Fig 2: Autophagy mediated the effects of ADSC-sEVs on tight junction-associated protein expression in LPS-treated PMVECs. A Representative western blots showing ZO-1 and claudin-5 expression in PMVECs. B Statistical analysis of ZO-1 and claudin-5 expression in PMVECs. LPS decreased the expression of ZO-1 and claudin-5 in PMVECs, which was significantly inhibited by ADSC-sEVs treatment. Autophagy inhibition weakened the effect of ADSC-sEVs on the expression of ZO-1 and claudin-5 (the relative expression of claudin-5, control, 0.95 ± 0.02 vs. LPS, 0.47 ± 0.01 vs. LPS + ADSC-sEV, 0.73 ± 0.03, LPS + ADSCsiATG5-sEV, 0.51 ± 0.01. the relative expression of ZO-1, control, 1.29 ± 0.05 vs. LPS, 0.75 ± 0.02 LPS + ADSC-sEV, 1.05 ± 0.03 vs. LPS + ADSCsiATG5-sEV 0.84 ± 0.04). The results are expressed as the mean ± SD of three independent experiments

Fig 3: The effect of ADSC-sEVs with or without autophagy inhibition on toll-like receptor 4 (TLR4)-nuclear factor (NF)-κB signaling in LPS-triggered PMVECs. A Representative western blots showing expressions of TLR4, MyD88 and total or phosphorylated(p)- NF-κB p65. B Statistical analysis of the relative expressions level of TLR4, MyD88 and phosphorylated(p)- NF-κB p65. LPS promoted the expression of TLR4, MyD88 and phosphorylated(p)-NF-κB p65. ADSC-sEVs treatment decreased expressions of all the aforementioned proteins. Autophagy inhibition by siATG5, however, significantly weakened the effect of ADSC-sEVs on inhibiting the TLR4/NF-κB signaling related proteins (the relative expressions level of TLR4, control, 0.28 ± 0.01 vs. LPS, 1.19 ± 0.03 vs. LPS + ADSC-sEV, 0.62 ± 0.03 vs. ADSCsiATG5-sEV, 0.91 ± 0.02. the relative expressions level of phosphorylated(p)- NF-κB p65, control, 0.19 ± 0.01 vs. LPS 0.97 ± 0.04 vs. LPS + ADSC-sEV, 0.49 ± 0.02 vs. ADSCsiATG5-sEV, 0.79 ± 0.03. the relative expressions level of MyD88, control, 0.25 ± 0.02 vs. LPS 1.05 ± 0.01 vs. LPS + ADSC-sEV, 0.55 ± 0.02 vs. ADSCsiATG5-sEV, 0.77 ± 0.02). The results are expressed as the mean ± SD of three independent experiments

Fig 4: Autophagy mediated the protective effect of ADSC-sEVs on LPS-induced PMVEC viability. A, B Representative fluorescence images and statistical analysis of LPS-induced endothelial cell death after incubation with sEVs derived from ADSC in the presence or absence of autophagy inhibition. LPS treatment significantly increased the percentage of dead cells, which were characterized by PI staining of the nuclei. Small extracellular vesicle pretreatment markedly alleviated LPS-induced cell death. However, autophagy inhibition markedly weakened sEV-mediated abrogation of cell death [the percentage of dead cells (%), control, 7.67 ± 1.27 vs. LPS, 33.57 ± 2.20 vs. LPS + ADSC-sEV, 21.67 ± 1.72 vs. LPS + ADSCsiATG5-sEV, 29.30 ± 1.51]. C MTT assays assessing the viability of PMVECs after LPS challenge. LPS significantly reduced cell viability, which was apparently alleviated by ADSC-sEVs. Autophagy inhibition reduced the effect of sEVs [the cell viability (OD), control, 2.94 ± 0.06 vs. LPS, 1.46 ± 0.21 vs. LPS + ADSC-sEV, 2.01 ± 0.14 vs. LPS + ADSCsiATG5-sEV, 1.49 ± 0.06]. The results are expressed as the mean ± SD of three independent experiments

Fig 5: The permeability of PMVECs was measured using a Transwell assay. LPS increased the permeability of endothelial cells, and this effect was significantly alleviated by ADSC-sEVs treatment. Autophagy inhibition weakened the protective effect of ADSC-sEVs against LPS-induced endothelial permeability. (the relative fluorescence at 6 h, control, 2407.67 ± 353.56 vs. LPS, 8251.00 ± 369.21 vs. LPS + ADSC-sEV, 4185.00 ± 396.32 vs. LPS + ADSCsiATG5-sEV, 6319.67 ± 421.00. the relative fluorescence at 12 h, control, 2260.33 ± 294.18 vs. LPS, 8190.00 ± 397.31, LPS + ADSC-sEV, 4251.00 ± 340.95 vs. LPS + ADSCsiATG5-sEV, 6228.33 ± 499.55). The experiment was repeated three times