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Atherosclerosis is a disease of the large arteries that is the cause of heart disease and stroke. It is a highly complex disorder with multiple genetic and environmental influences. The mouse model has proved very useful for studying atherosclerosis
because genetic analysis and planned genetic
Universityof California,LosAngeles,CA90095,USA. , ~ m o d i f i c a t i o n are feasible in this organism. In this Tel: +1 310 825 1595
Fax:+13107947345 [email protected]
brief review, s o m e recent findings are summarized and future prospects using m o u s e models to study atherosclerosis-related traits are discussed.
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THE mouse has become the most widely used animal model of atherosclerosis for several reasons ~2. First, the hundreds of strains of mice developed over the past century exhibit many of the same differences in disease susceptibility as do humans. In common with humans, essentially all of these differences (affecting lipoprotein metabolism, dietary responsiveness, immune functions, inflammatory functions, obesity and diabetes, and susceptibility to atherosclerotic lesions) are due to the effects of multiple genetic factors. Second, the mouse model ! been uniquely developed as a tool for genetic studies. A dense linkage map with more than 10000 genetic markers has been constructed, and many specialized stocks exhibiting mutations and chromosomal rearrangements have been collected. Third, techniques for directed genetic alterations are most advanced in the mouse. Transgenic mice carry copies of a gene introduced by microinjection into fertilized eggs, and gene targeted mice (also known as knockout mice) have specific mutations introduced by homologous recombination. © 1 9 q 5 , Elsevier Science Ltd
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The use of mice f o r genetic analysis of alherosclerosis began with the work of J. Thompson in the late 1960s. In the early 1981)s, B. Paigen and co-workers modified the methods of Thompson to quantitatively assess atherosclcrotic lesion development, and several laboratories began to examine lipoprotein metabolism in the mouse. These studies led to the identification of genetic variations in lipoprotein levels and lesion development. Subsequently, several laboratories, including those of Paigen and R. LcBoeuf, tentatively mapped several genes controlling high-density lipoprotcin (HDL) cholesterol levels and aortic lesion size (reviewed in Refs 1, 2). Beginning in the late 1980s, a number of laboratories began to use transgenic and gene targeted mice to study mechanisms involved in lipoprotein metabolism and the role of lipoproteins in atherosclerosis. Particularly significant were the findings from E. Rubin and co-workers that transgenic mice containing the gene for apolipoprotein AI (apoAl) exhibited dramatically reduced lesion development in response to an atherogenic diet, and from J. Brcslow's and N. Maeda's laboratories that apoE knockout mice exhibited hyperlipidemia and advanced atherosclerotic lesions similar to fibrous human lesions (reviewed in Refs 3, 4). In this review, we summarize some important findings from the past two years•
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Figure 1. Pathology of atherosclerotic lesions in mice. (a) A large lesion, occupying all layers of Atherosclerotic lesions the aortic wall, with calcification (arrow), in a naturally occurring susceptible mouse model (strain As mentioned above, previous studies have C57B!_/6J, atherogenic diet). (b) A similar lesion (arrow) with an early fibrous cap in an immuneshown that large genetic differences exist deficient mouse model (strain C57BL/6J, class I MHC knockout, atherogenic diet). (c) An atherosclerotic lesion with calcification (arrow) in a hypercholesterolemic mouse model (mixed genetic among inbred strains of mice in their suscepbackground, apoEknockout, chow). (d) Lipofuscin (dark brown pigment) deposition on the left tibility to the development of atherosclerotic ventricular side of the aortic valves in a naturally occurring susceptible strain (C57BI_/6J, atherolesions '-3 (Fig. 1). Naturally occurring suscepgenic diet). All sections were stained with Oil-Red-0, Hematoxylin and Fast green. Lipid-containing tible inbred strains develop lesions only when lesions stain red and calcified regions stain blue. maintained on a high-fat, high-cholesterol diet, whereas certain genetically engineered strains that exhibit high levels of plasma cholesterol also develop lesions on a low-fat diet. Lesions in mice differ not only influencing the levels and composition of plasma lipoproteins. At least in size, but also in composition. There are variations in the content of one gene may act at the level of lipoprotein oxidation (discussed below). Genetic studies with mice provided the first definitive evidence lipid, the presence or absence of fibrous elements and calcification, and the deposition of lipofuscin (a pigmented complex consisting of that arterial calcification is determined by genetic factors5. As yet the nature of these factors is unknown. oxidized lipids and proteins) -~(Fig. 1). Lipofuscin occurs in the cardiac valves and coronary arteries of Genetic crosses between various susceptible and resistant strains certain strains of mice but not others• A genetic cross between strains of mice provide a means of mapping and ultimately identifying genetic contributions to lesion development. Such studies suggested differing in deposition of coronary lipofuscin revealed co-segregation that increased expression of the apoAHgene, on chromosome 1, pro- of the trait with the gene encoding tyrosinase on chromosome 7. motes development of aortic lesions, presumably by influencing the Mutational studies of this gene confirmed that it determines lipoanti-atherogenic nature of HDL. This was confirmed using transgenic fuscin deposition 7. mice producing high levels of apoAII (Ref. 6). Several other putative chromosomal loci influencing aortic lesion size (designated Ath-l,Ath-2, Pathways involved in atherogenesis Ath-3, etc.) have been reported (reviewed in Refs 1-3), but these re- Lipoprotein metabolism Results fi'om studies with transgenic and knockout mice (Fig. 2) were quire confirmation, and the identities of the underlying genes are unknown. Some of these genes appear to contribute to atherosclerosis by consistent with the conclusion that high levels of apoB-containing
