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Genetic Differences in Endothelial Cells May Determine
Atherosclerosis Susceptibility
Jan L. Breslow, MD
A
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from fatty streaks to fibrous plaques to complicated lesions.3
In the diet-induced mouse atherosclerosis model, lesion
formation is largely limited to the aortic root, even after
feeding mice the Paigen diet for 14 weeks to 9 months. The
lesions are often small (only several hundred to a few
thousand square microns), and they consist almost entirely of
macrophage foam cells, with little evidence of progression to
fibrous plaques involving smooth muscle cells.
Another concern with the diet-induced model is that the
unphysiological diet required to produce lesions (extremely
high cholesterol content and the presence of cholic acid)
causes a chronic inflammatory state, with the expression of
inflammatory and oxidative stress genes (ie, MCP-1, CSFs,
heme oxygenase, SAA, and NF␬B activation) in the
atherosclerosis-sensitive mouse strains.4 Although the inflammation correlates with atherosclerosis in this model,5 it is
possible that some of the genetic differences between susceptible and resistant mouse strains pertain to the diet used,
rather than the atherogenic process as it is observed on
Western diets.
The diet-induced mouse atherosclerosis model has been
used in an attempt to identify genes for atherosclerosis
susceptibility. In Paigen et al’s6,7 original work, a locus
named Ath-1 was identified. Ath-1 denotes a phenotype in
which susceptible, but not resistant, mice respond to the
high-fat, high-cholesterol, cholate-containing diet with increased fatty streak lesions at the aortic root and reduced
HDL cholesterol levels. In these studies, they used recombinant inbred (RI) strains derived from C57Bl/6J (susceptible)
and C3H/HeJ (resistant) mice, as well as a cross between
C57Bl/6J and C3H/HeJ mice. This work indicated a locus
regulating both the atherosclerosis and the HDL cholesterol
phenotypes on mouse chromosome 1, near but separate from
the apolipoprotein A-II gene. In subsequent work in crosses
with a number of mouse strains, other atherosclerosis loci
were identified; they were designated Ath-2 thru Ath-8.8 –12
Ath-3 has been mapped to chromosome 7 and Ath-6 to
chromosome 12, but the others have not been mapped.
Another group was not able to confirm the map position of
Ath-1 in a reanalysis of the original RI strains that were used
or in a quantitative trait locus mapping experiment.13,14 None
of the genes corresponding to Ath-1 through Ath-8 have been
identified. An antioxidant gene was identified in the region of
Ath-1 (Aop2),15 but Paigen et al have not provided proof that
this gene is indeed Ath-1.
In a recent article describing Ath-8, the authors (including
Paigen) discussed the difficulty of finding genes in the
diet-induced model. They noted that “it is common to find no
lesions in an SM (atherosclerosis sensitive) mouse, despite its
having been fed an atherogenic diet. The fact that there is
therosclerotic cardiovascular disease is a complex
genetic disorder that involves many genes and has
significant gene-environment interactions. The extensive study of candidate genes in pathways relevant to
atherosclerotic cardiovascular disease risk factors has had
limited success in explaining the population’s susceptibility
to this disease. With the advent of the Genome Program, it is
now becoming possible to use positional cloning techniques
to reveal new genes involved in atherosclerosis susceptibility.
Studies in human populations are underway, but they may be
difficult because of the heterogeneity of human populations
and the very real possibility that large numbers of genes are
involved, each with very small effects. The probable difficulties of human genetic studies strongly suggest that parallel
approaches should be undertaken to identify genes for atherosclerosis susceptibility by positional cloning in animal
models. Genes identified through the study of animal models
can be used to identify human homologues. These can be
used to search for mutations associated with disease in human
populations and/or to identify important pathways involved in
atherogenesis that might lend themselves to the development
of novel therapeutic approaches.
