Atherosclerosis 207 (2009) 420427

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Atherosclerosis
journal homepage: www.elsevier.com/locate/atherosclerosis

A new insight into resveratrol as an atheroprotective compound: Inhibition of lipid peroxidation and enhancement of cholesterol efux
Hicham Berrougui a,b,c , Guillaume Grenier a,d , Soumaya Loued a,b , Genevive Drouin a,d , Abdelouahed Khalil a,b,
a

Research Center on Aging, Canada Department of Medicine, Geriatrics Service, Universit de Sherbrooke, Canada University of Sultan Moulay Slimane, Department of Biology, Beni Mellal, Morocco d Department of Orthopedic Surgery, Faculty of Medicine, Universit de Sherbrooke, Sherbrooke, QC, Canada
b c

a r t i c l e

i n f o

a b s t r a c t
Resveratrol, a polyphenolic constituent of red wine, is known for its anti-atherogenic properties and is thought to be benecial in reducing the incidence of cardiovascular diseases (CVD). However, the mechanism of action by which it exerts its anti-atherogenic effect remains unclear. In this study, we investigated the 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 that resveratrol inhibited copper- and irradiation-induced LDL and HDL oxidation as observed by a reduction in oxidation rate and an increase in the lag phase (p < 0.05). We used DPPH screening to measure free radical scavenging activity and observed that resveratrol (050 M) signicantly reduced the content of free radicals (p < 0.001). Respect to its effect on cholesterol homeostasis, resveratrol also enhanced apoA-1-mediated cholesterol efux (r2 = 0.907, p < 0.05, linear regression) by up-regulating ABCA-1 receptors, 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 (025 M) signicantly redressed this attenuation in a dose-dependent manner (p < 0.001). Resveratrol thus appears to be a natural antioxidant that enhances cholesterol efux. These properties make it a potential natural antioxidant that could be used to prevent and treat CVD. 2009 Elsevier Ireland Ltd. All rights reserved.

Article history: Received 19 February 2009 Received in revised form 16 April 2009 Accepted 14 May 2009 Available online 22 May 2009 Keywords: Atherosclerosis Resveratrol Antioxidant Cholesterol efux

1. Introduction Natural compounds have been used to regulate serum lipid concentrations to reduce the incidence of hyperlipidemia and atherosclerosis, which are responsible for cardiovascular diseases (CVD) [1]. There has been a recent focus on certain polyphenolic compounds as possible hypolipidemic agents. Resveratrol, a polyphenolic compound, has been reported to have atheroprotective properties [2,3]. Since resveratrol is a natural polyphenol present in red wine, it has been suggested that the antioxidant properties of resveratrol are responsible for the protective effect against CVD of consuming moderate amounts of red wine. A number of epidemiological and animal studies have conrmed the ability of red wine polyphenols to inhibit atherosclerotic progression, even

Corresponding author at: Research Center on Aging, 1036 rue Belvdre Sud, Sherbrooke, QC, Canada J1H 4C4. Tel.: +1 819 821 1170x45284; fax: +1 819 829 7141. E-mail address: Abdelouahed.Khalil@USherbrooke.ca (A. Khalil). 0021-9150/$ see front matter 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.atherosclerosis.2009.05.017

if alcohol intake it self raises high-density lipoprotein (HDL) levels [4]. Animal studies have provided stronger evidence of the positive effect of wine polyphenols on plasma lipids. For instance, nonalcoholized red wine increases the plasma concentration of HDL in rats [5]. In addition, red wine polyphenols have been shown to reduce total plasma cholesterol levels in hamsters [6]. Resveratrol has also been investigated for its antioxidant [7], platelet aggregation inhibition [8], smooth muscle cell proliferation inhibition [9], and plasma cholesterol level modulation activities [10]. Resveratrol also induces LXR- expression in human monocyte-derived macrophages and represses the expression of the lipid uptake genes LPL and SR-AII [11]. Macrophage cholesterol accumulation and foam cell formation are the hallmarks of early atherogenesis [12]. Low-density lipoprotein (LDL) can be taken up and oxidized by macrophages [13], resulting in a signicant increase in macrophage cholesterol mass [12]. Macrophages can also accumulate cholesterol by increasing the rate of cholesterol biosynthesis and/or decreasing the rate of HDL-mediated cholesterol efux. HDL is considered anti-

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atherogenic and plays a key role in protecting LDL against oxidation [14] and maintaining cholesterol homeostasis by reverse cholesterol transport (RCT). As suggested by Glomset [15], RCT involves the movement of cholesterol from peripheral tissues to the liver, which begins with the transfer of free cholesterol (FC) and phospholipids from peripheral tissue cells to lipid-poor or lipid free (unassociated) apolipoprotein A1 (apoA-1) and HDL3 [15,16]. This process is initiated by cholesterol efux, a mechanism by which HDL removes cholesterol excess from macrophages. FC efux occurs by three known pathways: (1) aqueous diffusion, which involves the desorption of FC molecules from the donor lipidwater interface and the diffusion of these molecules through the intervening aqueous phase until they collide with and are adsorbed by an acceptor; (2) scavenger receptor class B type I (SR-BI)-mediated FC ux in a bidirectional manner. Like the aqueous diffusion mechanism, the net movement of FC via SR-BI depends on the direction of the cholesterol gradient [17]; (3) ATP binding cassette-mediated cholesterol efux. ABCA1 promotes the transfer of cholesterol and phospholipids to lipid-poor apoA-1 [18]. In addition to cholesterol efux from arterial wall cells, ABCA1 is primarily responsible for the initiation of HDL formation, principally in the liver and, to a lesser extent, in the small intestine [19]. ABCG1 promotes cholesterol efux from macrophage foam cells and their transfer to HDL particles, but this activity has no inuence on overall HDL levels [20]. Despite several studies indicating that polyphenols such as resveratrol play a role in reducing and preventing the progression of atherosclerosis, little is known about their effect on RCT or their antioxidant mechanisms. Resveratrol was reported to protect LDL against ferrylmyoglobin, peroxynitrite, copper or AAPH-induced oxidation [21,22], however, the kinetic of resveratrol-antioxidant effect on the lipoproteins as well as on the cells oxidation system still poorly explored. Moreover, effect of resveratrol on RCT process still poorly investigated, since only one study of Sevov et al. [11], have reported that resveratrol modulated LXR, ABCA1 and ABCG1 mRNA levels in THP1-derived macrophage. The purpose of the present study was to elucidate the mechanism underlying the anti-atherogenic properties of resveratrol by investigating its antioxidant effect on lipoprotein particles, its free radical scavenging activity, and especially its effect on cholesterol homeostasis in several cell lines and the relationship between prevention of lipoproteins and cells from oxidation by resveratrol, and its impact on the cholesterol efux.

