EXPERIMENTAL AND MOLECULAR PATHOLOGY 65, 150-159 (1999) ARTICLE NO. MO982233

The Effect of Red Wine on Experimental Atherosclerosis: Lipid-Independent Protection ~

Prot~isio L. da Luz, 2 Carlos V. Serrano Jr.,'- Ana R Chacra, 2 Hugo R Monteiro, 3 Vanda M• Yoshida, 2 Mozart Furtado, z Silvia Ferreira, a Paulo Gutierrez, 2

and Ftilvio Pileggi 2

21nstituto do Carafdo-Divisao de Clhfica e Divisao de Experimentaf6o, and 3Fundaf6lo Prd-Sangue/Henlocentro de Sao Paldo, Hospital das Cl[nicas da Facuhlade de Medicina,

Universidade de Sao Paulo, 05403 Sdo Paulo, SP, Brazil

Received Decenlber 15, 1997, and in revised form September 15, 1998

To assess the effect of red wine on atherosclerosis, New Zealand rabbits were given 1% cholesterol diet for 12 weeks and compared to animals that received the diet plus either red wine or nonalcoholic wine products (NAWP). Diet induced marked increases in total and LDL cholesterol; yet no significant changes in HDL and triglyceride concentrations occurred. In the control group, plaque area was 69 +- 9% of the aortic surface, while in the wine and NAWP groups it was only 38 _+ 9 and 47 -+ 12%, respectively (P < 0.0001). The average intima/media thickness ratio was 0.60 __ 0.2 in control animals, 0.14 ~ 0.09 in the wine group, and 0.39 -+ 0.19 in the NAWP group (P < 0.0001). No significant differences were noted in LDL oxidizability among treatments. Thus, both red wine and NAWP can prevent plaque formation in hypercholesterolemic rabbits despite significant increases in LDL. We speculate that anti-platelet effect, blockade of expression of endothelial cell adhesion molecules, and/or NO stimulation by red wine flavonoids are possible explanations. © 1999 Aca- demic Press

INTRODUCTION

Moderate alcohol consumption is inversely related to the incidence of coronary artery disease in epidemiological studies (Criqui, 1986; Criqui et al., 1987; Langer et al., 1992; Moore et al., 1986; St. Leger et al., 1979; Stampfer et al., 1988; Suh et al., 1992; Ulbright

et al., 1991; Thun et al., 1997)• In particular, the expression "French paradox" refers to the lower incidence of coronary artery disease in France compared to other European countries with similar ingestion of animal fat; this paradox was attributed to wine drinking (Renaud et al., 1992)• A lower incidence of acute myocardial infarction also

was documented in American men who consumed one to three drinks/day of spirits, wine, or beer compared to those who drank less or none at all (Gaziano et al., 1993)• However, these evidences are mostly indirect. One of the few objective studies was performed by Klurfeld and Kritchevsky (1981)• By comparing the effects of different beverages they observed that red wine reduced aortic atherosclerosis significantly, while ethanol, white wine, and whisky caused only slight reductions and beer had no effect• Thus, it was not clear whether the alcohol component of wine is really necessary to cause the alleged protective benefit against atherosclerosis.

On the other hand several medhanisms could be responsible for these beneficial effects,

This work was supported in part by research grants from the "Funda~ao E.J. Zerbini," "Financiadora de Estudos e Projetos" (Grant SF 0752•94), and "Fundaqao de Amparo ~ Pesquisa do Estado de Sao Paulo" (Grant 94/1418-6).

150 0014-4800/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.

EFFECT Of RED WINE ON ATHEROSCLEROSIS 151

including HDL increases (Langer et al., 1992; Suh et al., 1992) decreased platelet aggrega- tion (Singleton et al., 1969; Seigneur et al., 1990), and antioxidation due mainly to

flavonoids (Frankel et al., 1993; Anderson et al., 1995, L996). Therefore, the main objectives of the present study were: (1) to objectively assess the

effect of red wine and nonalcoholic wine products upon atherosclerotic plaque formation

in hypercholesterolemic rabbits and (2) to assess the effects of the two treatment regimens upon plasma lipids and LDL susceptibility to oxidation.

MATERIAL AND METHODS

All experiments were performed in accordance with the "Position of the American Heart Association on Research Animal Use"; the protocol was aproved by the Ethics

Committee of the "Instituto do Cora~fio," University of S~o Paulo. For atherosclerotic plaque analysis, 31 New Zealand White rabbits, weighing an average

of 2.4 _ 0.9 kg at the beginning of the protocol, were used. Diet was prepared by adding 10 g of cholesterol (Sigma Chemical Co.; C8503) to each kilogram of Purina chow. Cholesterol was dissolved in sulfuric ether and 70% ethanol and then added to the diet.

