P

Em

VGa

b

c

d

e

a

ARR2AA

KFOSAO

1

afmthinefitbr

P

0d

ARTICLE IN PRESSG ModelEP-68188; No. of Pages 6

Peptides xxx (2010) xxx–xxx

Contents lists available at ScienceDirect

Peptides

journa l homepage: www.e lsev ier .com/ locate /pept ides

ndomorphin-1 prevents lipid accumulation via CD36 down-regulation andodulates cytokines release from human lipid-laden macrophages�

alerio Chiurchiùa,∗, Valerio Izzib, Fabiola D’Aquilioc, Daniela Vismarac, Felicia Carotenutod,iuseppina Catanzaroa,e, Mauro Maccarronea,e

Laboratory of Lipid Neurochemistry, European Center for Brain Research (CERC)/IRCCS Fondazione S.Lucia, Via del Fosso di Fiorano 65, 00143 Rome, ItalyDepartment of Experimental Medicine and Biochemical Sciences, University of Rome “Tor Vergata”, ItalyDepartment of Biology, University of Rome “Tor Vergata”, ItalyLaboratorio di Cardiologia Molecolare e Cellulare, Dipartimento di Medicina Interna, University of Rome Tor Vergata, ItalyDepartment of Biomedical Sciences, University of Teramo, Teramo, Italy

r t i c l e i n f o

rticle history:eceived 19 August 2010eceived in revised form4 September 2010ccepted 24 September 2010vailable online xxx

a b s t r a c t

CD36 is a scavenger receptor known to play a critical role in the development of atherosclerosis by medi-ating the uptake of oxidized low-density lipoproteins (oxLDL) by macrophages, thus leading to foam cellformation. 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 human

eywords:oam cellspioid peptidescavenger receptorstherosclerosisxidized lipoproteins

foam 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-based

oache

anti-atherosclerotic appr

. Introduction

The presence of lipid-laden macrophages is the first step oftherogenesis [5]. The transformation of these macrophages intooam cells is mediated by a dysregulated uptake of cholesterol-rich

odified low density lipoproteins (LDLs), due to scavenger recep-ors (ScRs) [5]. Studies of lipid metabolism in cultured macrophagesave led to the identification of several types of ScRs, includ-

ng CD36 and SR-A [8]. Unlike the native LDL receptor, ScRs areot down-regulated by increases of intracellular cholesterol lev-ls, resulting in a continued engulfment of modified LDL that isollowed by transformation of macrophages into foam cells [8]. It

Please cite this article in press as: Chiurchiù V, et al. Endomorphinmodulates cytokines release from human lipid-laden macrophages. P

s now generally recognized that atherosclerosis is an inflamma-ory disease, whose progression and consequences are determinedy dynamic interactions between inflammatory cells, recruited inesponse to sub-endothelial lipid accumulation, and the local repar-

� Valerio Chiurchiù wishes to dedicate this paper to the memory of his mentor,rof. Patrizia Morena Baldini, sorrowfully missed in 2008.∗ Corresponding author.

E-mail address: valchiu@yahoo.it (V. Chiurchiù).

196-9781/$ – see front matter © 2010 Published by Elsevier Inc.oi:10.1016/j.peptides.2010.09.024

s for the prevention and treatment of atherosclerosis.© 2010 Published by Elsevier Inc.

ative “wound-healing” response of surrounding vascular smoothmuscle cells [17,23]. Furthermore, even if our understanding of thepathogenesis of atherosclerosis has progressed significantly andthe use of statins seems to be the only strategy against this disease,there has been an increasing interest in developing novel therapiesdue 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,dynorphins and nociceptin [16]. These substances bind to and acti-vate several opioid receptors, among which the four major subtypesare the three classical MOP, DOP and KOP receptors and the nonclassical (nociceptin) NOP receptor [26,6]. Through the action ofthe three classical receptors, but above all of MOP, EM-1 is knownto induce hypotension [10], influence appetite [25], impair spatiallearning [27] and cause analgesia [19], as well as bearing reward-ing and addictive properties [24]. Furthermore, it has been recentlyreported that EM-1 can cross the blood-brain barrier [13], thus

