Assessment of LPS contamination in MPs. (A) HUVECs were incubated in serum-free conditions with the indicated amounts of MPs from LPS-treated THP-1 cells, LPS, or 10 ng/mL of TNF-α. Expression of VCAM-1 was detected by Western blot. (B) HUVECs were incubated with increasing concentrations of polymyxin B and then stimulated in serum-free conditions with MPs from LPS-treated THP-1 cells or in serum-containing media with LPS. VCAM-1 was detected by Western blot. (C) HUVECs were pretreated with the Toll like receptor-4 inhibitor TAK-242 or vehicle control (DMSO) and then stimulated with either 100 μg/mL of MPs derived from LPS-treated THP-1 cells in serum-free conditions or with 10 μg/mL of LPS in serum-containing media. HUVECs incubated with 10 ng/mL of TNF-α or left untreated served as controls. VCAM-1 expression in HUVECs was detected by Western blot. GAPDH served as a loading control. (D) Densitometry of Western blots shown in panel C. Data are mean ± SD; n = 4. *P < .01 (2-way ANOVA post-test).