Atherosclerosis is the primary cause of cardiovascular diseases and a leading cause of death worldwide. Diabetes prevalence is on the rise globally, with cardiovascular diseases (CVD) as the main cause of mortality among diabetic patients. Minimizing the risk of CVD is a critical clinical goal in the management of diabetic patients. In turn, hyperglicemia induces a large number of alterations in the vascular tissue at the cellular level that accelerate the atherosclerotic process. In fact, prolonged exposure to hyperglycemia is recognized as a major factor in the pathogenesis of atherosclerosis associated with diabetes. Endothelial cell (EC) dysfunction is a hallmark and initial step of atherosclerosis and is aggravated in diabetes. Nonetheless, there is a paucity in available treatments to target atherosclerosis associated with diabetes. Transcription factor-EB (TFEB), a crucial regulator of lysosomal biogenesis and autophagy, has beneficial effects in various diseases. Our systematic studies to address TFEB function in ECs uncovered that TFEB promotes autophagy and inhibits oxidative stress and inflammation in vitro. In models of CVD, we demonstrated that in EC-specific TFEB transgene promotes post-ischemic angiogenesis and plays an atheroprotective role in laminar flow. TFEB inhibits endothelial inflammation resulting in reduced atherosclerosis in vivo. We recently reported that EC- TFEB regulates plasma glucose. Our preliminary studies indicate that GPNMB may be a novel transcriptional target of TFEB mediating its downstream effects. Collectively, these data suggest that TFEB activation might contribute to ameliorate atherosclerosis in association with diabetes concurrently. Conjugated linoleic acid (CLA) is the preferential substrate for fatty acid nitration in humans. Gastric CLA nitration upon oral delivery of CLA and inorganic nitrite (NO2) renders NO2-CLA in the nanomolar range in humans and mice, making it an attractive intervention for CVD. Our preliminary data reveal that NO2-CLA regulates autophagy in a TFEB- dependent manner and enhances TFEB transcriptional activity in ECs. Based on these evidences, we will test the central hypothesis that direct activation of endothelial TFEB by NO2-CLA protects against atherosclerosis associated with diabetes through GPNMB. Through gain- and loss-of-function strategies in vitro and in vivo, using unique animal models generated specifically for these studies and NO2-CLA treatment, we will address two comprehensive specific aims. In Aim 1, we will demonstrate that NO2-CLA enhances TFEB-dependent protective effects in vitro via GPNMB and focus on the underlying mechanism, while showing the critical role of TFEB Cys212, a potential direct S-nitroalkylation site in TFEB. In Aim 2, we will establish that NO2-CLA inhibits atherosclerosis through TFEB and GPNMB in a diabetogenic atherosclerosis model in vivo. By establishing oral delivery of NO2-CLA as a feasible new therapeutic strategy operating throug...