-1 prevents lipid accumulation via CD36 down-regulation andeptides (2010), doi:10.1016/j.peptides.2010.09.024

entering the blood flow and possibly reaching several peripheralbody districts, where its concentrations vary from 10−12 to 10−6 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

INP

2 eptide

tdatsTtootcdsmdvahttCf

2

2

Acw(CDt(wlUcms[mpcmaigai(

2

LCii11bc

ARTICLEG ModelEP-68188; No. of Pages 6

V. Chiurchiù et al. / P

hrough opioid receptors. Consistently, opioid receptors are widelyistributed throughout the immune system [4]. In particular, thenti-inflammatory role of EM-1 on immune cells has been inves-igated by Azuma et al., who showed that it could significantlyuppress several innate and acquired immune functions fromHP-1 macrophages [1]. In addition, we recently demonstratedhat this endogenous opioid was able to inhibit the activationf lipopolysaccharide (LPS)-stimulated THP-1 monocytes and thenset of the monocytic hyporesponsive phenotype typical of sep-ic states [9]. Interestingly, following treatment with PMA, THP-1ells acquire the characteristics of mature macrophages, includingecreased native LDL receptor expression and increased expres-ion of scavenger receptors like CD36 [2]. Therefore, THP-1-derivedacrophages are widely accepted as a reliable model of foam cells,

ue to their high capacity of taking up oxLDL particles. Here, iniew of the purported role for EM-1 as a novel anti-inflammatorygent, we aimed at investigating the possible effects of EM-1 onuman macrophages-derived foam cells, focusing on the modula-ion of intracellular lipid accumulation. We also sought to evaluatehe potential role of EM-1 in regulating the level of expression ofD36-scavenger receptor, as well as in modulating cytokine release

rom atherosclerotic foam cells.

. Methods

.1. Cell culture and treatments

The human monocytic cell line THP-1 was obtained from themerican Type Culture Collection (ATCC, Rockville, MD), and wasultured in Falcon flasks with RPMI-1640 medium supplementedith 10% fetal bovine serum, l-glutamine (2 mM), sodium pyruvate

100 �g/ml), penicillin (100 U/ml) and streptomycin (100 �g/ml).ells were cultured at 37 ◦C in a humidified 5% CO2 atmosphere.ifferentiation of THP-1 monocytes into macrophages occurred in

he presence of 100 nM phorbol 12-myristate 13-acetate (PMA)Sigma, Italy) for 72 h. Peripheral blood mononuclear cells (PBMCs)ere obtained from four healthy donors (40 ml) and were iso-

ated by Ficoll-hystopaque gradient centrifugation (Pharmacia,ppsala, Sweden) on the day of blood donation (upon informedonsent). PBMCs were cultured at 106 cells/ml in the sameedium as THP-1 cells, plus 5% (vol/vol) heat-inactivated human

erum (Life Tecnologies, Grand Island, NY) and 10 mM HEPESN-2-hydroxyethylpiperazine-N′2-ethanesulfonic acid]. Using the

onocyte isolation kit (Miltenyi Biotec Gmbh, Germany), highlyurified human CD14+-cells were isolated by depletion of CD3+-Tells (negative selection). The purity of the enriched CD14+-onocytes was evaluated by flow cytometry and was consistently

bove 90%. Human peripheral monocytes were differentiatednto macrophages by incubation with 20 ng/ml M-CSF (Invitro-en Molecular Probes, Italy) for 6 days. Cell viability was routinelyssessed by Trypan blue exclusion method, using a Neubauer mod-fied chamber, and by flow cytometry with Live-Dead stainingInvitrogen).

.2. LDL oxidation and foam cell formation

LDL (Sigma) was reconstituted in deionized water (1.5 mgDL protein/ml), and oxidized at 37 ◦C by incubation with 5 �Mu2SO4 for 24 h. Lipoprotein oxidation was assessed by changes

Please cite this article in press as: Chiurchiù V, et al. Endomorphinmodulates cytokines release from human lipid-laden macrophages. P

n absorbance at 234 nm (due to conjugated dienes), and Cu2+

ons were removed by extensive dialysis. PMA-differentiated THP-macrophages cells and human macrophages were incubated with00 �g/ml of oxLDL for 18 h, in order to allow lipoproteins uptakey THP-1 macrophages, and hence their conversion into foamells.

