Atherosclerosis (AS) is a chronic inflammatory disease of medium and large arteries and remains the leading cause of death worldwide from its sequelae of heart attack, stroke and limb loss. During AS progression chronic activation of immune cells in the vessel wall, especially macrophages, leads to formation of lipid-loaded foam cells which become the major component of atherosclerotic plaque. The goal is to discover molecular mechanisms through which dyslipidemia and oxidative stress, which are major risk factors for AS, are linked to chronic macrophage activation via dysregulated function of fat cells (adipocytes). The proposal will explore how adipocyte-derived exosomes (Ad-Exo) carrying specific pro-atherogenic cargo such as miRNAs, are generated under oxidative stress, and how they activate macrophages. Focus is CD36, type II scavenger receptor highly expressed in adipocytes and macrophages that acts as a receptor for the atherogenic ligand oxidized LDL (oxLDL). The oxLDL/CD36 signaling axis creates an inflammatory paracrine loop between macrophages and adipocytes and facilitates oxidative stress and pro-inflammatory cytokine secretion through Src family kinase (SFK) activation. Na/K-ATPase (NKA) α1 subunit serves both as a CD36 co- receptor and a scaffold for the SFK, allowing conformational changes in the NKA to activate membrane bound SFK and initiate signaling cascades, a mechanism distinct from its well-understood pumping function. Hypothesis of this proposal is that activation of NKA/CD36 signaling in adipocytes produces oxidative stress that causes release of adipokines and exosomes (Ad-Exo). These released factors initiate and augment AS by stimulating macrophages. To test this hypothesis, 3 specific aims have been developed. The first will test the hypothesis that adipocyte NKA/CD36 signaling complex promotes AS through production of cellular oxidative stress leading to abnormal adipokines/exosome secretion. The approach will include use of the apoe null mouse model and lentiviral vectors to deliver cell-specific NaKtide, a peptide reagent derived from the NKA α1 domain that behaves as a specific inhibitor of the NKA/SFK pathway. Oxidant stress, AS plaque formation, and adipocyte gene expression will be assessed using state-of-the-art techniques, such as next generation RNASeq. The second aim will test whether Ad-Exo can modulate macrophage function in vitro and in vivo by characterizing the proteome, lipidome and miRNA profile of Ad-Exo released in response to oxidative stress and tracking their delivery to monocytes and AS plaque. A newly developed mouse model, GFP- CD63flox/Adipoq Cre mice will be used as an excellent Ad-Exo reporter animal model for these studies. The third aim will determine if Ad-Exo regulate macrophage lipid metabolism and mitochondrial function using sophisticated metabolic flux assays along with lipidomic and gene expression assays. Successful completion of this project will define an important concept that...