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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1995;15:349-358.)
© 1995 American Heart Association, Inc.

Articles

Relation of Vascular Oxidative Stress, -Tocopherol, and Hypercholesterolemia to Early Atherosclerosis in Hamsters

Rex A. Parker; Talal Sabrah; Michael Cap; Brian T. Gill

From the Department of Metabolic Diseases, Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, NJ.

Correspondence to Rex A. Parker, PhD, Department of Metabolic Diseases, Rm K-3111, Bristol-Myers Squibb Pharmaceutical Research Institute, PO Box 4000, Princeton, NJ 08543-4000.

	Abstract

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Abstract A model of early atherosclerosis in hamsters with moderate hypercholesterolemia (217 to 271 mg/dL) was established that was highly responsive to exogenous antioxidants. A key feature of this model was elevation of vascular oxidative stress by use of a diet deficient in nutritional antioxidants and supplemented with corn oil (10%) and cholesterol (0.2%, 0.4%, or 0.8%). After 10 weeks on the 0.4% cholesterol diet, mean plasma -tocopherol levels declined from 5.68±0.30 to 1.27±0.15 µg/mL, while monocyte-macrophage foam cell lesions in the aortic arch, as assayed by video microscopy/image analysis of oil red O–stained histological specimens, increased from undetectable at week 0 to 60 900±5400 µm² per specimen at week 10 (mean±SEM, n=36). -Tocopherol or probucol administered for 10 weeks markedly suppressed LDL oxidation ex vivo and profoundly inhibited aortic foam cell formation. However, the effects of antioxidants on aortic lesions were attenuated at higher plasma cholesterol levels, although LDL oxidation ex vivo was effectively inhibited. With a plasma cholesterol level at 250 mg/dL, the maximum effect of -tocopherol on lesion size reached 36% of control value, and the dose for half-maximal effect was 10 mg · kg^-1 · d^-1, which resulted in a plasma -tocopherol value of 20 µg/mL. Under these conditions a linear, inverse correlation of aortic lesion size and plasma -tocopherol concentration was observed (n=68, r=-0.581, P<.001). The data demonstrate that LDL oxidation is a significant component of early atherogenesis in this model but suggest that hyperlipidemia can outweigh the therapeutic effectiveness of antioxidants. The high sensitivity of aortic lesion initiation to -tocopherol in hamsters maintained on moderately hypercholesterolemic diets depleted of endogenous antioxidants demonstrates that vascular oxidative stress can be isolated from other causative factors in an in vivo model of atherosclerosis.

Key Words: -tocopherol • antioxidant • LDL oxidation • glutathione • atherosclerosis

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
Recent studies of the biological properties of oxidized LDL support the concept that LDL lipid peroxidation and unattenuated vascular oxidative stress contribute to the pathophysiology of atherosclerosis.^{1 2} Free-radical chain reaction–terminating antioxidants carried within LDL particles can curtail the formation of oxidized LDL in vitro, thus providing a paradigm for the therapeutic use of antioxidants in the treatment and prevention of atherosclerosis.^{2 3 4} However, oxidative stress in the vessel wall is not an isolated variable but exists in the context of hyperlipidemia, often complicated by hypertension and hyperglycemia, which confound direct tests of the impact of antioxidants on the initiation and progression of the disease. The principal objectives in the present studies were to establish a practical, relatively fast, and highly sensitive in vivo animal model of oxidant-dependent early atherosclerosis; to correlate lesion formation with ex vivo indices of LDL oxidation susceptibility; and to relate these parameters to plasma cholesterol and LDL concentrations. From this approach a means to quantitatively evaluate LDL antioxidant therapies as antiatherosclerosis agents might be found.

No published experimental models of atherosclerosis appear to have achieved sufficient sensitivity to low doses of antioxidants and plasma cholesterol levels within the typical human range. In several studies, probucol administered at 1% in the diet decreased atheroma size in Watanabe heritable hyperlipidemic (WHHL) or cholesterol-fed rabbits with plasma cholesterol levels greater than 700 mg/dL.^{5 6 7 8} The antioxidants N,N'-diphenylphenylenediamine and butylated hydroxytoluene, each at 1% in the diet, reduced atheromas in 1% cholesterol–fed rabbits with cholesterol levels greater than 1000 mg/dL.^{9 10} -Tocopherol at 0.5% in the diet for 10 weeks decreased aortic lesions in WHHL rabbits, although hypocholesterolemic effects were also noted.¹¹ A recent study of low-dose (0.025% in the diet) vitamin E and probucol in WHHL rabbits showed effects on LDL oxidation susceptibility but failed to detect significant inhibition of aortic atherosclerosis at 6 months; cholesterol levels in control animals exceeded 600 mg/dL.¹² Probucol (0.5% in a 4% cholesterol diet) inhibited aortic cholesterol accumulation without decreasing adherent monocytes in rats with plasma cholesterol levels of approximately 700 mg/dL.¹³ -Tocopherol was also recently shown to decrease carotid artery lesions in primates when given at a moderate dose for 3 years.¹⁴

