© 1999 American Heart Association, Inc.
Atherosclerosis and Lipoproteins |
Dietary ß-Carotene and 
-Tocopherol Combination Does Not Inhibit Atherogenesis in an ApoE–Deficient Mouse Model
From the Institute of Lipid and Atherosclerosis Research (A.S., P.K., H.L., D.H.) and Research Unit of Autoimmune Diseases (J.G., B.G.), Department of Medicine `B', Sheba Medical Center, Tel-Hashomer and Sackler Faculty of Medicine, Tel Aviv University, Israel.
Correspondence to Dror Harats, MD, Institute of Lipid and Atherosclerosis Research, Sheba Medical Center, Tel-Hashomer, 52621, Israel. E-mail dharats{at}post.tau.ac.il
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Abstract |
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Abstract—Although lipid oxidation plays a major role in atherogenesis, the role of antioxidants in the prevention and treatment of the process is not clear. Apolipoprotein (apo) E–deficient mice develop spontaneous atherosclerotic lesions in major arteries. The presence of oxidized lipoprotein epitopes in the lesion suggests that oxidation reactions are involved in atherogenesis in this mouse model, but the inhibitory effect of antioxidants on atherogenesis in the model is controversial. To test the effect of dietary antioxidants on atherogenesis, male apoE-deficient mice (n=15) were fed a standard chow diet supplemented with 0.05%
-tocopherol and 0.05%
all-trans ß-carotene. A control group (n=15) received no antioxidant
supplement. At the end of the trial, mice consuming vitamins had 5x
more plasma vitamin E but undetectable ß-carotene levels. However,
liver levels of the ß-carotene metabolite, retinyl palmitate, were
higher in antioxidant-treated mice compared with control mice. The
antioxidants had no effect on lipoprotein or on plasma
anti–oxidatively modified low density lipoproteins (anti-oxLDL)
antibody levels. The vitamins had a small but insignificant effect on
lipoprotein resistance to ex vivo oxidation, determined by a longer lag
period of conjugated diene formation. Atherosclerosis,
determined by the lesion size at the aortic sinus, was insignificantly
suppressed in antioxidant-treated mice (mean area±SE, 20 000±7129
versus 13 281±5861 µm2; P=0.40).
The aortic atherosclerotic lesion area was similar in both experimental
groups (2.55±0.65% and 2.08±0.5% of total aortic area in the
control and antioxidant group, respectively; P=0.58).
The results of the current study suggest that moderate levels of
synthetic antioxidant vitamins have no effect on atherogenesis in
apoE-deficient mice.
Key Words: atherosclerosis • oxidation • antioxidants • ß-carotene • -tocopherol • antibodies • mouse • apoE
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Introduction |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Early atherosclerotic lesions are characterized by lipid-laden foam cells in the arterial intima.1 A potential mechanism of foam cell formation is the unregulated uptake of modified forms of LDL by the macrophage scavenger receptor.2 3 This receptor(s) recognizes LDL modified by products of lipid peroxidation.4 5 6
Human consumption of vegetables and fruits rich in carotenoids is associated with a lower risk of atherosclerosis,7 although a recent study found that the synthetic all-trans ß-carotene was not effective.8 This may be because other naturally occurring antioxidants were omitted. Alternatively, antioxidants such as ß-carotene and vitamin E may affect only the early stages of atherogenesis, and thus be unable to prevent the progression of established lesions.
The influence of ß-carotene on LDL oxidation and experimental atherosclerosis is not well understood. ß-carotene rapidly quenches singlet oxygen and free radicals, and exhibits good radical-trapping antioxidant properties at low partial pressure of oxygen9 ; but reports on the ability of ß-carotene to protect LDL against oxidation are controversial.10 11 12 13 14 15 16 We found that pretreatment with ß-carotene inhibits atherosclerosis in rabbits without affecting the susceptibility of LDL to oxidation.14 This study is supported by Sun et al,15 who showed that ß-carotene reduces the atherosclerotic lesion area but has no effect on LDL oxidation ex vivo.
