Anti-inflammatory and barrier protective effects of FVIIa-released EEVs in vivo and dependency on miR10a. (A) C57WT/6J mice were given saline or FVIIa (0.25 mg/kg) via the tail vein. Two hours later, blood was collected, and the EVs were isolated from the plasma. An equal number of EEVs (2 × 10⁸) were used to determine miR10a expression levels by qRT-PCR. (B-D) An equal number of EVs (2 × 10⁸), isolated from the plasma of mice treated with saline or FVIIa, as in panel A, were incubated with murine peritoneal macrophages that had been transfected (T) with scr miR or anti-miR10a. The macrophages were then challenged with LPS (200 ng/mL) for 12 hours and the release of TNF-α (B), IL-6 (C), and IL-1β (D) was measured by ELISA. (E) EEVs, isolated from mice as in panel A, were allowed to incorporate into bEND.3 cells cultured in a transwell system and transfected (T) with scr miR or anti-miR10a. The cells were challenged with LPS (200 ng/mL), and barrier permeability was measured at 12 hours after the addition of LPS. (F) C57WT/6J mice were given a ROCK inhibitor, Y27632 (1 mg/kg), via the tail vein 1 hour before injection of saline or FVIIa (0.25 mg/kg) via the same route. Two hours after administration of FVIIa, EVs in the circulating blood were isolated and quantified by NTA. (G-I) C57WT/6J mice were given Y27632 and FVIIa, as described in panel F. At 2 hours after administration of FVIIa, LPS was administered (5 mg/kg, IP). Twelve hours after LPS challenge, plasma proinflammatory cytokines, TNF-α (G), IL-6 (H), and IL-1β (I) were measured by ELISA, and vascular permeability in the lungs (J) and heart (K) were evaluated as described in “Materials and methods.” *P < .05; **P < .01; ***P < .001; ****P < .0001; ns, not significant.