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Endomorphin-1 prevents lipid accumulation via CD36 down-regulation and modulates cytokines release from human lipid-laden macrophages

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Endomorphin-1 prevents lipid accumulation via CD36 down-regulation and modulates cytokines release from human lipid-laden macrophages
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  Please cite this article in press as: Chiurchiù V, et al. Endomorphin-1 prevents lipid accumulation via CD36 down-regulation andmodulates cytokines release from human lipid-laden macrophages. Peptides (2010), doi:10.1016/j.peptides.2010.09.024 ARTICLE IN PRESS G Model PEP-68188; No.of Pages6Peptides xxx (2010) xxx–xxx Contents lists available at ScienceDirect Peptides  journal homepage: www.elsevier.com/locate/peptides Endomorphin-1 prevents lipid accumulation via CD36 down-regulation andmodulates cytokines release from human lipid-laden macrophages  Valerio Chiurchiù a , ∗ , Valerio Izzi b , Fabiola D’Aquilio c , Daniela Vismara c , Felicia Carotenuto d ,Giuseppina Catanzaro a , e , Mauro Maccarrone a , e a Laboratory of Lipid Neurochemistry, European Center for Brain Research (CERC)/IRCCS Fondazione S.Lucia, Via del Fosso di Fiorano 65, 00143 Rome, Italy b Department of Experimental Medicine and Biochemical Sciences, University of Rome “Tor Vergata”, Italy c Department of Biology, University of Rome “Tor Vergata”, Italy d Laboratorio di Cardiologia Molecolare e Cellulare, Dipartimento di Medicina Interna, University of Rome Tor Vergata, Italy e Department of Biomedical Sciences, University of Teramo, Teramo, Italy a r t i c l e i n f o  Article history: Received 19 August 2010Received in revised form24 September 2010Accepted 24 September 2010Available online xxx Keywords: Foam cellsOpioid peptidesScavenger receptorsAtherosclerosisOxidized lipoproteins a b s t r a c t CD36isascavengerreceptorknowntoplayacriticalroleinthedevelopmentofatherosclerosisbymedi-atingtheuptakeofoxidizedlow-densitylipoproteins(oxLDL)bymacrophages,thusleadingtofoamcellformation. It is now generally recognized that the immune system has a pivotal role in the pathogenesisof atherosclerosis, whose progression is determined by ongoing inflammatory reactions. Recently, sev-eral studies pointed out that opioid peptides exert anti-inflammatory activities. Therefore the aim of thepresent study was to evaluate a possible endomorphin-1 (EM-1) immunomodulatory activity on humanfoam cells. Our results showed that EM-1 reduced Nile Red-stained lipid droplets content, decreased theexpression of CD36 receptor and modulated tumor necrosis factor-   (TNF-  ) and interferon-   (IFN-  )release from lipid-laden macrophages. Furthermore, Naloxone, an opioid receptors antagonist, revertedthe anti-atherogenic and anti-inflammatory observed effects of EM-1. These data demonstrated, for thefirst time, an unprecedented ability of EM-1 to act as a novel modulator for macrophage-to-foam celltransformation, and for inflammatory cytokines profile, suggesting possible novel endomorphin-basedanti-atherosclerotic approaches for the prevention and treatment of atherosclerosis.