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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
oxidative modification and subendothelial accumulation of lipids
and migration and proliferation of vascular smooth muscle cells with subsequent
synthesis of collagenous matrix
. In the advanced
stages disruption of the plaque causes superimposed thrombosis leading to unstable
angina pectoris, myocardial infarction or sudden death
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.
Some investigators have explained this beneficial mechanism
of alcohol consumption by reductions in platelet reactivity
and fibrinogen levels
superimposed on atherosclerosis. Recently Camargo et al
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
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,,17
but also non-human primates
, C57BL/6 mice
and recently LDL-receptor-knockout mice
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
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
Animal and biochemical studies on a special role for red
The hypothesis of a specific effect of red wine has had a consolidation
in experimental settings. In their study on rabbits, Klurfeld and Kritchevsky
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.
Dealcoholated red wine
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
and to reduce free radical
. The significance of this in
is however uncertain. Recently a study on apoE-deficient mice has been
conducted by Hayek et al
. 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
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
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
, 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
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
Ethanol has been reported to induce suppression of the T-cell-mediated
immune response in mice
, and flavonoids
to diminish formation of several proinflammatory mediators
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.
-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
. 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.
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
• 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
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
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
The comparison is by no means straight forward, because of very different rates
of alcohol metabolism and total oxidative metabolism in man and mice
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
which in contrast to bleeding from the cut tail, has been confirmed to provide
blood alcohol concentrations representative to the one exposing central tissues.
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.
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
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.
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
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
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