Mechanism of HO-1^hiinduction by heme-damaged HUVECs in PMos. (A) Following culturing of purified monocytes from HDs (n = 4) with hemin-pretreated HUVECs (20 µM) for 4 hours, frequency of HO-1^hi in nonadherent vs adherent monocytes was analyzed. (B) Frequencies of HO-1^hi–expressing PMos from HDs (n = 5) in the transwell culture system (blue bars) in which HUVECs exposed to hemin (20 μM) or not (“medium”) were placed in the bottom well and separated from purified monocytes on the top well. At the same time, as a control (“Control”), monocytes were also cocultured directly with HUVECs exposed to hemin (20 µM) or not (“medium”). Hemin (20 µM) exposed HUVEC was preincubated with annexin V or blocking antibodies anti-ICAM-1, VCAM-1, or isotype control for 30 minutes before addition of purified monocyte from (C) HDs (n = 5) and (D) SCD patients (n = 8). Frequencies of HO-1^hi–expressing PMos were then analyzed. Data represent mean ± SEM; means were compared using 2-tailed paired Student t test. *P < .05; **P < .01; ***P < .001. Ab, antibody.