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. 1999;19:2179-2188

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
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 Rong, J. X.
Right arrow Articles by Sevanian, A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Rong, J. X.
Right arrow Articles by Sevanian, A.
Related Collections
Right arrow Lipid and lipoprotein metabolism
Right arrow Oxidant stress
Right arrow Animal models of human disease
(Arteriosclerosis, Thrombosis, and Vascular Biology. 1999;19:2179-2188.)
© 1999 American Heart Association, Inc.

Atherosclerosis and Lipoproteins

Cholesterol Oxidation Products Induce Vascular Foam Cell Lesion Formation in Hypercholesterolemic New Zealand White Rabbits

James X. Rong; Lijiang Shen; Yi H. Chang; Arnis Richters; Howard N. Hodis; Alex Sevanian

From the Department of Pathology, School of Medicine (J.X.R., A.R., A.S.); the Department of Molecular Pharmacology and Toxicology, School of Pharmacy (L.S., Y.H.C., H.N.H., A.S.); the Division of Cardiology, School of Medicine (H.N.H.); and the Atherosclerosis Research Unit, School of Medicine (H.N.H., A.S.), University of Southern California, Los Angeles, Calif.

Correspondence to Alex Sevanian, Department of Molecular Pharmacology and Toxicology, USC School of Pharmacy, 1985 Zonal Ave, Los Angeles, CA 90033. E-mail asevan{at}hsc.usc.edu


*    Abstract
up arrowTop
 *Abstract
down arrowIntroduction
 down arrowMethods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Abstract—Circulating cholesterol oxidation products (ChOx) have long been implicated in the etiology of early atherosclerosis; however, direct in vivo evidence elucidating their role in atherogenesis is only recently becoming available. This study investigated ChOx effects on vascular lesion formation in New Zealand White rabbits under controlled hypercholesterolemic conditions. By closely monitoring plasma cholesterol levels and adjusting dietary cholesterol intake during a 78-day period, total plasma cholesterol exposures (cumulative plasma cholesterol levels over time) were controlled between 27 000 and 34 000 mg/dLxday (final plasma cholesterol concentration, 467±77 mg/mL), representing a threshold range for sudanophilic lesion formation in the aorta. Twenty injections of a ChOx mixture (70 mg per injection) were made bearing an oxysterol composition similar to that found in circulating oxidatively modified low density lipoprotein. At sacrifice, the ChOx-injected rabbits (n=5) had (1) significantly higher plasma ChOx levels, (2) significantly increased cholesterol content in the aortas, mainly as esterified cholesterol, and (3) significantly greater sudanophilic lesion size and frequency in the aortas compared with vehicle-injected control rabbits (n=5). The aortic cholesterol content and extent of sudanophilic lesion area were correlated significantly with total plasma ChOx exposure (P<0.003 and P<0.0001, respectively) but not with total cholesterol exposure. The results indicate that for moderate experimental hypercholesterolemia, a situation more relevant to physiological hypercholesterolemia in humans, circulating ChOx may play an important role in inducing formation of early atherosclerotic lesions. Because ChOx are often present in cholesterol-containing diets, foam cell lesion formation induced by ChOx rather than cholesterol cannot be overlooked.


Key Words: oxysterols • cholesterol feeding • foam cell lesions • hypercholesterolemia


*    Introduction
up arrowTop
 up arrowAbstract
 *Introduction
down arrowMethods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
There is growing evidence that cholesterol oxidation products (ChOx) contribute to the development of atherosclerosis.1 Studies specifically addressing this issue have shown that ChOx possess several characteristics that may promote atherosclerosis. These include cytotoxic/apoptotic potential to vascular cells such as fibroblasts,2 endothelial cells,3 4 5 and smooth muscle cells6 7 8 ; impairment of vascular endothelial barrier function9 10 ; inhibition of endothelial NO release11 and arterial relaxation12 ; inhibition of cholesterol synthesis or utilization13 14 ; perturbation of intracellular cholesterol trafficking15 16 17 ; and activation of acyl CoA:cholesterol acyltransferase (ACAT) activity.18 19 ChOx are abundant in oxidatively modified LDL (ox-LDL),20 and high levels of circulating ChOx are found in rabbits fed a cholesterol-containing diet.21 22 However, direct evidence linking circulating ChOx to early vascular lesion formation is only recently becoming available.23 24

Recently, we described the pharmacokinetic properties of ChOx in New Zealand White (NZW) rabbits25 by using a ChOx mixture with a composition similar to that found in hypercholesterolemic rabbit plasma21 and in circulating ox-LDL from hypercholesterolemic primates and humans.20 26 Our findings showed that consecutive intravenous injections caused a progressive increase in circulating ChOx levels in normocholesterolemic rabbits, increased vascular permeability, and increased cholesterol accumulation in the aorta. However, atherosclerotic/foam cell lesions were not observed, because plasma cholesterol levels remained in the normal range.

Diet-induced hypercholesterolemia in rabbits has been a widely used model system for studying the development of human atherosclerosis.27 To achieve rapid lesion development, exceedingly high plasma cholesterol levels (>1500 mg/dL)28 29 have been produced by feeding high levels of cholesterol (>=0.5% of diet) or by other dietary methods. As a consequence, the lesions that are usually produced are topographically and morphologically dissimilar to those seen in humans.30 This dissimilarity is due in part to the fact that humans usually do not ingest such large quantities of cholesterol; do not, in general, have plasma cholesterol levels exceeding 800 mg/dL31 ; and process and tolerate cholesterol intake better than do rabbits. However, recent studies employing low levels of dietary cholesterol (<0.5% by diet weight) produced moderate hypercholesterolemia (plasma cholesterol levels ranging from 200 to 800 mg/dL)32 33 34 with gradual lesion formation that more closely resembles early atherosclerosis in humans.33 35 36 Moreover, a number of studies have attempted to define the relationship between atherosclerotic lesion formation and diet-induced hypercholesterolemia35 37 38 39 in NZW rabbits fed low levels of cholesterol. A total plasma cholesterol exposure (cumulative plasma cholesterol levels over time) can be used to establish a threshold below which aortic lesions are minimal but above which the extent of aortic lesion formation is correlated positively with total plasma cholesterol exposure.37 39 On the basis of these studies, we examined the role of ChOx in the development of early atherosclerosis in NZW rabbits whose hypercholesterolemic status was controlled to approximate the threshold required for foam cell lesion formation. Our findings reveal a pivotal role of circulating ChOx in the development of early atherosclerosis under moderately hypercholesterolemic conditions that more closely approximate hypercholesterolemia in humans.