PRESSs xxx (2010) xxx–xxx

2.3. Confocal microscopy

The cellular content of lipid droplets was assessed by Nile redstaining [12]. Stock solutions of Nile red (5 mg/ml) in acetone wereprepared 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 for10 min. Intracellular localization of lipid droplets was visualizedby means of a Leica TCS SP confocal microscope and analyzed usingimage-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 24 h orwith �-opioid agonist DAMGO (Sigma) and antagonist naloxone(Sigma) [22]. After incubation, cells were washed 3 times with coldphosphate-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 × 106 cells/well, werewashed 3 times with cold PBS, incubated for 3 min 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 1 mM 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 of1:500 and then the appropriate horseradish peroxidase-conjugatedsecondary antibody (Santa Cruz) was added at a 1:5000 dilutionfor 1 h at room temperature. Proteins were detected by enhancedchemiluminescence (ECL; Amersham Pharmacia Biotech) and byexposure to X-ray film (Hyper ECL; Amersham Pharmacia Biotech),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 × 105cells/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 24 h 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 ofdetection were <2 pg/ml. The absorbance at 450 nm of each wellwas measured by microplate ELISA reader. Cytokines concentra-tions in each sample were calculated from the absorbance values ofthe samples, plotted on a standard calibration curve as percentageof controls.

-1 prevents lipid accumulation via CD36 down-regulation andeptides (2010), doi:10.1016/j.peptides.2010.09.024

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).

ARTICLE IN PRESSG ModelPEP-68188; No. of Pages 6

V. Chiurchiù et al. / Peptides xxx (2010) xxx–xxx 3

Fig. 1. Endomorphin-1 (EM-1) inhibits lipid-droplets accumulation in THP-1-derived foam cells via �-opioid receptor. THP-1 monocytes were differentiated into macrophageswith 100 nM phorbol 12-myristate 13-acetate for 3 days, and then were transformed into foam cells by adding 100 �g/ml oxLDL for 18 h. (A) Cells were incubated with EM-1a reated1 ed lip( ts. *p <

3

3c

aLratiwT(diwidtattesf1ooTasiwE

t different concentrations (10−6–10−10 M) for 24 h. (B) THP-1 foam cells were pre-t0−6 M for 2 h) and then were incubated with 10−6 M EM-1 for 24 h. Nile Red-stainB), as detailed in Section 2. Images are representative of 3 independent experimen

. Results

.1. EM-1 inhibits lipid-droplets accumulation in human foamells

Following treatment with PMA, THP-1 cells acquire the char-cteristics of mature macrophages, including decreased nativeDL receptor expression and increased expression of scavengereceptors like CD36 [2]. Therefore, THP-1-derived macrophagesre widely accepted as a reliable model of foam cells, due toheir high capacity of taking up oxLDL particles. In order tonvestigate whether EM-1 could modulate lipid accumulation

ithin macrophages, initial studies were performed by treatingHP-1-derived foam cells with different concentrations of EM-110−6–10−10 M) (Fig. 1A). After staining with Nile red, control cellsid not show any significant lipid-droplet accumulation, whereas

ncubation with 100 �g/ml of oxLDL generated many cells filledith large fluorescent, spherical cytoplasmic structures. Instead,

ncubation with native LDL did not affect the intracellular lipid-roplets content (data not shown). Dose–response curves showedhat EM-1 was able to reduce accumulation of lipid-droplets botht 10−6 and 10−8 M, with a maximal effect at the former concen-ration and no effect at all at 10−10 M (Fig. 1A). Also EM-1 used athe pharmacological concentration of 10−4 M exerted a significantffect on decreasing the lipid droplets content, but its effect wasimilar to that of 10−6 M; thus subsequent experiments were per-ormed using EM-1 at the highest physiological concentration of0−6 M. In addition, in order to elucidate the possible involvementf opioid receptors, in particular MOR, in the observed inhibitionf lipid accumulation, experiments were extended by pre-treatingHP-1-derived foam cells with naloxone, an opioid receptors