The effects of vitamin E on experimental atherosclerosis are equivocal.17 18 We found no evidence for inhibition of atherosclerosis in New Zealand White rabbits by vitamin E supplementation in their diet, although the LDL isolated from these animals was significantly more resistant to oxidation compared with the control group.14
The effect of antioxidants on atherogenesis in apoE-deficient mice is not clear. The antioxidant N, N'-diphenyl 1,4-phenylenediamine inhibited atherosclerosis in mice fed a high-fat diet,19 and licorice consumption reduced the incidence and extent of atherosclerotic lesions in the aortic arch.20 In contrast, the antioxidant probucol, which has been shown to inhibit atherogenesis in several animal models, accelerated atherogenesis in apoE-deficient mice.21
ß-carotene has been shown to act synergistically with
-tocopherol as a radical-trapping antioxidant in
membranes.22 This raised the question of whether the
combination of the 2 antioxidants would protect against atherogenesis
better than ß-carotene or -tocopherol alone. In the
present study we therefore examined the effect of the antioxidant
vitamins ß-carotene and -tocopherol on early
atherogenesis in apoE-deficient mice.
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Methods |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Animals and Diet
ApoE-deficient mice were fourth or fifth generation hybrids with a C57BL/6x129 OLA background. The mice were kindly provided by Dr Jan L. Breslow (Rockefeller University, NY) and bred in the local animal house (Sheba Medical Center, Tel-Hashomer, Israel). All procedures using animals were in accordance with Sheba Medical Center guidelines. Male, 4-week-old, apoE-deficient mice were randomly divided into 2 groups of 15 animals each. The control group was fed a normal chow diet containing 4.5% fat by weight (0.02% cholesterol). The antioxidant group was fed the same diet enriched with 500 mg all-trans ß-carotene and 500 mg racemic
-tocopherol (Sigma
Chemical Co). The experiment lasted 16 weeks. The antioxidants were
dissolved in hexane and added to the diet as described
previously.14 Mice fed a control diet were treated with
hexane alone. Diets were stored in the dark under vacuum at 4°C, and
the mice were fed daily to minimize oxidation and degradation of
antioxidants.
Cholesterol Level Determination
Total plasma cholesterol and
triglyceride (TG) levels were determined using an automated
enzymatic technique (Boehringer Mannheim). HDL
cholesterol levels were determined with an HDL
cholesterol reagent (Sigma Chemical Co).
Detection of Anti-Oxidized-LDL (oxLDL) Antibodies by ELISA
Polystyrene plates with 96 wells (Nunc Maxisorp) were coated
with either copper-oxLDL (10 µg/mL in PBS) or native LDL, overnight
at 4°C. After washing 4x with PBS containing 0.05% Tween and
0.001% aprotinin (Sigma Chemical Co) the plates were blocked with 2%
BSA for 2 hours at room temperature. Diluted (1:50) serum fractions
were added in PBS containing 0.05% Tween and 0.2% BSA. After
additional overnight incubation at 4°C the sera were washed, and
alkaline phosphatase-conjugated goat anti-mouse IgG (1:10 000 in PBS
containing 0.05% Tween and 0.2% BSA; Jackson Immuno-Research
Laboratory Inc) was added for 1 hour at room temperature. After
extensive washing, 1 mg/mL p-nitrophenyl phosphate (Sigma
Chemical Co) in 50 mmol/L carbonate buffer containing 1
mmol/L MgCl2, pH 9.8, was added as a substrate.
The reaction was stopped after 30 minutes by adding 1 mol/L of NaOH.
Absorbance was detected at 405 nm in a Titertek ELISA reader (S.L.T.
Laboratory Instruments) and results expressed as absorbance at 405 nm.
Anti-oxLDL levels were calculated as follows: binding to native LDL
subtracted from oxLDL binding.
Antioxidant Concentration
ß-carotene and -tocopherol levels in plasma and
liver were determined as previously described.14 Retinyl
palmitate levels in the liver were detected according to
Furr,23 using a C18 column (TP-54, 250 x
4.6 mm, 5 µm particle size; Vydac) with a linear
gradient over 10 minutes from acetonitrile-water (85:15, vol/vol) to
acetonitrile-dichloroethane (80:20, vol/vol, plus 0.1% cyclohexane)
with a 15-minute hold; flow rate 1.5 mL/minute.