*    Methods
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 *Methods
down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Rabbit Feeding
Ten male NZW rabbits ({approx}2.5 kg) were acquired from a local breeder (Irish Farms, Norco, Calif) and maintained by the USC vivaria in accordance with the National Institutes of Health guidelines.40 After a 7-day quarantine period, all rabbits were fed a cholesterol-containing diet prepared by dissolving Sigma-grade (99%+) cholesterol (Sigma Chemical Co) in distilled ether and spraying the solution onto the standard rabbit chow (Teklad No. 8630). The food was allowed to air-dry at room temperature (22°C) and was then stored in airtight containers at 4°C for no more than 2 weeks before use. ChOx content in the cholesterol-supplemented chow was below the limits of detection; however, detection was also limited by the sample size used for analysis. The final cholesterol content added to the chow was 0.1%, 0.2%, and 0.3% by weight, representing 3 levels of cholesterol-containing diets used in this study. Any food remaining in the feeders was replaced with fresh chow every 2 days.

Rabbit plasma cholesterol levels were elevated to and controlled at {approx}500 mg/dL by initially feeding the 0.3% cholesterol diet for 2 to 4 weeks and then closely monitoring plasma cholesterol levels and adjusting dietary cholesterol intake by using 1 of the 3 diets throughout the remainder of the study. The rabbits were randomized according to their original plasma cholesterol levels and responsiveness to cholesterol feeding and were divided into 2 groups consisting of 5 rabbits each.

ChOx Injection
The ChOx mixture was stored in ethanol (23.3 mg/mL), and its composition is shown in Table 1Down, being identical to that described previously.25 The purity of the individual ChOx was checked before use and was found to be >99% during the course of these studies. Injections were initiated 3 weeks after cholesterol feeding (plasma cholesterol level, 450.6±80.5 mg/dL). Before each injection, the ChOx solution (3 mL) was transferred to a sterile 15-mL polypropylene centrifuge tube containing lecithin (140 mg, Avanti Polar Lipids), and the ethanol was evaporated under N2. Saline (0.9% NaCl, 4.5 mL) was added to the residue, the tube was capped under Ar, and the contents were sonicated in a cup-horn sonicator (Heat Systems, Inc) at 4°C until a stable emulsion was obtained (usually requiring 5 minutes). The emulsion was stable for at least 3 hours at room temperature. The emulsion was immediately transferred to a syringe and injected into the rabbit by using a 23-gauge butterfly catheter via the lateral ear vein at a flow rate of 3 mL/min. All procedures were performed under sterile conditions. As shown in Figure 1Down (dotted arrows), animals were injected every other day, involving 3 series of 6 or 7 injections with a rest period of 7 days between each series of injection. Each animal received a total of 20 injections, amounting to 1400 mg of ChOx over 78 days. Controls rabbits were injected with the lecithin vehicle alone. No animals died throughout the procedures. The average weight of the rabbits at the end of the experiments was 4.11±0.13 kg, with no differences between the ChOx-injected and control groups.


View this table:
[in this window]
[in a new window]
  		Table 1. Percentage Composition of the ChOx Mixture



View larger version (26K):
[in this window]
[in a new window]
  		Figure 1. Animal feeding, injection, and blood sampling procedures. The days of ChOx/lecithin injection (solid arrows) and blood sampling (dashed arrows) are shown along the time scale. Three series of 6 or 7 injections were made, and the starting days of each series are indicated by the longer dashed arrows. Blood sampling was performed immediately before injection on days when both procedures were performed. *Day 0 represents the start of cholesterol feeding.

Blood Sampling
Blood was drawn from the central ear artery of each nonfasted animal at baseline, before, and once a week after initiating the cholesterol-feeding protocol (Figure 1Up, solid arrows). Blood was collected before ChOx or lecithin injection on days when sampling and injection regimens coincided. Plasma was separated from blood (collected with EDTA) by centrifugation (3000 rpm, 4°C, 20 minutes). A 100-µL aliquot was taken for cholesterol determination by use of an automated enzymatic procedure (Cobas Mira automatic analyzer, Roche Diagnostic Inc) that utilizes a Centers for Disease Control and Prevention (Atlanta, Ga) standardized protocol,41 and the remainder was stored under N2 (-70°C) until further analysis. The cholesterol levels were confirmed by gas chromatography (GC).

Chylomicron Isolation
Chylomicrons were separated from plasma by the method of Karpe and Hamsten42 as modified by Mero et al.43 In brief, solid NaCl (705 mg) was added to plasma (5.0 mL) to increase its density to 1.10 kg/L. A total volume of 4.0 mL of the plasma was transferred to a polyallomer ultracentrifuge tube (Ultra-Clear, 14x89 mm, Beckman Instruments). A density gradient consisting of 3.0 mL each of 1.065, 1.020, and 1.006 kg/L NaCl solutions was then carefully layered on top of the plasma. Ultracentrifugation was performed in a Beckman L8-55 ultracentrifuge equipped with an SW-40 Ti swinging-bucket rotor (40 000 rpm, 15°C, 32 minutes) The Sf >400 fraction representing chylomicrons was isolated by aspirating the top fraction of 1.0 mL and was then processed for cholesterol/ChOx determination as described below.

Determination of Cholesterol and ChOx in Chylomicrons, Plasma, and Liver
Cholesterol and ChOx contents in chylomicrons, plasma, and liver were determined by GC as previously described.21 In brief, total lipids from chylomicrons, plasma, or minced liver tissue were extracted by a modified Bligh-Dyer procedure44 and applied to "Diol" solid-phase extraction columns (VWR Scientific). The cholesterol and ChOx were collected, hydrolyzed by cold alkaline saponification, derivatized to trimethylsilyl ethers in autoinjector vials, and analyzed with a Shimadzu GC-14 gas chromatograph fitted with a DB-1 capillary column (J&W Scientific Inc) equipped with a flame ionization detector. Quantitative analysis of the biological samples was performed by the internal standard method to identify and quantify cholesterol and individual ChOx. Total ChOx was expressed as the sum of individual ChOx. ChOx content in rabbit chow was determined in a similar manner.

Morphometric Examination of Lesions
At the end of the experiments, the rabbits were killed by an overdose of pentobarbital. The aorta was removed and dissected from the aortic valve to 0.4 cm distal to the iliac bifurcation, and the adventitia was removed. The aorta was then opened, cut in half longitudinally as shown in Figure 2Down, and divided into segments comprising the aortic arch (from the aortic valve to 0.7 cm distal to the left subclavian branch), thoracic aorta (from 0.7 cm distal to the left subclavian branch to above the celiac branch), and abdominal aorta (from above the celiac branch to 0.4 cm distal to the iliac bifurcation). The left half of the aorta was pinned flat, fixed in 10% neutral buffered formalin (37°C, 16 hours), rinsed with water, stained with Sudan IV solution (optical density520 nm=0.22±0.01) in 38% isopropanol (vol/vol, in water) for 24 hours at room temperature, and rinsed with running water for 1 hour. The right half was stored under N2 (-70°C) for determination of cholesterol content. The stained aorta was placed under four 150-W reflector floodlight lamps (General Electric Co) and photographed with a Pentax K-1000 camera (Pentax Corp) on Kodak Ektachrome 160 film (Eastman Kodak Co). The photographic slides were then mounted on a microscope (Carl Zeiss, Inc) fitted with a 1x objective and equipped with an integrated Sony DXC-760 MD camera (Sony Corp) for image projection. The detected image was analyzed at a magnification of 50x (monitor micrometer image). The sudanophilic lesions were identified and quantified as a percentage of aortic segment surface area by using a Leica Quantimet-570 image analyzer (Leica, Inc). The aortic tissues were further processed for standard hematoxylin-eosin (H&E) staining.