When fed an appropriate diet, the hamster presents a more humanlike lipoprotein profile than do many other rodent species.^{15 16} Hypercholesterolemic diet–induced atherosclerosis in the hamster model is confined initially to a lesion-prone area along the inner curvature of the aortic arch. Atherogenesis in this region is characterized by the infiltration of monocytes, which become lipid-filled macrophage foam cells, the progenitor of the fatty streak.^{16 17} Within approximately 12 months these early aortic lesions can develop into complex advanced plaques resembling human lesions, with a fibrous cap of smooth muscle cells, connective tissue matrix, the presence of macrophage foam cells, and a necrotic core containing cholesterol crystals.¹⁸ In the studies presented in this article, quantitation by image analysis of the early macrophage foam cell lesions in hamster aortas served as a key end point for assessing the influence of antioxidants on atherogenesis under conditions of modest hypercholesterolemia.

A spectrum of oxidized LDL species appears to contribute to the pathobiology within the artery wall. Extensively oxidized LDL, containing alkyl hydroperoxides and aldehydes, cholesterol oxides, and chemically modified amino acid side-chain residues in apo B, provides a route for its own sustained uptake during foam cell formation by promoting expression of scavenger receptors on macrophages.^{19 20 21} Some products of oxidized LDL lipids adversely affect endothelial and smooth muscle cell function.²¹ Minimally oxidized LDL provokes expression of inflammatory mediators, including cytokines, adhesion molecules, and chemotactic factors, in cultures of endothelial and smooth muscle cells, activities that are consistent with monocyte influx and macrophage foam cell development in the artery wall.^{22 23 24 25} By analogy with the behavior of antioxidants in simple, single-phase systems,²⁶ the effect of antioxidants on the oxidation susceptibility of LDL can be estimated by monitoring the formation of lipid oxidation products, such as conjugated dienes, in vitro.^{27 28} With the assumption that the initial rate of LDL oxidation and the lag time for extensive oxidation in vitro reflect the in vivo tendency of LDL to undergo minimal and more extensive stages of oxidation, the effectiveness of antioxidants administered to animals can be quantitatively estimated by this ex vivo assay approach.

In the studies presented here, conditions that sensitize atherosclerosis to the therapeutic effects of antioxidants were explored in moderately hyperlipidemic hamsters. The hypothesis tested was that the relative contribution of oxidative stress to atherosclerosis depends on the degree of hypercholesterolemia. In the course of these experiments, the ability of low doses of -tocopherol to suppress atherogenesis was observed. The sizes of early aortic lesions were correlated with LDL oxidation susceptibility ex vivo and with plasma -tocopherol concentrations. These studies indicate that under appropriate experimental conditions, vascular oxidative stress can be effectively evaluated in an animal model of atherosclerosis.

	Methods

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Diets and Antioxidants
Male Syrian golden hamsters (initial weight, 100 to 110 g) were fed normal chow for 1 week, randomized into groups, and provided defined diets ad libitum for up to 10 weeks. In some studies a normal Purina Chow–based diet supplemented with corn oil and cholesterol was used, which provided a daily vitamin E intake of approximately 5.3 mg/kg body weight. A defined, synthetic, basal diet with no added vitamins E, K, and C; ß-carotene; and selenium was prepared (Research Diets, Inc) and stored under nitrogen at -20°C. The composition of the synthetic diet (in weight percent) was corn starch 50, casein 15, maltodextrin 10, cellulose 7.1, herbivore salt mixture minus selenium 6, standard herbivore vitamin mixture minus E and K 1, DL-methionine 0.3, and choline bitartrate 0.2. To this basal diet was added cholesterol (0.2%, 0.4%, or 0.8% as noted) and stripped corn oil (10%) obtained from Kodak. The daily vitamin E intake with this diet was less than 0.025 µg/kg body weight. Studies with -tocopherol used DL--tocopherol from Aldrich (vitamin E, No. 25802-4); probucol and coenzyme Q₁₀ (CoQ-10) were from Sigma; and ubiquinol-10 was prepared from CoQ-10 by sodium borohydride reduction.²⁹ Antioxidants were administered in the diet after they were formulated at a defined weight percent by dissolution in corn oil before being mixed into the diets. Dosages were calculated on the basis of average food consumption and body weights (155 g) at the end of the 10-week studies; the calculated dose of 60 mg · kg^-1 · d^-1 corresponds to 0.1% in the diet.

