© 1998 American Heart Association, Inc.
Original Contributions |
Arterial Injury by Cholesterol Oxidation Products Causes Endothelial Dysfunction and Arterial Wall Cholesterol Accumulation
From the Department of Pathology (J.X.R., A.S.), Division of Cardiology (R.D., H.N.H.), and Atherosclerosis Research Unit (H.N.H., A.S.), School of Medicine, and Department of Molecular Pharmacology and Toxicology, School of Pharmacy (L.S., Y.H.C., H.P., H.N.H., A.S.), University of Southern California, Los Angeles, Calif; and Department of Cell Biology, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, Ohio (S.R., G.M.C.).
Correspondence to Alex Sevanian, Department of Molecular Pharmacology and Toxicology, School of Pharmacy, University of Southern California, 1985 Zonal Ave, Los Angeles, CA 90033. E mail asevan@hsc.usc.edu
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Abstract |
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Abstract—Cholesterol oxidation products (ChOx) have been reported to cause acute vascular injury in vivo; however, the pharmacokinetics of ChOx after administration and the mechanisms by which they cause chronic vascular injury are not well understood. To further study the pharmacokinetics and atherogenic properties of ChOx, New Zealand White rabbits were injected intravenously (70 mg per injection, 20 injections per animal) with a ChOx mixture having a composition similar to that found in vivo during a 70-day period. Total ChOx concentrations in plasma peaked almost immediately after a single injection, declined rapidly, and returned to preinjection levels in 2 hours. After multiple injections, the ChOx concentrations rose gradually to levels 2- to 3-fold above baseline levels, increasing mostly in the cholesteryl ester fraction of LDL and VLDL. Rabbit serum and the isolated LDL/VLDL fraction containing elevated ChOx concentrations were cytotoxic to V79 fibroblasts and rabbit aortic endothelial cells. At the time of killing, cholesterol levels in the aortas from ChOx-injected rabbits were significantly elevated despite the fact that plasma cholesterol levels remained in the normal range. In addition, aortas from the ChOx-injected rabbits retained more 125I-labeled horseradish peroxidase, measured 20 minutes after intravenous injection. Transmural concentration profiles across the arterial wall also showed increased horseradish peroxidase accumulation in the inner half of the media from the thoracic aorta in ChOx-injected rabbits. In conclusion, ChOx injection resulted in accumulation of circulating ChOx and induced increased vascular permeability and accumulation of lipids and macromolecules. This study reveals that even under normocholesterolemic conditions, ChOx can cause endothelial dysfunction, increased macromolecular permeability, and increased cholesterol accumulation, parameters believed to be involved in the development of early atherosclerotic lesions.
Key Words: oxysterols • atherosclerosis • cytotoxicity • vascular injury • cholesteryl esters
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Introduction |
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There is growing evidence that cholesterol oxidation products (ChOx) are linked to development of atherosclerosis. ChOx are abundant in oxidatively modified LDL (ox-LDL),1 which is widely regarded as proatherogenic.2 Numerous studies have described the biological activity and atherogenicity of ChOx. Notable among these are the effects on vascular cell integrity and the ability to cause cell injury in a manner analogous to that of ox-LDL. ChOx are cytotoxic to the 3 major arterial wall cell types, endothelial cells,3 4 smooth muscle cells,5 6 and fibroblasts,7 whereas pure cholesterol has no demonstrable toxicity. There is increasing evidence that specific ChOx induce apoptosis in monocytes8 and smooth muscle cells.9 In addition, certain ChOx and ChOx-enriched LDL decrease vascular endothelial barrier function.10 11 ChOx administered to animals by intravenous injection, oral feeding, and gavage have been reported to cause vascular injury.12 13 14 15 16 17 However, the pharmacokinetics of ChOx are not well understood, nor are their vascular effects in the absence of hypercholesterolemia.
To address these questions, a mixture composed of the major ChOx found in circulating ox-LDL,1 aortic tissue,18 and macrophages isolated from aortas of atherosclerotic New Zealand White (NZW) rabbits19 was injected intravenously into NZW rabbits. The levels of injected ChOx were monitored to establish simple pharmacokinetic profiles. The plasma levels of ChOx were then correlated with the cytotoxicity of the rabbit serum, and the major lipoproteins were isolated after ChOx injection using V79 (Chinese hamster lung fibroblasts) cells and rabbit aortic endothelial cells (RAECs). In addition, vascular injury caused by ChOx was studied by measuring the accumulation of lipids and the penetration of horseradish peroxidase (HRP) into the aortic wall, and these effects were compared with the accumulation of cholesterol in the aortic wall. These studies were aimed at describing the role of elevated circulating ChOx in the development of early atherosclerotic lesions and accumulation of cholesterol in the vascular wall in the absence of elevated plasma cholesterol levels.
