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Articles

Novel Tocotrienols of Rice Bran Inhibit Atherosclerotic Lesions in C57BL/6 ApoE-Deficient Mice¹

Asaf A. Qureshi², Winston A. Salser^*, Rupal Parmar and Eugene E. Emeson

Advanced Medical Research, Madison, WI 53719; ^* Department of Molecular Cell and Developmental Biology and the Molecular Biology Institute, University of California, Los Angeles, CA 90024; and Department of Pathology, University of Illinois, Chicago College of Medicine, Chicago, IL 60612

²To whom correspondence and reprint requests should be addressed. E-mail: nqureshi{at}mhub.facstaff.wisc.edu.

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
We are studying novel tocotrienols, which have a number of activities that might interfere with the formation of atherosclerotic placques, including hypocholesterolemic, antioxidant, anti-inflammatory and antiproliferation effects. This study compared the effects of -tocopherol, the tocotrienol-rich fraction (TRF₂₅) and didesmethyl tocotrienol (d-P₂₅-T3) of rice bran on the pathogenesis of atherosclerotic lesions in C57BL/6 apolipoprotein (apo)E-deficient (-/-) mice. These mice are an excellent model because they become hyperlipidemic even when they consume a low fat diet and they develop complex atherosclerotic lesions similar to those of humans. These compounds were also tested in wild-type C57BL/6 apoE (+/+) and (+/-) mice fed low or high fat diets. When a high fat diet was supplemented with -tocopherol, TRF₂₅ or d-P₂₅-T3 and fed to mice (+/+) for 24 wk, atherosclerotic lesion size was reduced 23% (P = 0.33), 36% (P = 0.14) and 57% (P < 0.02), respectively, and in mice (+/-) fed for 18 wk, lesions were reduced by 19% (P = 0.15), 28% (P < 0.01) and 33% (P < 0.005), respectively, compared with mice fed a control diet. A low fat diet did not cause atherosclerotic lesions in these mice. The low fat diet supplemented with TRF₂₅ or d-P₂₅-T3 fed to apoE-deficient (-/-) mice for 14 wk decreased atherosclerotic lesion size by 42% (P < 0.04) and 47% (P < 0.01), respectively, whereas -tocopherol supplementation resulted in only an 11% (P = 0.62) reduction. These results demonstrate the superior efficacy of tocotrienols compared with -tocopherol. Although tocotrienols decreased serum triglycerides, total and LDL cholesterol levels, the decreases in atherosclerotic lesions seem to be due to the other activities. Serum tocol concentrations in various groups are also described. This is the first report of a significant reduction in the atherosclerotic lesion size in all three genotypes of apoE mice fed a novel tocotrienol (d-P₂₅-T3) of rice bran. Dietary tocotrienol supplements may provide a unique approach to promoting cardiovascular health.

KEY WORDS: • C57BL/6 apoE-deficient (-/-) mice • novel tocotrienols (TRF₂₅, d-P₂₅-T3) • atherosclerotic lesions • serum cholesterol • triglycerides

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Coronary heart disease is the result of a complex interaction among a number of different processes that affect either the acute phase of coronary disease or the initiation and growth of the atherosclerotic plaque (1 –4) . Lipoprotein metabolism is thought to be especially important in the initiation and growth of plaque, which involves complex cellular processes including the aggregation of "foam" cells, probably at a site of endothelial injury to initiate plaque formation (1 –6) . In the acute phase of coronary disease, aggregation of blood platelets, blood coagulation and fibrinolysis are of notable importance (1 ,2 ,4) . The cardiovascular risk of a given patient is dependent on the interactions of all of these factors (1 –6) .

Recent studies have indicated that administration of hypocholesterolemic and antioxidant drugs and compounds can restrict the development of early atherosclerotic lesions in the aorta in various experimental models (7 –11) . Inhibition of lesion development in the carotid arteries of cholesterol-fed primates has been reported after administration of vitamin E (-tocopherol) (12 –14) . The multitherapeutic properties of tocotrienols as hypocholesterolemic, antioxidant, antithrombotic, anticancer (antiproliferative) and anti-inflammatory agents in various experimental animal models and humans have been reported (15 –19) .

Vitamin E will be used in this report to refer to -tocopherol; however, it alternatively refers to a group of eight naturally occurring compounds with characteristic antioxidant activity (16 –18) . There are four tocopherols designated -, ß-, - and - and four corresponding tocotrienols. Tocotrienols differ from tocopherols (vitamin E) only in having three double bonds in the isoprene side chain. This unsaturation in the side chain is essential for inhibition of liver ß-hydroxy-ß-methylglutaryl coenzymeA (HMG-CoA)³ reductase (the rate-limiting enzyme in the synthesis of cholesterol) activity (16 –18) . Structure-function studies have revealed that the number and position of methyl substituents in different tocotrienols affect their hypocholesterolemic and antioxidant properties (16 –18) . -Tocotrienol (1 methyl group on benzene ring) is the most potent HMG-CoA reductase inhibitor among the four previously known tocotrienols (17) . On the other hand, -tocopherol has been shown to actually increase the activity of HMG-CoA reductase (17 ,20) . Recently, we isolated and identified two novel tocotrienols, [desmethyl (d-P₂₁-T3) and didesmethyl (d-P₂₅-T3) tocotrienols] from stabilized and heated rice bran (17) . These novel tocotrienols have superior efficacy in hypocholesterolemic, antioxidant, anti-inflammatory, antithrombotic and anticancer activities compared with the known tocotrienols and vitamin E (17 ,20 –22) . The pharmacokinetics and bioavailability of various tocotrienols under fed and fasted conditions in humans have been reported recently (23) .