Aortic Lesion Evaluation
The percentage of aortic intimal area covered by atherosclerotic
lesions was characterized as described elsewhere.24
Animals were killed with an overdose of ketamine; the aortae
were then rapidly dissected free from the ascending arch to the iliac
bifurcation and washed in ice-cold PBS containing 1 mmol/L EDTA.
The vessels were fixed overnight with formal-sucrose (4%
paraformaldehyde, 5% sucrose, 20 µmol/L
butylated hydroxytoluene, 2 µmol/L EDTA, pH 7.4) followed
by a 6-hour rinse in PBS. After adventitial tissue removal, the aortae
were rinsed for 1 minute in 70% ethanol, immersed for 15 minutes in a
filtered solution of 0.5% Sudan IV (Sigma Chemical Co) in 35% ethanol
and 50% acetone, and rinsed in 80% ethanol for 5 minutes. The stained
aortae were placed on a slide and photographed. The intimal area
covered with sudan-stained atherosclerotic lesions and the total aortic
area were determined from digitized photographs by a model GS-690
imaging densitometer.
Assessment of Atherosclerosis in the Aortic
Sinus
Quantification of atherosclerotic fatty streak lesions was
performed by calculating the lesion size in the aortic sinus. The heart
and upper section of the aorta were removed from the animals and the
peripheral fat cleaned carefully. The upper section was
embedded in O.C.T. compound (Miles Inc) and frozen. Every other section
(5 to 10 µm thick) throughout the aortic sinus (400 µm)
was taken for analysis. The distal portion of the aortic sinus
was identified by the 3 valve cusps that are the junctions of the aorta
to the heart. Sections were evaluated for fatty-streak lesions after
staining with oil-red O. Lesion areas per section were counted, using a
grid, by an observer unfamiliar with the tested specimen.
Immunohistochemistry
Immunohistochemical staining for CD4, CD8, and
macrophages was performed on 5-µm-thick frozen sections of
the aortic sinus. The sections were fixed for 4 minutes in methanol at
-20°C followed by 10 minutes incubation with ethanol at -20°C.
The sections were then blocked with nonimmune goat serum for 15 minutes
at room temperature, incubated with CAS blocking reagent (Zymed) for 30
minutes at room temperature, followed by incubation with biotinylated
antibodies. After washing, the slides were incubated in 0.3%
H2O2, followed by
additional rinses, and developed with peroxidase streptavidin complex.
Sections were counterstained with hematoxylin. Spleen sections were
used as a positive control. Staining in the absence of first or second
antibody was used as a negative control.
Antioxidant Concentrations in Plasma and Liver
The antioxidants -tocopherol and ß-carotene
were determined after extraction and separation using
high-performance liquid chromatography (HPLC),
as described.14
Lipoprotein Oxidation
Lipoproteins (d=1.063g/mL, top fraction) were
isolated from pooled plasma of 3 mice from each group and 3 pools of
lipoproteins from each group were analyzed. Lipoproteins were
incubated at a concentration of 50 µg/mL in PBS, pH 7.4, with 15
µmol/L CuSO4. Incubation was carried out at
37°C in the dark. Lipid oxidation was measured as diene conjugation
formation at 234 nm.25
Statistical Analysis
Student's t test and the Mann-Whitney U
test were used to compare independent values. P<0.05 was
accepted as statistically significant.
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Results |
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We studied the effect of the antioxidant vitamins ß-carotene and
-tocopherol on atherogenesis in an apoE-deficient mouse
model. Control animals were fed a standard diet and the antioxidant
group received a standard diet enriched with 500 mg/kg all-trans
ß-carotene and 500 mg/kg racemic -tocopherol. These
levels correspond to a daily intake of 
60 mg/kg body weight of each
antioxidant.
Body Weight and Plasma Cholesterol Levels
Fifteen mice in each group were initiated onto the experimental
protocol. One mouse in the antioxidant group and 2 in the control group
died. No significant difference in body weights was observed during the
experimental period (Figure 1
). The
antioxidant-enriched diet did not affect total cholesterol,
HDL cholesterol, or TG levels
(Table).
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) mice were measured every 2 weeks.