Please cite this article in press as: Chiurchiù V, et al. Endomorphinmodulates cytokines release from human lipid-laden macrophages. P

ntagonist which shows a greater affinity to MOR, or with the MOR-pecific agonist DAMGO, both used at 10−6 M for 2 h. As shownn Fig. 1B, confocal microscope images showed that pre-treatment

ith naloxone prevented the reduction of lipid-droplets induced byM-1, whereas pre-treatment with DAMGO was ineffective. On this

with selective �-opioid receptor antagonist naloxone or agonist DAMGO (both atid droplets were analyzed by fluorescence microscopy (A) or confocal microscopy0.01 versus Ctrl; #,�p < 0.01 versus oxLDL.

basis, it could be suggested that the ability of EM-1 to prevent intra-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 24 h (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 for18 h had a marked cell surface increase in CD36 with respect tountreated controls. However, after treatment with 10−6 M EM-1,the expression of CD36 was significantly reduced, compared to thatof untreated lipid-laden macrophages. In addition, the reduction ofCD36 expression by EM-1 was mimicked by treatment with MORagonist 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 assesswhether EM-1 could exert the same effect ex vivo. 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-one counteracted its effect on peripheral foam cells. The analysis ofthe levels of surface expressions of CD36 on both types of humanfoam cells is summarized in Table 1.

-1 prevents lipid accumulation via CD36 down-regulation andeptides (2010), doi:10.1016/j.peptides.2010.09.024

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

ARTICLE IN PRESSG ModelPEP-68188; No. of Pages 6

4 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 100 nMphorbol 12-myristate 13-acetate for 3 days, and then were transformed into foam cells by adding 100 �g/ml oxLDL for 18 h. Cells were pre-treated with selective �-opioidr n werW eporte#

sshatb

TL

F

eceptor antagonist naloxone or agonist DAMGO (both at 10−6 M for 2 h), and theestern blotting (A and B) or flow cytometry (C), as detailed in Section 2. Data are r

p < 0.01 versus oxLDL.

hows that both types of lipid-laden macrophages produced

Please cite this article in press as: Chiurchiù V, et al. Endomorphinmodulates cytokines release from human lipid-laden macrophages. P

ignificant amounts of TNF-� (THP-1 = 2660 ± 468 pg/ml, andMac-Fc = 1477 ± 100 pg/ml) and IFN-� (THP-1 = 1043 ± 58 pg/ml,nd hMac-Fc = 888 ± 84 pg/ml), whereas when treated with EM-1he release of TNF-� was remarkably inhibited (600 ± 172 pg/ml)ut that of IFN-� showed 60% and 75% increases, respectively,

able 1evels of surface expression of CD36 on human foam cells.

THP-1 FC hMac-FC

Ctrl 458 ± 56 993 ± 85oxLDL 1177 ± 105* 2462 ± 195*

EM-1a 533 ± 52# 1347 ± 95#

DAMGOb 520 ± 52# 1205 ± 87#

Naloxone + EM-1c 795 ± 118 2239 ± 182

C, Foam cells.a EM-1 = 10−6 M/24 h.b DAMGO = 10−6 M/2 h.c Naloxone = 10−6 M/2 h.* p < 0.05 versus Ctrl.# p < 0.05 versus oxLDL.

e incubated with 10−6 M EM-1 for 24 h. CD36 level of expression was assessed byd as mean ± SD of 4 independent experiments. In panel B, *p < 0.01 versus controls,

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 ± 59 pg/ml, and hMac-Fc = 163 ± 24 pg/ml) andIFN-� release (THP-1 = 1763 ± 77, and hMac-Fc = 1632 ± 48 pg/ml),whereas naloxone minimized the effect of EM-1 on therelease of both TNF-� (THP-1 = 1790 ± 305 pg/ml, and hMac-Fc = 1440 ± 80 pg/ml), and IFN-� (THP1 = 1072 ± 27 pg/ml, andhMac-Fc = 914 ± 52 pg/ml).