Histopathology
Hamsters were generally healthy and gained body weight (150% of their starting weight) during the 10-week duration of the atherosclerosis studies. A histological survey of several tissue types was conducted on hamsters after 12 weeks on the antioxidant-deficient, 0.4% cholesterol diet. The following differences from normal chow-fed control animals were noted: (1) liver: hepatocellular hypertrophy with vesiculation, diffuse lipidosis, and multifocal inflammation; mesenteric fat steatitis; (2) skeletal muscle: multifocal degeneration and necrosis, elevated plasma creatine kinase levels; and (3) skin: cellulitis. The liver abnormality was likely related to elevated dietary fat/cholesterol intake, whereas the steatitis and skin and muscle pathologies were symptomatic of elevated lipid peroxidation associated with the antioxidant vitamin deficiency.

Atherosclerosis Assay
Treatment and control groups (n=12 hamsters per group) for each 10-week study were processed within 5 days of each other. Macrophage foam cell formation, which was highly localized in the aortic arch region under these conditions, was assayed essentially as described by Kowala et al^{16 17} and Nunnari et al³⁰ using en face–mounted histological specimens of the aortic arch stained with oil red O. The area occupied by stained macrophage foam cells was determined by video microscopy/image analysis using a Nikon Microphot-FXA microscope with a 10x plan achromatic objective and an area-calibrated Hamamatsu Newvicon C2400 video camera linked to an Image-1 image analysis system (Universal Imaging). Six, seven, or eight image fields (480x512 pixels per field) were acquired per specimen. Virtually 100% of oil red O–stained areas was intracellular and contained within the foam cells. Total foam cell area was summed for each specimen, and the group mean±SEM values were calculated to compare effects in the study groups relative to the control groups. Significance of difference between group means was calculated by the standard, two-tailed Student's t test.

Ex Vivo Assays of LDL Oxidation
Plasma was separated from blood collected into EDTA (2 mmol/L) from 16-hour–fasted, pentobarbital sodium (Nembutal)–anesthetized hamsters. Plasma was pooled (two or three samples per pool) prior to LDL isolation. The plasma LDL fraction (d=1.019 to 1.063 g/mL) was prepared rapidly (in less than 6 hours) by sequential flotation at 16°C in a Beckman TL-120 rotor and TLX ultracentrifuge by a modification of the original procedure of Havel et al³¹ ; solid KBr was used to adjust the density. The isolated LDL was dialyzed into a 10 mmol/L HEPES (pH 7.2) and 0.15 mol/L NaCl buffer (buffer 1), and aliquots were added to 12-cell cuvettes at 100 µg protein per milliliter in a total volume of 200 µL in buffer 1. Oxidation reactions were initiated with CuSO₄ (2.5 µmol/L) at 20°C, and conjugated-diene absorbance at 234 nm was monitored continuously in a Beckman DU-7500 spectrophotometer for 10 hours.²⁷

Two parameters that characterize the oxidation susceptibility of hamster LDL ex vivo were obtained from Cu²⁺-dependent, conjugated-diene kinetics. By analogy with the behavior of antioxidants in simple, single-phase systems, the initial rate of LDL oxidation was taken as an estimate of the intrinsic activity of the antioxidant within LDL, and the lag time before extensive formation of oxidation products served as an index of the concentration of antioxidant within LDL.^{26 32} The initial rate of LDL oxidation was defined as the slope of the linear regression of absorbance at 234 nm versus time over the approximately linear initial phase of the reaction, ie, from 0 through 40 to 60 minutes; values are expressed in nanomoles of conjugated-diene equivalent per minute per milligram of LDL protein, with an assumed extinction coefficient of 28 000 cm^-1 for the conjugated diene. Lag time was defined as the time corresponding to the maximum of the first derivative of the 234-nm-absorbance versus time curve.

