MiR-10a is transferred from EC-EVs to monocytic cells. (A) Overexpression of the C elegans–specific miRNA, miR-39, in ECs results in a time-dependent accumulation of this miRNA in cocultured THP-1 cells (in the absence of cell-cell contact). MiR-39 expression level after 1-hour coculture was set to 1 because miR-39 was not detectable (ND) by qRT-PCR in THP-1 monoculture. Data are normalized to U6 expression. n = 3. (B) qRT-PCR was used to assess the expression of several miRNAs, including the EC-enriched miRNA, miR-126-3p, and several anti-inflammatory miRNAs (miR-10a, miR-146a/b, miR-147a, and miR-181b) in THP-1 monocytic cells treated with EC-EVs for 24 hours. Data are normalized to U6 expression. n = 5. (C) Addition of increasing concentrations of EC-EVs results in a dose-dependent increase in miR-10a in treated THP-1 cells. n = 2. (D) Kinetics of mature miR-10a and pri-miR-10a expression after treatment of THP-1 cells with EC-EVs (10 μg/mL). n = 2. (E) Levels of miR-10a and miR-126-3p are increased in THP-1 cells cocultured with ECs for 24 hours. n = 4. (F) Overexpression of miR-10a in ECs results in an increase in miR-10a expression in cocultured THP-1 cells. n = 4. (G) miR-10a expression is elevated in peritoneal leukocytes isolated from LPS-stimulated mice injected with EC-EVs compared with PBS injection control. n = 5. (H) EC-EV–dependent induction of miR-10a is resistant to actinomycin D (Act D) pretreatment of THP-1 cells, suggesting a transfer from EVs. Expression of TNF-α, a short-lived transcript, is reduced in actinomycin D–treated cells. n = 3.