See p 75
The most ideal mammal for such studies is the laboratory
mouse.1 Mice are small (25 to 40 g) and have short generation
times (9 to 10 weeks) and large litter sizes (5 to 10 pups); in
addition, there are many inbred strains available. However,
laboratory strains of mice fed the normal chow diet do not
develop atherosclerotic lesions. To circumvent this problem,
Paigen et al2 proposed a model in which mice are fed a diet
containing 15% fat, 1.25% cholesterol, and 0.5% cholic acid
to induce lesions. They used this diet to study 10 inbred
mouse strains and found large differences in atherosclerosis
susceptibility that did not correlate well with total plasma
cholesterol levels, implying other factors were involved in
susceptibility to this diet.
Unfortunately, serious limitations of the diet-induced
mouse atherosclerosis model exist. Human atherosclerotic
lesions typically occur at vessel branch points and progress
The opinions expressed in this editorial are not necessarily those of the
editors or of the American Heart Association.
From the Laboratory of Biochemical Genetics and Metabolism,
Rockefeller University, New York, NY.
Correspondence to Jan L. Breslow, MD, Laboratory of Biochemical
Genetics and Metabolism, The Rockefeller University, 1230 York Ave, Box
179, New York, NY 10021-6399. E-mail [email protected]
(Circulation. 2000;102:5-6.)
© 2000 American Heart Association, Inc.
Circulation is available at http://www.circulationaha.org
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Circulation
July 4, 2000
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incomplete penetrance reduces the power of linkage analysis
by weakening the correlation between genotype and phenotype.”12 The small lesion size in the diet-induced mouse
atherosclerosis model and the incomplete penetrance of
lesions add to the difficulty of isolating genes for atherosclerosis susceptibility using this model. Thus, it is clear that new
approaches will be needed with this model to identify the
genes responsible for the differences observed in susceptibility to atherosclerosis.
The article in the current issue of Circulation by Shi et al16
contributes to this goal. In this study, the authors build on
previous work with the diet-induced mouse atherosclerosis
model and take it in a new direction. They report a method for
culturing endothelial cells from the mouse thoracic aorta by
an explant technique. They then show that minimally modified LDL, a mildly oxidized form of LDL, induced higher
levels of inflammatory gene expression in C57Bl/6J-derived
compared with C3H/HeJ-derived cultured aortic endothelial
cells. These are the same 2 mouse strains originally used by
Paigen et al, which are atherosclerosis sensitive and resistant,
respectively. The authors conclude that their experiments
“provide strong evidence that genetic factors in atherosclerosis act at the level of the vessel wall.”
This article by Shi et al16 is important from several points
of view. It provides the first direct proof that factors operating
in the vessel wall, particularly endothelial cells, can serve as
atherosclerosis modifiers. This is reminiscent of clinical
experience in which patients with the same risk factors have
vastly different extents of disease, and it suggests a possible
mechanism for these clinical observations. The article demonstrates that the induction of an inflammation-related phenotype relevant to atherosclerosis susceptibility is preserved
ex vivo in tissue culture cells and is independent of the
relatively toxic diet used in the diet-induced mouse atherosclerosis model. The article also provides an in vitro tissue
culture system to compare endothelial cells from C57Bl/6J
and C3H/HeJ mice for differences in minimally modified
LDL binding and signal transduction. These cultures allow
one to design experiments to identify the pathway, specific
molecules, and perhaps even genes underlying the difference
in atherosclerosis susceptibility between the 2 strains. Finally,
the article by Shi et al16 provides a secondary marker for
atherosclerosis susceptibility that can be scored in quantitative trait locus mapping of atherosclerosis susceptibility
genes. Atherosclerosis is a complex phenotype, and it may be
more revealing in future mouse crosses using the diet-induced
mouse model to score secondary markers that might have a
simpler inheritance pattern. It may also be useful to score
endothelial responsiveness to minimally modified LDL in
ongoing crosses with other mouse atherosclerosis models,
such as the apoE knockout mouse.17
Shi et al16 provide another important step in the process of
using the powerful tools of mouse genetics to help unravel the
mysteries of atherosclerosis, a very complex human genetic
disease.