View larger version (21K):
[in this window]
[in a new window]
  		Figure 2. Diagram (not drawn to scale) showing bisection of the aorta, division of aortic segments for image analysis, and position of aortic tissue sampling for cholesterol analysis. 1 indicates the aorta; 2, brachiocephalic trunk; 3, common carotid artery (which sometimes branches from the brachiocephalic trunk); 4, left subclavian artery; 5, dorsal intercostal arteries; 6, celiac artery; 7, cranial mesenteric artery; 8, right and left renal arteries; 9, caudal mesenteric artery; and 10, iliac arteries.

Determination of Total Cholesterol (TC), Cholesteryl Esters (CEs), and Free Cholesterol (FC)
As indicated in Figure 2Up, the entire aortic arch, upper half of the thoracic aorta, and upper abdominal aorta (between the celiac and right renal branch) from the right half of the rabbit aorta were weighed and minced in a tissue homogenizer (Tekmar Instruments). Total lipids were extracted by a modified Bligh-Dyer procedure21 and evaporated to dryness under N2. TC was determined as described previously.21 Alternatively, CEs and FC were separated using the method of Kaluzny et al45 with some modifications. In brief, total lipids were redissolved in chloroform (0.5 mL), applied to an aminopropyl column (VWR Scientific) preconditioned by washing with hexane (4 mL), and allowed to dry under vacuum aspiration. The column was then eluted with chloroform/isopropanol (2:1 vol/vol, 4 mL total volume). The eluate (neutral lipids) was collected, reconstituted in hexane (0.2 mL), and applied to a new aminopropyl column prepared as above. Hexane (4 mL) was applied and the eluate (mainly unoxidized CEs) collected. Another new aminopropyl column was then attached in a piggyback fashion to the existing column, and hexane (6 mL) containing 1% diethyl ether and 10% methylene chloride was eluted through both columns. The eluate (triglycerides and oxidized CEs) was collected and pooled with the previous CE eluate. The columns were then eluted with methanol (8 mL) and the eluate (FC) collected. The CE and FC fractions were then evaporated to dryness and subjected to cold alkaline saponification for GC analysis (described above). TC is expressed as the sum of CEs and FC.

Statistics
Statistical analysis was performed using Origin 5.0 (Microcal Software, Inc). Quantitative results were reported as mean+SEM. Statistical evaluations for differences between paired observations were made with a 2-sample t test. The total plasma cholesterol/ChOx exposure over the course of the study was determined by applying a trapezoidal approximation to the cumulative plasma cholesterol/ChOx level versus time curve. The relationship between the percent sudanophilic lesion area and the total plasma cholesterol/ChOx exposure was determined by linear correlation analysis.


*    Results
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 *Results
down arrowDiscussion
 down arrowReferences
 
Control of Plasma Cholesterol Level and Total Plasma Cholesterol Exposure
As shown in Figure 3ADown, there was no difference between the cholesterol levels for ChOx-injected rabbits versus controls. Total plasma cholesterol exposure is defined as the area under the cumulative plasma cholesterol levels versus time curve. Figure 3BDown shows that there was no difference in total plasma cholesterol exposure between the 2 groups (P=0.69).



View larger version (28K):
[in this window]
[in a new window]
  		Figure 3. Rabbit plasma cholesterol levels (A) and total plasma cholesterol exposure (B) during the experiments. In both ChOx-injected rabbits (solid circle, n=5) and control rabbits (open circle, n=5), plasma cholesterol levels were elevated to {approx}500 mg/dL by feeding of the 0.3% cholesterol diet for 2 to 4 weeks and were maintained at this level by adjusting dietary cholesterol intake with 0% to 0.3% cholesterol–containing chow. Values are expressed as mean+SEM in A and B.

Plasma Total ChOx Levels and Total Plasma ChOx Exposure
As shown in Figure 4ADown, total ChOx levels increased in both groups due to cholesterol feeding. However, ChOx levels in the controls reached a plateau (21.9±1.4 mg/dL) as plasma cholesterol concentrations leveled off at 500 mg/dL, whereas ChOx levels in ChOx-injected rabbits increased continuously, reaching a maximum of 49.7±7.4 mg/dL. As shown in Figure 4BDown, the total plasma ChOx exposure was 1.8-fold greater in the ChOx-injected rabbits than in the controls (P<0.0002).



View larger version (28K):
[in this window]
[in a new window]
  		Figure 4. Rabbit plasma total ChOx levels (A) and total plasma ChOx exposure (B) during the course of cholesterol feeding and ChOx injection. The data showing SEs represent the means of 5 animals, whereas those not showing SEs (between days 0 and 20) were means of 2 animals. The arrow indicates the start of ChOx/lecithin injection (on day 21 of feeding). Values are expressed as mean+SEM in A and B. *P<0.0002 vs controls.

Contribution of Chylomicrons to Plasma Cholesterol/ChOx Exposure
As shown in Figure 5Down, at the end of the injection regimen and just before sacrifice, the chylomicron fraction contained slightly greater proportions of total plasma cholesterol in the controls than in the ChOx-injected rabbits. The proportion of ChOx in the chylomicron fraction was {approx}2-fold greater when expressed as a percentage of the plasma ChOx in the controls (78.3±10.9%) than in the ChOx-injected rabbits (40.2±6.3%, P<0.04 versus control), suggesting different origins for the plasma ChOx between the controls and the ChOx-injected rabbits. This finding also indicates that the diet served as a source of ChOx for both groups, but that a substantial portion of the circulating ChOx in the ChOx-injected group was, understandably, of nondietary origin.



View larger version (29K):
[in this window]
[in a new window]
  		Figure 5. Proportion of plasma cholesterol/ChOx in the chylomicron fraction. Results are presented as % plasma cholesterol/ChOx and expressed as mean+SEM, n=3 in each group. *P<0.04 vs controls.

ChOx Content in the Liver
The liver ChOx content at the conclusion of the experiments is presented in Figure 6Down. Livers from the ChOx-injected rabbits contained significantly greater (P<0.02) ChOx levels than did livers from pure cholesterol–injected rabbits, indicating possible involvement of the liver in eliminating or accumulating injected ChOx.



View larger version (31K):
[in this window]
[in a new window]
  		Figure 6. Total ChOx content in NZW rabbit livers at sacrifice. Results are presented as µg/g liver (wet weight) and are expressed as mean+SEM, n=5 in each group. *P<0.02 vs controls.

