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Arteriosclerosis, Thrombosis, and Vascular Biology. 1998;18:1885-1894

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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1998;18:1885-1894.)
© 1998 American Heart Association, Inc.

Original Contributions

Arterial Injury by Cholesterol Oxidation Products Causes Endothelial Dysfunction and Arterial Wall Cholesterol Accumulation

James X. Rong; Shanthini Rangaswamy; Lijiang Shen; Ravi Dave; Yi H. Chang; Hazel Peterson; Howard N. Hodis; Guy M. Chisolm; Alex Sevanian

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


*    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


*    Introduction
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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.


*    Methods
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Injection Procedure
Twenty-one male NZW rabbits ({approx}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 1Down. 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 1Down, 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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  		Table 1. Composition of the ChOx Mixture



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  		Figure 1. ChOx and pure cholesterol injection and blood sampling protocols. Days of injection are shown along the time scale. Dashed arrows indicate the start of each injection series. Solid arrows indicate blood sampling times. Note that except for the first injection, blood was sampled immediately before each injection. Rabbits were killed 10 days after the last injection.

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 {approx}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:

where both basal and induced release were determined by counting aliquots of the medium from cell cultures after treatment with rabbit serum obtained before injection (basal release) and from serum obtained after injection of the ChOx mixture (induced release). Maximum (100%) release was obtained by treating the cells with detergent (Triton X-100, 0.5%). The remaining medium was removed, and the cells were trypsinized and counted. The surviving fraction (SF) was expressed as follows:

Similarly, the cytotoxicity of the freshly isolated LDL/VLDL fraction from each animal was determined in 24-well Corning plates after addition to subconfluent RAEC cultures. Treatments used a 10% serum-equivalent concentration (the serum-equivalent concentration corresponds to the percentage of lipoprotein typically existing in serum) with 15% lipoprotein-deficient FBS (prepared as described previously1 ). After 24 hours, the medium was removed and the cells were trypsinized and counted. SF was expressed as described above. Alternatively, the LDL/VLDL fraction was added to V79 cells (Chinese hamster lung fibroblasts)22 at a 10% serum-equivalent concentration in DMEM containing 10% lipoprotein-deficient FBS, and SF was determined as described above. Details for measuring cytotoxicity by means of plating efficiency and SF assays are provided elsewhere.1

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 ({approx}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.


*    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 2Down). 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 {approx}70% of the baseline level (Table 2Down) during this period. For rabbits injected with pure cholesterol, plasma cholesterol levels peaked {approx}30 minutes after a single injection, reaching {approx}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 2Down and Table 2Down). 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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  		Figure 2. Pharmacokinetics of ChOx or pure cholesterol (chol) after intravenous injection. Concentrations of cholesterol and total ChOx were determined in plasma at various times during and after intravenous injection of ChOx or pure cholesterol as described in Methods. Baseline data are not shown. Determinations of plasma levels were initiated at the start of the first injection. Data obtained within 2 hours after the first injection were from 1 representative rabbit in the pure cholesterol or ChOx groups, and data obtained for intervals beyond 2 hours after the first injection represent the mean of 4 animals in each group. SEMs are shown at end points only. Arrows indicate the times at which serum was taken for cytotoxicity experiments.


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  		Table 2. Cholesterol and ChOx Concentrations in Plasma at Baseline and End Point of the Experiment (Killing)

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 3Down). 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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  		Table 3. Distribution of ChOx in LDL and VLDL Cholesterol Fractions at Killing

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 3Down, serum from ChOx-injected rabbits obtained 5 and 30 minutes after injection was toxic to RAECs, reducing the SF to {approx}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 2Up 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 {approx}50% of the 14C-adenine–derived radioactivity preincorporated into cells (Figure 4Down). 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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  		Figure 3. Cytotoxicity of rabbit serum to RAECs, expressed as the surviving fraction (SF). Subconfluent cells were treated for 24 hours with DMEM:M199 (80:20) containing 15% rabbit serum, which was obtained at various times after the first injection of the ChOx mixture (black bars) or pure cholesterol (white bars). The positive controls (hatched bar) were treated with cholestane triol (CT) at 20 µg/mL in complete medium. SF was determined as described in Methods. *P<0.01 vs baseline. **P<0.05 vs baseline.



