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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1995;15:1625-1630.)
© 1995 American Heart Association, Inc.

Articles

Effect of the Antioxidant N,N'-Diphenyl 1,4-Phenylenediamine (DPPD) on Atherosclerosis in ApoE-Deficient Mice

Rajendra K. Tangirala; Florencia Casanada; Elizabeth Miller; Joseph L. Witztum; Daniel Steinberg; Wulf Palinski

From the Department of Medicine, University of California San Diego, La Jolla, Calif.

Correspondence to Wulf Palinski, MD, Department of Medicine, 0682, University of California, San Diego, 9500 Gilman Dr, La Jolla, CA 92093-0682.

	Abstract

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Abstract Apolipoprotein (apo) E–deficient mice develop atherosclerotic lesions that contain epitopes formed during the oxidative modification of lipoproteins, and they demonstrate high titers of circulating autoantibodies against such epitopes, suggesting that this murine strain may provide a model to investigate the atherogenic mechanisms of oxidized lipoproteins (Palinski et al, Arterioscler Thromb. 1994;14:605-616). To test the hypothesis that lipoprotein oxidation contributes to lesion formation in apoE-deficient mice, we studied the effect of the antioxidant N,N'-diphenyl 1,4-phenylenediamine (DPPD) in mice fed a high-fat diet containing 0.15% cholesterol. Animals were divided into two subgroups matched for sex and plasma cholesterol levels, and DPPD (0.5% wt/wt) was added to the diet of one subgroup. Throughout the 6 months of intervention, DPPD treatment had no significant effect on plasma cholesterol. Plasma levels of DPPD at the end of the experiment were 33.1 µmol/L. As judged by resistance to loss of polyunsaturated fatty acids, lipoproteins (d <1.019 g/mL) from DPPD-treated animals showed greater resistance to copper-induced oxidation than lipoproteins from control animals. In addition, there was a greater than twofold prolongation of the lag time in the formation of conjugated dienes in the LDL and IDL fractions of DPPD-treated mice. Atherosclerosis was significantly reduced, by 36% in the DPPD-treated mice (14.0±4.53% of aortic surface area versus 21.9±11.6%; n=32; P<.02). These results are consistent with the hypothesis that lipoprotein oxidation contributes to atherogenesis in apoE-deficient mice. However, further studies with other antioxidants are needed to validate this hypothesis.

Key Words: lipoproteins, oxidized • antioxidants • mice, gene-targeted • apolipoprotein E • arteriosclerosis

	Introduction

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The oxidative modification of LDL is thought to be an important if not essential contributor to the formation of atherosclerotic lesions.^{1 2 3 4} Evidence for the in vivo occurrence of lipoprotein oxidation includes the immunocytochemical demonstration of epitopes formed during the oxidation of lipoproteins in atherosclerotic lesions^{5 6 7 8 9} and the fact that LDL extracted from lesions has physical and biologic properties of oxidized LDL (OxLDL).¹⁰ Furthermore, circulating autoantibodies against oxidized LDL are prevalent in several species,⁷ and immune complexes between such autoantibodies and OxLDL are present in lesions.¹¹ Strong evidence for the "oxidation hypothesis" is provided by antioxidant intervention studies in animal models of atherosclerosis.^{12 13 14 15 16 17 18} A large number of in vitro studies have documented that by-products of lipid peroxidation and apoprotein modification have atherogenic properties and that both early and late forms of OxLDL may contribute to atherogenesis by a number of mechanisms.^{1 2 3 4 19} However, the contribution of these postulated mechanisms to lesion formation in vivo remains to be established.

Recently, murine models such as apoE-deficient mice and LDL receptor–negative mice have been generated that develop extensive atherosclerosis. These models could prove invaluable in assessing the atherogenic relevance of factors involved in the oxidative modification of lipoproteins.^{20 21} ApoE-deficient mice are characterized by spontaneous and very pronounced hypercholesterolemia, even when fed a low-fat diet.^{22 23} Atherosclerotic lesions in apoE-deficient mice occur throughout the aortic tree and show many features typical of lesions found in other animal models of atherosclerosis.^{24 25 26} Recent immunocytochemical studies demonstrated the presence of epitopes formed during the oxidative modification of LDL and other lipoproteins ("oxidation-specific" epitopes) in their atherosclerotic lesions.²⁶ Additional evidence for the occurrence of oxidative modification in apoE-deficient mice is provided by their unusually high titers of circulating autoantibodies against oxidation-specific epitopes.²⁶ However, to date no evidence has been provided for a causative role of lipoprotein oxidation in atherogenesis in this or other murine models. In order to provide such evidence, we determined the effect of treatment with the antioxidant DPPD on aortic atherosclerosis in apoE-deficient mice.