Recently, the biological activities (antioxidant, antithrombotic, anti-inflammatory) of vitamin E (-tocopherol) were reviewed by a number of investigators (24 –29) . The role of -tocopherol in inhibiting the development of atherosclerotic lesions in the aorta has been attributed to the lower activity of protein kinase C (PKC) isoenzyme, which is caused by the higher concentration of -tocopherol (30 –33) . PKC plays an important role in cellular signal transduction and serves as the major intracellular receptor for tumor promotion, cellular growth, differentiation, secretion and cellular proliferation (30 ,31) . Control of the proliferation of aortic smooth muscle cells is especially important because hyperproliferation of these cells is associated with two vascular diseases, i.e., hypertension and atherosclerosis (32) . The inhibition of aortic lesions by -tocopherol in apolipoprotein (apo)E-deficient (-/-) mice has been reported to be due to inhibition of the activity of PKC by -tocopherol, and is not related to its antioxidant activity (34 ,35) . This reduction in lesions is achieved only by feeding a high level of -tocopherol (>1.0 mg/g in mice and 5–10 mg/g in rabbits) (9 , 33 –36) ; feeding a low level of -tocopherol (500 µg/g) does not significantly inhibit the lesions in apoE-deficient (-/-) mice (37) .

Several investigators reported recently that tocotrienols have greater antioxidant activity than -tocopherol (vitamin E), and protect more efficiently against some free radical–related diseases than does -tocopherol (12 ,38 –42) . However, there is no report of the effect of tocotrienols on the activity of PKC although tocotrienols and -tocopherol do share a common chromanol moiety in their structures (17) . Moreover, TRF₂₅ and didesmethyl tocotrienol (d-P₂₅-T3) are more potent inhibitors of cholesterol synthesis than the known tocotrienols (17) . Therefore, the present study was carried out to compare the effects of -tocopherol, TRF₂₅ and d-P₂₅-T3 on the pathogenesis of atherosclerotic lesions in C57BL/6 apoE-deficient (-/-) female mice fed a low fat diet with or without these compounds.

The normal rodent lipid profile is regarded as an HDL cholesterol model, because the HDL cholesterol level normally exceeds the level of LDL cholesterol, which is opposite to the human lipid profile (LDL cholesterol model). Recently, several useful models of atherosclerosis have been created by genetic alternation of lipid metabolism. The most widely used of these models involves a gene disruption of apolipoprotein E (43 –46) . Advantages of this model include the fact that the lesions develop at a much earlier age, exhibit more of the features of the so-called "complicated" lesions found in humans and can be induced by both low and high fat diets (47 ,48) . Unlike the wild-type mice [C57BL/6 apoE (+/+)] or heterozygous mice [C57BL/6 apoE (+/-)], the homozygous female mice [C57BL/6 apoE-deficient (-/-)] have elevated LDL cholesterol levels [consisting mainly of VLDL + intermediate density lipoproteins (IDL)], similar to the high LDL cholesterol level of humans (43 –48) . They are also an especially excellent model with which to study athersclerotic lesions because they become hyperlipidemic even when consuming a standard low fat rodent diet and develop large and complex atherosclerotic lesions that are similar to those of humans and 50 times larger than the lesions seen in wild-type mice fed a high fat diet (43 –46) .

The present study also compared the effects of -tocopherol, TRF₂₅ and d-P₂₅-T3 in wild-type, C57BL/6 apoE (+/+) and heterozygous, C57BL/6 apoE (+/-) female mice fed low and high fat diets with or without the test compounds. Both wild-type C57BL/6 apoE (+/+) and heterozygous (+/-) female mice fed a standard low fat diet have normal cholesterol levels and do not develop atherosclerosis. However, when they consume an atherogenic (high fat) diet, they develop spontaneous atherosclerotic lesions with extremely high serum total cholesterol levels (43 –48) .

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Sources of chemicals and diagnostic kits.

Sources of chemicals, substrates, and diagnostic kits have been identified previously (21) . d--Tocopherol was a gift from Archer Daniels Midland (Decatur, IL). Chemicals and solvents were of analytical grade. Sigma Diagnostic kits (Sigma Chemical, St, Louis, MO) were used to estimate serum total cholesterol and HDL cholesterol (kit 352; 500 nm), and triglycerides (kit 336; 500 nm).

Purification of -oryzanol–free TRF₂₅ and d-P₂₅-T3 by flash chromatography.

The purification of large quantities of TRF₂₅ (free from -oryzanols and most of -tocopherol) and d-P₂₅-T3 from stabilized and heated rice brans of ML-63 rice variety (supplied by M. Wells, Riviana Foods, Abbeville, LA) was carried out by flash chromatography as described recently (21) . The composition of various tocols in TRF₂₅ was 5.8% -tocopherol, 14.9% -tocotrienol, 1.9% ß-tocotrienol, 35.4% -tocotrienol, 4.1% -tocopherol, 5.3% -tocotrienol, 14.3% d-desmethyl tocotrienol (d-P₂₁-T3), 16.4% d-didesmethyl tocotrienol (d-P₂₅-T3) and 1.9% unidentified tocotrienols. The molecular structures of desmethyl (d-P₂₁-T3) and didesmethyl (d-P₂₅-T3) tocotrienols have been established as 3,4-dihydro-2-methyl-2-(4,8,12-trimethyltrideca-3'(E),7'(E),11'-trienyl)-2H-1-benzopyran-6-ol, and 3,4-dihydro-2-(4,8,12-trimethyltrideca-3'(E),7'(E),11'-trienyl)-2H-1-benzopyran-6-ol, respectively (17) .

