© 1997 American Heart Association, Inc.
Articles |
"Tall Oil"–Derived Phytosterols Reduce Atherosclerosis in ApoE-Deficient Mice
the Healthy Heart Program and Cardiovascular Research Laboratory, St. Paul's Hospital, and the Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, Canada. E-mail jifr@unixg.ubc.ca.
Correspondence to Jiri J. Frohlich, Department of Pathology and Laboratory Medicine, University of British Columbia, #180-1081 Burrard St, Vancouver, BC, Canada V6Z 1Y6.
 |
Abstract |
|---|
 Top Abstract IntroductionMethodsResultsDiscussionReferences |
|---|
We investigated the effects of a "tall oil"–derived phytosterol mixture (TODPM) on the formation of atherosclerotic lesions in apoE-deficient mice. TODPM was added at 2% (wt/wt) to the chow of nine mice; the control group had six animals. The diet of all animals contained 9% (wt/wt) fat and 0.15% (wt/wt) cholesterol. After 4 weeks, plasma total cholesterol levels were significantly reduced in the TODPM-treated mice (26.6 versus 42.0 mmol/L, P<.0001). The mean body weight of the TODPM-supplemented group was significantly higher at week 5 and throughout the study (29.4 versus 27.7 g, P<.05). The experiment was terminated at 18 weeks. Histological examination showed mature atherosclerotic lesions composed of foam cells underlying the endothelium, a mosaic of extracellular glycosaminoglycans, numerous apparently proliferative smooth muscle cells, and foci of cholesterol clefts in the control animals. By contrast, the TODPM-treated mice showed only early lesions containing mainly superficial foam cells. As assessed by morphometry, the lesion area in the aortic sinuses of TODPM-treated animals was less than half that of control animals (P<.0001). This reduced lesion area was accompanied by a substantial reduction in all lesional components, reflecting a delay in progression of atheromatous changes. A strong positive correlation (r=.69, P<.01) was found between plasma total cholesterol levels and lesion area in the aortic sinuses. TODPM also prevented the occurrence of xanthomatosis. We conclude that supplementation of a cholesterol-enriched diet with TODPM significantly lowers plasma cholesterol and retards development of atherosclerosis in apoE-deficient mice, suggesting a therapeutic potential for the mixture of phytosterols studied.
Key Words: cholesterol • phytosterols • apolipoprotein E–deficient mice • atherosclerosis
|
Introduction |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
It is well established that low levels of plasma cholesterol are associated with a decreased prevalence and incidence of atherosclerosis and, in particular, coronary artery disease.1 2 The addition of certain plant sterols to diets has been effective in decreasing plasma cholesterol levels in both animals and humans,3 4 5 6 7 and the addition of TODPM to diet significantly (P<.05) reduces TC and LDL cholesterol levels in rats.3 The cholesterol-lowering effects of phytosterols seem to be affected by various factors, including their solubility and rate of absorption and the cholesterol/phytosterol ratio in the intestinal lumen.8 9
In an effort to understand the pathogenesis of atherosclerosis and to evaluate the beneficial effects of different therapies in its prevention or treatment, investigators have used different animal models. Recently, considerable attention has been paid to transgenic murine models for the study of lipoprotein metabolism and atherogenesis. ApoE-deficient mice are a suitable model for the study of human atherosclerosis as they develop severe hypercholesterolemia and atherosclerotic lesions that are similar in appearance and distribution to those observed in humans.10 11 12 13 14 These animals also develop xanthomatosis when fed a cholesterol-enriched diet.15 Because apoE-deficient mice are useful in the study of the effects of compounds that might prevent atherosclerosis,16 we used them to assess the effects of TODPM on plasma lipid levels and on the quantity and quality of atherosclerotic lesions in this animal model.
|
Methods |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Animals
Nineteen 5-week-old male C57BL/6J apoE-deficient mice engineered at the University of North Carolina, Chapel Hill, were purchased from Jackson Laboratory. After 10 days of adaptation, animals were bled from tail veins, and their plasma cholesterol and TG levels were measured. The mice were then divided into two groups with similar baseline mean plasma cholesterol levels and body weights (10 mice in the treated group and nine in the control [untreated] group). They were housed individually throughout the experimental period. One animal in the treatment group developed dehydration during week 9 of the experiment. This animal was killed, and the reason for its decreased food intake and dehydration was found to be dental malocclusion. Three mice in the control group died unexpectedly during weeks 2, 11, and 13 of the experiment. Autopsy inspection and histological examination of their organs revealed no specific cause of the sudden death. Coronary ostial occlusion could not be excluded and represents one reasonable hypothesis regarding sudden death. Overall, 15 mice completed the study. The mice had ad libitum access to water and food; their body weights and food consumption were measured weekly.
