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(Circulation Research. 2001;89:102.)
© 2001 American Heart Association, Inc.

Editorial

Atherosclerosis Modified

Alan D. Attie

From the Departments of Biochemistry and Comparative Biosciences, University of Wisconsin-Madison, Madison, Wis.

Correspondence to Alan D. Attie, Department of Biochemistry, University of Wisconsin-Madison, 433 Babcock Dr, Madison, WI 53706. E-mail attie{at}biochem.wisc.edu

Key Words: modifier genes • inflammation • epistatis • dyslipidemia • hypercholesterolemia

For many years, I have taught medical students about dyslipidemia and atherosclerosis. Invariably, an alert student will ask how cholesterol causes atherosclerosis. The honest answer is that we do not know.

Genetic studies in humans and transgenic mice have established a strong link between dyslipidemia (typically high VLDL/LDL cholesterol or low HDL) and atherosclerosis. However, as a phenotype, atherosclerosis is a perfect example of a complex trait. In short, the relationship between genotype and phenotype is influenced by additional genes, termed modifier genes.^1,2

Two studies have identified genes that, when knocked out in transgenic mice, attenuate the atherosclerosis that would normally occur in the severely hyperlipidemic apoE-null mouse. First, Cyrus et al³ showed a diminution of atherosclerosis in the apoE-null mouse when the 12/15 lipoxygenase gene was also disrupted. More recently, a similar suppression of atherosclerosis in the apoE-null mouse was achieved by knocking out the fatty acid–binding protein gene, ap2.⁴ In an elegant study, Makowski et al⁴ showed that replenishment of macrophages of apoE^-/- ap2^+/+ with macrophages from ap2-knockout mice through bone marrow transplantation produced the same result, indicating a novel role for macrophage ap2 expression in atherogenesis. These two studies show that loss of function in either of two distinct loci can partially nullify the atherogenic risk posed by severe hypercholesterolemia.

How many other genes can modify atherosclerosis? Can a broad genetic approach be used to identify additional atherosclerosis modifier genes? In this issue of Circulation Research, Mehrabian et al⁵ in the Lusis laboratory answer the second question. In contrast to the well-studied C57BL/6J strain, Mus castaneus (CAST) mice are highly resistant to diet-induced atherosclerosis. Mehrabian et al⁵ carried out a mapping study in F2 mice generated from the two progenitor strains and obtained a highly significant link of atherosclerosis lesion area to a locus on chromosome 6. The chromosomal region containing this locus in the CAST mice was introgressed into LDL receptor–null C57BL/6J mice to generate a new congenic strain. This new strain was very resistant to lesion development compared with its background strain. This result establishes that the chromosome 6 locus from the CAST strain acts autonomously to modify atherosclerosis.

Replication of a phenotype of a founder strain by introgressing a small part of its genome into an unaffected strain is strong evidence that a single locus (or several closely spaced genes) is necessary and sufficient to produce the phenotype. By generating a series of congenic strains with overlapping recombinations within the confidence interval defined by the F2 screen, the Lusis team should be able to positionally clone the atherosclerosis modifier gene.⁶ Alternatively, there might be some obvious candidates in the region.

Plasma lipoprotein levels were unaltered by the modifier locus. Therefore, how might this gene be acting? There is a growing consensus that atherosclerosis is an inflammatory disease of the arterial wall.⁷ Thus, the discovery of modifiers of atherosclerosis that do not alter plasma lipoprotein levels can potentially provide new mechanistic clues to the process by which dyslipidemia contributes to atherogenesis. It might help to determine if there is a direct link between dyslipidemia and atherosclerosis (Figure) or if dyslipidemia contributes to inflammation.

View larger version (80K): [in this window] [in a new window]   Relationship between polygenes, modifier genes, and complex traits. Variation at numerous gene loci defines a composite genotype for a particular trait. For example, the expression of genes for lipoprotein receptors might produce a genotype likely to produce dyslipidemia. Modifier genes can act to alter the expression of a given phenotype. For example, genes controlling bile acid production or biliary cholesterol secretion might alter the phenotype normally associated with lipoprotein receptor dysfunction. If dyslipidemia contributes to atherogenesis solely through an inflammatory pathway, then modifiers of inflammation would be expected to modify the atherosclerosis normally associated with dyslipidemia.

