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 thi...