-Tocopherol Determination
-Tocopherol levels were determined by high-performance liquid chromatography of extracts of individual hamster plasma samples from blood collected into EDTA (2 mmol/L). Samples were prepared by addition of an -tocopherol acetate internal standard to 1.0-mL aliquots of plasma and then extracted twice at 20°C with two volumes of hexane. The extracts were evaporated under nitrogen and reconstituted in ethanol (120 µL), and aliquots (50 to 100 µL) were injected onto a C18 reverse-phase column (Rainin Microsorb: 5-µm particle size; column size, 250x4.6 mm) at a flow rate of 0.9 mL/min (1800 psi) under isocratic conditions and with a mobile phase consisting of 73.5% ethanol, 22.5% methanol, and 4% isopropanol. UV absorbance was monitored by a Beckman System Gold model 168 diode array detector at 284 nm, with spectral comparisons to confirm peak identities, and the -tocopherol concentration for each sample was calculated from peak area ratios with the internal standard.

Other Assays
The level of reduced glutathione (GSH) in hamster erythrocytes was determined by a commercially available colorimetric reagent system (GSH-400 from Bioxytech, SA). Erythrocytes isolated from EDTA-collected blood were extracted with 20% trichloroacetic acid at 4°C, and aliquots were used for the GSH assay. For all plasma lipid determinations, aliquots of EDTA-plasma samples from individual animals were assayed. Plasma total cholesterol and triglycerides were assayed with standard commercial enzyme kits on a COBAS-MIRA analyzer (Roche Diagnostic Systems). HDL cholesterol was determined after precipitation of apo B–containing lipoproteins from plasma with phosphotungstate reagent, and LDL+VLDL cholesterol (referred to as "LDL cholesterol" in the data tables) was calculated as the difference between total and HDL cholesterol.³³ Protein was determined by Coomassie assay (Pierce).

	Results

Top Abstract Introduction Methods Results Discussion References

 
Studies of hamsters that were fed chow-based, atherogenic diets failed to reveal significant effects of antioxidants on early atherosclerosis. Lipid-lowering drugs such as cholestyramine had previously been shown to reduce aortic fatty streak formation in hamsters fed a standard diet of chow plus 10% saturated fat (coconut oil) and 0.2% cholesterol for 10 weeks. Under these conditions, however, aortic lesions were not significantly affected by high doses of -tocopherol or probucol (60 mg · kg^-1 · d^-1; data not shown). The mean plasma cholesterol level exceeded 800 mg/dL at 10 weeks in this study, suggesting that excessive hypercholesterolemia can drive macrophage lipid accumulation and foam cell formation regardless of the susceptibility of LDL to oxidation.

The chow-based diet was then modified by substituting a source of polyunsaturated fat (corn oil) to obtain a more moderate cholesterol elevation and to elevate the oxidation susceptibility of LDL, as was previously shown with rabbits.³⁴ Compared with the saturated fat diet at 3 weeks, this diet increased linoleate and decreased palmitate levels within the hamster LDL fraction. The distributions of fatty acids in LDL cholesteryl esters for the corn oil–supplemented diet were 9% palmitate (16:0), 4% stearate (18:0), 28% oleate (18:1), and 59% linoleate (18:2). Values for the standard saturated fat diet were 17% 16:0, 4% 18:0, 32% 18:1, and 47% 18:2. For the corn oil diet, mean plasma cholesterol levels at 10 weeks were in the 330 to 358 mg/dL range (Table 1), substantially less than those from the saturated fat diet. In samples taken at 10 weeks, the lag phase for extensive Cu²⁺-mediated oxidation of LDL ex vivo was increased by -tocopherol (350% of control) and probucol (200% of control) (data not shown). However, mean aortic lesion size in either probucol or -tocopherol treatment groups was again not significantly different from control animals in two 10-week studies in hamsters (Table 1).

View this table: [in this window] [in a new window]   Table 1. Lack of Effect of Antioxidants on Aortic Foam Cell Formation in Hamsters Maintained on a Chow-Based Atherogenic Diet for 10 Weeks

