Donate Help Contact The AHA Sign In Home
American Heart Association
Arteriosclerosis, Thrombosis, and Vascular Biology
Search: search_blue_button Advanced Search
« Previous Article | Table of Contents | Next Article »
Arteriosclerosis, Thrombosis, and Vascular Biology. 1996;16:1454-1464

This Article
Right arrow Abstract Freely available
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kolodgie, F. D.
Right arrow Articles by Virmani, R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kolodgie, F. D.
Right arrow Articles by Virmani, R.
(Arteriosclerosis, Thrombosis, and Vascular Biology. 1996;16:1454-1464.)
© 1996 American Heart Association, Inc.

Articles

Hypercholesterolemia in the Rabbit Induced by Feeding Graded Amounts of Low-Level Cholesterol

Methodological Considerations Regarding Individual Variability in Response to Dietary Cholesterol and Development of Lesion Type

Frank D. Kolodgie; Andrew S. Katocs, Jr; Elwood E. Largis; Simeon M. Wrenn; J. Fredrick Cornhill; Edward E. Herderick; Sue J. Lee; Renu Virmani

the Department of Cardiovascular Pathology (F.D.K., S.J.L., R.V.), Armed Forces Institute of Pathology, Washington, DC; Cardiovascular Biological Research (A.S.K., E.E.L., S.M.W.), Medical Research Division, American Cyanamid Co, Lederle Laboratories, Pearl River, NY; the Biomedical Engineering Center (J.F.C., E.E.H.), The Ohio State University, Columbus; and the Department of Biomedical Engineering Research Institute (J.F.C.), The Cleveland Clinic Foundation, Cleveland, Ohio.

Correspondence to Renu Virmani, MD, Armed Forces Institute of Pathology, Cardiovascular Pathology, 14th and Alaska Aves NW, Washington, DC 20306-6000. E-mail virmani@email.afip.osd.mil.


*    Abstract
up arrowTop
 *Abstract
down arrowIntroduction
 down arrowMethods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
While a number of studies have presented detailed examinations of lesion development in the cholesterol-fed rabbit, individual variability in response to cholesterol feeding and type of lesion produced relative to the degree of cholesterol exposure is not well defined. This study analyzed such critical parameters in an attempt to further characterize the model and establish a baseline for future testing of treatments targeted at limiting atherosclerosis. For these experiments, male New Zealand White rabbits were fed atherogenic diets consisting of 0.05%, 0.10%, 0.15%, 0.20%, or 0.25% cholesterol dissolved in 6% peanut oil for 31 to 32 weeks. Raising dietary cholesterol from 0.05% to 0.15% resulted in a less than twofold stepwise increase in total plasma cholesterol (TPC) exposure (area under plasma cholesterol versus time curve), whereas further increases in cholesterol intake resulted in an exponential four- to fivefold increase in TPC exposure. Regression analysis of TPC exposure with aortic sudanophilia demonstrated a threshold of {approx}5000 cholesterol weeks; below this limit lesions were minimal, and above this value, the degree of plaque correlated with TPC exposure. Furthermore, a wide biological variability occurred among rabbits with respect to individual responsiveness to dietary cholesterol. In the aorta, various types of plaques, from fatty streaks to atheromatous lesions, were observed, depending on the degree of cholesterol intake. Diets consisting of <0.15% cholesterol resulted in the development of fatty streak lesions, while transitional lesions and atheromatous plaques were mostly found with higher cholesterol feeding. Coronary artery atherosclerosis was present in >50% of animals fed diets >=0.15% cholesterol. Despite the level of TPC exposure, coronary lesions in epicardial vessels were generally the fibrous type, whereas intramyocardial arteries demonstrated predominantly intimal foam cells. In conclusion, by adjusting dietary cholesterol intake and selecting rabbits with a similar responsiveness to cholesterol, the overall cholesterol exposure can be more closely controlled to minimize the inherent individual variability among animals in this model. The nature of the target lesion must also be carefully considered, because the efficacy of some treatments may depend on the type of atherosclerotic plaque.


Key Words: atherosclerosis • rabbit • cholesterol • acyl coenzyme A:cholesterol acyltransferase • sudanophilia


*    Introduction
up arrowTop
 up arrowAbstract
 *Introduction
down arrowMethods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Several animal models such as pigeon, swine, rabbit, and monkey have been used to study atherosclerosis and to test therapeutic strategies aimed at limiting the progression of the disease.1 2 3 4 5 Of these, the cholesterol-fed rabbit model is notable for rapid development of aortic lesions and low maintenance cost. A typical regimen for the induction of atherosclerosis involves cholesterol feeding (0.5% to 4% by weight) for about 8 to 16 weeks. Under these conditions, rabbits rapidly become hypercholesterolemic (serum cholesterol >1000 mg/dL), and the resulting lesions consist primarily of macrophage-derived foam cells. Although early foam-cell lesions in rabbits resemble human fatty streaks, these particular lesions are dissimilar to the fibrous or atheromatous plaques that are the hallmark of symptomatic atherosclerotic disease in humans. Long-term experiments in rabbits that use diets containing large amounts of cholesterol are discouraged because of the resultant hepatotoxicity and failure of the animal to thrive.6 Despite these limitations, a large number of studies have used this model for testing the efficacy of antiatherogenic drugs on fatty streak development, lesions which in humans are ubiquitous in all populations and are clinically insignificant. More recent studies have demonstrated that rabbits will tolerate lower doses of cholesterol over longer periods, thereby modifying lesions to resemble advanced human disease more closely.7 8 9 10 11

The goal of the present study was to further characterize the cholesterol-fed rabbit model in an effort to better define the individual variability in cholesterol exposure and to identify the types of plaques encountered in response to feeding various amounts of low-level dietary cholesterol (0.05% to 0.25%). The extent of topographic distribution, morphology, and biochemical composition of atherosclerotic lesions were assessed. Such information regarding cholesterol exposure, lesion type, and biological variability of individual responsiveness to dietary cholesterol will serve to establish a better model for the evaluation of therapeutic strategies designed to limit the progression of atherosclerotic disease in humans.


*    Methods
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 *Methods
down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Animals
Specific pathogen-free male New Zealand White rabbits (2.0 to 3.0 kg) were purchased from Hazleton Research Products. Rabbits were housed at an animal facility accredited by the American Association for the Accreditation of Laboratory Animal Care. Experiments were conducted in accordance with the guidelines of the American Heart Association and were approved by the Animal Care and Use Committee at the American Cyanamid Co.

Experimental Design
After 2 weeks of quarantine, 70 rabbits underwent a 1-week prescreening process to assess their response to cholesterol feeding before being randomized to the various dietary study groups. For this initial experiment, rabbits were fed standard rabbit chow (Purina) supplemented with 0.25% in weight of cholesterol (USP grade, anhydrous, Sigma Chemical Co) dissolved in 6% peanut oil (Planter's) (Table 1Down). The atherogenic diet was prepared by dissolving the cholesterol in the peanut oil and thoroughly coating the pellets of rabbit chow with this mixture. All animals received 125 g cholesterol feed/d; water was provided ad libitum. Food consumption was recorded daily, and body weight was measured before and after the test period. At the conclusion of the cholesterol screen, animals were phlebotomized through the marginal ear vein, and blood (10 mL) was collected in EDTA-containing tubes for determination of TPC.