Animals were divided into the following groups: control (n = 10), which received only 1% cholesterol diet; wine (n = 1 1), which received diet plus red wine on the dosage of 3.2 ml/kg/day; and nonalcoholic wine products (NAWP) (n = 10), which received NAWP,

in the same dosage. This dosage was chosen baded on extrapolation from human epidemio- logical data (Thun et al., 1997) suggesting that up to two glasses (250 ml) of wine/day

for adults is not harmful and may actually be beneficial; this dose represents the amount actually ingested by the rabbits. NAWP refers to all remaining substances once alcohol was removed from red wine. Alcohol was removed by evaporation, subjecting the wine

to 50°C for 30 min. This was done in order to assess whether alcohol was contributing to the effects of wine. After separation from wine, NAWP were stored at 4°C for no more

than 2 weeks. Wine and NAWP groups were given in the drinking water ad libitum; all animals drank avidly and no intolerance was observed. Fluid and food consumption were checked daily (see Table I). The wine used was Cabernet Cordilheira, Almaden Co.,

(Livramento, RS, Brazil); it contained 11.5% alcohol. Animals were fed I% cholesterol diet during 12 weeks. Rabbits were maintained in

silent, individual cages, at 23°C. In order to avoid undue stress, the same technicians

cared for the animals throughout the study.

TABLE I Plasma Lipids and Weight Profile

Control (n = 11) Wine (n = 12) NAWP (n = 12)

Initial Final Initial Final Initial Final

Total cholesterol 129 ~ 15 879 ± 38* 156 -+ 11 1103 - 41" 145 --- 10 824 __+ 50* LDL-cholesterol 59 - 11 839 ± 36* 111 - 13 1038 --+ 29* 93 - I 1 756 __. 49* HDL-cholesterol 28 _ 10 19 __. 4 20 - 5 17 ± 7 18 + 5 23 ___ 6 Triglycerides 107 _ 11 179 ± 11 134 ± 13 237 ± 13 174 - 12 223 _ 14 Weight 2.4 _ 1.2 3.1 ± 1.1 2.7 - 1.1 3.2 ± 1.2 2.6 ± 1.2 3.1 ± 1.1 Food intake (g) 181.1 _ 8.8 181.1 --- 12.8 188.1 ± 8.9 Fluid intake (g) 187.7 ± 9.8 191.1 - 12.1 188.6 - 8.8

Note. NAWP, nonalcoholic wine products. Lipid values are expressed in mg% and weight in kg. Food and fluid intakes were computed over 24 h. Values represent means _+ SEM.

* P < 0.02, Final vs Initial (intragroup).

152 DA LUZ ET AL.

Alcohol plasma levels. Alcohol plasma levels were determined by gas chromatography in 12 additional rabbits, 4 in each treatment ~oup, about 2 h after a 24-h ingestion period ad libitum.

Lipid measurements. Total and HDL serum cholesterol were measured by the enzy- matic CHOD-PAP method (Merck-PA). Serum HDL was measured after precipitation with phosphotungstic acid/magnesium chloride. LDL was calculated by the Friedwald formula; no sample exceeded 300 mg/dl of triglycerides (Friedwald et al., 1972). Triglycer- ides were measured in a Cobas Mira (Roche) machine by full-enzymatic color test (GPO- PAP method.

Lipoprotein analysis. Blood samples were obtained at the time of sacrifice, after ovemight fast, with the anticoagulant EDTA (1 mg/ml). The LDL fraction was isolated between densities of 1.019-1.063 g/ml by sequential ultracentrifugation and dialyzed ovemight at 4°C against saline containing the chelating resin Chelex (Havel et al., 1955). To inhibit proteolysis, benzamidine (2 mM), aprotinin (2 i~g/ml), and phenylmethylsulfonyl fluoride (1 raM) were added to the samples as soon as the plasma was separated from the blood cells. The protein content of the LDL preparation was determined according to the Lowry procedure (Lowry et al., 1951) with bovine serum albumin as a standard.