Diet
PicoLab mouse diet 20 (9% fat wt/wt) was purchased from Jamieson's Pet Food Distributors Ltd. The chow was finely ground by using a food processor. Cholesterol (Sigma Chemical Co) was added at 0.15% (wt/wt) to the diet and mixed well. Similarly, 2% (wt/wt) TODPM was added to the cholesterol-supplemented diet and used for the treatment group. The dietary mixture was repelleted by using a bakery syringe and air-dried.
TODPM
Phytosterols were extracted and purified from "tall-oil soap," a by-product of the British Columbia pulp and paper industry, in collaboration with Dr James P. Kutney's laboratory in the Department of Chemistry, University of British Columbia. The degree of purity and composition of the final product was determined by using gas chromatography with cholesterol as an internal standard. The phytosterol product was >95% pure and was composed of 69% ß-sitosterol, 15% campesterol, and 16% stigmastanol. The other components of the mixture were long-chain fatty alcohols, predominantly C24, C22, and a trace of C20.
Blood Sampling
Each mouse was restrained briefly in a 50-mL plastic Falcon tube, and blood was collected from the tail vein into heparinized capillary tubes (Fisher Scientific) and then transferred to 0.5-mL Eppendorf tubes. Blood samples were centrifuged for 3 minutes at 13 000 rpm by using an ICMCentra centrifuge. Aliquots of the plasma were used for lipid analyses. At the end of the experiment, the final blood sample was obtained from the right ventricle of the pentobarbital-anesthetized (60 mg/kg IP) animal.
Lipid Analyses and Histopathology
Plasma TC and TG levels were quantified by using enzymatic kits (Boehringer Mannheim).17 18 19
The hearts of pentobarbital-anesthetized animals were perfused slowly with 10 mL 10% buffered formalin solution through the left ventricle. The heart and aorta were removed and placed in 10% buffered formalin. Tissues surrounding the aorta including all fat were trimmed, and the aorta was cut transversely at the aortic arch. The heart was sectioned transversely at the level of the atria.20 The atrial portion of the heart was placed in 30% sucrose in 1% phosphate buffer overnight, after which it was embedded in OCT compound and sectioned at the level of the aortic valve cusps. Once the aortic sinuses appeared on sections, alternate sections were mounted on nine glass slides in such a way that slide No. 1 contained sections 1, 19, 37, 55, 73, and 91, slide No. 6 contained sections 11, 29, 47, 65, 83, and 101, and so on. Slides were stained with ORO and Movat's pentachrome. The formalin-fixed aortas were cut transversely into several short segments (each 
5 mm long) and used for both OCT and paraffin embedding followed by 10-µm OCT and 4-µm paraffin sectioning. The sections were stained with ORO, hematoxylin-eosin, and Movat's stains. Normal-appearing and thickened skin specimens were also fixed, sectioned, and stained with the aforementioned stains. Other organs were inspected and formalin-fixed.
Quantitative Analysis of Atherosclerotic Lesions
Six 10-µm sections from different levels of the aortic sinuses of each mouse mounted on slide No. 6 were stained with ORO. The lesional area was quantified by using a digitizing morphometry image-analysis system (Bioquant II, R&M Biometrics) with a calibration factor of x60 in a blinded fashion repeated three times; the means of individual measurements were used for statistical analysis. Because sections obtained from one animal were slightly too cephalad, such that the aortic sinuses were missed, data from this animal were excluded from the final analysis.