2.2. Measurement of free radical scavenging activity The free radical scavenging activity of resveratrol was measured using the DPPH method as previously described [23]. Briey, a 0.1 mM DPPH solution in ethanol was added to resveratrol dissolved in DMSO at various concentrations (050 M). Reactions were performed at room temperature and the absorbance at 518 nm was measured every 30 min for 3 h. Vitamin E (-tocopherol, 10 and 20 M) was used a positive control while DMSO was used as a negative control. The antioxidant activity was calculated using the following formula: AA% = 100 Abssample Abscontrol 100

2.3. Lipoprotein preparation Human plasma was collected from healthy volunteers (aged 2025) with normal blood pressure, glycemia, and lipid proles. The ethics committee of the Sherbrooke Geriatric University Institute approved the study, and all subjects provided written informed consent. The LDL and HDL3 subfractions were obtained by sequential ultracentrifugation as described previously [24]. Isolated lipoproteins were dialyzed overnight at 4 C in 10 mM sodium phosphate buffer (pH 7.0). Protein concentrations were measured using the Bradford method according to the manufacturers instructions (BioRad. Mississuga, Ont., Canada). 2.4. Lipoprotein oxidation 2.4.1. Copper-mediated lipoprotein oxidation The induction of LDL and HDL3 peroxidation was performed as previously described using transition metal ions as oxidizing agents [25]. Briey, lipoproteins (100 g/ml of LDL or 200 g/ml of HDL3 ) were suspended in 10 mM sodium phosphate buffer (pH 7.0) and were incubated in a 10 M CuSO4 solution containing 025 M resveratrol for 08 h. The reactions were stopped at 4 C adding 100 M EDTA. 2.4.2. LDL oxidation by -radiolysis The LDL was oxidized by exposure to oxygen free radicals produced by -radiolysis using a 60 cobalt gamma cell 220 (Atomic Energy of Canada, Mississauga, Ont., Canada) as previously described [25]. Water -radiolysis makes it possible to accurately estimate the nature and quantity of the free radicals that react with LDL, unlike commonly used techniques such as incubating cells with transition metal ions. The dose rate was 0.13 Gy/s [26]. Total radiation doses varied from 0 to 150 Gy. 2.4.3. Conjugated diene formation LDL or HDL3 oxidized alone or in the presence of various concentrations of resveratrol (01 M) were continuously monitored at 234 nm to detect the formation of conjugated dienes as described elsewhere [27]. 2.4.4. Kinetic prole of LDL oxidation The kinetic prole of lipid peroxidation was characterized using mathematical parameters such as the lag phase and the propagation phase, i.e., the phase with the maximum oxidation rate (Vmax ). These parameters were determined as previously described by Pinchuk and Lichtenberg [28]. 2.4.5. LDL electrophoresis The electrophoresis mobility of LDL was used as an indication of Apo-B oxidation and was measured using a Titan gel lipopro-

2. Materials and methods 2.1. Chemicals and cell lines Acetic acid, sulfuric acid, sodium phosphate, thiobarbituric acid, n-butanol, methanol, ethanol, n-isopropanol, and hexane were purchased from Fisher Scientic (Montreal, QC, Canada). 1,1,3,3Tetraethoxypropane, d--tocopherol, cupric sulfate (CuSO4 ), ethylenediaminetetraacetic acid (EDTA), 2--mercaptoethanol (2-ME), phorbol myristate acetate (PMA), 1, 2(n)-3 H cholesterol, l-glutamine, DPPH (1,1-diphenyl-2-picryl-hydrazyl), methylthiazoletetrazolium (MTT), apoprotein-A1, 8-Br-cyclic adenosine monophosphate (cAMP), bovine serum albumin (BSA), thioglycollate, and dimethylsulfoxide (DMSO) were from SigmaAldrich (St. Louis, MO, USA). Resveratrol was from Calbiochem (La Jolla, CA, USA). Dialysis bags were from Spectrum Medical Industries (Houston, TX, USA). The J774 and THP-1 cells were from the American Type Culture Collection (ATCC) (Manassas, VA, USA). The RPMI 1640 and Dulbeccos-modied medium (DMEM) were from Invitrogen Canada Inc (Burlington, Ont. Canada). The fetal bovine serum (FBS) was from Wisent Inc. (St-Bruno, QC, Canada).

