Effect And Mechanism Of Apolipoprotein A-I On Macrophages Inflammatory Response Via An ABCA1-dependent Manner

Atherosclerosis-related cardiovascular disease is one of the leading causes ofdeath in china. Thus, the prevention and treatment of atherosclerosis, which is usuallythought to be a complex pathological process, have become an important topic inmedical study areas.With advances in our understanding of the molecular mechanismsof atherosclerosis, inflammation is regarded as the main pathogenetic pathway of bothearly atherogenesis and advanced plaque rupture. Apolipoprotein A-Ⅰ (apoA-Ⅰ), themajor component of the high density lipoproteins (HDL), promotes cellularcholesterol efflux and participates in the reverse cholesterol transport (RCT), thuspreventing atherosclerosis. Recently, apoA-Ⅰ has been described to have variousantiinflammatory properties, which may provide new insights into the prevention ofatherosclerosis.ATP binding cassette transporter A1(ABCA1), an integrated membrane protein,mediates the transport of cholesterol across cellular membranes to lipid-poor apoA-Ⅰvia hydrolysis of ATP. Knockout of ABCA1in mice has been found to have increasedinflammatory cells infiltration in atherosclerosis plaque. In addition, knockout orknockdown of ABCA1abolished or decreased the antiinflammation effect of HDLand apoA-Ⅰ, suggesting ABCA1plays a key role in modulating inflammatory responseby HDL/apoA-Ⅰ. Previous studies have shown that cross-linking of apoA-Ⅰ to ABCA1was saturable and occurred at high affinity. After binding to ABCA1, apoA-Ⅰ rapidlyactivates a cascade of cellular signaling events such as janus kinase2(JAK2), cyclicadenosine monophosphate (cAMP) and Rho GTPase CDC42. Receptor-mediatedactivation of JAK2usually stimulates signal transducers and activators oftranscription3(STAT3), which plays a crucial role in suppressing inflammatory cytokines production in macrophages.For example, overexpression of STAT3or IL-10,an antiinflammation cytokine, have been found to suppress macrophage inflammationthrough JAK2/STAT3signaling pathway.Previous studies have shown that the STAT3docking sites in cytosolic domainsof plasma membrane receptors have a highly conserved YXXQ sequence. We foundthere are two motifs in the large cytosolic loops of ABCA1: at amino acids924-927(YEGQ) in NBD1and at residues1990-1994(YCPQ) in NBD2, suggesting theinteraction of apoA-Ⅰ and ABCA1may rapidly stimulate the JAK2/STAT3signalingpathway. Therefore, in this study, we firstly observed the effects of HDL as well as itsmajor components on LPS-stimulated macrophage inflammatory response, and theninvestigated the possible role and mechanism of apoA-Ⅰ and ABCA1in this process.In addtion, we observed the effect of apoA-Ⅰ on LPS-induced inflammatory responseand the development of atherosclerotic lesions in vivo. It will provide not only a novelmechanism for the apoA-Ⅰ-mediated inflammation suppression in macrophages butalso provide new insights for developing strategies for modulating vascularinflammation and atherosclerosis.Part Ⅰ: Effect and Mechanism of Apolipoprotein A-Ⅰ on LPSinduced Macrophage Inflammatory ResponseAims: To investigate the effect of apoA-Ⅰ and different components of HDL onLPS-induced macrophage inflammatory response and reveal the regulating manner ofapoA-Ⅰ on macrophage inflammatory response.Methods: Human THP-1monocytes were treated with phorbol12-myristate13-acetate (PMA)(160nmol/liter) for12h, and then the medium was replaced byserum-free medium containing oxLDL(50μg/ml) to become fully differentiatedmacrophage foam cells before their use in experiments. Cells was pretreated by nativeHDL(30μg/ml) and its major constituents, including apoA-Ⅰ(10μg/ml), apoB(10μg/ml), phosphatidylcholine(PC)(25μg/ml), or cholesterol (50ng/ml) for3h, and stimulated with LPS (10ng/ml) for6h. Protein was extracted and subjected to ELISAor Western-blotting analysis for tumor necrosis factor-α(TNF-α), interleukin-1β(IL-1β), IL-6, monocyte chemotactic