Experimental design.

Four experiments were carried out to study the effects of -tocopherol, the tocotrienol-rich fraction (TRF₂₅) and its pure component, d-P₂₅-T3 (didesmethyl tocotrienol) purified from stabilized and heated rice bran, on the pathogenesis of athersclerotic lesions in C57BL/6 apoE-deficient (-/-) female mice fed low fat specially prepared rodent diets. These compounds were also tested in wild-type C57BL/6 apoE (+/+) and heterozygous C57BL/6, apoE (+/-) female mice fed low and high fat diets.

Animals.

The protocol was reviewed and approved by the University of Illinois at Chicago College of Medicine Animal Care Committee and the animal care was in accordance with institutional guidelines. The wild-type C57BL/6 apoE (+/+), C57BL/6 apoE (+/-) and C57BL/6 apoE-deficient (-/-) female mice were obtained from Jackson Laboratories (Bar Harbor, MA). All mice were housed under pathogen-free conditions as previously described (49) . Female mice were used in the present study because in the C57BL/6 hyperlipidemic model, female mice more rapidly develop much larger atherosclerotic lesions than male mice (50) except in apoE-deficient (-/-) mice in which the lesions are approximately equal in males and females.

Animal diets.

The compositions of various diets are outlined in Table 1 . The normal control diet (TD-5015), fed during propagation of the mice and before commencing experimental low or high fat diets contains 110 g/kg fat (Table 1) . The standard low fat (Teklad Rodent Diet [W] 8604) mouse diet was purchased from Harlan/Teklad (Madison, WI). It has a crude fat concentration of 50 g/kg (Table 1) . The atherogenic diet (high fat; Teklad Cocoa Butter, TD-88051) was made by mixing a diet containing cocoa butter and cholesterol with the normal control diet in a ratio of 1:3 and then pelleting it. This diet has a total fat concentration of 150 g/kg and a cholesterol concentration of 125 g/kg. The diets were prepared by Harlan/Teklad. Each diet was administered in pellet form.

Wild-type C57BL/6 apoE (+/+) female mice were fed the normal control diet for 7 wk after weaning at 3 wk. When they were10 wk old, they were transferred to the experimental, standard low fat diet (n = 5/group) or a high fat atherogenic diet (n = 10/group), with or without -tocopherol, TRF₂₅ or d-P₂₅-T3 (100 µg/g) and fed these diets for 24 wk. At the end of this study and in the following experiments, the mice were deprived of food overnight and killed by severing their carotid arteries. Their aortas were removed for the quantitation of the areas of the ascending aorta occupied by atherosclerotic lesions. Blood samples were obtained from the mice after overnight food deprivation (12 h) by orbital puncture for the determination of serum lipids (total cholesterol, LDL cholesterol, HDL cholesterol and triglycerides).

Experiment 2: Heterozygous C57BL/6 apoE (+/-) female mice.

Heterozygous C57BL/6, apoE (+/-) female mice were fed the normal control diet (TD-5015) for 3 wk after weaning. At 6 wk of age, they were transferred to the experimental low fat (n = 5/group) or high fat (n = 10/group) diets as described above, with or without supplemental -tocopherol, TRF₂₅ or d-P₂₅-T3 (100 µg/g), and fed these diets for 18 wk. The mice were killed, their aortas and blood samples were collected as described in Experiment 1.

Experiment 3: Homozygous C57BL/6 apoE-deficient (-/-) female mice.

Mice (n = 10/group) were fed the standard low fat diet through weaning and up to 6 wk of age and then switched to the same low fat diet either without supplementation (control) or with one of the three supplements [-tocopherol, TRF₂₅ or d-P₂₅-T3 (100 µg/g)]. After consuming these diets for 14 wk, the mice were killed and sampled as described for Experiment 1.

Experiment 4: Attempt to reverse growth of atherosclerotic plaques.

C57BL/6 apoE-deficient (-/-) female mice were fed the standard low fat (5%) diet for 13 wk after weaning (3 wk) to permit atherosclerotic lesions to develop. Mice (n = 10/group) were then fed standard low fat diet supplemented with -tocopherol, TRF₂₅ or d-P₂₅-T3 (100 µg/g) for the next 10 wk to evaluate the effects of these additives on the progression of atherosclerotic lesions. After 10 wk, mice were killed and sampled as described above for Experiment 1.

Quantitative assessment of atherosclerotic lesions.

A slightly modified method of Paigen et al. (51) was used to quantitate the areas of aortic atherosclerosis for each mouse. These areas were measured by analytical morphometry in five 10-µm sections through the region of the aortic valve at 100-µm intervals. The mean of five sections was calculated instead of using the entire arterial tree. Lesions in wild-type mice C57BL/6 [apoE (+/+)] and heterozygous mice [apoE (+/-)] were found only in the aortic valve. Lesions in homozygous C57BL/6 apoE-deficient (-/-) mice had developed extensively throughout the aorta (52) . It has been reported that in C57BL/6 apoE-deficient (-/-) mice, there is a significant correlation between the extent of lesions in the entire aorta (measured as the percentage of surface area) and that at the aortic origin (measured as the averaged lesion area per cross section) (52) . The detailed procedure is described below.