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tein electrophoretic system (Helena Laboratories, Beaumont, TX, USA). Samples (2 l) were separated using 0.6% agarose gels in barbital buffer (pH 8.6) (Helena Laboratories, Montreal, QC, Canada) at a constant voltage (80 V) for 45 min. The gels were then oven dried at 75 C and stained with 0.1% (w/v) Fat Red 7B in 95% methanol. 2.5. Cell cultures Human THP-1 monocytes and J774 macrophages were grown in RPMI 1640 and DMEM medium, respectively. The media were supplemented with 10% heat-inactivated FBS, 50 mM 2ME (for THP-1), 2 mM L-glutamine, 1.5 mg/ml of glucose, and 100 U/ml of penicillin. The differentiation of THP-1 monocytes into macrophages was induced by plating the cells at a density of 105 cells/cm2 in the presence of 100 nM phorbol myristate acetate (PMA) for 96 h. Primary peritoneal macrophages were obtained from Balb/c mice. Five days after an intra-peritoneal injection of 2 ml of 4% (w/v) thioglycollate medium, the macrophages cells were harvested by injecting 10 ml of cold DMEM into the abdominal cavity [29]. The cells were pelleted and re-suspended in DMEM supplemented with 5% FBS and plated at a density of 105 cells/cm2 . They were allowed to adhere for 4 h and non-adherent cells were removed by rinsing with DMEM. The macrophage-enriched adherent cells were used for the experiments described below. 2.6. Measurement of cholesterol efux J774 macrophages were cultured for 48 h in RPMI medium supplemented with 1% FBS and 3 H cholesterol (2 Ci/ml). They were washed with serum free DMEM containing 0.2% BSA and then incubated with resveratrol (025 M) or 0.3 mM 8-Br-cAMP for 12 h to induce ABCA1 cells [30]. The macrophages were then incubated for 6 h with puried apoA-1 (25 g/ml), a specic acceptor of free cholesterol, to assess cholesterol efux. We also investigated the relationship between the antioxidant properties of resveratrol and its effect on HDL-mediated cholesterol efux. In one set of experiments, 3 H-cholesterol-labeled THP-1 and cAMP-stimulated J774 macrophages were incubated for 6 h with 50 g/ml of native or oxidized HDL3 (the HDL3 was previously oxidized in the presence or absence of 1 M resveratrol). In another set of experiments, ABCA1-enriched J774 and mouse peritoneal macrophages were subjected to oxidative stress by incubating them with 0.2 mM iron/ascorbate (Fe/Asc) in the absence or presence of 025 M resveratrol or 10 M vitamin E for 6 h. Cholesterol efux was assessed by incubating the cells with 50 g/ml of native HDL3 for 6 h at 37 C. The cells and medium were then separated by centrifugation (350 g for 10 min) and the cells were lysed in 0.1 M NaOH. The counts per minute (cpm) in the medium and cell lysates were determined separately using a liquid scintillation counter (model 1600 TR; Packard Instrument Company, Meriden, CT, USA). Cholesterol efux was measured by determining the percentage of radiolabeled cholesterol released (% cholesterol efux) using the following formula: (cpm in medium/cpm in the cell + medium) 100 [31]. 2.7. Measurement of cholesterol inux The effect of resveratrol on cholesterol inux was studied using J774 macrophages. The cells were incubated with 3 H-cholesterol (2 Ci/ml) for 24 h in the absence or presence of 025 M resveratrol. The medium was then removed from the culture dishes. The cells were washed twice with PBS and lysed. The cholesterol content was measured by determining the percentage of radiolabeled

cholesterol incorporated (% cholesterol inux) using the following formula: (cpm in the cell/cpm in the cell + medium) 100. 2.8. Statistical analysis Values are expressed as the means SEM. One-way analysis of variance (ANOVA) was used for multiple comparisons. Linear regression analysis was used to assess the association between two continuous variables. All statistical analyses were performed using GraphPad prism-5 softwareTM . 3. Results 3.1. Antioxidant effect of resveratrol Initial experiments were carried out to assess the antioxidant effect of resveratrol on LDL peroxidation. LDL oxidation was induced by copper ions or exposure to oxygen free radicals. Conjugated diene formation in the lipid fraction was measured to determine the extent of LDL peroxidation. 3.2. Resveratrol inhibits LDL oxidation Incubating native human LDL with CuSO4 resulted in the oxidation of LDL polyunsaturated fatty acids, as indicated by the formation of conjugated diene (Fig. 1A). The kinetic prole of the oxidation was characterized by an initial lag phase followed by a propagation phase, where the rate of conjugated diene formation was maximal, and then by a decomposition phase. The onset or lag phase, before appreciable peroxidation could be observed, was measured from the intercept of the initiation and propagation phases. Interestingly, progressively higher concentrations of resveratrol (0, 0.1, 0.5, and 1 M) inhibited the oxidation of LDL and reduced its susceptibility to lipid peroxidation, as observed by an increase in the lag phase and a reduction in the oxidation rate (Vmax ), respectively (Fig. 1A, upper and lower panels). At longer oxidation times (6 and 8 h), the antioxidant effect of resveratrol was signicant only at 1 M (p < 0.01 and 0.001, respectively). We also assessed the antioxidant effect of resveratrol in an ionfree system, where oxidation was induced by exposure to OH/O2 free radicals produced by -radioloysis. This method is superior to the CuSO4 method in that it is quantitative and highly selective in terms of free radical production [32], which made it possible to propose a mechanism for the antioxidant effect of resveratrol. LDL oxidized by exposure to OH/O2 free radicals produced using a range of radiation doses (0150 Gy) resulted in a parallel increase in the formation of conjugated dienes (p < 0.001), which was inhibited by resveratrol in a concentration-dependent manner (Fig. 1B). The protective effect of resveratrol on LDL oxidation was conrmed by the apoB electronegative charge measurements, which pointed to the oxidative modication of the LDL-protein moiety. Our results showed that oxidation of LDL alone increased its electrophoretic mobility, particularly at 75 and 150 Gy. This effect was attenuated when the LDL were oxidized in the presence of 5 M resveratrol (Fig. 1S). Taking in account that endothelial cells interact directly with lipoproteins and may exert an oxidative stress especially on the LDL particles, we have demonstrated that resveratrol signicantly inhibited endothelial cells (Eahy926)-induced LDL oxidation by reducing MDA formation (p < 0.05) (Fig. 2S). Moreover, effect of resveratrol on the glutathione system (glutathione peroxidase and reductase activities) was investigated in the THP-1 macrophages. Our results show that resveratrol protects glutathione system against H2 O2 -induced oxidation by preserving GPx and GR activities (Fig. 3S).

