Effect And Mechanism Of Advanced Oxidation Protein Products On ABCA1Expression And Cholesterol Efflux In Macrophages

Atherosclerotic cardiovascular disease is serious harm to human health. There isa lot of pathogenesis which is involved in abnormal lipid metabolism, inflammation,oxidative stress. A major event in the progression of atherosclerosis is thedifferentiation of monocytes to macrophages that accumulate lipoprotein-derivedcholesterol to form foam cells in the vessel wall. It has been demonstrated that theATP binding cassette transporter A1(ABCA1), which is an integrated membraneprotein, plays key regulatory roles in cellular cholesterol metabolism. ABCA1isinvolved in regulating cellular cholesterol homeostasis and mediates the efflux ofcellular cholesterol and phospholipids to an extracellular acceptor apolipoprotein A-I(apoA-I). Some studies have shown that ABCA1plays an important role in highdensity density lipoprotein (HDL) synthesis and reverse cholesterol transport (RCT).Also, ABCA1is a potential target to prevent atherosclerotic cardiovascular disease.Advanced oxidation protein products (AOPPs) is formed by reaction of plasmaproteins with chlorinated oxidants, and have been considered as novel markers ofoxidant-mediated protein damage. Both clinical data and basic science studies suggestthat AOPPs may contribute to the progression of atherosclerosis. AOPPs are highlycorrelated to atherosclerotic cardiovascular events, and increase level of AOPPs weredescribed as an independent risk factor for coronary artery disease. Although thesereports support a close relation between AOPPs and atherosclerosis, the molecularbasis of atherogenic effect of AOPPs remains largely unknown. Some studiesdemonstrate that AOPPs significantly increases athersclerosis lesion in animal models,and promotos oxidized low-density lipoprotein (ox-LDL) accumulation, macrophageinfiltration and smooth muscle cell proliferation in atherosclerotic plaques. These studies show that AOPPs is not just a marker of oxidation stress and may be related tolipid metabolism.The Janus kinase/signal transducers and activators of transcription (JAK/STAT)pathway is one of an important cellular signaling pathway. The JAK/STAT signalingpathway is considered a stress responsive signaling cascade that transduces signalsfrom cell surface receptors to the nucleus, thereby modulating gene expression andcompensatory mechanisms. So far, various vascular stress factors are describedlinking activation of the JAK/STAT signaling pathway to vascular diseases. TheJAK/STAT signaling pathway is also a key regulation pathway for atheroscleroticvascular damage, and associated with LXRα, ABCA1expression and cellularcholesterol efflux. Our previous data show that IFN-γ may decrease expression ofABCA1through the JAK/STAT1signaling pathway in THP-1macrophage-derivedfoam cells.This study is focus on ABCA1, which is a key factor of cholesterol-homostasis-regulation, and ABCG1-mediated cholesterol efflux and investigates the possible roleand mechanism of AOPPs in atherosclerosis. Using of agonist, antagonist or siRNA toinvestigate effect of AOPPs and signaling transduction mechansim on ABCA1expression and function. It will provide new experimental evidence for exploring therole of AOPPs in atherosclerosis and pathogenesis of atherosclerosis.Part I: Effect of Advanced Oxidation Protein Products onCholesterol Efflux and ABCA1Expressionin THP-1MacrophageAims: In order to observe effect of AOPPs on cholesterol efflux in in THP-1macrophage-derived foam cells and detect the change of ABCA1expression.Methods: Human THP-1monocytes were preincubated with Phorbol-12-myristate-13-acetate (PMA) and oxidized low density lipoprotein (oxLDL) to form foam cells. Cells was treated by AOPPs and observed the concentration-dependent ortime-dependent effect. Cellular cholesterol content and lipid droplet were measuredby High Performance Liquid Chromatography (HPLC) and Oil Red O stain,respectively. The cholesterol efflux was assessed by liquid scintillation counting. Theprotein and mRNA expression were examined by western immunoblotting assays andreal-time quantitative PCR, respectively.Results: THP-1macrophage-derived foam cells were treated with AOPPs (0,50,100, or200μmol/L) for24hours and cholesterol efflux was reduced by liquidscintillation counting assays. Cellular cholesterol content and lipid droplet, which wasdeterminated by HPLC and Oil Red O stain respectively, were increased byAOPPs-treated. Also, AOPPs significantly decreased the expression of ATP-bindingmembrane cassette transporter A-1(ABCA1). This effect of AOPPs was appearedwith concentration-dependent manner. Furthermore, cells were incubated with100μmol/L AOPPs for0,12,24,48hours, respectively. Data was shown that cellularcholesterol efflux was reduced by AOPPs. The cellular cholesterol content and lipiddroplet was increased in AOPPs-treatment groups. ABCA1expression wasdownregulated after AOPPs treated cells for different time. The effect of AOPPs wasappeared with time-dependent manner.Conclusion:①AOPPs increases accumulation of cellular cholesterol, andinhibits cellular cholesterol efflux with concentration-dependent and time-dependentmanner in THP-1macrophage-derived foam cells.