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Methods |
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Injection Procedure
Twenty-one male NZW rabbits (
2.5 kg) were acquired from a
local breeder (Irish Farms, Norco, Calif), maintained by the University
of Southern California vivaria in accordance with National Institutes
of Health guidelines,20 fed a
cholesterol-free rabbit chow diet, and quarantined for 1
week before treatment. The rabbits were divided into 2 study groups.
The test group was injected with a ChOx mixture (70 mg, Steraloids)
with lecithin (140 mg, Avanti Polar Lipids), and controls received an
injection of pure cholesterol (Sigma grade, 
99% pure, 70
mg, Sigma) with an equivalent amount of lecithin or lecithin alone. The
purity of the individual ChOx was checked before use and was >99%
during the course of these studies. The ChOx used in this study and the
composition of the mixture are provided in Table 1
. The ChOx mixture and
cholesterol were stored in ethanol (23.3 mg/mL at 4°C).
Before each injection, the ChOx or cholesterol solution (3
mL) was transferred to a 15-mL polypropylene centrifuge tube
containing lecithin (70 mg), and the ethanol was evaporated under
nitrogen. Saline (0.9% NaCl, 4.5 mL) was added to the residue, the
tube was capped under argon, and the contents were sonicated in a
cup-horn sonicator (Heat Systems, Inc) at 4°C until a stable emulsion
was obtained (usually 5 minutes). The resulting emulsion was stable for
at least 3 hours at room temperature. An aliquot (10 µL) was removed
and diluted with H2O (1 mL) to determine
ChOx/cholesterol and hydroperoxide contents, and the rest
was transferred to a syringe and injected into the rabbit via the
peripheral ear vein. The emulsion was injected using a
23-gauge butterfly catheter via the lateral ear vein at a flow rate of
3 mL/min. A second control group was injected with the sonicated
lecithin vehicle only under similar conditions. All procedures were
performed under sterile conditions. As shown in Figure 1, each animal was injected every other
day with 3 series of 6 to 7 injections each, and there was a rest
period of 10 days between each series of injections. Each animal
received a total of 20 injections amounting to 1400 mg of ChOx or pure
cholesterol over 70 days. No animals died during any of
these procedures. The average weight of the rabbits at the end of the
experiments was 3.6±0.11 kg, and there was no difference between the
ChOx-injected and control groups. Blood was drawn from the central ear
artery of each nonfasted animal at baseline before and 5, 10, 30, 60,
120, and 240 minutes after the first injection; immediately before each
of the remaining injections; and at killing. Plasma or serum was
separated from blood, collected with or without EDTA, respectively, by
centrifugation (3000 rpm, 4°C, 20 minutes), and
stored (N2, -70°C) until further
analysis.
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LDL/VLDL Preparation
For cell culture, the LDL/VLDL fraction was isolated from plasma
by density gradient ultracentrifugation using a Beckman
model TL-100 ultracentrifuge equipped with a TLA-100.2 rotor.
Rabbit plasma (0.5 mL) was added to centrifuge tubes
(11x34 mm, Beckman) containing 0.5 mL of sucrose solution (30%
wt/wt) and centrifuged (85 000 rpm, 5°C, 20 hours). The top
fraction (0.4 mL, d
1.063 mg/mL) was transferred to
dialysis tubes with a molecular weight cutoff of 40 000 kDa to remove
EDTA and then sterilized by passing through 0.2-µmol/L filters
(Corning Glass Works).
For ChOx determination, rabbit plasma (5 mL) was added to centrifuge tubes (14x89 mm, Beckman) containing NaBr (1.85 g) and Sudan black B (Sigma) in DMSO (0.2 mg/mL, 0.5 mL). NaBr solutions with densities of 1.250 mg/mL (1.6 mL) and 1.080 g/mL (2.0 mL) and water (3 mL) were then carefully layered into the tubes.1 The samples were centrifuged (285 000g, 4°C, 20 hours) in a Beckman L8 to 55 ultracentrifuge equipped with an SW-41 rotor. The bands (visible to Sudan black staining) with buoyant densities <1.063 g/mL were collected and concentrated for further analysis.
