Mechanosensing and Mechanotransduction in the Endothelial Nucleus

项目摘要/摘要 血管细胞不断受到与心脏节律活动相关的物理力的影响， 它与血管的个别几何形状相结合，进一步施加了振荡、扰动或层流剪切。 对内皮细胞的压力。这些血流动力学因素决定了其表型和基因表达谱。 动脉树不同区域的内皮细胞使它们具有动脉粥样硬化保护作用或易于发生动脉粥样硬化。由于 不同类型的血流在血管病理的发病中的影响，已经投入了大量的努力 识别与不同类型的切应力相关的机械感应蛋白质和转录图谱。在……里面 相比之下，较少强调细胞表面的力是如何传递到细胞核的， 影响血管系统中的核形状、核孔功能、染色质组织和完整性， 这是本应用程序的重点。我发现振荡的和扰动的水流具有渐进性的有害作用。 对核形状的影响，这种影响因波形蛋白的丧失而加剧。根据这一数据和其他初步数据，我 假设Vimentin对维持振荡区域的核形状和染色质完整性至关重要 并扰乱了内皮细胞的血流。利用体外培养的内皮细胞进行机制研究和波形蛋白缺失 对于小鼠的体内验证，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变形核中。我将从一个详细的描述开始 内皮细胞的核形态、核膜完整性和功能(通过核质转运分析) 在体内、主动脉内皮细胞和体外非层流条件下，使用活细胞成像和原子 力显微镜。我还将描述波形蛋白的动力学，包括它的翻译后修饰，在 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。我将研究Vimentin的后果 直接或间接作用于核骨架-细胞骨架复合体的蛋白质损失 与波形蛋白相互作用，并参与向细胞核的直接力传递(目标2)。最后，我将评估 应用ATAC测序技术研究波形蛋白缺失如何改变内皮细胞染色质完整性和表观遗传状态 提供证据证明波形蛋白在维持血管系统染色质平衡方面的关键作用(目标3)。 通过完成这项拟议的工作，我将极大地扩展我的技术、技能和 概念，同时积极有助于阐明物理力量对内皮细胞核的影响。我也是 我期待着拓宽我在处理大数据集方面的技能，包括分析、提炼信息 并衍生出新的问题。作为这一过程的一部分，我将寻求几个互动和科学联系 与我的机构内外的中心和合作者合作，这将有助于促进和 完成这项工作，我的科学训练，并为我的职业生涯的下一阶段做好准备。

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.