View this table:
[in this window]
[in a new window]
  		Table 1. Composition of the Various Experimental Diets

During the screening period, body weight and food consumption were similar among all animals; TPC values ranged between 150 and 550 mg/dL. For randomization, rabbits with the highest and lowest TPC levels were pooled so that cholesterol values were equally distributed among the five study groups. Animals were subsequently switched to atherogenic diets consisting of either 0.05% (n=10), 0.10% (n=10), 0.15% (n=10), 0.20% (n=10), or 0.25% (n=30) cholesterol. The diets were prepared as described above. Throughout the duration of the experiment (31 to 32 weeks), animals were bled and body weights were recorded every second week. Blood samples were analyzed for total serum cholesterol and liver enzymes (ALT, AST, and ALP). Elevated ALT and AST levels serve as indicators of hepatocellular injury; increased ALP indicates hepatobiliary obstructive disease.

Tissue Preparation
A final blood sample was obtained at the completion of the study, and rabbits were euthanized with an intravenous overdose of sodium pentobarbital (120 mg/kg). The thoracic cavity and abdomen were opened. Portions of the small intestine (jejunum) and liver were excised and frozen in liquid nitrogen for preparation of microsomes for analysis of ACAT activity. The aorta (ascending aorta to iliac bifurcation) was removed, trimmed of all adherent fat, cut open longitudinally, and pinned on a paraffin block with the endothelial surface exposed. The opened aorta was divided in half longitudinally along its ventral aspect with the transection line passing through the origins of the celiac, superior mesenteric, and inferior mesenteric arteries. The anatomic left half of the aorta was processed for evaluation of sudanophilia, and the right half was analyzed for lipid content and ACAT activity.

For evaluation of coronary atherosclerosis, the heart was removed, and sections were prepared by slicing the whole heart from the apex at 2-mm intervals to just above the aortic valve parallel to the atrioventricular sulcus.

Lipid Extraction and Analysis of Tissue and Plasma Cholesterol
TPC was measured by using enzymatic techniques with a Centrifichem System 400 Autoanalyzer (Union Carbide Co). Tissue samples from the liver, aortic arch (origin to first set of intercostal arteries), and thoracic aorta (first set of intercostal arteries to the level of the diaphragm) were gently blotted, and weights were determined. The tissue samples (0.06 to 0.28 g) were homogenized in 1.6 mL distilled water on ice with a Brinkman Polytron at full speed for 15 seconds. Total lipids were extracted from equivalent 0.8-mL aliquots according to the methods of Folch et al.12 Cholesterol was measured by using gas chromatography with cholesterol methyl ether (Sigma) as an internal standard. The total cholesterol content was measured after hydrolysis at 70°C for 45 minutes in 0.2 mL of 33% potassium hydroxide in water (wt/vol) and 2.0 mL of 95% ethanol. The concentration of esterified cholesterol was calculated by assessing the difference between the concentrations of unesterified and total cholesterol.

Analysis of ACAT Activity
Liver and Intestine
Microsomes from liver and intestine were prepared13 and stored at -70°C until analysis. ACAT activity was assayed in microsomes according to the methods of Hashimoto et al.14 Briefly, 30 µL of microsome suspension was added to 500 µL potassium hydrogen phosphate buffer (0.2 mol/L, pH 7.4) containing 0.2% bovine serum albumin. The reaction was initiated by adding 20 µL [14C]palmitoyl coenzyme A (55.5 mCi/mmol, New England Nuclear) and run for 4 minutes at 37°C in a shaker water bath. Enzymatic reactions were terminated by adding 1 mL ethanol, and lipids were extracted by vortexing with 3 mL petroleum ether. The lipids were separated by silica gel G thin-layer chromatography, and the CE band (Rf=0.94) was identified by using iodine vapor. The CEs were assayed by using scintillation counting. Recovery of the 14C-labeled CE added to microsomes was 89% to 95%.

Aortic Arch
The adventitia was removed, and the medial-intimal layers weighing approximately 70 to 200 mg were homogenized in 1 mL potassium phosphate buffer (154 mmol/L, pH 7.4) by using a Brinkman Polytron. Homogenates were centrifuged at 500g for 5 minutes, and the supernatant was assayed for ACAT activity. Aliquots of aortic homogenates were analyzed for protein content by using the Bio-Rad protein assay procedure. Measurement of ACAT activity was performed essentially as described above with the following changes. The aortic homogenate (20 µL) was preincubated for 15 minutes in 20 µL potassium phosphate buffer (0.2 mol/L, pH 7.4). The reaction was initiated by adding 10 µL [14C]palmitoyl coenzyme A and run for 10 minutes. Assays were terminated by adding ethanol (500 µL), and petroleum ether was added for lipid extraction. Separation of CEs followed the procedures described above.

Morphological Evaluation of Aortic Sudanophilia
For evaluation of aortic sudanophilia, the left half of the aorta was stretched, pinned on cork board, immersion-fixed in 10% buffered formalin, and stored at room temperature. Specimens were subsequently stained with Sudan IV for macroscopic determination of sudanophilia (fat content) and photographed. Sudanophilia, as shown by dark red regions corresponding to the uptake of Sudan IV by fat, was quantified by digital image analysis at Ohio State University by using the methods of Cornhill et al.15 In all cases, the degree and topographic distribution of aortic sudanophilia were measured from the ascending aorta to the branch of the celiac artery. In brief, digital images captured from 35-mm color slides were subdivided into triangular sections by using anatomic landmarks (ie, ostia) to outline the vertices; these were used to execute a linear transformation of the raw images to a standard template, thereby removing anatomic variation between vessels. Images were then edited to remove artifacts and were segmented into sudanophilic and nonsudanophilic areas by using an average boundary gradient algorithm to determine a threshold for image segmentation. Finally, probability-of-occurrence maps were generated by calculating the probability of sudanophilia occurring at each point on the entire aortic surface. These maps represent the percentage of animals with lesions at that particular site.15

Histomorphometry
Intimal and medial thickness measurements were determined on 3-mm transverse segments taken from four sites along the length of the entire aorta: (1) the ascending aorta, (2) the descending aorta between the third and fourth intercostal arteries, (3) just above the renal arteries, and (4) the aorta above the bifurcation of the iliac arteries. These segments are referred to as the ascending aorta, descending thoracic, thoracic abdominal, and abdominal aorta, respectively.

Light Microscopy
Aortic specimens were dehydrated in a graded series of alcohols and xylene and embedded in paraffin for light microscopic evaluation. Multiple 4-µm-thick sections were cut and stained with hematoxylin and eosin and Movat Pentachrome stains. The aortic sections were magnified (x45), and intimal and medial thickness measurements (in millimeters) were made by a skilled observer using computerized planimetry who was blinded to the various groups. Both intimal and medial thicknesses were measured at the sites showing maximal intimal thickening; the intimal-medial border was defined by parallel elastic lamellae. Intimal and medial thickness measurements were averaged for each study group. The intimal and medial thickness ratio was calculated as the intimal/medial thicknessx100 for each animal and was averaged for each study group.

