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Arteriosclerosis, Thrombosis, and Vascular Biology. 1999;19:1960-1968

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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1999;19:1960-1968.)
© 1999 American Heart Association, Inc.

Atherosclerosis and Lipoproteins

Genetic Background Determines the Extent of Atherosclerosis in ApoE-Deficient Mice

Hayes M. Dansky; Sherri A. Charlton; John L. Sikes; Simon C. Heath; Ronit Simantov; Lawrence F. Levin; Pei Shu; Karen J. Moore; Jan L. Breslow; Jonathan D. Smith

From the Laboratory of Biochemical Genetics and Metabolism (H.M.D., S.A.C., J.L.S., J.L.B., J.D.S.) and Laboratory of Statistical Genetics (S.C.H.), Rockefeller University, and the Division of Hematology-Oncology (R.S.) and Division of Cardiology (L.F.L.), Department of Medicine, Cornell University Medical College, New York, NY; and Millennium Pharmaceuticals, Inc (P.S., K.J.M.), Cambridge, Mass.

Correspondence to Jonathan D. Smith, Rockefeller University, Box 179, 1230 York Avenue, New York, NY 10021. E-mail smithj{at}rockvax.rockefeller.edu


*    Abstract
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 *Abstract
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Abstract—Two strains of ApoE-deficient mice were found to have markedly different plasma lipoprotein profiles and susceptibility to atherosclerosis when fed either a low-fat chow or a high-fat Western-type diet. FVB/NJ ApoE-deficient (FVB E0) mice had higher total cholesterol, HDL cholesterol, ApoA1, and ApoA2 levels when compared with C57BL/6J ApoE-deficient (C57 E0) mice. At 16 weeks of age, mean aortic root atherosclerotic lesion area was 7- to 9-fold higher in chow diet–fed C57 E0 mice and 3.5-fold higher in Western diet–fed C57 E0 mice compared with FVB E0 mice fed similar diets. Lesion area in chow diet–fed first-generation mice from a strain intercross was intermediate in size compared with parental values. The distribution of the lesion area in 150 chow diet–fed second-generation progeny spanned the range of the lesion area in both parental strains. There were no correlations between total cholesterol, non-HDL cholesterol, HDL cholesterol, ApoA1, ApoA2, ApoJ, or anti-cardiolipin antibodies and lesion area in the second-generation progeny. Thus, a genomic approach may succeed in identifying the genes responsible for the variation in atherosclerosis susceptibility in these 2 strains of ApoE-deficient mice, which could not be explained by measured plasma parameters.


Key Words: hypercholesterolemia • lipoproteins • apolipoproteins • intercross • oxidation


*    Introduction
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 *Introduction
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Family history is a powerful independent risk factor for the development of coronary heart disease. Inheritance patterns in most families with a high incidence of coronary heart disease suggest that atherosclerosis susceptibility is not transmitted as a single mendelian trait. The complexity of the disease has made it difficult to isolate genetic factors that are linked to atherosclerotic disease in humans. Performing human genetic linkage studies requires a large study group, and studies are confounded by difficulties in assignment of phenotype because of incomplete penetrance, by the presence of risk modifying diseases, and by differences in subject environmental exposure and lifestyle.

The availability of numerous inbred mouse strains provides an experimental approach in which to study the effect of genetic background on a disease phenotype. Mutant mice carrying disease genes can be backcrossed to wild-type inbred strains to generate lines of genetically inbred mutant (congenic) mice. The congenic mice can then be studied to determine the effect of genetic background on the disease phenotype, and to map and identify disease-modifying genes. This approach has already been used to study the effect of varying genetic background on obesity,1 2 diabetes,3 4 transplant rejection,5 hypertension,6 and immunological responses.7

Genetic studies of atherosclerosis, using mouse models, have mainly involved studies in which different inbred strains of mice are fed a high-fat, high-cholesterol, cholate-containing diet developed in the laboratory of Paigen et al.8 9 By using this diet, inbred strains were characterized as susceptible, resistant, or intermediate, based on quantitative lesion area measurements.10 Although no murine model fully recapitulates all of the features of human atherosclerosis, a potential drawback to using mice fed a high-fat, high-cholesterol, cholate-containing diet is that the diet itself is inflammatory when fed to certain strains of mice, and results in liver transaminase elevations, activation of hepatic NF{kappa}B, increases in acute-phase reactants such as serum amyloid A, and liberation of inflammatory cytokines.11 It is unclear whether this inflammation is similar or different from the inflammatory process present in human atherosclerotic lesions. In addition, atherosclerotic lesions in mice fed this diet are largely limited to foam cell lesions at the base of the aorta, and prolonged feeding of atherogenic diets is necessary to obtain fibroproliferative lesions.12