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Antioxidant Levels in Plasma and Liver
Plasma and liver levels of -tocopherol and
ß-carotene were measured at the end of the trial. A significant
increase was detected in both plasma and liver
-tocopherol levels in the antioxidant group (Figure 2). ß-carotene was undetectable in both
groups. However, liver levels of the ß-carotene metabolite, retinyl
palmitate, were significantly higher in the antioxidant group (Figure 3), suggesting that ß-carotene was
converted to its metabolites. These results indicate that both
ß-carotene and -tocopherol were absorbed by the
apoE-deficient mice.
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Lipoprotein Oxidation
The effect of antioxidants on lipoprotein oxidation was assayed
indirectly in vivo by measuring anti-oxLDL antibodies by ELISA and ex
vivo by oxidation with 15 µmol/L CuSO4.
The antioxidants had no effect on plasma anti-oxLDL antibodies. At the
end of the study the mean optical density value was
0.049+0.01 for the control group and 0.07+0.01 for the
antioxidant group. A slight, insignificant increase in lag phase of
lipoprotein oxidation was detected in the antioxidant group compared
with the control group (Figure 4;
69.5±5.33 versus 55.0±6.56 minutes for the antioxidant and control
groups, respectively).
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The Effect of Antioxidants on Atherosclerosis
The lesion area was measured in both the aortic sinus and aorta.
The atherosclerotic lesion area in the aortic sinus was smaller in the
antioxidant group than in the control,
4.6±1.6x105 versus
7.3±2.1x105
µm2 (Figure 5), but this decrease was not
statistically significant (P=0.268). The lesion area in the
aorta was similar in both groups, 2.55±0.65% and 2.08±0.5% of the
total aortic area in the control and antioxidant groups
(P=0.58). No differences were evident between the
experimental groups with respect to the density of macrophages
or T-lymphocytes.
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) and
antioxidant-treated mice (
) was quantified after 16 weeks of
treatment. Atherosclerotic lesion area in the sinus (A) was measured by
staining lesions with oil-red O and counting by a grid. The intimal
aortic area covered with Sudan IV-stained atherosclerotic lesions (B)
was determined from digitized photographs. |
Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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The results of the present study show that supplementation with the antioxidant combination of all-trans ß-carotene and
-tocopherol in the normal chow diet fed to
apoE-deficient mice does not inhibit atherogenesis in the aortic sinus
or the aorta. In a previous paper we suggested that ß-carotene
inhibits atherosclerosis in cholesterol-fed
rabbits by a novel pathway, independent of making LDL resistant
to oxidation.14 The hypothesis of the present paper
was that a synergistic effect on atherogenesis could be observed when
the 2 antioxidants, together, were given to the experimental
animals.
Both -tocopherol and ß-carotene were absorbed by the
mice. After 16 weeks of antioxidant feeding, the levels of
-tocopherol were about 6-fold higher in
antioxidant-treated animals than in control mice and liver
-tocopherol levels were 10x higher than in the
control mice. ß-carotene was undetectable in both plasma and liver.
However, retinyl palmitate levels in antioxidant-treated animals were
twice as high as those in the control group, suggesting that
ß-carotene is readily converted to its metabolite in apoE-deficient
mice. This is in accord with the results found in
rabbits,14 26 27 and in contrast to the fate of
ß-carotene in humans, where most of the ß-carotene is absorbed
intact and carried in plasma lipoproteins.28 29 These
results show that both -tocopherol and ß-carotene are
absorbed by the apoE-deficient mice, but the metabolism of
ß-carotene in the mice is different than that in humans.
Many lines of evidence suggest that oxidized LDL is
atherogenic.1 2 3 4 5 6 We assayed the protective effect of the
antioxidants against lipoprotein oxidation both in vivo and ex vivo.
