Atherosclerosis is a leading cause of death in the USA and globally due to myocardial infarction (MI) or stroke. Despite statin treatment to reduce LDL cholesterol, its incidence is on the rise due to the global epidemic of obesity, metabolic syndrome (MetS), and early onset type-2 diabetes (T2D). Indeed, more than half of the mortality in T2D is caused by cardiovascular complications. Human histopathological studies have shown that lesions prone to plaque rupture, with catastrophic thromboembolic events like MI or stroke, have a thin fibrous cap and a high CD68+/ACTA2+ [presumed MФ/smooth muscle cell (SMC)] cell ratio. These characteristics occur at a higher prevalence in atherosclerotic lesions of diabetics and in women versus men. However, we have a poor understanding of the mechanisms by which insulin resistance, hyperglycemia, and other metabolic abnormalities in T2D-MetS exacerbate atherosclerotic disease. We recently demonstrated that although multiple cell types contribute to formation of the ACTA2+ fibrous cap, long-term plaque stability is dependent on SMC. Moreover, we showed that aerobic glycolysis, a pathway dysregulated in T2D is required for transition of SMC to a beneficial myofibroblast (MF)-like state critical for formation and maintenance of a stable fibrous cap. Results are of major interest given studies of Clemmons and co-workers showing that SMC-selective knockout of insulin receptor substrate-1 (IRS1), a protein required for insulin and insulin- like growth factor-1 (IGF1) signaling, resulted in de-differentiation of SMC, hyper-proliferation, and increased neointimal formation following femoral artery injury. However, they did no atherosclerosis studies and did not consider that de-differentiated SMC could have beneficial or detrimental effects on lesion pathogenesis depending on the nature of their phenotypic transitions. Studies in this proposal will test the hypothesis that insulin-IGF1 resistance in SMC combined with metabolic abnormalities including the profound hyperglycemia and hyperlipidemia associated with T2D-MetD results in detrimental (plaque de-stabilizing) changes in SMC phenotype. Aim 1 will determine if insulin-IGF1 signaling in SMC is required for their investment into the fibrous cap and transition to a plaque stabilizing MF phenotype. Aim 2 will determine if global insulin resistance and the associated metabolic changes, including hyperglycemia, promote atherosclerosis development and late-stage lesion pathogenesis by inducing detrimental changes in SMC phenotype. Aim 3 will determine potential mechanisms by which impaired insulin-IGF1 signaling in SMC contributes to late-stage lesion pathogenesis in humans with T2D/MetS. Studies include: 1) use of our novel SMC lineage tracing atherosclerotic mice with global or SMC-specific insulin resistance with or without systemic metabolic dysfunction including hyperglycemia and hyperlipidemia; 2) rigorous analysis of indices of plaque stability and phenotypic tran...