Gene targeting and transgenic technology have provided mutant mouse models of hypercholesterolemia and atherosclerosis that do not require feeding high-fat, high-cholesterol, cholate-containing diets. When fed a low-fat, low-cholesterol chow diet, ApoE-deficient mice have spontaneous elevations in plasma cholesterol because of impaired clearance of cholesterol-rich remnant particles.13 Although the lipoprotein profile in the ApoE-deficient mouse is markedly different from the lipoprotein profile in the typical patient with hypercholesterolemia, the atherosclerotic lesions that develop in the ApoE-deficient mouse have many features in common with human atherosclerosis.13 Lesions in the ApoE-deficient mouse, as in humans, tend to develop at vascular branch points and progress from foam cell stage to the fibroproliferative stage with well-defined fibrous caps and necrotic lipid cores,14 although plaque rupture has not been observed in ApoE-deficient mice or in any other mouse model. Progression of lesions appears to occur at a faster rate than in humans atherosclerosis; however, the rapidity of lesion progression can be advantageous in many experimental situations.

In the present study, we report for the first time that genetic background has a major effect on atherosclerosis susceptibility in 2 strains of ApoE-deficient mice. C57Bl/6J ApoE-deficient (C57 E0) mice were much more susceptible to atherosclerosis than FVB/NJ ApoE-deficient (FVB E0) mice. Strain differences in plasma lipoproteins and markers of oxidation did not account for this difference in atherosclerosis susceptibility. The distribution of atherosclerotic lesion areas in second-generation progeny suggested that multiple genes are involved in conferring susceptibility to atherosclerosis in ApoE-deficient mice. This study sets the stage for future genomic studies aimed at identifying atherosclerosis-modifying genes, using mutant mouse models of hypercholesterolemia.


*    Methods
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Mice
Groups of mice were housed in cages with up to 5 mice per cage, and were maintained in a specific pathogen-free environment. All mice were weaned at 3 weeks and fed either a standard chow diet [PicoLab Rodent 20 (5053); 20% protein from plant and animals sources, 4.5% wt/wt fat, 0.02% wt/wt cholesterol, no casein, and no sodium cholate] or a Western-type diet (Teklad Adjusted Calories 88137; 21% wt/wt fat, 0.15% wt/wt cholesterol, 19.5% wt/wt casein, and no sodium cholate).

Genetic Background Assessments
The parental C57 and FVB E0 mice, used to generate F1 progeny, were subjected to a genome scan using polymorphic markers spaced at {approx}10-cM intervals as previously described.15

Quantitative Atherosclerosis Measurements
At 16 weeks of age, nonfasted mice were anesthetized and blood was collected via a left ventricular puncture into syringes containing ethylenediamine-tetraacetic acid (EDTA). The circulatory system was perfused with 0.9% NaCl by cardiac intraventricular canalization. The heart and ascending aorta including the aortic arch were removed, and the heart containing the aortic root was fixed in phosphate-buffered formalin and processed for the aortic root quantitative atherosclerosis assay as previously described.16

Plasma Cholesterol Analysis
Total plasma cholesterol and HDL cholesterol were isolated and measured as described previously.17 Non-HDL cholesterol was calculated as the difference between total and HDL cholesterol.

Measurement of Mouse ApoA1
ApoA1 in plasma and in HDL fractions was measured by an ApoA1 ELISA assay, which uses the same polyclonal antibody to capture and detect antigen as previously described.18 Rabbit anti-mouse ApoA1 (Biodesign) was purified on protein A affinity columns (Pierce) and an aliquot was biotinylated (Amersham). Purified antibody, diluted 1:400 in bicarbonate buffer, was applied to wells of Nunc Maxisorb ELISA plates and incubated overnight at 4°C. All subsequent incubations were performed at room temperature with gentle agitation. Wells were washed with PBS and blocked with casein blocker (Pierce) for 1 hour, followed by addition of plasma, diluted HDL fractions, or serum standards for 2 hours. Serum standards were calibrated by comparing with purified mouse ApoA1. Plates were washed with PBS/Tween (0.5%) and biotinylated antibodies, diluted 1:250 in PBS/Tween, were applied for 2 hours. After washing, streptavidin/HRP (Pierce) was applied for 1 hour. HRP enzyme was detected by incubation with TurboTMB substrate (Pierce). The reaction was terminated with 1 mol/L sulfuric acid, and the absorbance was measured at 450 nm on a SpectraMax plate reader (Molecular Devices).

