥样硬化。由于不同类型的流动在血管病理学开始时的影响，已将大量努力放在识别与不同类型的剪切应力相关的机械感应蛋白和转录曲线。在对比，较少的重点是如何将细胞表面的力传递到核，影响核形状，核孔功能和染色质组织以及脉管系统中的完整性，这是此应用程序的重点。我发现振荡和干扰的流动具有渐进的有害性核形状的效果会因波形蛋白的流失而加剧。基于此和其他初步数据，我假设波形蛋白对于在振荡性区域保持核形状和染色质完整性至关重要并在森植物中流动干扰。在体外使用Hothotherium细胞进行机械研究和波形蛋白null 用于体内验证的小鼠，我将完成核完整性和功能变化的深入表征与衰老和波形蛋白丧失相关的营养不良核中。我将从详细的表征开始核形状，核膜完整性和功能（通过EC中的核胞质转运分析）在非晶状体流动下，在体内，主动脉内皮中以及使用活细胞成像和原子化的体外体外力显微镜。我还将表征波形蛋白动力学，包括其后翻译后修饰，非线中流动（目标1）。为了评估转录和信号网络的变化，我将使用SCRNA测序和蛋白质组学以识别和验证波形蛋白相互作用组。我将研究波形蛋白的后果直接或间接的细胞骨架（LINC）复合物的核骨骼接头蛋白质损失与波形蛋白相互作用，并参与向核直接传播（AIM 2）。最后，我会评估波形蛋白的损失如何使用ATAC序列改变内皮染色质完整性和表观遗传状态提供波形蛋白在维持脉管系统中染色质稳态方面的关键功能的证据（AIM 3）。通过完成这项拟议的工作，我将大大扩展我的技术，技能和技能工具包概念在积极促进阐明物理力对内皮核us的影响的同时。我也是期待扩大我与大型数据集合作的技能，包括分析，信息蒸馏并提出新问题。作为此过程的一部分，我将进行几个互动和科学联系在我机构内外的中心和合作者将有助于进步和完成这项工作，我的科学培训，并为我的职业生涯下一个阶段做好准备。