A New Insight Into Resveratrol as an Atheroprotective Compound Inhibition Of

Atherosclerosis 207 (2009) 420427

Contents lists available at ScienceDirect

Atherosclerosis

journa l homepage: www.e lsev ier .com/ loc

A new teclipid pe ter

Hicham B ed a

Geneviva Research Centb Department oc University ofd Department o anada

a r t i c l

Article history:Received 19 February 2009Received in revised form 16 April 2009Accepted 14 May 2009Available online 22 May 2009

Keywords:AtherosclerosiResveratrolAntioxidantCholesterol ef

Resveratrol, a polyphenolic constituent of red wine, is known for its anti-atherogenic properties and isthought to be benecial in reducing the incidence of cardiovascular diseases (CVD). However, the mecha-nism of action by which it exerts its anti-atherogenic effect remains unclear. In this study, we investigatedthe relationship between the antioxidant effects of resveratrol and its ability to promote cholesterol efux.We measured the formation of conjugated dienes and the rate of lipid peroxidation, and observed thatresveratrol inhibited copper- and irradiation-induced LDL and HDL oxidation as observed by a reduc-tion in oxidation rate and an increase in the lag phase (p < 0.05). We used DPPH screening to measure

1. Introduc

Naturalconcentratiatheroscler(CVD) [1]. Tlic compoupolyphenoltective proppresent in reerties of resCVD of conepidemiolored wine po

CorresponSherbrooke, Q

E-mail add

0021-9150/$ doi:10.1016/j.as

ux

free radical scavenging activity and observed that resveratrol (050M) signicantly reduced the con-tent of free radicals (p < 0.001). Respect to its effect on cholesterol homeostasis, resveratrol also enhancedapoA-1-mediated cholesterol efux (r2 = 0.907, p < 0.05, linear regression) by up-regulating ABCA-1 recep-tors, and reduced cholesterol inux or uptake in J774 macrophages (r2 = 0.89, p < 0.05, linear regression).Incubation of macrophages (J774, THP-1 and MPM) with Fe/ascorbate ion, attenuated apoA-1 and HDL3-mediated cholesterol efux whereas resveratrol (025M) signicantly redressed this attenuation in adose-dependent manner (p < 0.001). Resveratrol thus appears to be a natural antioxidant that enhancescholesterol efux. These properties make it a potential natural antioxidant that could be used to preventand treat CVD.

2009 Elsevier Ireland Ltd. All rights reserved.

tion

compounds have been used to regulate serum lipidons to reduce the incidence of hyperlipidemia andosis, which are responsible for cardiovascular diseases

here has been a recent focus on certain polypheno-nds as possible hypolipidemic agents. Resveratrol, aic compound, has been reported to have atheropro-erties [2,3]. Since resveratrol is a natural polyphenold wine, it has been suggested that the antioxidant prop-

veratrol are responsible for the protective effect againstsuming moderate amounts of red wine. A number ofgical and animal studies have conrmed the ability oflyphenols to inhibit atherosclerotic progression, even

ding author at: Research Center on Aging, 1036 rue Belvdre Sud,C, Canada J1H 4C4. Tel.: +1 819 821 1170x45284; fax: +1 819 829 7141.ress: [email protected] (A. Khalil).

if alcohol intake it self raises high-density lipoprotein (HDL) levels[4]. Animal studies have provided stronger evidence of the positiveeffect of wine polyphenols on plasma lipids. For instance, non-alcoholized red wine increases the plasma concentration of HDLin rats [5]. In addition, red wine polyphenols have been shown toreduce total plasma cholesterol levels in hamsters [6]. Resveratrolhas also been investigated for its antioxidant [7], platelet aggre-gation inhibition [8], smooth muscle cell proliferation inhibition[9], and plasma cholesterol level modulation activities [10]. Resver-atrol also induces LXR- expression in human monocyte-derivedmacrophages and represses the expression of the lipid uptake genesLPL and SR-AII [11].

Macrophage cholesterol accumulation and foam cell forma-tion are the hallmarks of early atherogenesis [12]. Low-densitylipoprotein (LDL) can be taken up and oxidized by macrophages[13], resulting in a signicant increase in macrophage choles-terol mass [12]. Macrophages can also accumulate cholesterol byincreasing the rate of cholesterol biosynthesis and/or decreasingthe rate of HDL-mediated cholesterol efux. HDL is considered anti-

see front matter 2009 Elsevier Ireland Ltd. All rights reserved.therosclerosis.2009.05.017insight into resveratrol as an atheroproroxidation and enhancement of choles

errougui a,b,c, Guillaume Grenier a,d, Soumaya Loue Drouin a,d, Abdelouahed Khalil a,b,

er on Aging, Canadaf Medicine, Geriatrics Service, Universit de Sherbrooke, Canada

