Red Wine Intake Prevents Nuclear Factor-kB Activation in Peripheral Blood Mononuclear Cells of Healthy Volunteers During Postprandial Lipemia

Millán and Jesús EgidoMónica Ortego, Miguel Angel Hernández-Presa, Pilar Cancelas, Juan Gómez-Gerique, Jesús Luis Miguel Blanco-Colio, Mónica Valderrama, Luis Antonio Alvarez-Sala, Carmen Bustos,

Mononuclear Cells of Healthy Volunteers During Postprandial LipemiaB Activation in Peripheral BloodκRed Wine Intake Prevents Nuclear Factor-

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Red Wine Intake Prevents Nuclear Factor-kB Activation inPeripheral Blood Mononuclear Cells of Healthy Volunteers

During Postprandial LipemiaLuis Miguel Blanco-Colio, BSc; Monica Valderrama, MD; Luis Antonio Alvarez-Sala, MD;

Carmen Bustos, PhD; Monica Ortego, PhD; Miguel Angel Hernandez-Presa, PhD; Pilar Cancelas, PhD;Juan Gomez-Gerique, MD; Jesus Millan, MD; Jesus Egido, MD

Background—Several epidemiological studies have demonstrated the beneficial effect of red wine intake in reducing totaland cardiovascular mortality. This effect has been attributed in part to its antioxidant properties. Because themonocytes/macrophages and the nuclear transcription factorkB (NF-kB) are implicated in the pathogenesis ofatherosclerotic lesions, we examined the effect of red wine intake on the activation of NF-kB in peripheral bloodmononuclear cells.

Methods and Results—Sixteen healthy volunteers were studied 3 times each: after a moderate dose, a low dose, and nowine with a fat-enriched breakfast. Lipid profile and NF-kB activation (electrophoretic mobility shift assay) wereexamined in blood samples taken before and 3, 6, and 9 hours after wine intake. In addition, mononuclear cells wereincubated with VLDL in the presence of some antioxidants (quercetin anda-tocopherol succinate) contained in red wineto study their effects on NF-kB activation. Subjects receiving a fat-enriched breakfast had increased NF-kB activationin peripheral blood mononuclear cells coinciding with the augmentation in total triglycerides and chylomicrons. Redwine intake prevented NF-kB activity even though it induced a certain increase in serum lipids, particularly VLDL, thatdid not increase after the fat ingestion alone. However, another form of alcohol intake (vodka) did not modify the NF-kBactivation provided by postprandial lipemia. In cultured mononuclear cells, isolated human VLDL caused NF-kBactivation in a time-dependent manner that did not occur in the presence of the red wine antioxidants quercetin anda-tocopherol.

Conclusions—Our results provide a new potential mechanism to explain the beneficial effects of red wine intake in thereduction of cardiovascular mortality.(Circulation. 2000;102:1020-1026.)

Key Words: antioxidantsn blood cellsn molecular biologyn atherosclerosis

Many epidemiological studies have shown that moderatered wine intake decreases cardiovascular mortality and

incidence of ischemic stroke.1–3 Different classes of antioxi-dants present in red wine, such as quercetin, catechin, anda-tocopherol succinate,4 are able to modify the lipid peroxi-dation of LDL.5 Dietary supplementation with red wine hasbeen associated with increased resistance of plasma LDL tooxidation and an increment in human plasma antioxidantcapacity.6,7 The potent antioxidant activity of phenolic sub-stances, particularly in red wine, has been proposed as anexplanation of the “French paradox,”8 ie, the coexistence of ahigh-fat diet with a low incidence of coronary heart disease.9

A recent study demonstrated that red wine, catechin, orquercetin consumption exerts an inhibitory effect on thedevelopment of aortic lesions in atherosclerotic apolipopro-tein E–knockout mice.10

Nuclear factor (NF)-kB is a redox-sensitive transcriptionfactor that is involved in the transmission of various signalsfrom the cytoplasm to the nucleus of numerous cell types.11 Itis found as a trimer consisting of p50, p65, and IkB subunitsin the cytosol. The release of IkB from the trimer results inthe migration of the p50/p65 heterodimer to the nucleus andthe subsequent DNA binding.12 This process activates genesinvolved in the immune, inflammatory, or acute-phase re-sponse, such as cytokines (monocyte chemoattractantprotein-1, interleukin-8), adhesion molecules, and procoagu-lant proteins (tissue factor, plasminogen activator inhibitor 1).A variety of stimuli have been found to induce NF-kBactivation, including phorbol myristate acetate, oxidizedLDL, and cytokines such as tumor necrosis factor-a, lym-photoxin, and interleukin-1,13 whereas other agents, includingantioxidants,14 statins,15 and glucocorticoids, inhibit NF-kB

