Exercise-Intervened Circulating Extracellular Vesicles Alleviate Oxidative Stress in Cerebral Microvascular Endothelial Cells Under Hypertensive Plus Hypoxic Conditions

Figure 1. Exercise intervention stimulates cEV release and boosts the incorporation efficiency of cEVs into mBMECs. (A) Representative plots showing the size and distribution of nET-cEVs and ET-cEVs. (B) Summarized data on the level of the two groups of cEVs. Data are expressed as mean ± SD. * p < 0.05 vs. nET-cEV. n = 10/group. (C) Representative Western blot bands showing the expressions of EV-specific markers CD 63 and Tsg 101 (arrow). (D) Representative images showing the incorporation of cEVs with mBMECs after 24 h of co-culture. Blue: DAPI; red: PKH-26-labeled cEVs. Scale bar: 100 μm. 200× magnification. (E) Summary data of the uptake study. Data are expressed as mean ± SD. *** p < 0.01 vs. nET-cEV. n = 4/group.

Figure 2. Macropinocytosis and endocytosis modulate the uptake of ET-cEVs into mBMECs. (A) Representative image showing the incorporation of ET-cEVs into the cytoplasm of mBMECs pre-treated with or without pathway inhibitors. Blue: DAPI; red: PKH-26-labeled cEVs. Scale bar: 100 μm. 200× magnification. (B) Summary data of the uptake mechanism study. Data are expressed as mean ± SD. * p < 0.05 vs. ET-cEV only (no inhibitor pre-treatment). n = 4/group.

Figure 3. Migration assay of Ang II plus hypoxia-challenged mBMECs treated with cEVs. (A) Representative images of mBMEC migration at the baseline (0 h) and 24 h after the cEV treatment; 40× magnification. (B) Summary data depict the effects of cEVs on mBMEC migration following Ang II plus hypoxia injury. Data are shown as mean ± SD. ## p < 0.01 vs. control; * p < 0.05 vs. Ang II + hypoxia. n = 3/group.

Figure 4. DHE overexpression in Ang II plus hypoxia-challenged mBMECs treated with cEVs. (A) Representative image showing DHE expression in hypoxia plus Ang II-injured mBMECs. Scale bar: 100 μm. 200× magnification. (B) Summary data of the DHE fluorescence intensity of mBMECs. Data are shown as mean ± SD. ## p < 0.01 vs. control; * p < 0.05 vs. Ang II + hypoxia. n = 3/group.

Figure 5. Akt phosphorylation and Nox2 expression in Ang II plus hypoxia-challenged mBMECs treated with cEVs. (A1,A2) Representative bands and summary data showing the ratio of p-Akt/Akt in mBMECs. (B1,B2) Representative bands and summary data showing the expression of Nox2 in mBMECs. Data are shown as mean ± SD. ## p < 0.01 vs. control; * p < 0.05 vs. Ang II + hypoxia. n = 3/group.