| BackgroundMacrophages are the most prominent cell type in atherosclerotic lesions and are associated with two hallmarks of the disease, lipid deposition and inflammation. Recruitment of circulating monocytes to the subendothelial space is one of the earliest events in atherogenesis. Monocytes differentiate into macrophages, which subsequently internalize large amount of atherogenic, cholesterol-rich lipoprotein particles. In the early stages of atherosclerosis, most of the internalized cholesterol is stored as cholesteryl esters, resulting in "foam cell" formation. As a lesion becomes more advanced, however, there is a progressive decrease in the cholesteryl ester content and a reciprocal increase in the unesterified, or "free" cholesterol (FC) content. FC accumulation is a potent inducer of macrophage apoptosis and secondary necrosis, which is thought to contribute to necrosis and plaque disruption, leading to acute atherothrombotic cardiovascular events. Another key characteristic of advanced, or "vulnerable," atherosclerotic lesion is the presence of a variety of inflammatory cytokines, many of which are thought to be secreted by macrophages and triggered by FC accumulation.Atherosclerotic diseases are the leading cause of death in developed countries and part of developing countries. These facts highlight the need for effective therapies to manage atherosclerosis and thrombosis. In this regard, high-density lipoprotein (HDL)-based therapies have recently become the focus of attention. Apolipoprotein A-I (apoA-I) and apolipoprotein (apoE) are the main protein in HDL and exert diverse atheroprotective functions. However, both apoE and apoA-I are macromolecular protein (molecular weight of 28kD and 34kD, respectively) and can only be administered intravenously. In addition, difficult preparation and expensive cost limit their clinical application.Thus, their mimetic peptides which mimic the function of apolipoprotein with smaller molecular weight are increasingly considered as potential antiatherogenic strategy. Recently, a dual-domain peptide has been designed that possesses the arginine-rich domain (LRKLRKRLLR, 141-to 150-residue region of apo E) from apoE, covalently linked to the well-characterized class A amphipathic helical peptide 18A, high-affinity lipid-associating peptide (DWLKAFYDKVAEKL KEAF). The resulting peptide (Ac-hE-18A-NH2) has been shown to promote the rapid uptake and clearance of atherogenic apoB-containing lipoproteins in vitro and in dyslipidemic mouse models. In the Watanabe heritable hyperlipidemic (WHHL) rabbit, a single administration of the peptide Ac-hE18A-NH2 not only reduced plasma cholesterol levels but also restored endothelial function.Objective The aim of this study was to evaluate the effect of a dual-domain mimetic peptide, Ac-hE-18A-NH2 on the cholesterol efflux, apoptosis and inflammation response in RAW264.7 macrophages treated with oxidized low density lipoprotein(oxLDL), and to elucidate the possible mechanisms.MethodsRAW264.7 macrophages were divided into three groups:(1) Cholesterol efflux group:RAW264.7 macrophages were incubated in the medium containing various concentration of the peptide, Ac-hE-18A-NH2 (1,10,50 and 100μg/ml) for 24 hours. The intracellular cAMP level was determined by ELISA. ABCA1,LXRαand PPARγexpression in macrophages was quantitated by realtime RT-PCR and Western blot analysis. (2) Apoptosis group:The cells were exposed to 50μg/ml oxLDL for 48 hrs, and then the cells were incubated with the peptide Ac-hE-18A-NH2 with various concentrations. The apoptosis was detected using Annexin V-FITC staining and flow cytometric analysis. The caspase-3 activity and intracellular cholesterol content were measured using commercially available quantitation kits. Bcl-2 protein expression in macrophages was detected by Western blot analysis. In some experiments, the cells were co-treated withβ-cyclodextrin (a cholesterol efflux stimulator) or BFA (a cholesterol efflux blocker) for 