Mechanosensing and Mechanotransduction in the Endothelial Nucleus

内皮细胞核中的机械传感和机械转导

• 批准号: 10536215
• 负责人: Jocelynda Salvador
• 金额: $ 4.2万
• 依托单位: NORTHWESTERN UNIVERSITY AT CHICAGO
• 依托单位国家: 美国
• 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/10536215
• 关键词: ATAC-seq; Actins; Affect; Age; Aging; 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; 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; 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; XCL1 gene; age related; atheroprotective; base; 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

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.

项目总结/摘要 血管细胞不断地受到与心脏节律性活动相关的物理力的作用， 其与容器的单独几何形状相结合进一步施加振荡、扰动或层流剪切 对内皮细胞的压力。这些血流动力学的力量决定了表型和基因表达谱的 动脉树的不同区域中的内皮细胞使它们具有动脉粥样硬化保护性或动脉粥样硬化倾向性。由于 不同类型的血流在血管病变发生中的影响， 识别与不同类型剪切应力相关的机械感应蛋白和转录谱。在 相反，较少强调细胞表面的力如何传递到细胞核， 影响核形状、核孔功能以及染色质组织和脉管系统中的完整性， 这是本申请的焦点。我发现振荡和扰动流有一个渐进的有害 核形状的影响，这是由波形蛋白的损失加剧。根据这些数据和其他初步数据，我 假设波形蛋白对于维持振荡区域的核形状和染色质完整性至关重要， 以及内皮细胞中的血流紊乱。使用体外内皮细胞进行机制研究和波形蛋白无效 小鼠进行体内验证，我将完成对核完整性和功能变化的深入表征 在与衰老和波形蛋白丢失相关的畸形核中。我将开始详细描述 EC的核形状、核膜完整性和功能（通过分析核质转运） 在体内、主动脉内皮和体外使用活细胞成像的非层流下， 力显微镜我还将描述波形蛋白的动力学，包括其翻译后修饰， 非层流（目标1）。为了评估转录和信号网络的变化，我将使用scRNA测序， 和蛋白质组学来鉴定和验证波形蛋白相互作用组。我会检查波形蛋白的后果 核骨架与细胞骨架连接体（LINC）复合物的蛋白质损失， 与波形蛋白相互作用，并参与直接向细胞核的力传递（目的2）。最后，我将评估 波形蛋白的缺失如何改变内皮染色质的完整性和表观遗传状态， 提供了波形蛋白在维持血管系统中染色质稳态中的关键功能的证据（目的3）。 通过完成这项拟议的工作，我将大大扩展我的技术，技能， 概念，同时积极致力于澄清物理力对内皮细胞核的影响。我也是 我期待着扩大我的技能，在工作中与大型数据集，包括分析，提炼信息 并衍生新问题。作为这一过程的一部分，我将追求几个互动和科学联系 与我所在机构内外的中心和合作者合作， 完成这项工作，我的科学训练，并为我的职业生涯的下一个阶段做好准备。