Measurement of Mouse ApoA2
ApoA2 levels in plasma and in HDL fractions were measured by an ELISA assay, using the same protocol as in the ApoA1 assay with several modifications. Rabbit anti-mouse ApoA2 antibody (Biodesign) was purified on protein A affinity columns (Pierce) and an aliquot was biotinylated (Amersham). To obtain serum standards, HDL was isolated from the same mouse serum pool (Sigma). Apolipoproteins were separated and identified after SDS–polyacrylamide gel electrophoresis and Coomassie Blue staining. Western gels were scanned by laser densitometry, and ApoA2 concentrations in the HDL sample and in the mouse serum pool were calculated from the percentage of the total protein determined by the scan and the total protein concentration determined by a modified Lowry assay.19 The ApoA2 assay was performed as described for the ApoA1 ELISA assay, using the anti-mouse ApoA2 antibodies.

Measurement of Aryl Esterase and Paraoxonase
Mouse serum, which was never exposed to EDTA, was diluted in PBS and added to microtiter plates; 1 mmol/L phenyl acetate (aryl esterase) or 1 mmol/L paraoxon (paraoxonase) in 20 mmol/L Tris buffer, pH 8, was added and change in optical density (OD) at 270 nm (aryl esterase) or optical density at 405 nm (paraoxonase) was measured in a kinetic assay for 2 minutes in a SpectraMax plate reader at room temperature. Relative aryl esterase and paraoxonase activities were expressed as the slope ({delta}OD/sec).

Measurement of Plasma ApoJ Levels
Plasma ApoJ levels were assayed by ELISA as previously described.20 A mouse plasma pool was used to generate a standard curve and plasma ApoJ levels are reported in relative units.

Measurement of Plasma Anti-Cardiolipin Antibodies
Anti-cardiolipin (aCL) reactivity was measured by ELISA as described.21 In brief, polyvinyl chloride microtiter plates (Dynatech Laboratories) were coated with 50 µL of 50 µg/mL cardiolipin (Sigma) in ethanol and allowed to evaporate and oxidize in air by an overnight incubation at 4°C. After washing twice with PBS, pH 7.4, plates were blocked by incubation for 2 hours at 4°C with 2% BSA in PBS. After washing 3 times with PBS, mouse sera diluted 1:100 in 10% adult bovine serum (Sigma) was added to each well in triplicate and incubated for 2 hours at 4°C. Bound antibody was detected by using alkaline phosphatase–conjugated goat anti-mouse IgG and developed with p-nitrophenyl phosphate. The activity of aCL was expressed as a unit of a standard murine monoclonal aCL (provided by Dr Azzudin E. Gharavi, Morehouse School of Medicine, Atlanta, GA) as previously described.22

Statistics
Comparisons between groups were performed by using an unpaired Student's t test and an unpaired Student's t test with Welch's correction if the variances were unequal. The Kolmogorov–Smirnov test with Dalal and Wilkinson's approximation was used for testing whether distributions were gaussian. Statistical calculations were performed with Prism 2.0 software (GraphPad).


*    Results
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 up arrowIntroduction
 up arrowMethods
 *Results
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This study was designed to determine if genetic background affects plasma lipoproteins and atherosclerotic lesion size in ApoE-deficient mice. Two different strains of ApoE-deficient mice were used in this study. At the time the study was begun, no truly congenic strains of ApoE-deficient mice were available. ApoE-deficient mice,23 purchased from Jackson Laboratories in 1996, were reported to be on the C57BL/6 background. The coat color of these C57 E0 mice was gray, indicating that these mice were not truly congenic. A genome scan using 145 polymorphic markers (see Methods) indicated that the C57 E0 mice carried 92% of their genetic background derived from C57BL/6J. To our knowledge, atherosclerosis susceptibility of FVB/N mice had not been evaluated before this study. Because FVB/N mice have been widely used to create transgenic mice,24 ApoE-deficient mice were bred onto the FVB/N background. ApoE-deficient mice13 of mixed C57BL/6 and 129 genetic background were backcrossed to FVB/NJ wild-type mice by Dr Jeffrey Flier to yield ApoE-deficient mice that carry 91% of their genetic background derived from the FVB/NJ background.

