The Cardiovascular Protective Effect of Red Wine

Alfredo C Cordova, MD, La Scienya M Jackson, MD, David W Berke-Schlessel, Bauer E Sumpio, MD, PhD
Twenty-six years ago, St Legar and colleagues1 first drew attention to the inverse relationship between red wine consumption and mortality from ischemic heart disease in 18 different European and American countries. This report followed the observations of previous investigators of the association between alcohol consumption and the low incidence of coronary artery disease (CAD).2-4 In 1992, Renaud and associates5 introduced the term French Paradox to underscore the low mortality rate from ischemic heart disease among people in France despite the high amount of saturated fats in their diet,5-9 which is usually associated with increased mortality from CAD. They attributed this unusual occurrence to red wine consumption based on the findings of the MONICA (MONItoring system for CArdiovascular disease) project, a worldwide program organized by the World Health Organization. Collaborating researchers from 21 countries studied more than 7 million men and women (35 to 64 years of age) from 37 mostly European populations over a period of 10 years, from the mid-1980s to the mid-1990s. The investigators observed a lower mortality rate from CAD in France compared with that in the United Kingdom and the United States, despite the high consumption of saturated fats and similar serum cholesterol concentrations. In addition, other risk factors such as blood pressure, body mass index, and cigarette smoking were equivalent in France to what they were in other industrialized countries (Table 1).10,11 Although France and Italy have halved their wine consumption from what it was in the 1960s, and now average 67 and 57 L/capita/year, respectively, these countries still have a much higher intake than the United Kingdom or the United States,
This work was supported in part by an unrestricted grant from the North American Foundation for Limb Preservation. Received September 2, 2004; Revised October 22, 2004; Accepted October 22, 2004. From the Department of Vascular Surgery, Yale University School of Medicine, New Haven, CT. Correspondence address: Bauer E Sumpio, MD, Department of Surgery (Vascular Section), Yale University School of Medicine, 333 Cedar St, New Haven, CT 06520-8062.

where consumption is about 12 and 5 L/capita/year, respectively.12 Within France, alcohol intake is mostly in the form of red wine. This is particularly true in the south (Toulouse), where CAD is at its lowest; in the north (Strasbourg, Lille), they consume less wine and more spirits, and have a higher incidence of CAD (Table 1). Epidemiologic studies suggest that consumption of red wine at a level comparable to that of France (0.7 to 1.1 ounces of alcohol per day) can indeed reduce the risk of CAD5 by preventing arteriosclerosis. Several reports postulate that this can be attributed to the French consumption of three times more wine.9 But it is now widely accepted that regular, moderate intake of alcoholic beverages (1.1 to 1.8 ounces/day of alcohol) can also decrease the risk of CAD by at least 40%.5,13-16 Arteriosclerosis is the most significant condition contributing to the development of CAD.17 The process of atherogenesis is believed to be accelerated by oxidation of low density lipoprotein (LDL).18-23 Various biologic effects can lead to the lipid peroxidation of LDL, such as the oxidation of LDL polyunsaturated lipid components with reactive nitrogen and oxygen species, and enzyme systems such as the nicotinamide adenine dinucleotide phosphate, reduced form (NADPH) oxidase, 15-lipoxygenase, cytochrome p450, and myeloperoxidase.24 Accounting for nearly half the deaths in developed countries,25 arteriosclerosis has presented itself as a grave public health problem. There are a number of components of red wine that could have an effect on the cardiovascular system, preventing or delaying arteriosclerosis. Alcohol, which is present in up to 15% of the volume of red wine, is one of them. A number of epidemiologic studies have shown an inverse relationship between alcohol consumption and CAD.13-16 For example, a prospective study26,27 involving 51,529 men examined the relationship between alcohol consumption and risk of coronary disease among men without preexisting cardiovascular or cancer disorders. After a 12-year followup with food-frequency questionnaires, the investigators concluded that the consump-

2005 by the American College of Surgeons Published by Elsevier Inc.

428

ISSN 1072-7515/05/$30.00 doi:10.1016/j.jamcollsurg.2004.10.030

Vol. 200, No. 3, March 2005

Cordova et al

Cardiovascular Protective Effect of Red Wine

429

Abbreviations and Acronyms

CAD eNOS HUVEC NADPH NO PDGF SMC VSMC

coronary artery disease endothelial nitric oxide synthase human umbilical vein endothelial cells reduced nicotinamide adenine dinucleotide phosphate nitric oxide platelet derived growth factor smooth muscle cells vascular smooth muscle cell

tion of ethanol at least 3 to 4 days per week was inversely associated with the occurrence of myocardial infarction. This was observed even in people with a limited consumption of 0.35 to 0.42 ounces/day of alcohol, which is the equivalent of one drink, and in women.28 Even though this beneficial property of ethanol is described for low and moderate intake, high consumption of alcohol leads to increased morbidity and mortality. In this manner, ethanol consumption presents the J-shaped alcohol mortality relation, as shown by a study conducted in a Northern California Health Care Program involving 128,934 adults, with different drinking patterns, followed during a period of 20 years (Fig. 1).29 Red wine also contains a wide variety of polyphenols, most of which derive from grape solids (skin and seeds).9,30 A number of advantageous properties that help counteract arteriosclerosis have been attributed to polyphenols. Red wines polyphenolic aid inhibits ox-

idation of human LDL7,18,24,25,30-35 in vitro and in vivo through several different mechanisms, including scavenging reactive oxygen and nitrogen species, chelating transition metal ions, sparing LDLs associated antioxidants, and increasing or preserving serum paraoxonase activity.24 Red wine has also been shown to increase HDLs,36,37 modulate platelet aggregation,38-40 enhance vasorelaxation, and inhibit smooth muscle cell (SMC) proliferation and vascular hyperplasia.9,41,42 The question then arises whether red wine is any better than other alcoholic beverages in its attributed cardioprotective properties. This review will summarize relevant information and studies to help answer this question.
Arteriosclerosis

Arteriosclerosis is a multifactorial disease process and is the principal cause of myocardial infarction, stroke, and peripheral vascular disease, accounting for almost 40% of all mortality in the United States. Estimates by the American Heart Association indicate that it leads to more than 500,000 deaths from coronary artery disease and 170,000 deaths from stroke every year in this nation (American Heart Association, 2000). This entity is characterized by plaque formation and progressive narrowing of the arterials lumen diameter.9 Oxidation of LDL is a cause of early atherogenesis, so its presentation precipitates the cascade of events leading to plaque development.18-24 If the formation of such plaques compromises perfusion to a significant extent