References
1. Silver LM. Mouse Genetics. New York: Oxford University Press;
1995:362.
2. Paigen B, Morrow A, Brandon C, et al. Variation in susceptibility to
atherosclerosis among inbred strains of mice. Atherosclerosis. 1985;57:
65–73.
3. Ross R. Atherosclerosis: an inflammatory disease. N Engl J Med, 1999;
340:115–126.
4. Liao F, Andalibi A, deBeer FC, et al. Genetic control of inflammatory
gene induction and NF-kappa B-like transcription factor activation in
response to an atherogenic diet in mice. J Clin Invest. 1993;91:
2572–2579.
5. Liao F, Andalibi A, Qiao JH, et al. Genetic evidence for a common
pathway mediating oxidative stress, inflammatory gene induction, and
aortic fatty streak formation in mice. J Clin Invest. 1994;94:877– 884.
6. Paigen B, Mitchell D, Reue K, et al. Ath-1, a gene determining atherosclerosis susceptibility and high density lipoprotein levels in mice. Proc
Natl Acad Sci U S A. 1987;84:3763–3767.
7. Paigen B, Albee D, Holmes PA, et al. Genetic analysis of murine strains
C57BL/6J and C3H/HeJ to confirm the map position of Ath-1, a gene
determining atherosclerosis susceptibility. Biochem Genet. 1987;25:
501–511.
8. Paigen B, Nesbitt MN, Mitchell D, et al. Ath-2, a second gene determining atherosclerosis susceptibility and high density lipoprotein levels
in mice. Genetics. 1989;122:163–168.
9. Stewart-Phillips JL, Lough J, Skamene E. ATH-3, a new gene for atherosclerosis in the mouse. Clin Invest Med. 1989;12:121–126.
10. Paigen B. Genetics of responsiveness to high-fat and high-cholesterol
diets in the mouse. Am J Clin Nutr. 1995;62:458S– 462S.
11. Mu JL, Naggert JK, Svenson KL, et al. Quantitative trait loci analysis for
the differences in susceptibility to atherosclerosis and diabetes between
inbred mouse strains C57BL/6J and C57BLKS/J. J Lipid Res. 1999;40:
1328 –1335.
12. Pitman WA, Hunt MH, McFarland C, et al. Genetic analysis of the
difference in diet-induced atherosclerosis between the inbred mouse
strains SM/J and NZB/BINJ. Arterioscler Thromb Vasc Biol. 1998;18:
615– 620.
13. Hyman RW, Frank S, Warden CH, et al. Quantitative trait locus analysis
of susceptibility to diet-induced atherosclerosis in recombinant inbred
mice. Biochem Genet. 1994;32:397– 407.
14. Machleder D, Ivandic B, Welch C, et al. Complex genetic control of HDL
levels in mice in response to an atherogenic diet: coordinate regulation of
HDL levels and bile acid metabolism. J Clin Invest. 1997;99:1406 –1419.
15. Phelan SA, Johnson KA, Beier DR, et al. Characterization of the murine
gene encoding Aop2 (antioxidant protein 2) and identification of two
highly related genes. Genomics. 1998;54:132–139.
16. Shi W, Haberland ME, Jien M-L, et al. Endothelial responses to oxidized
lipoproteins determine genetic susceptibility to atherosclerosis in mice.
Circulation. 2000;101:75– 81.
17. Dansky H, Charlton SA, Sikes JL, et al. Genetic background determines
the extent of atherosclerosis in ApoE-deficient mice. Arterioscler Thromb
Vasc Biol. 1999;19:1960 –1968.
KEY
WORDS:
䡲 atherosclerosis
Editorials
䡲
endothelium
䡲
inflammation
䡲
genetics
Genetic Differences in Endothelial Cells May Determine Atherosclerosis Susceptibility
Jan L. Breslow
Circulation. 2000;102:5-6
doi: 10.1161/01.CIR.102.1.5
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