Plasma lipoproteins were measured in a cohort of 16-week chow-fed C57 E0 and FVB E0 mice at the time they were killed. FVB E0 mice had higher total and non-HDL cholesterol when compared with age- and sex-matched C57 E0 mice (Table 1Down). HDL cholesterol was 2-fold higher in FVB E0 mice compared with C57 E0 mice (Table 1Down). HDL cholesterol was higher in males than in females within each strain of ApoE-deficient mice.


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  		Table 1. Background Strain Effects on Plasma Lipids and Lipoprotein Values in ApoE-Deficient Mice

Because ApoA1 and ApoA2 play an important role in HDL particle composition, HDL metabolism, and atherosclerosis in mice, total ApoA1 and ApoA2 levels were measured in FVB E0 and C57 E0 mice. FVB E0 mice had {approx}4- to 5-fold higher ApoA1 levels compared with C57 E0 male mice (Table 1Up). Because of considerable variation in ApoA1 levels within each strain and the small sample size, the strain difference in ApoA1 levels did not reach statistical significance when male and female mice were analyzed separately (C57 E0 males versus FVB E0 males, P=0.08; C57 E0 females versus FVB E0 females, P=0.06). When both sexes were combined, the strain difference in ApoA1 levels was statistically significant. ApoA2 levels were 9-fold higher in FVB E0 mice compared with sex-matched C57 E0 mice. Although detailed HDL particle compositional analysis was not performed, the strain differences in plasma apolipoproteins and HDL cholesterol suggest that HDL particle composition is likely to be very different between the 2 parental strains.

To determine whether there are strain-dependent differences in markers of lipoprotein oxidation, we measured serum paraoxonase by assaying for paraoxonase and aryl esterase activities, using paraoxon and phenyl acetate as substrates, respectively. Paraoxonase activity was 24% higher in C57 E0 mice compared with FVB E0 mice. There were no significant differences in mean aryl esterase activity (Table 2Down). The levels of 2 other oxidation-related plasma parameters, ApoJ and aCL antibodies, were measured in C57 E0 and FVB E0 mice. ApoJ levels did not differ between the 2 strains of ApoE-deficient mice (Table 2Down). aCL antibody titer was higher in C57 E0 males than in FVB E0 males, but there were no strain differences in the females (Table 2Down).


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  		Table 2. Background Strain Effects on Oxidation-Related Parameters in ApoE-Deficient Mice

Male and female C57 E0 and FVB E0 mice were fed a chow diet and killed at 16 weeks of age for measurement of aortic root atherosclerosis. C57 E0 mice developed markedly larger lesions than did FVB E0 mice. Lesion size was {approx}7-fold higher in C57 E0 males than in FVB E0 males (mean±SD, 98 180±51 510 µm2 versus 13 650±5688 µm2, P<0.0001; Figure 1ADown). Lesion size in C57 E0 female mice was {approx}9-fold higher than in FVB E0 mice (mean±SD, 131 600±112 500 µm2 versus 14,250±6305 µm2, P=0.01; Figure 1BDown). The range of lesion areas from C57 E0 mice did not overlap with the range of lesion areas from FVB E0 mice. Distributions of lesion areas in parental mice were gaussian by the Kolmogorov–Smirnov test, and the coefficients of variation were 52%, 42%, 85%, and 44% for C57 E0 males, FVB E0 males, C57 E0 females, and FVB E0 females, respectively.



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  		Figure 1. Genetic background strain effects on atherosclerotic lesion area in ApoE-deficient mice. A cohort of chow diet–fed male (A) or female (B) FVB E0 mice, C57 E0 mice, F1 E0 mice, and F2 E0 mice were killed at 16 weeks of age for measurement of aortic root atherosclerosis. The horizontal line is the mean lesion area for each group. Statistical comparisons are stated in the text.

A high-fat diet accelerated atherosclerosis in both strains of ApoE-deficient mice. A cohort of male C57 E0 and FVB E0 mice were fed the Western-type diet and killed at 16 weeks for the measurement of aortic root atherosclerosis. Mean lesion area was {approx}3.5-fold larger in C57 E0 mice than in FVB E0 mice (mean±SD, 294 200±117 400 µm2 versus 80 840±17 690 µm2, P=0.0014). Distributions of lesion areas in parentals were gaussian by the Kolmogorov– Smirnov test, and the coefficients of variation were 40% and 22% for C57 E0 and FVB E0 mice, respectively.

