Abstract Body:
Hypertension is a predominant risk factor for cerebrovascular disease and events. It is estimated that ~60% of stroke and stroke-related morbidity is due to elevated blood pressure. However, factors that initiate, promote and accelerate hypertension-related cerebrovascular events are not completely understood. Extracellular vesicles, particularly endothelial cell-derived microvesicles (EMVs), have been linked to stroke risk, severity and outcome. We have previously demonstrated that circulating EMVs are elevated with hypertension and associated with vascular dysfunction. Part of a larger ongoing trial on hypertension and extracellular vesicle biology, the aim of this study was to determine the effect of circulating EMVs isolated from adults with hypertension on brain endothelial cell nitric oxide (NO) and endothelin (ET)-1 production in vitro. Sixteen sedentary adults were studied: 8 normotensive (6M/2F; age: 532 yr; BP: 117/74±2/2 mmHg; EMVs: 104±13 EMV/µL) and 8 hypertensive (6M/4F; 56±2 yr; 151/93±2/1 mmHg; 210±35 EMV/µL). All subjects were free of overt cardiometabolic diseases. EMVs (CD144+) were identified, sorted and collected from plasma by flow cytometry. Human cerebral microvascular endothelial cells (hCMECs) were cultured and treated with isolated EMVs from either normotensive or hypertensive adults for 24 hr. Intracellular protein expression was determined by capillary electrophoresis immunoassay. Total protein expression of endothelial nitric oxide synthase (eNOS) was significantly lower in cells treated with EMVs from hypertensive (130.3±7.2 AU) vs normotensive (344.1±13.5 AU). Phosphorylation is the primary posttranslational modification regulating eNOS enzyme activity. Expression of phosphorylated (p)-eNOS (Ser1177; primary activation) was lower (26.0±2.1 vs 60.2±4.8 AU; P<0.01) and p-eNOS (Thr495; primary inhibition) higher (10.5±0.5 vs 3.2±0.4 AU; P<0.01) in cells treated with EMVs from adults with hypertension. Consequently, NO production was markedly lower in cells treated with hypertension-related EMVs (6.5±0.2 vs 7.5±0.3 μmol/L; P=0.04). Cell expression of Big ET-1 (87.4±6.1 vs 66.1±4.4 AU; P=0.01) and ET-1 production (33.4±0.7 vs 25.4±2.4 pg/mL; AU; P<0.01) were higher in cells treated with EMVs from adults with hypertension. Reduced cerebral NO bioavailability and increased ET-1 production contribute to stroke risk. Thus, circulating EMVs represent a novel mechanistic factor underlying hypertension-related stroke risk.