ABSTRACT Glucose-6-phosphate dehydrogenase (G6PD) is a key enzyme in glucose metabolism and is also a master regulator of cellular redox signaling that plays a crucial role in the regulation of vascular function. We recently isolated and characterized long (G6PD545) and short (G6PD515) G6PD isoforms in vascular smooth muscle cells (VSMCs). Our preliminary data demonstrate that G6PD545 has a putative nuclear localization sequence (NLS) and is predominantly localized in the nucleus of synthetic VSMCs. We further show that nuclear G6PD545 (but not cytosolic G6PD515) represses expression of myocardin (MYOCD), the major switch for the VSMC contractile phenotype, thereby mediating VSMC reversion to a synthetic state, which is considered pathognomonic for a number of vascular complications, including atherosclerosis, transplant arteriopathy and bypass graft failure. Importantly, we found that arteries from rats, pigs and humans with metabolic syndrome all exhibit VSMC synthetic phenotype-associated atherosclerosis and contain high levels of nuclear G6PD545. Interestingly, individuals harboring a loss-of-function SNP within exon 6 of G6PD (Mediterranean-type G6PD mutation) exhibit 80% less G6PD activity and are less likely to develop cardiovascular disease. These observations lay the foundation for our hypothesis that up-regulation of a novel isoform of G6PD functions as a NADPH- dependent repressor of MYOCD expression that promotes a synthetic VSMC phenotype and pathogenic vascular remodeling. This novel hypothesis will be tested through a series of interrelated specific aims designed to elucidate the transcriptional control of MYOCD expression by G6PD545 and the role of G6PD545 in injury-induced neointimal vascular disease in normal and metabolic syndrome rats. Aim 1 will determine whether G6PD545 functions as a switch promoting a VSMC synthetic phenotype in balloon-injured rat carotid arteries in which G6PD545 is down-regulated through Cre/loxP knockdown or CRISPR-Cas9-mediated genome editing to model the Mediterranean-type G6PD mutation. The effects of G6PD545 ± NLS and G6PD515 vs. G6PD545 overexpression on VSMC phenotype will also be studied. Aim 2 will determine whether G6PD545 gain- of-function promotes a VSMC synthetic phenotype through repression of MYOCD expression in cultured VSMCs as well as vascular tissue from healthy vs. balloon-injured rat carotid arteries. Additionally, the effect of loss- and gain-of-function of MYOCD on G6PD545-induced changes in VSMC marker genes will be determined. Aim 3 will elucidate the mechanism of G6PD545 up-regulation and evaluate its role in switching VSMCs to a synthetic phenotype in models of pathological vascular remodeling (e.g., rat metabolic syndrome). This project will reveal a heretofore unrecognized relationship between G6PD545 and the regulation of VSMC phenotype and vascular remodeling in response to physical injury and metabolic disease. The results of these studies will have enormous applicability for development of new therapies to combat occlusive vascular diseases and perhaps other diseases in which MYOCD expression may be altered (e.g., asthma, hypertension).