The aorta was removed after the mice were killed, rinsed in saline to remove the blood and fixed in 4% formaldehyde. Each heart was cut just below the beginning of the aortic sinuses on a plane parallel with a plane formed by drawing a line between the tips of the atria; the lower portion of each heart was discarded. The upper portion was mounted on a cryostat, with the above identified plane parallel to the plane of cutting so as to obtain true cross sections of the aorta. Sections (10 µm) were then cut and discarded until we were able to locate (by examining unstained sections) the first section that showed the aortic valve sinus. Once this section was located, 10-µm sectioning was continued along the ascending aorta until the valve cusps were no longer visible; all sections were saved. This procedure usually includes 40–50 sections covering 400–500 µm. The sections were lined up and fixed on two groups of polylysine-coated slides, placing the odd-numbered sections (1,3,5,7 ...) on one series of slides and the even-numbered sections (2,4,6,8 ...) on the other. The first series of slides were stained with Oil Red O and counterstained with hematoxylin; the second was stained with hematoxylin and eosin (H & E). This approach permitted correlation of the lipid distribution in lesions stained with Oil Red O with the morphological details better appreciated with the H & E-stained sections. To quantitate the degree of atherosclerosis in each mouse, the areas of the atherosclerotic lesions of five of the sections stained by Oil Red O, sections 1, 6, 11, 16 and 24, were examined. These sections were 100 µm apart and covered a span of 400 µm of the aorta. If any of the designated sections were folded or torn, the section preceding it was used. The area of the lesions within each Oil Red O section was determined by point counting using a squared grid ocular graticule (Graticules, Townbridge, UK) at 40X magnification (53 ,54) . The two-dimensional area of the lesions per section were then determined by the following formula: a = p · µ², where a is the area in µ², p is the number of points falling within the lesions and µ is the distance between two neighboring points (i.e., µ² will equal the area associated with each point). In the present system, µ = 50 µm as determined by a reference grid; therefore, µ² = 2500 µm² at X40 magnification. The aortas of each mouse provided five independent data points for evaluation. The mean value of the five points was then used as the final value for each mouse. The morphometric studies were performed on frozen sections stained with Oil Red O and the photomicrographs were taken from frozen sections stained with H & E as described in detail previously (51) .

Serum lipid analyses.

Blood samples were obtained from the mice after overnight food deprivation (12 h) by orbital puncture under ether anesthesia. The serum total cholesterol, HDL cholesterol and triglycerides levels were estimated using Sigma Kits (Cat. #352–20 and 336–20), respectively. The LDL cholesterol (LDL chol), and VLDL cholesterol (VLDL chol) were precipitated using 400-µL aliquots of serum with 50 µL/L of a mixture of 9.7 mmol phosphotungstic acid and 0.4 mol MgCl₂ with gentle shaking for 10 min at room temperature, followed by centrifugation at 12,000 x g for 10 min. The supernatant, containing HDL chol, was analyzed with Sigma reagent (kit 352) (55) . LDL chol was estimated according to Friedewald’s formula by subtracting the total cholesterol from (HDL chol + triglycerides/5) (56) .

Estimation of tocols of serum.

The separation and quantitation of tocols (tocopherols and tocotrienols) of serum were carried out by HPLC as reported recently in cereals and serum (57) .

Expression of data and statistical analysis.

Treatment-mediated differences in atherosclerotic lesion size in aorta and serum lipid analyses were identified with one- or two-way ANOVA to compare group means of main effects by ANOVA; when the F-test indicated a significant effect, the differences between the means were analyzed by Fisher’s Protected Least Significant Difference (LSD) test (Abacus Concepts, Berkeley, CA). Differences were considered significant at P < 0.05 (58) .

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Atherosclerotic lesion size in aorta

    Experiment 1. The wild-type C57BL/6 apoE (+/+) female mice fed the low fat diet with or without -tocopherol, TRF₂₅ or d-P₂₅-T3 for 24 wk showed no lesions in the aortas (Table 2 ). However, lesions were observed in the mice fed the high fat diet with or without supplements. The control group had maximum lesions in the aorta, and supplementation of the high fat diet with d-P₂₅-T3 reduced (57%; P < 0.02) the size of the lesions compared with the control group (Table 2) . Supplementation of the high fat diet with -tocopherol or TRF₂₅ resulted in decreases of 23% (P = 0.33) and 36% (P = 0.14), respectively, compared with the control group (Table 2) .

Large lesions were found in heterozygous apoE (+/-) mice fed the high fat diet and supplementation with -tocopherol, TRF₂₅ or d-P₂₅-T3 reduced lesion size 19% (P = 0.15), 28% (P < 0.01) and 33% (P < 0.005), respectively, compared with the control group (Table 2) . The experimental period was 18 wk with these mice vs. 24 wk in the wild-type apoE (+/+) female mice, which may explain in part the smaller reduction of atherosclerotic plaques with d-P₂₅-T3 (33 vs. 57%).