	Methods

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Mice and Diets
A colony of homozygous apoE-deficient mice was generated from a breeding pair generously provided by Dr J. Breslow.²² These mice were hybrids with a C57 BL/6x129ola background. Two groups of 20 apoE-deficient mice, aged 3 months, were matched for sex, body weight, plasma cholesterol, and, as much as possible, litter. Both groups were fed a high-fat diet (Harlan Teklad 88137) containing 128 g/kg milk fat and 0.15% cholesterol but free of sodium cholate. One group was treated with DPPD; the other group served as a control. DPPD (0.5% wt/wt) (Aldrich Chemical Co) was added to the diet by dissolving it in diethylether, coating the chow with a fine mist of this solution, and allowing complete evaporation of the solvent, before use. The control group was fed chow treated in a similar way, except for the absence of DPPD. Mice were maintained on these diets for 6 months, during which time their body weight and total plasma cholesterol and triglyceride levels were monitored at approximately monthly intervals. In view of the large number of animals involved and the labor-intensive final procedures, the experiment was carried out in two time-staggered subgroups, under identical conditions. Comparison of body weights and plasma lipids revealed no differences between subgroups subjected to identical conditions, and data from these subgroups were therefore pooled.

Isolation of Lipoproteins and Analytical Methods
To monitor plasma cholesterol and triglyceride levels during the 6 months of dietary intervention, 100 µL of blood was collected in heparinized tubes from the retro-orbital venous plexus of anesthetized mice. At the end of the study 300 to 800 µL of blood was collected from the vena cava of anesthetized mice immediately before sacrifice, using heparinized syringes. Total plasma cholesterol and triglyceride levels were determined with an automated enzymatic procedure (Boehringer Mannheim Diagnostics). Because of the limited amounts of plasma that can be obtained from an individual mouse, terminal plasma samples from two to four mice were pooled for the determination of the fatty acid composition, the assessment of the susceptibility of lipoproteins to oxidation, and the determination of the plasma concentration of DPPD.

Lipoproteins (d <1.019 g/mL) were separated by ultracentrifugation (30 000 rpm, 5°C, 14 hours), using a Beckman L7-65 ultracentrifuge with a 50.3 rotor (Beckman Instruments). The density cutoff was chosen because VLDL and IDL account for most of the elevation in plasma lipoprotein levels in apoE-deficient mice on a high-fat diet.²² Protein concentrations of lipoprotein fractions were determined by the method of Lowry et al.²⁷ The total lipids of the (d <1.019 g/mL) plasma lipoprotein fractions were extracted using a modification of the method of Folch et al.²⁸ The lipids extracted from the lipoprotein fractions were then transmethylated²⁹ and analyzed by gas chromatography (model 3700, Varian Associates) using a column of 10% Silar 5CP on Gas Chrom QII, 100/120 mesh (Alltech Associates), as previously described.³⁰ The absolute quantity of total fatty acids in the sample was determined by addition of an internal standard of pentadecanoic acid (15:0) to each sample before lipid extraction. The amount of fatty acids with carbon chains longer than 20 was less than 3% of the total, so each run was truncated after elution of the C20 peaks.

Determination of DPPD Concentrations
The concentration of DPPD was determined both in plasma and in the lipoprotein fraction (d <1.019 g/mL). Aliquots of murine plasma or lipoprotein fractions were pooled from three to four mice to obtain sufficient material for an accurate determination. Because DPPD is a strongly basic compound, samples were acidified by addition of an equal volume of 0.05 mol/L sulfuric acid, and lipids were extracted by the method of Bligh and Dyer.³¹ Lipid extracts were resuspended in isopropanol and analyzed by reverse-phase high-performance liquid chromatography (Hitachi Instruments), using a Spherisorb C18 reverse-phase column (150x4.6 mm). Methanol/water/acetic acid (90:9.8:0.2 vol/vol/vol) was used for elution, and fluorescence was measured (excitation wavelength 310 nm, emission wavelength 405 nm) using an F-1050 fluorscence spectrophotometer (Hitachi). DPPD concentrations were determined using a standard curve obtained by adding DPPD to murine plasma from control mice and subjecting the plasma to the same extraction procedures.