Statistical Analysis
A two-tailed Student's t test assuming equal variances was used to assess the significance of differences between results in the two groups.
|
Results |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Body Weight and Lipid Analyses
The average body weight in both groups of animals was not significantly different during the first 4 weeks of the study, but by week 5 and throughout the rest of the study (except for weeks 7 and 9) animals in the TODPM group showed a significant (P<.05) increase in body weight compared with the control group (Fig 1
).
|
TC and TG levels were determined at baseline and at weeks 4, 11, and 18. Both groups of animals showed similar plasma TC levels at week 0 (15.5 mmol/L). The level of plasma TC was increased markedly after consumption of the 0.15% (wt/wt) cholesterol-enriched diet in both groups of mice. However, at 4 weeks, TODPM-treated animals showed a significantly lower plasma cholesterol level than the control group (26.62±2.86 versus 42.02±1.89 mmol/L, P<.0001). A significant difference was sustained until the end of the study (Fig 2). Both groups of mice had similar plasma TG levels.
|
Effect of TODPM on Atherosclerosis
Representative atherosclerotic lesions in the aortas and aortic sinuses of the treated and control groups of mice are illustrated in Fig 3 and Figs 4 through 6, respectively. Fig3A shows a section of normal-appearing aorta from a TODPM-treated mouse with normal intima and medial elastic laminae and associated smooth muscle cells; B and C show aortic sections from a control animal that developed prominent atherosclerotic lesions. Lipid-enriched foam cells are evident in the subendothelial space, but little increase in matrix is apparent. A similar lesion is present at the branch point of a secondary vessel arising from the thoracic aorta (Fig 3D and 3E). Advanced atherosclerotic lesions in the aortic root of control mice are shown in Figs 4C, 4D, 5B, 6A, and 6B. In these sections, in addition to foam cells, there is a significant extracellular matrix component. Corresponding sections from TODPM-treated animals stained with ORO or Movat's show not only a limited extent and severity of lesion involvement in the aortic sinuses (Fig 4A and 4B) but also that the origin of the epicardial coronary artery is free of lesions (Fig 5A, arrow). Marked differences between the two groups of animals in terms of extent and maturity of the lesions are reflected in the extent of superficial foam cells, underlying extracellular glycosaminoglycans, sheaths of apparently proliferative smooth muscle cells, and areas with many cholesterol clefts in the untreated animals. When a coronary ostium is visualized, the lesions in the most severely affected animals extend into the proximal coronary arteries (Fig 5B, curved arrow). In certain untreated animals, the coronary arteries show substantial fat deposits and lesion formation (Fig 6A, arrow) such that certain small branches of the coronary arteries were completely blocked. On ORO stains, the lipid richness of the foam cells was dramatic, as was the rather clustered superficial pattern of foam cell accumulation. TODPM treatment resulted in >50% reduction (1.96±0.8 versus 4.08±0.3 mm2, P<.0001) in the average area of atheromata compared with those in the control group (Fig 7).
|
|
|
|
|
Xanthomatosis
Three mice in the control group developed severe cholesterol "granulomas" in the skin during the experiment. Histological examination revealed thickened skin with fat deposits in subcutaneous tissue. Cholesterol clefts and xanthomas were very prominent in the mid-dermis and dermal-epidermal junction of affected skin. These lesions included a cellular reaction with eosinophils and histiocytes as well as xanthomatous cells. None of the animals in the TODPM-treated group developed skin lesions.
Concordance of Atherosclerotic Lesion Occurrence With Plasma Cholesterol Levels
Fig 8 shows a linear correlation (r=.69, P<.01) between mean plasma cholesterol during the experimental period and the area of atherosclerotic lesions in the aortic sinuses as measured by image analysis. This figure also demonstrates two clusters indicative of lesion size in the range of 1 to 2.5 mm2 for treated and 3.7 to 4.5 mm2 for control animals.