Lesions from 16-week chow diet–fed C57 E0 and FVB E0 mice were mainly the foam cell type (Figure 2ADown through 2D). Lesions in C57 E0 mice were relatively large (Figure 2ADown and 2CDown), and there was evidence of early development of fibrous caps in these mice (Figure 2CDown). Lesions in FVB E0 mice were relatively small with no fibrous component (Figure 2BDown and 2DDown). In Western diet–fed mice from both strains, fibroproliferative plaques were common (Figure 2EDown through 2H). Fibrous plaques from C57 E0 mice were larger in size and had larger necrotic cores compared with FVB E0 mice. Thus, lesions of both strains could progress to the fibrous stage, although this progression was accelerated in the C57 E0 mice.



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  		Figure 2. Oil Red O staining for lipid in aortic root lesions from 16-week-old chow- and Western diet–fed C57 E0 and FVB E0 mice. Left, C57 E0 mouse lesions. Right, FVB E0 mouse lesions. Lipid-filled lesions in chow-fed C57 E0 mice were relatively large (A, 4x objective) with evidence of early fibrous cap development in larger lesions (C, 20x objective). Lesions in chow-fed FVB E0 mice were small foam cell lesions (B, 4x objective) with no fibrous component (D, 20x objective). Fibroproliferative plaques were large in size in Western diet–fed C57 E0 mice (E, 4x objective) and had thick caps and large necrotic cores (G, 10x objective). Fibroproliferative lesions developed but were smaller in FVB E0 Western diet–fed mice (F, 4x objective) and had thinner caps without well-developed necrotic cores (H, 20x objective).

We performed a strain intercross to determine whether there is a genetic basis for the strain difference in atherosclerosis susceptibility. If genetic background plays a major role in atherosclerosis susceptibility in the 2 strains of mice, the distribution of lesion areas in the first-generation (F1) and second-generation (F2) progeny should provide insight as to the mode of inheritance and whether multiple genes are involved. One male C57 E0 mouse was bred with 3 female FVB E0 mice to yield all of the F1 progeny mice used in this study. The F1 mice were pooled, and a randomly selected group of male and female F1 mice were used to generate F2 mice. A second set of randomly selected F1 mice was not used in breedings and was killed at 16 weeks for measurement of aortic root atherosclerosis. Mean lesion area in F1 males (mean±SD, 51 440±17 890 µm2) was intermediate and significantly different from both male C57 E0 and FVB E0 mice (F1 versus C57, P=0.0024; F1 versus FVB, P=0.0007; Figure 1AUp). Mean lesion area in F1 females (mean±SD, 64 800±6402 µm2) was also intermediate and significantly different from female FVB E0 mice (F1 versus FVB, P<0.0001). There was some overlap in lesion areas from F1 female and C57 female mice, and mean lesion area was not significantly different (F1 versus C57, P=0.14; Figure 1BUp). Distributions of lesion areas in F1 mice were gaussian by the Kolmogorov–Smirnov test, and the coefficients of variation were 35% and 10% for males and females, respectively. Intermediate lesion size in the F1 progeny is consistent with inheritance of either 1 codominant atherosclerosis susceptibility gene or multiple genes influencing atherosclerosis susceptibility.

Male and female F1 mice were bred to generate F2 progeny, and aortic root lesion area was measured in 150 of these F2 mice (Figure 1AUp and 1BUp). Mean lesion areas in F2 males and females were 35 810±26 350 µm2 and 49 400±35 000 µm2. The coefficients of variation were 74% and 71% for F2 males and females, respectively. On visual inspection, the distribution of lesion area in F2 mice did not appear to fall into a gaussian distribution. P values for the Kolmogorov–Smirnov test were 0.032 and 0.0544 for F2 males and females, suggesting that the distributions were nongaussian. From using either untransformed or log-transformed data, variance in lesion area in the entire cohort of F2 mice was significantly higher than the variance in lesion area in F1 mice (F test, P<0.05).

The range of lesion areas in F2 progeny extended between and encompassed the range of parental values. There were no distinct clusters of lesion areas in the F2 distribution. The cohort of F1 mice used for breeding of F2 progeny was not killed for measurement of atherosclerosis. Therefore, an estimation of the relative contribution of genetic versus environmental factors to the variance seen in the F2 progeny was estimated by using the variance in lesion area in the nonbreeding F1 mice as a surrogate. This calculation was performed by using a formula for genetic determination (Variance F2-VarianceF1)/VarianceF2, as previously described25 ). The proportion of the total variance in lesion area in F2 males and F2 females attributed to genetic causes was 0.54 and 0.97, respectively. If the same calculation is performed by using the log10 of the lesion area, the proportions of the total variance in the lesion area attributable to genetic causes are 0.73, 0.98, and 0.87 for F2 males, females, and the combined F2 group, respectively.