Cytotoxicity Assays
RAECs were obtained from NZW rabbits, maintained in cultures as
described previously,3 and used between passages
21 and 25. The doubling time of the cells was 18 hours in complete
medium (Dulbecco's modified Eagle's medium [DMEM] and M199
[80:20] containing 15% FBS). Rabbit serum was used instead of plasma
because plasma containing EDTA was cytotoxic. Cells were plated in
48-well Corning culture plates in the presence of
14C-adenine (0.2 µCi/mL in complete medium, ICN
Pharmaceuticals, Inc). While in a subconfluent state (48 hours after
plating), cells were washed twice with medium and treated with 15%
rabbit serum (obtained from each of the experimental animals) in
DMEM:M199 (80:20). After 24 hours, an aliquot of the medium was
transferred to determine the amount of
14C-labeled product released as a measure of
cell injury.21 The toxicity of rabbit serum in
the wells was expressed as the percentage of specific release of
14C counts derived from adenine:
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Vascular Permeability Measurements
As previously described,21 23 HRP (50
mg/mL in PBS, 50 mg/kg body weight, Sigma) containing trace amounts of
125I-HRP (5 to 10 µCi) was injected as a bolus
over 30 to 60 seconds into the anesthetized rabbit via the
peripheral ear vein and allowed to circulate for 20
minutes. The rabbit was anesthetized by an intramuscular
injection of a 1.5-mL solution of ketamine (50 mg/mL) and
xylazine (10 mg/mL). Blood samples were taken at various times after
the HRP injection via a catheter in the right carotid artery to
determine the disappearance of 125I-HRP. This was
used to calculate the initial HRP concentration in plasma
(Cpo). The rabbit was then killed by a
lethal dose of pentobarbital (65 mg/mL, 6 mL). The peritoneal cavity
was opened, an incision was made in the vena cava, and ice-cold PBS
(50 mL) was perfused into the left ventricle to clear the
vasculature of blood. This was immediately followed by perfusion of
ice-cold glutaraldehyde (2.5% in PBS, Sigma), and
fixation in situ was continued for 20 minutes under a constant pressure
of 100 mm Hg. The aortic arch and descending thoracic aorta
(including the iliac bifurcation) were removed and cut in half
longitudinally. One half was processed for transmural HRP accumulation
by incubating the tissue with 3,3'-diaminobenzidine and
H2O2 and by quantifying HRP
accumulation in 4-µm-thick sections using an image-processing system
as described previously.23 HRP accumulation was
quantified by comparing the gray-scale values with those of standards
prepared by equilibrating (36 to 48 hours) fresh aortic tissue with
solutions of known HRP concentration and then processing the tissue
analogously. The HRP transmural concentration profile across the aorta
was determined according to the methods described by Penn et
al.23 The other half of the aorta was washed 10
times (2 minutes each time) in 50 mL of ice-cold saline, washed in 5%
KI in 0.9% saline, rinsed again with saline, and blotted dry. The
adventitia was carefully removed, and the sample was weighed.
125I content was measured using a Cobra-II gamma
counter (Packard Instruments) as described
previously.24 After counting, this half was
further processed for cholesterol and ChOx
determinations.
Separation of Cholesteryl Esters From LDL/VLDL Fraction
Cholesteryl esters (CEs) were separated using the method of
Kaluzny et al25 with some modification. In brief,
total lipids from the LDL/VLDL fraction (isolated from 5 mL of plasma
and concentrated to 1 mL) were extracted by a modified Bligh-Dyer
procedure,18 evaporated to dryness under
N2, dissolved 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, 4 mL). The eluate
(neutral lipids) was collected, reconstituted in hexane (0.2 mL), and
applied to a new aminopropyl column prepared in advance as above.
Hexane (4 mL) was applied, and the eluate (mainly unoxidized CEs) was
collected. Another new aminopropyl column was then attached in a
piggyback manner 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, pooled with the previous CE eluate, evaporated to dryness,
and subjected to cold alkaline saponification for analysis by
gas chromatography (GC) (described below).
Cholesterol and ChOx Determinations
Weighed rabbit aorta or liver tissue was minced using a tissue
homogenizer (Tekmar Instruments).
Cholesterol and ChOx contents in the injected emulsions,
rabbit plasma (1 mL), and minced tissues were determined as previously
described.18 In brief, total lipids were
extracted by a modified Bligh-Dyer procedure26
and then 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 after addition of 150 µL
each of dimethylformamide and
N,O-bis(trimethylsilyl)
trifluoroacetamide (Supelco). Samples were then analyzed by GC
using a Shimadzu GC-14 chromatograph fitted with a DB-1
capillary column (J&W Scientific) equipped with a flame ionization
detector. The injector was operated with a split ratio of 1:10 and set
at 290°C. Helium was used as the carrier gas at a flow rate of 1
mL/min, with an initial column temperature of 240°C. The flame
ionization detector temperature was set at 300°C. A programmed
temperature run was used with an initial temperature hold for 1 minute,
followed by a 3°C/min temperature ramp to 290°C, and a final
temperature hold for 25 minutes. Quantitative analysis of the
biological samples was performed by the internal standard method to
identify and quantify individual ChOx. Total ChOx were expressed as the
sum of individual ChOx.
