BMPER attenuates leukocyte adhesion in vitro and in vivo as a consequence of increased eNOS levels and decreased ICAM-1 and VCAM-1 expression. (A) HUVECs were exposed to recombinant BMPER and cell lysates were used for Western blotting with a human anti-eNOS antibody. β-tubulin served as a loading control. (B) BMPER enhances eNOS activity by phosphorylation at the serin 1177. HUVECs were exposed to indicated BMPER concentrations for 30 minutes and cell lysates were used for Western blotting. (C) BMPER accelerates the recovery of eNOS levels in cells exposed to TNFα. HUVECs were treated with TNFα (2 ng/mL) for 12 hours and afterward cells were exposed to cell culture medium in the presence of different BMPER concentrations. (D) BMPER inhibits TNFα induced ICAM-1 and VCAM-1 expression in HUVECs demonstrated by immuncytochemistry (green fluorescence). HUVECs were stimulated for 12 hours with TNFα (2 ng/mL) alone or in combination with human recombinant BMPER (500 ng/mL). DAPI (blue) was used for nuclear staining. (D) BMPER inhibits TNFα induced VCAM-1 protein quantified by Western blotting with anti–human VCAM-1 antibody (*P < .05 versus control). HUVECs were stimulated with TNFα (2 ng/mL) alone or in combination with human recombinant BMPER (doses as indicated). Cell lysates were used for Western blotting and β-tubulin served as loading control. (E) BMPER inhibits leukocyte adhesion to endothelial cells under dynamic flow. HUVECs were incubated with TNFα (2 ng/mL) alone or in combination with BMPER for 12 hours (doses as indicated). PMA-activated PBMCs attached on the endothelial surface (*P < .05 versus control; #P < .05 versus TNFα). (F) BMPER prevents TNFα-mediated leukocyte adhesion in C57BL/6 mice. Four hours before intravital microscopy TNFα (10 ng/g bw IP, n = 5) was injected alone or in combination with recombinant BMPER (0.33 μg/g bw, retro-orbital, n = 6). BMPER attenuates TNFα-induced leukocyte adhesion, confirming the potent anti-inflammatory effects of BMPER (*P < .05 versus TNFα).