4. Discussion

Over the last few decades, our understanding of the basic mech-anisms involved in atherosclerosis has progressed significantly.The hallmark of this disease is cholesterol accumulation withinmacrophages, which ultimately alters the biology of these cells

-1 prevents lipid accumulation via CD36 down-regulation andeptides (2010), doi:10.1016/j.peptides.2010.09.024

[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

ARTICLE IN PRESSG ModelPEP-68188; No. of Pages 6

V. Chiurchiù et al. / Peptides xxx (2010) xxx–xxx 5

Fig. 3. EM-1 down-regulates CD36 expression in human peripheral blood monocytes-derived foam cells. Human peripheral monocytes were differentiated into macrophagesby incubation with 20 ng/ml M-CSF and 10 ng/ml GM-CSF for 5 days, and then were transformed into foam cells by incubation with 100 �g/ml oxLDL for 18 h. Cells werep GO (be orted

itemigahrhsfsmawoiwIdbtpiaicTihato

re-treated with selective �-opioid receptor antagonist naloxone or agonist DAMxpression was assessed by means of Western blotting, as detailed in Section 2. Rep

n developing novel therapies aimed at reducing cholesterol con-ent as well as at exerting anti-inflammatory effects. Mountingvidence of the role of endomorphins in the modulation of inflam-atory processes led us to hypothesize a possible role for EM-1

n atherosclerosis. The present work was undertaken to investi-ate the effects of EM-1 on atherogenic-related processes suchs lipid accumulation and inflammatory mediators release fromuman foam cells. To the best of our knowledge, this is the firsteport showing the ability of EM-1 to inhibit lipid accumulation inuman foam cell-like macrophages. Since CD36 activity is respon-ible for the uptake of modified lipoproteins and the induction ofoam cells formation, the observed reduction of CD36 expressionhows that the mechanism by which EM-1 reduces lipid engulf-ent is by down-regulating this scavenger receptor. An interesting

spect of CD36 is that its expression on macrophages is increasedhen the cells are exposed to oxLDL [18]. Thus, endocytosed

xLDL can induce important and complex transcriptional changesn monocytes-derived macrophages, leading to a feed-forward loop

hich accelerates foam cells formation in the arterial neointima.n this context, it seems noteworthy that the ability of EM-1 toown-regulate the expression of CD36 might be instrumental inlunting such a regulatory loop. This result gains even bigger impor-ance if considered in the broader context of the atheroscleroticlaque, since it has been shown that CD36 expression triggers and

s also regulated by several inflammatory mechanisms that canctivate overlapping pathways responsible for the regulation ofmmune cell functions and for metabolic alterations [8]. Indeed,ytokines are key regulators of all phases of atherosclerosis, sinceNF-�, IFN-�, IL-1, IL-6, IL-12, IL-10 and TGF-� are highly expressed

Please cite this article in press as: Chiurchiù V, et al. Endomorphinmodulates cytokines release from human lipid-laden macrophages. P

n atherosclerotic regions by macrophages under conditions ofyperlipidemia, and most of them exhibit both pro- and anti-therogenic actions [21]. Our results uncover the ability of EM-1o significantly inhibit TNF-� release, while increasing the releasef IFN-� both from THP-1 macrophages-derived and peripheral

oth at 10−6 M for 2 h), and then were incubated with 10−6 M EM-1 for 24 h. CD36data are representative of 4 independent experiments.

macrophages-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, theuptake of oxLDL is reduced by IFN-�. However, the latter substancehas 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 oflipid 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 complexmolecular network of cell-mediated immunity, which is ultimatelyresponsible 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 suggestthat EM-1 is a novel modulator for macrophage-to-foam cell trans-formation via CD36 down-regulation, not only due to its capacity ofreducing the lipid accumulation but most notably due to its endow-ment in modulating the cell immune functions, which results in the

-1 prevents lipid accumulation via CD36 down-regulation andeptides (2010), doi:10.1016/j.peptides.2010.09.024

overset of the oxLDL-induced altered phenotype of foam cells. Thisacquires greater importance when observed on human peripheralmacrophages-derived foam cells, standing for a similar response ofthese 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