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Fig. 1. Resveratrol possesses antioxidant properties. (A) Kinetics of conjugated diene formation by CuSO4 -induced LDL oxidation in the absence or presence of resveratrol (01 M). Conjugated diene formation was followed by monitoring absorbance at 234 nm. The upper panel shows the effect of resveratrol on the lag phase of conjugated diene formation. The lower panel shows the effect of resveratrol on the maximal rate (Vmax ) of conjugated diene formation. (B) Formation of conjugated diene in LDL exposed to free radicals produced by -radiolysis of ethanolwater mixtures as a function of time (dose: 0.13 Gy/s). The LDL preparations were incubated with 010 M resveratrol. Preparations without resveratrol were used as negative controls. LDL (0.1 mg/ml) was prepared in oxygenated 10 mM sodium phosphate buffer (pH 7). Vitamin E (10 and 20 M) was used as a positive control. Conjugated diene formation was followed by monitoring absorption at 234 nm (234 nm = 27,000 M1 /cm). (C) DPPH free radical scavenging activity of resveratrol. DPPH was incubated with 050 M resveratrol for 180 min and the absorbance at 518 nm was monitored every 30 min. Vitamin E (10 and 20 M) was used as a positive control. The scavenging activity of resveratrol is expressed as the percentage of remaining DPPH. Results are expressed as the means SEM of at least three independent experiments.

3.3. Free radical scavenging activity of resveratrol To gain more insight into the ability of resveratrol to prevent LDL oxidation, we studied the kinetics of resveratrol free radical scavenging activity by allowing resveratrol to react with DPPH, a stable free radical. The DPPH assay measures the hydrogendonating ability of antioxidants over a relatively short period by monitoring the decrease in absorbance resulting from the reduction of the DPPH free radical form to the DPPH-H form. The effect of increasing concentrations of resveratrol (050 M) was compared to that of vitamin E (10 and 25 M). Both compounds acted rapidly to scavenge free radicals since over than 50% of their activity was observed in the rst 30 min, and at the remaining 50%, within 3 h. The antioxidant activity (AA%) of resveratrol at 5, 10, 25, and 50 M was 16 3.2, 21.6 1.8, 45.4 1.0, and 69.3 0.8, respectively. As shown in Fig. 1C, the reaction kinetics of the DPPH free radical and resveratrol concentrations were negatively correlated (r2 = 0.96, p = 0.0032). Interestingly, resveratrol and vitamin E at identical concentrations exhibited equivalent DPPH free radical scavenging activity. 3.4. Resveratrol enhances cholesterol efux

be used in the cholesterol homeostasis experiments. The IC50 was obtained with 114 1.3 M of resveratrol while the IC20 was obtained with 30 M (Fig. 4S). Effect of resveratrol on the apoA1-mediated cholesterol efux was also studied in the Eahy926 endothelial cells. Our results show that resveratrol signicantly enhanced the cholesterol efux (Fig. 5S-A). We next investigated the effect of resveratrol on ABCA1-dependent cholesterol efux pathways in J774 macrophages. Exposing 3 H-cholesterolloaded cells to apoA-1 (cAMP-free) for 6 h (time-range to react only with ABCA1) resulted in a low-cholesterol efux. However, when the macrophages were pretreated overnight with resveratrol (025 M), with 0.3 mM cAMP as a positive control, the apoA1-induced cholesterol efux was signicantly potentiated in a resveratrol concentration-dependent manner (r2 = 0.907, p < 0.05, linear regression) (Fig. 2A). To more clearly understand the mechanism of action of resveratrol on ABCA1-mediated cholesterol efux, we measured the effect of resveratrol on the ABCA1 protein expression in J774 macrophages. Incubating the macrophages in the presence of resveratrol (3, 6, 8, and 16 h) resulted in an increase in ABCA1 protein expression (data not shown). 3.5. Resveratrol reduces cholesterol inux

We assessed the effect of resveratrol (106 to 103 M) on J774 macrophage viability using the MTT colorimetric assay to determine the range of resveratrol concentrations that would

Cholesterol inux is dened as the movement of cholesterol molecules from the extra-cellular environment to cells. This pro-

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Fig. 3. Resveratrol protects macrophages against oxidation and promotes HDL3 mediated cholesterol efux. J774 macrophages (A) and mouse peritoneal macrophages (MPM) (B) were loaded with 3 H-cholesterol and allowed to equilibrate for 16 h. The macrophages were stressed with 0.2 mM iron/ascorbate (Fe/Asc) and cholesterol efux was assessed using 50 g/ml of HDL3 . The panels show the linear correlation between HDL3 -mediated cholesterol efux and resveratrol concentrations. Vitamin E (10 M) was used as a positive control.