24hours. (3) Inflammation group:Macrophages were incubated in the medium containing various concentrations of Ac-hE18A-NH2 (1-50μg/ml) with ox-LDL (50μg/ml) stimulated. The TNF-a level and intracellular cholesterol content were measured using commercially available quantitation kits. TNFαand ABCA1 mRNA expression were detected by Real-time PCR. ABCA1 and IκB protein expression in macrophages was determined by Western blot analysis. NF-κB activity was evaluated by Electrophoretic Mobility Shift Assay (EMSA).Results1. The peptide Ac-hE-18A-NH2 significantly increased the cholesterol efflux in concentration-and time-dependent manner. Compared with the control group,1.3-fold increase in 10μg/ml peptide Ac-hE-18A-NH2-mediated cholesterol efflux was seen, whereas 0.7-fold increase was observed in 1μg/ml Ac-hE-18A-NH2-mediated cholesterol efflux. The maximum cholesterol efflux mediated by the peptide was occurred at 50μg/ml (26.93±4.37%). Cholesterol efflux from macrophages at different time points was 10.86±1.46%(6 hrs),13.43±1.55%(12 hrs), 20.58±1.34%(18 hrs), and 26.93±4.37%(24 hrs), respectively.2. Concomitantly, Ac-hE-18A-NH2 increased intracellular cAMP level, ABCA1 mRNA and protein expression in dose-dependent manner, consistent with the changes of cholesterol efflux from macrophages.3.8-Br-cAMP was a potent activator of cholesterol efflux and ABCA1 expression during the process of cholesterol efflux mediated by Ac-hE-18A-NH2. Moreover, Ac-hE-18A-NH2 upregulated the expression of LXRa and PPARy. Addition of GW9662 markedly inhibited the increase of ABCA1 gene expression and Ac-hE-18A-NH2-mediated cholesterol efflux.4. After treated with 50μg/ml ox-LDL for various time points, the apoptotic rate of RAW264.7 macrophages increased in a time-dependent manner. Ox-LDL with increasing concentration induced macrophages apoptosis in a dose-dependent manner.5. The peptide Ac-hE-18A-NH2 with various concentrations (1μg/wl,10μg/ml, and 50μg/ml) inhibited the ox-LDL-mediated apoptosis in a concentration-dependent manner.6. The peptide also decreased the caspase-3 activity and increased bcl-2 expression in macrophages in a dose-dependent manner. It was accompanied by an increased rate of intracellular cholesterol efflux, and decreased total cholesterol levels in cells in a concentration-dependent manner.7. Moreover, blockage of cholesterol efflux by brefeldin A decreased the protective effect of Ac-hE-18A-NH2 on ox-LDL induced apoptosis While increase of the cholesterol efflux byβ-cyclodextrin led to a dramatic decrease in the apoptotic rate of cells.8. OxLDL stimulation induced a significant increase of TNFa secretion, mRNA expression, cholesterol accumulation and NF-κB activity in RAW264.7 macrophages.9. Ac-hE-18A-NH2 reduced TNFa secretion and mRNA expression, up-regulated the ABCA1 mRNA and protein expression, reduced the intracellular cholesterol content and inhibited NF-κB activation in a dose-dependent manner.10. Treatment with Ac-hE-18A-NH2 at 50μg/ml significantly suppressed TNFa secretion by 56%, reduced intracellular cholesterol content by 65%, inhibited NF-κB activation by 65.34%, upregulated ABCA1 mRNA and protein expression, and suppressed IκB-αphosphorylation as compared with ox-LDL stimulated group. Under the same condition and the same concentration, Ac-hE-18A-NH2 is more efficient than D-4F (apoA-I mimetic peptide) to inhibit the inflammatory response induced by ox-LDL in macrophages.Conclusions1. Ac-hE-18A-NH2 affects cholesterol efflux, cAMP level and ABCA1 expression of macrophages, and the cAMP is probably involved. Furthermore, the pathway of LXRα-PPARγ-ABCA1 may also be involved in this process.2. The mimetic peptide Ac-hE-18A-NH2 exerts a protective effect against macrophage apoptosis, through reducing the accumulation of cholesterol.3. Ac-hE-18A-NH2 could suppress TNFa secretion and mRNA expression in ox-LDL-stimulated RAW264.7 macrophages by IKBa-NF-KB signaling pathway.
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