The standard chow-based diets provided each hamster a daily -tocopherol intake of approximately 5.3 mg/kg body weight, as well as undetermined levels of carotenoids and other nutritional antioxidants. To amplify oxidant stress and provide a lower background for controlled studies of antioxidants, a defined, synthetic diet deficient in vitamins E, C, and K; carotenoids; and selenium was developed and then supplemented with corn oil and cholesterol as before (see "Methods"). Three-week maintenance of the hamsters on this diet resulted in elevated plasma total and LDL cholesterol levels in proportion to the amount of added dietary cholesterol (Table 2). The susceptibility of LDL to oxidation in the synthetic-diet groups was greater than that in the chow-based groups, as reflected in higher initial rates for LDL oxidation and lower lag times for full LDL oxidation ex vivo (Table 2). The levels of lipid peroxides initially present within these LDL samples were not detectably different. The initial rate of LDL oxidation, normalized to protein concentration, increased as plasma total and LDL cholesterol values increased in synthetic-diet groups, whereas lag times were the same. The effectiveness of the antioxidants -tocopherol, CoQ-10, ubiquinol-10 (the reduced form of CoQ-10), and probucol on LDL oxidation ex vivo was examined after treating the hamsters for 3 weeks with these compounds, which were added to the standard chow-based diet. CoQ-10, at 60 or 300 mg · kg^-1 · d^-1 and administered as CoQ-10 or as ubiquinol-10, resulted in only marginal effects on LDL oxidation susceptibility in the chow-based model (Table 3). In contrast, probucol (120 mg · kg^-1 · d^-1) moderately decreased the initial oxidation rate and increased the lag time, whereas -tocopherol (60 mg · kg^-1 · d^-1) was more effective on both parameters (Table 3). When administered as part of the synthetic diet, CoQ-10, probucol, and -tocopherol each had more of an effect on the LDL oxidation parameters compared with the chow diet. CoQ-10 was again least active and -tocopherol most active (Table 3).

View this table: [in this window] [in a new window]   Table 2. Plasma Lipid Levels and Ex Vivo LDL Oxidation Susceptibility in Hamsters Fed Chow-Based or Antioxidant-Deficient Synthetic Diets for 3 Weeks

View this table: [in this window] [in a new window]   Table 3. Effects of Coenzyme Q10 (CoQ-10), -Tocopherol, or Probucol on LDL Oxidation Susceptibility Ex Vivo in Hamsters After 3 Weeks on Chow-Based or Antioxidant-Deficient Synthetic Diets

Hamsters were maintained for 10 weeks on the antioxidant-deficient, synthetic diet supplemented with 0.2%, 0.4%, or 0.8% cholesterol, with or without added -tocopherol or probucol. Mean plasma total cholesterol levels in control animals at week 10 ranged from 217 to 271 mg/dL, with 43% to 45% of total cholesterol in the LDL fraction (Table 4). Plasma total cholesterol levels were not decreased by -tocopherol or probucol treatment, although minor increases in HDL cholesterol and a trend toward elevation of total cholesterol were noted (Table 4). Assays of LDL oxidation ex vivo at 10 weeks showed that -tocopherol and probucol each decreased the initial rate of LDL oxidation to less than 20% of control and markedly extended the lag phase, whereas -tocopherol had a greater effect than probucol on the latter parameter (Table 4). Histological evaluation revealed localized foam cell lesions in the aortic arch, with focal accumulations of mononuclear cells that stained intensely with oil red O (Fig 1A and 1B). Essentially all of the staining was intracellular within macrophages (Fig 1C and 1D), a finding consistent with an early stage of foam cell formation and the beginning of fatty streak development. Mean aortic foam cell area in control animals ranged threefold between the 0.2%, 0.4%, and 0.8% dietary cholesterol groups (Fig 2). Mean lesion size was significantly decreased in all -tocopherol and probucol treatment groups compared with control groups, but the effectiveness of the antioxidants declined as cholesterolemia increased (Fig 2). For example, mean lesion area in the 0.2% dietary cholesterol group was profoundly decreased to 13% of control (P=.010) by -tocopherol, but in the 0.8% cholesterol diet group, lesion size was only moderately inhibited, to 56% of control (P=.044), by the same dose of -tocopherol. Photomicrographs of aortic specimens from the -tocopherol groups showed markedly fewer foam cells than in controls, and foam cell clusters tended to be smaller (Fig 1E and 1F).

View this table: [in this window] [in a new window]   Table 4. Effects of -Tocopherol or Probucol on Plasma Lipids and LDL Oxidation Ex Vivo in Hamsters After 10 Weeks on an Antioxidant-Deficient, Synthetic Diet

View larger version (2K): [in this window] [in a new window]   Figure 1. Photomicrographs of aortic foam cell lesions from hamsters maintained for 10 weeks on an antioxidant-deficient, 0.4% cholesterol diet. Hamster aortic arch specimens were prepared and stained with oil red O as described in "Methods." A and B, Two representative control specimens (original magnification, x15.5 on film). C, Control specimen, with differential interference contrast (DIC) optics (original magnification, x187). D, Control specimen, with DIC optics. Note that oil red O staining is virtually entirely contained within foam cells (original magnification, x375). E and F, Two representative -tocopherol–treated specimens (60 mg · kg-1 · d-1 for 10 weeks), showing marked reduction in foam cells (original magnification, x15.5). Photomicrographs were taken with Fujichrome 100D 35-mm slide film, from which 3x5-in. Cibachrome prints were made.