3.6. Resveratrol protects against lipid peroxidation and promotes cholesterol efux
Fig. 2. Resveratrol increases cholesterol efux and reduces inux. (A) Effect of resveratrol on apoA1-mediated cholesterol efux. 3 H-cholesterol-loaded J774 cells were treated with various concentrations of resveratrol (025 M) and then incubated with 25 g/ml of apoA1. cAMP (300 M) was used as a positive control. The trace shows a linear correlation between apoA1-mediated cholesterol efux and resveratrol concentrations. All results are expressed as means SEM of at least three independent experiments. (B) J774 cells were labeled with 3 H-cholesterol in the absence or presence of various concentrations of resveratrol (025 M). Cholesterol inux is expressed as the difference in the cholesterol content of the medium and the cell lysates. Results are expressed as the means SEM of at least three independent experiments.

cess, which is mainly observed in macrophages, plays a regulatory role in cellular cholesterol homeostasis. The effect of resveratrol on cholesterol inux was investigated in J774 macrophages and we observed that the inux of 3 H-cholesterol in the presence of resveratrol was signicantly reduced in a dose-dependent manner (r2 = 0.89, p = 0.015) (Fig. 2B). Moreover, we show that resveratrol decreased signicantly cholesterol uptake by endothelial cells (Eahy926) from 3 H-cholesterol-enriched media (Fig. 5SB).

Oxidative damage to macrophages impairs cholesterol efux as previously demonstrated by an impairment of ABCA1 protein expression under Fe/Asc stress Marcil et al. [33]. We investigated the effect of resveratrol on cholesterol efux in J774 (Fig. 3A) and mouse primary macrophages (Fig. 3B) under oxidative stress induced by Fe/Asc. We observed that HDL3 -mediated cholesterol efux was signicantly impaired by Fe/Asc, but that the effect was restored by resveratrol in concentration-dependant manner (p < 0.05). This effect was also observed with THP-1 macrophages (Fig. 6S). The oxidation of HDL has been reported to signicant decrease their capacity to mediate cholesterol efux [34]. We thus assessed the capacity of resveratrol to preserve the functionality of HDL under oxidative stress conditions. ABCA1-enriched J774 cells previously loaded with cholesterol were incubated for 6 h with HDL3 oxidized in the absence (control) or in the presence of resveratrol (treated). Resveratrol protected HDL3 against Cu-induced oxidation in a concentration-dependent manner as shown by the decrease in conjugated diene formation (Fig. 4A). It also maintained the capacity of HDL3 to mediate cholesterol efux (p < 0.05) (Fig. 4B).

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Fig. 4. Resveratrol protects HDL3 from copper oxidation and increases cholesterol efux. (A) Conjugated diene formation in CuSO4 -induced HDL oxidation in the absence or presence of resveratrol as a function of time. HDL3 proteins were incubated with CuSO4 for 8 h (in a time course manner) in the absence or presence of 02 M resveratrol. (B) 3 H- cholesterol efux from human THP-1 macrophages incubated with 50 g/ml of copper-oxidized HDL3 for 0, 2, and 4 h in the presence or absence of 1 M resveratrol. Results are the means SEM of at least three independent experiments.

4. Discussion Dietary antioxidants have attracted considerable attention as preventive and therapeutic agents because of the oxidative theory of atherosclerosis [35]. A number of dietary antioxidant intake studies (epidemiological, casecontrol, and prospective and retrospective cohorts) have shown that antioxidants can prevent CVD progression [36]. Indeed, the consumption of foods rich in phenols and polyphenols has been positively correlated with CVD risk reduction by slowing atherosclerosis progression, principally by protecting lipoproteins from lipid peroxidation [37]. Because resveratrol is present in a variety of foods, including grapes, a primary impetus for research on resveratrol was the paradoxical observation that a low incidence of CVD could co-exist with a high-fat diet intake and moderate consumption of red wine, a phenomenon known as the French paradox [38]. Various mechanisms have been proposed to explain the anti-atherosclerotic proper-

ties of resveratrol, including inhibition of lipid peroxidation [22], modulation of platelet aggregation [8], inhibition of smooth muscle cell proliferation [9], and reduction of macrophage-induced inammation [39]. Despite numerous studies showing the effect of resveratrol on the lipid metabolism and the progression of atherosclerosis, little is known about the antioxidant properties of resveratrol and their effect on cholesterol homeostasis. A large body of evidence indicates that oxLDL plays a key role in both the early and more advanced inammatory stages of atherosclerosis lesions [40]. For example, oxLDL is present in atherosclerotic lesions [41], the progression of atherosclerosis is slowed when oxidation is inhibited [42,43], and there is a correlation between the ability of LDL to resist oxidation and the severity of coronary atherosclerosis [42]. However, LDL oxidation is not the sole factor involved in atherogenesis. In vivo oxidative stress impairs the anti-atherogenic properties of HDL, especially by impairing its antioxidant activity and its capacity to enhance cholesterol efux and promote RCT [34]. Moreover, oxidation can be directly induced in macrophages, which alters the expression of receptors involved in cholesterol ux, which in turn inuence foam cell formation and atherosclerosis development [33]. Our results showed that resveratrol prevented copper- and radiolysis-induced LDL peroxidation in a concentration-dependent manner by decreasing the formation of conjugated dienes, thus extending the lag phase and lowering the oxidation rate. It has also been reported that resveratrol decreases the rate of vitamin E disappearance and maintains the endogenous vitamin E content of LDL [44]. A wide variety of natural products exert their antioxidant effects by preventing the degradation of endogenous vitamins in lipoproteins. Resveratrol, which has three phenolic hydroxyl groups, is very lipophilic, enabling it to associate with the lipid moiety of lipoproteins and prevent the oxidation of their unsaturated fatty acids [44]. LDL oxidation is characterized by alterations in the structural and biological properties of the lipid fraction and the apolipoprotein moiety (apoB), including the early fragmentation of the protein moiety, which contains sensitive amino acid residues. This alteration is followed by cross-linking of reactive aldehydes and oxysterols (end products of lipid peroxidation). In the present study, we showed that resveratrol reduced the -radiolysis-induced alteration of apo-B, as shown by the decrease in the electrophoretic mobility of LDL. To clarify the mechanism by which resveratrol reduces LDL oxidation, we investigated the free radical scavenging activity of resveratrol, which may prevent chain-breaking and the alteration of apoB. Resveratrol may interact with free radicals to form relatively stable free radicals (SFR ) and non-radicals (NR). It may also reduce copper, resulting in the formation of a non-radical product and SFR that, under conditions of high-oxidative stress, quench another free radical. However, in addition to its broad range of activities in the lag phase, resveratrol also reduced the Vmax but had no effect on the ODmax , except at high concentrations. This antioxidant prole suggested that resveratrol might also act as an inhibitor of copper binding via interactions with apolipoproteins. This observation is in agreement with the results reported by Belguendouz et al. [7], who showed that resveratrol is a potent chelator of free copper ions and can also remove copper ions bound to apo-B. Our results are also in agreement with those of Zini et al. [45], who suggested that resveratrol inhibits the lipid peroxidation induced by Fenton reaction products. HDL plays an important anti-atherogenic role by mediating RCT [46]. This process, which is in part dependent on the physicochemical properties of HDL and on the oxidative state of the cells, enables cells to pump out excess free cholesterol. We, and others, have previously demonstrated that HDL oxidation signicantly affects the capacity of HDL to promote cholesterol efux from