Sultan Moulay Slimane, Department of Biology, Beni Mellal, Moroccof Orthopedic Surgery, Faculty of Medicine, Universit de Sherbrooke, Sherbrooke, QC, C

e i n f o a b s t r a c tate /a therosc leros is

tive compound: Inhibition ofol efux,b,

H. Berrougui et al. / Atherosclerosis 207 (2009) 420427 421

atherogenic and plays a key role in protecting LDL against oxidation[14] and maintaining cholesterol homeostasis by reverse choles-terol transport (RCT). As suggested by Glomset [15], RCT involves themovement of cholesterol from peripheral tissues to the liver, whichbegins withfrom periphapolipoprottiated by chcholesterolknown pathorption of Fthe diffusiophase untilscavenger rrectional mmovementterol gradieefux. ABClipids to lipfrom arteriation of HDLin the smallmacrophagactivity has

Despiteresveratrolof atherosclantioxidantagainst ferroxidation [effect on thstill poorlycess still po[11], have rABCG1 mRof the presethe anti-athantioxidanting activityin several clipoproteinon the chol

2. Materia

2.1. Chemic

Acetic acn-butanol,purchasedTetraethoxyethylenedia(2-ME), phol-glutamineiazoletetrazmonophospcollate, and(St. Louis, MCA, USA). D(Houston, TAmerican TRPMI 1640Invitrogen Cserum (FBS

2.2. Measurement of free radical scavenging activity

The free radical scavenging activity of resveratrol was measuredusing the DPPH method as previously described [23]. Briey, a

DPPO atat

red e) wae co

ing fo

100

popro

an) withcomed thhe LDntrifere(pHrd missis

popro

oppindu

usly drieyuspencubtrolED

LDL oLD

oducic Ened [

te thnlikeansion d

Conjuor H

tionsnm tere [

Kinetkine

matipha

. Thehuk

LDL eelec

-B othe transfer of free cholesterol (FC) and phospholipidseral tissue cells to lipid-poor or lipid free (unassociated)ein A1 (apoA-1) and HDL3 [15,16]. This process is ini-olesterol efux, a mechanism by which HDL removesexcess from macrophages. FC efux occurs by threeways: (1) aqueous diffusion, which involves the des-C molecules from the donor lipidwater interface andn of these molecules through the intervening aqueousthey collide with and are adsorbed by an acceptor; (2)

eceptor class B type I (SR-BI)-mediated FC ux in a bidi-anner. Like the aqueous diffusion mechanism, the netof FC via SR-BI depends on the direction of the choles-nt [17]; (3) ATP binding cassette-mediated cholesterolA1 promotes the transfer of cholesterol and phospho-id-poor apoA-1 [18]. In addition to cholesterol efuxl wall cells, ABCA1 is primarily responsible for the initia-formation, principally in the liver and, to a lesser extent,intestine [19]. ABCG1 promotes cholesterol efux from

e foam cells and their transfer to HDL particles, but thisno inuence on overall HDL levels [20].several studies indicating that polyphenols such asplay a role in reducing and preventing the progressionerosis, little is known about their effect on RCT or theirmechanisms. Resveratrol was reported to protect LDLylmyoglobin, peroxynitrite, copper or AAPH-induced

21,22], however, the kinetic of resveratrol-antioxidante lipoproteins as well as on the cells oxidation systemexplored. Moreover, effect of resveratrol on RCT pro-orly investigated, since only one study of Sevov et al.eported that resveratrol modulated LXR, ABCA1 and

NA levels in THP1-derived macrophage. The purposent study was to elucidate the mechanism underlyingerogenic properties of resveratrol by investigating itseffect on lipoprotein particles, its free radical scaveng-

, and especially its effect on cholesterol homeostasisell lines and the relationship between prevention of

s and cells from oxidation by resveratrol, and its impactesterol efux.

ls and methods

als and cell lines

id, sulfuric acid, sodium phosphate, thiobarbituric acid,methanol, ethanol, n-isopropanol, and hexane werefrom Fisher Scientic (Montreal, QC, Canada). 1,1,3,3-propane, d--tocopherol, cupric sulfate (CuSO4),minetetraacetic acid (EDTA), 2--mercaptoethanolrbol myristate acetate (PMA), 1, 2(n)-3H cholesterol,, DPPH (1,1-diphenyl-2-picryl-hydrazyl), methylth-olium (MTT), apoprotein-A1, 8-Br-cyclic adenosinehate (cAMP), bovine serum albumin (BSA), thiogly-dimethylsulfoxide (DMSO) were from SigmaAldrichO, USA). Resveratrol was from Calbiochem (La Jolla,

ialysis bags were from Spectrum Medical IndustriesX, USA). The J774 and THP-1 cells were from the

ype Culture Collection (ATCC) (Manassas, VA, USA). Theand Dulbeccos-modied medium (DMEM) were fromanada Inc (Burlington, Ont. Canada). The fetal bovine

) was from Wisent Inc. (St-Bruno, QC, Canada).