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exhibited marked reduction in levels of HDL cholesterol but did not show enhanced susceptibility to atherosclerosis when fcd a high-fat diet ~'. Thc results suggest that reduction of apoAl alonc does not cause atherosclerosis. The interpretation of the results, however, Intestine was complicated by the fact that the mice were of a mixed genetic background. It will be of interest to exanaine the efB48(+, fects of a deficiency of apoAl AI(+/-~'~N on the background of genetically engineered mice that are highly susceptible to lesion I Chylomicron development, such as apoE AIV(+)~ / knockout mice. ct(+)~,(+] The functional heterogeneity of HDL was strikingly demonCll(+) I LPL(+) strated by studies of apoAH transgenic mice". ApoAll is the FFA second most abundant protein of HDL (next to apoAI). In common with apoAltransgenic mice. apoAll transgenic mice BIER (+1-) exhibited increased levels of HDL cholesterol, but in cont ~ . ~ ! ~ "~,-: 2 ,----:: . . . . . . trast to apoAl transgenic mice, they showed dramatically increased susceptibility Figure 2. Major pathways for plasma lipid transport in humans. Genetically engineered mouse models that have been developed to examine lipoprotein metabolism are indicated: transgenic (+), knockout (-), both (+/-). The to atherosclerosis. These mice major classes of human plasma lipoproteins [chylomicrons, remnants, very low-density lipoproteins (VLDL), intershould be useful in dissecting mediate-density lipoproteins (IDL), low-density lipoproteins (LDL), high-density lipoproteins (HDL) and lipoprotein the properties of HDL that (a)] and component apolipoproteins [(a), AI, All, AIV, 1348,13100,CI, CII, CIII and E] are shown. Key receptors, transfer are reponsible for protection proteins and enzymes functioning in human lipid metabolism include: the LDL receptor (B/ER), the LDL-receptorrelated protein (LRP), lipoprotein lipase (LPL), hepatic lipase (HL), cholesteryl ester transfer protein (CETP), lecithinagainst atherosclerosis. cholesterol acyltransferase (LCAT), and cholesterol-7a-hydroxylase (7a OH). xLipids transported include free fatty The atherogenic potential acids (FFA), cholesteryl ester (CHOL E) and triacylglycerol (TG). For further reading, see Ref. 4. of the Lp(a) lipoprotein (associated with coronary artery disease in some but not all lipoproteins promote atherogenesis. Thus, apoEknockout mice, low- human epidemiological studies) was further studied in the mouse density lipoprotein [LDL (apoB/E)] receptor knockout mice and trans- model. Lp(a) is an LDL-like particle that contains, in addition to genic mice containing the human apoB gene all accumulated high apoB, a large polypeptide termed apo(a). Mice do not naturally proplasma levels of apoB-containing lipoproteins, and these mice devel- duce apo(a). Previous studies with human apo(a) transgenic mice oped much larger and more advanced lesions than those in naturally revealed an enhancement in atherosclerotic lesion development occurring susceptible strains 8-t°. These models will be valuable for (reviewed in Refs 2-4). These transgenic mice, however, failed to examining mechanisms contributing to the development of fibrous assemble intact Lp(a) particles owing to the inability of human and complex lesions, since the lesions in naturally occurring strains apo(a) to form complexes with mouse apoB. This problem has now been overcome by coexpression of human apoB as a transgene ~"JS. generally do not progress to advanced stages. The interaction of HDL and apoB-containing lipoproteins in ath- The combined human-apoB-human-apo(a) mice have high erosclerosis was studied in combined human apoAl-transgenic-apoE Lp(a) levels, but whether they will exhibit increased lesion developknockout mice. Previous studies had shown that apoAl transgenic ment remains to be tested. It will also be of interest to determine mice exhibited increased HDL-cholesterol levels and had reduced ath- whether there are differences in the atherogenic potentials of free erosclerosis in response to an atherogenic diet (reviewed in Refs 2-4). In apo(a) and Lp(a). the current studies, a dramatic protective effect of increased apoAI and HDL-cholesterol was observed in the apoEknockout model "J2. These Oxidation Oxidatively modified LDL occurs in both human and mouse results strongly support the concept that apoAI and HDL directly protect atherosclerotic lesions and it exhibits a variety of properties that are against atherosclerosis. In contrast to these results, apoA1knockout mice Dietary fat, cholesterol
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likely to be proathcrogenic, including ~, potent pro-inflammatory activity, cytotoxicity, rapid uptake by macrophages, and mitogenic activity'" (Fig. 3). Several recent genetic studies in mice support a role for oxidation in atherogenesis. First, the accumulation of aortic valve lipofuscin, a terminal oxidation complex, was associated with increased atherogencsis in BALB/cJ mice 7. Second, hypercholesterolemic apoE knockout mice, which develop relatively advanced atherosclerotic lesions, accumulated oxidized LDL in lesions and had high levels of antibodies specific for oxidized LDL'7, Third, differential inflammatory and oxidative responses to an atherogenic diet were observed between the atherosclerosis-susceptible strain C57BL/6J and the resistant strain C3H/HeJ (Fig. 4). Several genes induced by the diet in C57BL/6J but not in C3H/HeJ mice are likely to promote atherogenesis; for example, macrophage colony-stimulating factor (M-CSF) and monocyte chemotactic protein-1 (MCP-1) are probably important in the growth and recruitment of monocytes-macrophages in the artery wall. Precisely the same set of genes induced by the diet was also induced by injection of mice with minimally oxidized LDL. In genetic studies, both lipid oxidation and inflammatory gene activation segregated with aortic lesion development, suggesting a genetic link between the traits '~.
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The presence of lymphocytes and antibodies to oxidized LDL in atherosclerotic lesions has implicated both cellular and humoral immunity in atherogenesis. Recent studies in genetically susceptible C57BL/6J mice with a variety of immune deficiencies (including deficiencies of both cellular and humoral immune systems) resulted in lesion development that was greater than or comparable to that in normal C57BL/6J mice'". The finding of advanced lesions in class I MHC knockout mice maintained on an atherogenic diet (Fig. lb) was particularly interesting. The increase in lesions in these cytotoxicT-cell-deficient mice suggests that the immune system may actively suppress lesion development.
