FVIIa-derived EEVs promote anti-inflammation and endothelial barrier protection via miR10a transfer. (A) miR10a expression levels in THP-1 cells after the uptake of EEVs. An equal number of EVs (2 × 10⁸), released from endothelial cells treated with control vehicle (Con EVs) or FVIIa (FVIIa EVs), were incubated with THP-1 cells for 4 hours. Free EVs were removed, the cells were washed twice, and the miR10a level in the THP-1 cells was determined by qRT-PCR. (B) Increased miR10a level in THP-1 cells after EEV uptake resulted from the transfer of miR10a from EEVs to THP-1 cells and were not related to de novo transcription. THP-1 cells were treated with actinomycin D (Actn D; 10 µg/mL) for 8 hours before they were exposed to EEVs. The rest of the experimental procedure was the same as described in panel A. (C-E) The transfer of miR10a from EEVs to THP-1 cells confers anti-inflammatory phenotype to THP-1 cells. An equal number of EVs (2 × 10⁸), isolated from HUVECs transfected with scrambled miR (scr miR) or anti-miR10a and then treated with a control vehicle or FVIIa, were incubated with THP-1 cells (2 × 10⁶) for 4 hours to allow for the uptake of EEVs by THP-1 cells. Thereafter, THP-1 cells were challenged with LPS (200 ng/mL) for 12 hours, and the release of TNF-α (C), IL-6 (D), and IL-1β (E) was measured by ELISA. (F-H) Uptake by THP-1 cells of EEVs containing miR10a mimic reduced the elaboration of LPS-induced inflammatory cytokines. HUVECs were transfected with scr miR or miR10a mimic RNA (20 nM). EVs, isolated from the supernatant medium (2 × 10⁸), were left to fuse with the THP-1 cells for 4 hours. The cells were challenged with LPS for 12 hours, and the levels of TNF-α (F), IL-6 (G), and IL-1β (H) in the supernatant medium were determined. (I-J) miR10a-dependent endothelial barrier protection in target endothelial cells after the uptake of EEVs EVs containing scr miR, anti-miR10a, or miR10a mimic were generated as described in panels C to H. They were left for 4 hours to fuse with endothelial cells grown to confluence in a transwell system. The cells were challenged with LPS (200 ng/mL), and barrier permeability was assessed 12 hours after the LPS challenge. The barrier permeability (OD readings) observed in cells treated with LPS that were not exposed to EVs were taken as 100%. (K) Naive HUVECs fused with control- or FVIIa-EEVs containing scr miR, anti-miR10a, or miR10a mimic were treated with LPS (200 ng/mL) for 6 hours, and ZO-1 levels in the cell extracts were assessed by immunoblot analysis. *P < .05; **P < .01; ***P < .001; ****P < .0001; ns, not significant.