    Experiment 3. C57BL/6 apoE-deficient (-/-) mice fed the low fat diet with -tocopherol, TRF₂₅ or d-P₂₅-T3 for 14 wk had 11% (P = 0.62), 42% (P < 0.04) and 47% (P < 0.01) smaller lesions, respectively, compared with the control group (Table 2) . The average lesion size in the control group fed the low fat diet was similar to that of heterozygous apoE (+/-) mice fed the high fat diet.

    Experiment 4. The reductions of atherosclerotic lesions were 8% (P = 0.73), 22% (P = 0.42) and 24% (P = 0.31), respectively, compared with the control group (Table 2) in homozygous apoE-deficient (-/-) female mice fed the standard low fat diet for 16 wk and then the low fat diet supplemented with -tocopherol, TRF₂₅ or d-P₂₅-T3 for 10 wk, respectively. No samples were taken to measure the extent of plaques at 16 wk (the start of the therapy trial), but we speculate that the 22 or 24% reductions relative to 26-wk controls, if real, would represent a slowing of plaque growth rather than a reversal (Table 2) .

Morphological studies

Representative histologic sections from the control and d-P₂₅-T3 groups of heterozygous apoE (+/-) mice are illustrated in Figure 1 (Experiment 2). A low power view of a control lesion shows a large extensive covering (arrow head) over 95% of the circumference of the aortic wall (Fig. 1 A). A high power view of one of these lesions shows marked thickening of the subendothelial intimal space with extensive deposition of extracellular lipids and connective tissue matrix, and multiple scattered mononuclear cells, including foams cells (Fig. 1 B). In addition, a few scattered spindle-shaped cells with elongated nuclei suggestive of vascular smooth muscle cells or fibroblasts were seen. The representative lesions from the d-P₂₅-T3 group were somewhat smaller than those of the control group, covering (arrow head) 70% of the circumference of the aortic wall (Fig. 1 C). The histologic features of this lesion seen in high power were similar to those of the control group (Fig. 1 D).

View larger version (156K): [in this window] [in a new window]   Figure 1. Morphology of atherosclerotic lesions in heterozygous apolipoprotein (apo)E (+/-) mice in Experiment 2 fed high fat diets. (A) Low power view through the upper portion of the aortic valve of a control mouse fed only the high fat diet showing lesions (arrow head) involving >95% of the circumference of the aortic wall; hematoxylin and eosin (H & E)-stained, magnification X120. (B) High power view of one of the lesions showing marked thickening of the subendothelial intimal space, extensive deposition of extracellular lipids and connective tissue matrix and multiple scattered mononuclear cells, including foam cells. There are also a few scattered spindle-shaped cells with elongated nuclei suggestive of vascular smooth muscle cells or fibroblasts; H & E-stained, magnification X270. (C) Low power view through the upper portion of the aortic valve of a mouse fed the high fat diet supplemented with didesmethyl tocotrienol of stabilized and heated rice bran (d-P25-T3) showing lesions (arrow head) covering 70% of the circumference of the aortic wall; H & E-stained, magnification X90. (D) High power view of one of the lesions showing changes similar to those in (B); H & E-stained, magnification X270. _art>

View larger version (166K): [in this window] [in a new window]   Figure 2. Morphology of atherosclerotic lesions in apolipoprotein (apo)E-deficient (-/-) mice in Experiment 3 fed low fat diets. (A) Low power view through the upper portion of the aortic valve of a control mouse fed the standard low fat diet showing multiple lesions (arrow head) covering >90% of the circumference of the aortic wall; hematoxylin and eosin (H & E)-stained, magnification X100. (B) High power view of one of the lesions displaying marked thickening of the subendothelial intimal space, extensive deposition of extracellular lipids and connective tissue matrix and multiple scattered mononuclear cells, including foam cells. There are also a few scattered spindle-shaped cells with elongated nuclei suggestive of vascular smooth muscle cells or fibroblasts; H & E-stained, magnification X300. (C) Low power view through the upper portion of the aortic valve of a mouse fed the standard low fat diet plus didesmethyl tocotrienol of stabilized and heated rice bran (d-P25-T3) showing lesions (arrow head) covering 60% of the circumference of the aortic wall; H & E-stained, magnification X 90. (D) High power view of one of the lesions showing changes similar to those in (B); H & E-stained, magnification X270. _art>

    Experiment 1. Feeding the wild-type C57BL/6, apoE (+/+) female mice the high fat diet for 24 wk resulted in a twofold higher serum total cholesterol level than in those fed the low fat diet in the control groups (Table 3 ). Supplementing either diet with -tocopherol did not affect serum total cholesterol. When TRF₂₅ or d-P₂₅-T3 was added to the low or high fat diets fed for 24 wk, total serum cholesterol was 5–7% lower (P < 0.05) (Table 3) .

    Experiments 3 and 4. The decrease in serum total cholesterol levels relative to controls in C57BL/6 apoE-deficient (-/-) female mice fed the low fat diet supplemented with -tocopherol for 14 wk was 12% (P < 0.003), with TRF_25, 32% (P < 0.001) and with d-P₂₅-T3, 31% (P < 0.0001) (Table 3) . These components had no effect in the mice fed the low fat diet for 10 wk (Table 3) .