New insights into lipid metabolism The mechanism of chylomicron remnant uptake ApoE is a crucial ligand in remnant uptake, and various mutations of apoE dramatically influence remnant accumulation. However, individuals with defects in the LDL (apoB/E) receptor do not exhibit impaired remnant uptake, suggesting the involvement of some other receptor in clearance of these lipoprotein particles. To test the involvement of the LDL-receptor-related protein (LRP), which is capable
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of binding apoE and is present primarily in liver, LRP knockout mice were constructed. Unfortunately, the homozygous knockout mice died hz utero, making studies of lipoprotein metabolism impossible (although suggesting some novel and unexpected functions for LRP). This problem was ingeniously overcome by transiently overexpressing the receptor-associated protein (RAP) in mice using an adenoviral vector:". RAP associates with LRP and inhibits its ability to bind apoE-containing lipoproteins. Whereas mice overproducing RAP exhibited only moderately decreased remnant uptake, overproduction
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Regulation oJ"Lp(a) Transgenic mice have proved useful for examining factors regulating gene expression. For example, previous work has defined elements involved in the tissue-specific expression of the apoAl and apoE genes. The ability to construct transgenic mice with large cloned fragments, such as PI phage clones and yeast artificial chromosome (YAC) clones, has now made analysis of the regulation of large genes such as human apoB and human apo(a) feasible. Recently, a series of YAC-carrying transgenic mice containing the entire apo(a) genc plus extensive 5' and 3' flanking sequences were generated and used to demonstrate dramatic eft'eels of sex hormones on the expression of the gene-'L Castration of male transgenic mice caused a dramatic increase in the expression of apo(a), at the level of both mRNA and plasma protein, and administration of testosterone to the castrated mice decreased the levels to those observed before castration. The importance of hormones on human lipoprotein metabolism remains to be studied. These recent studies of apo(a) also showed that, in contrast to previous speculation, apo(a) is not an acute-phase reactant. This latter point is of interest with respect to the interpretation of human
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Figure 4. Differential inflammatory and oxidative responses of an atherosclerosis-susceptible strain, C57BU6J, and a resistant strain, C3H/HeJ, to feeding of an atherogenic diet. Mice were maintained on a low-fat, low-cholesterol chow diet or a high-fat, high-cholesterol atherogenic diet. The relative expression of inflammatory genes (hepatic serum amyloid A mRNA, and hepatic NF-KB activity), an oxidative stressgene (hepatic herne oxygenase mRNA) and genes involved in rnonocyte-rnacrophage growth and chemotaxis [serum macrophage colony stimulating factor (M-CSF)and hepatic rnonocyte chemotactic protein-1 (MCP-1)mRNA] are indicated in arbitrary units. The sizes of lipid-staining aortic lesions were determined using serial sections of the tissue. Levels of conjugated dienes in liver were determined as a measure of lipid oxidation. In contrast to the other traits examined, plasma cholesterol levels in plasma high-density lipoproteins (HDL) decreased in C57BU6J mice following challenge with the atherogenic diet. Data are taken from Ref. 18.
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Figure 5. Quantitative trait loci (QTLs) and candidate genes for atherosclerosis-related traits in the mouse. QTLs, represented by colored rectangles, are indicated as 20 cM regions centered around the locations of peak LOD scores z3. (A LOD score is a statistical measure of the likelihood that a gene contributing to a particular trait resides near a given genetic locus. A LOD score of >3 is considered to be strong evidence of linkage, with a probability of false positives of <0.05.) The candidate genes, including published and unpublished loci, are indicated to the rnght of the schematic chromosomes; recombination distances (cM) relative to the centromere are shown to the left. Genes assigned to a chromosome but not mapped are indicated below the corresponding chromosome. Data from a variety of mouse crosses were combined with respect to anchor loci to form this composite map. Gene names and locations, with the exception of unpublished data, are as reported in Ref. 38. The QTLs are described in Refs 7, 25, 26, 34.
epidemiological results. Most of the early studies, which revealed a strong correlation between heart disease and Lp(a) levels, were retrospective (they examined control individuals and affected individuals subsequent to myocardial infarction). These studies raised the possibility that Lp(a) levels were increased as a result of the infarct. Several recent prospective studies (in which individuals are studied before the clinical event) have failed to observe a significant association between Lp(a) levels and myocardial infarction. The studies with mice suggest that this apparent discrepancy is probably not explained by the possibility that Lp(a) is an acute-phase reactant that is induced by the clinical event.
The role of hepatic lipase The physiological functions of hepatic lipase in lipoprotein metabolism have been poorly understood. Recent studies of hepatic lipase knockout mice revealed, surprisingly, little or no impairment in the clearance of triacylglycerol-rich lipoprotein particles'-'. However, relative to normal mice, the knockout mice exhibited an accumulation
of a subclass of HDL particles, indicating that hepatic lipase is important in the remodeling and clearance of HDL. It should be noted that whereas hepatic lipase in humans is bound to the sinusoidal endothelium of liver, in mice it is largely present in the circulation. Thus, the specific role of hepatic lipase may differ between mice and humans.
ApoB and vitamin transport An unexpected result of studies with apoB knockout mice was the finding that homozygous knockout mice exhibited embryonic lethality, possibly owing to a failure of vitamin E transport~J. Human disorders resulting in very low levels of apoB (abetalipoproteinemia and some forms of hypobetalipoproteinemia) are associated with defective vitamin transport but are not lethal. Genetic dissection of complex traits related to atherosclerosis
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to humans. Seven separate loci contributing to multifactorial obesity (which may be more similar to human obesity) were revealed recently using the QTL mapping approach '-~'~4 (Fig. 5). Interestingly, three of these QTI.z overlap mendelian mutations (chromosomes 2, 6 and 7), suggesting that different alleles of the mendelian genes may contribute to common multifactorial obesity. The effects of four of the loci (chromosomes 6, 7, 12 and 15) on the percentage of body fat in a cross between strains C57BL/6J and Mus sprems are depicted in Fig. 6. The loci exhibit nonadditive interactions with respect to body fat. Certain loci (chromosomes 6 and 7) influence plasma cholesterol levels, whereas others (chromosomes 12 and 15) do not. The loci also exhibit striking differences with respect to the regional distribution of body fat '-'.