View larger version (26K): [in this window] [in a new window]   Figure 2. Bar graph showing effect of probucol or -tocopherol (-tocoph) on aortic foam cell lesions in hamsters after 10 weeks on an antioxidant-deficient diet containing three levels of cholesterol (chol). Data correspond to the study in Table 4. Values are mean±SEM, n=12 hamsters per group. Significance vs control group means are indicated above each bar.

The relationship between mean total lesion area and ex vivo LDL oxidation for control, -tocopherol–, and probucol-treated hamsters in the 0.2% and 0.8% dietary cholesterol groups is shown in Fig 3. LDL oxidation susceptibility was strongly affected by the antioxidants under both dietary conditions, but lesion size was markedly more responsive to antioxidants and the lower cholesterol diet. Fig 4 depicts the relationship between lesion size and plasma cholesterol level for control and -tocopherol groups and includes mean±SEM values from five separate studies with three levels of dietary cholesterol. The intercept of the regression line on the plasma cholesterol axis was 237 mg/dL for the -tocopherol groups compared with 186 mg/dL for control groups. This suggests that in the presence of -tocopherol, the cholesterol threshold for lesion initiation was approximately 50 mg/dL higher than in control. The slopes of the regression lines were parallel, suggesting that above a certain threshold, the dependence of lesion formation on cholesterol concentration with or without -tocopherol was similar (Fig 4).

View larger version (20K): [in this window] [in a new window]   Figure 3. Plots showing the relation between aortic lesion size and ex vivo LDL oxidation parameters. Aortic lesions and ex vivo LDL oxidation were assayed after 10-week treatment with the antioxidant-deficient diet containing 0.2% (upper panel) or 0.8% (lower panel) cholesterol. -Tocopherol was given at 60 mg · kg-1 · d-1, probucol at 120 mg · kg-1 · d-1. Data are mean±SEM, n=12 hamsters per group. init. indicates initial.

View larger version (24K): [in this window] [in a new window]   Figure 4. Plot and regression lines showing relation of aortic lesion size in control and -tocopherol–treated hamsters to plasma cholesterol levels. Hamsters were maintained for 10 weeks on the antioxidant-deficient diet containing one of three levels of cholesterol as indicated by the symbol type. Data are mean±SEM for n=12 hamsters per group. Open symbols indicate control animals; closed symbols, animals treated with -tocopherol at 60 mg · kg-1 · d-1.

Changes in plasma -tocopherol concentration and erythrocyte GSH content were determined as markers of the onset of vascular oxidative stress in this model. Fig 5 relates the fall in plasma -tocopherol and erythrocyte GSH levels over time to the appearance of aortic lesions. The mean plasma -tocopherol concentration after 10 weeks of treatment with the synthetic diet (0.4% cholesterol) was 1.27±0.45 µg/mL, approximately 20% of the concentration in normal chow-fed animals. The time course showed a linear decrease in plasma -tocopherol level through the seventh week, which leveled off by the 10th week (Fig 5). Erythrocyte GSH content decreased significantly, to 1.7±0.1 µmol/g at week 7, 74% of its initial level (P<.03). (Ten-week samples for GSH analysis were not obtained.) During this period, aortic foam cell formation, undetectable at week 0, increased approximately linearly at weeks 5 through 10. As described in "Methods," pathological changes in several tissues at week 12 of the antioxidant-deficient diet suggested that more generalized tissue damage arises with continued antioxidant deficiency, indicating that the practical limits for atherosclerosis studies lie within the 10-week time frame.

View larger version (19K): [in this window] [in a new window]   Figure 5. Time course plot of plasma -tocopherol () and erythrocyte reduced glutathione (GSH) () depletion and increases in aortic lesion size () in hamsters on the antioxidant-deficient, 0.4% cholesterol diet. Data are mean±SEM for n=12 hamsters per group.