Serum lipids: LDL cholesterol

    Experiment 1. The decreases in the serum total cholesterol levels in these mice were also reflected in the serum LDL cholesterol (mainly VLDL + IDL) levels (Table 3) . The wild-type C57BL/6 apoE (+/+) female mice fed the low fat diet supplemented with -tocopherol, TRF₂₅ or d-P₂₅-T3 for 24 wk had 2% (P = 0.25), 11% (P = 0.14) and 30% (P < 0.004) lower levels, respectively, than the control group (Table 3) . The decreases in those fed the high fat diet (24 wk) supplemented with these components were only 5% (P = 0.10), 9% (P < 0.03) and 6% (P = 0.10), respectively (Table 3) .

    Experiment 2. Similar decreases of 9% (P = 0.17), 19% (P < 0.02) and 23% (P < 0.02) in serum LDL cholesterol were also observed when the low fat diet supplemented with -tocopherol, TRF₂₅ or d-P₂₅-T3, respectively, was fed for 18 wk to C57BL/6 apoE (+/-) female mice. The decreases with the high fat diet (18 wk) supplemented with these components were 1% (P = 0.3), 6% (P < 0.02) and 8% (P < 0.02), respectively (Table 3) .

    Experiments 3 and 4. The C57BL/6 apoE-deficient (-/-) female mice fed the low fat diet (14 wk) supplemented with -tocopherol, TRF₂₅ or d-P₂₅-T3 decreased serum LDL cholesterol levels 12% (P = 0.17), 32% (P < 0.001) and 31% (P < 0.001), respectively (Table 3) . These components had no effect in the mice fed the low fat diet for 10 wk (Table 3) .

Other serum lipids

    Experiments 1–4. Serum HDL cholesterol was not affected in mice fed the low or high fat diets containing -tocopherol, TRF₂₅ or d-P₂₅-T3 (Table 4 ). The HDL cholesterol levels in all three genotypes of apoE female mice fed control and experimental diets (0.84–1.26 mmol/L) are similar to those of humans (1.29 mmol/L HDL cholesterol), despite the fact that the normal rodent pattern is the high HDL cholesterol model (HDL cholesterol level > 3.62–4.65 mmol/L).

Serum tocols (tocopherols + tocotrienols)

    Experiments 1–4. The HPLC analyses of serum samples for tocols obtained from the wild-type mice [apoE (+/+)] and heterozygous mice [apoE (+/-)] fed the control low fat or high fat diets or supplemented with -tocopherol showed the presence of -, -, and -tocopherols only (Tables 5 and 6). The samples of the -tocopherol–supplemented diets had 25% higher concentrations of these tocopherols compared with samples from the control groups fed each diet (Tables 5 , 6) . There were also 20–25% higher concentrations of these tocopherols in mice fed the high fat diets compared with those fed the low fat diets (Tables 5 , 6) . Similar results were also observed with homozygous apoE-deficient (-/-) mice fed the low fat diet for 14 and 10 wk, supplemented with -tocopherol (Table 7 ).

The HPLC analyses of serum samples obtained from all three genotypes of mice after feeding each diet (low or high fat) supplemented with d-P₂₅-T3 showed the presence of only -, - and -tocopherols plus d-P₂₅-T3 (Tables 5 6 7) . The concentrations observed with this treatment were similar to their control groups. The concentrations of tocols (tocopherols and tocotrienols) in serum in all of the experimental groups were 25–200% higher than those of their respective control groups (Table 5 6 7) .

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
A striking result of these experiments is that the novel tocotrienol d-P₂₅-T3 can substantially reduce the growth of atherosclerotic plaques in all three of the tested mouse genotypes and diet combinations that produce plaques. For instance, wild-type mice [apoE (+/+)] fed a high fat diet showed a 57% reduction, heterozygous mice [apoE (+/-)] fed a high fat diet showed a 33% reduction, and homozygous defective mice [apoE (-/-)] fed a low fat diet showed a 47% reduction in plaque size. Intervention before the plaques form or early in their growth is suggested to be important because when the treatment of apoE (-/-) mice was delayed from 6 to 16 wk, the reduction in plaque amount dropped from 47% (P < 0.01) [Experiment 3, treatment from 6 to 20 wk (Table 2) ] to 24% (P = 0.31) [Experiment 4, treatment from 16 to 26 wk (Table 2) ].

TRF₂₅ also had substantial effects. TRF₂₅ is a mixture of tocols, enriched to obtain a high tocotrienol/tocopherol ratio, purified from rice bran. The TRF₂₅ preparation used in these experiments contains >88% tocotrienols and only 10% tocopherols. It contains 16.4% d-P₂₅-T3; thus the mice supplemented with 100 µg TRF₂₅/g were receiving only one sixth the amount given to the groups supplemented with pure d-P₂₅-T3. Yet, TRF₂₅ also substantially reduced atherosclerotic plaque formation. Comparing the effects with TRF₂₅ vs. d-P₂₅-T3, we observed 26 vs. 57% for (+/+) mice (high fat), 28 vs. 33% for (+/-) mice (high fat), and 42 vs. 47% for (-/-) mice (low fat) (Table 2) . We doubt that these TRF₂₅ results are due only to the 600% reduced amount of d-P₂₅-T3 and suggest that the other tocotrienols in the TRF₂₅ mixture probably also played important roles.

