MiR-10a negatively regulates NF-κB signaling. (A) Luciferase assays in bovine aortic ECs. BTRC, a known miR-10a target, is included as a positive control. Overexpression of miR-10a suppresses the activity of mouse and human IRAK4 3′ UTR–containing luciferase constructs, but LTBP1 (another miR-10a predicted target gene) is not suppressed. n = 3. (B) EC-EV treatment suppresses BTRC and MAP3K7 (known miR-10a target genes), as well as IRAK4 mRNA expression in LPS-stimulated (2 hours) THP-1 cells, whereas effects on known miR-146a (TRAF6) or miR-181b (Importin-A3) targets are not observed. n = 4. (C) Western blotting confirms that IRAK4 protein is suppressed by EC-EV treatment. Densitometry is indicated above. A representative experiment of 4 is shown. (D) MiR-10a overexpression (OE) in THP-1 monocytic cells represses BTRC, MAP3K7, and IRAK4 mRNA levels. n = 4. (E) MiR-10a overexpression in THP-1 cells represses IRAK4 protein as assessed by western blot. Densitometry is indicated above. A representative experiment of 3 is shown. (F) NF-κB activity is repressed in THP-1 cells overexpressing miR-10a. Shown is a representative experiment of 4 with triplicate determinations. (G) Model of how ECs suppress monocytic inflammatory responses and promote an immunomodulatory phenotype through the secretion of EC-EVs that contain antiinflammatory miRNAs, including miR-10a. Suppression of NF-κB signaling is mediated in part by the targeting of IRAK4, TAK1/MAP3K7, and β-TRC by miR-10a, whereas IRF5 is downregulated by EC-EVs in an miR-10a–independent manner (data not shown). MiR-126 and miR-181b (which are present in EC-EVs) can suppress proinflammatory responses when overexpressed in monocytic cells. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.