Abstract Body: Phenotypic modulation of smooth muscle cells (SMCs) is a significant driver of cardiovascular diseases. Similar to tumor cells, modulated SMCs can have an altered metabolic profile of increased glycolysis and decreased oxidative phosphorylation (OXPHOS). Metabolism and differentiation are closely linked: our data shows that siRNA-mediated knockdown of electron transport chain Complex IV component Cox4i1 leads to dedifferentiation of wild-type (WT) SMCs. However, the mechanisms linking SMC phenotype and metabolism are poorly understood. Ten-eleven translocation 2 (TET2) is an established regulator of SMC differentiation. TCA cycle metabolite 2-hydroxygluterate (2HG) competitively inhibits α-keto-gluterate-(αKG)-dependent dioxygenases, including TET2. TET2 oxidizes 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), leading to DNA demethylation and subsequent gene activation. We hypothesize that accumulation of 2HG in SMCs with high glycolytic activity leads to inhibition of TET2 and subsequent epigenetic changes that decrease expression of SMC differentiation genes. Our prior work established that treatment with nicotinamide riboside (NR), a NAD+ analog, increases OXPHOS and decreases glycolysis; this metabolic reprogramming in Acta2^R179C/+ SMCs drives increased RNA and protein levels of SMC differentiation genes. Glucose flux experiments using 1,2-¹³C₂-glucose tracer identified higher accumulation of 2HG in Acta2^R179C/+ SMCs than WT SMCs, which was significantly reduced with NR treatment. Subsequently, chromatin immunoprecipitation (ChIP) assays determined that TET2 binding to SMC differentiation genes is decreased in mutant cells and increased with NR. We also confirmed global and site-specific decreases in 5mC and increases in 5hmC with NR. To determine if these findings represent a generalizable mechanism linking SMC metabolism with differentiation, WT SMCs were treated with 2HG, which induced dedifferentiation and decreased 5hmC and TET2 levels. Knockdown of the enzyme L2HGDH, which metabolizes 2HG back to αKG, leads to accumulation of 2HG in WT SMCs, and as a result, key SMC markers are repressed due to changes in 5hmC via TET2 inhibition. Taken together, these data support that a metabolic switch from OXPHOS to glycolysis drives SMC dedifferentiation via the 2HG-TET2 axis. This work suggests novel metabolic treatment targets to restore SMC differentiation and prevent pathologic phenotypic modulation in cardiovascular diseases.