Smooth muscle O-GlcNAcylation and vascular aging in metabolic syndrome - Vascular aging, which leads to structural and functional changes in blood vessels as we age, is a major risk factor for cardiovascular diseases. Metabolic syndrome (MetS), a cluster of modifiable cardiovascular risk factors that often co-exist with advancing age, significantly accelerates early vascular aging. However, molecular mechanisms by which MetS promotes vascular aging are incompletely understood. MetS stimulates vascular smooth muscle cell (VSMC) migration and proliferation, hallmarks of VSMC de-differentiation, contributing to vascular dysfunction. Growing evidence indicates that hyperglycemia, obesity and dyslipidemia, key features of MetS, independently increase O-GlcNAcylation, a posttranslational modification linked to vascular disease. O- GlcNAcylation involves addition of N-acetylglucosamine (GlcNAc) to serine and threonine residues on proteins and is mediated via O-GlcNAc transferase (OGT), a key regulator of protein O-GlcNAcylation. While recent studies, including our preliminary data, support the notion that OGT promotes VSMC de-differentiation, the spatiotemporal role of OGT as a driver of vascular aging and VSMC plasticity in MetS and underlying molecular mechanisms are poorly understood. Guided by preliminary data, we hypothesize that OGT-mediated O- GlcNAcylation of YY1 modulates SRF-dependent pathways and binding partners (ELK1), triggering VSMC cell cycle progression and lineage switch to diseased phenotypes prompting vascular aging in MetS. Using our newly generated VSMC-restricted conditional Ogt knockout combined with VSMC-specific lineage tracing reporter mice (both using Itga8-CreERT2) on MetS agouti KKAy+/- background, we will i) determine O-GlcNAcylation-dependent molecular signals that regulate VSMC cell cycle progression in MetS, and ii) determine whether smooth muscle O-GlcNAcylation drives vascular aging and VSMC fate switch in MetS. Ogt loss-of-function and rescue strategies will be employed coupled with cellular, molecular, proteomics and transcriptomics approaches as well as Atomic Force Microscopy (vessel biomechanics), Treadmill Stress Test (cardiometabolic fitness) and echocardiography ± contrast (PWV, coronary blood flow). We will also use murine aortic SMC (MASMC) primary cultures from above mice and glucose-stimulated human coronary artery SMC and wild-type MASMC ± lentiviral-mediated Ogt knockdown. We will determine the effect of a key CRISPR-edited O-GlcNAc site in YY1 on VSMC cell cycle progression, phenotypic changes, and vascular aging in MetS KKAy+/- mice. Using VSMC-specific ectopic Myocd-overexpressing KKAy+/- ± smOgtKO mice, we will assess the competition between YY1 and MYOCD for binding to SRF during vascular aging. Finally, to ascertain the clinical relevance of our murine findings, aortic tissue from MetS patients of varying ages, undergoing coronary artery bypass grafting (CABG), will be used to determine how aging impacts aortic proteome, transcriptome, and O-GlcNAcylation in MetS-induced vasculopathy. Overall, this proposal will uncover novel O-GlcNAcylation-dependent molecular targets of MetS- induced vascular aging, opening new therapeutic avenues to prevent or treat vascular aging in MetS.