Future directions Identification of new genes
The identification of chromosomal loci contributing to complex traits relevant to Genotype atherosclerosis is only beginning and numerous new loci will doubtless be identified in the next decade. But will it be feasible to Figure 6. Interactions of four obesity loci in a mouse model for multifactorial obesity. Mincefrom a identify many of the underlying genes and backcross of (C57BI_]6J × Mus spretus) F1 × C57BI.J6J exhibit varying levels of obesity, ranging pathways? The identification of new genes from less than a few percent to more than 50 percent body fat. Median body fat (%) of mice with on the basis of position is currently very each of the 16 possiblecombinationsof alleles at four obesityloci (chromosomes 7, 6, 12 and 15) are indicated by red bars. Mice heterozygous for Mus spretus and C57BI_/IM alleles are represented laborious, and only about 40 human genes, by white boxes; mice homozygous for C57BL/6J alleles are represented by blue boxes. Each nearly all for mendelian disorders, have been genotypic group included between 13 and 29 mice. Data are taken from Ref. 25. successfully identified by positional cloning (and the majority of these studies were aided by chromosomal disruptions). However, as involving multiple genetic factors as well as environmental influ- increasingly dense expression maps (which identify chromosomal ences. The chromosomal regions involved can be identified by analysis locations of expressed genetic sequences) are constructed in humans of genetic crosses using a complete linkage map approach (also and mice, a positional candidate-gene approach will largely replace termed quantitative trait locus or QTL mapping)-". Such studies have a pure positional cloning strategy. Using this approach, QTLs and revealed a number of chromosomal regions (QTLs) that are impor- other linked loci are scanned for relevant candidate genes; the canditant in determining lipoprotein levels (Fig. 5), as well as the changes date genes can be tested directly, accelerating the identification of the in lipoprotein levels in response to high-fat diets ~''-'. Some, but not all, genes underlying the quantitative traits. A current map of QTLs and of these QTLs correspond to the locations of known candidate genes, candidate genes for atherosclerosis-related traits is shown in Fig. 5. indicating that new genes affecting lipoprotein metabolism remain to The complementation of mouse and human genetic studies in the disbe discovered. Recent studies have revealed that genetic differences section of complex genetic traits such as atherosclerosis is illustrated in the absorption of cholesterol, or the conversion of cholesterol into in Fig. 7. bile acids, are responsible, in part, for differences in dietary responsiveness among inbred strains of mice -'7. Disease models and gene therapy The ability to perform planned genetic modifications in mice has Obesity and diabetes allowed the creation of a number of mendelian disease models relObesity and diabetes are strong risk factors for cardiovascular dis- evant to atherosclerosis". These include models for familial hypercholease. Six mendelian mutations resulting in obesity and diabetes in esterolemia (FH)9 and homocyst(e)inemia35. Such mice offer attractive mice have been identified over the years (Fig. 5). Three of the under- models for the study of genetic and environmental interactions conlying genes, obese (ob), agouti (a) and [at have now been cloned~% tributing to atherosclerosis and for the development of suitable The product of the ob gene is particularly interesting as it has been delivery vehicles and expression vectors for gene therapy. Although shown to influence appetite, food intake and energy balance by inter- gene therapy trials for FH have been initiated 3~, it is clear that imporaction with the diabetes (db) gene 3'-33. Clinical trials with the product tant basic problems relating to gene delivery and expression remain. of the ob gene, leptin, are ongoing. Discovery of the ob gene pro- Transplantation of bone marrow from normal mice into apoE knockvides an example of how studies with mouse models can be applied out mice demonstrated that bone-marrow-derived cells (presumably chr. 15
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The suitability of particular mouse models for atherosclerosis continues to stir debate. It human , c h r o m o s o m a l synteny= I mouse loci [ has been argued that the diet-induced lesions that occur in certain naturally occurring susceptible strains are poor models for atheropositional cloning or breeding~ sclerosis since the required diet is artificially positional candidate high in cholesterol and the lesions are at an positional gene congenic strains early stage of development. This reasoning has candidate led to the widespread use of certain models, gene particularly the apoE knockout mouse, as a positional cloning or background for physiological and genetic positional candidate gene studies. Although feasible, the expense of breeding mice onto the background of genetically engineered models is considerable. In adhuman gene sequence_ conservation= mouse gene dition, although the lesions in naturally occurring susceptible strains (such as C57BL/6J) are J human or mouse much smaller and less advanced, these strains biochemistry transgenics, provide a means for studying the early stages gene targeting, of atherogenesis. It will be interesting to biochemistry determine whether the same physiological and genetic factors influence lesion development in the various models. In mice, as in rabbits, the aorta has been used as a convenient large artery for mutation ~ clinical analysis "X~ls J screening drug examining atherogenesis. However, a recent study revealed that susceptibility to coronary lesions in mice is determined, in part, by genetic factors distinct from those for aortic [diagnostics] therapeutics[ lesions 5. Since coronary lesion development is of primary clinical interest, one may Figure7. Schematicdiagram showing how mouse models can accelerateidentificationof genes question whether scoring of aortic lesions contributing to complex human genetic traits. The left side of the diagram indicatesthe steps provides the most relevant information for involved in cloning a gene for a complex trait directly in humans and the right side indicatesthe human disease. steps in the mouse model.The searchesin miceand humans are bridged by chromosomalsynteny It is important to note that the mouse has (a conservationof linkagegroups in the two species)and sequenceconservation(allowingidentification of the homologous gene by cross-hybridization). A 'congenic strain' contains a small significant limitations for atherosclerosis chromosomal region derived from one strain that has been placed onto the genetic background research. There are, for example, several of a second strain by breeding. The use of congenic strains allows the isolation of individual fundamental differences in lipoprotein genescontributing to complextraits. metabolism between mice and humans, such as the absence in mice of cholesteryl ester transfer protein and Lp(a). Also, the small size of the mouse complicates studies of artery wall metabolism. atherosclerosis. It is interesting to note that, whereas the mouse Clearly, it will be important to continue studies with other estab- hardly deserved mention in atherosclerosis research a decade ago, it lished animal models such as non-human primates, pigs, rats and rab- was the subject of nearly half of the presentations at the 1995 Gordon bits; and results from studies with mice should be extrapolated to Research Conference on Atherosclerosis. What will the next ten years bring? Most of the significant candidate genes will be knocked humans with caution. out or overexpressed and new genes underlying complex traits related to atherosclerosis will be mapped and cloned. These studies Concludingremarks This review highlights only a small fraction of the recent obser- will reveal new pathways and generate new hypotheses that can then vations made in the mouse that are relevant to the problem of be tested in human subjects.
