© 1999 American Heart Association, Inc.
Atherosclerosis and Lipoproteins |
Genetic Background Determines the Extent of Atherosclerosis in ApoE-Deficient Mice
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
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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
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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
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.
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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 
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 (
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).
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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 1
). HDL
cholesterol was 2-fold higher in FVB E0 mice compared with
C57 E0 mice (Table 1). HDL cholesterol was higher in
males than in females within each strain of ApoE-deficient mice.
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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 4-
to 5-fold higher ApoA1 levels compared with C57 E0 male mice (Table 1
). 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 2). 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 2). aCL antibody titer was higher in C57 E0 males than in FVB E0
males, but there were no strain differences in the females (Table 2).
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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 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 1A). Lesion size in C57 E0 female mice
was 9-fold higher than in FVB E0 mice (mean±SD,
131 600±112 500 µm2 versus
14,250±6305 µm2, P=0.01;
Figure 1B). 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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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 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 2A through
2D). Lesions in C57 E0 mice were relatively large (Figure 2A and 2C), and there was evidence of early development of fibrous caps in
these mice (Figure 2C). Lesions in FVB E0 mice were relatively
small with no fibrous component (Figure 2B and 2D). In Western
diet–fed mice from both strains, fibroproliferative plaques were
common (Figure 2E 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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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 1A). 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 1B). 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 1A and 1B). 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 3). 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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Discussion |
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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.
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Acknowledgments |
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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.
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C. A. Reardon, L. Blachowicz, J. Lukens, M. Nissenbaum, and G. S. Getz Genetic Background Selectively Influences Innominate Artery Atherosclerosis: Immune System Deficiency as a Probe Arterioscler. Thromb. Vasc. Biol., August 1, 2003; 23(8): 1449 - 1454. [Abstract] [Full Text] [PDF]
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G. Schonfeld Familial hypobetalipoproteinemia: a review J. Lipid Res., May 1, 2003; 44(5): 878 - 883. [Abstract] [Full Text] [PDF]
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P. Cullen, R. Baetta, S. Bellosta, F. Bernini, G. Chinetti, A. Cignarella, A. von Eckardstein, A. Exley, M. Goddard, M. Hofker, et al. Rupture of the Atherosclerotic Plaque: Does a Good Animal Model Exist? Arterioscler. Thromb. Vasc. Biol., April 1, 2003; 23(4): 535 - 542. [Abstract] [Full Text] [PDF]
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 C. Colombo, M. Haluzik, J. J. Cutson, K. R. Dietz, B. Marcus-Samuels, C. Vinson, O. Gavrilova, and M. L. Reitman Opposite Effects of Background Genotype on Muscle and Liver Insulin Sensitivity of Lipoatrophic Mice. ROLE OF TRIGLYCERIDE CLEARANCE J. Biol. Chem., January 31, 2003; 278(6): 3992 - 3999. [Abstract] [Full Text] [PDF]
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J. D. Smith, D. James, H. M. Dansky, K. M. Wittkowski, K. J. Moore, and J. L. Breslow In Silico Quantitative Trait Locus Map for Atherosclerosis Susceptibility in Apolipoprotein E-Deficient Mice Arterioscler. Thromb. Vasc. Biol., January 1, 2003; 23(1): 117 - 122. [Abstract] [Full Text] [PDF]
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P. C. R. Hopkins, Y. Huang, J. G. McGuire, and R. E. Pitas Evidence for differential effects of apoE3 and apoE4 on HDL metabolism J. Lipid Res., November 1, 2002; 43(11): 1881 - 1889. [Abstract] [Full Text] [PDF]
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D. G. Kuhel, B. Zhu, D. P. Witte, and D. Y. Hui Distinction in Genetic Determinants for Injury-Induced Neointimal Hyperplasia and Diet-Induced Atherosclerosis in Inbred Mice Arterioscler. Thromb. Vasc. Biol., June 1, 2002; 22(6): 955 - 960. [Abstract] [Full Text] [PDF]
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M. R. Bennett Breaking the Plaque: Evidence for Plaque Rupture in Animal Models of Atherosclerosis Arterioscler. Thromb. Vasc. Biol., May 1, 2002; 22(5): 713 - 714. [Full Text] [PDF]
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H. Williams, J. L. Johnson, K. G. S. Carson, and C. L. Jackson Characteristics of Intact and Ruptured Atherosclerotic Plaques in Brachiocephalic Arteries of Apolipoprotein E Knockout Mice Arterioscler. Thromb. Vasc. Biol., May 1, 2002; 22(5): 788 - 792. [Abstract] [Full Text] [PDF]