During oxidation of lipoproteins, malondialdehyde can form covalent
bonds with lysine residues on LDL apoB, and autoantibodies against
oxLDL can be detected in the plasma of
hypercholesterolemic animals and humans.30
Moreover, hyperimmunization of rabbits31 32 and
apoE-deficient mice33 with homologous
malondialdehyde-modified LDL (MDA-LDL) leads to the production
of high titers of antibodies against MDA-LDL and suppresses
atherosclerosis. The results of the present work
show that antioxidant treatment has no effect on oxLDL plasma levels in
apoE-deficient mice. Neuzil et al34 showed that vitamin E
in apoE-deficient mice is associated with VLDL and LDL. Because vitamin
E is a potent inhibitor of lipoprotein oxidation, and
plasma -tocopherol levels increased to very high levels
in the antioxidant group (Figure 2
), we measured its effect on
VLDL and LDL oxidation ex vivo. The small increase in the lag phase of
diene conjugation formation (Figure 3) was not statistically
significant. Moreover, as suggested by Fruebis et al,35
LDL resistance to oxidation must reach threshold levels before there is
a significant protection against atherogenesis. ß-carotene has been
shown to act synergistically with -tocopherol as a
radical-trapping antioxidant in membranes.22 This
observation raised the question of whether the combination of the 2
antioxidants could protect against LDL oxidation more effectively than
ß-carotene or -tocopherol alone. However, the rapid
conversion of ß-carotene to its metabolite in the mouse model ruled
out this possibility. In humans, on the other hand, increased levels of
both antioxidants may protect better than ß-carotene or
-tocopherol alone.
The inhibitory effect of antioxidants on atherogenesis in
apoE-deficient mice and other mouse models is not consistent.
The antioxidants N, N'-diphenyl-1,4-phenylenediamine
(DPPD)19 and licorice20 inhibited
atherosclerosis in apoE-deficient mice, but the
antioxidant probucol, which has been shown to inhibit atherogenesis in
several animal models,36 37 38 paradoxically accelerated
atherogenesis in apoE-deficient mice.22 The inhibition of
atherogenesis in DPPD-treated and licorice-treated mice is explained by
the greater resistance to copper-induced oxidation of lipoprotein
isolated from DPPD-treated mice. The protection effect was achieved at
very high levels of the antioxidants, 5 g/kg DPPD, which is equivalent
to 19.2 mmol/kg diet, whereas -tocopherol and
ß-carotene concentrations in the present work were only 1.16 and
0.9 mmol/kg, respectively. The resistance of lipoproteins to
oxidation in the probucol trial has not been assayed. In the
present work we found no effect on oxidation and atherogenesis. The
failure to inhibit atherogenesis by -tocopherol is in
agreement with results obtained in C57BL/6 mice fed a high-fat
high-cholesterol diet.39 In that study,
vitamin E reduced total cholesterol and HDL
cholesterol levels. In our work, the vitamin combination
had no effect on lipoprotein profile (Table). It is not clear
whether vitamin E in other animal models inhibits atherogenesis by its
antioxidative effect because suppression of atherogenesis by vitamin E
in animal models is associated with decreased serum
cholesterol levels.40 41 42
The paradoxical effect of probucol on atherogenesis in apoE-deficient
mice and the failure to inhibit atherogenesis by ß-carotene and
-tocopherol combination raises the issue of the
appropriateness of apoE-deficient mice as a model to study the role of
oxidation and antioxidants on atherogenesis. There are 2 possible
explanations for the inability to suppress atherogenesis in
apoE-deficient mice: First, the major circulating lipoproteins in
apoE-deficient mice are chylomicrons and ß-VLDL remnants, and most of
the antioxidants are carried by those lipoproteins.34 The
role of oxidation in atherogenesis induced by ß-VLDL is uncertain
because native ß-VLDL converts macrophages into foam
cells.42 Second, the concentrations of antioxidants used
in our study are too low to affect the atherogenesis process. Indeed,
during the preparation of the revised manuscript, Pratico et
al43 published a paper demonstrating that considerably
higher levels of vitamin E suppress isoprostane generation and reduce
atherosclerosis in apoE-deficient mice. This
observation implies that threshold levels of vitamin E may need to be
achieved to obtain a beneficial effect on atherogenesis.
In conclusion, we have found that moderate dietary vitamin supplementation does not influence the extent of atherosclerosis in the apoE-deficient mouse, despite higher plasma and tissue antioxidant levels.
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Acknowledgments |
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This work was supported by BSF grant No. 93 to 00193/3.
Received September 29, 1998; accepted December 1, 1998.
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