LDL oxidation assay. For evaluation of LDL susceptibility to oxidation 47 additional rabbits were used; of these, 7 were fed normally and the remaining received cholesterol diet (n = 13) diet + wine (n = 15) or diet + NAWP (n = 12) for 2 to 6 weeks. LDL oxidizability was assessed using a spectrophotometric technique described by Esterbauer et al., (1992). Native LDL was diluted to 150 txg/ml (final concentration in PBS) and incubated with 100 I-d of a 500 I~M CuSO4 stock solution in a final volumne of 1 ml. LDL oxidation was assessed by using ultraviolet spectrophotometric absorbance at 234 nm in a 1-cm quartz cuvette as an index of conjugated diene formation. Absorbance was measured at 5-rain intervals for 480 rain at 37°C. The lag time was defined as the interval between initiation of oxidation by the addition of CuSO4 (time 0) and the tangent intercept of the slope of the absorbance curve during the propagation phase.

Aortic lesion quantification. Atherosclerotic lesion quantification was performed ac- cording to previously described techniques (Kita et al., 1987; Klurfeld and Kritchevsky, 1981; Freyschuss et al., 1993). For macroscopic analysis animals were sacrificed and their aortas excised. Immediately upon excision, aortas were fixed in formaldehyde and then immersed in Sudan IV solution. Atherosclerotic plaques were clearly identified by their pinkish color; the whole specimen was photographed. Plaque-area was quantitated by planimetry using the Quantimet-500 system adapted to a Leica optical system and expressed as percentage of the total aortic surface. Microscopic analysis: after macroscopi- cal measurements, 0.5-cm-thick cross sections of the aortas were taken at each 3 cm from the beginning of the aorta to its bifurcation (levels 0, 3, 6, 9, and 12 cm), subjected to regular histological handling, and embedded in paraffin. Four-micrometer-thick sections were obtained and stained with hematoxylin and eosin. The slides were examined by one of the authors (P.G.), who was unaware of the group to which each sample belonged. The sections were analyzed subjectively and afterward subjected to a morphometric study using the Quantimet 500 software adapted to a Leica optical system with a 2.5 × objective magnification. Starting at one of the ends of the sections, intimal and medial layer thicknesses were measured at each 2131.37 I-~m (corresponding to the length of the screen). Therefore the longer sections had more measurements than the shorter ones. The mean thickness of both intimal and medial layers, as well as the ratio between these two values,

EFFECT Of RED WINE ON ATHEROSCLEROSIS 153

was calculated for each section and finally for the whole animal (each of them comprising

five slides).

Statistical analysis. Data are presented as means ± SEM. Statistical analysis was performed by two-factor analysis of variance (ANOVA) with Tukey's Studentized range test for macroscopic and microscopic evaluations. A P value lower than 0.05 was conven- tionally assumed to indicate statistical significance. When ANOVA revealed a statistical significance, Tukey's test was used. When indicated Student 's t test, paired or unpaired, as used for all other data.

RESULTS

Red wine and NAWP were given in the drinking water and were well tolerated by all animals with no signs of any undesirable side effects. Plasma alcohol concentration was 0.32 g/L in four wine-treated rabbits, zero in four control animals, and trace in four NAWP rabbits. A moderate and similar increase in total weight was observed in all groups (see Table I). No animal died during the experimental period.

Macroscopic analysis: extent of aortic involvement by plaques. In the control group, 69 -+ 9% of the aortic surface was covered by plaques while in the wine group only 38 --- 9% of the aorta was involved; in the NAWP group, this involvement was only 47 -+ 12%. These differences among the three groups are significant (P < 0.0001) by ANOVA. However, the difference between wine and NAWP groups is not significant. Figure 1 illustrates these findings, in representative specimens. Figure 2A shows aver- age results.

FIG. 1. Representative aortas stained with Sudan IV solution. Atherosclerotic plaques are identified by their pinkish color. Aortas from the control group had greater atherosclerotic compromise than aortas from the wine and NAWP groups. NAWP, nonalcoholic wine products.

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DA LUZ ET AL.

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FIG. 2. (A) Extent of aorta involvement by atherosclerotic plaques in control, wine-treated, and nonalcoholic wine product (NAWP)-treated rabbits. Plaque area was obtained by planimetry of Sudan IV-stained plaques and expressed as percentage of total aortic surface. (B) Average intima/media thickness ratio in aortas of the three study groups. *P < 0.001, control vs wine and NAWP, by ANOVA. *P < 0.05, wine vs NAWE Data are means _ SEM.

Histological analysis: intimal/media thickness ratio. The medial layer showing typical lamellar units of continuous elastic sheets surrounding smooth muscle cells and extracellu- lar matrix composed mostly by collagen and proteoglycans was identified in all cases. In some of them zones of cartilaginous metaplasia were present. The intima varied from a virtual coat consisting of a single layer of endothelial cells to a thick band containing foamy cells, mononuclear cells, and extracellular l ipid deposition (the variation between the cases is well characterized by the morphometric study); in a few ~ases there was a small fibrous nodule at the subendothelium. No particular features were noted in the aventitia.