ARTICLE ING ModelPEP-68188; No. of Pages 6

6 V. Chiurchiù et al. / Peptide

Fig. 4. EM-1 inhibits cytokines release in human foam cells via �-opioid receptor.Both THP-1 monocytes (A) or peripheral blood monocytes (B) were differentiatedinto macrophages by incubation with 100 nM 12-myristate, 13-acetate (PMA) for3 days or with 20 ng/ml M-CSF and 10 ng/ml GM-CSF for 5 days, respectively, andthen were transformed into foam cells by incubation with 100 �g/ml oxLDL for 18 h.Both cell types were pre-treated with selective �-opioid receptor antagonist nalox-one or agonist DAMGO (both at 10−6 M for 2 h), and then were incubated with 10−6 MEM-1 for 24 h. The release of TNF-� and IFN-� was determined by means of ELISA,as detailed in Section 2. Data are reported as mean ± SD of 4 independent exper-iments. (A) *p < 0.001 versus TNF-� releasing CTRL cells, *′p < 0.0001 versus IFN-�releasing CTRL cells, #p < 0.05 versus TNF-� releasing oxLDL cells, # ′p < 0.01 versusIFN-� releasing oxLDL cells, ##p < 0.01 versus TNF-� releasing oxLDL cells, �p < 0.05versus TNF-� releasing EM-1 cells, � ′p < 0.01 versus IFN-� releasing EM-1 cells. (B)*p < 0.001 versus TNF-� releasing CTRL cells, *′p < 0.1 versus IFN-� releasing CTRLcor

totadd

A

2

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

ells, #p < 0.001 versus TNF-� releasing oxLDL cells, # ′p < 0.01 versus IFN-� releasingxLDL cells, �p < 0.001 versus TNF-� releasing EM-1 cells, � ′p < 0.001 versus IFN-�eleasing EM-1 cells.

he standards for the identification of new mechanisms of actionf opioid peptides in macrophage lipid metabolism and chiefly forhe carrying out of novel endomorphin-based anti-atheroscleroticpproaches for the prevention and treatment of this widespreadisease and, possibly, also for other related cardiovascular disor-ers.

Please cite this article in press as: Chiurchiù V, et al. Endomorphinmodulates cytokines release from human lipid-laden macrophages. P

cknowledgement

This work was partly supported by Fondazione TERCAS (Project009–2012) to M.M.

[

[

PRESSs xxx (2010) xxx–xxx

References

[1] Azuma Y, Ohura H. Endomorphins 1 and 2 inhibit IL-10 and IL-12 productionand innate immune functions, and potentiate NF-kB DNA binding in THP-1differentiated to macrophage-like cells. Scand J Immunol 2002;56:260–9.

[2] Banka CL, Black AS, Dyer CA. Curtiss LK THP-1 cells form foam cells in responseto coculture with lipoproteins but not platelets. J Lipid Res 1991;32:35–43.

[3] Beltowski J, Wojcicka G, Jamroz-Wisniewska A. Adverse effects of statins-mechanisms and consequences. Curr Drug Saf 2009;4:209–28.

[4] Chuang TK, Killam Jr KF, Chuang LF, Kung HF, Sheng WS, Chao CC, et al. Mu opi-oid receptor gene expression in immune cells. Biochem Biophys Res Commun1995;216:922–30.

[5] Collot-Teixeira S, Martin J, McDermott-Roe C, Poston R, McGregor JL. CD36 andmacrophages in atherosclerosis. Cardiovascular Res 2007;75:468–77.

[6] Dietis N, Guerrini R, Calo G, Salvadori S, Rowbotham DJ, Lambert DG. Simulta-neous targeting of multiple opioid receptors: a strategy to improve side-effectprofile. Br J Anaesth 2009;103:38–49.

[7] Geng YJ, Hansson GK. Interferon-gamma inhibits scavenger receptor expres-sion and foam cell formation in human monocyte-derived macrophages. J ClinInvest 1992;89:1322–30.

[8] Greaves DR, Gordon S. The macrophage scavenger receptor at 30 years of age:current knowledge and future challenges. J Lipid Res 2009;50:282–6.