Immunocytochemistry
Semiquantitative analysis was performed on serial sections immunostained with the muscle actin–specific monoclonal antibody HHF35 (dilution 1:200) and the rabbit macrophage-specific monoclonal antibody RAM 11 (dilution 1:100; Dako).16 Lesions were classified as fatty streak (macrophage-derived foam cells with little intervening extracellular matrix); transitional (SMCs interspersed with macrophages, proteoglycans with or without the rare presence of cholesterol clefts); or atheromatous (presence of an SMC-rich fibrous cap, cholesterol clefts, and necrotic core).

Evaluation of Coronary Atherosclerosis
Each myocardial section (base to apex, five slices per heart) was processed for light microscopy and stained with Movat Pentachrome. Myocardial sections were evaluated by an observer unaware of the dietary group. The number of cases demonstrating intimal thickening in epicardial or intramyocardial vessels (ie, frequency of animals with lesions) was determined. For the ratio of coronary vessels with lesions, the total number of both epicardial and intramyocardial muscular arteries from the base and middle myocardial sections, respectively, was counted. The number of arteries demonstrating atherosclerotic plaque was then determined and divided by the total number of the respective vessels present in each myocardial section.

Statistical Analysis
Statistical comparisons between groups were made by using ANOVA and linear correlation between two variables (StatView, version 4.01, for the Macintosh, Abacus Concepts, Inc). A quantitative estimate of the cumulative total exposure to cholesterol for individual animals was determined by plotting the biweekly total cholesterol values and calculating the areas under the curves using the trapezoidal rule.17 Quantitative results are reported as mean±SEM, and statistical differences between groups were determined by using Scheffe`s test. Calculations of confidence intervals at 95% for TPC exposure and estimates of variability in individual responsiveness to dietary cholesterol were performed by using Statistical Analysis Software (SAS Institute). Differences among dietary groups were considered significant at a probability value of .05 or less.


*    Results
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 *Results
down arrowDiscussion
 down arrowReferences
 
Rabbits receiving 0.05% to 0.15% dietary cholesterol remained healthy throughout the experimental protocol. In animals fed 0.20% and 0.25% dietary cholesterol, food consumption began to decrease at 24 and 18 weeks, respectively. This decrease led to a significant overall loss in body weight gain by the end of the study in rabbits fed 0.25% dietary cholesterol (P<.03 versus the lower cholesterol dietary groups). Plasma ALT and AST transaminases in animals fed <=0.15% cholesterol remained stable throughout the protocol. In rabbits fed >0.15% cholesterol, liver enzymes were essentially normal until the 12th week of cholesterol feeding, when a gradual rise was observed; ALT and AST levels at euthanasia in these dietary groups were elevated approximately twofold (the borderline upper limit of normal18 ) over precholesterol diet values. ALP levels were similar among dietary groups throughout the duration of cholesterol feeding. Three animals in the 0.25% dietary cholesterol group demonstrated a severe loss of appetite and died during the final 8 weeks of the study. Gross examinations of major organs revealed hepatomegaly with evidence of bile stasis. The TPC level in these three animals at the time of death was 3257±266 mg/dL, and cholesterol data from these rabbits were not included in the final analysis.

TPC and Arterial Wall Cholesterol Exposure
After the 1-week prescreen, during which all rabbits were fed 0.25% cholesterol, animals were grouped such that TPC values were similar (306±38, 310±38, 310±34, 302±31, and 304±20 mg/dL [0.05%, 0.10%, 0.15%, 0.20%, and 0.25% cholesterol, respectively]). After randomizing the rabbits to the various dietary regimens, TPC in animals fed 0.05% to 0.15% dietary cholesterol gradually decreased over the first 6 to 8 weeks of feeding and then stabilized at a quantity below the starting value (Fig 1Down). In animals fed <=0.15% dietary cholesterol, no significant differences were noted in plasma cholesterol among groups at any time. In animals receiving >0.15% dietary cholesterol, TPC rose steadily throughout the study (Fig 1Down). Statistical differences in plasma cholesterol in rabbits fed 0.20% and 0.25% dietary cholesterol relative to the 0.05% cholesterol group were apparent by the first and second weeks of randomized feeding (P=.02 and .002, respectively) and remained significantly elevated throughout the experiment (Fig 1Down). At euthanasia, plasma cholesterol levels were 70±7, 109±15, 186±30, 1457±347, and 1706±161 mg/mL for the 0.05%, 0.10%, 0.15%, 0.20%, and 0.25% dietary cholesterol groups, respectively.



View larger version (26K):
[in this window]
[in a new window]
  		Figure 1. Line graph shows time-related changes in TPC in animals fed varying amounts of low-level dietary cholesterol (0.05% to 0.25%) for 31 to 32 weeks. Each data point represents the average TPC from the individual dietary groups. Note the significant increase in TPC in the 0.20% and 0.25% dietary cholesterol groups relative to animals fed <=0.15% cholesterol.

The exposure of the arterial wall to cholesterol over the duration of the experiment (cumulative cholesterol-week values) in individual rabbits was estimated from the area under the curve of the TPC versus duration of cholesterol feeding. The cumulative cholesterol-week values in animals receiving 0.05% to 0.15% dietary cholesterol did not differ statistically, suggesting that the degree of cholesterol exposure between these three groups was similar. Cumulative cholesterol-week values in rabbits receiving 0.20% and 0.25% dietary cholesterol were also not statistically different from each other. Increasing dietary cholesterol from 0.05% to 0.15% resulted in a less than twofold stepwise increase in TPC exposure, whereas the cholesterol-week values in animals receiving 0.20% and 0.25% cholesterol were significantly higher (four- to fivefold increase; P<.008) relative to those values in the 0.05% to 0.15% cholesterol groups.

Sudanophilic Area and Distribution
The degree of aortic sudanophilia increased with escalating levels of dietary cholesterol (Fig 2Down). Raising dietary cholesterol from 0.05% to 0.15% resulted in a modest increase in aortic sudanophilia (1.7±0.6% to 13.9±4.5%), whereas lesion area increased exponentially with cholesterol intakes >0.15% (lesion area was 47.7±10.4% and 64±4.9% in the 0.20% and 0.25% cholesterol groups, respectively).



View larger version (23K):
[in this window]
[in a new window]
  		Figure 2. Bar graph shows effects of varying amounts of low-level dietary cholesterol on the percentage of total aortic intimal surface positive for Sudan IV; TPC exposure as reported in cholesterol weeks (see "Methods" for definition) is shown in parentheses. Values are mean±SEM. *P<.03, **P<.0001 vs 0.05% to 0.15% dietary cholesterol groups.

Tissue Biochemistry
Tissue Cholesterol
Total tissue cholesterol accumulated progressively with increasing dietary cholesterol, with maximal deposition in the aortic arch region relative to the thoracic aorta. Esterified cholesterol was present only in rabbits fed >=0.15% dietary cholesterol (Fig 3Down). In all groups, the free (unesterified) cholesterol/CE ratio was never <1 except in animals receiving 0.25% dietary cholesterol, demonstrating that there was generally more free cholesterol than CE.