Received February 2, 2000; revision received April 3, 2000; accepted April 7, 2000.From the Research Laboratory, Instituto de Investigacio´n Medica, Fundacion Jimenez Dıaz, Universidad Autonoma de Madrid (L.M.B.-C., C.B., M.O.,

M.A.H.-P., P.C., J.G.-G., J.E.), and the Lipid Research Unit, Hospital Gregorio Maran˜on (M.V., L.A.A.-S., J.M.), Madrid, Spain.Correspondence to Professor Jesus Egido, Renal and Vascular Laboratory, Fundacion Jimenez Dıaz, Avenida Reyes Catolicos 2, 28040 Madrid, Spain.

E-mail [email protected]© 2000 American Heart Association, Inc.

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activation. Recent data strongly suggest that NF-kB could beinvolved in the pathogenesis of atherosclerosis.16 NF-kB ispresent in the human atherosclerotic lesions in the nuclei ofmacrophages and endothelial cells17 and participates in dys-regulation of vascular smooth muscle cells in human athero-sclerosis.18 Conversely, accumulating evidence suggests thatpostprandial lipemia is strongly associated with a risk ofdevelopment of atherosclerotic lesions.19 In this study, wehave shown that a fat-enriched breakfast increases triglycer-ides and chylomicrons, whereas the simultaneous consump-tion of red wine was associated with an increment of totaltriglycerides, chylomicrons, and VLDL triglycerides. Post-prandial lipemia was correlated with an increment of NF-kBactivation in peripheral blood mononuclear cells (PBMCs)that was prevented by red wine intake. An intake of anotherform of alcohol, vodka, did not prevent the activation of thistranscription factor provided by postprandial lipemia. Be-cause VLDLs were the only lipoproteins that were augmentedwith red wine intake but not after the fat ingestion alone, wetested the effect of VLDLs on NF-kB activation. VLDLselicited an increase in NF-kB activation in human mononu-clear Thp-1 cells that was prevented by coincubation withquercetin anda-tocopherol succinate, 2 antioxidants con-tained in red wine. Because NF-kB regulates many genesinvolved in the pathogenesis of coronary artery disease, theseresults provide a new explanation of the potential beneficialeffects of moderate consumption of red wine in humanbeings.

MethodsStudy SubjectsEight men and 8 women, 22 to 33 years old, were included in thisstudy. Participants (medical students or young doctors) were exam-ined to exclude any pathological disorder confirmed by blood test,including an abnormal lipid profile. Those who had plasma choles-terol levels.220 mg/dL or plasma triglyceride levels.200 mg/dLor were hypertensive or smokers were excluded. Weight and heightof subjects were measured, and body surface index was calculated bythe Dubois formula. All participants gave written informed consentfor the study.

Study DesignOn 3 different days, all participants received the same fatty breakfast.On each occasion, a different dose of red wine was given: moderateintake, low intake, and no intake (see below). In the 3 situations, thecaloric intake was maintained constant, with sugar added when thewine consumption was low or none. The 3 phases in each case wereseparated by 2 to 4 weeks. In addition, in a new group, 11 of 16volunteers received vodka in amounts equal to the low dose of redwine. In the meantime, participants were randomly assigned to theiracute fat load in a crossover design. Blood samples were obtainedafter a 10-hour fast and at 3, 6, and 9 hours after breakfast. No foodwas allowed until the end of the study.

DietThe standard fat-enriched breakfast contained 602 kcal/m2, with 342kcal/m2 (56.8%) as fat. It was prepared with common solid and liquidfoods: white bread, ham, margarine, coffee, and whole milk. The redwine was aged in the barrel for 3 years. In men, wine represented anextra 140 kcal/m2 (20 g/m2) in the moderate dose and 84 kcal/m2 (12g/m2) in the low dose. According to National Cholesterol EducationProgram recommendations, women received a lower dose (40%reduction), 84 kcal/m2 (12 g/m2) and 50 kcal/m2 (7.2 g/m2), respec-tively. As a whole, male participants received a total of 742 kcal/m2

and female participants 686 kcal/m2. A calculated amount of sugarwas added to the milk in the case of no wine or a low dose of wineto keep the total amount of calories constant. In all cases, saccharinewas allowed for sweetening of the liquid intake.

