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Atherosclerosis in apoE-deficient mice:
role of red wine.

Examine the isolated and combined effects of red wine components
in atherosclerosis-prone apoE-deficient mice.

Atherosclerosis is by far the most frequent cause of ischemic heart disease (IHD) and ischemic stroke, the first and third most common cause of death in North America and Europe. It is a complex disease of, mainly, the tunica intima, involving several pathological processes, including inflammation and T-cell mediated immune response in the vascular wall[1]; oxidative modification and subendothelial accumulation of lipids [2], and migration and proliferation of vascular smooth muscle cells with subsequent synthesis of collagenous matrix[3]. In the advanced stages disruption of the plaque causes superimposed thrombosis leading to unstable angina pectoris, myocardial infarction or sudden death[4].

Alcohol and IHD
A vast body of ecological, case-control and prospective studies have consistently, and in the course of a 70 year period, shown moderate alcohol consumption to be negatively associated with the risk of IHD. This association has been established in diverse populations, for men and women, middle-aged and elderly, smokers as well as non-smokers and is likely to be causal in origin.[5]
Some investigators have explained this beneficial mechanism of alcohol consumption by reductions in platelet reactivity[6] and fibrinogen levels[7] inhibiting thrombosis superimposed on atherosclerosis. Recently Camargo et al[8], however, demonstrated an inverse association between alcohol and not only myocardial infarction but also stable angina pectoris. As stable angina pectoris is not attributed to thrombosis, but to the direct narrowing action of an atherosclerotic plaque, this gives reason to believe that the causal pathway is partly an anti-atherogenic one.

Animal studies linking alcohol to IHD
The hypothesis of an anti-atherogenic effect of alcohol has been investigated in only strikingly few animal studies, involving mainly rabbits, in which results have been contradictive[9],[10],17, but also non-human primates[11], C57BL/6 mice[12] and recently LDL-receptor-knockout mice[13]. Though interesting, the nonhuman primate study lacked statistical strength, and the applications of the remainder of the studies are limited, because the atherosclerotic lesions of the employed animals fail to progress beyond fatty streak stage. Another concern of previous studies is the highly nonphysiological diets that are necessary to induce atherosclerotic lesions.

Alcohol - or red wine ?
The thought of a protecting effect of alcohol consumption against IHD is widely accepted, even though the mechanisms are not fully understood and no experimental studies with IHD as endpoint have been presented. In contrast no agreement has been reached on the subject of which alcoholic beverage to prefer. Ecological studies distinguishing between types of alcoholic beverage generally attribute the full association to red wine consumption, but prospective studies have failed to reproduce this finding. Of ten prospective studies reviewed by Rimm et al., four showed a negative association between IHD and red wine, four between IHD and beer, and four between IHD and spirits[14].

Animal and biochemical studies on a special role for red wine
The hypothesis of a specific effect of red wine has had a consolidation in experimental settings. In their study on rabbits, Klurfeld and Kritchevsky[15] showed that red wine ingestion significantly reduced aortic atherosclerosis, whereas other beverages caused only slight non-significant reductions. Such protecting properties of red wine against the development of atherosclerosis are regularly referred to the antioxidant capacity of flavonoids; a subclass of phenols of red wine synthesised in the skin of grapes. Flavonoids are present in a number of fruits and vegetables, but could be of greater bioavailability in wine because of the presence of alcohol.[16] Dealcoholated red wine[17] was shown by Frankel et al to inhibit Cu2+-induced oxidation of LDL in vitro, and isolated flavonoids have been demonstrated in vitro to inhibit LDL-oxidation by macrophages[18] and to reduce free radical processes[19]. The significance of this in vivo is however uncertain. Recently a study on apoE-deficient mice has been conducted by Hayek et al[20]. In this experiment the intake of red wine (1,1 % ethanol) and flavonoids (catechin, quercetin) in (1,1 %)-ethanol-suspension inhibited the development of early fatty streaks and the ex vivo oxidation of LDL, when compared to mice drinking the (1,1 %)-ethanol-solution only.