View larger version (21K):
[in this window]
[in a new window]
  		Figure 3. Bar graph shows lipid composition of the aortic arch region in animals fed varying amounts of low-level dietary cholesterol. Values are mean±SEM and are expressed as cholesterol content in milligrams per gram wet weight. *P<.05, **P<.05 vs 0.05% to 0.15% dietary cholesterol groups.

ACAT Activity
Increasing dietary cholesterol from 0.05% to 0.15% resulted in modest increases in aortic ACAT activity (1.7±0.3 to 10.3±4.5 pmol·min-1·mg protein-1, respectively). In animals fed >0.15% cholesterol, ACAT activity increased markedly (63.6±11.9 and 73.7±14 pmol·min-1·mg protein-1 in the 0.20% and 0.25% dietary groups, respectively).

In the intestine, increasing dietary cholesterol from 0.05% to 0.25% progressively increased ACAT activity (109±9.4 to 288±50.4 pmol·min-1·protein-1, respectively). Liver ACAT activity at euthanasia in rabbits fed 0.05% cholesterol was 65.5 pmol·min-1·protein-1. Levels peaked in animals fed 0.15% cholesterol (191.2±26.1 pmol·min-1·protein-1) and remained elevated with higher cholesterol intake (0.20% and 0.25%).

Correlation of TPC Exposure With Aortic Sudanophilia
The extent of Sudan IV staining in each rabbit aorta as a function of cumulative TPC exposure is presented in Fig 4Down. Linear regression analysis for the individual dietary groups showed a positive correlation between TPC exposure and sudanophilia in animals fed >=0.10% cholesterol. In addition, a threshold level of TPC exposure was apparent at {approx}5000 cholesterol weeks. Below this level lesions were minimal or absent, and above it lesions correlated with the degree of cholesterol exposure. The strongest correlation of Sudan IV staining with TPC exposure was observed in rabbits fed 0.15% cholesterol (R2=.88). Increasing the cholesterol content in the diet weakened this relationship (R2=.51 and .28 in the 0.20% and 0.25% dietary cholesterol groups, respectively).



View larger version (24K):
[in this window]
[in a new window]
  		Figure 4. Line plots show correlation of cholesterol weeks (see "Methods" for definition) with extent of aortic sudanophilia as measured by percent surface area. Regression lines are shown for all data points except for B, in which the point marked by the asterisk is an outlier and was excluded from regression analysis. Note the wide range in cholesterol exposure within the various dietary groups. C indicates dietary cholesterol.

Individual Variability Among Animals in Response to the Cholesterol Diets
The variation among individual rabbits in response to the different cholesterol diets is presented in Table 2Down. The mean percent difference in TPC exposure of two hypothetical experimental groups to satisfy statistical significance based on the observed means of the reported dietary groups was calculated. Given the individual variability in response to dietary cholesterol in the present study, a >50% difference in mean TPC exposure value is predicted for two hypothetical groups consisting of 10 animals each to become statistically significant. This required difference in mean cholesterol values is reduced to approximately 30% when the number of animals is increased to 26, as shown in the experimental group fed 0.25% cholesterol.


View this table:
[in this window]
[in a new window]
  		Table 2. Individual Variability in Response to Cholesterol Feeding

Plaque Distribution and Intimal Thickness
The probability distribution of sudanophilia for the ascending aorta to the celiac branch in the individual dietary groups is shown in Fig 5Down. All animals demonstrated the typical atherosclerotic lesion distribution and characteristics of the cholesterol-fed rabbit model. In rabbits fed 0.05% to 0.15% dietary cholesterol, the greatest probability (>=50%) of lesion formation was confined to the greater curvature of the aorta along the dorsal surface. With increasing dietary cholesterol (>0.15%), high probability regions were observed throughout the arch to the distal thoracic aorta.



View larger version (31K):
[in this window]
[in a new window]
  		Figure 5. Composite of probability-of-occurrence maps of sudanophilic area in the aorta (arch to celiac branch) of animals fed varying degrees of low-level cholesterol. All maps are displayed in banded incidence isopleths according to the scale shown at the bottom. Blood flow is from right to left. High probability regions for development of atherosclerosis are predominantly located in the ascending aorta in the 0.05% to 0.15% dietary cholesterol groups. Animals fed >0.15% dietary cholesterol demonstrate high-probability regions throughout the ascending and descending thoracic aorta.

The mean intimal/medial thickness ratios in the various aortic regions are shown in Table 3Down. In animals fed 0.05% to 0.15% cholesterol, intimal thickening as a result of cholesterol feeding was mostly negligible, ranging from zero to <=0.15±0.09. In animals fed >0.15% dietary cholesterol, the intimal/medial thickness ratio was markedly increased relative to those of the lower dietary cholesterol groups. In the 0.20% to 0.25% dietary cholesterol groups, intimal/medial thickness ratios ranged from 0.18±0.12 to 2.00±0.28. Intimal/medial thickness ratios were never >1 except for animals in the 0.25% dietary cholesterol group.


View this table:
[in this window]
[in a new window]
  		Table 3. Lesion Size in Various Regions of Interest in the Aortas of Rabbits Fed Increasing Amounts of Low-Level Cholesterol

Plaque Morphology
Aorta
Most of the aortic sections from animals fed >=0.1% cholesterol had microscopically detectable lesions and displayed various lesion morphologies (Fig 6Down). Atherosclerotic plaques in rabbits fed <0.15% cholesterol were generally fatty streaks, consisting predominantly of intimal macrophage-derived foam cells, and demonstrated few, if any, SMCs. Atherosclerotic plaques in rabbits fed >=0.15% cholesterol consisted of transitional and advanced lesions, with an increased frequency of advanced lesions in the higher dietary cholesterol groups (Table 4Down). Transitional plaques showed SMCs scattered among foamy macrophages, with evidence of extracellular matrix formation consisting of proteoglycans. In transitional lesions, macrophages were the predominant cell type, comprising {approx}50% of the plaque; SMC composition was {approx}30% (Table 5Down). Lesions identified as atheromatous plaques were characterized by an evolving lipid-rich necrotic core filled with cellular debris and cholesterol clefts covered by a well-developed fibrous cap consisting of SMCs, extracellular matrix, and focal foam-cell infiltrate. In atheromatous plaques, {approx}70% of the lesion consisted of acellular material (proteoglycans, collagen, and necrotic debris), while SMCs and macrophages were equally present. Intimal calcification was not observed in any of the lesions examined (Table 5Down).



View larger version (116K):
[in this window]
[in a new window]
  		Figure 6. Photomicrographs showing range of lesion morphologies in the ascending aorta from rabbits fed low levels of dietary cholesterol (0.15% to 0.25%). A-C, Aortic sections stained with Movat Pentachrome stain. D-I, Immunoperoxidase stains in which the corresponding tissue sections were reacted with either the antimacrophage monoclonal antibody Ram-11 (D-F) or monoclonal antibody HHF-35, directed against muscle cell–specific actin (G-I). A, Fatty streak lesion containing multiple layers of foam cells; B, transitional lesion showing SMCs scattered among macrophage-derived foam cells with interspersed proteoglycan matrix; and C, atheromatous plaque showing SMC-rich fibrous cap overlying a necrotic core filled with cellular debris and cholesterol clefts (arrows). D-I were counterstained with hematoxylin. Internal elastic lamina is identified in the bottom left of each panel (arrowhead) (bar=200 µm).