© 2010 Published by Elsevier Inc. 1. Introduction The presence of lipid-laden macrophages is the first step of atherogenesis [5]. The transformation of these macrophages intofoamcellsismediatedbyadysregulateduptakeofcholesterol-richmodified low density lipoproteins (LDLs), due to scavenger recep-tors(ScRs)[5].Studiesoflipidmetabolisminculturedmacrophageshave led to the identification of several types of ScRs, includ-ing CD36 and SR-A [8]. Unlike the native LDL receptor, ScRs arenot down-regulated by increases of intracellular cholesterol lev-els, resulting in a continued engulfment of modified LDL that isfollowed by transformation of macrophages into foam cells [8]. Itis now generally recognized that atherosclerosis is an inflamma-torydisease,whoseprogressionandconsequencesaredeterminedby dynamic interactions between inflammatory cells, recruited inresponsetosub-endotheliallipidaccumulation,andthelocalrepar-  Valerio Chiurchiù wishes to dedicate this paper to the memory of his mentor,Prof. Patrizia Morena Baldini, sorrowfully missed in 2008. ∗ Corresponding author. E-mail address:  valchiu@yahoo.it (V. Chiurchiù). ative “wound-healing” response of surrounding vascular smoothmusclecells[17,23].Furthermore,evenifourunderstandingofthe pathogenesis of atherosclerosis has progressed significantly andtheuseofstatinsseemstobetheonlystrategyagainstthisdisease,therehasbeenanincreasinginterestindevelopingnoveltherapiesdue to the many adverse effects of the existing ones [3].Endomorphin-1 (EM-1) is a member of the endogenous opi-oid peptides family, which also includes endorphins, enkephalins,dynorphinsandnociceptin[16].Thesesubstancesbindtoandacti-vateseveralopioidreceptors,amongwhichthefourmajorsubtypesare the three classical MOP, DOP and KOP receptors and the nonclassical (nociceptin) NOP receptor [26,6]. Through the action of  the three classical receptors, but above all of MOP, EM-1 is knownto induce hypotension [10], influence appetite [25], impair spatial learning [27] and cause analgesia [19], as well as bearing reward- ingandaddictiveproperties[24].Furthermore,ithasbeenrecentlyreported that EM-1 can cross the blood-brain barrier [13], thus entering the blood flow and possibly reaching several peripheralbodydistricts,whereitsconcentrationsvaryfrom10 − 12 to10 − 6 M.Coherently, EM-1 has been identified in the rat spleen and thy-mus as well as in spleens from human patients [20], thus havingthe physiological potential to influence immunological function 0196-9781/$ – see front matter © 2010 Published by Elsevier Inc.doi:10.1016/j.peptides.2010.09.024  Please cite this article in press as: Chiurchiù V, et al. Endomorphin-1 prevents lipid accumulation via CD36 down-regulation andmodulates cytokines release from human lipid-laden macrophages. Peptides (2010), doi:10.1016/j.peptides.2010.09.024 ARTICLE IN PRESS G Model PEP-68188; No.of Pages62  V. Chiurchiù et al. / Peptides xxx (2010) xxx–xxx throughopioidreceptors.Consistently,opioidreceptorsarewidelydistributed throughout the immune system [4]. In particular, the anti-inflammatory role of EM-1 on immune cells has been inves-tigated by Azuma et al., who showed that it could significantlysuppress several innate and acquired immune functions fromTHP-1 macrophages [1]. In addition, we recently demonstratedthat this endogenous opioid was able to inhibit the activationof lipopolysaccharide (LPS)-stimulated THP-1 monocytes and theonset of the monocytic hyporesponsive phenotype typical of sep-tic states [9]. Interestingly, following treatment with PMA, THP-1cells acquire the characteristics of mature macrophages, includingdecreased native LDL receptor expression and increased expres-sionofscavengerreceptorslikeCD36[2].Therefore,THP-1-derivedmacrophagesarewidelyacceptedasareliablemodeloffoamcells,due to their high capacity of taking up oxLDL particles. Here, inview of the purported role for EM-1 as a novel anti-inflammatoryagent, we aimed at investigating the possible effects of EM-1 onhuman macrophages-derived foam cells, focusing on the modula-tionofintracellularlipidaccumulation.Wealsosoughttoevaluatethe potential role of EM-1 in regulating the level of expression of CD36-scavengerreceptor,aswellasinmodulatingcytokinereleasefrom atherosclerotic foam cells. 