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Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:119-126

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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:119-126.)
© 1997 American Heart Association, Inc.

Articles

"Tall Oil"–Derived Phytosterols Reduce Atherosclerosis in ApoE-Deficient Mice

Mohammed H. Moghadasian; Bruce M. McManus; P. Haydn Pritchard; Jiri J. Frohlich

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
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 *Abstract
down arrowIntroduction
 down arrowMethods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
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
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 up arrowAbstract
 *Introduction
down arrowMethods
 down arrowResults
 down arrowDiscussion
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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
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 up arrowAbstract
 up arrowIntroduction
 *Methods
down arrowResults
 down arrowDiscussion
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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 {approx}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
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 up arrowAbstract
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 *Results
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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 1Down).



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  		Figure 1. Line graph shows body weight at baseline and at weekly intervals throughout the experiment. Values are mean±SD. *P<.05.

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 ({approx}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 2Down). Both groups of mice had similar plasma TG levels.



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  		Figure 2. Plasma levels of TC in the control and TODPM-treated mice at baseline and during the study. Number of animals is shown in parentheses. Values are mean±SD. Probability values are shown for between-group differences at specified times.

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 3Down and Figs 4Down through 6, respectively. Fig3ADown 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 3EDownDown). Advanced atherosclerotic lesions in the aortic root of control mice are shown in Figs 4C, 4D, 5B, 6A, and 6BDownDownDownDownDown. 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 4BDownDown) but also that the origin of the epicardial coronary artery is free of lesions (Fig 5ADown, 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 5BDown, curved arrow). In certain untreated animals, the coronary arteries show substantial fat deposits and lesion formation (Fig 6ADown, 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 7Down).



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  		Figure 3. Photomicrographs of transverse sections of aorta from TODPM-treated (A) and untreated (B through E) mice. A has no visible intima but has normal intact musculoelastic layers in the media. In B a localized intimal lesion is shown; at a higher power (C) numerous foam cells are evident, and the overlying endothelium appears to be intact. There is disruption of the internal-most elastic lamina with projection of certain foam cells into the superficial media. D and E, At the bifurcation of a probable segmental (intercostal) branch of the aorta there is an atheromatous lesion that appears to virtually occlude the mouth of the small branch vessel. The nature of this lesion is more complex than that in B and C, with apparent intimal smooth muscle cells as well as numerous foam cells. The media is variably, and focally very severely, disrupted (Movat's pentachrome stains, original magnification x25 [A, B, and D], x330 [C and E]).



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  		Figure 4. Photomicrographs of serial transverse sections at the level of the aortic valve cusps taken from one TODPM-treated (A and B) and one untreated (C and D) mouse. The volume of the lesions in the treated animal is markedly reduced, and the lesions are less complex. Movat's pentachrome stain (A and C) highlights the interstitial matrix as well as outlining cholesterol clefts and foam cells. Corresponding ORO-stained sections (B and D) emphasize the prominence of neutral lipid, most dramatically in the untreated animal (D) (original magnification x25).



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  		Figure 5. Photomicrographs of transverse sections at the level of the aortic valve cusps show severity of atheromatous lesions in TODPM-treated (A) and untreated (B) mice. Compare lack of ostial narrowing of a major epicardial coronary artery in the treated group (arrow) with virtual occlusion by atheromatous change in the untreated animal (curved arrow) (ORO stain, original magnification x25).



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  		Figure 6. Photomicrographs of transverse sections of aortic root at the level of the aortic valve cusps show involvement of proximal epicardial coronary arteries (A, arrow) and the severity of foam cell formation in underlying matrix and cellular components (B) in an untreated mouse (ORO stain, original magnification x25 [A], x330 [B]).



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  		Figure 7. Bar graph shows average lesion area in aortic sinuses measured in ORO-stained sections from each aorta using an image-analysis system with a calibration factor of x60. Sections were taken from six different levels representative of {approx}100 to 1010 µm of the aortic root length. Values are mean±SD (n=9 treated and n=5 control animals). *P<.0001.

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 8Down 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.



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  		Figure 8. Plot shows relationship of total area of atherosclerotic lesions in the aortic sinuses to plasma TC in treated ({blacktriangleup}) and control ({bullet}) mice.


*    Discussion
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 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 up arrowResults
 *Discussion
down arrowReferences
 
Effects of TODPM on Plasma Lipids and Body Weight
ApoE-deficient mice had very high plasma TC levels ({approx}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 2Up). 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-{alpha}-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 {approx}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 8Up 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
 
OCT = optimal cutting temperature
ORO = oil red O
TC = total cholesterol
TG = triglyceride
TODPM = "tall oil"–derived phytosterol mixture


*    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;
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 up arrowMethods
 up arrowResults
 up arrowDiscussion
 *References
 

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