Lipoprotein Profile and VLDL IsolationLipoprotein profile was measured by standard techniques,20–23 andVLDLs were isolated from pooled plasma from healthy blood bankdonors. Potential endotoxin contamination of lipoproteins was mon-itored with the chromogenic limulus amoebocyte lysate assay (Bio-Whitaker) usingEscherichia coliendotoxin supplied with the kit forthe standard curve. Samples with endotoxin.2.5 pg/mg of proteinwere discarded.

Cell CultureThp-1 cells (human monocytic cell line) were obtained from theAmerican Type Culture Collection (TIB 202) (ATCC) and culturedin RPMI medium (BioWhitaker) containing penicillin (100 U/mL),streptomycin (100mg/mL), and glutamine (2 mmol/L) with 10%FBS (BioWhitaker). Quercetin anda-tocopherol succinate wereobtained from Sigma Chemical Co and DMSO from Merck. Quer-cetin was dissolved in DMSO anda-tocopherol succinate in ethanol.

Isolation of PBMCsThe blood samples were diluted 1:1 in PBS, and cells were separatedin 5 mL Ficoll gradient (lymphocyte isolation solution, Rafer) bycentrifugation at 2000gfor 30 minutes. PBMCs were collected,washed twice with cold PBS, and resuspended in buffer A (seeProtein Extraction). Approximately 95% of the cells were mononu-clear cells (flow cytometry, not shown).

Protein Extraction and Electrophoretic MobilityShift AssaysProteins of Thp-1 and PBMCs were extracted as described.24 Briefly,Thp-1 cells were made quiescent for 24 hours in 0.5% FBS medium,and then 43106 cells were incubated with the stimuli for differentperiods of time. Peripheral blood mononuclear or Thp-1 cells werecollected, washed with cold PBS, and resuspended in 5 cell-pelletvolumes in buffer A (in mmol/L: HEPES 10 [pH 7.8], KCl 15,MgCl2 2, EDTA 0.1, DTT 1, and PMSF 1) and homogenized. Nucleiwere centrifuged at 1000gfor 10 minutes and resuspended in 2volumes buffer A. Then 3 mol/L KCl was added drop by drop toreach 0.39 mol/L KCl. Nuclear proteins were extracted for 1 hour at4°C and centrifuged at 100 000gfor 30 minutes. Supernatant wasdialyzed in buffer C (mmol/L: HEPES 50 [pH 7.8], KCl 50, PMSF1, EDTA 0.1, DTT 1, and 10% glycerol), then cleared by centrifu-gation and stored at280°C. Protein concentration was determinedby the bicinchoninic assay method (Pierce).

Gel shift assays were performed with a commercial kit accordingto the instructions of the manufacturer (Promega). Briefly, NF-kBconsensus oligonucleotide (59-AGTTGAGGGGACTTTCC-CAGGC-39) was end-labeled with32P by incubation with 10 U T4polynucleotide kinase (Promega) in a reaction containing 10mCi[g-32P]ATP (3000 Ci/mmol) (Amersham), 70 mmol/L Tris-HCl,10 mmol/L MgCl2, and 5 mmol/L DTT. The reaction was stopped bythe addition of EDTA to a final concentration of 0.05 mol/L. Nuclearproteins (5mg) were equilibrated in a binding buffer containing 4%glycerol, 1 mmol/L MgCl2, 0.5 mmol/L EDTA, 0.5 mmol/L DTT,50 mmol/L NaCl, 10 mmol/L Tris-HCl (pH 7.5), and 50mg/mLpoly(dI-dC) (Pharmacia LKB). When competition assays were per-formed, the cold probe was added to this buffer 10 minutes beforethe addition of the labeled probe. The labeled probe (0.35 pmol) wasadded to the reaction. For supershift assays, 1mg anti-p65 (SantaCruz Biotechnology Inc) or anti-p50 (Chemikon) antibodies wereadded and incubated for 1 hour. Nuclear extracts of HeLa cells wereused as positive control. The gel was dried and exposed to x-ray film.The autoradiograph was subjected to densitometry with an ImageQuant densitometric scanner (Molecular Dynamics). Percentageswere calculated in relation to the basal value in each subject andsituation.