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  		Figure 4. Cytotoxicity of rabbit serum to RAECs, determined by specific 14C release. Subconfluent cells preloaded with 14C-adenine were treated with 15% rabbit serum, which was prepared from blood drawn from rabbits at various times after the first injection of the ChOx mixture (black bars) or pure cholesterol (white bars). Cells were treated with DMEM:M199 (80:20) for 24 hours. Positive controls (left hatched bar) were treated with cholestane triol at 20 µg/mL in complete medium. Specific 14C release was determined as described in Methods. Release of counts from cells using serum obtained at baseline was set as 0%. Maximum release (100%, right hatched bar) was obtained by lysing the cells with Triton X-100 and counting the radioactivity in the total sample (medium plus cell lysates).

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 5Down, 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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  		Figure 5. Cytotoxicity of the LDL/VLDL fraction from rabbit plasma to RAECs (drawn after the first injection). Freshly isolated LDL/VLDL fractions from rabbit plasma were drawn at various times after the first injection of the ChOx mixture (black bars) or pure cholesterol (white bars). The lipoproteins were incubated with subconfluent cells at a 10% serum-equivalent dose in 15% LDL/VLDL-deficient FBS for 24 hours. Untreated controls (UC, right hatched bar) were treated with complete medium. The positive controls (left hatched bar) were treated with cholestane triol (CT) at 20 µg/mL in complete medium. The surviving fractions (SFs) were determined as described in Methods. *P<0.01 vs the baseline value of the same ChOx-injected rabbit.

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 1Up). Cytotoxicity assays were then performed using V79 fibroblasts. As shown in Figure 6Down, 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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  		Figure 6. Cytotoxicity of the LDL/VLDL fraction from rabbit plasma to V79 fibroblasts (drawn after multiple injections). LDL/VLDL fractions were isolated from rabbit plasma drawn at baseline and the end of each injection series (24 hours after the last injection in each series) of the ChOx mixture (black bars) or pure cholesterol (white bars). The lipoproteins were incubated with subconfluent cells at a 10% serum-equivalent dose in 10% LDL/VLDL-deficient FBS for 24 hours. Positive controls (left hatched bar) were treated with cholestane triol at 20 ng/mL in DMEM containing 10% FBS. Untreated controls (right hatched bar) were treated with DMEM containing 10% FBS. Surviving fractions (SFs) were determined as described in Methods. *P<0.01 vs the baseline value of the same ChOx-injected rabbit and vs the LDL/VLDL fraction from the cholesterol-injected rabbit after the same number of injections.

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 4Down). Aortas from the ChOx-injected rabbits had significantly higher tissue cholesterol levels (P<0.01) than cholesterol- and lecithin-injected rabbits (Table 4Down), 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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  		Table 4. Cholesterol or Total ChOx Content in Rabbit Aorta and Liver at Killing

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 7Down, 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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  		Figure 7. 125I-HRP retained in rabbit aortas injected with ChOx (black bar, n=7), pure cholesterol (white bar, n=5), and lecithin (hatched bar, n=3). After each group received 20 injections, 5 to 10 µCi 125I-HRP was injected intravenously into rabbits and allowed to circulate for 20 minutes before killing. Aortas were removed, and retained radioactivity was determined as described in Methods. *P<0.01 vs rabbits injected with pure cholesterol and lecithin.

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 8Down. 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 8Down, inset). The difference between the cholesterol- and lecithin-injected control groups was not significant; thus, the data were combined as shown in Figure 8Down.



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  		Figure 8. HRP transmural concentration profiles across the aortic wall of the ChOx-injected rabbits (n=3) or controls (pure cholesterol-injected, n=2; lecithin, n=2). After each group received 20 injections, HRP (50 mg/kg body weight) was injected intravenously into rabbits and allowed to circulate for 20 minutes before killing. Transmural HRP concentration profiles were determined as described in Methods and plotted against the normalized distance from the endothelium, ie, intima (0.0), to the adventitia (1.0). Data were plotted as mean+SEM for ChOx-injected rabbits and mean-SEM for controls. Where SE bars are not shown, the SE is within the symbols. *P<0.05 vs controls.


*    Discussion
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 up arrowMethods
 up arrowResults
 *Discussion
down arrowReferences
 
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{alpha}-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-{alpha}-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 2Up and 3Up). 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 4Up). 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 3Up). 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{alpha}-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{alpha},6{alpha}-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 4Up). 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.


*    Acknowledgments
 
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.


*    References
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 up arrowAbstract
 up arrowIntroduction
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 *References
 

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