factor-1(MCP-1), interferon-γ(IFN-γ) andcyclooxygenase-2(COX-2). Human primary macrophages were treated with LPS for6h with or without pretreatment of apoA-Ⅰ for3h, and TNF-αlevels were determinedby Western blots. TNF-αpromoter fragment fused upstream to a chloramphenicolacetyltransferase(CAT) reporter gene was constructed and transfected to THP-1macrophages, which was used to observed the effect of apoA-Ⅰ on the transcriptionalactivity of TNF-α promoter with ELISA. THP-1macrophage-derived foam cellswere treated with LPS alone, or with apoA-Ⅰ for3h, the phosphorylation andconcentration of IkBαand nuclear p65NF-kB were measured by Western-blottinganalyses.Cells were treated with different conditions for3h followed by treatment ofact D (5μg/ml) to stop the transcription. After the indicated time points(0,30,60,120min), RNA was extracted and subjected to real-time quantitative PCR forinflammatory cytokines mRNA analysis.Results: native HDL and apoA-Ⅰ significantly suppressed the LPS-inducedupregulation of TNF-α, IL-1β, IL-6, IFN-γ, and MCP-1, but it had no obviouseffects on COX-2expression. Other components of the HDL including apoB, PC, andCHO had no obvious effects on the LPS-induced upregulation of these inflammatorycytokines. ApoA-Ⅰ did not significantly influenced the transcriptional activity of TNF-α promoter induced by LPS. The LPS-induced expression of phosphorylated-IkBαand nuclear p65NF-kB were also not influenced by apoA-Ⅰ. The results of RT-PCRindicated that apoA-Ⅰ reduces the mRNA levels of TNF-α, IL-6, IL-1β, and MCP-1at the different time points after treatment of act D, suggesting apoA-Ⅰ increased TNF-α, IL-6, IL-1β, and MCP-1mRNAdegradation.Conclusion:①apoA-Ⅰ significantly suppress the macrophage-derived foam cellsinflammatory response induced by LPS.②apoA-Ⅰ suppresses the expression of someinflammatory cytokines induced by LPS via a specific post-transcriptional regulationprocess, namely mRNA destabilization, in macrophage-derived foam cells. Part Ⅱ: Molecular Mechanism of Apolipoprotein A-Ⅰ OnLPS-Induced Macrophage Inflammatory ResponseAims: To investigate the molecular mechanism of apoA-Ⅰ on LPS-inducedinflammatory response in THP-1macrophage-derived foam cells.Methods: Human THP-1macrophage-derived foam cells and human primarymacrophages were cultured and treated with apoA-Ⅰ and (or) LPS as described above.Total protein extracts from cells were subjected to Western-blotting analyses fortristetraprolin(TTP), Human antigen R(HuR), phosphor-JAK2and phosphor-STATs.Cells were treated with JAK2antagonist AG490, siRNA of ABCA1,TTP and STAT3.Protein and RNA were extracted and subjected to ELISA or Western-blotting orRT-PCR analysis for inflammatory cytokines. To directly address the apoA-Ⅰ effect onTTP binding to3`-UTR AREs of TNF-α, the RNA-protein complex formation wasperformed in EMSA experiments. RNA-protein complex contents were calculated bydensitometry using Labworks analysis software.Results: apoA-Ⅰ treatment substantially increased the expression of TTP, but hadno effect on HuR expression in LPS-stimulated THP-1macrophage-derived foamcells and human primary macrophages.Treatment with siRNA for TTP significantlydown-regulated apoA-Ⅰ-induced TTP protein expression in LPS-treated THP-1cells,which further abolished the inhibition effect of apoA-Ⅰ on inflammatory cytokinesexpression induced by LPS. RT-PCR results showed that the incomplete apoA-Ⅰ-mediated inhibition of cytokine production in TTP siRNA cells resulted fromdecreased mRNA decay of cytokines. RNA-EMSA results showed that proteinsextracted from THP-1cells treated by apoA-Ⅰ promoted the formation of RNA-proteincomplex. The RNA-protein bands were further retarded (supershifted) after theprotein extracts were preincubated with different levels of monoclonal TTP antibody,indicating that TTP forms a complex with the sequence of TNF-α3`-untranslatedregion (3`-UTR) AU-rich elements (AREs). In THP-1macrophages, apoA-Ⅰ rapidlyphosphorylated JAK2and STAT3, and