0.1 mMin DMSformedmeasu20Mnegativfollow

AA% =

2.3. Li

Hum2025ethicsapprovsent. Tultraceteins wbufferBradfoRad. M

2.4. Li

2.4.1. CThe

previo[25]. Bwere swere iresvera100M

2.4.2.The

cals pr(AtomdescribestimaLDL, uwith trradiati

2.4.3.LDL

centraat 234elsewh

2.4.4.The

mathegation(Vmax)by Pinc

2.4.5.The

of ApoH solution in ethanol was added to resveratrol dissolvedvarious concentrations (050M). Reactions were per-

room temperature and the absorbance at 518 nm wasvery 30 min for 3 h. Vitamin E (-tocopherol, 10 and

s used a positive control while DMSO was used as antrol. The antioxidant activity was calculated using thermula:

(

AbssampleAbscontrol

) 100

tein preparation

plasma was collected from healthy volunteers (agednormal blood pressure, glycemia, and lipid proles. The

mittee of the Sherbrooke Geriatric University Institutee study, and all subjects provided written informed con-L and HDL3 subfractions were obtained by sequential

ugation as described previously [24]. Isolated lipopro-dialyzed overnight at 4 C in 10 mM sodium phosphate7.0). Protein concentrations were measured using theethod according to the manufacturers instructions (Bio-suga, Ont., Canada).

tein oxidation

er-mediated lipoprotein oxidationction of LDL and HDL3 peroxidation was performed asescribed using transition metal ions as oxidizing agents, lipoproteins (100g/ml of LDL or 200g/ml of HDL3)nded in 10 mM sodium phosphate buffer (pH 7.0) andated in a 10M CuSO4 solution containing 025Mfor 08 h. The reactions were stopped at 4 C adding

TA.

xidation by -radiolysisL was oxidized by exposure to oxygen free radi-ed by -radiolysis using a 60cobalt gamma cell 220

ergy of Canada, Mississauga, Ont., Canada) as previously25]. Water -radiolysis makes it possible to accuratelye nature and quantity of the free radicals that react with

commonly used techniques such as incubating cellstion metal ions. The dose rate was 0.13 Gy/s [26]. Totaloses varied from 0 to 150 Gy.

gated diene formationDL3 oxidized alone or in the presence of various con-of resveratrol (01M) were continuously monitored

o detect the formation of conjugated dienes as described27].

ic prole of LDL oxidationtic prole of lipid peroxidation was characterized using

cal parameters such as the lag phase and the propa-se, i.e., the phase with the maximum oxidation ratese parameters were determined as previously describedand Lichtenberg [28].

lectrophoresistrophoresis mobility of LDL was used as an indication

xidation and was measured using a Titan gel lipopro-

422 H. Berrougui et al. / Atherosclerosis 207 (2009) 420427

tein electrophoretic system (Helena Laboratories, Beaumont, TX,USA). Samples (2l) were separated using 0.6% agarose gels in bar-bital buffer (pH 8.6) (Helena Laboratories, Montreal, QC, Canada)at a constant voltage (80 V) for 45 min. The gels were then ovendried at 75methanol.

2.5. Cell cul

Humanin RPMI 1were suppME (for TH100 U/ml ointo macrosity of 105 cacetate (PMobtained frotion of 2 mlcells wereabdominalin DMEMsity of 105

non-adheremacrophagments desc

2.6. Measur

J774 macplementedwashed witbated withinduce ABCfor 6 h withcholesterol,

We alsopropertiesterol efuxTHP-1 andfor 6 h witwas previoresveratrol)and mousestress by inthe absencemin E for 6cells withand medium10 min) andminute (cpseparatelyPackard Inswas measucholesteroling formula[31].

2.7. Measur

The effecJ774 macro(2Ci/ml) fatrol. The mcells were wtent was m

cholesterol incorporated (% cholesterol inux) using the followingformula: (cpm in the cell/cpm in the cell + medium) 100.