Sudanophilic Lesions in Rabbit Aortas
At sacrifice, sudanophilic lesions were present proximal to the brachiocephalic, common carotid, and left subclavian branches in the aortic arch, as well as the celiac, cranial mesenteric, and renal branches in the abdominal aorta in both the ChOx-injected rabbits and controls. Little or no lesions were found in the thoracic and lower abdominal aortas. Microscopically, H&E staining (Figure 7Down) indicated that the prominent lesions consisted of foam cells, degenerative smooth muscle, and clear spaces, which were mostly lipid-filled, as confirmed by oil red O staining (data not shown). To determine the extent of the lesions morphometrically, lesion surface percent was used because aortic thickening was only minimally present and volumetric quantification could be subject to a number of errors. Owing to the site-prone distribution of the lesions, the aorta was divided into aortic arch, thoracic aorta, and abdominal aorta segments, and lesion areas were quantified as described in Methods. Comparisons of percent lesion area in aortic segments from both groups of rabbits are shown in Figure 8Down. In the aortic arch, the ChOx-injected rabbits had a 2-fold greater lesion area coverage than did controls (P<0.02). This result accounted for the significantly greater percent lesion area for the whole aorta from the ChOx-injected rabbits (P<0.002 versus the controls), since no significant differences were observed in the thoracic (P=0.15) and abdominal (P=0.51) aortic segments.



View larger version (121K):
[in this window]
[in a new window]
  		Figure 7. H&E staining of prominent sudanophilic lesion from a ChOx-injected rabbit aorta. Note the presence of foam cells (*), clear spaces (arrowheads), and degenerative smooth muscle (arrow) in the thickened intima. L indicates lumen; I, intima; and M, media. Magnification x40.



View larger version (29K):
[in this window]
[in a new window]
  		Figure 8. Percentage area coverage of the aortic wall by sudanophilic lesion on the left half of rabbit aortas at sacrifice. Rabbit aortas were divided into aortic arch, thoracic aorta, and abdominal aortic segments, and lesion areas were quantified as described in Methods. Values are expressed as mean+SEM. *P<0.02, **P<0.002 vs the same aortic segment in controls.

The extent of sudanophilic lesions in rabbit aortas as a function of total plasma cholesterol/ChOx exposure is presented in Figure 9Down. There was a poor correlation (R=0.28, P<0.44) between lesion area and total plasma cholesterol exposure (Figure 9ADown). In contrast, a strong correlation (R=0.93, P<0.0001) was found between lesion area and total plasma ChOx exposure (Figure 9BDown).



View larger version (17K):
[in this window]
[in a new window]
  		Figure 9. Plots showing the correlation between the extent of aortic sudanophilic lesions by percentage surface area coverage as a function of plasma cholesterol exposure (A) and ChOx exposure (B). Regression lines are shown for all plotted data points, which include pooled data from ChOx-injected (n=5) and lecithin-injected (control) (n=5) rabbits.

TC, CE, and FC Contents in Aortic Segments
The comparison of TC, CE, and FC contents in aortic segments from both groups of animals is shown in Table 2Down (CE and FC were not determined in the aortic arch owing to the limited availability of tissue.) In ChOx-injected rabbits, the aortic arch contained the greatest levels of TC, followed by the upper abdominal and thoracic aortic segments. Interestingly, in the lecithin-injected controls, cholesterol levels appeared to be greatest in the upper abdominal aorta among the analyzed segments. This is consistent with the finding that, in the controls, slightly higher percentages of sudanophilic lesion areas were present in the upper abdominal aorta than in the aortic arch (data not shown). ChOx-injected rabbit aortas contained significantly greater levels of TC (P<0.01, aortic arch; P<0.05, thoracic and upper abdominal aorta) than did control aortas. The CE content in upper abdominal and thoracic aortic segments in the ChOx-injected rabbits was greater (P<0.07 in abdominal aorta, P<0.05 in thoracic aorta.) than in controls, whereas no differences in FC were observed. This indicated that the increased cholesterol content in the ChOx-injected rabbit aorta was mainly associated with esterified cholesterol.


View this table:
[in this window]
[in a new window]
  		Table 2. TC, CE, FC Contents in Aortic Segments

The TC content in the rabbit aortic arch as a function of total plasma cholesterol/ChOx exposure is presented in Figure 10Down. There was a poor correlation (R=0.30, P<0.40) between tissue TC content and total plasma cholesterol exposure (Figure 10ADown). In contrast, a strong correlation (R=0.83, P<0.003) was observed between the aortic tissue TC content and ChOx exposure (Figure 10BDown).



View larger version (19K):
[in this window]
[in a new window]
  		Figure 10. Plots showing the correlation between cholesterol content in the aortic arch and cholesterol (A) and ChOx (B) exposure. Regression lines are shown for all data points that were pooled from ChOx-injected (n=5) and lecithin-injected (n=5) rabbits.


*    Discussion
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 up arrowResults
 *Discussion
down arrowReferences
 
This study was based on previous findings25 that consecutive intravenous injections of a ChOx mixture with a composition similar to that found in circulating ox-LDL20 led to a progressive increase in circulating ChOx levels in normocholesterolemic NZW rabbits and also to an increase in vascular permeability. The vascular injury was accompanied by increased cholesterol accumulation in the aorta. In the present study, we administered the same mixture under similar conditions, except that the animals were fed a cholesterol-containing diet that produced hypercholesterolemia under controlled conditions. This led to a significant elevation of circulating ChOx levels. Although the relevance of the ChOx levels achieved in the present study to the ChOx levels under real physiological conditions is not clear, our results indicate that elevations in plasma ChOx levels lead to a significant increase in lesion area and cholesterol accumulation in the arterial wall under moderate hypercholesterolemic conditions.