Varying the dose of -tocopherol administered to hamsters on a 10-week synthetic diet resulted in marked dose-dependent increases in mean plasma -tocopherol values, which were associated with graded effects on mean lesion size (Fig 6). At the highest dose used in this study (180 mg · kg^-1 · d^-1, or 0.3% in the diet), mean plasma levels of -tocopherol were 35-fold higher than in control and nearly eight times greater than the basal level at week 0. From these data it may be estimated that approximately 10 mg · kg^-1 · d^-1 of -tocopherol resulted in half-maximal inhibition of lesion formation, with the maximum reaching 36% of control (Fig 6). Regression analysis of data from control and all -tocopherol–treated animals at 10 weeks revealed a significant, inverse, linear correlation of aortic lesion size with plasma -tocopherol concentration (n=68, r=-.581, P<.001) (Fig 7). The half-maximal inhibition of lesion size correlated with a plasma -tocopherol concentration of approximately 20 µg/mL.

View larger version (21K): [in this window] [in a new window]   Figure 6. Plot of plasma -tocopherol concentration () and aortic lesion size () in response to increasing doses of -tocopherol in hamsters on the antioxidant-deficient, 0.4% cholesterol diet. Data are mean±SEM for n=12 hamsters per group. Treatment time was 10 weeks.

View larger version (24K): [in this window] [in a new window]   Figure 7. Scatterplot and regression analysis of aortic lesion size vs plasma -tocopherol concentration (-T) in the hamster model. Hamsters were maintained for 10 weeks on a synthetic diet (0.4% cholesterol) with -tocopherol dosages indicated by the symbols.

The possibility that fluctuations in lipoprotein levels in hamsters given -tocopherol contributed to the lesion effects were further investigated. In no study were mean plasma total cholesterol or triglyceride levels in -tocopherol treatment groups significantly lower than in control groups. However, small but significant decreases in mean LDL cholesterol and corresponding increases in mean HDL cholesterol level were observed in some of the -tocopherol relative to control groups (Tables 3 and 4). This finding was also noted for the synthetic diet but not the chow-based diet groups (data not shown). Regression analysis of data from all control and -tocopherol–treated hamsters at 10 weeks on the synthetic diet showed a weak although significant correlation of aortic lesion size with HDL cholesterol level (n=105, r=-.282, P<.001) (Fig 8).

View larger version (27K): [in this window] [in a new window]   Figure 8. Scatterplot and regression analysis of aortic lesion size vs HDL cholesterol concentration in the hamster model. Hamsters were maintained for 10 weeks on a synthetic diet (0.4% cholesterol) with -tocopherol (-T) dosages indicated by the symbols.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
The present work was undertaken to determine whether antioxidants could be quantitatively and rapidly demonstrated to be antiatherosclerotic agents in vivo in a small-animal model. This study required development of a practical lesion assay with greater sensitivity to the reference agents -tocopherol and probucol than had been previously demonstrated in the literature. Hamsters fed cholesterol- and unsaturated fat–containing diets were selected for these studies on the basis of their lipoprotein profile; the well-defined, quantifiable aortic lesions obtained; and the practical advantages of a small species. With early aortic lesion size as the end point, oxidant-dependent atherosclerosis was clearly revealed only when natural dietary sources of antioxidants were depleted. This scheme required use of a synthetic diet that magnified the oxidation susceptibility of LDL and resulted in significant differences between reasonably small study groups (n=12). These findings are in contrast to the inability of similar doses of tocopherol or probucol to result in significant inhibition of aortic lesions, as found in a recent study using small groups of WHHL rabbits with plasma cholesterol levels exceeding 500 mg/dL.¹²

Elevation of the susceptibility of LDL to oxidation ex vivo in the synthetic-diet model and the strong effects of administered -tocopherol and probucol on ex vivo LDL oxidation parameters suggest that the antioxidants diminished formation of minimally modified and highly oxidized LDL in vivo. If one assumes that plasma -tocopherol levels are in equilibrium with tissue reservoirs under the conditions of this model, then the decrease in plasma -tocopherol concentration over time in controls suggests that a variety of tissues and cells, including the vessel wall and monocytes, became depleted in -tocopherol and experienced elevated oxidative stress. This notion is consistent with the observed decline in erythrocyte GSH as well as the histopathologic evaluations. Substantial evidence indicates that GSH, maintained in the reduced form by an NADPH-dependent reductase, is directly active in quenching reactive oxygen species and in serving as a substrate for enzymes that reduce lipid peroxides and other oxidation products.³⁵ Therefore, in addition to providing antioxidant protection through its effect on LDL, -tocopherol administration in the hamster may have directly diminished lesion formation by preventing radical-mediated injury and inflammatory reactions at the endothelium and in the aortic intima. This possibility is consistent with a recent report, which showed that -tocopherol and probucol directly diminished U-937 monocyte adhesion to inflammatory cytokine-stimulated endothelial cells in tissue culture.³⁶