Table 1. Mortality from Coronary Artery Disease and Relative Risk Factors (MONICA Populations; 35 64 Years)
Annual CAD mortality/ 100,000 population Men Women Mean serum cholesterol (mmol/L) Men Women Mean systolic blood pressure (mmHg) Men Women Proportion of regular cigarette smokers (%) Men Women

MONICA center

Body mass index (kg/m2) Men Women

Japan* Beijing, China Toulouse, France Strasbourg, France Lille, France Friuli, Italy A. Brianza, Italy Halifax, Canada Stanford, CA, USA Belfast, UK Glasgow, UK

33 46 53 80 89 99 103 176 163 280 332

9 26 9 17 16 18 21 38 51 79 114

4.5 5.8 6.0 5.8 5.9 5.9 5.6 5.4 5.9 6.1

4.5 5.6 5.9 5.8 5.7 5.9 5.8 5.3 5.9 6.1

131 125 135 135 140 131 130 129 135 133

130 117 127 129 134 127 126 119 129 126

64 24 23 33 29 34 32 23 29 41

9 22 15 17 22 23 25 19 25 41

24.1 26.1 27.3 26.4 26.9 26.4 27.5 26.9 26.3 26.8

24.5 24.5 26.2 26.4 25.8 25.5 27.6 26.6 25.6 26.9

Data from Tunstall-Pedoe et al10 and Kuulasmaa et al.11 *Population not part of MONICA project. CAD, coronary artery disease; MONICA, monitoring system for cardiovascular disease.

430

Cordova et al

Cardiovascular Protective Effect of Red Wine

J Am Coll Surg

J-Curve: Alcohol Mortality Relation All Causes

1.6 1.5 Relative Risk 1.4 1.3 1.2 1.1 1 0.9 0.8 1 2 3 4 Ethanol Consumption 1.1 1 0.9 0.9 1.1 1.6 1.5 1.4

men

women

total

J-Curve: Alcohol Mortality Relation Cardiovascular Causes

2 1.9 1.8 1.7 1.6 1.5 1.4 1.3 1.2 1.1 1 0.9 0.8 1 1.9 1.6 1.5 1.2 1 0.9 2 1 3 1.2 1.1

that play a role in producing such reactive species.24 The oxidized LDL then attracts macrophages that proceed to ingest these cholesterol molecules, forming foam cells, which release factors that recruit fibroblasts, macrophages, and other inflammatory cells. Subsequently, proliferating smooth muscle cells, lipids, and connective tissue become incorporated into the maturing plaque. Finally, formation of a fibrous cap, which might have areas of cell necrosis and calcification beneath it, causes narrowing of the arterial lumens diameter.24,33 To prevent such a chain of events, serum paraoxonase, an HDL-associated esterase, protects against LDLs oxidation by hydrolyzing lipid peroxidases.44,45 The exact mechanism of red wines cardioprotection is not fully elucidated, but a number of pathways through which it prevents arteriosclerosis have been postulated (Table 2). These include increased antioxidant serum activity, increased resistance of LDL to peroxidation,7,18,24,25,30-35 increased activity of high
Table 2. Putative Mechanisms of Red Wines Cardioprotective Effects Inhibition LDL oxidation Free radical scavenger Metal ion chelator Sparing of antioxidants (ie, vitamin E, carotenoid) Increase or preserve paraoxonase activity Inhibition SMC proliferation and vascular hyperplasia Cell cycle arrest DNA strand breakage Decrease cyclin A expression Inhibition ICAM-1 and VCAM-1 Inhibition PDGF Inhibition P13-K and p38 MAPK Inhibition MMP 9 Apoptosis Inhibition platelet aggregation Inhibition cyclo-oxygenase pathway Inhibition thromboxane B-2 synthesis Potentiate prostaglandin |2 Increase phosphodiesterases (cAMP, cGMP) Increase HDL Increased apolipoproteins A-1 & A-2 C 20:5 (omega 3) Vasorelaxation eNOS expression NO release
eNOS, endothelial nitric oxide synthase; ICAM 1, intercellular adhesion molecule 1; MMP, matrix metalloproteinase 9; P13-K, phosphatidylinositol 13-kinase; p38 MAPK, p38 mitogen activated protein kinase; PDGF, platelet derived growth factor; VCAM 1, vascular cell adhesion molecule-1.

Relative Risk

men

women

total
4

Ethanol Consumption

Figure 1. Adjusted risk of death from all causes and cardiovascular causes according to total alcohol consumption compared with abstinence.1, 1 drink/month; 2, 12 drinks/day; 3, 35 drinks/day; 4, 6 drinks/day. Black line, men; pink line, women; yellow line, total. Data from Klatsky and colleagues.29

(occluding more than 70% of the vessel), it can lead to tissue ischemia, necrosis, and in severe cases, to death. To understand the effects of red wine on CAD, it is important to recognize the different steps involved in the formation of a harmful arteriosclerotic plaque. A longitudinal accumulation of lipids (fatty streak) first occurs within the arterial wall, which is surrounded by proliferating smooth muscle. LDL enters the endothelium in the area of this fatty streak, where it becomes oxidized by several types of cells including endothelial cells, smooth muscle cells, and monocytederived macrophages.24,43,44 Under conditions of oxidative stress, reactive oxygen and nitrogen species are produced by cellular oxygenases, which are also capable of oxidizing LDL. There are a number of enzymatic systems, mentioned previously,

Vol. 200, No. 3, March 2005

Cordova et al

Cardiovascular Protective Effect of Red Wine

431

density-lipoprotein cholesterol,5,7,18,25,38,46 and by enhancing serum paraoxonase activation.45 In addition, it serves its cardiovascular protective role by acting as a platelet aggregation modulator,38-40,46 producing NO-mediated vasorelaxation,47-49 and through inhibition of smooth muscle cell proliferation9,41 and intimal hyperplasia.42 These favorable effects of red wine are attributed to its polyphenolic constitution, which has been the motive for multiple studies.
Red wines ethanol component

volume implied a reduction in risk of 0.72 with respect to deaths from cardiovascular and cerebrovascular diseases. But consumption of spirits was associated with an increased risk in deaths from all causes and in deaths from vascular diseases, suggesting the relative benefits of wine consumption over other alcoholic drinks.
Red wines polyphenolic aid