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macrophages [27,34]. The oxidation of HDL3 alters the uidity of the phospholipidic layer, modies the structure of apoproteins, and decreases paraoxonase 1 activity [34,47]. All these modications have an effect on the normal interactions between HDL3 components and membrane receptors, including ABCA1, ABCG1, and SR-BI [48]. When J774 macrophages were incubated with resveratrol, apoA1-mediated cholesterol efux increased, suggesting that resveratrol up-regulates this process by up-regulating ABCA1. A number of mechanisms have been proposed to explain this effect, including direct binding of apoA-1 to ABCA1, ABCA1-mediated changes to the plasma membrane that stimulate apoA1 binding, the availability of phospholipids, and cholesterol efux [49]. Over-expression of ABCA1 alters the morphology of the plasma membrane [18] and increases apoA-1 binding [50] and cholesterol oxidase activity, which point to changes in cholesterol concentrations or distribution within the external leaet of the plasma membrane. Our results showed that resveratrol-stimulated ABCA1 expression in J774 macrophages, which is in agreement with those of Sevov et al. [11], who showed that resveratrol induces LXR- and elevated ABCA1 and ABCG1 mRNA levels. This suggests that the up-regulation of ABCA1 protein expression may be one of the pathways involved in the resveratrol-mediated cholesterol efux process, which would be consistent with the increased HDL levels [51] and reduced atherosclerosis seen following polyphenol consumption. We also showed that resveratrol reduces the inux of cholesterol into J774 macrophages in a dose-dependent manner, which ties in with the fact that resveratrol down-regulates lipoprotein lipase (LPL) and SR-AII, which promote increased lipid uptake by macrophages [11]. Oxidative stress is a continuous process in all physiological systems, and results in the oxidation of various molecules and the impairment of their functions. HDL can also be oxidized in vivo, which causes a loss of its anti-atherogenic proprieties [52,53]. Oxidative stress also inuences cholesterol efux in macrophages [33]. In the present study, we also investigated the effect of resveratrol on cholesterol efux in J774 macrophages stressed by a Fe/Asc complex, which induces lipid peroxidation [54] and reduces cholesterol efux. While great care should be taken in extrapolating the results we obtained with J774 macrophages, they are in agreement with those of a previous study using mouse primary peritonealderived macrophages [33]. Oxidative stress can also reduce the expression of ABCA1, LXR, and PPAR, but has no effect on SR-BI. Incubating macrophages with Fe/Asc in the presence of resveratrol signicantly restored cholesterol efux from macrophages to HDL3 , probably by suppressing the effect of Fe/Asc on the cell surface receptors involved in this process. This effect has also been reported with vitamin E and butylhydroxytoluene (BHT), two other antioxidants [33]. The oxidation of HDL results in increased levels of lipid peroxidation markers, including conjugated dienes, lipid hydroperoxides, thiobarbituric acid reactive substances (TBARS), and aldehydes. Compositional changes are associated with alterations in the physico-chemical properties of HDL, including uidity, molecular order, and electric charges [55]. Compositional and structural modications are also associated with changes in the biological activities of HDL. For instance, oxidizing HDL in vitro decreases its ability to promote the RCT process [56]. In the present study, we showed that resveratrol inhibited the oxidation of HDL3 and helped to maintain its capacity to mediate cholesterol efux. This effect may be related to the preservation of the physico-chemical properties of HDL and the integrity of protein moieties like apoA-1 and PON1. We contributed to a better understanding of the potential benets of consuming foods rich in polyphenols by showing that resveratrol protects against lipoprotein oxidation and foam cell formation and promotes cholesterol efux from macrophages. Our