View this table:
[in this window]
[in a new window]
  		Table 4. Morphological Evaluation of Aortic Lesion Stage Among the Various Dietary Cholesterol Groups


View this table:
[in this window]
[in a new window]
  		Table 5. Plaque Composition in the Three Lesion Stages

Coronary Arteries
The frequency of epicardial and intramyocardial arteries demonstrating coronary atherosclerosis from rabbits among the various dietary groups is shown in Table 6Down. Coronary atherosclerosis was only observed in rabbits fed >=0.1% cholesterol and, when present, was localized to both the epicardial and intramyocardial arteries; however, it was more extensive in the latter. Epicardial coronary atherosclerosis in animals fed 0.10% cholesterol was rare (2 of 10 cases examined); however, the frequency of detectable epicardial lesions increased with escalating levels of dietary cholesterol, such that the majority of rabbits fed 0.25% dietary cholesterol demonstrated coronary lesions in at least one vessel. In contrast to aortic findings, atherosclerotic plaques in coronary arteries displayed two distinct histological profiles. Atherosclerotic lesions in epicardial coronary arteries consisted mostly of SMCs interspersed with a small number of macrophage-derived foam cells. Lesions in intramyocardial arteries consisted of macrophage-derived foam cells along with proteoglycans and occasional foci of necrosis.


View this table:
[in this window]
[in a new window]
  		Table 6. Frequency of Coronary Atherosclerosis in Rabbits Fed Varying Degrees of Low-Level Cholesterol


*    Discussion
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 up arrowResults
 *Discussion
down arrowReferences
 
The present study further characterized the effect of varying degrees of low-level cholesterol feeding on aortic and coronary atherosclerosis in the rabbit. A wide variation in cholesterol exposure among individual rabbits occurred in response to cholesterol feeding. Nevertheless, we have identified a threshold level at {approx}5000 cholesterol weeks, below which lesion development is minimal or absent and above which lesion area is associated with TPC exposure. Intake of >=0.15% dietary cholesterol was associated with marked elevations in aortic wall esterified cholesterol content, ACAT activity, topographic distribution, and intimal thickness relative to the groups fed lower amounts of cholesterol. In the aorta, lesion type was similarly affected by the degree of dietary cholesterol; rabbits fed >=0.15% cholesterol showed predominantly transitional lesions (advanced fatty streaks) and atheromatous fibrous plaques. This relationship, however, was not observed in coronary arteries, where lesion type was unchanged by increased cholesterol feeding. Taken together, these data suggest that a number of critical variables should be carefully considered when using the cholesterol-fed rabbit model to assess the efficacy of potential antiatherogenic drugs. Efforts to minimize the individual variability in response to dietary cholesterol by preselecting animals with similar responsiveness to cholesterol as well as an awareness of the type of lesion produced by varying degrees of cholesterol exposure should be accounted for in the experimental design.

Diet-Induced Atherosclerosis in the Rabbit
Diet-induced hypercholesterolemia in the rabbit is caused by an accumulation of exogenous cholesterol. Rabbits are sensitive to atherosclerosis induced by dietary cholesterol because they are unable to increase sterol excretion, resulting in enhanced liver export of cholesteryl ester–rich lipoproteins into the circulation.19 20 21 Under these conditions, the principal carriers of cholesterol in the circulation are LDL and ß-VLDL, the latter being the major cholesterol transport vehicle when TPC values reach approximately 700 to 800 mg/dL.22 23 Atherogenic lipoproteins are markedly increased; consequently, the number of lipoprotein receptors is reduced, prolonging the residence time of circulating lipids.24 In cholesterol-fed rabbits, lesion-prone areas selectively accumulate these atherogenic lipoproteins from the circulation, resulting in lesion development.

Cholesterol Exposure and Sudanophilia
In humans, the influence of plasma cholesterol on the degree of aortic and coronary artery atherosclerosis is well established.25 26 27 In the cholesterol-fed rabbit, earlier reports have demonstrated a general relationship of either TPC with aortic cholesterol content and/or aortic atherosclerosis scores, without statistical correlation.22 28 More recent studies suggest a statistically significant correlation of average plasma cholesterol with aortic cholesterol content and/or intimal lesion area.7 29 30 31 32 However, few studies have systematically correlated varying dietary regimens to the extent of lesion formation in the cholesterol-fed rabbit. In a study by Bocan et al,31 a significant correlation was observed in pooled data from rabbits fed increasing amounts of cholesterol/fat (0% to 2.0%) for 9 weeks. However, the validity of an analysis of "pooled" data is in question, since one would have to assume that similar degrees of cholesterol exposure would produce the same amount of sudanophilia independent of the amount of cholesterol in the diet.

In the present study, rabbits fed dietary cholesterol of 0.05% and 0.1% failed to show a good correlation of TPC exposure with atherosclerotic plaque, possibly due to the kinetics of disease progression or a necessity to achieve some arbitrary threshold for atherosclerosis to become evident; such a threshold was evident in our data at {approx}5000 cholesterol weeks. Similarly, cholesterol diets >0.15% may induce factors that may lower the predictive value of circulating cholesterol, such as the saturation of an atherogenic lipoprotein species, whereby levels above a certain threshold do not proportionally increase atherosclerosis.

Although cholesterol exposure can be controlled by modifying the amount of cholesterol in the diet, this technique alone has limited value due to the inherent variability in individual responsiveness to cholesterol feeding in the rabbit, as exemplified by our data. Using the observed mean values for TPC exposure among rabbits in the reported dietary groups, we predict that a hypothetical group would require a >50% mean difference in cholesterol exposure to become statistically different, assuming an equivalent number of 10 animals per group. One method to control for this variability intrinsic to the rabbit model is to increase the number of animals in each group. For instance, a hypothetical group consisting of 26 animals would require only an approximate >30% difference in mean TPC exposure for statistical significance, as shown by our study. Of course, maintenance of large numbers of animals minimizes the cost effectiveness of the rabbit model. A better approach to manage this variability more closely may require not only restricting cholesterol exposure but also carefully selecting rabbits with similar responsiveness to the diet prior to experimentation.