2. Methods  2.1. Cell culture and treatments The human monocytic cell line THP-1 was obtained from theAmerican Type Culture Collection (ATCC, Rockville, MD), and wascultured in Falcon flasks with RPMI-1640 medium supplementedwith10%fetalbovineserum, l -glutamine(2mM),sodiumpyruvate(100  g/ml), penicillin (100U/ml) and streptomycin (100  g/ml).Cells were cultured at 37 ◦ C in a humidified 5% CO 2  atmosphere.Differentiation of THP-1 monocytes into macrophages occurred inthe presence of 100nM phorbol 12-myristate 13-acetate (PMA)(Sigma,Italy)for72h.Peripheralbloodmononuclearcells(PBMCs)were obtained from four healthy donors (40ml) and were iso-lated by Ficoll-hystopaque gradient centrifugation (Pharmacia,Uppsala, Sweden) on the day of blood donation (upon informedconsent). PBMCs were cultured at 10 6 cells/ml in the samemedium as THP-1 cells, plus 5% (vol/vol) heat-inactivated humanserum (Life Tecnologies, Grand Island, NY) and 10mM HEPES[ N  -2-hydroxyethylpiperazine- N  ′ 2-ethanesulfonic acid]. Using themonocyte isolation kit (Miltenyi Biotec Gmbh, Germany), highlypurified human CD14 + -cells were isolated by depletion of CD3 + -Tcells (negative selection). The purity of the enriched CD14 + -monocytes was evaluated by flow cytometry and was consistentlyabove 90%. Human peripheral monocytes were differentiatedinto macrophages by incubation with 20ng/ml M-CSF (Invitro-gen Molecular Probes, Italy) for 6 days. Cell viability was routinelyassessedbyTrypanblueexclusionmethod,usingaNeubauermod-ified chamber, and by flow cytometry with Live-Dead staining(Invitrogen).  2.2. LDL oxidation and foam cell formation LDL (Sigma) was reconstituted in deionized water (1.5mgLDL protein/ml), and oxidized at 37 ◦ C by incubation with 5  MCu 2 SO 4  for 24h. Lipoprotein oxidation was assessed by changesin absorbance at 234nm (due to conjugated dienes), and Cu 2+ ions were removed by extensive dialysis. PMA-differentiated THP-1macrophagescellsandhumanmacrophageswereincubatedwith100  g/ml of oxLDL for 18h, in order to allow lipoproteins uptakeby THP-1 macrophages, and hence their conversion into foamcells.  2.3. Confocal microscopy The cellular content of lipid droplets was assessed by Nile redstaining[12].StocksolutionsofNilered(5mg/ml)inacetonewereprepared and stored protected from light. Briefly, EM-1-treatedTHP-1 foam cells were fixed on coverglasses and were permeabi-lized with 0.1% Triton-X100, and then stained with Nile red for10min. Intracellular localization of lipid droplets was visualizedbymeansofaLeicaTCSSPconfocalmicroscopeandanalyzedusingimage-acquisition LAS AF program (Leica Microsystems, Wetzlar,Germany).  2.4. Cytofluorimetry Both foam-like THP-1 cells and human macrophages-derivedfoam cells (hMac-Fc) were incubated or not with EM-1 for 24h orwith   -opioid agonist DAMGO (Sigma) and antagonist naloxone(Sigma)[22].Afterincubation,cellswerewashed3timeswithcoldphosphate-buffered saline (PBS), collected by means of accutaseand then stained with FITC conjugated anti-CD36 specific anti-body. Flow cytometry analysis was performed on a FACSCaliburcytofluorimeter (Beckton Dickinson, NJ, USA).  