ECs secrete extracellular vesicles (EC-EVs) that inhibit monocyte activation and modulate polarization. (A) Nanoparticle analysis of EVs isolated from HUVEC media by ultracentrifugation. A representative experiment of 3 is shown. The mode particle size is 142 ± 2.6 nm. (B) Western blotting of the exosomal marker, CD63, in lysates from ECs and isolated EC-EVs (2 independent preparations). (C) EC-EVs (10 μg/mL) isolated from HUVECs suppress the activation of THP-1 monocytic cells by LPS (as assessed by qRT-PCR of proinflammatory genes) and promote their polarization (as assessed by qRT-PCR of immunomodulatory markers); n = 5. (D) HUVEC-derived EC-EVs (10 μg/mL) polarize the response of primary monocytes to LPS treatment. ND, not detected. n = 5. (E) Protein levels of IL-12p40 in the medium of EC-EV–treated THP-1 cells and primary monocytes, as measured by ELISA. Cells were either unstimulated or treated with LPS for 8 hours. n = 5. (F) EC-EVs isolated from human CAECs polarize monocyte activation in response to LPS treatment. n = 3.