Susceptibility of Lipoproteins to Oxidation
The susceptibility of freshly isolated lipoproteins to oxidation was assessed as follows. Immediately after isolation, duplicate aliquots of the nondialyzed d <1.019 g/mL lipoprotein fractions (50 µg protein, corresponding to 299 µg cholesterol) were incubated with 10 µm CuSO₄ in phosphate-buffered saline (PBS) (500 µL final volume) for 3.5, 7, and 16 hours at 37°C. After the incubation, lipids were extracted as described above. The initial concentration of each fatty acid before addition of copper was defined as 100%, and the susceptibility of lipoproteins to oxidation was measured in terms of the percentage decrease of total polyunsaturated fatty acids (linoleate and arachidonate) over time during oxidation.

Because the amount of plasma available from the mice used in the intervention study was insufficient for both measurement of the degradation of polyunsaturated fatty acids and measurement of the lag times in the formation of conjugated dienes, the latter was measured subsequently in two additional groups of four apoE-deficient mice each, fed the same diets with and without DPPD. After 2 months, the LDL (1.019 <d <1.063 g/mL), IDL (1.006 <d <1.019 g/mL), and VLDL (d <1.006 g/mL) were prepared by sequential ultracentrifugation from each individual plasma sample. The lipoprotein fractions were extensively dialyzed against PBS (without EDTA) to remove the heparin, adjusted to 100 µg protein/mL with PBS, and CuSO₄ was added at a final concentration of 5 µmol/L. The formation of conjugated dienes was determined in a Uvikon 810 spectrophotometer at 234 nm.³² Absorption at the beginning of the reaction was set to zero. Lag times were determined graphically as the time point at which the tangent to the curve during the maximum slope of the propagation phase intercepted the time axis.

Quantification of Aortic Atherosclerosis
After 6 months of dietary intervention, aortas of the apoE-deficient mice were prepared as previously described.²⁶ After collection of the terminal blood sample from the vena cava, the animals were sacrificed. The aortic tree was perfused for 20 minutes with PBS containing 20 µmol/L butylated hydroxytoluene (BHT) and 2 mmol/L EDTA, pH 7.4, using a cannula inserted into the left ventricle and allowing free efflux from an incision in the vena cava. For an initial fixation of the aorta, perfusion was continued for an additional 20 minutes with formal-sucrose (4% paraformaldehyde, 5% sucrose, 20 µmol/L BHT, 2 mmol/L EDTA, pH 7.4). Adventitial tissue was removed as much as possible, in situ. The aorta was opened longitudinally, from the heart to the iliac bifurcation, pinned out on a black wax surface in a dissecting pan, fixed with formal-sucrose for an additional 12 hours, and stained with Sudan IV.

Image analysis was performed as described in detail in Reference 33³³ . Three partial images of each stained aorta were captured with a Sony DXC-960MD three-chip color video camera. Image analysis was performed on 24-bit color images, using OPTIMAS 4.0 image analysis software (Bioscan), an Oculus TCX color frame grabber with 4 megabytes of frame buffer memory (Coreco), and a separate VGA image monitor for the processed image. After retouching of the needle holes in the captured image, the extent of atherosclerosis was determined by the software in a largely operator-independent fashion, using a selection of threshold ranges in the three basic colors. This threshold was initially determined in a few randomly selected arteries by matching the lesion areas and shapes highlighted on the processed image to those of the actual arteries (observed through a stereo microscope). The selection was verified by a second investigator (W.P.) blinded for the identity of the image analyzed. The selected threshold could be used for the subsequent analysis of most arteries. Nevertheless, during image analysis, the aortas were always used for reference, to ensure that the selected threshold truly reflected atherosclerotic lesions. The extent of atherosclerosis was then determined by the software and expressed as percent of the aortic surface area covered by lesions.

Analytical and Statistical Analysis
Results were expressed as mean±SEM. Body weights, cholesterol levels, and extent of atherosclerosis were analyzed by ANOVA and Student's unpaired two-tailed t test. Multi-way ANOVA for repeated determinations was used to evaluate the decrease of unsaturated fatty acids during copper oxidation.

