HO-1 regulates human and murine DAF expression on vascular ECs. HUVECs were treated for 48 hours with (A) VEGF (25 ng/mL), (B) TNF-α (10 ng/mL), and IFN-γ (500 U/mL) (T/I), in the presence or absence of ZnPPIX (15 μM) or with vehicle alone (UT). DAF expression was measured by flow cytometry using mAb 1H4 and presented as RFI (mean ± SEM), derived by dividing the mean fluorescent intensity (MFI) with test mAb by the MFI obtained with an isotype-matched irrelevant mAb (n = 3 experiments). (C) HUVECs were transfected with a DAF promoter luciferase reporter construct pGL3-DAF or pGL3-basic before treatment with hemin (0.2 μM) and analysis of luciferase activity. Data are expressed as relative to untreated ECs. (D) Murine cardiac ECs isolated from Hmox1^−/− and Hmox1^+/+ mice were lysed, transblotted to polyvinylidene difluoride membranes, and immunoblotted with rat anti–mouse DAF mAb MD1. Equal loading was confirmed by reprobing with anti–α-tubulin. (E) Aortic ECs from Hmox1^−/− and Hmox1^+/+ mice were lysed and immunoblotted for expression of HO-1 and HO-2. (F) Livers from Hmox1^−/− and Hmox1^+/+ mice were homogenized, and expression of DAF was detected by immunoblotting using rat anti–mouse DAF mAbs MD1 and 3D5. Equal loading was confirmed by reprobing with antiactin. In panels D and F, relative levels of protein expression were quantified using image analysis and expressed as the DAF/control Ab ratio (n = 4 experiments). (G) HUVECs were left untreated (UT) or transfected with control siRNA (Ctrl) or validated HO-1–specific siRNA. DAF mRNA levels were quantified by real-time PCR after 48 hours. *P < .05; **P < .01.