Lesion Morphology
It has become increasingly apparent that lesion morphology, at least in rabbits, may be selectively altered by titrating the level of dietary cholesterol and duration of feeding. Short-term feeding of dietary cholesterol in the rabbit (>=2%) yields high levels of circulating cholesterol, resulting in macrophage-derived foam-cell lesions.29 33 34 35 Conversely, reducing the level of dietary cholesterol has been reported to produce lesions containing large numbers of SMCs, cholesterol clefts, and necrotic debris, a model more representative of human atherosclerotic disease.7 8 9 10 11 More advanced complicated lesions that are characteristic of human atherosclerosis are also observed with intermittent, rather than continuous, feeding of an atherogenic diet.36

Few studies describe a range of lesions (fatty streaks to advanced fibrous plaques) with cholesterol feeding in the rabbit.16 37 38 Only in the study by Daley et al38 did animals develop aortic lesions demonstrating a progression of atherosclerosis from fatty streaks to atheromatous lesions. In their study, rabbits were fed low-level cholesterol (0.125% to 0.5% by weight) for 6 months. However, it is unclear in this investigation how cholesterol exposure influenced lesion type. Other studies have used casein diets supplemented with either 0.2% cholesterol16 or 19% butter fat37 and longer periods of feeding (6 to 60 months). Our study clearly showed the development of advanced atherosclerotic lesions with low-level (0.20% to 0.25%) cholesterol feeding for 8 months. Diets of 0.05% to 0.15% cholesterol produced mostly fatty streaks and transitional lesions. As in the aforementioned studies, lesion type in our rabbits was more precisely characterized by using monoclonal antibodies to identify the composition of macrophages and SMCs within plaques. In fatty streaks and transitional lesions, the percentage of macrophages versus SMCs was greater, whereas more advanced lesions showed a relative increase in acellular areas and SMC number.

Some studies suggest that the development of advanced lesions in the cholesterol-fed rabbit may depend on the age of the animal. An increased prevalence of aortic fibroatheromatous plaques has been described in aged (3 to 4.5 years) rabbits versus young (4-month-old) animals fed low-level cholesterol (0.1% or 2%) for 18 months.9 34 These findings are in contrast with the present study, in which atheromatous lesions were observed within 8 months of cholesterol feeding in young rabbits (4 to 8 months of age) with escalating levels of dietary cholesterol. Similarly, Daley et al38 found that >70% of plaques sampled along the entire aorta from young rabbits (aged 6 months) fed 0.125% to 0.5% dietary cholesterol for 6 months were advanced lesions.

Lesion Topography
Computerized planimetry was used to precisely assess the extent and topographic distribution of intimal surface involved by atherosclerotic plaque in animals fed varying degrees of low-level cholesterol. Lesion-prone areas were confined to the upper thoracic aorta only in animals fed <0.15% cholesterol. In rabbits fed >0.15% cholesterol, regions of highest probability were found throughout the entire length of the vessel, with frequent extension well into the abdominal aorta. In rabbits fed 0.125% to 0.5% cholesterol for 6 months, Daley et al39 report abdominal lesions covering 39% of intimal surface area, much greater than previously reported percentages of surface area involvement of abdominal aorta in cholesterol-fed rabbits.40 41 42 Nevertheless, the topography of aortic lesions in the rabbit is clearly influenced by the level and duration of circulating cholesterol; with sufficient cholesterol exposure, significant sudanophilic lesions can be observed at all anatomic locations of the aorta.

Esterified Cholesterol and Lesion Severity
Cholesterol esterification by cells of the arterial wall has been demonstrated in various species, including humans.43 44 CEs are localized within the intima and are found both extracellularly and intracellularly within monocyte/macrophages or smooth muscle–derived foam cells.45 46 In rabbits maintained on diets containing <0.15% cholesterol, no evidence of CE formation in the arterial wall was observed. However, the esterified cholesterol content of the lesions was evident at cholesterol diets of 0.15% and was substantially increased when animals were fed higher amounts of dietary cholesterol. CE deposition within the plaque may, at least in part, influence the morphological appearance of lesions. Studies of human plaques as well as experimental atherosclerosis models demonstrate increased lesion severity with a rise in arterial wall esterified cholesterol content.47 48 In addition to affecting plaque morphology, increased CE content may weaken the stability of the plaque (lipid in the form of CE softens plaque), thereby increasing the risk of rupture and thrombosis.49

An essential protein responsible for the esterification of cholesterol is the microsomal enzyme ACAT. Previous studies report low basal ACAT activity in normal arteries from various animal species and humans.50 51 However, ACAT activity is enhanced severalfold after experimental induction of atherosclerosis in animals and in human atherosclerotic tissue.50 The susceptibility of a species to atherosclerosis does not appear to correlate with increased ACAT activity; the rat, one of the species most resistant to induction of atherosclerosis, demonstrates high levels of microsomal cholesterol esterifying activity in the aorta relative to atherosclerosis-sensitive species such as the rabbit.52 Rather than producing an increased vulnerability to plaque development, induction of ACAT through the formation of CEs may strongly affect the conversion of a foam-cell lesion to a more advanced stage of atherosclerosis. In the present study induction of aortic wall ACAT activity with increased presence of CEs was associated with advanced atherosclerotic lesions.

Coronary Atherosclerosis
Hypercholesterolemia in the rabbit is associated with the development and progression of coronary atherosclerosis.1 5 53 Intramyocardial plaques are relatively common, and lesions consist primarily of macrophage-derived foam cells. However, unlike lesions in intramyocardial vessels, atherosclerotic plaques in the main epicardial coronary arteries in the cholesterol-fed rabbit have been reported as rare.1 5 However, Hunt and Duncan53 report extensive atherosclerosis in the epicardial coronary arteries of Dutch Belted rabbits fed a diet of 0.06% cholesterol, 14% hydrogenated coconut oil, and 20% casein. Similarly, the prevalence of epicardial coronary artery disease in our study was also significant; increasing levels of dietary cholesterol consistently produced epicardial lesions in virtually all animals fed >0.15% cholesterol. In our study, atherosclerotic lesions in epicardial vessels were rich in SMCs, unlike intramyocardial plaques, which consisted primarily of macrophage-derived foam cells. Interestingly, in contrast to the aorta, lesion type in coronary arteries was not affected by the level of cholesterol exposure. The mechanism of why foam-cell lesions in intramyocardial vessels are resistant to conversion to a more advanced stage of plaque development is unclear.

Conclusions
By selecting rabbits with similar responsiveness to cholesterol and adjusting cholesterol intake, the overall cholesterol exposure can be more closely controlled to produce various degrees and stages (early fatty streaks to fibrous plaques) of atherosclerotic lesions. Thus, we recommend that animals be screened to select those with similar responses to cholesterol. Careful consideration must also be given to the nature of the target lesion, because the efficacy of some treatments depends on the stage of plaque development.


*    Selected Abbreviations and Acronyms
 
ACAT = acyl coenzyme A:cholesterol O-acyltransferase
ALP = alkaline phosphatase
ALT = aspartate leucine transferase
ASP = aspartate serine transferase
CE = cholesterol ester
SMC = smooth muscle cell
TPC = total plasma cholesterol


*    Footnotes
 
The opinions and assertions contained herein are the private views of the authors and are not to be construed as official or as reflecting views of the US Army, Navy, Air Force, or Department of Defense.