The effect of alcohol on the cardiovascular system is varied and complex. Ethanol can act as a fuel source, alter cholesterol composition, impair fluid balance, and alter the activity of xenobiotic-metabolizing enzymes (p-450 cytochrome family, glutathione S-transferase superfamily, N-acetyl-transferases, etc).50,51 Moderate consumption of ethanol by itself can increase plasma concentrations of HDL and decrease the adhesiveness of platelets. Some researchers have speculated that the presence of ethanol in red wine can increase polyphenol availability by improving intestinal absorption,51 accomplishing this by delaying excretion or by affecting its flow through the xenobiotic excretion pathways.51,52 There is some additional evidence that consumption of ethanol has a prooxidant effect.24,25 Polyphenols in alcoholic beverages counteract the potential prooxidant effect of ethanol. Beverages such as white wine and beer, which have low levels of phenolic compounds, may have a prooxidant effect; red wine has net antioxidant activity. Nonetheless, multiple studies have shown that in comparison to other alcoholic beverages such as spirits, beer, and white wine, red wine is the most beneficial in reducing the risks of CAD and mortality in general.53-56 A prospective study56 involving 6,051 men and 7,234 women aged 30 to 70 years, reported the association between consumption of different types of alcoholic beverages and mortality, after 10 to 12 years of followup. This association was assessed among subjects who consumed different volume ranges of wine, beer, and spirits. The risk of death from all causes decreased with a higher consumption of wine, with a relative risk of 0.51 (95% confidence interval) and by 0.44 for deaths from cardiovascular or cerebrovascular disease for subjects who consumed 3 to 5 glasses a day, compared with a relative risk of 1.00 for those who never drank wine. In the case of beer consumption, the intake of the same amounts of

Red wine has been part of human culture, serving dietary and socioreligious functions for more than 6,000 years. Its chemical composition has changed throughout history, influenced by oenologic techniques, the grape cultivar from which it originated, and environmental factors. Red wine is composed of more than 500 compounds, of which only a few are present at concentrations 100 mg/L. These include water, ethanol, glycerol, sugar, and organic acids, which are the primary components responsible for the taste and mouth-feel sensation of wine. Wine usually contains 0.8 to 1.2 g/L of aromatic compounds, most of which are fusel oils, fatty acid esters, and volatile acids. Fusel oils such as isoamyl, isobutyl, and propyl alcohol are higher alcohol products of yeast fermentation activity and represent 50% of the total amount of aromatic compounds. In lower concentrations and presenting specific sensory characteristics related to its fragrance, red wine is composed of phenols, carbonyls, hydrocarbons, acetals, lactones, sulfur, and nitrogen compounds. The majority of the compounds in wine, of which 160 are esters, are present in concentrations ranging from 10 1to 10 6 mg/L. Because of their low levels, individually they play a small role, if any at all, in the human organoleptic perception (taste), although collectively their role might be significant (Table 3).57 Red wine contains a wide variety of polyphenolic substances, most of which are derived from the solid components of the grape berry, the skin and seeds. Polyphenols provide the tastes of bitterness and astringency of wine and are responsible for the color. These compounds are potent antioxidants and as such, are wines main preservative, allowing for a long aging process.30 Polyphenols are categorized as flavonoids and nonflavonoids. Among the flavonoids are the flavonols (quercitin and myricetin), flavanols or flavan-3-ols (catechin and epicatechin), and the anthocyannins. The nonflavonoids include the stilbenes (resveratrol), hydroxynnamates (caffeic, caftaric, and coutaric acids), and the hydroxybenzoates (Table 4).30 Red wine is produced by fermenting the grape juice in

432

Cordova et al

Cardiovascular Protective Effect of Red Wine

J Am Coll Surg

Table 3. Chemical Composition of Wine

Dessert wines Component White Red White Table wines Red

Water (by difference) Sugars (mostly glucose and fructose) Ethanol Other volatiles Extract Glycerol Acids Pectins Amino acids Ash Phenols* Fats, terpenoids Vitamins, etc Total

76 8 14 0.05 10.1 0.9 0.5 0.25 0.2 0.2 0.01 0.01 0.01 100

74 10 14 0.05 12.2 0.9 0.05 0.25 0.2 0.2 0.1 0.02 0.01 100

87 0.05 10 0.04 2.6 1.1 0.7 0.3 0.25 0.2 0.01 0.01 0.01 100

87 0.05 10 0.04 2.7 1.1 0.6 0.3 0.25 0.2 0.2 0.02 0.01 100

Estimates of typical gross composition (% weight). *Phenols constitute the major compositional difference between table and dessert red and white wines. Adapted from Soleas and colleagues.57

the presence of the berrys solids. White wine, on the other hand, is made by pressing the juice away from the grapes solids and then allowing it to ferment.9,30 Because the majority of polyphenols in wine come from the extraction derived from the skin and seeds during the fermentation process, red wines have a much higher concentration of polyphenols than white wines, so red wines have a higher level of antioxidant activity.18,58 In addition, the fermentation process for red wine lasts up to 2
Table 4. Typical Wine Phenolic Levels
Phenol class Source*

weeks, providing a longer time for the extraction of much of the phenolics. The presence of ethanol also facilitates the process because alcohols are good solvents for polyphenol extraction. Polyphenolic composition varies among different wines according to the type of berry used, vivification process used, type of yeast that participates in the fermentation, and whether or not there were grape solids present in the maceration process. When the same type

White wine Young Aged

Red wine Young Aged

Flavonoids Flavonol monomers (ie, catechin) Proanthocyanidins and condensed tannins Flavonols (ie, quercitin) Anthocyanins (ie, malvin-3-glucoside) Others Total, mg/L Nonflavonoids Cinnamates derivatives Benzene derivatives Hydrolyzable tannins (from oak) Stilbenes (ie, resveratrol) Total, mg/L Total, all phenols, mg/L

g g,d g,d g g,d,e,m

25 20 45 154 10 0 0.5 164.5 209.5

15 25 40 130 15 100 0.5 245.5 285.5

200 750 100 400 50 1,500 165 60 0 7 232 1,732

100 1,000 100 90 75 1,365 60 60 250 7 377 1,742

g,d g,d,e,m e g,m

*g, grapes; d, degradable product; e, environment, cooperage; m, microbes, yeast. Young refers to new wine, less than 6 months of age, not having been aged or fermented in oak barrels. Aged implies about 1 year for white, about 2 years for red, and some oak barrel aging (or other oak contact). Adapted from Waterhouse AL.30