FVB E0 mice had higher total cholesterol, HDL cholesterol, ApoA1, and ApoA2 levels than did C57 E0 mice. To determine if these parental strain lipid and apolipoprotein differences play a significant role in determining the strain difference in atherosclerosis susceptibility, these parameters were measured in the 150 F2 progeny, and correlations between these lipoprotein parameters and the atherosclerotic lesion area were calculated. If a given parameter contributes significantly to the strain difference in atherosclerosis, then there should be a significant correlation between the parameter and lesion area in the F2 progeny. If no correlation exists, then the measured lipid or apolipoprotein difference between the 2 strains is not likely to contribute significantly to the difference in atherosclerosis susceptibility. The following parameters were evaluated for correlations with lesion area in the male and female F2 progeny: total cholesterol, non-HDL cholesterol, HDL cholesterol, plasma ApoA1 and HDL ApoA1, plasma ApoA2 and HDL ApoA2, HDL ApoA1/HDL ApoA2 ratio, HDL ApoA1/HDL cholesterol ratio, HDL ApoA2/HDL cholesterol ratio, and aCL antibodies. Except for the ratio of HDL ApoA1/HDL ApoA2, there were no significant correlations between any of these parameters and lesion area (Table 3Down). The ratio of HDL ApoA1/HDL ApoA2 comprised 7.6% of the lesion variance in F2 females. Furthermore, the HDL ApoA1/HDL ApoA2 correlation with lesion area in F2 mice was a positive correlation, which would not be predicted based on the influence of ApoA1 and ApoA2 on atherosclerosis in mice.16 26 27 When data from the 2 F2 females with the highest HDL ApoA1/HDL ApoA2 ratios were excluded from the analysis, the correlation between HDL ApoA1/HDL ApoA2 was no longer significant. In addition, there was no significant correlation between HDL ApoA1/HDL ApoA2 ratio and lesion area in F2 males.


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  		Table 3. Univariate Correlations With Aortic Root Lesion Area in F2 Progeny


*    Discussion
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 up arrowIntroduction
 up arrowMethods
 up arrowResults
 *Discussion
down arrowReferences
 
The use of the ApoE-deficient mouse model of hypercholesterolemia and atherosclerosis has yielded substantial insight into the pathogenesis of atherosclerosis. Previous experiments in which ApoE-deficient mice were bred to various knockout/transgenic mice have provided information on the role of HDL apolipoproteins,16 monocytes,28 lymphocytes,17 29 lipoprotein-processing enzymes,30 and clotting factors31 32 in atherosclerotic lesion formation. The present study is the first to examine the effect of varying genetic background on plasma lipoproteins and atherosclerosis in ApoE-deficient mice. In this study, we chose to measure atherosclerosis by using the aortic root atherosclerosis assay, originally developed by Paigen et al.33 The aortic root assay is widely used in murine studies of atherosclerosis, allows for the coincident inspection of lesion histology, and is amenable in studies using large numbers of mice. Alternative measures of atherosclerosis, such as the en face method, correlate with aortic root measurements34 ; however, these methods are less amenable for studies using large numbers of mice and do not allow for inspection of lesion histology.

Two strains of ApoE-deficient mice, C57 E0 and FVB E0, were generated by successive backcrosses with wild-type inbred mice. Although these mice were not completely inbred, genetic background had a dramatic effect on atherosclerotic lesion size. As noted in studies using the C57BL/6 wild-type mice fed a high-fat, high-cholesterol, cholate-containing diet, the C57BL/6 strain was found to be highly susceptible to atherosclerosis when bred onto the ApoE-deficient background. Chow diet–fed C57 E0 mice had 7- to 9-fold larger aortic root lesions than did chow-fed FVB E0 mice. The Western diet accelerated atherosclerosis in both strains of mice, and C57 E0 mice had 3.5-fold larger lesions than did Western diet–fed FVB E0 mice. However, there were no strain differences in the ability of lesions to progress to fibroproliferative plaques. Therefore, it is likely that the genetic differences between the 2 strains involve genes that affect the temporal aspects of atherosclerosis. The identification of genes that affect the rate of lesion development would have significant clinical importance, as they could serve as targets for the development of pharmaceutical agents that would attenuate lesion development.