|
) and control (
) mice. |
Discussion |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Effects of TODPM on Plasma Lipids and Body Weight
ApoE-deficient mice had very high plasma TC levels (
15.5 mmol/L) when fed mouse chow.21 After consuming a 0.15% cholesterol–enriched diet, the level of plasma cholesterol increased markedly.22 23 This further rise in plasma cholesterol was significantly inhibited by addition of TODPM (2% wt/wt) to the diet (Fig 2
). Similar effects of TODPM have been observed by Ling and Jones3 in cholesterol-fed (1% wt/wt) rats. Specifically, rats fed TODPM (1% wt/wt) had a significant decrease in LDL cholesterol accompanied by an increase in HDL cholesterol. These changes in lipoprotein levels were associated with a significant increase in plasma levels of campesterol and ß-sitosterol compared with controls. Similarly, when rats were fed a phytosterol mixture (24 mg/d), significant (P<.001) increases in the levels of campesterol and ß-sitosterol in hepatic tissue were noticed compared with control groups.24 The increment in plasma or hepatic phytosterol levels was dependent on the phytosterol content of the diet. Ikeda et al25 have shown that orally administered sitosterol inhibits cholesterol absorption by up to 57% in rats. These investigators and others26 27 have demonstrated that the cholesterol-lowering effect of phytosterols is due to the interference of plant sterols with cholesterol absorption at two major steps: micelle formation and uptake of cholesterol at the brush border of the microvilli. This concept is supported by Miettinen et al,7 who have shown that sitostanol ester added to margarine significantly decreases plasma cholesterol levels in a mildly hypercholesterolemic population. In this study7 the decrease in plasma cholesterol correlated significantly with a decrease in plasma campesterol, a marker of decreased cholesterol absorption.
Although it is believed that the cholesterol-lowering effect of plant sterols is mainly due to an effect on intestinal cholesterol absorption, several lines of evidence suggest that phytosterols may regulate lipid metabolism in organs other than the intestine. For example, dietary phytosterol (24 mg/d) exerts a significant inhibitory effect on the activity of acetyl coenzyme A carboxylase (P<.001) and malic enzyme (P<.05) in the liver of rats.24 Administration of ß-sitosterol (6 g/d) to hypercholesterolemic subjects resulted in a significant (P<.01) increase in lecithin:cholesterol acyltransferase activity.28 The activities of other enzymes involved in lipid metabolism, such as acyl coenzyme A:cholesterol acyltransferase, 3-hydroxy-3-methylglutaryl–coenzyme A reductase, and cholesterol-7-
-hydroxylase, are also affected by high levels of plasma plant sterols in humans or animals.29 30 31
A significant (P<.05) gain in body weight was noted in the TODPM group compared with control animals. Because body weight gain in apoE-deficient mice is independent of plasma cholesterol levels,22 it seems unlikely that the differences in average body weight in the present study were due only to decreased plasma cholesterol levels. The weekly food consumption was significantly (P<.05) higher in the treated than the control animals during weeks 4, 5, 9, 10, 11, and 13 of the experiment. Since the additional fatty alcohols of TODPM contributed to <0.1% of total calories, and the level of physical activity appeared similar in both groups of mice, the higher food intake appears to be the best explanation for the differences in body weight between the groups.
Effects of TODPM on the Development of Atherosclerosis
We observed advanced atherosclerotic lesions and cutaneous xanthomatosis in control animals. The atherosclerotic lesion areas included not only foam cells but also prominent glycosaminoglycan-rich extracellular matrix and proliferative smooth muscle cells, with numerous cholesterol clefts. Atherosclerotic lesion progression was associated with severe cutaneous xanthomatosis in three of six control animals. TODPM treatment significantly reduced the development of atherosclerotic lesions and also effectively prevented the occurrence of cutaneous xanthomatosis. Although the cellular composition of atherosclerotic lesions in apoE-deficient mice fed regular mouse chow has not yet been fully defined, aortic lesions in these mice fed either regular or cholesterol-enriched diets are similar histologically except for the lesion size.11 In the present study we found that TODPM reduced both atherosclerotic lesion size and complexity.
Inhibition of intestinal cholesterol absorption may be one of the major mechanisms of action of TODPM in reducing lesion area in aortic sinuses and in the prevention of cutaneous xanthomatosis in treated animals. The reduction of plasma cholesterol in treated animals most likely resulted in the absence of cholesterol clefts in the attenuated atherosclerotic lesions of the treated animals. This slowing in the progression of atherosclerosis was reflected in the absence of advanced lesions either in the arterial system or skin of treated animals.