Vol. 200, No. 3, March 2005

Cordova et al

Cardiovascular Protective Effect of Red Wine

433

of berry is involved, phenolic content may differ depending on the type of soil, weather variations (temperature, rain, humidity), and other biologic effects (fungi, insecticides, fertilizers).59 Water deficit decreases the amount of flavonols and greatly increases the rate of loss during fruit ripening. Polyphenols also decrease as the fruit matures.60 On the other hand, exposure of grape skins to sunlight strongly favors high levels of some flavonoids.61 A typical commercial bottle of red wine contains approximately 1.8 g/L of total polyphenols; a typical bottle of white wine contains only about 0.2 to 0.3 g/L of total polyphenols (Table 4).9 So the total amount of polyphenols found in a glass of red wine is about 200 mg in comparison to only 30 mg in a glass of white wine. Flavonoids constitute the majority of these phenols in red wine. In typical wine making, about half of the phenolics are extracted during the maceration process. The amount extracted is strongly affected by the types of seed extracting methods used and are higher with the use of extended maceration techniques. The amounts of polyphenols in a wine decrease with prolonged storage because of the formation of polymers, which precipitate in response to their insoluble states. This could also be attributed to some oxidation reactions.30 There are many attempts to enhance the polyphenol content of wine through the production process. Exposing white wine for increasing periods of time to the grape mash has led to white wines with increased polyphenolic concentrations, which can be further enhanced in the presence of alcohol.18 Incubation for 18 hours of whole squeezed grapes with skin contact results in white wines with up to a 41% increase in polyphenol content; if it is also stored with 18% alcohol, the polyphenol content increases by 60%, a level that results in antioxidant effects similar to those of red wine. Given their control over the processing of grapes to reach a specific flavor, winemakers are ultimately the critical determinants of a wines polyphenol content. Polyphenols have varying levels of antioxidant characteristics that depend on their chemical structures.24,62-63 The phenol group is a strong electron donor and is readily oxidized.30 Wine is also rich in catechol-containing compounds, which also have antioxidant activity, although less than polyphenols. For instance, although the OH groups on quercitin and catechin possess similar arrangements, quercitin inhibits LDL oxidation to a greater extent, because of its

2-to-3 double bond and the 4-oxo structure in its C ring (Fig. 2).58 Conversely, anthocyannins and polymerized tannins are very poor antioxidants.18 Even though tannins are the most abundant of the wine polyphenols, they are not absorbed because of their large molecular size, so they impart no health effects, except perhaps in the gut.30
Polyphenol inhibition of LDL oxidation

LDL oxidation is a key event in atherogenesis.17 Polyphenols reduce LDL sensitivity to peroxidation. In one study,34 subjects consumed 375 mL/day of red wine (cabernet sauvignon) for 2 weeks and LDL and plasma polyphenols were quantified. Lipid peroxides decreased by 40%, thiobarbituric acid-reactive substances by 44%, and conjugated dienes by 48%, although the lag phase before oxidation increased 380%. The antioxidant effect was associated with an increase in the polyphenols plasma concentration and was not secondary to vitamin E or -carotene content. The susceptibility of LDL to copper-mediated oxidative modifications was not influenced by the dealcoholized red wine, as determined by the lag time before and after the intake of red or white wine. These results confirmed earlier studies from Nigdikar and associates,25 following a similar protocol. Both studies demonstrated that the consumption of white wine actually has a prooxidant effect; there was minimal to no increase in the lag time, and there was a 21% to 28% increase in thiobarbituric acid-reactive substances in the subjects who drank white wine. The reason for this phenomenon is unclear, but this effect could be explained by the prooxidative properties of alcohol. In fact, the investigators were able to gradually increase the capacity of white wine to scavenge free radicals and to inhibit LDLs copper ion-induced oxidation when the fruit juices were macerated with grape solids in the presence of increasing levels of alcohol. Oxidation was inhibited up to 87% when the juices were exposed for 18 hours with 18% alcohol, similar to the 94% inhibition seen with red wines.18 This led to the conclusion that red wine, but not white wine, has important antioxidant activity because of the high content of polyphenols. In support of this hypothesis, it can be shown that an alcohol-free powder of a red wine phenolic extract has similar effects as red wine, showing an antioxidant effect both in plasma and in LDL.25 This suggests that although alcohol may contribute to the cardioprotective effect, the main antioxidant protection is from the

434

Cordova et al

Cardiovascular Protective Effect of Red Wine

J Am Coll Surg

Structure

General Type Flavonoids:

Flavanols (catechin, epicatechin, gallocatechin, condensed tannins, procyanidins)
Flavonols (quercitin, myrecitin, kaempferol)

Anthocyanins (cyanidin, peonidin, delphinidin, petunidin, malvidin)

Non-Flavonoids:
Hydroxinnamic Acids (caffeic acid, ferulic acid, cholorogenic acid, coumaric acid, caftaric acid)
Benzoic Acids (gallic acid, vanillic acid, benzoic acid, protocatechuic acid, hydrolizable tannins)
Stilbenes (resveratrol)

Figure 2. Polyphenols structures. Adapted from Soleas and associates57 and Waterhouse AL.30

polyphenolic aid by decreasing the susceptibility of LDL to undergo lipid peroxidation in vivo. There are several potential mechanisms by which LDL is protected from oxidation by phenolics. The first is as a free radical scavenger, with polyphenols assuming the role of reducing agents, of hydrogen atom-donating molecules, and as singlet quenchers. Another mechanism is through the chelation of transition metal ions by

phenolics, consequently diminishing the capacity of the metal to generate free radicals. A third potential pathway would be sparing of vitamin E and carotenoids in the LDL particle, resulting in increased levels and subsequent enhanced antioxidant effect. Finally, by protecting or increasing serum paraoxonase activity, hydrolysis of arterial cells and LDL associated lipid peroxides is promoted.24 Polyphenols inhibitory effects on LDL ox-

Vol. 200, No. 3, March 2005

Cordova et al

Cardiovascular Protective Effect of Red Wine

435

idation appeared to be more marked for oxidation induced by copper metal ion than for that induced by aqueous peroxyls,33 implying that both active copper binding and free radical trapping are associated with phenolic compounds. Redox reactive copper ions can be found in atherosclerotic lesions and ceruloplasmin, and in this manner, oxidatively affect LDL.64 Polyphenols can also potentially attenuate LDL oxidation by reducing macrophage oxidative stress through inhibition of cellular oxygenases such as NADPH oxidase, 15-lipoxygenase, cytochrome p450, and myeloperoxidase, or by activating cellular antioxidants such as the glutathione system.24 In human subjects, red wine polyphenols are absorbed efficiently and bind LDL, protecting it from peroxidation.24,25,34 As mentioned previously, polyphenols have varying effects depending on their chemical structures. Cathecol-like structured compounds are better at inhibiting LDLs peroxyl-initiated oxidation than monoprotric phenols,33 perhaps because of the formed antioxidant radicals ability to be resonance stabilized. These compounds are found in cathecins, which have been demonstrated to inhibit LDLs copper catalyzed oxidation.65 In dose-response studies, caffeic acid appears to be the most active antioxidant, with radical trapping as its main mechanism for protection, consequently sparing alpha tocopherol from oxidation.33
Polyphenol inhibition of vascular smooth muscle cell proliferation and migration