A major finding of these studies is the diminution of atherogenesis by vitamin E supplementation alone to an animal depleted of other nutritional antioxidants, which suggests that -tocopherol possesses quantitatively sufficient antioxidant activity that is localized to appropriate tissues and compartments in vivo. The effectiveness of -tocopherol exceeded that of probucol and ubiquinol-10 in all aspects of the current studies. Ubiquinol-10, administered in the diet as either the active quinol or the more stable quinone form, was essentially ineffective in suppressing LDL oxidation ex vivo in this model, despite recent data showing that ubiquinol-10 can be a more active antioxidant than -tocopherol in human LDL.³⁷ Because ubiquinol-10 was effective as an inhibitor of LDL oxidation under similar assay conditions after its incorporation into LDL in vitro (data not shown), it is likely that poor absorption or bioavailability on oral dosing explains the present results. On the other hand, the high effectiveness of -tocopherol is consistent with recent evidence for a hepatic -tocopherol transfer protein that discriminates between isoforms of tocopherol, enriches liver membranes with -tocopherol, and in effect serves to enhance levels of -tocopherol in nascent VLDL and therefore, in LDL and most tissues.^{38 39} Although the tocopherol content of LDL itself was not directly measured due to insufficient sample quantity, it would be expected to be reflected in the plasma -tocopherol levels over the 10-week duration of these studies. In these experiments, half-maximal inhibition of atherogenesis was associated with approximately 20 µg -tocopherol per milliliter plasma, which required a daily intake of approximately 10 mg -tocopherolper kilogram of body weight in hamsters. Although it is impossible to extrapolate the dose or the corresponding effective plasma -tocopherol level to other species, it is striking that in a recent human study, comparable levels of plasma -tocopherol were found after 3-week dosing with a range of -tocopherol levels; the highest dose, 800 mg (1200 IU vitamin E), resulted in a plasma -tocopherol level of approximately 25 µg/mL, which was twice the baseline value.²⁸

The magnitude of the effect of -tocopherol and probucol on aortic lesion size was dependent on the plasma total cholesterol level, which was directly manipulated by varying the amount of dietary cholesterol. Higher levels of plasma total cholesterol masked the effects of antioxidants on early atherosclerosis in this model. Thus, at plasma cholesterol levels of approximately 250 mg/dL, as much as 65% of foam cell generation was oxidant dependent. In the presence of -tocopherol, the plasma total cholesterol threshold for lesion initiation was approximately 50 mg/dL higher than in controls. The idea that hypercholesterolemia could overwhelm antioxidant efficacy is compatible with the long-established role of cholesterol and LDL in atherogenesis. The remarkable effectiveness of -tocopherol in moderate hypercholesterolemia suggests that -tocopherol as the sole lipophilic antioxidant can substantially suppress vascular oxidative stress and atherogenesis. These conclusions suggest that antioxidants may prove to be therapeutically more effective antiatherosclerotic agents when given in combination with bile acid sequestrants or hydroxymethylglutaryl coenzyme A reductase inhibitors to decrease plasma cholesterol levels.

The possibility that changes in lipoprotein levels contributed to the effects on lesions was addressed. Mean plasma total cholesterol and triglyceride levels in -tocopherol or probucol groups were not significantly lower than control values in any of these studies. However, small but significant decreases in LDL cholesterol and corresponding increases in HDL cholesterol in -tocopherol groups were observed in some experiments using the synthetic diets, whereas the normal chow-based diets showed no such effects. Potential interferences with LDL precipitation assays in samples with higher than typical -tocopherol levels were ruled out by using ultracentrifugal methods for LDL isolation. The underlying explanation for this phenomenon is unknown; one possibility is hepatic abnormalities due to antioxidant deficiency, which may have affected lipoprotein metabolism in controls. In studies to be published elsewhere, other potent antioxidants elicited antiatherosclerotic effects in this model in the absence of changes in plasma LDL or HDL levels.

The present results indicate that vascular oxidative stress can be isolated as an experimental variable in an in vivo model of atherosclerosis. This approach may provide a relatively rapid, quantitative, experimental system for comprehensive evaluation of antioxidants in vivo in the pathogenesis of atherosclerosis. The remarkable activity of -tocopherol on LDL oxidation susceptibility and atherogenesis as revealed by these studies suggests that it will be a significant reference agent in the search for potent new antioxidant, antiatherosclerotic drugs.

Received October 6, 1994; accepted December 12, 1994.

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