Hypercholesterolemia increases atherosclerosis susceptibility in numerous mouse models of atherosclerosis. A high-fat diet accelerates atherosclerosis in both ApoE-deficient mice and in LDL receptor–deficient mice.35 In the present study, 16-week FVB E0 mice had higher total cholesterol, non-HDL cholesterol, HDL cholesterol, ApoA1, and ApoA2 levels than did 16-week C57 E0 mice. One might expect that the higher total cholesterol, non-HDL cholesterol, and ApoA2 levels in FVB E0 mice would be proatherogenic and the higher HDL cholesterol and ApoA1 levels would inhibit atherosclerosis. Notwithstanding these opposing effects, FVB E0 mice has smaller lesions than did C57 E0 mice. To determine whether any 1 of the above lipoprotein parameters contributes significantly to the strain difference in atherosclerosis susceptibility, total cholesterol, HDL cholesterol, total and HDL ApoA1, and total and HDL ApoA2 were measured in the F2 mice. There were no strong correlations between lipoproteins and lesion area in the F2 progeny. Therefore, although diet induced increases in non-HDL cholesterol and genetic increases in ApoA1 and HDL cholesterol are known to be correlated with, respectively, larger and smaller lesions in ApoE-deficient mice,13 16 the observed strain differences in total cholesterol, non-HDL cholesterol, HDL cholesterol, ApoA1, or ApoA2 are not likely to explain the interstrain variation in lesion area.

Lipoprotein oxidation is thought to play an important role in the development of the atherosclerotic lesion. Autoantibodies to oxidized LDL have been detected in the plasma of hypercholesterolemic patients36 and in ApoE-deficient mice.37 Palinski et al38 and Horkko et al39 also found high titers of antioxidized LDL and aCL antibodies in the plasma of ApoE-deficient mice. Navab et al20 demonstrated that levels of the HDL-associated antioxidant enzyme paraoxonase were diminished in the serum of ApoE-deficient mice, compared with serum from wild-type mice. The ratio of ApoJ/paraoxonase was elevated in ApoE-deficient mice and in patients with coronary artery disease.20 In addition, targeted disruption of the gene encoding paraoxonase accelerated atherosclerosis in high-fat, high-cholesterol, cholate diet–fed mice.40 In the present study, ApoJ, paraoxonase, and aCL antibodies were measured in C57 E0 and FVB E0 mice. There were no differences in mean ApoJ levels in the 2 strains of ApoE-deficient mice. There were also no strain differences in aryl esterase activity, which is often used as a measure of paraoxonase. Mean paraoxonase levels, assayed with paraoxon as the substrate, were slightly higher in the C57 E0 mice. Because C57 E0 mice were more susceptible to atherosclerosis, strain differences in paraoxonase would not be likely to explain the strain differences in atherosclerosis susceptibility. We observed a high degree of variation in aCL antibody titer in FVB E0 and C57 E0 mice. aCL antibody titer was higher in male C57 E0 mice than in male FVB E0 mice; however, this strain difference was not seen in female mice. Furthermore, there was no correlation between aCL titer and lesion area in the F2 mice. These data suggest that strain differences in the measured lipoprotein oxidation parameters do not contribute significantly to the strain differences in atherosclerosis susceptibility. However, this does not exclude the possibility that strain differences in other lipoprotein oxidation parameters, such as indices of vessel wall oxidation, play a role in the interstrain differences in atherosclerosis.

The parental mice used in this study were >90% inbred but were not completely congenic. However, the parental strains were the best available at the time the study was initiated. Despite the fact that the mice were not congenic, there were dramatic and nonoverlapping differences in lesion areas between the strains. The coefficients of variation in lesion area in the parental ApoE-deficient mouse strains ranged from 22% to 85%. This variation can be attributed to a multitude of factors including stochastic events involved in atherosclerotic lesion formation, assay variability, environmental factors, and genetic impurity of our mouse strains. By using previously published reports of inbred wild-type strains fed a high-fat, high-cholesterol, cholate-containing diet,10 41 42 we calculated coefficients of variation, based on the stated number of mice, mean, and standard errors reported for 5 different cohorts of mice. The coefficients of variation ranged from 40% to 60%, similar to the range reported in the present study. In addition, we have recently measured aortic root atherosclerosis in a cohort of 16-week-old chow-fed 10th generation congenic C57BL/6 ApoE-deficient mice (more recently available from Jackson Laboratories). The coefficient of variation was 59% (Dansky et al, unpublished data, 1998) in this male cohort of pure-bred C57BL/6 ApoE-deficient mice (n=12). Therefore, even in atherogenic diet–fed C57BL/6 wild-type and chow diet–fed congenic C57BL/6 ApoE-deficient mice, aortic root atherosclerosis lesion area varies between individual mice of a given strain. Therefore, it is likely that a significant proportion of the variance in lesion area in the present study is caused by environmental factors, stochastic events, or assay variation, rather than caused by genetic impurity.