A study similar to the present one tested the effects of the antioxidant N,N'-diphenyl-1,4-phenylenediamine (DPPD) on the development of atherosclerosis in apoE-deficient mice.16 The overall antiatherogenic effects of this antioxidant were similar to our observations. However, DPPD, unlike TODPM, has no cholesterol-lowering properties and may have toxic effects, as it results in a significant decrease in the body weights of experimental animals. At the end of our study, the mean plasma TC level of treated mice was decreased by 20% compared with the control group, while the mean atherosclerotic lesion area was reduced by >50% in the aortic sinuses of treated mice compared with the control group. These observations are roughly similar to the "1% to 2% rule" in regard to the relationship of changes in plasma cholesterol and the likelihood of cardiac events in human trials.32 Regression analysis of plasma cholesterol levels and lesion areas showed a significant positive correlation (r=.69, P<.01). Fig 8 shows a clustering of the data reflective of the two groups: the treated animals with lower cholesterol levels and lesion areas and untreated animals with correspondingly higher plasma cholesterol levels and lesion areas. Due to the small number of animals we cannot comment on the apparent discontinuity between the groups, but the data suggest a continuous relationship between lipid levels and lesion areas.
The observed antiatherogenic effect of TODPM may be due not only to its cholesterol-lowering properties alone but also to other mechanisms, such as its antioxidant properties. It is of interest that dietary olive oil (50 g/d) significantly (P<.01) increased LDL sitosterol content in 10 healthy men; this alteration was associated with a significant (P<.01) decrease in the sensitivity of their LDL to in vitro oxidation and a significant (P<.01) reduction in macrophage uptake of LDL.33 Moreover, the LDL of apoE-deficient mice is 360 times more susceptible to in vitro oxidation than that of controls.34 Therefore, perhaps some plant sterols are also absorbed and incorporated into lipoproteins, especially LDL and VLDL, which may significantly alter their properties. One of these alterations could be related to the sensitivity of lipoproteins to oxidation. Thus, if LDL is resistant to oxidation, it becomes less atherogenic, with a resultant reduction in the area and complexity of lesions.
The findings of our study demonstrate the effectiveness of TODPM in preventing xanthomatosis, decreasing plasma cholesterol levels, and retarding the development of atherosclerotic lesions. The lack of toxicity of these substances, their abundance in nature, and their low cost make their potential use in the prevention and treatment of human hypercholesterolemia attractive.
|
Selected Abbreviations and Acronyms |
|---|
|
|
Acknowledgments |
|---|
This work was supported by Forbes Medi-Tech Inc, Vancouver, BC. We are thankful to Dr R. Riesen, Department of Chemistry, University of British Columbia, for his help in extracting and purifying the phytosterol mixture; to Dr R.C. LeBoeuf (DK35816), Department of Medicine, University of Washington, Seattle, for her assistance in sectioning the mouse aortic roots; and to Dr A. Magil, Department of Pathology and Laboratory Medicine, St. Paul's Hospital, for his help in digitizing the aortic sinus lesions. The support of A.F. Ayyobi is also appreciated.
Received April 25, 1996;
revision received July 14, 1996;
|
References |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
1. Gould AL, Rossouw JE, Santanello NC, Heyse JF, Furberg CD. Cholesterol reduction yields clinical benefit: a new look at old data. Circulation. 1995;91:2274-2282.
2.
Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults. Summary of the second report of the National Cholesterol Education Program (NCEP) expert plan on detection, evaluation, and treatment of high blood cholesterol in adults (adult treatment panel II). JAMA. 1993;269:3015-3023.
3. Ling WH, Jones PJH. Enhanced efficacy of sitostanol-containing versus sitostanol-free phytosterol mixtures in altering lipoprotein cholesterol levels and synthesis in rats. Atherosclerosis. 1995;118:319-331.[Medline] [Order article via Infotrieve]
4. Laraki L, Pelletier X, Debry G. Effects of dietary cholesterol and phytosterol overload on Wistar rat plasma lipids. Ann Nutr Metab. 1991;35:221-225.[Medline] [Order article via Infotrieve]
5. Becker M, Staab D, von Bergmann K. Treatment of severe familial hypercholesterolemia in childhood with sitosterol and sitostanol. J Pediatr. 1993;122:292-296.[Medline] [Order article via Infotrieve]
6. Heinemann T, Leiss O, von Bergmann K. Effects of low dose sitostanol on serum cholesterol in patients with hypercholesterolemia. Atherosclerosis. 1986;61:219-223.[Medline] [Order article via Infotrieve]
7.