2.8. Statistical analysis

ues aanceion a

uousad p

ults

tioxi

ial exf resper

forment o

svera

batiof L

ion oion wagatias mphaseasuasestrol

duceease

, resptimeantalso

stemdicalSO4 mof fre a md byf rad

matieratprotby t

d tosultsreticatedtrol

ing iteinrticleed eng Mtrol

ase asults

t H2Oig. 3SC and stained with 0.1% (w/v) Fat Red 7B in 95%

tures

THP-1 monocytes and J774 macrophages were grown640 and DMEM medium, respectively. The medialemented with 10% heat-inactivated FBS, 50 mM 2-P-1), 2 mM L-glutamine, 1.5 mg/ml of glucose, and

f penicillin. The differentiation of THP-1 monocytesphages was induced by plating the cells at a den-ells/cm2 in the presence of 100 nM phorbol myristateA) for 96 h. Primary peritoneal macrophages werem Balb/c mice. Five days after an intra-peritoneal injec-of 4% (w/v) thioglycollate medium, the macrophages

harvested by injecting 10 ml of cold DMEM into thecavity [29]. The cells were pelleted and re-suspendedsupplemented with 5% FBS and plated at a den-cells/cm2. They were allowed to adhere for 4 h andnt cells were removed by rinsing with DMEM. Thee-enriched adherent cells were used for the experi-ribed below.

ement of cholesterol efux

rophages were cultured for 48 h in RPMI medium sup-with 1% FBS and 3H cholesterol (2Ci/ml). They wereh serum free DMEM containing 0.2% BSA and then incu-resveratrol (025M) or 0.3 mM 8-Br-cAMP for 12 h toA1 cells [30]. The macrophages were then incubatedpuried apoA-1 (25g/ml), a specic acceptor of freeto assess cholesterol efux.investigated the relationship between the antioxidant

of resveratrol and its effect on HDL-mediated choles-. In one set of experiments, 3H-cholesterol-labeledcAMP-stimulated J774 macrophages were incubatedh 50g/ml of native or oxidized HDL3 (the HDL3usly oxidized in the presence or absence of 1M. In another set of experiments, ABCA1-enriched J774peritoneal macrophages were subjected to oxidative

cubating them with 0.2 mM iron/ascorbate (Fe/Asc) inor presence of 025M resveratrol or 10M vita-

h. Cholesterol efux was assessed by incubating the50g/ml of native HDL3 for 6 h at 37 C. The cells

were then separated by centrifugation (350 g forthe cells were lysed in 0.1 M NaOH. The counts per

m) in the medium and cell lysates were determinedusing a liquid scintillation counter (model 1600 TR;trument Company, Meriden, CT, USA). Cholesterol efuxred by determining the percentage of radiolabeledreleased (% cholesterol efux) using the follow-: (cpm in medium/cpm in the cell + medium) 100

ement of cholesterol inux

t of resveratrol on cholesterol inux was studied usingphages. The cells were incubated with 3H-cholesterolor 24 h in the absence or presence of 025M resver-edium was then removed from the culture dishes. Theashed twice with PBS and lysed. The cholesterol con-

easured by determining the percentage of radiolabeled

Valof variregresscontinGraphP

3. Res

3.1. An

Initeffect oby copdienethe ext

3.2. Re

Incudationformatoxidata proption wor lagwas mtion phresveraand rean incr(Vmax)dationsignic

Wefree syfree rathe Cutermsproposoxidizerange othe forby resv

ThermedpointeOur retrophoattenuresvera

TaklipoproLDL painhibitreduciresverareductOur reagainsities (Fre expressed as the means SEM. One-way analysis(ANOVA) was used for multiple comparisons. Linearnalysis was used to assess the association between twovariables. All statistical analyses were performed usingrism-5 softwareTM.

dant effect of resveratrol

periments were carried out to assess the antioxidantveratrol on LDL peroxidation. LDL oxidation was inducedions or exposure to oxygen free radicals. Conjugatedation in the lipid fraction was measured to determinef LDL peroxidation.

trol inhibits LDL oxidation

ng native human LDL with CuSO4 resulted in the oxi-DL polyunsaturated fatty acids, as indicated by thef conjugated diene (Fig. 1A). The kinetic prole of theas characterized by an initial lag phase followed by

on phase, where the rate of conjugated diene forma-aximal, and then by a decomposition phase. The onsete, before appreciable peroxidation could be observed,red from the intercept of the initiation and propaga-. Interestingly, progressively higher concentrations of(0, 0.1, 0.5, and 1M) inhibited the oxidation of LDL

d its susceptibility to lipid peroxidation, as observed byin the lag phase and a reduction in the oxidation rateectively (Fig. 1A, upper and lower panels). At longer oxi-s (6 and 8 h), the antioxidant effect of resveratrol was

only at 1M (p < 0.01 and 0.001, respectively).assessed the antioxidant effect of resveratrol in an ion-, where oxidation was induced by exposure to OH/O2

s produced by -radioloysis. This method is superior toethod in that it is quantitative and highly selective in

ee radical production [32], which made it possible toechanism for the antioxidant effect of resveratrol. LDLexposure to OH/O2 free radicals produced using a

iation doses (0150 Gy) resulted in a parallel increase inon of conjugated dienes (p < 0.001), which was inhibitedrol in a concentration-dependent manner (Fig. 1B).ective effect of resveratrol on LDL oxidation was con-he apoB electronegative charge measurements, whichthe oxidative modication of the LDL-protein moiety.showed that oxidation of LDL alone increased its elec-mobility, particularly at 75 and 150 Gy. This effect waswhen the LDL were oxidized in the presence of 5M(Fig. 1S).n account that endothelial cells interact directly withs and may exert an oxidative stress especially on thes, we have demonstrated that resveratrol signicantly

ndothelial cells (Eahy926)-induced LDL oxidation byDA formation (p < 0.05) (Fig. 2S). Moreover, effect ofon the glutathione system (glutathione peroxidase andctivities) was investigated in the THP-1 macrophages.