2.5. Western blotting  THP-1-derived foam cells, seeded at 3 × 10 6 cells/well, werewashed 3 times with cold PBS, incubated for 3min with accutaseand collected for cell lysis. Then, cells were lysed in PBS containing1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% sodium dode-cylsulfate, phenylmethylsulfonyl fluoride (100  g/ml), aprotinin(30  l/ml; Sigma) and 1mM sodium orthovanadate. The proteincontent of each sample was determined by the Bradford methodbefore storage at  − 80 ◦ C. Protein samples were separated by elec-trophoresis through 7% acrylamide gradient gels, and then weretransferred to nitrocellulose by standard electroblotting proce-dures [12]. The blots were then first incubated overnight withthe CD36 primary antibody (Santa Cruz, CA, USA) at a dilution of 1:500andthentheappropriatehorseradishperoxidase-conjugatedsecondary antibody (Santa Cruz) was added at a 1:5000 dilutionfor 1h at room temperature. Proteins were detected by enhancedchemiluminescence (ECL; Amersham Pharmacia Biotech) and byexposuretoX-rayfilm(HyperECL;AmershamPharmaciaBiotech),quantitating protein band intensity by densitometry analysis ina ChemiImager 4400 apparatus (Alpha Innotech, San Leandro,CA).  2.6. ELISA THP-1 foam cells or hMac-Fc were plated at 1 × 10 5 cells/welland were left untreated or were pre-treated with 10 − 6 M EM-1, orwith DAMGO or naloxone, each used at 10 − 6 M. Cell supernatantswere collected 24h after treatment. Cytokines levels were deter-mined by ELISA, using appropriate kits (Pierce Endogen IL, USA),according to the manufacturer’s instructions and whose limits of detection were <2pg/ml. The absorbance at 450nm of each wellwas measured by microplate ELISA reader. Cytokines concentra-tionsineachsamplewerecalculatedfromtheabsorbancevaluesof the samples, plotted on a standard calibration curve as percentageof controls.  2.7. Statistical analysis Statistical analysis of the data was performed by means of thenon-parametric one-way ANOVA test through Prism 4 (GraphPADSoftware for Science, San Diego, CA).  Please cite this article in press as: Chiurchiù V, et al. Endomorphin-1 prevents lipid accumulation via CD36 down-regulation andmodulates cytokines release from human lipid-laden macrophages. Peptides (2010), doi:10.1016/j.peptides.2010.09.024 ARTICLE IN PRESS G Model PEP-68188; No.of Pages6 V. Chiurchiù et al. / Peptides xxx (2010) xxx–xxx  3 Fig.1.  Endomorphin-1(EM-1)inhibitslipid-dropletsaccumulationinTHP-1-derivedfoamcellsvia  -opioidreceptor.THP-1monocytesweredifferentiatedintomacrophageswith100nMphorbol12-myristate13-acetatefor3days,andthenweretransformedintofoamcellsbyadding100  g/mloxLDLfor18h.(A)CellswereincubatedwithEM-1at different concentrations (10 − 6 –10 − 10 M) for 24h. (B) THP-1 foam cells were pre-treated with selective  -opioid receptor antagonist naloxone or agonist DAMGO (both at10 − 6 M for 2h) and then were incubated with 10 − 6 M EM-1 for 24h. Nile Red-stained lipid droplets were analyzed by fluorescence microscopy (A) or confocal microscopy(B), as detailed in Section 2. Images are representative of 3 independent experiments. *  p <0.01 versus Ctrl;  #,   p <0.01 versus oxLDL. 3. Results  3.1. EM-1 inhibits lipid-droplets accumulation in human foamcells Following treatment with PMA, THP-1 cells acquire the char-acteristics of mature macrophages, including decreased nativeLDL receptor expression and increased expression of scavengerreceptors like CD36 [2]. Therefore, THP-1-derived macrophagesare widely accepted as a reliable model of foam cells, due totheir high capacity of taking up oxLDL particles. In order toinvestigate whether EM-1 could modulate lipid accumulationwithin macrophages, initial studies were performed by treatingTHP-1-derived foam cells with different concentrations of EM-1(10 − 6 –10 − 10 M) (Fig. 1A). After staining with Nile red, control cellsdid not show any significant lipid-droplet accumulation, whereasincubation with 100  g/ml of oxLDL generated many cells filledwith large fluorescent, spherical cytoplasmic structures. Instead,incubation with native LDL did not affect