Lipid Hydroperoxide Measurements
Measurements of lipid hydroperoxide levels in injected lipid
emulsions were based on the method of Auerbach et
al27 with minor modifications. Aliquots from the
lipid emulsion were diluted with phosphate buffer (1:10 and 1:40
dilutions), and then 40-µL aliquots were added to a 96-well
microtiter plate containing ethanol (10 µL) and N-benzoyl
leucomethylene blue (LMB) color reagent (100 µL). The LMB reagent was
prepared with 5 mg of LMB (Tokyo Kasei Kogyo) dissolved in 8 mL of
N,N-dimethyl-formamide and 90 mL of 0.05 mol/L
potassium phosphate buffer, pH 5, 1.4 g of Triton X-100, and 5.5
mg of hemoglobin. Linoleic acid hydroperoxide
(13-hydroperoxy-9,11-octadecadienoic acid, ie, LOOH, ranging from 1 to
20 nmol in 10 µL ethanol) was added as a standard to wells containing
saline (40 µL) and LMB. After 40 minutes at room temperature, the
standards and samples were read at 650 nm using a microtiter plate
reader (Cambridge Technology). A standard curve for absorbency versus
LOOH concentration was generated, and the hydroperoxide levels in
samples were determined from this curve.
Statistics
For cytotoxicity studies, the mean±SEM was determined from 2
independent experiments in which all samples were analyzed in
triplicate. All statistical evaluations for differences between paired
observations were made using a 2-sample t test.
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Results |
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Oxidation of Lipids During Preparation of Liposome Emulsions
Initially, we examined the extent of lecithin and cholesterol oxidation during preparation of the liposomal emulsions that were injected into rabbits because the oxidation products derived from these lipids may confound interpretation of results. Aliquots from the emulsions were taken after sonication, and hydroperoxides were determined by LMB color reaction, and ChOx were measured by gas chromatographic analysis. Less than 0.11% of the lecithin and <0.4% of the cholesterol were oxidized in all liposome emulsions. This represents <0.4% of the mass of ChOx injected into rabbits at any time, and the total peroxide levels that would be introduced to plasma after injection is estimated to be <1.4 µmol/L.
Pharmacokinetics of Cholesterol and ChOx
Twenty injections of pure cholesterol or the ChOx
mixture were made via the peripheral ear vein of each
rabbit. After a single injection of the ChOx mixture, total ChOx in
plasma peaked within 2 minutes, the shortest time required to finish
injection and blood sampling, and reached concentrations >30-fold
above baseline levels. ChOx levels then declined rapidly and returned
to baseline levels in 2 hours (Figure 2).
All the individual ChOx that were injected in this mixture were cleared
at a similar rate (data not shown). However, after 20 injections, ChOx
levels increased gradually to >2.5-fold above baseline levels,
representing the maximum levels measured at the time of
killing. Plasma cholesterol levels decreased to 70% of
the baseline level (Table 2) during this
period. For rabbits injected with pure cholesterol, plasma
cholesterol levels peaked 30 minutes after a single
injection, reaching 1.4-fold above the baseline level, and then
rapidly declined. Plasma ChOx levels also increased transiently to
>2.5-fold above the baseline level after injection of pure
cholesterol. However, after multiple injections, the ChOx
levels returned to baseline and, along with the plasma
cholesterol level, remained relatively unchanged (Figure 2
and Table 2). This suggests that pure cholesterol
injections contributed virtually no ChOx to the plasma ChOx pool and
that adventitious oxidation of cholesterol was
negligible.
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Distribution of ChOx in LDL/VLDL Cholesterol Fractions
at End Points
At the end of the injection regimen and just before killing, the
LDL/VLDL fractions from ChOx-injected rabbits contained slightly higher
proportions of total plasma cholesterol and ChOx as
compared with rabbits injected with pure cholesterol (Table 3). The absolute levels of ChOx in the
LDL/VLDL fractions from ChOx-injected rabbits were twice those measured
in control rabbits injected with pure cholesterol
(P<0.07), whereas the level of ChOx in the CE fraction was
3-fold greater in ChOx-injected rabbits than in controls
(P<0.05).
