Atherothrombosis, resulting from rupture or erosion of unstable atherosclerotic plaques, is the leading cause of death worldwide. However, the mechanisms that regulate the stability of late stage atherosclerotic lesions remain poorly understood. Recent studies from our lab showed that smooth muscle cell (SMC) conditional knockout of the platelet-derived growth factor receptor-β (PdgfrbSMC-Δ/Δ) in ApoE-/- mice was associated with nearly complete failure of SMC to invest into lesions or the fibrous cap. However, surprisingly we observed no changes in lesion size or indices of plaque stability, including the thickness of the Acta2+ fibrous cap, following 18 weeks of Western diet (WD) feeding. Further investigation provided novel insights regarding the mechanisms underlying these changes. Key findings included: 1) contrary to long-standing dogma that Acta2+ fibrous cap cells are derived almost exclusively from SMC, we showed that they account for only 60-70% of Acta2+ cells in advanced ApoE-/- brachiocephalic (BCA) or human coronary artery lesions with the remainder coming from endothelial cell-to-mesenchymal-to-myofibroblast transitions (EndoMT-MFT, 20-25%) and macrophage-to-myofibroblast transitions (MMFT), 10-15%) respectively; 2) loss of SMC investment into lesions with SMC PDGFRB KO was associated with large adaptive increases in EndoMT-MFT and MMFT; 3) increased EndoMT-MFT, and MMFT did not sustain indices of stability when WD feeding was extended to 26 weeks suggesting there may be qualitative differences in Acta2+ fibrous cap cells depending on their origin; 4) RNA-seq analysis on the BCA of 18-week WD-fed PdgfrbSMC-Δ/Δ ApoE-/- mice versus control littermate mice showed that energy metabolism pathways were the top ten upregulated pathways suggesting that metabolic reprogramming may be required for SMC-MF, EndoMT-MF, and MMF transitions; and 5) inhibition of aerobic glycolysis in cultured SMC prevented their transition to a MF state following treatment with PDGF and TGFβ. Studies in this proposal will test the hypothesis that SMC, EC, and macrophage adaptive responses for maintaining plaque stability require major shifts in energy metabolism and that the metabolic state of these cells can be manipulated to stimulate beneficial changes in the phenotype of lesion cells and overall increases in plaque stability. Aim 1 will determine if genetic knockout or pharmacologic inhibition of aerobic glycolysis in SMC and EC lineage tracing ApoE-/- mice with advanced atherosclerosis is associated with evidence of reduced plaque stability. Aim 2 will determine if genetic or pharmacologic promotion of aerobic glycolysis in SMC and EC lineage tracing ApoE-/- lineage tracing mice with advanced atherosclerosis is associated with increased plaque stability. Studies for both aims will include human validation studies using scRNAseq and histological data from stable asymptomatic versus unstable symptomatic carotid endarterectomy samples. All studies will assess se...