The inhibition of vascular smooth muscle cell (VSMC) proliferation and migration should theoretically retard or inhibit the appearance of arteriosclerosis because it constitutes an important component of atherogenesis.9,41 The abnormal proliferation of VSMC in the arterial intima is thought not only to be a key step in the formation of the atherosclerotic plaque but also in the pathogenesis of restenosis.41 In a rabbit endothelial denudation model,42 rabbits fed a high-dose resveratrol diet (4 mg/kg/day) developed less intimal hyperplasia than control rabbits. The number of SMCs in the thickened intima was considerably reduced in the resveratroltreated animals. These results correlate with our investigations,9 in which bovine aortic SMCs were treated with growth media supplemented with dealcoholized red wine, red wine polyphenol extract, or resveratrol for as long as 48 hours. By assessing DNA synthesis, we ob-

served that red wine and red wine polyphenols inhibit SMC proliferation in a dose-dependent manner. The antiproliferative effect of red wine on SMC can involve several pathways (Table 2). It retards cell cycle progression at the S phase of mitosis.66,67 The mechanism of this action is not fully elucidated, but it is known that in the presence of copper ions, resveratrol is able to cause DNA strand breakage;68 resveratrol that is bound to cellular DNA forms a complex with Cu (II) reducing it to Cu (I), causing redox cycling of copper.9 This leads to the production of reactive oxygen species that act as DNA cleaving agents. So resveratrols antiproliferative activity would be enhanced by the consumption of red wine because red wine is a great source of dietary copper. Another potential mechanism involves cyclin A, a cell cycle regulator known to play a critical role in DNA replication at the G1/S transition, and in the S and G2/M phases. These antiproliferative effects on VSMC have been associated with decreased expression of cyclin A gene through the downregulation of transcription factor cAMP, responsive element-binding protein (CREB), and activating transcription factor-1 (ATF-1).69 Cyclin A genes expression downregulation is believed to contribute to the antiproliferative effects of red wine polyphenols through the inhibition of transcription factor expression.70 Platelet derived growth factor (PDGF), which is a potent chemoattractant for SMCs, induces cellular motility through activation of the phosphatidylinositol 3=kinase (PI3-K) and p38MAPK pathways.49 Red wine polyphenols have been shown to dependently inhibit induced PDGF and serum SMC migration through the inhibition of the activity of these two signaling pathways.69 In addition, PI3-K is associated with cell cycle regulation, as PI3-K inhibitors induce G1 arrest through upregulation of the cyclic-dependent kinase inhibitor p27Kip1, and so might represent another mechanism for SMC growth regulation by red wine. Quercitin is believed to have antiatherogenic effects on vascular SMC by inhibiting the expression of matrix metalloproteinase 9, an enzyme associated with the development of intimal formations and plaque rupture in arterial lesions. It has also been reported that quercitin can arrest the cell cycle at G1. The transcription factor NF- B and the apolipoprotein A-1 on tumor necrosis factor- -induced human aortic SMCs are postulated to be responsible for quercitins efficacy in inhibiting this G1-to-S transition, and inhibiting metalloproteinase 9 expression and cell proliferation.71 Of note, resveratrol

436

Cordova et al

Cardiovascular Protective Effect of Red Wine

J Am Coll Surg

has been shown to inhibit the expression of cell adhesion molecules ICAM1 and VCAM1 on the endothelium, reducing monocyte and granulocyte adhesion.72 Inhibition of endothelial cell ICAM expression has been seen through a reduction of nuclear factor B activity, suggesting a role for red wine in the maintenance of vascular structure integrity and in the functional restoration of damaged endothelial cells.70 Resveratrol may perhaps cause some degree of apoptosis, limiting cell proliferation.42
Polyphenol modulation of platelet aggregation

Polyphenol effects on vasomotor tone

Decreased platelet aggregation is frequently associated with low incidence and prevalence of CAD. Drugs that attenuate platelet aggregability, such as aspirin, prostacyclin, or the nitric oxide donor nitroprusside, are protective for ischemic heart disease.73 Polyphenols have also been shown to inhibit platelet aggregation, and this may account for their cardioprotective properties (Table 2). In vitro experiments demonstrate polyphenols antiaggregatory effect on human platelets, with resveratrol and quercitin having the greatest effects.39 Other studies indicate that aggregation of fresh washed human platelets by ADP and thrombin and the synthesis of thromboxane B2 are strongly inhibited by red wine.38 Quercitin has also been found to potentiate prostaglandin I2 by increasing levels of cAMP. It has been shown to increase cAMP and cGMP phosphodiesterases, raising the platelet levels of cAMP and cGMP, and causing a decrease in the levels of platelet cytosolic calcium and lower activity levels of platelets.39 In human volunteers, 375 mL/day of red wine for 2 to 4 weeks decreased ADP-induced platelet aggregation40 and plasma thromboxane B2 concentration by 59.4%,38 respectively. The cyclooxygenase pathway of the arachidonate metabolism was also inhibited. In a Folts model of stenosed canine coronary arteries and intimal damage,73 administration of red wine intravenously or intragastrically, at a dose of 1.6 and 4.0 mL/kg, respectively, eliminated the cyclic flow reductions in coronary blood flow because of the periodic formation of acute plateletmediated thrombi. The same effect was observed with the administration of higher quantities of grape juice, but not with white wine, suggesting that polyphenols, which are present in higher levels in red wine and grape juice than in white wine, are the reason for these platelet inhibitory properties.

Endothelial nitric oxide is considered to have vasoprotective and antiatherosclerotic effects.74 Postmenopausal women have an increased risk of CAD. Although an imbalance of estrogen and progesterone are central to the pathogenesis, the role of estrogen replacement therapy is controversial.75 Expression of endothelial nitric oxide synthase (eNOS) has been shown to be upregulated by estrogen, and this may play a contributory role. Resveratrol can upregulate eNOS expression and consequently increase eNOS-derived nitric oxide (NO) production. This effect was shown,48 in a time- and concentration-dependent manner, in human umbilical vein endothelial cells (HUVEC) incubated for 24 to 72 hours with up to 10 mol/L of resveratrol or in culture medium treated with red wine.This exposure resulted in an enhanced endothelial-dependent NO-mediated vasorelaxation, showing an upregulation of eNOS mRNA expression of up to 2.8-fold, and an increase in eNOS protein levels and eNOS derived NO expression. When HUVECs were exposed to 7.4 mol/L of resveratrol, eNOS mRNA expression increased approximately 150% compared with controls; red wine led to an increase of 230%. We can conclude that although resveratrol has evident enhancing effects on eNOS-mediated NO expression, it is not the sole agent responsible for red wines vasorelaxation protective property. On the other hand, quercitin has also been found to produce a marked coronary vasorelaxation effect that is endothelial independent. This effect was seen to be significantly greater in resistance vessels (80 to 150 um, internal diameter) than in conductance vessels (300 to 525 um, internal diameter), evoking a greater than 80% vasorelaxation in resistance vessels.47 At concentrations of 5.6, 8, and 30 M, quercitin significantly relaxed resistance in 32%, 47%, and 82%, respectively. Conductance was also relaxed 20%, 32%, and in 70% for the same doses, respectively. At moderate alcohol consumption concentrations (14 mmol/L), neither red wine nor ethanol alone showed this effect. These findings still demand that we take into consideration the vasorelaxation property of quercitin, although it is not present at moderate red wine consumption levels. In this manner, these reports describe red wine polyphenols beneficial property to induce NO release with the subsequent effect of vasorelaxation, resulting in a cardioprotective action.