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W. Shi, X. Wang, K. Tangchitpiyanond, J. Wong, Y. Shi, and A. J. Lusis Atherosclerosis in C3H/HeJ Mice Reconstituted With Apolipoprotein E-Null Bone Marrow Arterioscler. Thromb. Vasc. Biol., April 1, 2002; 22(4): 650 - 655. [Abstract] [Full Text] [PDF]
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J. W. Furbee Jr., J. K. Sawyer, and J. S. Parks Lecithin:Cholesterol Acyltransferase Deficiency Increases Atherosclerosis in the Low Density Lipoprotein Receptor and Apolipoprotein E Knockout Mice J. Biol. Chem., January 25, 2002; 277(5): 3511 - 3519. [Abstract] [Full Text] [PDF]
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E. Sehayek, J. G. Ono, E. M. Duncan, A. K. Batta, G. Salen, S. Shefer, L. B. Neguyen, K. Yang, M. Lipkin, and J. L. Breslow Hyodeoxycholic acid efficiently suppresses atherosclerosis formation and plasma cholesterol levels in mice J. Lipid Res., August 1, 2001; 42(8): 1250 - 1256. [Abstract] [Full Text] [PDF]
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C. L. Welch, S. Bretschger, N. Latib, M. Bezouevski, Y. Guo, N. Pleskac, C.-P. Liang, C. Barlow, H. Dansky, J. L. Breslow, et al. Localization of atherosclerosis susceptibility loci to chromosomes 4 and 6 using the Ldlr knockout mouse model PNAS, July 3, 2001; 98(14): 7946 - 7951. [Abstract] [Full Text] [PDF]
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C. J. McGillicuddy, M. J. Carrier, and P. D. Weinberg Distribution of Lipid Deposits Around Aortic Branches of Mice Lacking LDL Receptors and Apolipoprotein E Arterioscler. Thromb. Vasc. Biol., July 1, 2001; 21(7): 1220 - 1225. [Abstract] [Full Text] [PDF]
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 N. Ishimori, K. Iwabuchi, S. Fujii, K. Watano, C. Iwabuchi, M. Ato, H. Chiba, S. Tanaka, A. Kitabatake, and K. Onoé Mixed allogeneic chimerism with wild-type strains ameliorates atherosclerosis in apolipoprotein E-deficient mice J. Leukoc. Biol., May 1, 2001; 69(5): 732 - 740. [Abstract] [Full Text]
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K. Aalto-Setala, K. Laitinen, L. Erkkila, M. Leinonen, M. Jauhiainen, C. Ehnholm, M. Tamminen, M. Puolakkainen, I. Penttila, and P. Saikku Chlamydia pneumoniae Does Not Increase Atherosclerosis in the Aortic Root of Apolipoprotein E-Deficient Mice Arterioscler. Thromb. Vasc. Biol., April 1, 2001; 21(4): 578 - 584. [Abstract] [Full Text] [PDF]
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C. P. Sparrow, C. A. Burton, M. Hernandez, S. Mundt, H. Hassing, S. Patel, R. Rosa, A. Hermanowski-Vosatka, P.-R. Wang, D. Zhang, et al. Simvastatin Has Anti-Inflammatory and Antiatherosclerotic Activities Independent of Plasma Cholesterol Lowering Arterioscler. Thromb. Vasc. Biol., January 1, 2001; 21(1): 115 - 121. [Abstract] [Full Text] [PDF]
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 D. T. Eitzman, R. J. Westrick, Z. Xu, J. Tyson, and D. Ginsburg Plasminogen activator inhibitor-1 deficiency protects against atherosclerosis progression in the mouse carotid artery Blood, December 15, 2000; 96(13): 4212 - 4215. [Abstract] [Full Text] [PDF]
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T. Mazzone Scavenger Receptors in Atherosclerosis : New Answers, New Questions Arterioscler. Thromb. Vasc. Biol., December 1, 2000; 20(12): 2506 - 2508. [Full Text] [PDF]
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J. W. Knowles and N. Maeda Genetic Modifiers of Atherosclerosis in Mice Arterioscler. Thromb. Vasc. Biol., November 1, 2000; 20(11): 2336 - 2345. [Abstract] [Full Text] [PDF]
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G. Friedman, A. Ben-Yehuda, Y. Dabach, G. Hollander, S. Babaey, M. Ben-Naim, O. Stein, and Y. Stein Macrophage Cholesterol Metabolism, Apolipoprotein E, and Scavenger Receptor AI/II mRNA in Atherosclerosis-Susceptible and -Resistant Mice Arterioscler. Thromb. Vasc. Biol., November 1, 2000; 20(11): 2459 - 2464. [Abstract] [Full Text] [PDF]
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A. Daugherty, S. C. Whitman, A. E. Block, and D. L. Rateri Polymorphism of class A scavenger receptors in C57BL/6 mice J. Lipid Res., October 1, 2000; 41(10): 1568 - 1577. [Abstract] [Full Text]
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C. D. Sigmund Viewpoint: Are Studies in Genetically Altered Mice Out of Control? Arterioscler. Thromb. Vasc. Biol., June 1, 2000; 20(6): 1425 - 1429. [Abstract] [Full Text] [PDF]
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W. Shi, N. J. Wang, D. M. Shih, V. Z. Sun, X. Wang, and A. J. Lusis Determinants of Atherosclerosis Susceptibility in the C3H and C57BL/6 Mouse Model : Evidence for Involvement of Endothelial Cells but Not Blood Cells or Cholesterol Metabolism Circ. Res., May 26, 2000; 86(10): 1078 - 1084. [Abstract] [Full Text] [PDF]
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M. Mehrabian, J. Wong, X. Wang, Z. Jiang, W. Shi, A. M. Fogelman, and A. J. Lusis Genetic Locus in Mice That Blocks Development of Atherosclerosis Despite Extreme Hyperlipidemia Circ. Res., July 20, 2001; 89(2): 125 - 130. [Abstract] [Full Text] [PDF]
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W. Shi, X. Wang, K. Tangchitpiyanond, J. Wong, Y. Shi, and A. J. Lusis Atherosclerosis in C3H/HeJ Mice Reconstituted With Apolipoprotein E-Null Bone Marrow Arterioscler. Thromb. Vasc. Biol., April 1, 2002; 22(4): 650 - 655. [Abstract] [Full Text] [PDF]
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