the intracellular lipid-droplets content (data not shown). Dose–response curves showedthat EM-1 was able to reduce accumulation of lipid-droplets bothat 10 − 6 and 10 − 8 M, with a maximal effect at the former concen-tration and no effect at all at 10 − 10 M (Fig. 1A). Also EM-1 used atthe pharmacological concentration of 10 − 4 M exerted a significanteffect on decreasing the lipid droplets content, but its effect wassimilar to that of 10 − 6 M; thus subsequent experiments were per-formed using EM-1 at the highest physiological concentration of 10 − 6 M. In addition, in order to elucidate the possible involvementof opioid receptors, in particular MOR, in the observed inhibitionof lipid accumulation, experiments were extended by pre-treatingTHP-1-derived foam cells with naloxone, an opioid receptorsantagonistwhichshowsagreateraffinitytoMOR,orwiththeMOR-specific agonist DAMGO, both used at 10 − 6 M for 2h. As shownin Fig. 1B, confocal microscope images showed that pre-treatmentwithnaloxonepreventedthereductionoflipid-dropletsinducedbyEM-1,whereaspre-treatmentwithDAMGOwasineffective.Onthisbasis,itcouldbesuggestedthattheabilityofEM-1topreventintra-cellular lipid accumulation was mediated by  -opioid receptors.  3.2. EM-1 down-regulates CD36 expression in human foam cells Uptake of oxidized low density lipoproteins (oxLDLs) via CD36leads to intracellular lipid-droplets accumulation [2]. Therefore,we further investigated whether EM-1 could modulate the levelof expression of CD36 scavenger receptor. The effect of EM-1on CD36 expression in THP-1 foam cells was first examined byWestern blotting, showing that CD36 over-expression caused byoxLDLs intracellular accumulation was significantly suppressedwhen foam cells were treated with 10 − 6 M EM-1 for 24h (Fig. 2Aand B). A more detailed evaluation of EM-1 effect on CD36expression on THP-1 foam cells was performed by means of flowcytometry, investigating also the involvement of MOR. Fig. 2Cshows that macrophages incubated with 100  g/ml of oxLDL for18h had a marked cell surface increase in CD36 with respect tountreated controls. However, after treatment with 10 − 6 M EM-1,theexpressionofCD36wassignificantlyreduced,comparedtothatofuntreatedlipid-ladenmacrophages.Inaddition,thereductionof CD36 expression by EM-1 was mimicked by treatment with MOR agonist DAMGO, and was almost erased by naloxone. We repeatedthe analysis of the cell surface CD36 expression also on foam cellsderived from human peripheral macrophages, in order to assesswhetherEM-1couldexertthesameeffectexvivo.Similarly,10 − 6 MEM-1 significantly reduced CD36 surface expression on hMac-Fc(Fig. 3). Again, DAMGO mimicked the activity of EM-1, and nalox-onecounteracteditseffectonperipheralfoamcells.Theanalysisof the levels of surface expressions of CD36 on both types of humanfoam cells is summarized in Table 1.  3.3. EM-1 modulates cytokines release from human foam cells Next, we ascertained the possible impact of EM-1 on therelease of inflammatory cytokines from foam cells. Fig. 4  Please cite this article in press as: Chiurchiù V, et al. Endomorphin-1 prevents lipid accumulation via CD36 down-regulation andmodulates cytokines release from human lipid-laden macrophages. Peptides (2010), doi:10.1016/j.peptides.2010.09.024 ARTICLE IN PRESS G Model PEP-68188; No.of Pages64  V. Chiurchiù et al. / Peptides xxx (2010) xxx–xxx Fig. 2.  