Received September 5, 1995; revision received April 4, 1996;
*    References
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 up arrowResults
 up arrowDiscussion
 *References
 

  1. Clarkson TB. Atherosclerosis: spontaneous and induced. Adv Lipid Res. 1963;1:211-252.[Medline] [Order article via Infotrieve]
  2. Stehbens WE. An appraisal of cholesterol feeding in experimental atherogenesis. Prog Cardiovasc Dis. 1986;29:107-128.[Medline] [Order article via Infotrieve]
  3. Vesselinovitch D. Animal models and the study of atherosclerosis. Arch Pathol Lab Med. 1988;112:1011-1017.[Medline] [Order article via Infotrieve]
  4. Jokinen MP, Clarkson TB, Prichard RW. Animal models in atherosclerosis research. Exp Mol Pathol. 1985;42:1-28.[Medline] [Order article via Infotrieve]
  5. Wissler RW, Vesselinovitch D. Experimental models of human atherosclerosis. Ann N Y Acad Sci. 1968;149:907-922.[Medline] [Order article via Infotrieve]
  6. Prior JT, Kurtz DM, Ziegler DD. The hypercholesterolemic rabbit: an aid to understanding arteriosclerosis in man? Arch Pathol. 1961;71:672-684.[Medline] [Order article via Infotrieve]
  7. Adams CW, Miller NE, Morgan RS, Rao SN. Lipoprotein levels and tissue lipids in fatty-fibrous atherosclerosis induced in rabbits by two years' cholesterol feeding at a low level. Atherosclerosis. 1982;44:1-8.[Medline] [Order article via Infotrieve]
  8. Walker LN, Reidy MA, Bowyer DE. Morphology and cell kinetics of fatty streak lesion formation in the hypercholesterolemic rabbit. Am J Pathol. 1986;125:450-459.[Abstract]
  9. Spagnoli LG, Orlandi A, Mauriello A, Santeusanio G, de Angelis C, Lucreziotti R, Ramacci MT. Aging and atherosclerosis in the rabbit, I: distribution, prevalence and morphology of atherosclerotic lesions. Atherosclerosis. 1991;89:11-24.[Medline] [Order article via Infotrieve]
  10. Bocan TM, Mueller SB, Uhlendorf PD, Ferguson E, Newton RS. Dietary and mechanically induced rabbit iliac-femoral atherosclerotic lesions: a chemical and morphologic evaluation. Exp Mol Pathol. 1991;54:201-217.[Medline] [Order article via Infotrieve]
  11. Lupu F, Danaricu I, Simionescu N. Development of intracellular lipid deposits in the lipid-laden cells of atherosclerotic lesions: a cytochemical and ultrastructural study. Atherosclerosis. 1987;67:127-142.[Medline] [Order article via Infotrieve]
  12. Folch J, Lees M, Sloane Stanley GH. A simple method for the isolation and purification of total lipids from animal tissues. J Biol Chem. 1957;226:494-509.
  13. Largis EE, Wang CH, DeVries VG, Schaffer SA. CL 277,082: a novel inhibitor of ACAT-catalyzed cholesterol esterification and cholesterol absorption. J Lipid Res. 1989;30:681-690.[Abstract]
  14. Hashimoto S, Dayton S, Alfin-Slater RB, Bui PT, Baker N, Wilson L. Characteristics of the cholesterol-esterifying activity in normal and atherosclerotic rabbit aortas. Circ Res. 1974;34:176-183.[Abstract/Free Full Text]
  15. Cornhill JF, Herderick EE, Stary HC. Topography of human aortic sudanophilic lesions. Monogr Atheroscler. 1990;15:13-19.[Medline] [Order article via Infotrieve]
  16. Tsukada T, Rosenfeld M, Ross R, Gown AM. Immunocytochemical analysis of cellular components in atherosclerotic lesions: use of monoclonal antibodies with the Watanabe and fat-fed rabbit. Arteriosclerosis. 1986;6:601-613.[Abstract]
  17. Shanks ME, Gambill R. Calculus of the Elementary Functions. New York, NY: Holt, Rinehart and Winston, Inc; 1969:313.
  18. McLaughlin RM, Fish RE. Clinical biochemistry and hematology. In: Manning PJ, Ringler DH, Newcomer CE, eds. The Biology of the Laboratory Rabbit. 2nd ed. San Diego, Calif: Academic Press; 1994:111-127.
  19. Thompson KH, Zilversmit DB. Plasma very low density lipoprotein (VLDL) in cholesterol-fed rabbits: chylomicron remnants or liver lipoproteins? J Nutr. 1983;113:2002-2010.
  20. Mackinnon AM, Savage J, Gibson RA, Barter PJ. Secretion of cholesteryl ester-enriched very low density lipoproteins by the liver of cholesterol-fed rabbits. Atherosclerosis. 1985;54:145-155.[Medline] [Order article via Infotrieve]
  21. Kroon PA, Thompson GM, Chao Y. ß-very low density lipoproteins in cholesterol-fed rabbits are of hepatic origin. Atherosclerosis. 1985;56:323-329.[Medline] [Order article via Infotrieve]
  22. Brattsand R. Distribution of cholesterol and triglycerides among lipoprotein fractions in fat-fed rabbits at different levels of serum cholesterol. Atherosclerosis. 1976;23:97-110.[Medline] [Order article via Infotrieve]
  23. Schwenke DC, Carew TE. Initiation of atherosclerotic lesions in cholesterol-fed rabbits, I: focal increases in arterial LDL concentration precede development of fatty streak lesions. Arteriosclerosis. 1989;9:895-907.[Abstract/Free Full Text]
  24. Kovanen PT, Brown MS, Basu SK, Bilheimer DW, Goldstein JL. Saturation and suppression of hepatic lipoprotein receptors: a mechanism for the hypercholesterolemia of cholesterol-fed rabbits. Proc Natl Acad Sci U S A. 1981;78:1396-1400.[Abstract/Free Full Text]
  25. Pathobiological Determinants of Atherosclerosis in Youth (PDAY) Research Group. Relationship of atherosclerosis in young men to serum lipoprotein cholesterol concentrations and smoking: a preliminary report from the Pathobiological Determinants of Atherosclerosis in Youth (PDAY) research group. JAMA. 1990;264:3018-3024.[Abstract]
  26. Pathobiological Determinants of Atherosclerosis in Youth (PDAY) Research Group. Natural history of aortic and coronary atherosclerotic lesions in youth: findings from the PDAY study. Arterioscler Thromb. 1993;13:1291-1298.[Abstract/Free Full Text]
  27. Solberg LA, Strong JP. Risk factors and atherosclerotic lesions: a review of autopsy studies. Arteriosclerosis. 1983;3:187-198.[Abstract/Free Full Text]
  28. Brattsand R. The effect of niceritrol (pentaerythritoltetranicotinate) on experimental atherosclerosis in cholesterol-fed rabbits at different levels of plasma cholesterol. Acta Pharmacol Toxicol. 1975;36:39-55.[Medline] [Order article via Infotrieve]
  29. Ross AC, Minick CR, Zilversmit DB. Equal atherosclerosis in rabbits fed cholesterol-free, low-fat diet or cholesterol-supplemented diet. Atherosclerosis. 1978;29:301-315.[Medline] [Order article via Infotrieve]
