Heme Oxygenase-1 Expression And Function In Macrophage-derived Foam Cells

During both early and late atherosclerotic lesions, macrophage-derived foam cells, accumulating in the form of atherosclerotic plaques, are prodigious secretary cells that can produce a number of cytokines with the potential to either initiate or dampen immune responses. While there is ample evidence that pro-inflammatory cytokines and growth factors secreted from foam cells play a role in atherogenesis, information regarding the potential role of anti- inflammatory factors is limited.Heme oxygenase (HO) is a rate-limiting enzyme in heme catabolism, leading to the generation of biliverdin, free iron and carbon monoxide (CO). Three mammalian HO isoforms have been identified, one of which, HO-1, is a stress-responsive protein induced by various oxidative agents. Previous studies also showed that HO-1 is expressed in apoliprotein (Apo) E-deficient mice, acting in an anti-inflammatory and anti-apoptotic manner. Recent studies also focused on the anti-inflammatory role of CO derived from HO-1 in the mechanism of the therapeutic effects on atherosclerosis.Hemin is a strong inducer of HO-1 expression in a variety of cell types in vitro and in vivo. In this study we examined the regulatory effects of Hemin on inflammatory reactions in human monocyte line, U937 cells, associated with the increase of HO-1 levels, by using hemin (HO-1 activator) and zinc protoporphyrin IX (ZnPP IX, HO-1 inhibitor) to determine the effect of HO-1 on the regulation of cytokine expressions including pro-inflammatory cytokines, IL-1β, TNF-α, as well as anti-inflammatory cytokine IL-10.The following results were gained in this study:1. HO-1 and cytokine expressions induced by oxLDL in U937 foam cellsOur previous study has showed that macrophage-derived foam cells can be obtained in vitro from U937 cells after incubated with oxLDL. The U937 foam cells were identified after treated with 0.3% Oil red O, in which many red pellets were found in the plasma of the U937 cells.Cytokine levels of U937 foam cells were both determined by ELISA kits. Protein levels of IL-ip, TNF-a, IL-10 protein expressions in foam cells were significant increased in a dose dependent manner compared with control U937 cells. Especially, at the dose of oxLDL 100 mg/L, IL-ip, TNF-a, and IL-10 protein level increased to 41-fold, 25-fold, 19-fold of control U937 cells separately.It has been showed that HO-1 can be up regulated by oxLDL in macrophages. In present study on U937 foam cells, associated with cytokines up-regulation, HO-1 protein expressions were enhanced by oxLDL in a dose dependent manner. Compared to the control U937 cells, HO-1 level was increased to be 1.9-fold of control induced by oxLDL at the dose of 100 mg/L.2. Effect of HO-1 activator Hemin on secretions of cytokines in U937 foam cellsHO-1 activator Hemin was added to U937 foam cells in order to evaluate the role of HO-1 in macrophage-derived foam cell functions. It has been reported that IL-ip and TNF-a secreted from foam cells play central roles in inflammatory reactions in atherosclerosis. Therefore, we firstly determined the effects of hemin on the pro-inflammatory cytokines IL-ip and TNF-a levels. ELISA results indicated significantly inhibitory effects of Hemin from dose O.lumol/L to lOumol/L on pro-inflammatory cytokine expressions in U937 foam cells. At the dose of Hemin 10u.mol/L, IL-ip level decreased 73% and TNF-a level decreased 75% compared to U937 foam cells.In addition, we further investigated the effect of Hemin on anti-inflammatory cytokine IL-10 levels. The supernatant was measured to determine IL-10 secretion in U937 foam cells by ELISA. The production of anti-inflammatory cytokine IL-10 was increased significantly by Hemin at the dose from lumol/L to lOumol/L in U937 foam cells. In sharp contrast, IL-10 production was enhanced by 2.9-fold of foam cells with addition of Hemin 10 umol/L. These data confirmed that HO-1 activator, Hemin, could enhance IL-10 levels in U937 foam cells.3. Effect of HO-1 inhibitor ZnPPIX on secretions of cytokines in U937 foam cellsIn contrast with the modulation effects of HO-1 activator Hemin on secretions of cytokines in U937 foam cells, the role of HO-1 inhibitor ZnPPIX in cytokine productions were analyzed. Withthe addition of ZnPPIX, neither pro-inflammatory cytokines IL-ip and TNF-a nor anti-inflammatory cytokine IL-10 were changed significantly compared with U937 foam cell group, although HO-1 was significantly inhibited by ZnPPIX. These data indicated that inhibition of HO-1 did not affect the inflammatory reactions in U937 foam cells.4. HO-1 was up-regulated in AS plaques in human aortaFor an in vivo study, we choose 3 human aorta samples for determination in human AS plaques. From HO-1 expression patterns determined by fluorescence-immunohistochemistry, we found significant up-regulation of HO-1 in human AS plaques, which are accumulated in foam cells. The Western Blot results also confirmed the enhanced HO-1 expression in AS plaques compared with normal human aorta tissues.Our results of the present study confirmed the anti-inflammatory roles of HO-1 induction in U937 foam cells through inhibiting pro-inflammatory cytokines IL-ip and TNF-a and enhancing anti-inflammatory cytokine IL-10. Therefore, our present results suggest a new approach by activating HO-1 for altering inflammatory responses.