Mechanisms Of THP-1-derived Foam Cells Formation Induced By Visfatin

Background Adipokines have also been found to be expressed within atherosclerotic plaques, suggesting local and endocrine effects of these mediators on atherosclerotic lesions. One of the most recently identified adipokines is visfatin, originally identified as pre-B-cell colony-enhancing factor-1 and nicotinamide phosphoribosyltransferase. Visfatin has been found that it is an adipose-derived hormone proposed to exert insulin-mimicking effects and play a positive role in attenuating macrophage foam cell formation and atherosclerosis. Recently it has been found that visfatin expression is increased in unstable coronary plaques and carotid lesions in humans. In macrophages, LOX-1, ABCA1 and ABCG1 are involved in cholesterol metabolism, which play an important part in the formation of foam cells. However, the mechanisms underlying effects of visfatin on macrophage foam cell formation and atherosclerosis remain unknown.Objective To determine whether visfatin modulate cholesterol metabolism related gene and protein (LOX-1, ABCA1 and ABCG1) by accelerating human monocytic cell line (THP-1)-derived foam cell formation and to further evaluate its mechanism of atherosclerosis in vitro. Methods The human monocytic cell line THP-1 was strongly induced to foam cell conversion of these macrophages in RPMI-1640 with 160nmol/L phorbol myristate acetate (PMA) for 48h and further incubated in serum-free medium with recombinant human visfatin in a dose-dependent and time-dependent manner. THP-1-derived macrophages were randomly divided into several groups:(1) dose-dependent effects of visfatin,①control group:only with 100mg/L LDL for 24h,②ox-LDL group:only with 100mg/L ox-LDL for 24h,③intervention groups at different dose:THP-1-derived macrophages were treated with increasing concentrations of (1×10-7mol/L,1×10-6mol/L,2×10-6mol/L, 1×10-5mol/L) visfatin containing 100mg/L LDL for 24h. (2) time-dependent effects of visfatin,①control group:without LDL and visfatin,②intervention groups at different time: THP-1-derived macrophages were stimulated with the same concentration (2×10-6 mol/L) of visfatin containing 100mg/L LDL from 12h to 36h. Different group was stained with Oil red O to visualize lipid droplet accumulation. Macrophages were incubated with tetramethyl rhodamine isothiocyanate (TRITC)-labeled ABCA1 or fiuorescein isothiocyanate (FITC)-labeled ABCG1 to observe their changes in fluorescence microscopy. Reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting were performed to determine the expressions of LOX-1, ABCA1 and ABCG1 mRNA and protein.Results Higher concentrations of visfatin for 24h resulted in the large accumulation of lipid droplets in THP-1-derived macrophages when co-cultured with LDL. Intracellular red O-positive droplets accumulated in the cytosol with time increasing. The cytoplasm of cells with ABCA1 polyclonal antibody labeled with TRITC and ABCG1 polyclonal antibody labeled with FITC exhibited weak fluorescence of red and green with visfatin in a dose-dependent manner. TRITC-positive of red and green FITC-positive of cells was clearly observed in a time-dependent manner. Visfatin was demonstrated to enhance macrophage foam cell formation by up-regulating the expression of LOX-1 mRNA and protein in THP-1 macrophages pre-treated with LDL(P<0.05). The results also demonstrated that visfatin activated foam cell formation via simultaneously down-regulating ABCA1 and ABCGl in a dose concentration (P<0.05). We found that these results suggested that visfatin promote lipid accumulation in human macrophages through its up-regulating LOX-1 expression in a dose-dependent manner (P<0.05). Compared with control group, we found an increase in the expressions of ABCA1 and ABCG1 with visfatin from macrophages-derived foam cells of different time group (P<0.05).Conclusion Our study implys visfatin caused significant changes of several key cholesterol-related genes in THP-1 cells. Thus, we could verify that the increased levels of LOX-1 and decreased levels of ABCAl and ABCG1 by visfatin in macrophages actually correlated with foam cells formation. Background Peroxisome proliferator-activated receptor y(PPARy) which is blocked by GW9662 is the key nuclear hormone receptor in the lipid metabolism of macrophages.In previous study, we examined macrophages play a central role in the pathogenesis of atherosclerosis by accumulating cholesterol down-regulating PPARy, leading to foam cell formation.Objective To explore the role of PPARy agonist in the regulation of THP-1 derived foam cells of LOX-1, ABCA1 and ABCG1 by stimulated visfatin in vitro.Methods We observed that monocytes were transformed into macrophages with PMA in the serum-free RPMI 1640 medium. Macrophages cultured in the presence or absence of 2×10-6mol/L recombinant human visfatin after exposing the cells to PPARy antagonist for an additional 24h. Effect of PPARy blocker with increasing