Fig. 5
EVARDSAlk5Runx1p66 increase proliferation of LPS-treated ECs. Biotin/neutrAvidin-Alexa Fluor 594 labeling of EVs isolated from a human healthy donor (a, EVCtrl) and an ARDS subject (b, inset; EVARDS). Arrows in a indicate several donut-shaped EVCtrl. Bars: 1 μm (a, and b inset), 2 μm (b). n = 3. BrdU labeling of ECCtrl (c), ECLPS (d) and ECLPS exposed to EVARDSAlk5Runx1p66 isolated from the blood of a long-term ARDS survivor (e) and ECLPS exposed to EVARDSAlk5Runx1p52 isolated from the blood of a non-surviving ARDS patient (f). A dose of 1.6 × 103 EVARDS/1 ml growth medium was used for the micrographs shown. Bars: 50 μm (c–f). g Quantification of BrdU-positive cells. The results are expressed as BrdU-positive ECs/high power field (50 power field images/experimental condition). Data are representative of three different experiments and were normalized per total number of cells counted. Values are shown as mean ± SEM. n = 3; *p < 0.05; **p < 0.01. h MTT assay and cell counting of ECCtrl and ECLPS exposed for 3 days to EVCtrl and EVARDS, as above (ratio 2:1 EVARDS:EVCtrl). Data are normalized to day 1 ECCtrl. Values are shown as mean ± SEM. n = 3; *p < 0.05; **p < 0.01. I ECCtrl (a bars), ECLPS (b bars), ECLPS + 5 μg EVMSC (c bars) and ECLPS + 10 μg EVMSC (d bars) were counted 3 days post-EVMSC exposure. Briefly, EVMSC released by 3 × 106 MSCs, at day 5, day 7 and day 8, were resuspended in 50 μl sterile PBS; 1 dose of 10 μg/ml protein was found efficient in inducing proliferation of ECLPS. All data labels are included for each bar. Values are shown as mean ± SEM. n = 3 experiments (triplicate wells each); *p < 0.01 vs. ECCtrl