Vol. 200, No. 3, March 2005

Cordova et al

Cardiovascular Protective Effect of Red Wine

437

Polyphenol effects on HDL

Red wines are also capable of increasing HDL (Table 2). This lipid prevents cholesterol accumulation in peripheral cells and is protective against arteriosclerosis by transporting cholesterol from the arteries to the liver for its subsequent metabolism and excretion. Alcohol consumption increases the levels of HDL and may explain, at least partially, wines protective effect.5,25,36,37 Previous reports34 in which healthy men received 400 mL/day of wine during 2 weeks indicated an increase of HDL with intake of red wine, but not with white wine. In other studies,36,76 dose-dependent increases in HDL and in Apo A-1 levels were observed. Based on the ECTIM study (Enquete Cas-Temoins de IInfarctus du Myocarde), which involved 561 men with myocardial infarction and 643 healthy men from France and Northern Ireland, Marques-Vidal and colleagues76 reported a significant increase in HDL cholesterol, from 0.47 to 0.59 g/L, in men who consumed 2.3 ounces/day of alcohol, mostly in the form of red wine, compared with nondrinkers. Apolipoproteins A-I and A-II also increased significantly. Perret and associates36 observed an increase in HDL levels in people drinking 1.7 ounces of wine per day. Isolated HDL displayed an increase of 27% in all cholesteryl ester molecule species. This effect was associated with enrichment of the HDL particles in polyunsaturated phospholipids, in those containing arachidonic acids ( 30%) and eicosapentaenoic acids ( 90%), and especially in those containing C 20:5 (omega 3), which is thought could be beneficial by itself against CAD. In conclusion, CAD is the major cause of morbidity and mortality in developed countries. Red wine and its polyphenolic aid present many beneficial characteristics against cardiovascular disease. These are associated with their capability to lipid peroxidize LDL, inhibit SMC proliferation, modulate platelet adhesiveness, enhance HDL serum levels, and produce vasorelaxation (Table 2). Attempts to determine which of the 8,000 specific polyphenolic compounds known to exist in nature59 are responsible for these effects have been the impetus for a multitude of studies. Caffeic acid and protocatechuic acid seem to have the most potent antioxidant effects, resveratrol and quercitin have the highest activity in platelet adhesiveness modulation, and resveratrol may have the strongest antiproliferative potential. Additional research to determine the exact compounds and doses required, perhaps acting synergistically, to improve their

cardioprotective effects is needed. It may be that the total spectrum of red wine phenolics is necessary, perhaps in the presence of ethanol. This combination has already been proved to share activity in modulating platelet aggregation and elevating the levels of HDL cholesterol. Although phenolics may undergo chemical modifications once absorbed into the bloodstream, such as glycosylation, methylation, or glucoronidation,7,77 their availability and capability to exert biologic activity still remain. In this manner, red wine is capable of playing an important cardiovascular protective role. Its beneficial aspects do not seem to be limited to the cardiovascular system. Recent studies have found potential use in cellular membrane protection,31-34 delay of Alzheimers disease progression,78 and also as an anticancer agent;31,79-81 it has been observed to have antiproliferative effects on various cancer cell lines including squamous cell carcinoma, human breast cancer, and different human leukemias. A better understanding of the health benefits of red wine and perhaps the specific polyphenolic extracts with the described properties would be a great contribution to society. Meanwhile, the evidence is highly compelling that moderate and regular red wine consumption of one or two glasses per day can lower our risk of suffering from cardiovascular disease.
REFERENCES 1. St Legar AS, Moore F, Cochrane AL. Factors associated with cardiomortality in developed countries with particular reference to consumption of wine. Lancet 1979;1:10171020. 2. Klatsby AL, Friedman GD, Siegalub AB. Alcohol consumption before myocardial infarction. Ann Intern Med 1974;81:294 301. 3. Hennekens CH, Robner B, Cole D. Daily alcohol consumption and fatal coronary heart disease. Am J Epidemiol 1978;107: 196200. 4. Yano K, Rhoads GG, Kagan A. Coffee, alcohol and risk of coronary heart disease among Japanese men living in Hawaii. N Engl J Med 1977;297:405409. 5. Renaud S, de Lorgeril M. Wine, alcohol, platelets, and the French paradox for coronary heart disease. Lancet 1992;339: 15231526. 6. Teissedre PL, Waterhouse AL. Inhibition of oxidation of human low density lipoproteins by phenolic substances in different essential oils varieties. J Agric Food Chem 2000;48:38013805. 7. Howard A, Chopra M, Thurnham D, et al. Red wine consumption and inhibition of LDL oxidation: what are the important components? Med Hypotheses 2002;59:101104. 8. Saucier CT, Waterhouse AL. Synergetic activity of catechin and other antioxidants. J Agric Food Chem 1999;47:44914494. 9. Araim O, Ballantyne J, Waterhouse A, Sumpio BE. Inhibition of

438

Cordova et al

Cardiovascular Protective Effect of Red Wine

J Am Coll Surg

10.

11.

12. 13. 14. 15. 16. 17.

18.

19.

20. 21.

22. 23. 24. 25.