increased the expression of STAT3in the nucleus, but had no effect on phosphorylation of STAT1and STAT6. AG-490, a JAK2inhibitor, and siRNA for STAT3significantly inhibited the nuclear translocation ofSTAT3as well as the expression of TTP. The ability of apoA-Ⅰ to stimulatephosphorylation of STAT3and TTP expression were also significantly impaired inABCA1siRNA-treated THP-1macrophage derived foam cells. The rate ofapoA1-induced TNF-α mRNA decay in ABCA1siRNA cells was also markedlydecreased.Conclusion:①apoA-Ⅰ increased expression of TTP through a JAK2/STAT3signaling pathway-dependent manner in LPS-stimulated human macrophage derivedfoam cells.②apoA-Ⅰ increased some inflammatory cytokines mRNA decay viapromoting the expression of TTP in LPS-stimulated THP-1macrophage derived foamcells.Part Ⅲ: Effects and Mechanisms of Apolipoprotein A-Ⅰ onAtherosclerotic Lesion and Plaque Inflammation in LPS-Challenged Apolipoprotein E Knockout MiceAims: To observe the effect and mechanism of apoA-Ⅰ on atherosclerotic lesion,inflammatory cells accumulation and inflammatory cytokines expression in LPSchallenged apoE-KO mice.Methods: Male20-week-old apoE-KO mice were randomly divided into fourgroups (n=20): control group, LPS group, LPS+apoA-Ⅰ group and LPS+GFP group.All of the mice were fed a chow diet. The control group were subcutaneouslyadministered with PBS; The LPS group were subcutaneously administered withLPS(25μ g/mice); The LPS+apoA-Ⅰ and LPS+GFP groups were injectedintravenously with rAAV2-apoA-Ⅰ or rAAV2-GFP(1×1011v.p./mice) besides LPStreatment.After4weeks, animals were killed for detection of human apoA-Ⅰ mRNAand protein expression in liver using RT-PCR and Western-blotting. Blood wascollected for the detection of plasma apoA-Ⅰ, lipid, and inflammatory cytokines expression via immunoturbidimetry, commercially enzymatic methods and ELISA,respectively. Quantification of lesions in aortic sinus was tested by HE stain. Lipidaccumulation in aorta and aortic sinus were evaluated by Oil Red O stain. Theexpression of CD68, a marker for macrophage, in lesions was tested byimmunohistochemistry. The mRNA and (or) protein expression of ABCA1, TTP,p-JAK2and p-STAT3in aorta and abdominal cavity macrophage were detected byRT-PCR and Western-blotting, respectively.Results: After intravenous administration of rAAV2-apoA-Ⅰ-GFP for four weeks,the GFP was detected in liver of mice by fluorescence microscope. Human apoA-ⅠmRNA and protein were detected in livers by RT-PCR and Western-blotting.Immunoturbidimetry results showed that plasma human apoA-Ⅰ level was increased inrAAV2-apoA-Ⅰ-GFP group, but not in rAAV2-GFP group. Although the total serumcholesterol except for HDL-cholesterol have not obviously changed in LPS treatmentcompared with PBS, the levels of TNF-α, IL-1β and MCP-1have significantlyincreased in LPS treatment group. The areas, lipid content and the amount ofinflammatory cells in the aortic sinus plaques were increased in mice treated with LPS.The levels of HDL-cholesterol and total cholesterol were increased in apoE-KO miceinjected with rAAV2-apoA-Ⅰ-GFP, which, however, developed less advanced lesionsin the aortic sinus, including decreased lipid content, reduced macrophagesaccumulation and decreased inflammatory cytokines levels in plasma and aorta.According to the results of immunohistochemistry, RT-PCR and Western-blotting, theamount of CD68+cells was decreased, however, the expression of ABCA1, p-JAK2,p-STAT3and TTP were increased in aortic sinus plaques and abdominal cavitymacrophage in rAAV2-apoA-Ⅰ-GFP group but not in rAAV2-GFP group.Conclusion:①Overexpression of human apoA-Ⅰ inhibits the progress ofatherosclerosis in LPS-challenged apoE-KO mice.②ApoA-Ⅰ decreased the plasmainflammatory cytokines levels and the accumulation of macrophages in plaques ofLPS-challenged apoE-KO mice.③ApoA-Ⅰ up-regulates the expression of TTP andinflammatory cytokines mRNA decay via activating the JAK2/STAT3pathway andupregulating the expression of ABCA1.