②The ABCA1expression isdownregulated by AOPPs with concentration-dependent and time-dependent manner.Part Ⅱ: Mechanism of ABCA1Expression inhibited byAdvanced Oxidation Protein Productsin THP-1MacrophageAims: To investigate the possible mechanism of ABCA1expression and cellularcholesterol efflux were inhibited by AOPPs in THP-1macrophage-derived foam cells. Methods: Human THP-1monocytes were preincubated with Phorbol-12-myristate-13-acetate (PMA) and oxidized low density lipoprotein (ox-LDL) to formfoam cells. Cells were treated with AOPPs and (or) intervention, such as agonist,antagonist or siRNA of LXRα and some key factors of JAK/STAT signaling pathway.Cellular cholesterol efflux was assessed by liquid scintillation counting. The proteinand mRNA expression were examined by western immunoblotting assays andreal-time quantitative PCR, respectively.Results: THP-1macrophage-derived foam cells were treated with LXRα agonist22(R)-Hch and LXRα siRNA respectively. As demonstrated, ABCA1expression wassignificantly up-regulated in cells treated by22(R)-Hch compared with BSA.Down-regulation of ABCA1expression by AOPPs was almost totally compensated byaddition of22(R)-Hch. At the same time, cellular cholesterol efflux in cells treated bythe combination of22(R)-Hch and AOPPs was siginificantly increased as comparedwith these treated by AOPPs alone. ABCA1and LXRα expression were down-regulated by treated with siRNA for LXRα. At the same time, cellular cholesterolefflux in cells treated by the combination of LXRα siRNA and AOPPs wassiginificantly decreased as compared with these treated by AOPPs alone. Tyrosine(Tyr701) phosphorylation of STAT1was detected15min after addition of AOPPs andit was further enhanced up to60minutes by Western immunoblotting analysis.Treatment with siRNA for STAT1up-regulated ABCA1mRNA and proteinexpression compared with these treated by AOPPs only. At the same time, cellularcholesterol efflux in cells treated by STAT1siRNA was siginificantly increased ascompared with these treated by AOPPs alone. JAK inhibitor AG-490increased theexpression of ABCA1mRNA and protein, and increased the cellular cholesterolefflux compared with only AOPPs-treated group. NADPH oxidase inhibitor DPIincreased the expression of ABCA1detected by real-time quantitative PCR andWestern immunoblotting analysis, compared with these treated by AOPPs alone.Cellular cholesterol efflux in cells treated by DPI was increased as compared withthese treated by AOPPs alone. Conclusion:①AOPPs inhibits the expression and function of ABCA1throughregulating LXRα expression in THP-1macrophage-derived foam cells.②AOPPsaffect the activation of JAK/STAT signaling pathway. Down-regulation of ABCA1expression induced by AOPPs was mediated by JAK/STAT signaling pathway.③NADPH oxidase are involved in AOPPs-induced activation of JAK/STAT signalingpathwayPart Ⅲ: Effects of Advanced Oxidation Protein Products onAtherosclerotic lesion and ABCA1Expressionin Apolipoprotein E Knockout MiceAims: To observe the effect of AOPPs on ABCA1expression, lipid accumulationand atherosclerosis lesion in apoE-KO mice. Furthermore, the mechanism of these isto preliminaryly study.Methods: Male8-week-old apoE-KO mice were randomly divided into forgroups (n=10). All of the mice were fed a high-fat/high-cholesterol diet (15%fatwt/wt,0.25%cholesterol wt/wt). Group1, the control group, only was fed withhigh-fat/high-cholesterol diet; group2received intraperitoneal injections of5mg/kgAOPPs once every other day; group3received intraperitoneal injections of5mg/kgAG-490once every other day; group4was received the same volume of AOPPs andAG-490once every other day. Control animals received intraperitoneal injections ofthe same dose of normal saline. After8weeks, plasma lipid was determined bycommercially enzymatic methods. The lipid accumulation and atherosclerosis lesionwere shown by HE and Oil Red O stain, respectively. The protein and mRNAexpression were examined by western immunoblotting assays, immunohistochemistryand real-time quantitative PCR, respectively.Results: Treatment of apoE-KO mice intraperitoneal injected with AOPPs led toincrease in plasma TC, TG, LDL-C and HDL-C level, while decreased in mice treatedwith AG-490, compared with control group. The plasma lipid level were decreased in mice treated with AOPPs and AG-490compared to the AOPPs group. The lipidcontent in the aortic sinus plaques and livers were more in mice treated with AOPPs,and were less in the AG-490group mice compared with control group mice.ApoE-KO mice at AOPPs-treatment group developed advanced lesions in the aorticsinus, compared with control group. The AG-490-treatment groups were shown with areduction of aortic atherosclerotic lesion area compared with control group. Inaddition, the lesions in the aortic were decreased in mice treated with AOPPs andAG-490compared to the AOPPs group. ABCA1, LXRα expression of aortic, liver andintestine was significantly downregulated in AOPPs group, while the expressionswere up-regulated in the AOPPs and AG-490group mice in compared to the AOPPsgroup.Conclusion:①AOPPs increase accumulation of lipid and exacerbateatherosclerosis in apoE-KO mice.②ABCA1, LXRα expression of aortic, liver andintestine was significantly downregulated by AOPPs.③JAK inhibitor AG490mayinhibit regulation of AOPPs-induced ABCA1.