In the present study, the strain difference in the rate of atherosclerotic lesion development is clearly genetic. The genetic determination estimates indicate that >50% of the variance in the F2 mice is caused by genetic factors. The absence of distinct clusters of lesion areas in the F2 distribution suggests that multiple genetic factors explain the interstrain variation in lesion size.

The lack of correlation between aortic root lesion area and plasma lipoproteins in the F2 progeny suggests that the genes responsible for interstrain difference in atherosclerosis are distinct from the genes responsible for interstrain differences in plasma lipoprotein levels. Thus, other genetic factors besides those genes controlling lipoproteins and oxidative factors may explain the strain difference in atherosclerosis susceptibility. Two likely sites for atherosclerosis susceptibility gene products to act are the monocyte/macrophage and the vascular wall. Monocyte/macrophages were shown to play an important role in foam cell lesion formation as ApoE-deficient mice lacking macrophage colony–stimulating factor-1 (op) had dramatic decreases in atherosclerosis compared with control ApoE-deficient mice.28 43 ApoE-deficient mice lacking the macrophage scavenger receptor A had a 58% decrease in atherosclerosis.44 The vascular wall is implicated in studies using cultured endothelial and/or smooth muscle cells, which suggests that differences in lipoprotein retention,45 adhesion molecule expression,46 and subendothelial matrix components47 48 would be likely to affect foam cell lesion formation.

This study ruled out several lipoprotein and oxidative parameters as candidates for the large difference in atherosclerosis susceptibility between C57 and FVB ApoE-deficient mice. The advent of genomic technology has allowed the mapping of disease susceptibility loci based on phenotypic differences between mouse strains without a priori knowledge of putative candidate genes. Disease susceptibility loci for hypertension,49 50 51 obesity,52 53 54 and diabetes55 56 have been identified in rodents by using genomic techniques. Previous studies that have examined the effect of genetic background on atherosclerotic lesion formation in different strains of mice have used mice fed a high-fat, high-cholesterol, cholic acid–containing diet.9 With this diet, the first atherosclerosis susceptibility loci, termed Ath-1, was mapped by using recombinant inbred lines created from a diet-induced susceptible strain, C57BL/6, and C3H- or BALB/c-resistant strains.57 Other atherosclerosis susceptibility loci were inferred, such as Ath-2 and Ath-3, using recombinant inbred lines created by using C57BL/6 and other resistant strains.58 59 Semiquantitative assays of lesion size were used to obtain Ath-1 and Ath-2, and Ath-1 was initially mapped to chromosome 1, nearby but distinct from the ApoA2 gene.42 57 60 61 When lesion size in the RI strains were reanalyzed by using a quantitative aortic root atherosclerosis assay and quantitative trait loci analysis, no significant genetic locus was associated with lesion area.41 In addition, quantitative trait loci analysis of a large F2 intercross derived from the C57BL/6 and C3H parental strains also yielded no significant gene locus associated with atherosclerosis susceptibility.62 The most recent atherosclerosis susceptibility locus inferred was named Ath-8 to explain the difference in diet-induced atherosclerotic lesion size between the atherosclerosis-susceptible SM/J and atherosclerosis-resistant NZB/BINJ strains. However, no significant genetic locus was identified that associated with lesion size among RI strains derived from these parental strains.63 Besides Ath-1, which has not been independently confirmed, the only other Ath gene that has been assigned a genetic location is Ath-3, mapped to chromosome 7.59 Therefore, there is a need for additional gene mapping studies aimed at identifying atherosclerosis-modifying genes that, as in the current study, use the more recently created mutant mouse models of hypercholesterolemia and atherosclerosis.


*    Acknowledgments
 
The authors wish to thank Dr Jeffrey Flier for kindly providing the FVB E0 mice for this study. This research was supported by an Established Investigatorship from the American Heart Association to J.D.S., and by grant PO1 HL54591 from the NIH. H.M.D. was supported by a cardiovascular training grant (Institutional NRSA) from the Mount Sinai School of Medicine.

Received August 13, 1998; accepted January 4, 1999.


*    References
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