show that resveratrol protects glutathione system2-induced oxidation by preserving GPx and GR activ-).


Fig. 1. Resvera(01M). Condiene formatiexposed to freresveratrol. Pr(10 and 20Mscavenging act20M) was usat least three i

3.3. Free ra

To gainLDL oxidatiscavenginga stable fredonating abmonitoringtion of the Dof increasinpared to thrapidly to sity was obswithin 3 h.25, and 50respectivelyfree radicallated (r2 = 0at identicalscavenging

3.4. Resvera

We asseJ774 macrodeterminetrol possesses antioxidant properties. (A) Kinetics of conjugated diene formation by Cujugated diene formation was followed by monitoring absorbance at 234 nm. The upper

on. The lower panel shows the effect of resveratrol on the maximal rate (Vmax) of cone radicals produced by -radiolysis of ethanolwater mixtures as a function of time (deparations without resveratrol were used as negative controls. LDL (0.1 mg/ml) was prepa) was used as a positive control. Conjugated diene formation was followed by monitoringivity of resveratrol. DPPH was incubated with 050M resveratrol for 180 min and the aed as a positive control. The scavenging activity of resveratrol is expressed as the percentndependent experiments.

dical scavenging activity of resveratrol

more insight into the ability of resveratrol to preventon, we studied the kinetics of resveratrol free radicalactivity by allowing resveratrol to react with DPPH,e radical. The DPPH assay measures the hydrogen-ility of antioxidants over a relatively short period bythe decrease in absorbance resulting from the reduc-PPH free radical form to the DPPH-H form. The effectg concentrations of resveratrol (050M) was com-

at of vitamin E (10 and 25M). Both compounds actedcavenge free radicals since over than 50% of their activ-erved in the rst 30 min, and at the remaining 50%,The antioxidant activity (AA%) of resveratrol at 5, 10,M was 16 3.2, 21.6 1.8, 45.4 1.0, and 69.3 0.8,. As shown in Fig. 1C, the reaction kinetics of the DPPHand resveratrol concentrations were negatively corre-.96, p = 0.0032). Interestingly, resveratrol and vitamin Econcentrations exhibited equivalent DPPH free radicalactivity.

trol enhances cholesterol efux

ssed the effect of resveratrol (106 to 103 M) onphage viability using the MTT colorimetric assay tothe range of resveratrol concentrations that would

be used inwas obtainwas obtainapoA1-medendothelialenhancedgated the eefux pathloaded cellonly withever, whenresveratrolthe apoA1-ated in a rep < 0.05, linstand the mcholesterolABCA1 promacrophagresulted inshown).

3.5. Resvera

Cholestemolecules fSO4-induced LDL oxidation in the absence or presence of resveratrolpanel shows the effect of resveratrol on the lag phase of conjugatedjugated diene formation. (B) Formation of conjugated diene in LDLose: 0.13 Gy/s). The LDL preparations were incubated with 010Mred in oxygenated 10 mM sodium phosphate buffer (pH 7). Vitamin Eabsorption at 234 nm (234 nm = 27,000 M1/cm). (C) DPPH free radicalbsorbance at 518 nm was monitored every 30 min. Vitamin E (10 andage of remaining DPPH. Results are expressed as the means SEM of

the cholesterol homeostasis experiments. The IC50ed with 114 1.3M of resveratrol while the IC20ed with 30M (Fig. 4S). Effect of resveratrol on theiated cholesterol efux was also studied in the Eahy926cells. Our results show that resveratrol signicantly

the cholesterol efux (Fig. 5S-A). We next investi-ffect of resveratrol on ABCA1-dependent cholesterol

ways in J774 macrophages. Exposing 3H-cholesterol-s to apoA-1 (cAMP-free) for 6 h (time-range to reactABCA1) resulted in a low-cholesterol efux. How-

the macrophages were pretreated overnight with(025M), with 0.3 mM cAMP as a positive control,induced cholesterol efux was signicantly potenti-sveratrol concentration-dependent manner (r2 = 0.907,ear regression) (Fig. 2A). To more clearly under-echanism of action of resveratrol on ABCA1-mediatedefux, we measured the effect of resveratrol on the

tein expression in J774 macrophages. Incubating thees in the presence of resveratrol (3, 6, 8, and 16 h)

an increase in ABCA1 protein expression (data not

trol reduces cholesterol inux

rol inux is dened as the movement of cholesterolrom the extra-cellular environment to cells. This pro-