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Cytotoxicity of Rabbit Serum to RAECs
Rabbit serum was drawn at different time intervals after injection
of pure cholesterol or the ChOx mixture and used to treat
subconfluent cells for 24 hours. This was followed by determinations of
cytotoxicity, as measured by cell SF and specific release of
14C-adenine–derived radioactivity. As shown in
Figure 3, serum from ChOx-injected
rabbits obtained 5 and 30 minutes after injection was toxic to RAECs,
reducing the SF to 60% at a 15% rabbit serum-equivalent dose. When
plasma ChOx levels decreased (ie, 240 minutes after injection), the SF
of treated cells increased to 80%. Serum from rabbits injected with
liposomes containing pure cholesterol was not cytotoxic.
The approximate plasma ChOx levels at the sampling intervals used to
obtain serum for cytotoxicity experiments are indicated in Figure 2
as
the second (5 minutes), third (30 minutes), and seventh (240 minutes)
open circles (ChOx injected) or triangles (pure cholesterol
injected). Consistent with this observation, serum from
ChOx-injected rabbits containing elevated plasma ChOx levels caused the
release of 50% of the 14C-adenine–derived
radioactivity preincorporated into cells (Figure 4). When ChOx levels in plasma decreased,
the extent of 14C-adenine release also decreased.
Serum from rabbits injected with pure cholesterol induced
little or no 14C-adenine release. Interestingly,
treatment of RAECs with liposomal emulsions containing the ChOx mixture
or pure cholesterol at concentrations comparable to the
levels of ChOx achieved in plasma after injection of the ChOx produced
no cytotoxicity compared with untreated controls (data not shown).
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Effect of Plasma ChOx Elevation on LDL/VLDL-mediated
Cytotoxicity
To study the effect of short-term elevations in plasma ChOx levels
resulting from a single injection, the LDL/VLDL fraction isolated from
rabbit plasma after the first injection of cholesterol or
the ChOx mixture was incubated with RAECs. As shown in Figure 5, the LDL/VLDL fraction from
ChOx-injected rabbits containing elevated ChOx levels (5 and 8 minutes
after injection) significantly decreased SF (P<0.01) as
compared with treatments using LDL/VLDL isolated from rabbits before
injection of the ChOx mixture. In contrast, no difference in SF was
observed for LDL/VLDL fractions isolated from rabbits injected with
pure cholesterol.
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Plasma ChOx levels increased gradually after multiple injections. To
study the effect of LDL/VLDL isolated from plasma after longer
intervals between injections, the lipoproteins were isolated from
plasma drawn at baseline and the end of each injection series (see
Figure 1). Cytotoxicity assays were then performed using V79
fibroblasts. As shown in Figure 6, LDL/VLDL fractions from the ChOx-injected rabbits were substantially
more cytotoxic than fractions obtained at baseline or isolated from
rabbits injected with pure cholesterol. Moreover, when the
ChOx level in plasma gradually increased with continued injection, the
cytotoxicity increased in the same gradual manner.
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Cholesterol or ChOx Content in Liver and Aorta
After the 20-injection, 70-day treatment period, livers from the
ChOx-injected rabbits contained significantly higher
(P<0.01) ChOx than livers from rabbits injected with pure
cholesterol, indicating possible involvement of the liver
in eliminating injected ChOx. No measurements were made of
metabolic products derived from the injected ChOx;
therefore, the levels in liver, plasma, and aorta may underestimate the
total amounts of these compounds at any given time. No differences were
found in either total plasma ChOx or cholesterol among
cholesterol- and lecithin-injected control groups (Table 4). Aortas from the
ChOx-injected rabbits had significantly higher tissue
cholesterol levels (P<0.01) than
cholesterol- and lecithin-injected rabbits (Table 4),
whereas tissue ChOx levels were minimally increased. These findings
indicate that sustained elevations in plasma ChOx levels result in an
increase in cholesterol levels in the aortic wall.
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125I-Labeled HRP Retained in Aortic Tissue
The ability of rabbit aorta to retain macromolecules was
determined by injecting 5 to 10 µCi of
125I-labeled HRP intravenously 20
minutes before killing and measuring the radioactivity retained by the
aortic tissue at the time of killing. As shown in Figure 7, 125I-HRP was
retained in aortas from the ChOx-injected rabbits to a significantly
greater extent than in rabbits injected with pure
cholesterol or lecithin (P<0.01). The
difference between the latter 2 control groups was not significant. On
average, aortas from the ChOx-injected rabbits retained twice as much
125I-HRP as those from control animals.
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HRP Transmural Concentration Profiles Across the Aortic
Wall
To further demonstrate the effect of injected ChOx on
endothelial and vessel wall permeability, the transport
of HRP across the aortic intima to the media was determined.