EM-1 down-regulates CD36 expression in THP-1-derived foam cells via   -opioid receptor. THP-1 monocytes were differentiated into macrophages with 100nMphorbol 12-myristate 13-acetate for 3 days, and then were transformed into foam cells by adding 100  g/ml oxLDL for 18h. Cells were pre-treated with selective  -opioidreceptor antagonist naloxone or agonist DAMGO (both at 10 − 6 M for 2h), and then were incubated with 10 − 6 M EM-1 for 24h. CD36 level of expression was assessed byWesternblotting(AandB)orflowcytometry(C),asdetailedinSection2.Dataarereportedasmean ± SDof4independentexperiments.InpanelB,*  p <0.01versuscontrols, #  p <0.01 versus oxLDL. shows that both types of lipid-laden macrophages producedsignificant amounts of TNF-   (THP-1=2660 ± 468pg/ml, andhMac-Fc=1477 ± 100pg/ml) and IFN-   (THP-1=1043 ± 58pg/ml,and hMac-Fc=888 ± 84pg/ml), whereas when treated with EM-1the release of TNF-   was remarkably inhibited (600 ± 172pg/ml)but that of IFN-   showed 60% and 75% increases, respectively,  Table 1 Levels of surface expression of CD36 on human foam cells.THP-1 FC hMac-FCCtrl 458 ± 56 993 ± 85oxLDL 1177 ± 105 * 2462 ± 195 * EM-1 a 533 ± 52 # 1347 ± 95 # DAMGO b 520 ± 52 # 1205 ± 87 # Naloxone+EM-1 c 795 ± 118 2239 ± 182FC, Foam cells. a EM-1=10 − 6 M/24h. b DAMGO=10 − 6 M/2h. c Naloxone=10 − 6 M/2h. *  p <0.05 versus Ctrl. #  p <0.05 versus oxLDL. compared to the level of oxLDL-laden macrophages. In addi-tion, DAMGO mimicked the activity of EM-1 in terms of bothTNF-  (THP-1=272 ± 59pg/ml,andhMac-Fc=163 ± 24pg/ml)andIFN-  release (THP-1=1763 ± 77, and hMac-Fc=1632 ± 48pg/ml),whereas naloxone minimized the effect of EM-1 on therelease of both TNF-   (THP-1=1790 ± 305pg/ml, and hMac-Fc=1440 ± 80pg/ml), and IFN-   (THP1=1072 ± 27pg/ml, andhMac-Fc=914 ± 52pg/ml). 4. Discussion Overthelastfewdecades,ourunderstandingofthebasicmech-anisms involved in atherosclerosis has progressed significantly.The hallmark of this disease is cholesterol accumulation withinmacrophages, which ultimately alters the biology of these cells[5], and a key role for inflammation at all stages of atherosclero-sis is now widely recognized [16]. Since conventional therapiesagainst atherosclerosis show several adverse side effects, includ-ing myopathy, hepatotoxicity, peripheral neuropathy and evenautoimmune diseases [3], there has been an increasing interest  Please cite this article in press as: Chiurchiù V, et al. Endomorphin-1 prevents lipid accumulation via CD36 down-regulation andmodulates cytokines release from human lipid-laden macrophages. Peptides (2010), doi:10.1016/j.peptides.2010.09.024 ARTICLE IN PRESS G Model PEP-68188; No.of Pages6 V. Chiurchiù et al. / Peptides xxx (2010) xxx–xxx  5 Fig.3.  EM-1down-regulatesCD36expressioninhumanperipheralbloodmonocytes-derivedfoamcells.Humanperipheralmonocytesweredifferentiatedintomacrophagesby incubation with 20ng/ml M-CSF and 10ng/ml GM-CSF for 5 days, and then were transformed into foam cells by incubation with 100  g/ml oxLDL for 18h. Cells werepre-treated with selective   -opioid receptor antagonist naloxone or agonist DAMGO (both at 10 − 6 M for 2h), and then were incubated with 10 − 6 M EM-1 for 24h. CD36expression was assessed by means of Western blotting, as detailed in Section 2. Reported data are representative of 4 independent experiments. in developing novel therapies aimed at reducing cholesterol con-tent as well as at exerting anti-inflammatory effects. Mountingevidence of the role of endomorphins in the modulation of inflam-matory processes led us to hypothesize a possible role for EM-1in atherosclerosis. The present work was undertaken to investi-gate the effects of EM-1 on atherogenic-related processes suchas lipid accumulation and inflammatory mediators release fromhuman foam cells. To the best of our knowledge, this is the firstreport showing the ability of EM-1 to inhibit lipid accumulation inhuman foam cell-like macrophages. Since CD36 activity is