Fig. 2. Resverresveratrol onwere treated wbated with 25trace shows aresveratrol conindependent eabsence or preinux is exprescell lysates. Reexperiments.

cess, whichrole in celluon cholestewe observeresveratrol(r2 = 0.89, patrol decrecells (EahyB).atrol increases cholesterol efux and reduces inux. (A) Effect ofapoA1-mediated cholesterol efux. 3H-cholesterol-loaded J774 cellsith various concentrations of resveratrol (025M) and then incu-g/ml of apoA1. cAMP (300M) was used as a positive control. Thelinear correlation between apoA1-mediated cholesterol efux andcentrations. All results are expressed as means SEM of at least threexperiments. (B) J774 cells were labeled with 3H-cholesterol in thesence of various concentrations of resveratrol (025M). Cholesterolsed as the difference in the cholesterol content of the medium and thesults are expressed as the means SEM of at least three independent

is mainly observed in macrophages, plays a regulatorylar cholesterol homeostasis. The effect of resveratrol

rol inux was investigated in J774 macrophages andd that the inux of 3H-cholesterol in the presence ofwas signicantly reduced in a dose-dependent manner= 0.015) (Fig. 2B). Moreover, we show that resver-

ased signicantly cholesterol uptake by endothelial926) from 3H-cholesterol-enriched media (Fig. 5S-

Fig. 3. Resvermediated chomacrophagesbrate for 16 h.and cholesterolinear correlatcentrations. V

3.6. Resveracholesterol e

Oxidativas previousexpressioneffect of resprimary maFe/Asc. Wesignicantlby resverateffect wasoxidation ocapacity tocapacity ofoxidative stloaded witdized in th(treated). Rin a concenconjugatedity of HDL3atrol protects macrophages against oxidation and promotes HDL3-lesterol efux. J774 macrophages (A) and mouse peritoneal

(MPM) (B) were loaded with 3H-cholesterol and allowed to equili-The macrophages were stressed with 0.2 mM iron/ascorbate (Fe/Asc)l efux was assessed using 50g/ml of HDL3. The panels show theion between HDL3-mediated cholesterol efux and resveratrol con-itamin E (10M) was used as a positive control.

trol protects against lipid peroxidation and promotesfux

e damage to macrophages impairs cholesterol efuxly demonstrated by an impairment of ABCA1 proteinunder Fe/Asc stress Marcil et al. [33]. We investigated theveratrol on cholesterol efux in J774 (Fig. 3A) and mousecrophages (Fig. 3B) under oxidative stress induced byobserved that HDL3-mediated cholesterol efux was

y impaired by Fe/Asc, but that the effect was restoredrol in concentration-dependant manner (p < 0.05). Thisalso observed with THP-1 macrophages (Fig. 6S). Thef HDL has been reported to signicant decrease theirmediate cholesterol efux [34]. We thus assessed theresveratrol to preserve the functionality of HDL underress conditions. ABCA1-enriched J774 cells previously

h cholesterol were incubated for 6 h with HDL3 oxi-e absence (control) or in the presence of resveratrolesveratrol protected HDL3 against Cu-induced oxidationtration-dependent manner as shown by the decrease indiene formation (Fig. 4A). It also maintained the capac-to mediate cholesterol efux (p < 0.05) (Fig. 4B).


Fig. 4. Resverefux. (A) Coabsence or prebated with Cuof 02M resincubated witence or absenindependent e

4. Discussi

Dietarypreventiveory of athestudies (epispective coprogressionnols and prisk reductiby protectiresveratrolprimary imobservationhigh-fat dienomenon khave been

ties of resveratrol, including inhibition of lipid peroxidation [22],modulation of platelet aggregation [8], inhibition of smooth mus-cle cell proliferation [9], and reduction of macrophage-induced

mation [39]. Despite numerous studies showing the effecterat

sclertrol

argebot

roscscler

whn bey ofthe simpaairin

terolctly

ors inmatiinamof resvatheroresvera

A lrole inof atheatheroslowedrelatioseveritis notstressby impcholesbe direreceptcell foratrol protects HDL3 from copper oxidation and increases cholesterolnjugated diene formation in CuSO4-induced HDL oxidation in thesence of resveratrol as a function of time. HDL3 proteins were incu-SO4 for 8 h (in a time course manner) in the absence or presenceveratrol. (B) 3H- cholesterol efux from human THP-1 macrophagesh 50g/ml of copper-oxidized HDL3 for 0, 2, and 4 h in the pres-ce of 1M resveratrol. Results are the means SEM of at least threexperiments.

