Abstract Body: Factors that initiate, promote and accelerate obesity-related vascular dysfunction and disease are not completely understood. With increased adiposity there is an apparent pathologic shift in the phenotype of adipose tissue and its secretome. Indeed, interest in adipocyte-derived extracellular vesicles (Ad-EVs) has intensified due to their potential as a circulating biomarker and mediator of cardiometabolic health and disease. We have shown that circulating Ad-EVs are elevated with obesity and associated with endothelial dysfunction; although, the underlying mechanisms are unknown. The experimental aim of this study was to determine the effect of Ad-EVs isolated from adults with obesity on endothelial cell oxidative stress and nitric oxide (NO) production. Twenty-four sedentary, otherwise healthy mid-life adults (43-65 years) were studied: 12 normal weight (6M/6F; age: 57+2 yr; BMI: 22.1+0.5 kg/m2; Ad-EVs: 119+11 Ad-EV/µL) and 12 obese (7M/5F; 56+2 yr; 32.0+0.7 kg/m2; 442+51Ad-EV/µL). Ad-EVs (perilipin A+ vesicles) were identified, enumerated, and isolated from plasma by flow cytometry. Human coronary artery endothelial cells (HCAECs) were cultured and treated with Ad-EVs from either normal weight or obese adults. Intracellular reactive oxygen species (ROS) production was significantly higher (~50%) in HCAECs treated with Ad-EVs from obese (150+14%) vs normal weight (99+9%) adults. Concordantly, expression of antioxidant proteins SOD-1 (220.2+28.1 vs 347.5+34.7 AU) and catalase (58.2+7.2 vs 89.4+8.5 AU) were ~35% lower (P<0.01) in cells treated with obesity-related Ad-EVs. Phosphorylation is the primary posttranslational modification regulating endothelial nitric oxide synthase (eNOS) enzyme activity. Phosphorylation of Ser1177 confers the greatest activation of eNOS; whereas, phosphorylation at Thr495 reduces eNOS activation. Expression of p-eNOS (Ser1177) was lower (29.2±2.6 vs 42.1±3.1 AU; P=0.001) and p-eNOS (Thr495) higher (16.8±1.1 vs 8.1±0.5 AU; P<0.001) in cells treated with Ad-EVs from obese vs normal weight adults. As a result, NO production was markedly lower (5.7±0.2 vs 6.8±0.2 μmol/L; P=0.02) in cells treated with Ad-EVs from obese adults. Increased oxidative stress and decreased eNOS activity and NO production renders the vascular endothelium prone to atherosclerosis and thrombosis. Circulating Ad-EVs likely contribute to the proatherogenic endothelial phenotype and, in turn, the increased incidence of cardiovascular disease with obesity.