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The outstanding questions • Mice differ from humans in several aspects of lipoprotein metabolism and probably other pathways contributing to atheroselerosis. How important will such differences be in extrapolating results in the mouse to humans? • Which mouse models (e.g. naturally occurring strains or genetically engineered animals) will be most useful in examining mechanisms contributing to atherosclerosis? • What strategies will be most useful in the identification of new genes contributing to atherosclerosis? • What is the nature of the genes determining differences in atherosclerosis susceptibility among inbred strains of mice? Will these genes be important in determining susceptibility variations in human populations? • How do risk factors such as hyperlipidemia, diabetes, hypertension and high homocysteine levels interact to promote atherogenesis?
Acknowledgements. Work in the authors' laboratory was supported, in part, by NIH grants HL30568, HIA2488 and HL28481. D.M.S. was supported, in part, by NIH training grant 5T32DK07688-02. We are grateful to C. Hedrick,A. Fyfe and J-H. Qiao for help in the preparationof the manuscript;and J. Berliner and A. Watson for preparation of Fig. 3.
References 1 Paigen, B. et al. (1990) Atherosclerosis susceptibility differences among progenitors of recombinant inbred strains of mice, Arteriosclerosis I0, 316-323 2 Lusts, AJ. (1993) The mouse model for atherosclerosis, Trends Cardiovasc. MetL 3, 135-143 3 Paigen, B., Plump, A.S. and Rubin, EM. (1994) The mouse as a model for human cardiovascular disease and hyperlipidemia, Curr. Opm. Lipidol. 5, 258-264 4 Breslow, J.L. (1994) Lipoprotein metabolism and atherosclerosis susceptibility in transgenic mice, Curr. Opin. LtpidoL 5, 175-184 5 Qiao, J-H. et aL (1994) Pathology of atheromatous lesions in inbred and genetically engineered mice: genetic determination of arterial calcification, Arterioscler. Tbromb. 14, 1480-1497 6 Warden, C.H. et aL (1993) Atherosclerosls in trunsgenic mice overexpressing apolipoprotein All, Science 261,469--472 7 Qiao, J-H. et aL (1993) Involvement of the tyrosiuase gone in deposition of cardiac lipofuscin in mice: association with aortic fatty streak development, J. Clin. Invest. 92, 2386-2393 8 Nakashima, Y. et al. (1993) ApoE-deficient mice develop lesions of all phases of atherosclerosis throughout the arterial tree, Arterwscler. Thromb. 14, 133-140 9 lshibashi,S. et aL (1994) Massive xanthomatosis and atherosclerosis in cholesteralfed low density lipoprotein receptor-negative mice, J. Clin. hwest. 93, 1885-1893 10 PurcelI-Huynh, D.A. et al. (1995) Transgeuic mice expressing high levels of human apolipoproteiu B develop severe atherosclerotic lesions in response to a high-fat diet, J. Clin. Invest. 95, 2246-2257 11 Paszty, C., Maeda, N., Verstuyfl, J. and Rubin, E.M. (1994) Apolipoprotein AI transgene corrects apolipoprotein E deficiency-induced atheroosclerosis in mice, J. Clin. Invest. 94, 899-903 12 Plump, A.S., Scott, C. and Breslow, J.L. (1994) Human apoAl gone expression increases high density lipoprotein and suppresses atherosclerosis in the apolipoprotein E-deficient mouse, Proc. NatlAcad. Sci. USA 91, 9607-9611 13 Li, H., Reddick, R.L. and Maeda, N. (1993) Lack of apoAl is not associated with increased susceptibilityto atheresderosis in mice, Arterioscler. Thromb. 13, 1814-1821 14 Linton, M.E et al. (1993) Transgenic mice expressing high plasma concentrations of human apolipoprotein B100 and lipoprotein(a), J. Clin. Invest. 92, 3029-3037
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15 Callow. M.J.. Slolzfus, L.I.. I~lwn. R.M. and Rubin, E.M (1t~94) Expression of human apolipoprotein B and assembly of lipoprotein(a) in transgenic mice. Proe. Natl Acad. Set. USA 91. 2130-2134 16 Berliner,J.A et at. (1995) Atherosclerosis: basic mechanisms, oxidation, inflammation, and genetics. Cwculatton 91, 2488-2496 17 Palinski,W. ct at. (1994) ApoE-deficient mice are a model of lipoprotein oxidation in atherogenesis: demonstration of oxidation specific epitopes in lesions and high titers of autoantibodies to malondialdehyde-lysine in serum, Artertosch, r. TIv'omb. 14, 605-616 18 Liao, E et al. (1994) Genetic evidence for a common pathway mediating oxidative stress, inflammatory, gene induction, and aortic fatty streak formation in mice, J. CIm. Invest. 94. 877-884 19 Fyfe,A.I., Qtan, J-H. and Lusts,A.J. (1994) Immune deficient mice develop typical atherosclerotic fatty streaks when fed an atherogenic diet, J. Clin. Invest. 94, 2516-2520 20 Willnow,T.E., Sheng, Z., lshibashi,S and Herz.J. (1994) Inhibition of hepatic chylomicron remnant uptake by gene transfer of a receptor agonlst,Science 264, 1471-1464 21 Frazer, K.A., Narla, G., Zhang, J.L. and Rubin, E.M (1994) The apolipoprotein(a) gene is regulated by sex hormones and acute-phase inducers in YAC transgenic mice, Nat. Genet. 9, 424--431 22 Homanics, G.E. et aL (1995) Mild dyslipidemia in mice following targeted inactivation of the hepatic lipase gone. J. Biol. Chem. 270, 2974-2980 23 Farese, R.V, Jr et al. (1995) Knockout of the mouse apolipoprotein B gone results in embryonic lethality in homozygutes and protection against diet-induced hypereholesterolemia in heterozygotes. Proc. NatlAcad. Set. USA 92, 1774-1778 24 Lander, E.S. and Schork, N.J. (1994) Genetic dissection of complex traits, Scieace 265, 2037-2048 25 Warden. C.H. et at. (1995) Identification of four chromosomal loci determining obesity in a multifactorial mouse model, J. Clin. Invest. 95, 1545-1552 26 PurcelI-Huynh, D.A. et al. Genetic factors in lipoprotein metabolism: analysis of a genetic cross between inbred mouse strains NZB/BINJ and SM/J using a complete linkage map approach, J. Chn. Invest. (ra press) 27 Kirk. E.A. et al. (1995) Hyper- and hypo-responsiveness to dietary fat and cholesterol among inbred mice: search for level and variability genes. J. Ltptd Res. 36, 1522-1532 28 Zhang, Y. et al. (1994) Positional cloning of the mouse obese gene and its human homologue, Nature 372, 425--432 29 Bultman, S.J., Michand, E.J. and Woychik R.P. (1992) Molecular characterization of the mouse agouti locus, Cell 71, 1195-1204 30 Naggert, J.K. et at. (1995) Hyperproinsulinaemia in obese fat/fat mice associated with a carboxypeptidase E mutation which reduces enzyme activity, Nat. Genet. 10, 135-142 31 Pelleymounter, M.A. el aL (1995) Effects of the obese gene product on body weight regulation in ob/ob mice, Science 269, 540-543 32 Hallas, J.L. et aL (1995) Weight-reducing effects ofthe plasma protein encoded by the obese gene, Science 269, 543-546 33 Campfield, L.A. et at. (1995) Recombinant mouse ob protein: evidence for a peripherul signal linking adiposity and central neural networks, Science 269, 546-549 34 West, D.B., Goudey-Lefevre,J., York, B. and Truett, G. (1994) Dietary obesity linked to genetic loci on chromosomes 9 and 15 in a polygenic mouse model, J. Clia. hn,est. 94, 1410-1416 35 Watanabe,M. et al. (1995) Mice deficient in cystathionine I~-synthase:animal models for mild and severe homocyst(e)inemia, Proc. NadAcad. Sci. USA 92, 1585-1589 36 Grossman, M. et al. (1994) Successful ex vivo gene therapy directed to liver in a patient with familial hypereholesterolemia, Nat. Genet. 6, 335-341 37 Linton, M.E, Atkinson, J.B. and Fazio, S. (1995) Prevention of atherosclerosis in apolipoprotein E-deficient mice by hone marrow transplantation, Science 267, 1034-1037 38 Silver, L.M., Nadeau, J.H. and Goodfellow, P.N., eds (1994) Mammalian Genome 5 (Suppl.), Springer-Verlag