The average intimal/media thickness ratio was 0.60 "- 0.2 in control animals; this ratio was redued to 0.14 ___ 0.09 in the wine group and to 0.4 -L-_ 0.2 in the NAWP group. The differences among groups are significant at the P < 0.0001 level by ANOVA. The difference between wine and NAWP is also significant at the P < 0.05 level by the Tukey test. Figure 2B illustrates the histologic findings.

Plasma lipids. Table I depicts fluid and food intake as well as the initial and final values for total cholesterol, HDL, LDL, and triglycerides in the three groups of rabbits used for plaque analysis. Initial l ipid values were in the normal range in all groups. The 1% cholesterol diet caused major increases in total cholesterol and LDL in all treatment groups. However, in the wine group this increase was even greater than in controls and NAWP, although the final values were not statistically different. On the other hand, there

EFFECT Of RED WINE ON ATHEROSCLEROSIS 155

were no significant changes in HDL concentration, in any of the groups. Triglyceride levels increased slightly, but not significantly in the three groups.

LDL suscept ibi l i ty to oxidation. The lag phase of diene'formation was tested in animals treated for 2 and 6 weeks. Since the results did not differ significantly in these periods, the data were pooled. Lag time was 114.9 --_ 23.6 rain in 7 normally fed rabbits; it decreased to 73.8 ___ 4.2 rain in 13 animals that received only 1% cholesterol diet; it was 84 _ 13.2 min in 15 animals of the hypercholesterolemic diet plus wine group and 72.3 ± 6.4 min in 12 of the cholesterol diet plus NAWP group; these differences, however, are not statistically significant. For comparison, probucol was given to 4 additional nor- mally fed rabbits and 4 rabbits treated with 1% cholesterol diet for 2 weeks. Probucol did not change lag time in normally fed rabbits but increased it significantly to 250.3 ± 15.2 min in cholesterol-fed rabbits (P < 0.05, Student's t test).

DISCUSSION

This study shows that both red wine and NAWP protect against atherosclerotic plaque formation in hypercholesterolemic rabbits; such effect is independent of plasma lipids. It is noteworthy that pure red wine is somewhat more effective than NAWP. The findings are an objective demonstration of a beneficial effect of red wine and NAWP upon the development of atherosclerosis and complement previous epidemiological evidence that suggests a protective role of the so-called Mediterranean diet, of which wine is a regular component (Rimm et al., 1995). The results are in part confirmatory of the work of Klurfeld and Kritchevsky (1981), who also found protection by red wine while other beverages were ineffective, in a similar experimental model.

The mechanisms underlying the protective effect of red wine and NAWP are not clear. Interiestingly, protection occurred despite major increases in total cholesterol and LDL and no statistically significant changes in HDL and triglycerides. Our results showed somewhat higher lipid levels than those found by Klurfeld and Kritchevsky (1981); such differences are most likely due to a higher dietary cholesterol concentration in the present study (1% vs 0.5%); also, animals in their study lost weight while weight was gained in ours. Although LDL cholesterol increased slightly more in the wine groups than in controls and the NAWP group, protection against atherosclerosis was more pronounced precisely in the wine group. This may indicate that the alcohol component of wine exerts a particular, albeit undefined, protective role. These findings point to other mechanisms involved in plaque formation as key determinants of lesion development; interactions between blood elements, adhesion molecules, and the vessel wall, as suggested by others (Berliner et

al., 1995; Jang et al., 1994; Libby, 1995), are probably of fundamental importance. On the other hand, prolonged alcohol ingestion increases HDL in humans CLanger et al.,

1992; Suh et al., 1992; Gaziano et al., 1993). In the present study no such augmentation was found; our results are consistent with those of Hayek et al. (1997), who also did not find changes in HDL or LDL after giving red wine or the polyphenols quercetin or catechin to mice; note, however, that these animals were apolipoprotein E deficient, a model different from ours. Hence the reasons for this finding remain speculative. However, we did not measure liver weight, which is a limitation in interpreting the lipid data.