	Results

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Body Weight, Plasma Cholesterol, and Triglyceride Levels
During the 6 months of intervention, the body weight of both groups of mice increased significantly, although the mean body weight of DPPD-treated mice was slightly but significantly less than that of control mice at the end of the experiment (40.8 versus 34.7 g, P<.05) (Table). In addition, more animals died in the DPPD group than in the control group (5 of 20 versus 1 of 20). Furthermore, two DPPD-treated animals died from accidental anesthesia overdose, during one of the monthly blood samplings. Thus, the number of animals available for evaluation was 19 in the control group and 13 in the DPPD group. Determinations of plasma cholesterol and triglyceride levels showed that feeding of diets containing 0.15% cholesterol raised the plasma cholesterol and triglyceride levels within the first month of intervention, and that they remained high and relatively stable until the end of the 6 months of intervention (Table). The time-averaged plasma cholesterol levels in the DPPD-treated and control animals were not significantly different (Table). The DPPD-treated animals had slightly higher nonfasting triglyceride levels at the terminal point.

View this table: [in this window] [in a new window]   Table 1. Body Weight, Total Plasma Cholesterol Levels, and Triglyceride Levels in ApoE-Deficient Mice Fed a High-Fat Diet With or Without 0.5% N,N'-Diphenyl 1,4-Phenylenediamine (DPPD)

DPPD Levels
Plasma levels of DPPD were determined at the end of the study, in four samples pooled from two to three DPPD-fed animals each, and four pooled samples from control animals. The average DPPD concentration in the treated group was 33.1 µmol/L (pooled samples ranged from 20.4 to 44.4 µmol/L). Plasma samples from control mice contained no measurable concentration of DPPD. The plasma concentrations obtained in mice fed 0.5% DPPD were approximately one half of the plasma concentration reported in rabbits fed a diet containing 0.5% cholesterol and 1% DPPD for 2 months (59 µmol/L).¹⁵ The DPPD concentration in the pooled lipoprotein fractions (d <1.019 g/mL) was 59.2 µmol/L, or 8.3 nmol/mg cholesterol (n=3).

Effect of Diet on Fatty Acid Composition of Lipoproteins
Because the total fatty acid content of lipoproteins affects their susceptibility to oxidative modification,^{34 35} we analyzed the fatty acid composition of pooled plasma lipoprotein fractions (d <1.019 g/mL) obtained from DPPD-treated and control animals at the end of the 6 months of intervention. As shown in Fig 1, oleate was the predominant unsaturated fatty acid, accounting for 35% of total fatty acids. DPPD treatment had no significant effect on the fatty acid composition.

View larger version (22K): [in this window] [in a new window]   Figure 1. Bar graph shows fatty acid composition of the d <1.019 g/mL lipoprotein fraction isolated from the pooled plasma samples of DPPD-treated and control mice fed a high-fat diet, at the end of the 6 months of intervention. Results are expressed as the percent of total fatty acids. Less than 3% of the total fatty acids had carbon chains longer than 20 and are not shown. Each sample was pooled from two to three animals, and data represent the mean±SEM of four pooled samples. Error bars not shown were smaller than 0.1%.

Effect of Diet and DPPD Treatment on Lipoprotein Oxidation
The effect of DPPD on the susceptibility of lipoproteins to oxidation was initially assessed by determining the decrease in polyunsaturated fatty acids (linoleic acid and arachidonic acid) during progressive copper-induced oxidation.³⁰ Since the amount of plasma that can be obtained from an apoE-deficient mouse is small and contains predominantly VLDL and IDL (rather than LDL), we used the d <1.019 g/mL lipoprotein fraction isolated from pooled plasma samples to provide a measure of antioxidant protection conveyed by DPPD to apoB-containing lipoproteins. As shown in Fig 2, DPPD clearly protected the d<1.019 g/mL lipoproteins from oxidation, as indicated by a slower rate of loss of polyunsaturated fatty acids over time. The differences between the DPPD-treated group and the control group were significant for both arachidonic and linoleic acid (P<.05).