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Atherosclerosis is a disease of the large arteries that is the cause of heart disease and stroke. It is a highly complex disorder with multiple genetic and environmental influences. The mouse model has proved very useful for studying atherosclerosis
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Universityof California,LosAngeles,CA90095,USA. , ~ m o d i f i c a t i o n are feasible in this organism. In this Tel: +1 310 825 1595
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THE mouse has become the most widely used animal model of atherosclerosis for several reasons ~2. First, the hundreds of strains of mice developed over the past century exhibit many of the same differences in disease susceptibility as do humans. In common with humans, essentially all of these differences (affecting lipoprotein metabolism, dietary responsiveness, immune functions, inflammatory functions, obesity and diabetes, and susceptibility to atherosclerotic lesions) are due to the effects of multiple genetic factors. Second, the mouse model ! been uniquely developed as a tool for genetic studies. A dense linkage map with more than 10000 genetic markers has been constructed, and many specialized stocks exhibiting mutations and chromosomal rearrangements have been collected. Third, techniques for directed genetic alterations are most advanced in the mouse. Transgenic mice carry copies of a gene introduced by microinjection into fertilized eggs, and gene targeted mice (also known as knockout mice) have specific mutations introduced by homologous recombination. © 1 9 q 5 , Elsevier Science Ltd
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The use of mice f o r genetic analysis of alherosclerosis began with the work of J. Thompson in the late 1960s. In the early 1981)s, B. Paigen and co-workers modified the methods of Thompson to quantitatively assess atherosclcrotic lesion development, and several laboratories began to examine lipoprotein metabolism in the mouse. These studies led to the identification of genetic variations in lipoprotein levels and lesion development. Subsequently, several laboratories, including those of Paigen and R. LcBoeuf, tentatively mapped several genes controlling high-density lipoprotcin (HDL) cholesterol levels and aortic lesion size (reviewed in Refs 1, 2). Beginning in the late 1980s, a number of laboratories began to use transgenic and gene targeted mice to study mechanisms involved in lipoprotein metabolism and the role of lipoproteins in atherosclerosis. Particularly significant were the findings from E. Rubin and co-workers that transgenic mice containing the gene for apolipoprotein AI (apoAl) exhibited dramatically reduced lesion development in response to an atherogenic diet, and from J. Brcslow's and N. Maeda's laboratories that apoE knockout mice exhibited hyperlipidemia and advanced atherosclerotic lesions similar to fibrous human lesions (reviewed in Refs 3, 4). In this review, we summarize some important findings from the past two years•
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Figure 1. Pathology of atherosclerotic lesions in mice. (a) A large lesion, occupying all layers of Atherosclerotic lesions the aortic wall, with calcification (arrow), in a naturally occurring susceptible mouse model (strain As mentioned above, previous studies have C57B!_/6J, atherogenic diet). (b) A similar lesion (arrow) with an early fibrous cap in an immuneshown that large genetic differences exist deficient mouse model (strain C57BL/6J, class I MHC knockout, atherogenic diet). (c) An atherosclerotic lesion with calcification (arrow) in a hypercholesterolemic mouse model (mixed genetic among inbred strains of mice in their suscepbackground, apoEknockout, chow). (d) Lipofuscin (dark brown pigment) deposition on the left tibility to the development of atherosclerotic ventricular side of the aortic valves in a naturally occurring susceptible strain (C57BI_/6J, atherolesions '-3 (Fig. 1). Naturally occurring suscepgenic diet). All sections were stained with Oil-Red-0, Hematoxylin and Fast green. Lipid-containing tible inbred strains develop lesions only when lesions stain red and calcified regions stain blue. maintained on a high-fat, high-cholesterol diet, whereas certain genetically engineered strains that exhibit high levels of plasma cholesterol also develop lesions on a low-fat diet. Lesions in mice differ not only influencing the levels and composition of plasma lipoproteins. At least in size, but also in composition. There are variations in the content of one gene may act at the level of lipoprotein oxidation (discussed below). Genetic studies with mice provided the first definitive evidence lipid, the presence or absence of fibrous elements and calcification, and the deposition of lipofuscin (a pigmented complex consisting of that arterial calcification is determined by genetic factors5. As yet the nature of these factors is unknown. oxidized lipids and proteins) -~(Fig. 1). Lipofuscin occurs in the cardiac valves and coronary arteries of Genetic crosses between various susceptible and resistant strains certain strains of mice but not others• A genetic cross between strains of mice provide a means of mapping and ultimately identifying genetic contributions to lesion development. Such studies suggested differing in deposition of coronary lipofuscin revealed co-segregation that increased expression of the apoAHgene, on chromosome 1, pro- of the trait with the gene encoding tyrosinase on chromosome 7. motes development of aortic lesions, presumably by influencing the Mutational studies of this gene confirmed that it determines lipoanti-atherogenic nature of HDL. This was confirmed using transgenic fuscin deposition 7. mice producing high levels of apoAII (Ref. 6). Several other putative chromosomal loci influencing aortic lesion size (designated Ath-l,Ath-2, Pathways involved in atherogenesis Ath-3, etc.) have been reported (reviewed in Refs 1-3), but these re- Lipoprotein metabolism Results fi'om studies with transgenic and knockout mice (Fig. 2) were quire confirmation, and the identities of the underlying genes are unknown. Some of these genes appear to contribute to atherosclerosis by consistent with the conclusion that high levels of apoB-containing