The authors suggest that peroxisome proliferator–activated receptor- (PPAR-) is a positional candidate. Its expression is reduced in the macrophages from the congenic strain, and there is a sequence polymorphism within the promoter of the gene. Moreover, the logarithm of odds peak for atherosclerosis coincides with a logarithm of odds peak for fasting insulin, a marker of insulin resistance. PPAR- has been linked with insulin sensitivity in two ways: first, PPAR- agonists are used therapeutically as insulin sensitizers,⁸ and second, mice with one null allele of PPAR- are (paradoxically) more insulin sensitive.⁹ Chawla et al¹⁰ have recently proposed that PPAR- regulates macrophage cholesterol efflux through a transcriptional cascade. PPAR- induces the transcription factor liver X receptor- (LXR), which in turn induces the transcription of ABCA1. The latter protein mediates apoA1-specific phospholipid and cholesterol efflux from macrophages. This scheme incorporates lipid agonists in two ways: first, hydroxylated cholesterol metabolites are agonists for LXR¹¹; second, arachidonate and linoleate-derived metabolites (13-hydroxyoctadecadienoic acid and 15-hydroxyeicosatetraenoic acid, respectively) are agonists for PPAR-.^12,13 This pathway extends the web of polygenes and modifier genes (Figure) that might affect susceptibility to atherosclerosis to include enzymes that produce lipid agonists, lipid transporters, and lipid-activated transcription factors, among many other known genes. Interestingly, PPAR- regulates ap2 in macrophages as it does in adipose tissue.⁴

Is this case closed; ie, is PPAR- the CAST chromosome 6 atherosclerosis modifier? Candidate genes are notoriously nympholeptic. With complex diseases like atherosclerosis, a strong case can be made for numerous categories of genes. For example, less than one centimorgan from PPAR- on mouse chromosome 6 is the gene encoding 5-lipoxygenase, an enzyme involved in leukotriene biosynthesis. Notwithstanding the case for PPAR-, might there be a case for 5-lipoxygenase? Definitive gene identification awaits additional genetic evidence in the form of a gene knockout or complementation experiment.

Modifier genes are presently one of the most exciting and challenging areas of mammalian genetics.^1,2 Hobbs et al¹⁴ found evidence of a dominant suppressor of familial hypercholesterolemia. Knoblauch et al¹⁵ mapped a locus that lowers blood cholesterol in patients with heterozygous familial hypercholesterolemia. It is uncertain if this new locus acts on the function of the LDL receptor pathway (for example, see García et al¹⁶) or acts in an additive fashion through a separate pathway.

The rewards that come with the identification of modifier genes are illustrated by the following dramatic now-classic example. Dietrich et al¹⁷ showed that the number of intestinal tumors caused by mutations at the apc locus in the Min mouse was dependent on mouse strain background. They proceeded to identify the modifier locus as secretory phospholipase A₂ and a second closely linked gene.¹⁸ The identification of the PLA₂ gene led to the novel hypothesis that arachidonate-derived lipids might play a role in this process and identified a potentially "druggable" target for treatment of intestinal neoplasia.¹⁹ The CAST modifier gene mapped by Mehrabian et al⁵ has the potential for a similarly dramatic outcome.

As we gain more understanding of factors necessary for the development of atherosclerosis, we will develop a scheme of subphenotypes for this complex disease. Then broad genetic screens like the one carried out by the Lusis team can be carried out to map modifiers of these subphenotypes. These traits might be more robust than arterial lesion size, thus enhancing the power to detect linkage. With the completion of the human genome sequence rough draft and the imminent completion of the mouse genome sequence, the pace of positional cloning projects has greatly accelerated. With high throughput genotyping, a full positional cloning project can be carried out within the time span of a doctoral dissertation or a postdoctoral fellowship. This would have been unrealistic just a few years ago.

Footnotes

The opinions expressed in this editorial are not necessarily those of the editors or of the American Heart Association.