on

antioxidants have attracted considerable attention asand therapeutic agents because of the oxidative the-rosclerosis [35]. A number of dietary antioxidant intakedemiological, casecontrol, and prospective and retro-horts) have shown that antioxidants can prevent CVD

[36]. Indeed, the consumption of foods rich in phe-olyphenols has been positively correlated with CVDon by slowing atherosclerosis progression, principallyng lipoproteins from lipid peroxidation [37]. Becauseis present in a variety of foods, including grapes, apetus for research on resveratrol was the paradoxical

that a low incidence of CVD could co-exist with at intake and moderate consumption of red wine, a phe-nown as the French paradox [38]. Various mechanismsproposed to explain the anti-atherosclerotic proper-

Our resuradiolysis-imanner byextendingalso been rE disappearof LDL [44]dant effectsin lipoprotgroups, is veety of lipopfatty acids [

LDL oxidand biologimoiety (aptein moietyalteration ioxysterols (we showedation of apmobility of

To clarifoxidation, wresveratrol,of apoB. Retively stablereduce copand SFR thanother freeities in theeffect on thprole suggcopper bindtion is in ag[7], who shions and caare also in athat resverareaction pro

HDL pla[46]. This pchemical prenables celers, have praffects therol on the lipid metabolism and the progression ofosis, little is known about the antioxidant properties ofand their effect on cholesterol homeostasis.body of evidence indicates that oxLDL plays a key

h the early and more advanced inammatory stageslerosis lesions [40]. For example, oxLDL is present inotic lesions [41], the progression of atherosclerosis isen oxidation is inhibited [42,43], and there is a cor-tween the ability of LDL to resist oxidation and thecoronary atherosclerosis [42]. However, LDL oxidationole factor involved in atherogenesis. In vivo oxidativeirs the anti-atherogenic properties of HDL, especiallyg its antioxidant activity and its capacity to enhanceefux and promote RCT [34]. Moreover, oxidation can

induced in macrophages, which alters the expression ofvolved in cholesterol ux, which in turn inuence foamon and atherosclerosis development [33].lts showed that resveratrol prevented copper- and -

nduced LDL peroxidation in a concentration-dependentdecreasing the formation of conjugated dienes, thus

the lag phase and lowering the oxidation rate. It haseported that resveratrol decreases the rate of vitaminance and maintains the endogenous vitamin E content

. A wide variety of natural products exert their antioxi-by preventing the degradation of endogenous vitamins

eins. Resveratrol, which has three phenolic hydroxylry lipophilic, enabling it to associate with the lipid moi-roteins and prevent the oxidation of their unsaturated44].ation is characterized by alterations in the structural

cal properties of the lipid fraction and the apolipoproteinoB), including the early fragmentation of the pro-, which contains sensitive amino acid residues. This

s followed by cross-linking of reactive aldehydes andend products of lipid peroxidation). In the present study,that resveratrol reduced the-radiolysis-induced alter-o-B, as shown by the decrease in the electrophoreticLDL.y the mechanism by which resveratrol reduces LDLe investigated the free radical scavenging activity of

which may prevent chain-breaking and the alterationsveratrol may interact with free radicals to form rela-

free radicals (SFR) and non-radicals (NR). It may alsoper, resulting in the formation of a non-radical productat, under conditions of high-oxidative stress, quenchradical. However, in addition to its broad range of activ-

lag phase, resveratrol also reduced the Vmax but had noe ODmax, except at high concentrations. This antioxidantested that resveratrol might also act as an inhibitor ofing via interactions with apolipoproteins. This observa-

reement with the results reported by Belguendouz et al.owed that resveratrol is a potent chelator of free coppern also remove copper ions bound to apo-B. Our resultsgreement with those of Zini et al. [45], who suggestedtrol inhibits the lipid peroxidation induced by Fentonducts.

ys an important anti-atherogenic role by mediating RCTrocess, which is in part dependent on the physico-operties of HDL and on the oxidative state of the cells,ls to pump out excess free cholesterol. We, and oth-eviously demonstrated that HDL oxidation signicantlycapacity of HDL to promote cholesterol efux from


macrophages [27,34]. The oxidation of HDL3 alters the uidity ofthe phospholipidic layer, modies the structure of apoproteins, anddecreases paraoxonase 1 activity [34,47]. All these modicationshave an effect on the normal interactions between HDL3 compo-nents and m[48].