  30. de la Llera-Moya M, Rothblat GH, Glick JM, England JM. Etoposide treatment suppresses atherosclerotic plaque development in cholesterol-fed rabbits. Arterioscler Thromb. 1992;12:1363-1370.[Abstract/Free Full Text]
  31. Bocan TM, Mueller SB, Mazur MJ, Uhlendorf PD, Brown EQ, Kieft KA. The relationship between the degree of dietary-induced hypercholesterolemia in the rabbit and atherosclerotic lesion formation. Atherosclerosis. 1993;102:9-22.[Medline] [Order article via Infotrieve]
  32. Adelman SSJ, Chandrasekaran A, Jayo J, St. Clair RW. Effect of 17{alpha}-dihydroequilin sulfate, a conjugated equine estrogen, and ethynylestradiol on atherosclerosis in cholesterol-fed rabbits. Arterioscler Thromb Vasc Biol. 1995;15:837-846.[Abstract/Free Full Text]
  33. McGill HC Jr. Atherosclerosis: problems in pathogenesis. Atheroscler Rev. 1977;2:27-59.
  34. Orlandi A, Mauriello A, deAngelis C, Ramacci MT, Spagnoli LG. Age-related differences in the distribution and occurrence of atherosclerotic aortic lesions in the hyperlipemic rabbit. Arch Gerontol Geriatr. 1992;16:295-302.
  35. Kritchevsky D, Tepper SA, Vesselinovitch D, Wissler RW. Cholesterol vehicle in experimental atherosclerosis, II: peanut oil. Atherosclerosis. 1971;14:53-64.[Medline] [Order article via Infotrieve]
  36. Constantinides P, Booth J, Carlson G. Production of advanced cholesterol atherosclerosis in the rabbit after cessation of cholesterol feeding. Arch Pathol. 1960;70:712-724.[Medline] [Order article via Infotrieve]
  37. Wilson RB, Miller RA, Middleton CC, Kinden D. Atherosclerosis in rabbits fed a low cholesterol diet for five years. Arteriosclerosis. 1982;2:228-241.[Abstract/Free Full Text]
  38. Daley SJ, Klemp KF, Guyton JR, Rogers KA. Cholesterol-fed and casein-fed rabbit models of atherosclerosis, II: differing morphological severity of atherogenesis despite matched plasma cholesterol levels. Arterioscler Thromb. 1994;14:105-114.[Abstract/Free Full Text]
  39. Daley SJ, Herderick EE, Cornhill JF, Rogers KA. Cholesterol-fed and casein-fed rabbit models of atherosclerosis, I: differing lesion area and volume despite equal plasma cholesterol levels. Arterioscler Thromb. 1994;14:95-104.[Abstract/Free Full Text]
  40. Mirhadi SA, Singh S, Gupta PP. Effect of garlic supplementation to cholesterol-rich diet on development of atherosclerosis in rabbits. Indian J Exp Biol. 1991;29:162-168.[Medline] [Order article via Infotrieve]
  41. Lacombe C, Corraze G, Nibbelink M. Increases in hyperlipoproteinemia, disturbances in cholesterol metabolism and atherosclerosis induced by dietary restriction in rabbits fed a cholesterol-rich diet. Lipids. 1983;18:306-312.[Medline] [Order article via Infotrieve]
  42. Restori G, Boiardi L, Barbagallo M, Novo S, Passeri M, Strano A. Progression of induced aortic atherosclerosis in rabbits fed a high cholesterol diet. Int Angiol. 1990;9:263-265.[Medline] [Order article via Infotrieve]
  43. Bottcher CJF. Chemical constituents of human atherosclerotic lesions. Proc R Soc Med. 1964;57:792-795.[Medline] [Order article via Infotrieve]
  44. Brecher PI, Chobanian AV. Cholesteryl ester synthesis in normal and atherosclerotic aortas of rabbits and rhesus monkeys. Circ Res. 1974;35:692-701.[Abstract/Free Full Text]
  45. Kruth HS. Filipin-positive, oil red O-negative particles in atherosclerotic lesions induced by cholesterol feeding. Lab Invest. 1984;50:87-93.[Medline] [Order article via Infotrieve]
  46. Simionescu N, Vasile E, Lupu F, Popescu G, Simionescu M. Prelesional events in atherogenesis: accumulation of extracellular cholesterol-rich liposomes in the arterial intima and cardiac valves of the hyperlipidemic rabbit. Am J Pathol. 1986;123:109-125.[Abstract]
  47. Scott RF, Florentin RA, Daoud AS, Morrison ES, Jones RM, Hutt MS. Coronary arteries of children and young adults: a comparison of lipids and anatomic features in New Yorkers and East Africans. Exp Mol Pathol. 1966;5:12-42.[Medline] [Order article via Infotrieve]
  48. Smith EB, Evans PH, Downham MD. Lipids in the aortic intima: the correlation of morphologic and chemical characteristics. J Atheroscler Res. 1967;7:171-186.[Medline] [Order article via Infotrieve]
  49. Loree HM, Tobias BJ, Gibson LJ, Kamm RD, Small DM, Lee RT. Mechanical properties of model atherosclerotic lesion lipid pools. Arterioscler Thromb. 1994;14:230-234.[Abstract/Free Full Text]
  50. Suckling KE, Strange EF. Role of acyl-CoA:cholesterol acyltransferase in cellular cholesterol metabolism. J Lipid Res. 1985;26:647-671.[Medline] [Order article via Infotrieve]
  51. Proudlock JW, Day AJ. Cholesterol esterifying enzymes of atherosclerotic rabbit intima. Biochim Biophys Acta. 1972;260:716-723.[Medline] [Order article via Infotrieve]
  52. Morisaki N, Matsuoka N, Shiral K, Sato Y, Kumagai A. Studies on acyl-CoA synthetase in rat arterial wall. Atherosclerosis. 1980;37:439-447.[Medline] [Order article via Infotrieve]
  53. Hunt CE, Duncan LA. Hyperlipoproteinaemia and atherosclerosis in rabbits fed low-level cholesterol and lecithin. Br J Exp Pathol. 1985;66:35-46.[Medline] [Order article via Infotrieve]



This article has been cited by other articles:


Home page
 J. Nutr.Home page
N. Ota, S. Soga, T. Hase, I. Tokimitsu, and T. Murase
Dietary Diacylglycerol Induces the Regression of Atherosclerosis in Rabbits
J. Nutr., May 1, 2007; 137(5): 1194 - 1199.
[Abstract] [Full Text] [PDF]

Home page
 J. Lipid Res.Home page
A. Ritsch, I. Tancevski, W. Schgoer, C. Pfeifhofer, R. Gander, P. Eller, B. Foeger, U. Stanzl, and J. R. Patsch
Molecular characterization of rabbit scavenger receptor class B types I and II: portal to central vein gradient of expression in the liver
J. Lipid Res., February 1, 2004; 45(2): 214 - 222.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
C. Castro, A. Diez-Juan, M. J. Cortes, and V. Andres
Distinct Regulation of Mitogen-activated Protein Kinases and p27Kip1 in Smooth Muscle Cells from Different Vascular Beds. A POTENTIAL ROLE IN ESTABLISHING REGIONAL PHENOTYPIC VARIANCE
J. Biol. Chem., February 7, 2003; 278(7): 4482 - 4490.
[Abstract] [Full Text] [PDF]

<