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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1999;19:1470-1475.)
© 1999 American Heart Association, Inc.

Atherosclerosis and Lipoproteins

Dietary ß-Carotene and -Tocopherol Combination Does Not Inhibit Atherogenesis in an ApoE–Deficient Mouse Model

Aviv Shaish; Jacob George; Boris Gilburd; Pnina Keren; Hana Levkovitz; Dror Harats

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

	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

	Introduction

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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.

	Methods

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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.

	Results

Top Abstract Introduction Methods Results Discussion References

 
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).

View larger version (15K): [in this window] [in a new window]   Figure 1. Body weight in control and antioxidant-treated mice. Body weight determined in control (•) and antioxidant-treated () mice were measured every 2 weeks.

View this table: [in this window] [in a new window]   Table 1. Cholesterol and Triglyceride Concentrations in Control Mice and Antioxidant-Treated Mice

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.

View larger version (18K): [in this window] [in a new window]   Figure 2. Vitamin E concentration in plasma and liver. The concentrations of vitamin E in plasma (A) and liver (B) were determined by reversed-phase HPLC analysis at the end of the experiment, as described in Methods.

View larger version (14K): [in this window] [in a new window]   Figure 3. Retinyl palmitate concentration in liver. The concentrations of retinyl palmitate in liver were determined by reversed-phase HPLC analysis at the end of the experiment, as described in Methods.

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).

View larger version (11K): [in this window] [in a new window]   Figure 4. Oxidation of lipoprotein from control and antioxidant-treated mice. Lipoproteins (d=1.063g/mL, top fraction) were isolated at the end of the trial from the control group (•) and the antioxidant-treated group (). Conjugated diene formation during incubation of 50 µg/mL protein with 15 µmol/L CuSO4 was measured at 234 nm.

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.

View larger version (15K): [in this window] [in a new window]   Figure 5. Extent of atherosclerosis in control and antioxidant-treated mice. The extent of atherosclerosis in controls () 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

Top Abstract Introduction Methods Results Discussion References

 
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 rabbits^{31 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.

	Acknowledgments

 
This work was supported by BSF grant No. 93 to 00193/3.

Received September 29, 1998; accepted December 1, 1998.

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