Atherosclerosis is a multi-faceted vascular disease that involves maladaptation of several cell types in the arterial wall responding to systemic and local factors. During the last two funding cycles, we have used bioinformatics and system biology approaches together with in vitro and in vivo experimental validations to study the cellular and molecular mechanisms by which atheroprotective and atheroprone flows regulate the vascular endothelial cell (EC) in health and disease. Our results demonstrate the crucial roles of flow-regulated EC epigenomes and transcriptomes in the atheroprotective and athero-prone phenotypes. Emerging evidence suggests that the focal nature of atherosclerosis is linked to EC heterogeneity resulting from interplay between intrinsic EC properties and extrinsic shear forces. To further advance our understanding on EC heterogeneity in relation to atherosclerosis, we hypothesize that mediators (e.g., MED-1) coordinate with lineage-dependent transcription factors (LDTFs, e.g., KLF4) and signal-dependent transcription factors (SDTFs, e.g., SMAD2) to regulate the spatiotemporal networks of mechanotransduction. The five specific aims proposed to test this novel hypothesis are: Aim 1. To delineate the spatiotemporal changes in flow-mediated EC epigenomes and transcriptomes with single-cell resolution; Aim 2. To elucidate the effect of shear stress on interactions between ECs and vascular smooth muscle cells (SMCs) or macrophages (MØs) with spatial resolution; Aim 3. To characterize the transcriptomes and the regulating epigenomes in the arterial wall in vivo with spatial resolution; Aim 4. To employ system biology approaches to compute and integrate data for the construction of temporal and spatial regulatory networks; Aim 5. To validate the shear stress-regulated EC heterogeneity at the disease level using mouse atherosclerosis models and human artery disease specimens. With the use of multi-omics platform at single-cell resolutions, this renewal proposal will decipher the shear stress regulations of the EC heterogeneity and the consequential phenotypical changes of ECs and neighboring cell types (SMCs and MØs) relevant to atherosclerosis.