Rabbits fed a low cholesterol–containing diet (<0.5% by weight) have been studied more frequently in recent years as an atherosclerotic animal model.33 35 36 In this model, plasma cholesterol levels comparable to those found in human hypercholesterolemia can be achieved.32 33 34 The early foam cell lesions more closely resemble the human fatty streak than lesions in animals fed a high-cholesterol diet (>=0.5% by weight), and the advanced lesions consist primarily of smooth muscle cells, lipid-laden foam cells, and a fibromuscular cap covering a core composed of extracellular lipid and necrotic debris,35 38 46 which is a hallmark of advanced atherosclerosis in humans. There have been a number of attempts to define the relationship between atherosclerotic lesion formation and diet-induced hypercholesterolemia35 37 38 39 in low cholesterol–fed rabbits. Bocan et al37 reported that plasma cholesterol levels >700 mg/dL, ie, a total plasma cholesterol exposure >31 868 mg/dL day, represented a threshold level necessary for consistent development of foam cell lesions. Similarly, Kolodgie et al39 reported a total plasma cholesterol exposure threshold of 5000 mg/dLxweek (35 000 mg/dLxday), below which lesions were minimal but above which the extent of sudanophilic lesions was correlated with total plasma cholesterol exposure. Consistent with these findings, our studies showed that a total plasma cholesterol exposure in the range of 27 000 to 34 000 mg/dLxday was sufficient to produce a small number of early sudanophilic lesions. When rabbits were fed an adjusted diet containing 0.1% to 0.3% cholesterol, final plasma cholesterol levels and total plasma exposure were controlled at 470±80 mg/dL and 30 600±900 mg/dLxday, respectively (Figure 3AUp and 3BUp). Under these circumstances, a poor correlation (r=0.28, P<0.44) between total plasma cholesterol exposure and percentage of sudanophilic lesion area was observed (Figure 9AUp). This is consistent with the findings by Kolodgie et al,39 who classified cholesterol exposure according to different dietary cholesterol levels and reported that under 35 000 mg/dLxday, no correlation could be found, although others37 38 have reported that foam cell lesions in rabbit aortas are correlated with total plasma cholesterol exposure over a wide range.

Total plasma ChOx exposure has not been taken into consideration in previous cholesterol feeding studies, although it has been shown that cholesterol feeding leads to a substantial increase in circulating ChOx levels.21 22 24 47 This increase may result from direct intake of ChOx in cholesterol-supplemented diets21 22 24 and/or from endogenous ChOx production associated with hypercholesterolemia.47 In the present study, plasma total ChOx levels in both groups increased with cholesterol feeding. Although the ChOx levels in the diet were below the limits of detection, large intakes of dietary cholesterol with minimal oxidation could lead to substantial ChOx accumulation in the gut. Though with controversy,48 rapid49 and continuous50 absorption of ChOx has been reported. In addition, cholesterol in the gastrointestinal tract could also be oxidized like other lipids.51 Alternatively, the increase can also be due to postabsorption cholesterol oxidation. The liver has been implicated in this process by other investigators,13 52 and the liver ChOx content in these rabbits was substantially higher than that in normocholesterolemic rabbits.25 In addition, in situ cholesterol oxidation by vascular cells or local oxidants may also be possible. ChOx levels in the lecithin-injected control group reached a plateau when plasma cholesterol levels were controlled, whereas the ChOx levels in ChOx-injected rabbits continued to rise (Figure 4AUp). The significantly greater ChOx content in livers from ChOx-injected rabbits indicate the involvement of this organ in clearing injected ChOx. However, the liver may also contribute to the release of ChOx into the circulation. Lipid oxidation products have been reported to be processed and repackaged into VLDL by the liver ex vivo.53 This is consistent with our previous findings25 in which ChOx were administered to normocholesterolemic rabbits. Under these conditions, there was a rapid clearance of plasma ChOx, followed by a gradual increase after multiple ChOx injections, and the increased ChOx content in the circulation was mainly associated with the LDL-VLDL fraction. In the present study, after 7 weeks there was a marked increase in total plasma ChOx exposure in the ChOx-injected rabbits (Figure 4BUp). This increase is considered an important criterion for exposure to and toxicity resulting from treatments with many toxic agents, and the continuous exposure to elevated ChOx is postulated to cause injury to the vessel wall as well as perturbations in cholesterol metabolism.

Evidence demonstrating the effects of ChOx on vascular lesions in vivo varies considerably. Cook et al54 fed male NZW rabbits cholestane triol at 0.1% diet weight and observed sudanophilic lesions in the aorta after only 27 days. Matthias et al 55 failed to reproduce Cook's findings in rats but reported angiotoxicity resulting from cholestane triol ingestion. Jacobson et al56 found a significant increase in coronary luminal stenosis in pure cholesterol plus cholestane triol–fed White Carneau pigeons compared with pure cholesterol–fed controls, but no difference in aortic lipids was found. Imai et al57 reported grossly visible thickening in the major branches of rabbit pulmonary arteries after 3 consecutive intravenous injections of cholestane triol or 25-hydroxycholesterol. By increasing plasma cholesterol levels to >1100 mg/dL during a 12-week period, Staprans et al24 found that dietary ChOx accelerated the development of aortic atherosclerosis in cholesterol-fed rabbits. Similarly, Mahfouz et al23 reported that dietary ChOx caused more severe atherosclerotic lesions than did pure cholesterol at plasma cholesterol levels >1400 mg/dL over 11 weeks. By contrast, Higley et al58 found that cholesterol-fed NZW rabbits exhibited 6-fold more arterial lesions than did rabbits fed a cholesterol-free ChOx mixture. The discrepancy between these studies may be due to different responses to ChOx in different animal species and/or to different biological effects and potencies of the various ChOx mixtures versus individual ChOx.1 59 60

Based on these findings, a ChOx mixture resembling that found in hypercholesterolemic rabbits was used in the present study, and this mixture represented the ChOx found in plasma,21 atherosclerotic lesions,61 and in vivo circulating ox-LDL.20 Injections of the ChOx mixture led to significantly greater foam cell lesion formation in these moderately hypercholesterolemic animals. This is consistent with the findings by Mahfouz et al 23 and Staprans et al,24 who used very high plasma cholesterol levels. Although most previous studies reported that ChOx administration caused vascular lesions resulting from chronic or prolonged exposure,54 55 56 57 no attempt was made to define the relationship between the lesion and plasma ChOx levels or total plasma ChOx exposure. The present findings show that the increased ChOx level and exposure in ChOx-injected rabbits are associated with increased sudanophilic lesion formation (Figure 8Up). The relatively small total aortic coverage (<6%) indicates very early stage lesions. The positive and strong correlation between the sudanophilic lesion area and total plasma ChOx exposure (Figure 9BUp) verifies that when plasma cholesterol concentrations approximate the threshold levels required for fatty streak formation, ChOx levels are better determinants for the progression of lesion formation.

The effects of ChOx on lesion formation are further demonstrated by the TC contents in aortic tissues (Table 2Up). Due to hypercholesterolemia, aortic TC contents in both groups increased substantially; however, the aortic segments from ChOx-injected rabbits contained 1.3 to 1.7-fold more TC than in controls. This is supported by our previous findings25 that ChOx injection led to aortic cholesterol accumulation in normocholesterolemic rabbits. The positive and significant correlation between TC in the aortic arch and ChOx exposure (Figure 10BUp) indicates that ChOx play an important role in facilitating the accumulation of aortic cholesterol under mild to moderate hypercholesterolemic conditions.