Plaque size and plaque composition
In summary, both ethanol and flavonoids have been associated with inhibition of fatty streak development, although in the case of ethanol in animal models with nonphysiological diets and in the case of flavonoids without water as control. By scavenging free radicals and increasing resistance of LDL to oxidation, flavonoids are hypothesised to reduce oxidative modification of LDL, thereby diminishing the uptake and accumulation of lipids in macrophages and smooth muscle cells. The same mechanism may apply to ethanol[21] although the ability of ethanol to raise HDL-cholesterol probably is of greater importance. But increases in HDL-cholesterol can only account for about half of the association[22], and the mechanism by which HDL may reduce atherosclerosis is not understood in detail..
Protective effects against fatty streak development do not exclusively explain the epidemiological data, since most adult people already have these lesions, and the mechanism probably involves a component of shorter termed impact[23]. Additionally, the presence of a stable fibrous atherosclerotic plaque may not be hazardous, whereas a plaque with a relatively large atheromatous core and thin fibrous cap with ongoing inflammation is highly prone for disruption causing thrombosis4.
Ethanol has been reported to induce suppression of the T-cell-mediated immune response in mice[24], and flavonoids to diminish formation of several proinflammatory mediators[25]. Thus, ethanol and red wine solids might each on their own be able to reduce both the relative size of the atheromatous core and the degree of fibrous cap inflammation. This may lead to plaques less vulnerable to disruption. Whether such speculative effects of red wine components work in addition when drinking red wine could provide information about the mechanisms involved. There has been no studies addressing the impact of red wine, ethanol, and red wine solids on advanced plaque development and plaque composition, because of the lack of a proper animal model.

ApoE-deficient mice
ApoE-/--mice, with a genetic background of C57BL/6 and 129 strains, develop all the phases of atherosclerotic lesions from fatty streaks to advanced fibrous plaques on a western type diet at 15-20 weeks of age[26]. These lesions are characteristic in appearance and distribution to those found in humans.
In the present study, the separate and combined effects of ethanol and red wine solids on development and composition of mature atherosclerotic plaques are evaluated in atherosclerosis-prone apoE-deficient mice.

Pilot study
A pilot study on 50 C57BL/6 mice, lasting 2 weeks, has been carried out to estimate the voluntary consumption of water, suspended red wine solids, ethanol (5,75 %) and red wine (5,75 % ethanol), and the impact of these substances on weight gain and well being of the mice.
The voluntary intake of suspended red wine solids and water were significantly higher than the intake of alcoholic beverage. (Water: 241,3 ± 18,9 g/kg·day; red wine solids: 219,2±17,9 g/kg·day; ethanol: 185,0±16,7 g/kg·day; red wine: 174,8±9,0 g/kg·day, all presented as ingested fluid in g/day·kg body weight of mouse).
Nonetheless, no discomfort was observed and after a transient (non-significant) weight loss in the alcohol consuming groups no significant differences in weight gain were observed. Two groups of alcohol fed groups receiving 11,5 % ethanol and non-diluted red wine were part of the pilot study, but proved to have very low fluid intake. Blood was sampled by puncture of the right ventricle to obtain blood alcohol concentrations, but has as yet not been analysed.

Sixty mice, 7 weeks of age, homozygous for the inactivated apoE gene (C57BL/6 apoEm1Unc) are divided into four groups of 15 mice each.

• Control (Water) n=15 • Ethanol (6%) n=15 • Red wine solids (Clarét) n=15 • Red wine (6%) n=15

All are fed a western type diet, altromin C1057, which contains only small amounts of antioxidants α-tocopherol (75 μg/g) and vitamin C (50 μg/g), and so cannot mask an antioxidant effect of the ingested flavonoids. The chosen red wine is Cahors, Domaine de Paillas, 1996, selected for its high content of phenols, and the fact that it is not matured in oak tree casks, which could add additional oak-specific phenols to the wine. Ethanol concentration of the red wine is 12 %, but is diluted to 6 % in the experiment. Red wine solids is available through the recent commercial introduction of Clarét, a spray dried extract of unspecified red wine, kindly provided by Poul Olsen, PO medical. Clarét is suspended in water. The concentration of the Clarét suspension is calculated to provide equal concentrations of phenols in the red wine solids and red wine groups.
Red wine and Clarét solutions are supplied in light protected bottles.