View larger version (18K): [in this window] [in a new window]   Figure 2. Graphs show percent of arachidonic acid (20:4) (left) and linoleic acid (18:2) (right) remaining during progressive oxidative modification of the d <1.019 g/mL lipoprotein fraction, induced in vitro by incubation with 10 µmol/L of copper at 37°C. The concentration of each fatty acid before addition of copper was defined as 100%. Lipoprotein fractions were isolated from four pooled plasma samples of animals fed a high-fat diet and were the same samples used for the analysis of fatty acid composition (see Fig 1). Data are mean±SEM.

The antioxidant protection of lipoprotein fractions by DPPD was also determined by measuring the lag times in the formation of conjugated dienes. For these determinations, plasma was obtained from four DPPD-treated and four control mice after 2 months of intervention in a separate study. In contrast to the pooled samples used to determine the fatty acid degradation, lipoproteins were isolated and tested from individual animals. Fig 3 shows the formation of conjugated dienes under standardized conditions (100 µg protein, 5 µmol/L Cu²⁺ ions). The mean lag time for LDL (1.019 < d <1.063 g/mL) was 386±30 minutes (mean±SD, n=4) for control mice versus 678±68 minutes for three DPPD-treated mice and greater than 1000 minutes for a fourth animal (Fig 3A). Because most lipoproteins of apoE-deficient mice fed a cholesterol-enriched diet are found in the IDL/VLDL density range, we also isolated these fractions. However, the VLDL (d <1.006 g/mL) had too much turbidity to be used for the spectrophotometric determination. The antioxidant protection of the IDL (1.006 <d <1.019 g/mL) was very similar to that of the LDL. The mean lag time was 351±21 minutes in control mice (n=4) versus 746±107 minutes in DPPD-treated animals (n=4) (Fig 3B). The mean cholesterol content of the lipoprotein fractions from DPPD-treated animals was similar to that of control mice (LDL, 2.67±0.13 mg cholesterol/mg protein in control animals versus 2.74±0.18 mg cholesterol/mg protein in DPPD-treated animals; IDL, 6.00±0.45 in control animals versus 6.03±0.53 in treated animals). The lag-time measurements confirmed the results obtained by measuring the degradation of polyunsaturated fatty acids and indicated a good antioxidant protection of the lipoproteins by DPPD.

View larger version (54K): [in this window] [in a new window]   Figure 3. Graphs show formation of conjugated dienes in LDL (1.019 <d <1.063 g/mL) (A) and in IDL (1.006 <d <1.019 g/mL) (B) isolated from apoE-deficient mice fed a high-fat diet with and without 0.5% DPPD for 2 months. Lipoproteins were isolated from four control animals and four DPPD-treated animals as described in "Methods," and conjugated diene formation during incubation of 100 µg/mL protein with 5 µmol/L Cu2+ was measured at 234 nm. LDL from a control animal (Control-LDL) and from a DPPD-treated animal (DPPD-LDL) incubated without Cu2+ served as controls.

Effect of DPPD on Atherosclerosis
After 6 months of dietary intervention with the high-fat diet, the extent of atherosclerosis in the aortic tree of control and DPPD-treated mice was evaluated by image analysis. In all animals atherosclerotic lesions were present throughout the aortic tree. However, aortas from DPPD-treated animals showed less extensive lesions than aortas from control animals. Morphometric quantitation of the extent of aortic atherosclerosis clearly demonstrated that DPPD treatment significantly reduced atherosclerosis in mice on the high-fat diet (DPPD group, 14.0±4.53% of total aortic surface area covered by lesions; control group, 21.9±11.6%; n=32; P<.02) (Fig 4). The relative reduction of atherosclerosis by DPPD (36.1%) was even more pronounced (47.6%) when the data for the heavily involved aortic arch were excluded.

View larger version (25K): [in this window] [in a new window]   Figure 4. Bar graph shows percent of the aortic surface covered by atherosclerotic lesions after 6 months of intervention. Data represent results obtained in 32 apoE-deficient mice fed a high-fat diet (19 controls, 13 DPPD-treated). The extent of lesions is reported for the total aorta (cross-hatched bars), and for the thoracic and abdominal aorta without the arch (open bars). The reduction in atherosclerosis in DPPD-treated animals vs controls was statistically significant (*P<.05, **P<.02). Values are mean±SEM.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
Transgenic and gene-targeted animal models may be invaluable to study the in vivo relevance of factors involved in lipoprotein oxidation and the impact of these factors on atherogenesis. ApoE-deficient mice^{22 23 24 25 26} and LDL receptor–negative mice^{36 37} are two such models. These mice develop extensive atherosclerosis in major arteries, and the distribution of lesions and their cellular composition closely resemble that of other animal species and of humans. Using immunocytochemistry, we previously documented the presence of epitopes of oxidized lipoproteins in atherosclerotic lesions of apoE-deficient mice and developed a morphometric method that allows for the accurate quantification of the extent of atherosclerosis in murine aortas.²⁶ In the present study, we demonstrated that antioxidant intervention significantly reduces lesion formation in apoE-deficient mice. These results support the hypothesis that lipoprotein oxidation contributes to atherogenesis in this model.