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Statistical AnalysisResults are expressed as mean6SD (unless specified). Significancewas established with GraphPDH InStat (GraphPAD Software).Student’st test and 2-way ANOVA were used to compare the data.Differences were considered significant whenP,0.05.

ResultsRed Wine and Postprandial LipemiaTo test the effect of red wine on postprandial lipemia, bloodsamples of healthy volunteers were collected before and 3, 6,and 9 hours after a fat-enriched breakfast with or without redwine intake. Lipid profile revealed an increment of totaltriglycerides and chylomicrons after a fat-enriched breakfast(Figure 1), and red wine intake was associated with a furtheraugmentation in total triglycerides at 3 hours (P,0.05, nowine versus moderate dose) and at 6 and 9 hours (P,0.05, nowine and low versus moderate dose), chylomicrons at 3 hours(P,0.05, no wine versus moderate dose), and VLDL triglyc-erides at 3 hours (P,0.05, no wine versus low and moderatedose) and at 6 hours (P,0.05, no wine and low versusmoderate dose). The consumption of red wine did not causevariations in other lipidic parameters studied (total, LDL, andHDL cholesterol, not shown).

Postprandial Lipemia and NF-kBTo examine whether postprandial lipemia could activateNF-kB, blood samples of healthy volunteers were taken

before and 3, 6, and 9 hours after a fat-enriched breakfast. Asshown in Figure 2, electrophoretic mobility shift assay(EMSA) of nuclear proteins from PBMCs showed a retardedband that was increased at 6 and 9 hours (2.5- and 3-foldversus basal;P,0.05). This band was displaced with a 100molar excess of unlabeled NF-kB oligonucleotide (Figure 3),whereas no displacement was observed with the unlabeledand unrelated oligonucleotide AP-1. A supershifted band wasrevealed by incubation of nuclear extracts with anti-p50 oranti-p65 antibodies but not with anti–c-Rel antibody. Theseresults indicated that the heterodimer p50/p65 was implicatedin the activation of NF-kB in PBMCs.

Red Wine Intake and NF-kB ActivationTo study the effects of red wine intake on NF-kB activity,blood samples of volunteers were taken before and 3, 6, and9 hours after a fat-enriched breakfast with 2 different doses ofred wine; PBMCs were extracted; and nuclear proteins wereobtained. As shown in Figure 2, red wine intake preventedNF-kB activation at 6 and 9 hours (P,0.05, no wine versuslow and moderate dose).

Vodka Intake and NF-kB ActivationTo test whether alcohol intake could prevent NF-kB activa-tion, blood samples of volunteers were taken before and after

Figure 1. Lipid profile of volunteers. A, Total triglycerides. Red wine intake was associated with an increment of total triglycerides at 3hours (*P,0.05, no wine vs moderate dose) and 6 and 9 hours (1P,0.05, moderate vs no wine and low dose). B, Chylomicrons tri-glycerides. Red wine consumption increases chylomicrons at 3 hours (*P,0.05, no wine vs moderate dose). C, VLDL triglycerides.VLDL increases after red wine intake at 3 hours (*P,0.05, no wine vs low and moderate dose) and 6 hours (1P,0.05, no wine and lowvs moderate dose). Results are mean of all volunteers.

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a fat-enriched breakfast with another form of alcohol intake(vodka) in an amount equal to that of the low dose of redwine. Lipid profiles did not reveal significant changes inrelation to the low dose of red wine. However, we observed(Figure 2) that vodka intake did not modify the increment ofNF-kB activation provided by postprandial lipemia (P,0.05vodka versus low and moderate dose).

VLDL, Wine Antioxidants, and NF- kB ActivationVLDL is the only lipoprotein that increases in serum after redwine intake but not after the fat ingestion alone. In this sense,we examined the effect of native VLDL on NF-kB activity inthe human mononuclear cell line Thp-1. Those cells weremade quiescent for 24 hours in 0.5% FBS, and then 43106

cells were incubated in the presence of 10mg/mL humanVLDL (a concentration similar to that found in humanplasma). Cells were collected at 0, 3, 6, and 9 hours, andnuclear proteins were extracted. As shown in Figure 4,EMSA revealed a retarded band in the nuclear extracts fromPBMCs. The intensity of the retarded band, indicated by an

arrow, was markedly increased at 6 and 9 hours of incubationwith VLDL (3.5- and 5-fold, respectively;P,0.05 versusbasal).