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exhibited marked reduction in levels of HDL cholesterol but did not show enhanced susceptibility to atherosclerosis when fcd a high-fat diet ~'. Thc results suggest that reduction of apoAl alonc does not cause atherosclerosis. The interpretation of the results, however, Intestine was complicated by the fact that the mice were of a mixed genetic background. It will be of interest to exanaine the efB48(+, fects of a deficiency of apoAl AI(+/-~'~N on the background of genetically engineered mice that are highly susceptible to lesion I Chylomicron development, such as apoE AIV(+)~ / knockout mice. ct(+)~,(+] The functional heterogeneity of HDL was strikingly demonCll(+) I LPL(+) strated by studies of apoAH transgenic mice". ApoAll is the FFA second most abundant protein of HDL (next to apoAI). In common with apoAltransgenic mice. apoAll transgenic mice BIER (+1-) exhibited increased levels of HDL cholesterol, but in cont ~ . ~ ! ~ "~,-: 2 ,----:: . . . . . . trast to apoAl transgenic mice, they showed dramatically increased susceptibility Figure 2. Major pathways for plasma lipid transport in humans. Genetically engineered mouse models that have been developed to examine lipoprotein metabolism are indicated: transgenic (+), knockout (-), both (+/-). The to atherosclerosis. These mice major classes of human plasma lipoproteins [chylomicrons, remnants, very low-density lipoproteins (VLDL), intershould be useful in dissecting mediate-density lipoproteins (IDL), low-density lipoproteins (LDL), high-density lipoproteins (HDL) and lipoprotein the properties of HDL that (a)] and component apolipoproteins [(a), AI, All, AIV, 1348,13100,CI, CII, CIII and E] are shown. Key receptors, transfer are reponsible for protection proteins and enzymes functioning in human lipid metabolism include: the LDL receptor (B/ER), the LDL-receptorrelated protein (LRP), lipoprotein lipase (LPL), hepatic lipase (HL), cholesteryl ester transfer protein (CETP), lecithinagainst atherosclerosis. cholesterol acyltransferase (LCAT), and cholesterol-7a-hydroxylase (7a OH). xLipids transported include free fatty The atherogenic potential acids (FFA), cholesteryl ester (CHOL E) and triacylglycerol (TG). For further reading, see Ref. 4. of the Lp(a) lipoprotein (associated with coronary artery disease in some but not all lipoproteins promote atherogenesis. Thus, apoEknockout mice, low- human epidemiological studies) was further studied in the mouse density lipoprotein [LDL (apoB/E)] receptor knockout mice and trans- model. Lp(a) is an LDL-like particle that contains, in addition to genic mice containing the human apoB gene all accumulated high apoB, a large polypeptide termed apo(a). Mice do not naturally proplasma levels of apoB-containing lipoproteins, and these mice devel- duce apo(a). Previous studies with human apo(a) transgenic mice oped much larger and more advanced lesions than those in naturally revealed an enhancement in atherosclerotic lesion development occurring susceptible strains 8-t°. These models will be valuable for (reviewed in Refs 2-4). These transgenic mice, however, failed to examining mechanisms contributing to the development of fibrous assemble intact Lp(a) particles owing to the inability of human and complex lesions, since the lesions in naturally occurring strains apo(a) to form complexes with mouse apoB. This problem has now been overcome by coexpression of human apoB as a transgene ~"JS. generally do not progress to advanced stages. The interaction of HDL and apoB-containing lipoproteins in ath- The combined human-apoB-human-apo(a) mice have high erosclerosis was studied in combined human apoAl-transgenic-apoE Lp(a) levels, but whether they will exhibit increased lesion developknockout mice. Previous studies had shown that apoAl transgenic ment remains to be tested. It will also be of interest to determine mice exhibited increased HDL-cholesterol levels and had reduced ath- whether there are differences in the atherogenic potentials of free erosclerosis in response to an atherogenic diet (reviewed in Refs 2-4). In apo(a) and Lp(a). the current studies, a dramatic protective effect of increased apoAI and HDL-cholesterol was observed in the apoEknockout model "J2. These Oxidation Oxidatively modified LDL occurs in both human and mouse results strongly support the concept that apoAI and HDL directly protect atherosclerotic lesions and it exhibits a variety of properties that are against atherosclerosis. In contrast to these results, apoA1knockout mice Dietary fat, cholesterol
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likely to be proathcrogenic, including ~, potent pro-inflammatory activity, cytotoxicity, rapid uptake by macrophages, and mitogenic activity'" (Fig. 3). Several recent genetic studies in mice support a role for oxidation in atherogenesis. First, the accumulation of aortic valve lipofuscin, a terminal oxidation complex, was associated with increased atherogencsis in BALB/cJ mice 7. Second, hypercholesterolemic apoE knockout mice, which develop relatively advanced atherosclerotic lesions, accumulated oxidized LDL in lesions and had high levels of antibodies specific for oxidized LDL'7, Third, differential inflammatory and oxidative responses to an atherogenic diet were observed between the atherosclerosis-susceptible strain C57BL/6J and the resistant strain C3H/HeJ (Fig. 4). Several genes induced by the diet in C57BL/6J but not in C3H/HeJ mice are likely to promote atherogenesis; for example, macrophage colony-stimulating factor (M-CSF) and monocyte chemotactic protein-1 (MCP-1) are probably important in the growth and recruitment of monocytes-macrophages in the artery wall. Precisely the same set of genes induced by the diet was also induced by injection of mice with minimally oxidized LDL. In genetic studies, both lipid oxidation and inflammatory gene activation segregated with aortic lesion development, suggesting a genetic link between the traits '~.