[9] Izzi V, Chiurchiù V, D’Aquilio F, Martino A, Tresoldi I, Modesti A,et al. Endomorphin-1 inhibits the activation and the development ofa hyporesponsive-like phenotype in lipopolysaccharide-stimulated THP-1monocytes. Int J Immunopathol Pharmacol 2008;21:833–43.

10] Kwok EH, Dun NJ. Endomorphins decrease heart rate and blood pres-sure possibly by activating vagal afferents in anesthetized rats. Brain Res1998;803:204–7.

11] Nakagawa T, Nozaki S, Nishida M, Yakub JM, Tomiyama Y, Nakata A, et al.Oxidized LDL increases and interferon-gamma decreases expression of CD36in human monocyte-derived macrophages. Arterioscler Thromb Vasc Biol1998;18:1350–7.

12] Oddi S, Fezza F, Pasquariello N, De Simone C, Rapino C, Dainese E, et al. Evi-dence for the accumulation of anandamide in adiposomes. Cell Mol Life Sci2008;65:840–50.

13] Pan W, Kastin AJ. From MIF-1 to endomorphins: the Tyr-MIF-1 family of pep-tides. Peptides 2007;28:2411–34.

14] Panousis CG, Zuckerman SH. Regulation of cholesterol distributionin macrophage-derived foam cells by interferon-gamma. J Lipid Res2000;41:75–83.

15] Panousis CG, Zuckerman SH. Interferon-gamma induces downregulation ofTangier disease gene (ATP-binding-cassette transporter 1) in macrophage-derived cells. Arterioscler Thromb Vasc Biol 2000;20:1565–71.

16] Przewłocki R, Przewłocka B. Opioids in chronic pain. Eur J Pharmacol2001;429:79–91.

17] Ross R. Atherosclerosis: an inflammatory disease. N Engl J Med1999;340:115–26.

18] Shiffman D, Mikita T, Tai JT, Wade DP, Porter JG, Seilhamer JJ, et al. Largescale gene expression analysis of cholesterol-loaded macrophages. J Biol Chem2000;275:37324–32.

19] Stone LS, Fairbanks CA, Laughlin TM, Nguyen HO, Bushy TM, Wessendorf MW,et al. Spinal analgesic actions of the new endogenous opioid peptides peptidesendomorphin-1 and -2. Neuroreport 1997;8:3131–5.

20] Straub RH, Wolff C, Fassold A, Hofbauer R, Chover-Gonzalez A, Richards LJ, et al.Antiinflammatory role of endomorphins in osteoarthritis, rheumatoid arthritis,and adjuvant-induced polyarthritis. Arthritis Rheum 2008;58:456–66.

21] Tedgui A, Mallat Z. Cytokines in atherosclerosis: pathogenic and regulatorypathways. Physiol Rev 2006;86:515–81.

22] Vukojevic V, Ming Y, D’Addario C, Hansen M, Langel U, Schulz R, et al. Mu-opioidreceptor activation in live cells. FASEB J 2008;22:3537–48.

23] Wick G, Knoflach M, Xu Q. Autoimmune and inflammatory mechanisms inatherosclerosis. Annu Rev Immunol 2004;22:361–403.

24] Wilson AM, Soignier RD, Zadina JE, Kastin AJ, Nores WL, Olson RD, et al. Disso-ciation of analgesic and rewarding effects of endomorphin-1 in rats. Peptides2000;21:1871–4.

25] Xia FZ, Lu YL, Chen Y, Gu T, Zhang HX, Yu J, et al. Peripheral endomorphin-1 lev-els are suppressed in diabetic patients. Diabetes Res Clin Pract 2010;87:200–3.

-1 prevents lipid accumulation via CD36 down-regulation andeptides (2010), doi:10.1016/j.peptides.2010.09.024

26] Zadina JE, Hackler L, Ge LJ, Kastin AJ. A potent and selective endogenous agonistfor the mu opioid receptor. FEBS Lett 1997;439:152–6.

27] Zangen A, Ikemoto S, Zadina JE, Wise RA. Rewarding and psychomotorstimulant effects of endomorphin-1: anteroposterior differences within theventral tegmental area and lack of effect in nucleus accumbens. J Neurosci2002;22:7225–33.