The mechanisms by which ChOx facilitate vascular cholesterol accumulation remain to be elucidated. ChOx can induce endothelial dysfunction12 or endothelial injury.4 Previous studies have shown that certain ChOx and ChOx-enriched LDL decreased vascular endothelial barrier functions, leading to increased transendothelial macromolecule transfer,9 10 62 63 and that some ChOx strongly inhibit gap junction formation in fibroblasts.64 Alternatively, apoptosis induced by ChOx leads to increased endothelial cell turnover, which is associated with increased endothelial transcytosis or permeability.65 66 Our previous studies with rabbits25 demonstrated that ChOx injection led to increased vascular permeability. Therefore, circulating ChOx may increase arterial wall permeability and retention of particles such as LDL and VLDL, thus depositing more cholesterol and CEs into the vessel walls. This is consistent with the finding that the increased aortic cholesterol content in ChOx-injected rabbits was mostly in the form of CEs (Table 2Up). Another possible mechanism could involve increased uptake4 or retention15 67 of cholesterol by vascular cells associated with activation of ACAT,18 19 analogous to ACAT activation by hypercholesterolemia.36 39 68 Recent studies in our laboratory (A.S., personal communication) as well as in others17 have shown that ChOx inhibit cholesterol efflux, either by disrupting intracellular cholesterol trafficking16 or by inhibiting cholesterol transfer to HDL.15 In addition, ChOx have been shown to damage lysosomes, similar to the effects of ox-LDL and core aldehydes,69 which could inhibit CE hydrolases and other lysosomal enzymes and facilitate CE accumulation.70

Because ChOx are invariably present in cholesterol-containing diets, foam cell formation may be substantially determined by ChOx content rather than by cholesterol. Our findings indicate that whereas elevated cholesterol levels are required for the development of atherosclerotic foam cell lesions, the ChOx associated with hypercholesterolemia mediate lesion formation.


*    Acknowledgments
 
This work was supported by grant ES03466 from the National Institutes of Health, Bethesda, Md (to A.S.). We express our gratitude for the excellent technical assistance of Drs Russell P. Sherwin and Juliana Hwang and the generosity of Steraloids, Inc, for providing various cholesterol oxidation products at a substantially reduced cost.

Received August 11, 1998; accepted February 3, 1999.


*    References
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 up arrowResults
 up arrowDiscussion
 *References
 