References

1. Nadeau JH. Modifier genes in mice and humans. Nat Rev Genet. . 2001; 2: 165–174.[Medline] [Order article via Infotrieve]
2. Dipple KM, McCabe ER. Phenotypes of patients with "simple" mendelian disorders are complex traits: thresholds, modifiers, and systems dynamics. Am J Hum Genet. . 2000; 66: 1729–1735.[Medline] [Order article via Infotrieve]
3. Cyrus T, Witztum JL, Rader DJ, Tangirala R, Fazio S, Linton MF, Funk CD. Disruption of the 12/15-lipoxygenase gene diminishes atherosclerosis in apo E-deficient mice. J Clin Invest. . 1999; 103: 1597–1604.[Medline] [Order article via Infotrieve]
4. Makowski L, Boord JB, Maeda K, Babaev VR, Uysal KT, Morgan MA, Parker RA, Suttles J, Fazio S, Hotamisligil GS, Linton MF. Lack of macrophage fatty-acid-binding protein aP2 protects mice deficient in apolipoprotein E against atherosclerosis. Nat Med. . 2001; 7: 699–705.[Medline] [Order article via Infotrieve]
5. Mehrabian M, Wong J, Wang X, Jiang Z, Shi W, Fogelman AM, Lusis AJ. Genetic locus in mice that blocks the development of atherosclerosis despite extreme hyperlipidemia. Circ Res. . 2001; 89: 125–130.[Abstract/Free Full Text]
6. Darvasi A. Experimental strategies for the genetic dissection of complex traits in animal models. Nat Genet. . 1998; 18: 19–24.[Medline] [Order article via Infotrieve]
7. Ross R. Atherosclerosis: an inflammatory disease. N Engl J Med. . 1999; 340: 115–126.[Free Full Text]
8. Olefsky JM. Treatment of insulin resistance with peroxisome proliferator-activated receptor agonists. J Clin Invest. . 2000; 106: 467–472.[Medline] [Order article via Infotrieve]
9. Kubota N, Terauchi Y, Miki H, Tamemoto H, Yamauchi T, Komeda K, Satoh S, Nakano R, Ishii C, Sugiyama T, Eto K, Tsubamoto Y, Okuno A, Murakami K, Sekihara H, Hasegawa G, Naito M, Toyoshima Y, Tanaka S, Shiota K, Kitamura T, Fujita T, Ezaki O, Aizawa S, Nagai R, Tobe K, Kimura S, Kadowaki T. PPAR mediates high-fat diet-induced adipocyte hypertrophy and insulin resistance. Mol Cell. . 1999; 4: 597–609.[Medline] [Order article via Infotrieve]
10. Chawla A, Boisvert WA, Lee CH, Laffitte BA, Barak Y, Joseph SB, Liao D, Nagy L, Edwards PA, Curtiss LK, Evans RM, Tontonoz P. A PPAR -LXR-ABCA1 pathway in macrophages is involved in cholesterol efflux and atherogenesis. Mol Cell. . 2001; 7: 161–171.[Medline] [Order article via Infotrieve]
11. Janowski BA, Grogan MJ, Jones SA, Wisely GB, Kliewer SA, Corey EJ, Mangelsdorf DJ. Structural requirements of ligands for the oxysterol liver X receptors LXR and LXRß. Proc Natl Acad Sci U S A. . 1999; 96: 266–271.[Abstract/Free Full Text]
12. Nagy L, Tontonoz P, Alvarez JG, Chen H, Evans RM. Oxidized LDL regulates macrophage gene expression through ligand activation of PPAR. Cell. . 1998; 93: 229–240.[Medline] [Order article via Infotrieve]
13. Huang JT, Welch JS, Ricote M, Binder CJ, Willson TM, Kelly C, Witztum JL, Funk CD, Conrad D, Glass CK. Interleukin-4-dependent production of PPAR- ligands in macrophages by 12/15-lipoxygenase. Nature. . 1999; 400: 378–382.[Medline] [Order article via Infotrieve]
14. Hobbs HH, Leitersdorf E, Leffert CC, Cryer DR, Brown MS, Goldstein JL. Evidence for a dominant gene that suppresses hypercholesterolemia in a family with defective low density lipoprotein receptors. J Clin Invest. . 1989; 84: 656–664.
15. Knoblauch H, Muller-Myhsok B, Busjahn A, Ben Avi L, Bahring S, Baron H, Heath SC, Uhlmann R, Faulhaber HD, Shpitzen S, Aydin A, Reshef A, Rosenthal M, Eliav O, Muhl A, Lowe A, Schurr D, Harats D, Jeschke E, Friedlander Y, Schuster H, Luft FC, Leitersdorf E. A cholesterol-lowering gene maps to chromosome 13q. Am J Hum Genet. . 2000; 66: 157–166.[Medline] [Order article via Infotrieve]
16. García CK, Wilund K, Arca M, Zuliani G, Fellin R, Maioli M, Calandra S, Bertolini S, Cossu F, Grishin N, Barnes R, Cohen JC, Hobbs HH. Autosomal recessive hypercholesterolemia caused by mutations in a putative LDL receptor adaptor protein. Science. . 2001; 292: 1394–1398.[Abstract/Free Full Text]
17. Dietrich WF, Lander ES, Smith JS, Moser AR, Gould KA, Luongo C, Borenstein N, Dove W. Genetic Identification of Mom-1, a major modifier locus affecting Min-induced intestinal neoplasia in the mouse. Cell. . 1993; 75: 631–639.[Medline] [Order article via Infotrieve]
18. Cormier RT, Bilger A, Lillich AJ, Halberg RB, Hong KH, Gould KA, Borenstein N, Lander ES, Dove WF. The Mom1AKR intestinal tumor resistance region consists of Pla2g2a and a locus distal to D4 Mit64. Oncogene. . 2000; 19: 3182–3192.[Medline] [Order article via Infotrieve]
19. Jacoby RF, Marshall DJ, Newton MA, Novakovic K, Tutsch K, Cole CE, Lubet RA, Kelloff GJ, Verma A, Moser AR, Dove WF. Chemoprevention of spontaneous intestinal adenomas in the Apc Min mouse model by the nonsteroidal anti-inflammatory drug piroxicam. Cancer Res. . 1996; 56: 710–714.[Abstract/Free Full Text]

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