When JapoA1-medresveratrolnumber ofincluding dchanges toing, the avaOver-expremembraneoxidase acttrations ormembrane.expressionof Sevov etand elevatethe up-regupathways inprocess, wh[51] and resumption. Wcholesterolwhich ties itein lipase (by macroph

Oxidativtems, and rimpairmenwhich causOxidative s[33]. In theatrol on chocomplex, wterol efux.results we owith thosederived maexpressionIncubatingtrol signicHDL3, probaface receptreported wiantioxidant

The oxiddation markthiobarbituCompositiophysico-cheorder, and ecations areof HDL. Forpromote thresveratrolits capacityto the presethe integrit

We contets of conresveratrolmation and

ndings on the intracellular signaling pathways modulating thecholesterol efux process will help lay the foundation for devel-oping new therapeutic approaches to preventing and treatingatherosclerosis and cardiovascular diseases. Although, our study

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774 macrophages were incubated with resveratrol,iated cholesterol efux increased, suggesting thatup-regulates this process by up-regulating ABCA1. A

mechanisms have been proposed to explain this effect,irect binding of apoA-1 to ABCA1, ABCA1-mediatedthe plasma membrane that stimulate apoA1 bind-

ilability of phospholipids, and cholesterol efux [49].ssion of ABCA1 alters the morphology of the plasma[18] and increases apoA-1 binding [50] and cholesterolivity, which point to changes in cholesterol concen-distribution within the external leaet of the plasmaOur results showed that resveratrol-stimulated ABCA1

in J774 macrophages, which is in agreement with thoseal. [11], who showed that resveratrol induces LXR-

d ABCA1 and ABCG1 mRNA levels. This suggests thatlation of ABCA1 protein expression may be one of thevolved in the resveratrol-mediated cholesterol efuxich would be consistent with the increased HDL levels

duced atherosclerosis seen following polyphenol con-e also showed that resveratrol reduces the inux of

into J774 macrophages in a dose-dependent manner,n with the fact that resveratrol down-regulates lipopro-LPL) and SR-AII, which promote increased lipid uptakeages [11].e stress is a continuous process in all physiological sys-esults in the oxidation of various molecules and the

t of their functions. HDL can also be oxidized in vivo,es a loss of its anti-atherogenic proprieties [52,53].

tress also inuences cholesterol efux in macrophagespresent study, we also investigated the effect of resver-lesterol efux in J774 macrophages stressed by a Fe/Aschich induces lipid peroxidation [54] and reduces choles-While great care should be taken in extrapolating thebtained with J774 macrophages, they are in agreementof a previous study using mouse primary peritoneal-crophages [33]. Oxidative stress can also reduce theof ABCA1, LXR, and PPAR, but has no effect on SR-BI.macrophages with Fe/Asc in the presence of resvera-antly restored cholesterol efux from macrophages tobly by suppressing the effect of Fe/Asc on the cell sur-

ors involved in this process. This effect has also beenth vitamin E and butylhydroxytoluene (BHT), two others [33].ation of HDL results in increased levels of lipid peroxi-ers, including conjugated dienes, lipid hydroperoxides,

ric acid reactive substances (TBARS), and aldehydes.nal changes are associated with alterations in themical properties of HDL, including uidity, molecular

lectric charges [55]. Compositional and structural modi-also associated with changes in the biological activities

instance, oxidizing HDL in vitro decreases its ability toe RCT process [56]. In the present study, we showed thatinhibited the oxidation of HDL3 and helped to maintainto mediate cholesterol efux. This effect may be relatedrvation of the physico-chemical properties of HDL and

y of protein moieties like apoA-1 and PON1.ributed to a better understanding of the potential ben-suming foods rich in polyphenols by showing that

protects against lipoprotein oxidation and foam cell for-promotes cholesterol efux from macrophages. Our

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gements

rk was supported by the Canadian Institute of HealthIHR). G. Grenier and A. Khalil are recipients of a Junior 1igator award, respectively, from the Fonds de RechercheQubec (FRSQ).

. Supplementary data

entary data associated with this article can be found, inersion, at doi:10.1016/j.atherosclerosis.2009.05.017.

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A new insight into resveratrol as an atheroprotective compound: Inhibition of lipid peroxidation and enhancement of cholesterol effluxIntroductionMaterials and methodsChemicals and cell linesMeasurement of free radical scavenging activityLipoprotein preparationLipoprotein oxidationCopper-mediated lipoprotein oxidationLDL oxidation by gamma-radiolysisConjugated diene formationKinetic profile of LDL oxidationLDL electrophoresis

Cell culturesMeasurement of cholesterol effluxMeasurement of cholesterol influxStatistical analysis

ResultsAntioxidant effect of resveratrolResveratrol inhibits LDL oxidationFree radical scavenging activity of resveratrolResveratrol enhances cholesterol effluxResveratrol reduces cholesterol influxResveratrol protects against lipid peroxidation and promotes cholesterol efflux

DiscussionAcknowledgementsSupplementary dataReferences

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A New Insight Into Resveratrol as an Atheroprotective Compound Inhibition Of