concentrations,①visfatin group, only with 100mg/L LDL for 24h,②intervention groups of GW9662 at different dose:macrophages were incubated with or without PPARy inhibitor (GW9662) in a dose-dependent manner(1μM,5μM,10μM,20μM), then added to 2×10-6mol/L recombinant human visfatin and 100mg/L LDL for 24h,③LDL group:only with 100mg/L LDL for 24h. Differentiated THP-1 cells staining with Oil red O to visualize lipid accumulation. The expressions of LOX-1, ABCA1 and ABCG1 at mRNA and protein levels were measured by RT-PCR and Western blotting, respectively.Results The current studies showed staining positive lipids with Oil red O were increased when co-cultured with GW9662. Administration of GW9662, these results provided evidence that visfatin up-regulated expressions of LOX-1, and down-regulated expressions of ABCA1/ABCG1 through PPARy pathway in THP-1 macrophage-derived foam cells (P<0.05).Conclusion In conclusion, we observed that PPARy antagonist GW9662 significantly induced the effects of eccessive lipid accmulation in foam cell formation, suggesting a PPARy-independent mechanism is critical to maintaining lipid homeostasis in the macrophage. These findings thus demonstrated effects of PPARγantagonist to activate expression of LOX-1 and suppress expression of ABCA1 and ABCGl in the formation of foam cells induced by visfatin.Background Mitogen-activated protein kinase pathway(MAPK) plays important roles in the progression of atherosclerosis by regulating cholesrerol metabolism. In previous studies shows that visfatin exerted insulin-mimetic effects. Insulin is closely associated with the formation of THP-1-derived foam cells, it can activate the MAPK pathway to play anti-atherosclerosis effect.Objective To investigate the effect of visfatin on expressions of LOX-1, ABCA1 and ABCG1 of MAPK pathways and explore the blocking roles of mitogen-activated protein kinase(SB203528), extracellular regulated protein kinase(PD98059); c-Jun N-terminal Kinase (SP600125) in the regulation of THP-1 macrophage-derived foam cells in vitro. We investigated whether inhibition of MAPK pathway could attenuate THP-1 macrophage-derived foam cells stimulated by visfatin.Methods After culturing in the presence of PMA, THP-1 monocytes differentiated to macrophages. THP-1-derived macrophages cultured with increasing concentrations of SB203528, PD98059 and SP600125, then with or without 2×10-6mol/L recombinant human visfatin were added for 24h. THP-1-derived macrophages were allocated into three groups with MAPK antagonists:(1)effects of p38 MAPK antagonist with increasing concentrations.①visfatin group:only with 100mg/L LDL for 24h,②intervention groups of SB203528 at different dose:Macrophage-derived foam cells were treated with (1μM,5μM,10μM,20μM) SB203528, an inhibitor of p38 MAPK, followed by the addition of 2×10-6mol/L visfatin and 100mg/L LDL for 24h to initiate cholesterol efflux.③LDL group:only with 100mg/L LDL for 24h. (2) effects of ERK1/2 antagonist with increasing concentrations,①visfatin group:macrophages were incubated only with 100mg/L LDL for 48h after propagated with recombinant human visfatin,②intervention groups of PD98059 at different dose: macrophage-derived foam cells were treated with (5μM, 10μM,20μM,50μM) PD98059, an inhibitor of ERK1/2, followed by the addition of 2×10-6mol/L visfatin and 100mg/L LDL for 24h.③LDL group:only with 100mg/L LDL for 24h. (3) effects of JNK antagonist with increasing concentrations,①visfatin group:only with 100mg/L LDL for 24h,②intervention groups of JNK at different dose:macrophage-derived foam cells were treated with (5μM, 10μM,20μM,50μM) SP600125, an inhibitor of JNK, followed by the addition of 2×10-6mol/L visfatin and 100mg/L LDL for 24h.③LDL group:only with 100mg/L LDL for 24h. Gene and protein expressions of LOX-1, ABCA1 and ABCG1 were determined using RT-PCR and Western blotting.Results Lipid droplets in LDL-treated macrophages induced by visfatin were reduced in the presence of increasing doses of SP600125 and PD98059. The massive accumulation of lipids was not changed in macrophages in the presence of increasing doses of SB203528. Administration of increasing SP600125, which decreased LOX-1 and increased expression of both ABCA1 and ABCG1 in vitro (P<0.05). Macrophage sterol efflux of LOX-1, ABCA1 and ABCG1 was not mostly blocked by SB203528 in a dose-dependent manner (P<0.05).Conclusion In summary, the present study demonstrated that visfatin mediated ERK1/2 and JNK effects by key receptors reducing cholesterol accumulation and increasing cholesterol efflux. Induction of LOX-1 expression by visfatin was abolished by ERK1/2 and JNK inhibitors. Although visfatin markedly upregulated ABCA1 and ABCG1 expression in THP-Ⅰcells by JNK antagonist, it had no effect on ABCA1 and ABCG1 expression by p38 MAPK and ERK1/2 inhibitors. These effects of visfatin were abrogated by MAPK antagonists, which contribute to novel therapeutics that target atherosclerosis at the level of cholesterol.