Because these results suggested that red wine was able tointercept lipid-mediated NF-kB activation, we next studiedthe effect of some wine antioxidants on Thp-1 cells treatedwith VLDL. The treatment of Thp-1 with VLDL (10mg/mL)and the antioxidants contained in red wine, such as quercetinand a-tocopherol succinate (100mmol/L), inhibited VLDLactivation of NF-kB (Figure 4). Neither ethanol nor DMSOhad any effect on NF-kB activation (not shown).

DiscussionIn this article, we analyze the effect of red wine intake onpostprandial lipemia and NF-kB activation after a fat-enriched breakfast. NF-kB is a ubiquitous transcription factorthat participates in various transcriptionally controlled pro-cesses, such as cytokine responsiveness, inflammation, andcell growth control. Recent studies strongly suggest that theinducible NF-kB is involved in the pathogenesis of athero-

Figure 2. NF-kB activation in PBMCs. A fat-enriched breakfast increases NF-kB activation in atime-dependent manner (*P,0.05 vs basal), andred wine intake decreases this activation with 2doses tested (*P,0.05 basal vs low and moderatedose). However, vodka intake did not modify acti-vation of NF-kB induced by postprandial lipemia(*P,0.05 vs basal and 2 doses of red wine). Aand B, EMSA of nuclear proteins from PBMCs. C,Densitometer quantification of NF-kB activity inPBMCs. Results are expressed in arbitrary units(generated by densitometer) and as percentage ofbasal (0 hours) at each time point. Results aremean of all volunteers.

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sclerosis. The presence of NF-kB in human atheroscleroticlesions in the nuclei of macrophages, vascular smooth musclecells, and endothelial cells has recently been demonstrated,whereas little or no active NF-kB is detected in normalvessels.17 Therefore, it has been suggested that NF-kB acti-vation could be involved in the pathogenesis of atheroscle-rosis, because numerous proinflammatory genes are regulatedby this transcription factor.24 Furthermore, an increasedexpression of numerous genes known to be regulated byNF-kB has been found in the atherosclerotic lesion,18 andNF-kB is selectively and markedly activated in humans withunstable angina pectoris.25 Conversely, postprandial lipemiarepresents the state of absorption during which the metaboliccapacity of triglycerides is challenged. Postprandial hypertri-glyceridemia has been shown to be a coronary risk marker.19

We observed that a fat-enriched breakfast caused a post-prandial lipemia, particularly related to triglyceride-rich li-poproteins, and that red wine intake increased this postpran-dial lipemia. In addition, a fat-enriched breakfast increasedNF-kB activation in a time-dependent manner in PBMCs asa result of the translocation of p50 and p65 subunits.Interestingly, the simultaneous consumption of red wine inmoderate amounts avoided the activation of NF-kB despitethe increment in chylomicrons and VLDL and total triglyc-erides. However, another form of alcohol intake (vodka) didnot prevent the NF-kB activation, suggesting that alcoholcould not regulate the activation of NF-kB. It has just beendemonstrated that red wine that is aged in the barrel exerts avasodilatory effect within a concentration rate that could bereached in vivo by moderate wine consumption,26 and mod-erate alcohol intake has been associated with a significantdecrease in total mortality.27 Furthermore, monocytes areinvolved in the progression of atherosclerosis and are potentactivators of blood coagulation through their ability to syn-thesize procoagulant factors (plasminogen activator inhibi-tor-1, tissue factor) that are regulated by NF-kB. Conversely,it is known that oxidants increase NF-kB activation, whereassuch antioxidants as pyrrolidine dithiocarbamate andN-acetylcysteine inhibit NF-kB activation.14 Because of the redoxregulation of NF-kB, it is possible that the antioxidantscontained in red wine were the cause of the inhibition ofNF-kB activation. In this sense, Feng et al28 demonstratedthat red wine intake inhibited monocyte chemoattractantprotein-1 expression in cholesterol-fed rabbits, a proteinregulated by NF-kB, and this effect might be partly attributedto its antioxidant effects. In addition, catechin and vitamin Eprevent the development of fatty streak in hypercholesterol-emic hamsters29 and attenuate early lesion development inrabbits.30 Moreover, red wine and nonalcoholic wine productscan prevent plaque formation in hypercholesterolemic rabbitsdespite significant increases in LDL.31 Also, red wine poly-phenols inhibit proliferation of vascular smooth musclecells32 and reduce the susceptibility of low-density lipopro-

