© 1999 American Heart Association, Inc.
Atherosclerosis and Lipoproteins |
Cholesterol Oxidation Products Induce Vascular Foam Cell Lesion Formation in Hypercholesterolemic New Zealand White Rabbits
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
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Abstract |
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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
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Introduction |
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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.
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Methods |
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Rabbit Feeding
Ten male NZW rabbits (
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 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 1
, 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 1 (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.
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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 1
, 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 2, 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.
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Determination of Total Cholesterol (TC), Cholesteryl
Esters (CEs), and Free Cholesterol (FC)
As indicated in Figure 2, 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.
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Results |
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Control of Plasma Cholesterol Level and Total Plasma Cholesterol Exposure
As shown in Figure 3A
, 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 3B
shows that there was no difference in total plasma
cholesterol exposure between the 2 groups
(P=0.69).
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Plasma Total ChOx Levels and Total Plasma ChOx Exposure
As shown in Figure 4A, 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 4B, the total plasma ChOx exposure was 1.8-fold greater in the
ChOx-injected rabbits than in the controls (P<0.0002).
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Contribution of Chylomicrons to Plasma Cholesterol/ChOx
Exposure
As shown in Figure 5, 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
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.
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ChOx Content in the Liver
The liver ChOx content at the conclusion of the experiments is
presented in Figure 6. 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.
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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 7) 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 8. 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.
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The extent of sudanophilic lesions in rabbit aortas as a function of
total plasma cholesterol/ChOx exposure is presented
in Figure 9. There was a poor correlation
(R=0.28, P<0.44) between lesion area and total
plasma cholesterol exposure (Figure 9A). In
contrast, a strong correlation (R=0.93,
P<0.0001) was found between lesion area and total plasma
ChOx exposure (Figure 9B).
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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 2 (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.
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The TC content in the rabbit aortic arch as a function of total plasma
cholesterol/ChOx exposure is presented in Figure 10. There was a poor correlation
(R=0.30, P<0.40) between tissue TC content and
total plasma cholesterol exposure (Figure 10A). In
contrast, a strong correlation (R=0.83, P<0.003)
was observed between the aortic tissue TC content and ChOx exposure
(Figure 10B).
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Discussion |
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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 3A and 3B). 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 9A). 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 4A). 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 4B). 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 8). 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 9B) 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 2). 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 10B) 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 2). 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.
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Acknowledgments |
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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.
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