Extensive data on the antiatherogenic effect of several potent lipophilic antioxidants exist in other animal models. Carew and colleagues demonstrated that probucol, a lipid-lowering drug with strong antioxidant effect, inhibited atherosclerosis in LDL receptor–deficient rabbits by a mechanism independent of its cholesterol-lowering effect.¹² Very similar results were obtained with probucol in rabbits by Kita et al,¹³ Daugherty et al,¹⁶ and more recently in nonhuman primates by Sasahara et al.¹⁸ Similar data were also obtained with other potent antioxidant compounds that do not lower plasma cholesterol levels, such as BHT, DPPD, and a probucol analogue.^{14 15 17}

Preliminary experiments preceding this study indicated that probucol has a remarkable lipid-lowering effect in apoE-deficient mice (as previously reported for other murine strains³⁸ ), which would have complicated the assessment of its antioxidant effect on atherosclerosis, and that BHT was toxic for this particular murine strain. We therefore used DPPD for our intervention study. DPPD has mutagenic effects in some species, but no pertinent toxicological data were available to predict the pharmacokinetic properties or potential toxicity of DPPD in apoE-deficient mice. During the 6 months of the intervention, weight gain in the DPPD-treated group was slightly less than in the control animals, and more DPPD-treated animals died than did control animals. Despite the fact that the DPPD-treated mice gained weight less rapidly, their plasma cholesterol levels were not different from control animals. A similar situation occurred when cholesterol-fed rabbits were given DPPD.¹⁵ This may indicate toxicity but could also be related to a relative aversion of the mice to the DPPD-chow. Because the DPPD-treated animals continued to gain weight, albeit at a slower rate, and because their plasma cholesterol levels were not different from untreated control animals, we do not believe that toxicity was responsible for the decrease in atherosclerosis, but we cannot exclude this possibility. We do not know the cause of death in the animals that died. Atherosclerosis in apoE-deficient mice often involves the media,^{25 26} and we have occasionally observed death from aortic aneurysms in apoE-deficient mice. However, aortas of surviving animals in the DPPD group gave no indication of such aneurysms.

Our data demonstrate that lipoproteins from DPPD-treated animals were better protected against oxidation than lipoproteins from control animals (Figs 2 and 3) and that DPPD treatment was effective in reducing atherogenesis (36.1% reduction of lesions in the entire aorta, 47.6% reduction excluding the arch) (Fig 4). Taken together with the presence of "oxidation-specific" epitopes in lesions and the presence of high titers of circulating autoantibodies to OxLDL,²⁶ these data support the hypothesis that oxidation of lipoproteins contributes significantly to atherogenesis in this murine model. This suggests that the apoE-deficient mouse may indeed be a valid model in which to study the role of oxidative processes. However, in view of the fact that DPPD may have some toxicity in apoE-deficient mice, it will be important to test the antiatherogenic efficacy of other antioxidants in these mice and in other murine models of atherosclerosis.

	Selected Abbreviations and Acronyms

 

DPPD = N,N'-diphenyl 1,4-phenylenediamine IDL = intermediate-density lipoprotein LDL = low-density lipoprotein Ox-LDL = oxidized LDL VLDL = very-low-density lipoprotein

	Acknowledgments

 
These studies were supported by NHLBI grant HL14197 (La Jolla Specialized Center of Research in Arteriosclerosis). We thank Drs A. Plump and J. Breslow for supplying the apoE-deficient mice that enabled us to establish our own colony. We also thank Jennifer Pattison, Richard Elam, Joe Juliano, and Frank Peralta for excellent technical assistance.

Received October 6, 1994; accepted August 2, 1995.

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