Miettinen TA, Puska P, Glyying H, Vanhanen H, Vartiainen E. Reduction of serum cholesterol with sitostanol-ester margarine in a mildly hypercholesterolemic population. N Engl J Med. 1995;333:1308-1312.
8. Heinemann T, Axtmann G, von Bergmann K. Comparison of intestinal absorption of cholesterol with different plant sterols in man. Eur J Clin Invest. 1993;23:827-831.[Medline] [Order article via Infotrieve]
9.
Vahouny GV, Connor WE, Subramaniam S, Lin DS, Gallo LL. Comparative lymphatic absorption of sitosterol, stigmasterol, and fucosterol and differential inhibition of cholesterol absorption. Am J Clin Nutr. 1983;37:805-809.
10.
Nakashima Y, Plump AS, Raines EW, Breslow JL, Ross R. Apo E–deficient mice develop lesions of all phases of atherosclerosis throughout the arterial tree. Arterioscler Thromb. 1994;14:133-140.
11. Plump AS, Smith JD, Hayek T, Aalto-Setala K, Walsh A, Verstuyft JG, Rubin EM, Breslow JL. Severe hypercholesterolemia and atherosclerosis in apolipoprotein E-deficient mice created by homologous recombination in ES cells. Cell. 1992;71:343-353.[Medline] [Order article via Infotrieve]
12.
Zhang SH, Reddick RL, Piedrahita JA, Maeda N. Spontaneous hypercholesterolemia and arterial lesions in mice lacking apolipoprotein E. Science. 1992;258:468-471.
13.
Palinski W, Ord VA, Plump AS, Breslow JL, Steinberg D, Witztum J. Apo E–deficient mice are a model of lipoprotein oxidation in atherogenesis: demonstration of oxidation-specific epitopes in lesions and high titers of autoantibodies to malondialdehyde-lysine in serum. Arterioscler Thromb. 1994;14:605-616.
14.
Plump AS, Scott CJ, Breslow JL. Human apolipoprotein A-I gene expression increases high density lipoprotein and suppresses atherosclerosis in apolipoprotein E-deficient mouse. Proc Natl Acad Sci U S A. 1994;91:9607-9611.
15. van Ree JH, Gijbels MJJ, van den Broek WJAA, Hofker MH, Havekes LM. Atypical xanthomatosis in apolipoprotein E-deficient mice after cholesterol feeding. Atherosclerosis. 1995;112:237-243.[Medline] [Order article via Infotrieve]
16.
Tangirala RK, Casanada F, Miller E, Witztum JL, Steinberg D, Palinski W. Effects of the antioxidant N,N'-diphenyl 1,4-phenylenediamine (DPPD) on atherosclerosis in apoE-deficient mice. Arterioscler Thromb Vasc Biol. 1995;15:1625-1630.
17.
Dobiasova M, Stribrna J, Sparks DL, Pritchard PH, Frohlich JJ. Cholesterol esterification rates in very low-density lipoprotein– and low-density lipoprotein–depleted plasma: relation to high density lipoprotein subspecies, sex, hyperlipidemia, and coronary artery disease. Arterioscler Thromb. 1991;11:64-70.
18. Allain CC, Poon LS, Chan CS, Richmond W. Enzymatic determination of total serum cholesterol. Clin Chem. 1974;29:470-475.
19. Bucolo G, David H. Quantitative determination of serum triglycerides by the use of enzymes. Clin Chem. 1973;19:476-482.[Abstract]
20. Paigen B, Morrow A, Holmes PA, Mitchell D, Williams RA. Quantitative assessment of atherosclerotic lesions in mice. Atherosclerosis. 1987;68:231-240.[Medline] [Order article via Infotrieve]
21. Paszty C, Maeda N, Verstuyft J, Rubin EM. Apolipoprotein AI transgene corrects apolipoprotein E deficiency-induced atherosclerosis in mice. J Clin Invest. 1994;94:899-903.
22. Zhang SH, Reddick RL, Burkey B, Maeda N. Diet-induced atherosclerosis in mice heterozygous and homozygous for apolipoprotein E gene disruption. J Clin Invest. 1994;94:937-945.