  1. Smith LL. Review of progress in sterol oxidations: 1987–1995. Lipids. 1996;31:453–487.[Medline] [Order article via Infotrieve]
  2. Sevanian A, Peterson AR. The cytotoxic and mutagenic properties of cholesterol oxidation products. Food Chem Toxicol. 1986;24:1103–1110.[Medline] [Order article via Infotrieve]
  3. Sevanian A, Berliner J, Peterson H. Uptake, metabolism and cytotoxicity of isomeric cholesterol-5,6-epoxides in rabbit aortic endothelial cells. J Lipid Res. 1991;32:147–155.[Abstract]
  4. Sevanian A, Hodis HN, Hwang J, Peterson H. Involvement of cholesterol oxides in the atherogenic action of oxidized LDL. In: Bellomo G, Finardi G, Maggi E, Rice-Evans C, eds. Free Radicals, Lipoprotein Oxidation and Atherosclerosis. 1st ed. London, England: Eichelieu Press; 1995:139–161.
  5. Palladini G, Finardi G, Bellomo G. Disruption of actin microfilament organization by cholesterol oxides in 73/73 endothelial cells. Exp Cell Res. 1996;223:72–82.[Medline] [Order article via Infotrieve]
  6. Peng SK, Tham P, Taylor CB, Mikkelson B. Cytotoxicity of oxidation derivatives of cholesterol on cultured aortic smooth muscle cells and their effect on cholesterol biosynthesis. Am J Clin Nutr. 1979;32:1033–1042.[Abstract/Free Full Text]
  7. Hughes H, Mathews B, Lenz ML, Guyton JR. Cytotoxicity of oxidized LDL to porcine aortic smooth muscle cells is associated with the oxysterols 7-ketocholesterol and 7-hydroxycholesterol. Arterioscler Thromb. 1994;14:1177–1185.[Abstract/Free Full Text]
  8. Nishio E, Watanabe Y. Oxysterols induced apoptosis in cultured smooth muscle cells through CPP32 protease activation and bcl-2 protein downregulation. Biochem Biophys Res Commun. 1996;226:928–934.[Medline] [Order article via Infotrieve]
  9. Boissonneault GA, Hennig B, Wang Y, Ouyang CM, Krahulik K, Cunnup L, Oeltgen PR. Effect of oxysterol-enriched low-density lipoprotein on endothelial barrier function in culture: low-density lipoproteins. Ann Nutr Metab. 1991;35:226–232.[Medline] [Order article via Infotrieve]
  10. Boissonneault GA, Hennig B, Ouyang CM. Oxysterols, cholesterol biosynthesis, and vascular endothelial cell monolayer barrier function. Proc Soc Exp Biol Med. 1991;196:338–343.[Abstract]
  11. Deckert V, Brunet A, Lantoine F, Lizard G, Brussel EM, Monier S, Lagrost L, David-Dufilho M, Gamber P, Devynck M-A. Inhibition by cholesterol oxides of NO release from human vascular endothelial cells. Arterioscler Thromb Vasc Biol. 1998;18:1054–1060.[Abstract/Free Full Text]
  12. Deckert V, Persegol L, Viens L, Lizard G, Athias A, Lallemant C, Gambert P, Lagrost L. Inhibitors of arterial relaxation among components of human oxidized low-density lipoproteins: cholesterol derivatives oxidized in position 7 are potent inhibitors of endothelium-dependent relaxation. Circulation. 1997;95:723–731.[Abstract/Free Full Text]
  13. Tamasawa N, Hayakari M, Murakami H, Matsui J, Toshihiro S. Reduction of oxysterol levels up-regulates HMG-CoA reductase activity in rat liver. Atherosclerosis. 1997;131:237–242.[Medline] [Order article via Infotrieve]
  14. Taylor FR, Kandutsch AA. Use of oxygenated sterols to probe the regulation of 3-hydroxy-3-methylglutaryl-CoA reductase and sterologenesis. Methods Enzymol. 1985;110:9–19.[Medline] [Order article via Infotrieve]
  15. Kilsdonk EPC, Morel DW, Johnson WJ, Rothblat GH. Inhibition of cellular cholesterol efflux by 25-hydroxycholesterol. J Lipid Res. 1995;36:505–516.[Abstract]
  16. Fielding CJ, Bist A, Fielding PE. Caveolin mRNA levels are up-regulated by free cholesterol and down-regulated by oxysterols in fibroblast monolayers. Proc Natl Acad Sci U S A. 1997;94:3753–3758.[Abstract/Free Full Text]
  17. Gelissen IC, Brown AJ, Mander EL, Kritharides L, Dean RT, Jessup W. Sterol efflux is impaired from macrophage foam cells selectively enriched with 7-ketocholesterol. J Biol Chem. 1996;271:17852–17860.[Abstract/Free Full Text]
  18. Cheng D, Chang CCY, Qu X-M, Chang T-Y. Activation of acyl-coenzyme A:cholesterol acyltransferase by cholesterol or by oxysterol in a cell-free system. J Biol Chem. 1995;270:685–695.[Abstract/Free Full Text]
  19. Brown MS, Dana SE, Goldstein JL. Cholesterol ester formation in cultured human fibroblasts: stimulation by oxygenated sterols. J Biol Chem. 1975;250:4025–4027.[Abstract/Free Full Text]
  20. Hodis HN, Kramsch DM, Avogaro P, Bittolo-Bon G, Cazzolato G, Hwang J, Peterson H, Sevanian A. Biochemical and cytotoxic characteristics of an in vivo circulating oxidized LDL. J Lipid Res. 1994;35:669–677.[Abstract]
  21. Hodis HN, Crawford DW, Sevanian A. Cholesterol feeding increases plasma and aortic tissue cholesterol oxide levels in parallel: further evidence for the role of cholesterol oxidation in atherosclerosis. Atherosclerosis. 1991;89:117–126.[Medline] [Order article via Infotrieve]
  22. Hodis HN, Chauhan A, Hashimoto S, Crawford DW, Sevanian A. Probucol reduces plasma and aortic wall oxysterol levels in cholesterol fed rabbits independently of its plasma cholesterol lowering effect. Atherosclerosis. 1992;96:125–134.[Medline] [Order article via Infotrieve]
  23. Mahfouz MM, Kawano H, Kummerow FA. Effect of cholesterol-rich diets with and without added vitamins E and C on the severity of atherosclerosis in rabbits. Am J Clin Nutr. 1997;66:1240–1249.[Abstract/Free Full Text]
  24. Staprans I, Pan X-M, Rapp JH, Feingold KR. Oxidized cholesterol in the diet accelerates the development of aortic atherosclerosis in cholesterol-fed rabbits. Arterioscler Thromb Vasc Biol. 1998;18:977–983.[Abstract/Free Full Text]
  25. Rong JX, Rangaswamy S, Shen L, Dave R, Chang YH, Hazel P, Hodis HN, Chisolm GM, Sevanian A. Arterial injury by cholesterol oxidation products causes endothelial dysfunction and arterial wall cholesterol accumulation. Arterioscler Thromb Vasc Biol. 1998;18:1885–1894.[Abstract/Free Full Text]
  26. Sevanian A, Bittolo-Bon G, Cazzolato G, Hodis H, Hwang J, Zamburlini A, Maiorino M, Ursini F. LDL- is a lipid hydroperoxide-enriched circulating lipoprotein. J Lipid Res. 1997;38:419–428.[Abstract]
  27. Clarkson TB, Lehner NDM, Bullock BC. Specialized research applications, I: arteriosclerosis research. In: Weisbroth SH, Flatt RE, Kraus AL, eds. The Biology of the Laboratory Rabbit. 5th ed. San Diego, Calif: Academic Press Inc; 1993:155–164.
  28. Parker F, Peterson N, Odland GF. A comparison of cholesterol-ester fatty acid patterns in the blood and in evolving xanthoma and atheroma during cholesterol-feeding of rabbits. J Invest Dermatol. 1966;47:253–259.[Medline] [Order article via Infotrieve]
  29. Newman HAI, Zilversmit DB. Accumulation of lipid and nonlipid constituents in rabbit atheroma. J Atheroscler Res. 1964;4:261–271.
  30. Jokinen MP, Clarkson TB, Prichard RW. Recent advances in molecular pathology: animal models in atherosclerosis research. Exp Mol Pathol. 1985;42:1–28.[Medline] [Order article via Infotrieve]
  31. Evan AS, Myers GL. Lipids, lipoproteins, and apolipoproteins. In: Burtis CA, Ashwood ER, eds. Tietz Textbook of Clinical Chemistry. 2nd ed. Philadelphia, Pa: WB Saunders Co; 1994:1002–1093.
  32. Holvoet P, Collen D. ß-VLDL hypercholesterolemia relative to LDL hypercholesterolemia is associated with higher levels of oxidized lipoproteins and a more rapid progression of coronary atherosclerosis in rabbits. Arterioscler Thromb Vasc Biol. 1997;17:2376–2382.[Abstract/Free Full Text]
  33. Adams CWM, 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]
  34. Feldman DL, Mogelesky TC, Liptak BF, Gerrity RG. Leukocytosis in rabbits with diet-induced atherosclerosis. Arterioscler Thromb. 1991;11:985–994.[Abstract/Free Full Text]