ndings on the intracellular signaling pathways modulating the cholesterol efux process will help lay the foundation for developing new therapeutic approaches to preventing and treating atherosclerosis and cardiovascular diseases. Although, our study is only contribution on some mechanisms by which resveratrol may exert its benecial effect, human studies are needed to conrm the benecial effect of resveratrol and its impact in clinical setting. Acknowledgements This work was supported by the Canadian Institute of Health Research (CIHR). G. Grenier and A. Khalil are recipients of a Junior 1 and 2 investigator award, respectively, from the Fonds de Recherche en Sant du Qubec (FRSQ). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.atherosclerosis.2009.05.017. References
[1] Superko HR. Drug therapy and the prevention of atherosclerosis in humans. Am J Cardiol 1989;64:31G8G. [2] Das S, Das DK. Resveratrol: a therapeutic promise for cardiovascular diseases. Recent Patents Cardiovasc Drug Discov 2007;2:1338. [3] Norata GD, Marchesi P, Passamonti S, et al. Anti-inammatory and antiatherogenic effects of cathechin, caffeic acid and trans-resveratrol in apolipoprotein E decient mice. Atherosclerosis 2007;191:26571. [4] Wang Z, Zou J, Cao K, et al. Dealcoholized red wine containing known amounts of resveratrol suppresses atherosclerosis in hypercholesterolemic rabbits without affecting plasma lipid levels. Int J Mol Med 2005;16:53340. [5] Araya J, Rodrigo R, Orellana M, Rivera G. Red wine raises plasma HDL and preserves long-chain polyunsaturated fatty acids in rat kidney and erythrocytes. Br J Nutr 2001;86:18995. [6] Auger C, Caporiccio B, Landrault N, et al. Red wine phenolic compounds reduce plasma lipids and apolipoprotein B and prevent early aortic atherosclerosis in hypercholesterolemic golden Syrian hamsters (Mesocricetus auratus). J Nutr 2002;132:120713. [7] Belguendouz L, Fremont L, Linard A. Resveratrol inhibits metal ion-dependent and independent peroxidation of porcine low-density lipoproteins. Biochem Pharmacol 1997;53:134755. [8] Wang Z, Huang Y, Zou J, et al. Effects of red wine and wine polyphenol resveratrol on platelet aggregation in vivo and in vitro. Int J Mol Med 2002;9:779. [9] Araim O, Ballantyne J, Waterhouse AL, Sumpio BE. Inhibition of vascular smooth muscle cell proliferation with red wine and red wine polyphenols. J Vasc Surg 2002;35:122632. [10] Avellone G, Di Garbo V, Campisi D, et al. Effects of moderate Sicilian red wine consumption on inammatory biomarkers of atherosclerosis. Eur J Clin Nutr 2006;60:417. [11] Sevov M, Elneh L, Cavelier LB. Resveratrol regulates the expression of LXR-alpha in human macrophages. Biochem Biophys Res Commun 2006;348:104754. [12] Lusis AJ, Atherosclerosis. Nature 2000;407:23341. [13] Aviram M, Rosenblat M. Macrophage-mediated oxidation of extracellular low density lipoprotein requires an initial binding of the lipoprotein to its receptor. J Lipid Res 1994;35:38598. [14] Cherki M, Berrougui H, Isabelle M, et al. Effect of PON1 polymorphism on HDL antioxidant potential is blunted with aging. Exp Gerontol 2007;42:81524. [15] Glomset JA. The plasma lecithins:cholesterol acyltransferase reaction. J Lipid Res 1968;9:15567. [16] Bortnick AE, Rothblat GH, Stoudt G, et al. The correlation of ATP-binding cassette 1 mRNA levels with cholesterol efux from various cell lines. J Biol Chem 2000;275:2863440. [17] de La Llera-Moya M, Connelly MA, Drazul D, et al. Scavenger receptor class B type I affects cholesterol homeostasis by magnifying cholesterol ux between cells and HDL. J Lipid Res 2001;42:196978. [18] Wang N, Silver DL, Costet P, Tall AR. Specic binding of ApoA-I, enhanced cholesterol efux, and altered plasma membrane morphology in cells expressing ABC1. J Biol Chem 2000;275:330538. [19] Brunham LR, Kruit JK, Iqbal J, et al. Intestinal ABCA1 directly contributes to HDL biogenesis in vivo. J Clin Invest 2006;116:105262. [20] Kennedy MA, Barrera GC, Nakamura K, et al. ABCG1 has a critical role in mediating cholesterol efux to HDL and preventing cellular lipid accumulation. Cell Metab 2005;1:12131. [21] Zou J, Huang Y, Chen Q, et al. Effects of resveratrol on oxidative modication of human low density lipoprotein. Chin Med J (Engl) 2000;113:99102.