respon-sible for the uptake of modified lipoproteins and the induction of foam cells formation, the observed reduction of CD36 expressionshows that the mechanism by which EM-1 reduces lipid engulf-mentisbydown-regulatingthisscavengerreceptor.Aninterestingaspect of CD36 is that its expression on macrophages is increasedwhen the cells are exposed to oxLDL  [18]. Thus, endocytosedoxLDL can induce important and complex transcriptional changesinmonocytes-derivedmacrophages,leadingtoafeed-forwardloopwhich accelerates foam cells formation in the arterial neointima.In this context, it seems noteworthy that the ability of EM-1 todown-regulate the expression of CD36 might be instrumental inbluntingsucharegulatoryloop.Thisresultgainsevenbiggerimpor-tance if considered in the broader context of the atheroscleroticplaque, since it has been shown that CD36 expression triggers andis also regulated by several inflammatory mechanisms that canactivate overlapping pathways responsible for the regulation of immune cell functions and for metabolic alterations [8]. Indeed,cytokines are key regulators of all phases of atherosclerosis, sinceTNF-  ,IFN-  ,IL-1,IL-6,IL-12,IL-10andTGF-  arehighlyexpressedin atherosclerotic regions by macrophages under conditions of hyperlipidemia, and most of them exhibit both pro- and anti-atherogenic actions [21]. Our results uncover the ability of EM-1 to significantly inhibit TNF-  release, while increasing the releaseof IFN-   both from THP-1 macrophages-derived and peripheralmacrophages-derived foam cells. It should be recalled that TNF-  is widely accepted as a pro-atherogenic marker, and that IFN-  , known to be a pro-inflammatory agent, also displays severalanti-inflammatory properties like the reduction of LDL oxidationor inhibition of CD36 expression [7,11]. As a consequence, the uptakeofoxLDLisreducedbyIFN-  .However,thelattersubstancehas been also shown to decrease cholesterol efflux by inhibitingATP-binding cassette transporter A1 (ABCA1) [15], which initi-ates reverse cholesterol transport in macrophages; furthermore,it increases the expression of ACAT, an enzyme which promotesintracellular cholesterol storage [14]. Furthermore, the observeddirect modulation of CD36 demonstrates how this opioid peptidecan exert a crucial role in the overall balance of the inflamma-tory processes involved in the formation of the altered phenotypeof foam cells acting both directly in the negative regulation of lipid influx or indirectly in the promotion of the anti-atherogenicrole of several cytokines or soluble factors. Our data regardingthe cytokines profile of foam cells, candidate EM-1 as an impor-tant anti-inflammatory agent capable of blunting the complexmolecularnetworkofcell-mediatedimmunity,whichisultimatelyresponsible for the tissue damage and the progression of theatherosclerotic plaque. Taken together, these findings call for fur-ther studies aimed at understanding the mode of action of EM-1in reverse cholesterol transport as well as in the molecular mech-anism of inflammatory processes. At any rate, our results suggestthatEM-1isanovelmodulatorformacrophage-to-foamcelltrans-formationviaCD36down-regulation,notonlyduetoitscapacityof reducingthelipidaccumulationbutmostnotablyduetoitsendow-mentinmodulatingthecellimmunefunctions,whichresultsintheoversetoftheoxLDL-inducedalteredphenotypeoffoamcells.Thisacquires greater importance when observed on human peripheralmacrophages-derivedfoamcells,standingforasimilarresponseof these cells upon EM-1 which is not only limited to the THP-1  invitro  model of foam cells. On this basis, presented data might set
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