FVIIa-released EEVs prevent LPS-induced inflammation via miR10a-dependent downregulation of the TAK1-NF-ĸB signaling axis. (A) miR10a putative binding site within the 3'-UTR of TAK1. (B) An equal number of EVs (2 × 10⁸), isolated from the conditioned medium of HUVECs that were transfected with scr miR or anti-miR10a and then treated with control vehicle or FVIIa, were left to fuse with THP-1 for 4 hours. The levels of TAK1 protein in THP-1 cells were analyzed by immunoblot analysis (top), and the band intensities were quantified by densitometric analysis (bottom). (C) EVs (2 × 10⁸), generated from HUVECs transfected with scr miR or miR10a mimic, were added to THP-1 cells. Four hours later, the expression of TAK1 was analyzed by immunoblot analysis (top), and the band intensities were quantified by densitometric analysis (bottom). (D) THP-1 cells that incorporated control-EEVs or FVIIa-EEVs containing scr miR or anti-miR10a were treated with LPS for 30 minutes. The nuclei were isolated, and the levels p65 were determined by immunoblot analysis (top). Histone H3 was used as a control for the loading of nuclear proteins and used for normalization in densitometric analysis (bottom). (E) Control EEVs and control EEVs containing scr miR or miR10a mimic were incubated with THP-1 cells for 4 hours. The cells were treated with LPS for 30 minutes, and p65 levels in nuclei were analyzed as described in panel D. (F) Schematic representation of how miR10a, transferred via FVIIa-EEVs, downregulates the NF-ĸB–mediated inflammatory pathway in monocytes. An inflammatory stimulus, such as LPS, induces TAK1 activation, which in turn, induces IĸBα phosphorylation and its subsequent degradation to release the NF-ĸB p65 subunit to enter the nucleus to induce the expression of proinflammatory genes, such as TNF-α, IL-1β, and IL-6. miR10a, transferred from FVIIa-EEVs, regulates the TAK1-NF-ĸB signaling pathway by targeting TAK1 (F). **P < .01; ***P < .001; ****P < .0001; ns, not significant.