The effect of red wine upon LDL oxidizability also was explored. However, the lag time for diene formation was not different in LDL obtained from the three study groups. Admittedly, the test to assess LDL oxidizability is ex vivo and may not reflect the real situation in the living animal. It also could be argued that LDL oxidizability was tested in animals treated for 2 to 6 weeks, and not for 3 months. However, four animals were

156 DA LUZ ET AL.

given probucol, a known anti-oxidant (Hodis et al., 1992), for the same period of time in order to ascertain whether an anti-oxidant effect could be detected under these experimental conditions. A significant lag time prolongation was indeed documented, thus proving the adequacy of the test. Hence, protection against LDL oxidation is not the explanation for the results found. Our findings are at variance with those of Sasahara et al. (1994), who observed an inverse correlation between LDL resistance to oxidation and plaque area in nonhuman primate. The antioxidant effect of red wine seems in fact inconsistent, even in human subjects; for instance, Frankel et al. (1993) and Fuhrman et al. (1995) observed it, while Sharpe et al. (1995) did not. Thus, our findings could indicate that protection against atherosclerosis may occur by means other than antioxidation.

Red wine and NAWP may have decreased platelet adhesiveness and aggregability as suggested by several reports (Beretz et al., 1981; Corvazier et al., 1985; Ferrel et al., 1979; Gryglewski et al., 1987; Landolfi et al., 1984; Mower et al., 1984; Slane et al.,

1994). Flavonoids present in red wine, such as quercetin, resveratrol, and rutin, do so through cAMP phosphodiesterase inhibition (Ferrel et al., 1979; Ruchstuhl et al., 1979). In addition, Demrow and Folts (1995), using the Folts model of coronary thrombosis, demonstrated that red wine and purple grape juice given either intravenously or intragas- trically to dogs eliminate cyclic coronary flow variations and decreased platelet aggregabil- ity; in contrast, white wine had no effect on coronary flow. Although in our study platelet function was not studied, similar anti-platelet effects may in part explain the protective effect encountered, since platelet deposition contributes to plaque growth by liberating growth factors (Berliner et al., 1995).

Red wine also may have acted by inducing NO production. As shown by Fitzgerald et al. (1993) red wine phenolic compounds increase NO production and induce vasorelaxa- tion of aortic strips in rats. Since NO is also an antiproliferative substance, its increased production may contribute to reduce plaque formation, as previously shown (Cayatte et al. , 1994).

Another possible explanation is the effect of flavonoids upon the expression of endothe- lial cell adhesion molecules. According to Gerritsen et al. (1995) flavonoids, especially apigenin, inhibit intercellular adhesion molecule-l, vascular cell adhesion molecule-l, and selectin expression in cultured human endothelial cells. Since in the early phase of atherosclerotic plaque formation, adhesion molecules play a major role by mediating leukocyte adherence to the endothelium, such blockade could be a protective mechanism against atherosclerosis (Berliner al. , 1995; Jang et al. , 1994; Libby, 1995).

As mentioned above, an interesting feature of the present investigation is that both pure red wine and wine without alcohol induce protection, although pure red wine was somewhat more efficient. Considering the well-known undesirable side effects of heavy alcohol drinking, this finding is of potential importance and adds additional support to the idea that flavonoids are important active compounds in red wine. In fact, protective effects against coronary events were attributed to dietary flavonoids in epidemiological studies (Hertog et al., 1993; Knekt et al. , 1996).

CONCLUSIONS

The present data offer a clear message: both red wine and NAWP can protect against plaque formation, in the rabbit model of atherosclerosis. Remarkably, this protection occurred despite major increases in plasma LDL and was not due to increased resistance to LDL oxidation. Anti-platelet effect, blockade of expression of endothelial cell adhesion molecules, and NO induction are plausible although unproven explanation. An implication

EFFECT Of RED WINE ON ATHEROSCLEROSIS 157

o f the s tudy is tha t m e c h a n i s m s o the r than h y p e r l i p i d e m i a are o f f u n d a m e n t a l i m p o r t a n c e

for the d e v e l o p m e n t o f a the rosc le ro t i c p laques ; accord ingly , t he rapeu t i c s t ra teg ies d i rec ted

at these m e c h a n i s m s , such as endo t he l i a l ce l l / b lood in te rac t ions , s h o u l d be tested. Finally,

no d i rec t c l in ical app l i ca t ions shou ld be de r ived f rom these f ind ings , due to obv ious

d i f f e rences in species . H o w e v e r , the da ta sugges t that an appropr i a t e c l in ica l s tudy to

assess the role o f red w ine and f l a v o n o i d s as p ro tec t ive agen ts aga ins t the d e v e l o p m e n t

o f a the rosc le ros i s m a y be jus t i f ied . .

A C K N O W L E D G M E N T S

We hank Neusa R. Dini and Sueide Cassinelli for secretarial aid and Dalia Ballas and Julia T. Sukushima for statistical assistance in the preparation of this report. We also thank Dr. ilma Leyton for alcohol measurements.

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