At this dose, -tocopherol did not significantly reduce plaque with any genotype/diet combination. The apoE-deficient (-/-) mice fed a low fat diet are perhaps of greatest importance because of the resemblance of this model to the human disease. Here -tocopherol produced a reduction of only 11% compared with 42 and 47% with TRF₂₅ and d-P₂₅-T3, respectively. Thus, these results clearly demonstrate a superior efficacy of tocotrienols compared with -tocopherol (vitamin E) (Table 2) .

This is the first report to describe the significant reduction in atherosclerotic lesions in C57Bl/6 apoE-deficient (-/-) mice after feeding TRF₂₅ and a novel tocotrienol (d-P₂₅-T3) of rice bran. As mentioned earlier, apoE-deficient (-/-) mice provide an excellent model because they become hyperlipidemic even when consuming a standard low fat mouse diet (47 ,48) . These mice have increased oxidation-specific autoantibodies and develop complex atherosclerotic lesions that are similar to those of humans (59 ,60) . The normal serum cholesterol level is four times greater in these mutant mice than normal mice, which show a high LDL cholesterol (consisting of VLDL + IDL) pattern atypical of normal mice. The progression of lesion formation is rapid in the apoE-deficient (-/-) mice, even when they consume a low fat diet; they develop foam cell–rich depositions in their proximal aorta by age 3 mo, after which lesions progress spontaneously and completely clog the coronary artery by 8 mo (43 –48) .

The early termination of treatment in Experiment 3 [20 wk for apoE-deficient (-/-) mice compared with 34 wk for wild-type apoE (+/+) mice in Experiment 1] was dictated in part by the aggressiveness of stenosis in the apoE-deficient (-/-) mice. At close to 34 wk, the untreated apoE-deficient (-/-) mice would be reaching total occlusion of their coronary arteries (45 ,46) . It would be interesting to carry out further experiments in which the treatment interval in Experiment 4 was changed to start at weaning (3 wk) and continue until either 20 or 28 wk. Would the 3- to 20-wk treatments with TRF₂₅ or d-P₂₅-T3 reduce atherosclerotic lesions more than what was seen with the 6- to 20-wk treatment interval in Table 2 , suggesting that a large amount of damage has already occurred by 6 wk? Would the 3- to 28-wk treatment period reduce the amount of damage (as a percentage of the control group) more than the 3- to 20-wk treatment, suggesting the degree to which TRF₂₅ or d-P₂₅-T3 can retard the progression of development of atherosclerotic plaques? Finally, if the results of the preceding experiment are positive, it would be interesting to carry out a survival experiment, extending the treatment of apoE-deficient (-/-) mice fed the low fat diet indefinitely with either no supplement or with TRF₂₅ or d-P₂₅-T3 to measure either survival time or time to an objective measure of advanced heart disease. Moreover, the standard deviations are quite large in most of the estimations (Table 2 ; 30–50%), which might be avoided by using recently reported better and more sensitive methods (35 ,37 ,61 ,62) .

The present results indicate a much higher efficacy of tocotrienols than -tocopherol for the reduction of atherosclerotic plaques in all three mouse genotypes tested. This may be due to greater absorption of tocotrienols. It has been demonstrated that tocotrienols are transported via the lymphatic system after oral absorption in rats (63) . Recently, the pharmacokinetics and bioavailability of various tocotrienols in postprandial and fasting humans were reported (23) . The absorption of each tocotrienol was 100% greater in the fed state, and their elimination half-life (t_1/2) was found to be relatively short compared with tocopherols (23) . The plasma concentrations of all tocotrienols were increased markedly in the fed state and were achieved between 3 and 5 h.

The bioavailability of tocotrienols in fasting and postprandial humans was compared using the parameters peak plasma concentration (C_max), time to reach peak plasma concentration (T_max) and total area under the plasma concentration-time curve (AUC_0-) (23) . The mean C_max and AUC_0- values of all three tocotrienols in the fed state were higher than the values in the fasting state (23) . Moreover, the mean apparent volume of distribution (V_d/f) values under fed conditions were significantly smaller than those of the fasting state, which could be attributed to increased absorption of the tocotrienols in the fed state (23) . Experimental values of various tocotrienols compared with -tocopherol of the above-mentioned parameters and the pharmacokinetic results in humans were reported recently (23) .

The treatment of hypercholesterolemia with diet and lipid-lowering agents has been the mainstay of the treatment of atherosclerosis (7 –11) . There are a number of possible mechanisms that could prevent the development of atherogenesis (1 –4 ,38 ,40 ,41 ,45 ,46) . Atherosclerosis is a disease of injury, chronic inflammation, accumulation of cholesterol and foam cells within the arterial wall and altered lipoprotein cholesterol metabolism (1 –4) . Physiologic and genetic factors also contribute to the progression of the fatty streaks into an athersclerotic lesion (3) . The development of atheroscletic lesions could be prevented by modifying the response to injury or the response to retention of lipoproteins, or by the oxidative modification of lipoproteins (5 ,64 –66) .

Endothelial injury causes the formation of fatty streaks by increasing the permeability of the endothelium to lipoproteins and macrophages (3) . The increased endothelial permeability leads to the accumulation of lipoproteins; subsequently, foam cells within the subendothelial space start forming atherosclerotic plaque (5 ,64 –66) . The oxidation of lipoproteins, particularly LDL, is responsible for the inflammatory response seen in atherosclerosis (9 ,64) . Most lipoprotein oxidation occurs within the arterial wall (9 ,64) . There is growing evidence that some immunological factors may also play a role and that heat shock proteins or oxidized lipoproteins may be targets of an autoimmune response (10 ,67) . Recent reports indicate that atherosclerotic lesions are due primarily to the proliferation of smooth muscle cells in the arterial intima where they accumulate, surrounded by connective tissue, lipid-loaded macrophages and lymphocytes, and are responsible for vascular occlusions (10 ,67 –69) .

