Age-related circulating endothelial extracellular vesicles promote cerebral microvascular cell dysfunction

The aim of this study was to determine, in vitro, the effect of endothelial cell-derived extracellular vesicles (EEVs) from older adults on brain microvascular endothelial cell oxidative stress, inflammation, nitric oxide (NO) and endothelin (ET)-1 production, as well as tissue-type plasminogen activator (t-PA) release. Circulating EEVs (CD144⁺ extracellular vesicles) were enumerated and isolated (flow cytometry) from the plasma of 30 healthy, sedentary, nonobese adults: 15 young (age: 21-35 yr; 7 M/8 F) and 15 older (55-80 yr; 7 M/8 F). Human cerebral microvascular endothelial cells (hCMECs) were cultured and treated with EEVs from either group. EEVs from older adults induced higher reactive oxygen species (ROS; 140 ± 51 vs. 99 ± 16% of control; P = 0.02) production and lower expression of catalase [13.7 ± 3.7 vs. 23.2 ± 7.4 arbitrary units (AU); P < 0.01] and superoxide dismutase-1 (SOD-1; 147.6 ± 41.1 vs. 281.2 ± 78.3; P < 0.01) in hCMECs than EEVs from young adults. EEVs from older adults did not induce cellular inflammation. Expression of phosphorylated (p)-endothelial nitric oxide synthase (eNOS) (Ser1177) was significantly lower (61.6 ± 13.0 vs. 77.7 ± 17.6 AU), p-eNOS (Thr495) was significantly higher (49.9 ± 16.4 vs. 38.0 ± 10.9 AU), and NO production (5.9 ± 1.6 vs. 7.3 ± 1.3 µmol/L) was significantly lower in hCMECs treated with EEVs from older adults. Big ET-1 (95.3 ± 17.1 vs. 69.4 ± 20.6 AU; P < 0.001) and endothelin-converting enzyme (ECE; 164.8 ± 26.0 vs. 128.4 ± 18.5 AU; P < 0.001) expression as well as ET-1 production (27.3 ± 9.1 vs. 21.2 ± 5.5 pg/mL; P = 0.03) were significantly higher in hCMECs treated with EEVs from older adults. t-PA release in response to thrombin was significantly lower (∼30%) in hCMECs treated with EEVs from older adults. Circulating EEVs represent a potential mechanistic factor contributing to increased stroke risk with aging.NEW & NOTEWORTHY Aging is the preeminent risk factor for ischemic stroke. The mechanisms underlying the age-related increase in stroke risk and incidence are complex, diverse, and not completely understood. This study provides novel data implicating circulating endothelial cell-derived extracellular vesicles (EEVs) as a novel mediator of age-related cerebrovascular disease and events. EEVs from older adults increased oxidative stress, compromised nitric oxide bioavailability, enhanced ET-1 production, and impaired t-PA release in brain microvascular endothelial cells in vitro.