26. 27. 28.

vascular smooth muscle cell proliferation with red wine and red wine polyphenols. J Vasc Surg 2002;35:12261232. Tunstall-Pedoe H, Kuulasmaa K, Mahonen M, et al. Contribution of trends in survival and coronary-event rates to changes in coronary heart disease mortality: 10-year results from 37 WHO MONICA Project populations. Lancet 1999;353:15471557. Kuulasmaa K, Tunstall-Pedoe H, Dobson A, et al. Estimation of contribution of changes in classic risk factors to trends in coronary-event rates across the MONICA Project populations. Lancet 2000;355:675687. Yarnell JW. The Mediterranean diet revisited-toward resolving the (French) paradox. Q J Med 2000;93:783785. Kozararevic D, Mc Gee D, Vojvodic N, Racic Z, et al. Frequency of alcohol consumption and morbidity and mortality. Lancet 1980;1:613616. Marmot MG, Rose G, Shipley MJ, Thomas BJ. Alcohol and mortality: explaining the U-shaped curve. Lancet 1981;1:580 583. Shaper AG, Wattamethee G, Walker M. Alcohol and mortality in British men: explaining the U-shaped curve. Lancet 1988;1: 1267273. Boffetta P, Garfinkel L. Alcohol drinking and mortality among men enrolled in an American Cancer Society prospective study. Epidem 1990;1:337339. Sumpio BE, Pradhan S. Artherosclerosis. Biological and surgical considerations. In: Ascher E, Hollier L, Strandness DE, eds. Haimovicis vascular surgery. 5th ed. Malden, MA: Blackwell Science, Inc; 2004:137163. Fuhrman B, Volkova N, Suraski A, Aviram M. White wine with red wine-like properties: increased extraction of grape skin polyphenols improve the antioxidant capacity of the derived white wine. J Agric Food Chem 2001;49:31643168. Aviram M. Interaction of oxidized low-density lipoprotein with macrophages in atherosclerosis and the anti-atherogenicity of antioxidants. Eur J Clin Chem Clin Biochem 1996;34:599 608. Steinberg D. Low density lipoprotein oxidation and its pathobiological significance. J Biol Chem 1997;272:2096320966. Kaplan M, Aviram M. Oxidized low density lipoprotein: atherogenic and proinflamatory characteristics during macrophage foam cell formation. An inhibitory role for nutritional antioxidants and serum paraoxonase. Clin Chem Lab Med 1999;37: 777787. Berliner JA, Heinecke JW. The role of oxidized lipoproteins in atherosclerosis. Free Radic Biol Med 1996;20:707727. Parthasarathy S, Santanam N, Auge N. Oxidized low-density lipoprotein, a two-faced janus in coronary artery disease? Biochem Pharmacol 1988;56:279284. Fuhrman B, Aviram M. Flavonoids protect LDL from oxidation and attenuate atherosclerosis. Curr Opin Lipidol 2001;12:41 48. Nigdikar SV, Williams NR, Griffin BA, Howard AN. Consumption of red wine polyphenols reduces the susceptibility of low-density lipoproteins to oxidation in vivo. Am J Clin Nutr 1998;68:258265. Mukamal KJ, Conigraves KM, Mittleman MA, et al. Roles of drinking pattern and type of alcohol consumed in coronary heart disease. N Engl J Med 2003;348:109118. Rimm EB, Giovannucci FL, Willet WC, et al. Prospective study of alcohol consumption and risk of coronary disease in men. Lancet 1991;338:464486. Stampfer MJ, Colditz GA, Willet WC, et al. A prospective study

29. 30. 31. 32. 33.

34.

35. 36.

37. 38. 39.

40. 41. 42. 43. 44. 45.

46.

47.

of moderate alcohol consumption and the risk of coronary disease and stroke in women. N Engl J Med 1998;319:267273. Klatsky AL, Friedman GD, Armstrong MA, Kipp H. Wine, liquor, beer, and mortality. Am J Epidemiol 2003;158:585595. Waterhouse AL. Wine phenolics. Ann NY Acad Sci 2002;957: 2136. Brenna O, Pagliarini E. Multivariate analysis of antioxidant power and polyphenolic composition in red wines. J Agric Food Chem 2001;49:48414844. Waterhouse AL, German J, Walzem R, et al. Is it time for a wine trial? Am J Clin Nutr 1998;68:220221. Abu-Amsha R, Croft KD, Puddey IB, et al. Phenolic content of various beverages determines the extent of inhibition of human serum low-density lipoprotein oxidation in vitro: identification and mechanism of action of some cinnamic acid derivatives from red wine. Clin Sci (Lond) 1996;91:449458. Fuhrman B, Lavy A, Aviram M. Consumption of red wine meals reduces the susceptibility of human plasma and low-density lipoprotein to lipid peroxidation. Am J Clin Nutr 1995;61:549 554. Zou J, Huang Y, Chen Q, et al. Effect of resveratrol on oxidative modification of human low density lipoprotein. Chin Med J (Engl) 2000;113:99102. Perret B, Ruidavets JB, Vieu C, et al. Alcohol consumption is associated with enrichment of high-density lipoprotein particles in polyunsaturated lipids and increased cholesterol esterification rate. Alcohol Clin Exp Res 2002;26:11341140. 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;86:189195. Pace-Asciak CR, Rounova O, Hahn SE, et al. Wines and grape juices as modulators of platelet aggregation in healthy human subjects. Clin Chim Acta 1996;246:163182. Pace-Asciak CR, Hahn S, Diamandis EP, et al. The red wine phenolics trans-resveratrol and quercitin block human platelet aggregation and eicosanoid synthesis: implications for protection against coronary heart disease. Clin Chim Acta 1995;236: 207219. Seigneur M, Bonnet J, Dorian B, et al. Effect of the consumption of alcohol, white wine, and red wine on the platelet function and serum lipids. J Appl Cardiol 1990;5:215222. Rivard A, Andres V. Vascular smooth muscle cell proliferation in the pathogenesis of atherosclerotic cardiovascular diseases. Histol Histopathol 2000;15:557571. Zou J, Huang Y, Cao K, et al. Effect of resveratrol on intimal hyperplasia after endothelial denudation in an experimental rabbit model. Life Sci 2000;68:153163. Aviram M, Rosenblat M. Macrophage mediated oxidation of extracellular low density lipoprotein requires an initial blinding of the protein to its receptor. J Lipid Res 1994;35:385398. Parthasarathy S, Printz DJ, Boyd D, et al. Macrophage oxidation of low density lipoprotein generates a modified form recognized by the scavenger receptor. Arteriosclerosis 1986;6:505510. Aviram M, Rosenblat M, Bisgaier CL, et al. Paraoxonase inhibits high-density lipoprotein oxidation and preserves its functions. A possible peroxidative role of paraoxonase. J Clin Invest 1998; 101:15811590. Halpern MJ, Dahlgren AL, Laakso I, et al. Red-wine polyphenols and inhibition of platelet aggregation: possible mechanisms, and potential use in health promotion and disease prevention. J Int Med Res 1998;26:171180. Rendig SV, Symons JD, Longhurst JC, Amsterdam EA. Effects

Vol. 200, No. 3, March 2005

Cordova et al

Cardiovascular Protective Effect of Red Wine

439

48.