The four groups are housed in a ventilated, temperature and humidity controlled environment with a 12h light-dark cycle. Diet and drink are available ad libitum. Fluid consumption are recorded and the fluids changed every other day. Weight gain is measured on a weekly basis. A pair feeding protocol is not used because the pilot study assured that all groups, after transient weight loss in the alcohol fed groups, gained weight at similar rates. Calories of the supplied ethanol are not substituted with glucose in the water and Clarét groups, because of the ad libitum feeding regimen and the doubtful influence of ethanol calories on weight gain[27].
To match the phenol ingestion between the red wine solids group, which in the pilot study had significantly higher fluid intake, to the red wine group, it may be necessary to further dilute the Clarét suspension. This will be decided 2 weeks into the study. The alcohol fed groups are supplied with alcoholic beverage as their only fluid source to obtain high internal validity. In this regimen inter-individual variance in compliance with alcohol drinking is reduced by the need for fluid.
The voluntarily ingested dose of red wine (5,75 %), measured in the pilot study of approximately 4,8 ml/d, is by some authors estimated to be analogous to 9-10 standard drinks of human consumption[28]. The comparison is by no means straight forward, because of very different rates of alcohol metabolism and total oxidative metabolism in man and mice[29].
Blood for determining blood alcohol concentration levels are sampled at the end of the dark cycle after 2 weeks from all alcohol fed mice as well as some red wine solids and water fed mice. Blood is collected from the orbit of the eye according to the method of Riley[30], which in contrast to bleeding from the cut tail, has been confirmed to provide blood alcohol concentrations representative to the one exposing central tissues.[31] After 3 months the animals are anaesthetised and blood samples are obtained by puncture of the right ventricle for determination of blood alcohol concentrations, total cholesterol, and HDL-cholesterol.

Pathoanatomic examinations
The animals are then sacrificed and perfused at ~100 mm Hg with phosphate-buffered 4 % formaldehyde (pH 7.2) via the left ventricle and then immersed in the fixative overnight. The heart, including the ascending aorta, is removed, weighed, and cut transversely as described by Paigen et al[32] followed by paraffin embedding. The half containing the aortic root is sectioned serially at 4 μm interval. The unstained 4-μm sections are checked frequently by microscopic examination to identify the beginning of the aortic root. Once the aortic sinus appear, every other section is collected on glass slides. Five sections taken from five different levels 80 μm apart and thus spanning 320 μm of the aortic root are stained (orcein for elastic tissue) and evaluated microscopically. Total plaque size and size of the different plaque components are measured blindly using computer-assisted image analysis equipment (Olympus BX50 light microscope, Sony DXC-151P color video camera, Imagraph Precision frame grapper, and SigmaScan Pro from Jandel Scientific Software), and the mean total plaque area and plaque composition is calculated.

Animal welfare and ethics
Inactivation of the gene coding for apoE results in hypercholesterolemia and spontaneous development of atheroslerosis. The mice are fertile, they grow normally, appear healthy and do not, by any means, suffer; no increase in morbidity or mortality have been reported. The mice are housed and cared for according to the national guidelines for the care and use of experimental animals. Our laboratory complies with the national regulations regarding the containment and use of genetically modified organisms.

Practical considerations
All necessary facilities for animal housing, pathoanatomic examinations and lipoprotein measurements are available at Aarhus University Hospital. Measurements of blood alcohol concentrations will be performed at the Institute of Forensic Chemistry, Aarhus University. Wine analyses is carried out at University Victor Segalen Bordeaux II.

Jacob Bentzon, medical student, Aarhus University, Erik Skovenborg, MD, associate professor, Department of General Practice, Erling Falk, MD, PhD, Research Professor, Coronary Pathology Research (IECR) and Department of Cardiology, Aarhus University Hospital, Skejby, Carsten Hansen, associate professor, MD, Department of Forensic Medicine, Section for Toxicology and Drug Analysis, Aarhus University.

RED WINE DOES NOT REDUCE MATURE ATHEROSCLEROSIS IN APOE-DEFICIENT MICE Bentzon J, Skovenborg E, Hansen C, Saint-Cricq de Gaulejac N, Falk E.Department of Cardiology, Aarhus University Hospital, 8200 Aarhus N, DenmarkRed wine and alcohol reduce fatty streak formation in various animal models. These experimental results support the observation that alcoholic beverages protect against ischemic heart disease. It is, however, questionable if fatty streaks reflect the pathology of mature and clinically relevant atheroclerosis. The present study examined the effects of red wine, red wine antioxidants, and alcohol on mature atherosclerosis in apoE-deficient mice.

Methods: Mice, 7 weeks of age, were randomized to receive water, red wine (diluted to 6% ethanol v/v), 6% ethanol v/v, or a non-alcoholic red wine powder in water, with antioxidant capacity comparable to that of the diluted red wine (8.5 mM and 12.7 mM TEAC, respectively). All mice were fed chow. At 26 weeks of age the mice were killed and the aortic roots were sectioned and quantitated.

Results: Paradoxically, red wine substances lowered plasma antioxidant capacity as measured with the kit from Randox Laboratories. No differences were seen in the amount of atherosclerosis.

Conclusion: Neither red wine, red wine antioxidants, or alcohol affected the development of mature atherosclerosis in the aortic root of apoE-deficient mice.

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Erik Skovenborg Hjemmeside <>