PROJECT SUMMARY The overall goal of this proposal is to define the role of the canonical Wnt signaling pathway in endothelial cells in the development and progression of atherosclerosis. Exciting preliminary data generated through genomic sequencing experiments show that endothelial GR up-regulates canonical Wnt signaling and that suppression of Wnt signaling can improve atherosclerosis, quiet inflammation, and restore central endothelial metabolic defects. The Wnt pathways have recently been implicated in the pathogenesis of atherosclerosis and other states of vascular inflammation though have not been well-studied in cell-specific environments. To investigate the effects of endothelial-cell specific canonical Wnt signaling suppression we propose the following Aims: In Aim 1, we will characterize the atherosclerotic phenotype of a novel double-mutant mouse model that lacks the expression of low-density lipoprotein receptor-related protein 5 and 6 (LRP5/LRP6) in endothelial cells. These are key proteins necessary for canonical Wnt signaling. In addition, we will analyze endothelial cell inflammation, vascular function, and nitric oxide production in this model. We will also examine markers of fibrosis and systemic blood pressure. In Aim 2, our broad goal is to analyze central endothelial metabolism and gene expression in the presence and absence of Wnt signaling. In Aim 2.1 we plan to evaluate the in vitro metabolic phenotypes of endothelial cells with suppressed Wnt signaling by assaying fatty acid oxidation, cellular lipid metabolism, mitochondrial bioenergetics, and fuel preference. In Aim 2.2, we will examine endothelial cell heterogeneity by performing single-cell RNA-seq from aortas of mice with augmented and suppressed Wnt signaling. These studies will be extended by examining chromatin capture conformation in key differentially-regulated metabolic genes. These data sets will be integrated to conclude how chromatin conformation affects the functionality of key genes of interest.