Concentration profiles of HRP were obtained as a function of the radial
distance through the media of the aortic wall 20 minutes after
injection of HRP. The results for all animals are shown in Figure 8. Accumulation of HRP in the
arterial media was expressed relative to
Cpo to allow data from multiple animals to
be averaged. The intimal accumulation of HRP in aortas of ChOx-injected
animals was similar to accumulation in controls (0.053±0.030 versus
0.037±0.024 relative to Cpo,
P=0.7). However, HRP penetrated deeper and in significantly
greater amounts (P<0.05) across the media in the
ChOx-injected rabbits compared with controls. In the luminal half of
the media, there was a 2.5-fold greater accumulation of HRP in
ChOx-injected rabbits as compared with controls (Figure 8, inset). The
difference between the cholesterol- and lecithin-injected
control groups was not significant; thus, the data were combined as
shown in Figure 8.
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Discussion |
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The studies described herein were based on previous findings18 that cholesterol feeding of NZW rabbits led to a heavy ChOx burden under hypercholesterolemic conditions and that the levels of the various ChOx greatly exceeded the cytotoxic levels for any individual ChOx.3 6 18 We used a ChOx mixture with a composition similar to that found in circulating ox-LDL from hypercholesterolemic animals.18 In contrast to Imai et al,14 we used a ChOx mixture rather than individually administered ChOx because the effects of the mixture should be more representative of ChOx found in plasma,18 atherosclerotic lesions,19 or in vivo circulating ox-LDL.1 The cytotoxic action of the ChOx mixture has also been reported to be greater than that of an individual ChOx at comparable dosage levels.4 28 Our results indicate that multiple ChOx injections lead to accumulation of circulating plasma ChOx levels and that the levels achieved under these conditions injure vascular cells in vivo, as shown by the increased vascular permeability to macromolecules. It is noteworthy that under these normocholesterolemic conditions, ChOx injection caused the cholesterol levels in rabbit aortic tissues to significantly increase.
We chose the intravenous route of administration because
oral administration can result in uncertainties related to absorption,
biotransformation, retention, and excretion of these compounds. Each
injection contained 70 mg of total sterols, sufficient to more than
double the plasma total sterol levels as compared with
normocholesterolemic rabbits. The dose of ChOx
administered was developed from preliminary studies and attempted to
simulate plasma levels achieved when rabbits were fed chow containing
0.5 to 1% USP-grade cholesterol,18
which is usually contaminated with 1% to 10% ChOx as
impurities.29 30 Rapid clearance of administrated
ChOx from plasma has been reported in a number of studies. Krut et
al31 reported that
2H-7
-hydroxycholesterol and
2H-7ß-hydroxycholesterol injected
intravenously into rats were cleared from the circulation
within the first 5 minutes by 99% and 92%, respectively, and that the
residual ChOx decreased along a single exponential curve with a
t1/2 of 86 minutes. Bowden et
al32 administered
14C--cholesterol epoxide to mice
by dermal absorption and found that after 18 hours, 64% of the
recovered label was found in the feces. Peng et
al33 fed squirrel monkeys a single dose of
3H-25-hydroxycholesterol, and after
24 hours, the residual specific activity in plasma was 200-fold lower
than peak levels after feeding. Rapid clearance was also reported for
CEs injected intravenously as chylomicron-like emulsion
into rats and humans. Approximately 90% of the sterols were cleared
from plasma in 10 to 20 minutes.34 However,
controversial findings have also been reported. Peng et
al35 found that gavage feeding of ChOx to rabbits
resulted in peak plasma levels within 24 hours and that nearly 80% of
the compounds remained in the circulation after 48 hours. Osada et
al36 studied the lymphatic absorption of ChOx fed
to rats over a 24-hour period and found that ChOx accumulated in lymph
at a relatively constant rate. Thus, the pharmacokinetics of ChOx
appear to be variable among different species, but there is a
general tendency for rapid absorption and rapid initial clearance
followed by a prolonged decay curve with substantial amounts remaining
in the blood for several hours.
ChOx accumulation in plasma after long-term treatment with
cholesterol or ChOx has received little attention thus far.
The 20 injections administered over a 70-day period in this study
amounted to a total of 1400 mg of ChOx being injected into each rabbit.
This resulted in a 2.5- and 2-fold increase in plasma and LDL/VLDL
total ChOx levels, respectively (Tables 2 and 3). The underlying
mechanism for the secondary or prolonged increase, as distinguished
from the primary or immediate increase after injection, remains
unknown. Our findings suggest that the liver is likely responsible for
the rapid clearance of injected ChOx (Table 4). After multiple
injections, the capacity of the liver to eliminate these compounds may
become saturated or impaired. Another plausible mechanism may involve
uptake of ChOx by various tissues after injection and gradual release
from these depots back into the circulation (a slow 2-compartment
exchange). This could also apply to the liver, which can assimilate the
ChOx and then release them as components of VLDL. This was previously
suggested by Kosykh et al,37 who showed that
administered ChOx stimulated VLDL secretion by hepatocytes.