Figure 3. Competition and supershift assays. EMSA of nuclearproteins from PBMCs shows a retarded band (Control) that wasdisplaced with 100 molar excess of unlabeled NF-kB (Comp.)but not with 100 molar excess of unrelated unlabeled oligonu-cleotide (Comp. AP-1). Incubation of nuclear extract with anti-p50 or anti-p65 antibody shows a supershift band (see p50 andp65 with a time-exposure film) but not with anti–c-Rel. EMSA ofnuclear extracts from HeLa cells revealed a retarded band(HeLa) that is displaced with 100 molar excess of unlabeledNF-kB (HeLa1comp.). This result is representative of 3 indepen-dent experiments.

Figure 4. NF-kB activation in mono-nuclear cells. NF-kB activityincreases in Thp-1 in a time-dependent manner (lanes 1 to 4)with VLDL (10 mg/mL), P,0.05 vsbasal. Quercetin and a-tocopherolsuccinate inhibited this activation at9 hours, P,0.05 vs basal. A, EMSAof nuclear proteins from Thp-1 cells.Representative gel of 3 independentexperiments. B, Densitometer quan-tification of NF-kB activity in Thp-1cells. Cells were incubated withVLDL (open bars), VLDL1quercetin(stippled bars), or VLDL1a-tocoph-erol succinate (hatched bars) at dif-ferent periods of time, as indicated.Results are expressed in arbitraryunits and as percentage of basal (0hours) at each time point. Values aremean6SD of 3 independentexperiments.

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teins to oxidation in vitro33 and in vivo.34 However, inpatients at high risk for cardiovascular events, treatment onlywith vitamin E had no apparent effect on cardiovascularoutcomes,35 at least during the years of this study.

Conversely, VLDL was the only lipoprotein that increasedwith red wine intake but not after the fat ingestion alone inour study. Because of its potential in vivo effect on thephenomenon described, we also studied the effect of VLDLon NF-kB activation in cultured mononuclear cells becausethey have been implicated in the early events of the patho-genesis of atherosclerosis. The treatment of Thp-1 cells withnative VLDL increased NF-kB activation in a time-dependent manner. Furthermore, other authors have observedthat native VLDLs induce NF-kB activation in endothelialcells36 and that oxidized VLDLs also induce the activation ofthis transcription factor in PC12 cells.37 These results couldprovide an explanation of NF-kB activation by postprandiallipemia. In addition, 2 antioxidant compounds present in redwine, quercetin anda-tocopherol succinate, inhibited thisactivation, and ethanol did not modify NF-kB activationinduced by VLDL (not shown). In this sense,a-tocopherolsuccinate, a vitamin E derivative, inhibits NF-kB activationin Thp-1 cells induced by lipopolysaccharide,38 and quercetininhibits interleukin-8 and monocyte chemoattractantprotein-1 expression in synoviocytes.39 However, Wadsworthet al40 reported that quercetin and resveratrol did not inhibitlipopolysaccharide-induced activation of NF-kB in RAW264.7 cells. It is possible that different cells, activators ofNF-kB, and incubation conditions of antioxidants couldprovide different results. Suzuki et al41 demonstrated thatantioxidants did not block NF-kB activation induced by someactivators of this transcription factor. These results correlatewith the data obtained in vivo and support the hypothesis thatthe beneficial effects of red wine are a result of its antioxidantcapacity.

In conclusion, red wine intake, but not another form ofalcohol beverage intake (vodka), prevents NF-kB activationin PBMCs elicited in healthy volunteers by postprandiallipemia. Because NF-kB activation is involved in the patho-genesis of atherosclerotic lesions, the inhibitory effect of redwine on NF-kB activation provides a further explanation ofthe beneficial effects of red wine intake in cardiovasculardisease.

AcknowledgmentsThis work was supported in part by grants 96/0559, 98/0402, and99/0425 from the Fondo de Investigaciones Sanitarias, InstitutoCarlos III of the Spanish Ministry of Health, and Fundacion Ramo´nAreces. L.M. Blanco-Colio is a recipient of a Fundacion ConchitaRabago fellowship. We thank Pablo Alvarez and Bodegas Alio´n(Valladolid, Spain), who generously donated the red wine, and L.Gulliksen for her secretarial assistance.

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