23. van Ree JH, van den Broek WJAA, Dahlmans VEH, Groot PHE, Vidgeon-Hart M, Frants RR, Wieringa B, Havekes LM, Hofker MH. Diet-induced hypercholesterolemia and atherosclerosis in heterozygous apolipoprotein E-deficient mice. Atherosclerosis. 1994;111:25-37.[Medline] [Order article via Infotrieve]
24. Laraki L, Pelletier X, Mourot J, Debry G. Effects of dietary phytosterols on liver lipids and lipid metabolism enzymes. Ann Nutr Metab. 1993;37:129-133.[Medline] [Order article via Infotrieve]
25. Ikeda I, Tanaka K, Sugano M, Vahouny GV, Gallo LL. Inhibition of cholesterol absorption in rats by plant sterols. J Lipid Res. 1988;29:1573-1582.[Abstract]
26. Heinemann T, Kullak-Ublick GA, Pietruck B, von Bergmann K. Mechanisms of action of plant sterols on inhibition of cholesterol absorption: comparison of sitosterol and sitostanol. Eur J Clin Pharmacol. 1991;40:S59-S63.
27. Ikeda I, Tanaka K, Sugano M, Vahouny GV, Gallo LL. Discrimination between cholesterol and sitosterol for absorption in rats. J Lipid Res. 1988;29:1583-1591.[Abstract]
28. Weisweiler P, Heinemann V, Schwandt P. Serum lipoproteins and lecithin:cholesterol acyltransferase (LCAT) activity in hypercholesterolemic subjects given ß-sitosterol. Int J Clin Pharmacol. 1984;22:204-206.
29. Boberg KM, Åkerlund J, Bjorkhem I. Effect of sitosterol on the rate-limiting enzymes in cholesterol synthesis and degradation. Lipids. 1989;24:9-12.[Medline] [Order article via Infotrieve]
30.
Shefer S, Hauser S, Lapar V, Mosbach EH. Regulatory effects of sterols and bile acids on hepatic 3-hydroxy-3-methylglutaryl CoA reductase and cholesterol 7-hydroxylase in the rat. J Lipid Res. 1973;14:573-580.[Abstract]
31.
Shefer S, Salen G, Bullock J, Nguyen LB, Ness GC, Vhao Z, Belamarich PF, Chowdhary I, Lerner S, Batta AK, Tint GS. The effect of increased hepatic sitosterol on the regulation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase and cholesterol 7-hydroxylase in the rat and sitosterolemic homozygotes. Hepatology. 1994;20:213-219.[Medline]
[Order article via Infotrieve]
32.
Lipid Research Clinics Program. The Lipid Research Clinics Coronary Primary Prevention Trial results, II: the relationship of reduction in incidence of coronary heart disease to cholesterol lowering. JAMA. 1984;251:365-374.
33. Aviram M, Eias K. Dietary olive oil reduces low-density lipoprotein uptake by macrophages and decreases the susceptibility of the lipoprotein to undergo lipid peroxidation. Ann Nutr Metab. 1993;37:75-84.[Medline] [Order article via Infotrieve]
34. Hayek T, Oiknine J, Brook JG, Aviram M. Increased plasma and lipoprotein lipid peroxidation in apo E-deficient mice. Biochem Biophys Res Commun. 1994;201:1567-1574.[Medline] [Order article via Infotrieve]
This article has been cited by other articles:
 |
|
 |
|
  S. Helske, T. Miettinen, H. Gylling, M. Mayranpaa, J. Lommi, H. Turto, K. Werkkala, M. Kupari, and P. T. Kovanen Accumulation of cholesterol precursors and plant sterols in human stenotic aortic valves J. Lipid Res., July 1, 2008; 49(7): 1511 - 1518. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 O. Weingartner, D. Lutjohann, S. Ji, N. Weisshoff, F. List, T. Sudhop, K. von Bergmann, K. Gertz, J. Konig, H.-J. Schafers, et al. Vascular effects of diet supplementation with plant sterols. J. Am. Coll. Cardiol., April 22, 2008; 51(16): 1553 - 1561. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. A. Othman, M. Suh, G. Fischer, N. Azordegan, N. Riediger, K. Le, D. S. Jassal, and M. H. Moghadasian A comparison of the effects of fish oil and flaxseed oil on cardiac allograft chronic rejection in rats Am J Physiol Heart Circ Physiol, March 1, 2008; 294(3): H1452 - H1458. