Abstract Body: Despite an improved understanding of many of the pathologic consequences associated with human obesity, factors that initiate, promote and accelerate cerebrovascular events with increased adiposity are not completely understood. Circulating endothelial-cell derived microvesicles (EMVs) have been shown to be elevated with obesity and associated with stroke risk; however, their direct effect on cerebral endothelial cells is unknown. The experimental aim of this study was to determine the effect of EMVs isolated from adults with obesity on on human cerebral microvascular endothelial cells (hCMECs) nitric oxide (NO) and endothelin (ET)-1 production, tissue-type plasminogen activator (t-PA) release.Circulating EMVs (CD144-PE) were isolated (flow cytometry) from 24 sedentary, midlife and older adults (45-71 yr) were studied: 12 normal weight (6 M/6 F; 55±5 yr; body mass index (BMI) >18.5 and < 25 kg/m²) adults and 12 adults with obesity (6 M/6 F; 57±8 yr; BMI >30.0 kg/m²). hCMECs were cultured and treated with isolated EMVs from either normal weight or obese adults for 24 hr. Expression of phosphorylated (p)-eNOS (Ser1177) was ~20% lower (31.5±5.6 vs 39.1±7.9 AU; P=0.01) and p-eNOS (Thr495) was markedly higher (~40%; 47.3±13.2 vs 33.4±10.0 AU; P<0.001) in hCMECs treated with EMVs from adults with obesity. Consistent with the p-eNOS expression pattern, NO production was significantly lower (~20%; P<0.001) in hCMECs treated with EMVs from obese (4.9±0.4 µmol/L) compared with normal weight (5.9±0.5 µmol/L) adults. In addition, expression of both Big ET-1 (317.8±51.8 vs 241.6±65.0 AU; P=0.004) and ECE (838.3±160.8 vs 631.8±126.3 AU; P=0.002) as well as ET-1 production (21.9±1.7 vs 18.0±3.9 pg/mL) were higher in hCMECs treated with obesity-related EMVs. In response to thrombin stimulation, there was a significant decrease (~20%; P=0.008) in intracellular t-PA protein expression in hCMECs treated with EMVs from normal weight (from 78.1±11.3 to 63.7±17.5 AU) adults; but not cells treated with EMVs from obese adults (from 65.2±11.6 to 68.5±14.4 AU). Concordantly, t-PA release in response to thrombin was ~20% higher (P=0.04) in hCMECs treated with EMVs from normal weight (from 52.3±8.8 to 62.0±8.1 pg/mL) compared with obese (from 49.3±6.6 to 52.3±8.2 pg/mL) adults. Circulating EMVs associated with obesity adversely affects brain microvascular endothelial cells and may contribute to the increased risk of cerebrovascular dysfunction and events with increased adiposity.