H. Berrougui et al. / Atherosclerosis 207 (2009) 420427 [22] Fremont L, Belguendouz L, Delpal S. Antioxidant activity of resveratrol and alcohol-free wine polyphenols related to LDL oxidation and polyunsaturated fatty acids. Life Sci 1999;64:251121. [23] Mensor LL, Menezes FS, Leitao GG, et al. Screening of Brazilian plant extracts for antioxidant activity by the use of DPPH free radical method. Phytother Res 2001;15:12730. [24] Sattler W, Mohr D, Stocker R. Rapid isolation of lipoproteins and assessment of their peroxidation by high-performance liquid chromatography postcolumn chemiluminescence. Methods Enzymol 1994;233:46989. [25] Khalil A, Fortin JP, LeHoux JG, Fulop T. Age-related decrease of dehydroepiandrosterone concentrations in low density lipoproteins and its role in the susceptibility of low density lipoproteins to lipid peroxidation. J Lipid Res 2000;41:155261. [26] Fricke H. The chemical action of Rntgen rays on dilute ferrosulfate solutions as measure of dose. Am J Roentgenol Radium Ther Nucl Med 1927;18:42932. [27] Berrougui H, Cloutier M, Isabelle M, Khalil A. Phenolic-extract from argan oil (Argania spinosa L.) inhibits human low-density lipoprotein (LDL) oxidation and enhances cholesterol efux from human THP-1 macrophages. Atherosclerosis 2006;184:38996. [28] Pinchuk I, Lichtenberg D. The mechanism of action of antioxidants against lipoprotein peroxidation, evaluation based on kinetic experiments. Prog Lipid Res 2002;41:279314. [29] Linton MF, Atkinson JB, Fazio S. Prevention of atherosclerosis in apolipoprotein E-decient mice by bone marrow transplantation. Science 1995;267: 10347. [30] Hajj Hassan H, Blain S, Boucher B, et al. Structural modication of plasma HDL by phospholipids promotes efcient ABCA1-mediated cholesterol release. J Lipid Res 2005;46:145765. [31] Berrougui H, Isabelle M, Cloutier M, Grenier G, Khalil A. Age-related impairment of HDL-mediated cholesterol efux. J Lipid Res 2007;48:32836. [32] Bonnefont-Rousselot D. Gamma radiolysis as a tool to study lipoprotein oxidation mechanisms. Biochimie 2004;86:90311. [33] Marcil V, Delvin E, Sane AT, Tremblay A, Levy E. Oxidative stress inuences cholesterol efux in THP-1 macrophages: role of ATP-binding cassette A1 and nuclear factors. Cardiovasc Res 2006;72:47382. [34] Girona J, LaVille AE, Sola R, Motta C, Masana L. HDL derived from the different phases of conjugated diene formation reduces membrane uidity and contributes to a decrease in free cholesterol efux from human THP-1 macrophages. Biochim Biophys Acta 2003;1633:1438. [35] Gugliucci A, Menini T. Three different pathways for human LDL oxidation are inhibited in vitro by water extracts of the medicinal herb Achyrocline satureoides. Life Sci 2002;71:693705. [36] Kaliora AC, Dedoussis GV, Schmidt H. Dietary antioxidants in preventing atherogenesis. Atherosclerosis 2006;187:117. [37] Xu BJ, Yuan SH, Chang SK. Comparative studies on the antioxidant activities of nine common food legumes against copper-induced human low-density lipoprotein oxidation in vitro. J Food Sci 2007;72:S5227. [38] Tunstall-Pedoe H, Kuulasmaa K, Mahonen M, et al. Contribution of trends in survival and coronary-event rates to changes in coronary heart disease mor-

427

[39]

[40] [41]

[42]

[43]

[44] [45] [46] [47]

[48]

[49]

[50] [51]

[52] [53]

[54]

[55] [56]

tality: 10-year results from 37 WHO MONICA project populations. Monitoring trends and determinants in cardiovascular disease. Lancet 1999;353:154757. Tsai SH, Lin-Shiau SY, Lin JK. Suppression of nitric oxide synthase and the downregulation of the activation of NFkappaB in macrophages by resveratrol. Br J Pharmacol 1999;126:67380. de Lorgeril M, Salen P. Diet as preventive medicine in cardiology. Curr Opin Cardiol 2000;15:36470. Haberland ME, Fong D, Cheng L. Malondialdehyde-altered protein occurs in atheroma of Watanabe heritable hyperlipidemic rabbits. Science 1988;241:2158. Regnstrom J, Nilsson J, Tornvall P, Landou C, Hamsten A. Susceptibility to low-density lipoprotein oxidation and coronary atherosclerosis in man. Lancet 1992;339:11836. Heinecke JW. Oxidants and antioxidants in the pathogenesis of atherosclerosis: implications for the oxidized low density lipoprotein hypothesis. Atherosclerosis 1998;141:115. Belguendouz L, Fremont L, Gozzelino MT. Interaction of transresveratrol with plasma lipoproteins. Biochem Pharmacol 1998;55:8116. Zini R, Morin C, Bertelli A, Bertelli AA, Tillement JP. Effects of resveratrol on the rat brain respiratory chain. Drugs Exp Clin Res 1999;25:8797. Fredenrich A, Bayer P. Reverse cholesterol transport, high density lipoproteins and HDL cholesterol: recent data. Diab Metab 2003;29:2015. Jaouad L, Milochevitch C, Khalil A. PON1 paraoxonase activity is reduced during HDL oxidation and is an indicator of HDL antioxidant capacity. Free Radic Res 2003;37:7783. Pirillo A, Uboldi P, Kuhn H, Catapano AL. 15-Lipoxygenase-mediated modication of high-density lipoproteins impairs SR-BI- and ABCA1-dependent cholesterol efux from macrophages. Biochim Biophys Acta 2006;1761:292300. Oram JF, Heinecke JW. ATP-binding cassette transporter A1: a cell cholesterol exporter that protects against cardiovascular disease. Physiol Rev 2005;85:134372. Vaughan AM, Oram JF. ABCA1 redistributes membrane cholesterol independent of apolipoprotein interactions. J Lipid Res 2003;44:137380. Tsang C, Higgins S, Duthie GG, et al. The inuence of moderate red wine consumption on antioxidant status and indices of oxidative stress associated with CHD in healthy volunteers. Br J Nutr 2005;93:23340. Francis GA. High density lipoprotein oxidation: in vitro susceptibility and potential in vivo consequences. Biochim Biophys Acta 2000;1483:21735. Panzenboeck U, Raitmayer S, Reicher H, et al. Effects of reagent and enzymatically generated hypochlorite on physicochemical and metabolic properties of high density lipoproteins. J Biol Chem 1997;272:2971120. Trudel K, Sinnett D, James RW, et al. Iron-ascorbic acid-induced oxidant stress and its quenching by paraoxonase 1 in HDL and the liver: comparison between humans and rats. J Cell Biochem 2005;96:40411. Ferretti G, Bacchetti T, Negre-Salvayre A, et al. Structural modications of HDL and functional consequences. Atherosclerosis 2006;184:17. Rici VA, Khachadurian AK. Oxidation of high density lipoproteins: characterization and effects on cholesterol efux from J774 macrophages. Biochim Biophys Acta 1996;1299:8794.