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Q. Chen, L. Steinhauer, J. Hammerlindl, W. Keller, and J. Zou Biosynthesis of Phytosterol Esters: Identification of a Sterol O-Acyltransferase in Arabidopsis Plant Physiology, November 1, 2007; 145(3): 974 - 984. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Plat, I. Beugels, M. J. J. Gijbels, M. P. J. de Winther, and R. P. Mensink Plant sterol or stanol esters retard lesion formation in LDL receptor-deficient mice independent of changes in serum plant sterols J. Lipid Res., December 1, 2006; 47(12): 2762 - 2771. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. M. C. Dupasquier, A.-M. Weber, B. P. Ander, P. P. Rampersad, S. Steigerwald, J. T. Wigle, R. W. Mitchell, E. A. Kroeger, J. S. C. Gilchrist, M. M. Moghadasian, et al. Effects of dietary flaxseed on vascular contractile function and atherosclerosis during prolonged hypercholesterolemia in rabbits Am J Physiol Heart Circ Physiol, December 1, 2006; 291(6): H2987 - H2996. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. Nashed, B. Yeganeh, K. T. HayGlass, and M. H. Moghadasian Antiatherogenic Effects of Dietary Plant Sterols Are Associated with Inhibition of Proinflammatory Cytokine Production in Apo E-KO Mice J. Nutr., October 1, 2005; 135(10): 2438 - 2444. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. R. Wilund, L. Yu, F. Xu, G. L. Vega, S. M. Grundy, J. C. Cohen, and H. H. Hobbs No Association Between Plasma Levels of Plant Sterols and Atherosclerosis in Mice and Men Arterioscler. Thromb. Vasc. Biol., December 1, 2004; 24(12): 2326 - 2332. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. B. Katan, S. M. Grundy, P. Jones, M. Law, T. Miettinen, R. Paoletti, and Stresa Workshop Participants Efficacy and Safety of Plant Stanols and Sterols in the Management of Blood Cholesterol Levels Mayo Clin. Proc., August 1, 2003; 78(8): 965 - 978. [Abstract] [PDF]
|
|
|
|
|
|
O. L. Volger, H. van der Boom, E. C. M. de Wit, W. van Duyvenvoorde, G. Hornstra, J. Plat, L. M. Havekes, R. P. Mensink, and H. M. G. Princen Dietary Plant Stanol Esters Reduce VLDL Cholesterol Secretion and Bile Saturation in Apolipoprotein E*3-Leiden Transgenic Mice Arterioscler. Thromb. Vasc. Biol., June 1, 2001; 21(6): 1046 - 1052. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y.-Y. Fan, K. S. Ramos, and R. S. Chapkin Dietary {{gamma}}-Linolenic Acid Suppresses Aortic Smooth Muscle Cell Proliferation and Modifies Atherosclerotic Lesions in Apolipoprotein E Knockout Mice J. Nutr., June 1, 2001; 131(6): 1675 - 1681. [Abstract] [Full Text]
|
|
|
|
|
|
C. P. Sparrow, C. A. Burton, M. Hernandez, S. Mundt, H. Hassing, S. Patel, R. Rosa, A. Hermanowski-Vosatka, P.-R. Wang, D. Zhang, et al. Simvastatin Has Anti-Inflammatory and Antiatherosclerotic Activities Independent of Plasma Cholesterol Lowering Arterioscler. Thromb. Vasc. Biol., January 1, 2001; 21(1): 115 - 121. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
W. M. N. Ratnayake, M. R. L’Abbé, R. Mueller, S. Hayward, L. Plouffe, R. Hollywood, and K. Trick Vegetable Oils High in Phytosterols Make Erythrocytes Less Deformable and Shorten the Life Span of Stroke-Prone Spontaneously Hypertensive Rats J. Nutr., May 1, 2000; 130(5): 1166 - 1178. [Abstract] [Full Text]
|
|
|
|
 |
|
 M. H. Moghadasian, B. M. McManus, D. V. Godin, B. Rodrigues, and J. J. Frohlich Proatherogenic and Antiatherogenic Effects of Probucol and Phytosterols in Apolipoprotein E–Deficient Mice : Possible Mechanisms of Action Circulation, April 6, 1999; 99(13): 1733 - 1739. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||