Vitamin E (-tocopherol) protects against free radical damage and inhibits aortic smooth muscle cell proliferation and platelet aggregation by decreasing the activity of PKC (25 ,70 ,71) . Although the exact mechanism of inhibition of atherosclerotic lesions by tocotrienols has not been elucidated, there is evidence suggesting that tocotrienols affect several distinct steps in the pathways leading to formation of complex atherosclerotic lesions. The effect of tocotrienols on the activity of PKC has not been reported; recently, however, we and several other investigators reported that tocotrienols have greater antioxidant activity than -tocopherol (vitamin E), and are more effective than -tocopherol in protecting against some free radical–related diseases (15 –18) . Moreover, tocotrienols are also beneficial for the prevention of oxidative LDL modification and are potent hypocholesterolemic and anti-inflammatory agents (15 ,19 ,22) .

Pretreatment with novel tocotrienols reduced the induction of tumor necrosis factor (TNF) in response to Escherichia coli lipopolysaccharide (Re-LPS) in mice (19) . The inhibition of TNF levels in serum was 72 and 82% with TRF₂₅ and d-P₂₅-T3, respectively, compared with the control group. A corresponding rise was observed in the plasma levels of corticosterone and adrenocorticotropic hormone (19) . These results suggest that treatment of mice with tocotrienols blocked the rapid and transient rise in TNF caused by Re-LPS. TRF₂₅ and d-P₂₅-T3 also lowered arachidonic acid in various tissues of hereditary hypercholesterolemic swine (17 ,22) . Thus, there is an overall reduction in prostaglandins and leukotrienes, both of which are synthesized from arachidonic acid, and thus a possible reduction in interleukin-1.

The inhibition of TNF by novel tocotrienols is accompanied by a decrease in inflammation, by inhibition of the respiratory burst of neutrophils or by free radical scavenging; the decrease in the secretion of TNF by tocotrienols could be due to the rise in endogenous corticosteroids, which modulate the synthesis of inflammatory cytokines. This property of tocotrienols might be effective in reducing acute and chronic inflammation, and in reducing the size of atherosclerotic lesions in the arteries of humans.

Now it is of great interest to study the effect of various tocotrienols on the activity of PKC to understand its role in the inhibition of development of atherosclerotic lesions. As mentioned earlier, atherosclerotic lesions are due primarily to the proliferation of smooth muscle cells (67 –69) . Tocotrienols, particularly novel tocotrienols, have significantly greater potency in inhibiting proliferation of tumor cells compared with -tocopherol (17) . It is possible that tocotrienols inhibit the proliferation of vascular smooth muscle cells by interfering with signal transduction events involving PKC, and this effect might not be related to their antioxidant properties. Thus, the inhibition of PKC and vascular smooth muscle cell proliferation by tocotrienols might represent a physiologic mechanism for the inhibition of atherosclerotic lesions.

The present results reveal that all C57BL/6 apoE (+/+, or +/-) mice fed a high fat diet and (-/-) mice fed a low fat diet plus TRF₂₅ and a novel tocotrienol (d-P₂₅-T3) of rice bran showed significant reductions in the size of atherosclerotic lesions. These decreases were reflected in their serum total and LDL cholesterol levels. The decrease in these lipids was not as great in this model as that found in chickens and hereditary hypercholesterolemic swine (17 ,21) . These novel tocotrienols may be used to prevent or reverse blood clots and lesions that may lead to diseases such as myocardial infarction, stroke and other blood system thromboses.

	ACKNOWLEDGMENTS

 
The authors thank Sajjad Nasir, L. He, H. Mo and Suzanne G. Yu for their technical assistance, literature search and proofreading of the manuscript. We thank M. Wells (Riviana Foods, Abbeville, LA) for supplying rice brans and Bradley C. Pearce and J. J. Kim Wright of Bristol-Myers Squibb Pharmaceutical Research Institute for their helpful discussions and kind provision of pure tocols. We also thank Martin L. Andreas (Archer Daniels Midland, Decatur, IL) for the gift of d--tocopherol, and R. H. Lane (BioNutrics, Phoenix, AZ) for his helpful and constructive discussions throughout this study.

	FOOTNOTES

 
¹ Supported in part by BioNutrics, Phoenix, AZ 85016.

³ Abbreviations used: apo, apolipoprotein; chol, cholesterol; d-P₂₁-T3, desmethyl tocotrienol of stabilized and heated rice bran; d-P₂₅-T3, didesmethyl tocotrienol of stabilized and heated rice bran; H & E, hematoxylin and eosin; HMG-CoA, ß-hydroxy-ß-methylglutaryl coenzyme A; IDL, intermediate density lipoproteins; PKC, protein kinase C; Re-LPS, Escherichia coli lipopolysaccharide; TNF, tumor necrosis factor; TRF₂₅, tocotrienol-rich fraction from stabilized and heated rice bran.

Manuscript received March 20, 2001. Initial review completed May 4, 2001. Revision accepted July 19, 2001.

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