  35. Daley SJ, Klemp KF, Guyton JR, Rogers KA. Cholesterol-fed and casein-fed rabbit models of atherosclerosis, part 2: differing morphological severity of atherogenesis despite matched plasma cholesterol levels. Arterioscler Thromb. 1994;14:105–114.[Abstract/Free Full Text]
  36. Bocan TMA, Mueller SB, Uhlendorf PD, Newton RS, Krause BR. Comparison of CI-976, an ACAT inhibitor, and selected lipid-lowering agents for antiatherosclerotic activity in iliac-femoral and thoracic aortic lesions: a biochemical, morphological, and morphometric evaluation. Arterioscler Thromb. 1991;11:1830–1843.[Abstract/Free Full Text]
  37. Bocan TMA, 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]
  38. Daley SJ, Herderick EE, Cornhill JF, Rogers KA. Cholesterol-fed and casein-fed rabbit models of atherosclerosis, part 1: differing lesion area and volume despite equal plasma cholesterol levels. Arterioscler Thromb. 1994;14:95–104.[Abstract/Free Full Text]
  39. Kolodgie FD, Katocs AS, Largis EE, Wrenn SM, Cornhill JF, Herderick EE, Lee SJ, Virmani R. 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. Arterioscler Thromb Vasc Biol. 1996;16:1454–1464.[Abstract/Free Full Text]
  40. Institute of Laboratory Animal Resources, National Research Council. Guide for the Care and Use of Laboratory Animals. 7th ed. Washington, DC: National Academy Press; National Institutes of Health, Dept of Agriculture and Dept of Human Affairs. 1996.
  41. Lipid Research Clinics Program: The Manual of Laboratory Operations: Lipid and Lipoprotein Analysis. Bethesda, Md: National Institutes of Health; Dept of Health, Education and Welfare publication. 1974.
  42. Karpe F, Hamsten A. Determination of apolipoproteins B-48 and B-100 in triglyceride-rich lipoproteins by analytical SDS-PAGE. J Lipid Res. 1994;35:1311–1317.[Abstract]
  43. Mero N, Syvanne M, Rosseneu M, Labeur C, Hilden H, Taskinen M-R. Comparison of three fatty meals in healthy normolipidaemic men: high post-prandial retinyl ester response to soybean oil. Eur J Clin Invest. 1998;28:407–415.[Medline] [Order article via Infotrieve]
  44. Bligh EG, Dyer WJ. A rapid method of total lipid extraction and purification. Can J Biochem Physiol. 1959;37:911–917.
  45. Kaluzny MA, Duncan LA, Merritt MV, Epps DE. Rapid separation of lipid classes in high yield and purity using bonded phase columns. J Lipid Res. 1985;26:135–144.[Abstract]
  46. 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;16:601–613.
  47. Watabe T, Isobe M, Kanai M. Cholesterol diet increases plasma and liver concentrations of cholesterol epoxides and cholestanetriol. J Pharmacobiodyn. 1980;3:553–556.[Medline] [Order article via Infotrieve]
  48. Vine DF, Croft KD, Beilin LJ, Mamo JCL. Absorption of dietary cholesterol oxidation products and incorporation into rat lymph chylomicrons. Lipids. 1997;32:887–893.[Medline] [Order article via Infotrieve]
  49. Krut LH, Yang JW, Schonfeld G, Ostlund REJ. The effect of oxidizing cholesterol on gastrointestinal absorption, plasma clearance, tissue distribution, and processing by endothelial cells. Arterioscler Thromb Vasc Biol. 1997;17:778–785.[Abstract/Free Full Text]
  50. Osada K, Sasaki E, Sugano M. Lymphatic absorption of oxidized cholesterol in rats. Lipids. 1994;29:555–559.[Medline] [Order article via Infotrieve]
  51. Kaur M, Kaur J, Ojha S, Mahmood A. Ethanol effects on lipid peroxidation and glutathione-mediated defense in rat small intestine: role of dietary fats. Alcohol. 1998;15:65–69.[Medline] [Order article via Infotrieve]
  52. Smith LL. The oxidation of cholesterol. In: Peng S-K, Morin RJ, eds. Biological Effects of Cholesterol Oxides. Boca Raton, Fla: CRC Press Inc; 1992:7–31.
  53. Staprans I, Rapp JH, Pan XM, Feingold KR. Oxidized lipids in the diet are incorporated by the liver into very low density lipoprotein in rats. J Lipid Res. 1996;37:420–430.[Abstract]
  54. Cook RP, MacDougall JDB. Experimental atherosclerosis in rabbits after feeding cholestane triol. Br J Exp Pathol. 1968;49:265–271.[Medline] [Order article via Infotrieve]
  55. Matthias D, Becher C-H, Godicke W, Schmidt R, Ponsold K. Action of cholestane-3ß,5{alpha},6ß-triol on rats with particular reference to the aorta. Atherosclerosis. 1987;63:115–124.[Medline] [Order article via Infotrieve]
  56. Jacobson MS, Price MG, Shamoo AE, Heald FP. Atherogenesis in white Carneau pigeons: effects of low-level cholestane-triol feeding. Atherosclerosis. 1985;57:209–217.[Medline] [Order article via Infotrieve]
  57. Imai H, Werthessen NT, Subramanyam V, Lequosne PW, Soloway AH, Kanisawa M. Angiotoxicity of oxygenated sterols and possible precursors. Science. 1980;207:651–653.[Abstract/Free Full Text]
  58. Higley NA, Beery JT, Taylor SL, Porter JW, Dziuba JW, Lalich JJ. Comparative atherogenic effects of cholesterol and cholesterol oxides. Atherosclerosis. 1986;62:91–104.[Medline] [Order article via Infotrieve]
  59. Smith LL. Cholesterol autoxidation 1981–1986. Chem Phys Lipids. 1987;44:87–125.[Medline] [Order article via Infotrieve]
  60. Peng SK, Hu B, Morin RJ. Angiotoxicity and atherogenicity of cholesterol oxides. J Clin Lab Anal. 1991;5:144–152.[Medline] [Order article via Infotrieve]
  61. Hulten LM, Lindmark H, Diczfalusy U, Bjorkhem I, Ottosson M, Liu Y, Bondjers G, Wiklund O. Oxysterols present in atherosclerotic tissue decrease the expression of lipoprotein lipase messenger RNA in human monocyte-derived macrophages. J Clin Invest. 1996;97:461–468.[Medline] [Order article via Infotrieve]
  62. Hennig B, Boissonneault GA. Cholestane-3ß,5{alpha},6ß-triol decrease barrier function of cultured endothelial cell monolayers. Atherosclerosis. 1987;63:115–124.
  63. Ramasamy S, Boissonneault GA, Hennig B. Oxysterol-induced endothelial cell dysfunction in culture. J Am Coll Nutr. 1992;11:532–538.[Abstract]
  64. Chang CC, Jone C, Trosko JE, Peterson AR, Sevanian A. Effect of cholesterol epoxides on the inhibition of intercellular communication and on mutation induction in Chinese hamster V79 cells. Mut Res. 1988;206:471–478.[Medline] [Order article via Infotrieve]
  65. Davies PF, Selden SC, Schwartz SM. Enhanced rates of fluid pinocytosis during exponential growth and monolayer regeneration by cultured arterial endothelial cells. J Cell Physiol. 1980;102:119–127.[Medline] [Order article via Infotrieve]
  66. Lin SJ, Jan KM, Weinbaum S, Chien S. Transendothelial transport of low density lipoprotein in association with cell mitosis in rat aorta. Arteriosclerosis. 1989;9:230–236.[Abstract/Free Full Text]
  67. Cao J, Fales HM, Schaffner CP. Cellular sterol accumulation stimulated by cholesterol 5ß,6ß-epoxide in J774 macrophages (43896). Proc Soc Exp Biol Med. 1995;209:195–204.[Abstract]
  68. St Clair RW. Cholesterol ester metabolism in atherosclerotic arterial tissue. Ann N Y Acad Sci. 1976;275:228–237.
  69. Hoppe G, Ravandi A, Herrera D, Kuksis A, Hoff HF. Oxidation products of cholesteryl linoleate are resistant to hydrolysis in macrophages, form complexes with proteins, and are present in human atherosclerotic lesions. J Lipid Res. 1997;38:1347–1360.[Abstract]
  70. Hoff HF, Hoppe G. Structure of cholesterol-containing particles accumulating in atherosclerotic lesions and the mechanisms of their derivation. Curr Opin Lipidol. 1995;6:317–325.[Medline] [Order article via Infotrieve]



This article has been cited by other articles:


Home page
 J. Exp. Biol.Home page
Y. Jiang, J. Jiang, J. Xiong, J. Cao, N. Li, G. Li, and S. Wang
Homocysteine-induced extracellular superoxide dismutase and its epigenetic mechanisms in monocytes
J. Exp. Biol., March 15, 2008; 211(6): 911 - 920.
[Abstract] [Full Text] [PDF]

Home page
 J. Nutr.Home page
X.-j. Zhang, D. L. Chinkes, A. Aarsland, D. N. Herndon, and R. R. Wolfe
Lipid Metabolism in Diet-Induced Obese Rabbits Is Similar to That of Obese Humans
J. Nutr., March 1, 2008; 138(3): 515 - 518.
[Abstract] [Full Text] [PDF]