Oxidized fatty acids in chylomicrons have been reported to be
repackaged and secreted in VLDL.38 Peng et
al35 showed that injected ChOx were selectively
transported by VLDL and LDL, whereas only minute amounts were found in
HDL. This is consistent with the view that ChOx accumulate in
the liver after chronic exposure and are then released along with VLDL
cholesterol and is supported by our finding that esterified
ChOx levels in the LDL/VLDL fraction from ChOx-injected rabbits were
3-fold higher than in rabbits injected with pure
cholesterol (Table 3). This is likely due to esterification
of the injected ChOx by liver acyl-coenzyme A:cholesterol
acyltransferase (ACAT). ChOx have been reported to be substrates for
ACAT in hepatocytes39 and other cell
types,40 although ChOx esterification by serum
lecithin:cholesterol acyltransferase may also be
possible.41 42 The extent to which the latter
contributes to esterification of plasma ChOx is being investigated.
Results of our studies indicate that the ratio of
cholesterol to other sterols in organic-phase extracts of
rabbit feces was not changed after multiple ChOx injections (data not
shown), suggesting that the ChOx were not excreted directly into the
bile. This is consistent with other findings. Krut et
al31 found that 5 minutes after
intravenous injection of
14C-7-hydroxycholesterol into
rats, only 23% of the radioactivity remained in plasma and the rest
was distributed among the liver (19%), red cells (13%), muscle
(10%), fat (7%), lung (4%), spleen (2%), and other tissues (22%).
Bascoul et al43 reported that 75% of the
3H-cholesterol-5,6-epoxide
injected into Wistar rats was retained for at least 2 days and proposed
an enterohepatic recycling and retention of the oxysterol. Erickson
et al44 reported that
3H-7-ketocholesterol administered to
rats or perfused into rat liver was rapidly metabolized to more polar
derivatives and rapidly excreted in bile. Metabolism of
these compounds before excretion could thereby prevent identification
of products and lead to an underestimate of the proportion of ChOx
retained. It appears from our studies that elimination of these
compounds is neither rapid nor complete after injection and that
considerable amounts are assimilated by the liver, incorporated into
lipoproteins, and released into the bloodstream.
The serum from rabbits injected with ChOx is markedly toxic to RAECs and to other types of cells. When applied to cultures individually or as a mixture in an ethanol vehicle, ChOx are cytotoxic to fibroblasts,7 monocytes and macrophages,45 endothelial cells,3 4 and smooth muscle cells.5 6 Recent studies have shown that some ChOx induce apoptosis in monocytes8 and smooth muscle cells9 ; however, these effects may be dose-related, because at high concentrations, some ChOx are acutely toxic and cause disruption of cell membranes, cell lysis,7 and tissue necrosis.46 In contrast, pure cholesterol at similar concentrations is not toxic. Our findings also show that ChOx in liposomal emulsions are essentially not toxic and that residual amounts in the blood after injection likely do not contribute directly to the cytotoxicity of serum from ChOx-injected rabbits. It is possible that ChOx in stable emulsions have limited access to cells as compared with treatments in the presence of serum and serum lipoproteins. The mechanism by which ChOx exert cytotoxic effects is thought to be related to perturbations in cholesterol metabolism4 and trafficking47 and/or to disruption of ion homeostasis in the cells,48 49 particularly effects on intracellular calcium levels. The present findings show that ChOx enrichment of the major lipoproteins in rabbits markedly increases the cytotoxicity of these lipoproteins. Endothelial injury was caused by ChOx-enriched lipoproteins, as evidenced in vivo by increased permeation across the vascular wall and accumulation of macromolecules and lipids. Previous in vitro studies by Boissonneault et al10 11 Hennig and Boissonneault,50 and Ramasamy et al51 showed that certain ChOx and ChOx-enriched LDL decreased vascular endothelial barrier functions, leading to increased transendothelial albumin transfer. This was attributed to ChOx-induced alterations in membrane structure and function. In our in vivo study, we used a 44-kDa macromolecular marker, HRP, and found a 2-fold increase in the aortic retention of 125I-HRP in ChOx-injected rabbits. This effect was similar to that found in a pilot study, in which a 3-fold increase in 125I-rabbit albumin retention (molecular weight, 40 to 50 kDa) was found in ChOx-injected rabbits (data not shown).