49. 50. 51. 52. 53. 54. 55. 56. 57. 58. 59.

60. 61. 62. 63. 64. 65.

of red wine, alcohol, and quercitin on coronary resistance and conductance arteries. J Cardiovasc Pharmacol 2001;38:219 227. Wallerath T, Deckert G, Ternes T, et al. Resveratrol, a polyphenolic phytoalexin present in red wine, enhances expression and activity of endothelial nitric oxide synthase. Circulation 2002; 106:16521658. Iijima K, Yoshizumi M, Hashimoto M, et al. Red wine polyphenols inhibit vascular smooth muscle migration through two distinct signaling pathways. Circulation 2002;105:24042410. Goldberg DM, Hahn SE, Parkes JG. Beyond alcohol: beverage consumption and cardiovascular mortality. Clin Chim Acta 1995;237:155187. Bell J, Donnovan J, Wong R, et al. ( )-catechin in human plasma after ingestion of a single serving of reconstituted red wine. Am J Clin Nutr 2000;71:103108. Ruf JC, Berger JL, Renaud S. Platelet rebound effect of alcohol withdrawal and wine drinking in rats. Arterioscler Thromb Vasc Biol 1995;15:140144. Criqui MH, Ringel BL. Does diet or alcohol explain the French Paradox? Lancet 1994;344:17191723. Klatsky AL, Armstrong MA. Alcoholic beverage choice and risk of coronary heart disease mortality: do red wine drinkers fare best? Am J Cardiol 1993;71:467469. Klatsky AL, Armstrong MA. Freedman GD. Relation of alcoholic beverage use to subsequent coronary heart disease hospitalization. Am J Cardiol 1986;58:710714. Gronbaek M, Deis A, Sorensen TI, et al. Mortality associated with moderate intakes of wine, beer, or spirits. Br Med J 1995; 310:11651169. Soleas GJ, Diamandis EP, Goldberg DM. Wine as a biological fluid: history, production, and role in disease prevention. J Clin Lab Anal 1997;11:287313. Vinson JA, Hontz BA. Phenol antioxidant index: Comparative antioxidant effectiveness of red and white wines. J Agric Food Chem 1995;43:401403. Alvarez-Sala L, Slowing K, Gomez-Serranillos P, et al. Variabilidad del contenido de polifenoles de distintos tipos de vino y su potencial aplicacin al conocimiento de sus efectos biologicos. Med Clin (Barc) 2000;114:331332. Kennedy JA, Matthews MA, Waterhouse AL. Changes in grape seed polyphenols during fruit ripening. Phytochem 2000;55: 7785. Price SF, et al. Cluster sun exposure and quercitin in Pinot noir grapes and wine. Am J Enol Viticulture 1995;46:18794. Van Acker S, Van-den Berg DJ, Tromp M, et al. Structural aspects of antioxidant activity of flavonoids. Free Radic Biol Med 1996;20:331342. Rice-Evans CA, Miller NJ, Paganga G. Structure-antioxidants activity relationships of flavonoids and phenolic acids. Free Radic Biol Med 1996;20:933956. Ehrenwald E, Chisolm GM, Fox PL. Intact human ceruloplasmin oxidatively modifies low density lipoprotein. J Clin Invest 1994;93:14941501. Mangiapane H, Thomson J, Salter A, et al. The inhibition of the

66. 67.

68. 69.

70.

71.

72.

73.

74.

75. 76. 77. 78. 79. 80. 81.

oxidation of low density lipoprotein by ( ) catechin, a naturally occurring flavonoid. Biochem Pharmacol 1992;43:445450. Ragione FD, Cucciolla V, Borriello A, et al. Resveratrol arrests cell division cycle at S/G2 phase transition. Biochem Biophys Res Comm 1998;250:5358. Park JW, Choi YJ, Jang MA, et al. Chemopreventive agent resveratrol, a natural product derived from grapes, reversible inhibits progression through S and G2 phases of the cell cycle in U937 cells. Cancer Lett 2001;163:4349. Fukuhara K, Miyata N. Resveratrol as a new type of DNA cleaving agent. Bioorg Med Chem Lett 1998;8:31873192. Iijima K, Yoshizumi M, Hashimoto M, et al. Red wine polyphenols inhibit proliferation of vascular smooth muscle cells and downregulate expression of cyclin A gene. Circulation 2000; 101:805811. Iijima K, Yoshizumi M, Ouchi Y. Effect of red wine polyphenols on vascular smooth muscle cell functionmolecular mechanism of the French Paradox. Mech Aging Dev 2002;123: 10331039. Moon SK, Cho GO, Jung SY, et al. Quercitin exerts multiple inhibitory effects on vascular smooth muscle cells: role of ERK 1/2, cell-cycle regulation, and matrix metalloproteinase-9. Biochem Biophys Res Commun 2003;301:10691078. Carluccio MA, Siculella L, Ancora MA, et al. Olive oil and red wine antioxidant polyphenols inhibit endothelial activation: antiatherogenic properties of Mediterranean diet phytochemicals. Arterioscler Thromb Vasc Biol 2003;23:622629. Demrow HS, Slane PR, Folts JD. Administration of wine and grape juice inhibits in vivo platelet activity and thrombosis in stenosed canine coronary arteries. Circulation 1995;91:1182 1188. Sumpio BE, Oluwole B, Wang X, Awolesi M. Regulation of nitric oxide synthase expression and activity by hemodynamic forces. In: Rubanyi GM, ed. The pathophysiology and clinical applications of nitric oxide, Chapter 10. Reading, United Kingdom: Hardwood Academic Publishers; 1998:171193. Pradhan S, Sumpio B. Do the estrogen effects on blood vessels translate into clinically significant atheroprotection? J Am Coll Surg 2004;198:462474. Marques-Vidal P, Cambou JP, Nicaud V, et al. Cardiovascular risk factors and alcohol consumption in France and Northern Ireland. Atherosclerosis 1995;115:225232. Hollman P, Katan MB. Health effects and bioavailability of dietary flavonols. Free Radical Res 1999;31:S75S80. Russo A, Palumbo M, Aliano C, et al. Red wine micronutrients as protective agents in Alzheimer-like induced insult. Life Sci 2003;72:23682379. Mgbonyebi OP, Russo J, Russo IH. Antiproliferative effect on synthetic resveratrol on human breast epithelial cells. Int J Oncol 1998;12:865869. Elattar TM, Virgi AS. The effect of red wine and its components on human oral squamous carcinoma cells. Anticancer Res 1999; 5:407414. Jang M, Udeani GO, Slowing KV, et al. Cancer chemopreventive activity of resveratrol, a natural product derived from grapes. Science 1997;275:218220.