In these normocholesterolemic rabbits, ChOx injection
led to increased aortic cholesterol content (Table 4). This
may be related to the compromised permeability of the
arterial wall to macromolecules and particles such as LDL
or VLDL. Therefore, we postulate that circulating ChOx promote the
accumulation of cholesterol in the arterial
wall. This appears to be an early event in vascular foam cell formation
that is aggravated by hypercholesterolemia.
Although no differences were found in ChOx content in aortas between
ChOx-injected and control rabbits, this apparent discrepancy may have
been due to the low abundance of ChOx in LDL relative to its
cholesterol content or to local metabolism of
ChOx. Relatively small increases in ChOx levels could perturb the
influx versus efflux of lipoprotein cholesterol, leading to
a rapid and extensive accumulation of cholesterol and CEs
in vascular tissues.39 52 53 It is tempting to
speculate that the increased transport of albumin and HRP from
plasma to medium reflects the penetration of larger particles such as
LDL. Stemerman et al54 showed that
125I-LDL concentrations were 47 times higher in
regions of normocholesterolemic rabbit aorta where
permeability to HRP was enhanced. However, Fry et
al55 found that the extent of
125I-albumin and
125I-LDL uptake by the aortic
endothelium was similar in areas with and without
lesions. The spatial location–averaged uptake rate of
125I-albumin in
normocholesterolemic minipig aortas was 7 times higher
than that of 125I-LDL. HRP, albumin, and
LDL are likely transported across normal endothelium
from plasma by transcytosis56 and open junctions
with gap widths of 30 to 450 nm between adjacent
endothelial cells.57 Aside from
ChOx-induced membrane alterations, increased macromolecule passage
across endothelium may be related to enhanced vesicular
transport in proliferating cells58 and increased
movement through open junctions formed at sites of cell
injury59 and in proliferating
cells.60 It should be noted that some of the ChOx
have been shown to strongly inhibit gap-junction formation in
fibroblasts.61 Using methods similar to ours,
Rangaswamy et al21 infused Sprague-Dawley rats
with a single dose of ox-LDL (80 mg cholesterol/300 g body
weight) over 30 minutes and 48 hours later observed a 2- to 3-fold
increase in intimal and medial accumulation of HRP compared with
infusions with native LDL.
The angiotoxicity of ChOx observed in our studies was not as severe as reported by Imai et al,14 who found grossly visible thickening in major branches of rabbit pulmonary artery after 3 consecutive intravenous injections of cholestane triol or 25-hydroxycholesterol at 5 mg/kg per day. They also found microscopic lesions in minor branches of the pulmonary artery injected with 7-ketocholesterol or cholesterol-5,6-epoxide. These studies used younger rabbits, which may be more sensitive to cholesterol-induced atherosclerosis62 and possibly ChOx. Furthermore, the higher degree of angiotoxicity may have been due to a different method of administration. Imai et al14 used an intravenous injection of suspended crystals of ChOx in saline, in contrast to the lecithin–ChOx liposome vehicle used in our study. Thus, no animal died throughout the course of our experiments, whereas in the studies of Imai et al,14 1 in 5 rabbits died immediately after injection of pure cholesterol at 10 mg/kg.
ChOx can be assimilated as common components of foods. They have been shown to be absorbed by rat intestine,43 are present in rat lymph36 after gavage feeding, and are carried by chylomicrons in humans after a meal rich in fat and ChOx.63 This is consistent with the reported uptake and metabolism of other oxidized lipids in food. Oxidized fatty acids have been shown to be delivered to the liver by chylomicrons after a peroxide-enriched meal,64 repackaged in the liver, and released in VLDL.38 This feeding regimen accelerated the development of fatty streak lesions.65 However, the extent to which in vivo oxidation versus dietary uptake of oxidized fats contributes to the levels of circulating ChOx is unknown.
Our findings show a direct role of circulating ChOx in mediating the development of early atherosclerotic lesions under normocholesterolemic conditions, causing vascular cell injury and accumulation of cholesterol in the aortic wall. The degree to which elevated ChOx contribute to cholesterol accumulation and foam cell lesion formation is currently under study using moderately hypercholesterolemic rabbits.
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Acknowledgments |
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We express our gratitude for the excellent technical assistance of Juliana Hwang and the generosity of Steraloids, Inc, for providing various cholesterol oxidation products at a reduced cost. This work was supported by grants ES03466 (to A.S.) and HL29582 (to G.M.C.) from the National Institutes of Health, Bethesda, Md.
Received January 9, 1998; accepted May 20, 1998.
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