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The presence of lymphocytes and antibodies to oxidized LDL in atherosclerotic lesions has implicated both cellular and humoral immunity in atherogenesis. Recent studies in genetically susceptible C57BL/6J mice with a variety of immune deficiencies (including deficiencies of both cellular and humoral immune systems) resulted in lesion development that was greater than or comparable to that in normal C57BL/6J mice'". The finding of advanced lesions in class I MHC knockout mice maintained on an atherogenic diet (Fig. lb) was particularly interesting. The increase in lesions in these cytotoxicT-cell-deficient mice suggests that the immune system may actively suppress lesion development.
New insights into lipid metabolism The mechanism of chylomicron remnant uptake ApoE is a crucial ligand in remnant uptake, and various mutations of apoE dramatically influence remnant accumulation. However, individuals with defects in the LDL (apoB/E) receptor do not exhibit impaired remnant uptake, suggesting the involvement of some other receptor in clearance of these lipoprotein particles. To test the involvement of the LDL-receptor-related protein (LRP), which is capable
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of binding apoE and is present primarily in liver, LRP knockout mice were constructed. Unfortunately, the homozygous knockout mice died hz utero, making studies of lipoprotein metabolism impossible (although suggesting some novel and unexpected functions for LRP). This problem was ingeniously overcome by transiently overexpressing the receptor-associated protein (RAP) in mice using an adenoviral vector:". RAP associates with LRP and inhibits its ability to bind apoE-containing lipoproteins. Whereas mice overproducing RAP exhibited only moderately decreased remnant uptake, overproduction
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Regulation oJ"Lp(a) Transgenic mice have proved useful for examining factors regulating gene expression. For example, previous work has defined elements involved in the tissue-specific expression of the apoAl and apoE genes. The ability to construct transgenic mice with large cloned fragments, such as PI phage clones and yeast artificial chromosome (YAC) clones, has now made analysis of the regulation of large genes such as human apoB and human apo(a) feasible. Recently, a series of YAC-carrying transgenic mice containing the entire apo(a) genc plus extensive 5' and 3' flanking sequences were generated and used to demonstrate dramatic eft'eels of sex hormones on the expression of the gene-'L Castration of male transgenic mice caused a dramatic increase in the expression of apo(a), at the level of both mRNA and plasma protein, and administration of testosterone to the castrated mice decreased the levels to those observed before castration. The importance of hormones on human lipoprotein metabolism remains to be studied. These recent studies of apo(a) also showed that, in contrast to previous speculation, apo(a) is not an acute-phase reactant. This latter point is of interest with respect to the interpretation of human
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Figure 4. Differential inflammatory and oxidative responses of an atherosclerosis-susceptible strain, C57BU6J, and a resistant strain, C3H/HeJ, to feeding of an atherogenic diet. Mice were maintained on a low-fat, low-cholesterol chow diet or a high-fat, high-cholesterol atherogenic diet. The relative expression of inflammatory genes (hepatic serum amyloid A mRNA, and hepatic NF-KB activity), an oxidative stressgene (hepatic herne oxygenase mRNA) and genes involved in rnonocyte-rnacrophage growth and chemotaxis [serum macrophage colony stimulating factor (M-CSF)and hepatic rnonocyte chemotactic protein-1 (MCP-1)mRNA] are indicated in arbitrary units. The sizes of lipid-staining aortic lesions were determined using serial sections of the tissue. Levels of conjugated dienes in liver were determined as a measure of lipid oxidation. In contrast to the other traits examined, plasma cholesterol levels in plasma high-density lipoproteins (HDL) decreased in C57BU6J mice following challenge with the atherogenic diet. Data are taken from Ref. 18.
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Figure 5. Quantitative trait loci (QTLs) and candidate genes for atherosclerosis-related traits in the mouse. QTLs, represented by colored rectangles, are indicated as 20 cM regions centered around the locations of peak LOD scores z3. (A LOD score is a statistical measure of the likelihood that a gene contributing to a particular trait resides near a given genetic locus. A LOD score of >3 is considered to be strong evidence of linkage, with a probability of false positives of <0.05.) The candidate genes, including published and unpublished loci, are indicated to the rnght of the schematic chromosomes; recombination distances (cM) relative to the centromere are shown to the left. Genes assigned to a chromosome but not mapped are indicated below the corresponding chromosome. Data from a variety of mouse crosses were combined with respect to anchor loci to form this composite map. Gene names and locations, with the exception of unpublished data, are as reported in Ref. 38. The QTLs are described in Refs 7, 25, 26, 34.
epidemiological results. Most of the early studies, which revealed a strong correlation between heart disease and Lp(a) levels, were retrospective (they examined control individuals and affected individuals subsequent to myocardial infarction). These studies raised the possibility that Lp(a) levels were increased as a result of the infarct. Several recent prospective studies (in which individuals are studied before the clinical event) have failed to observe a significant association between Lp(a) levels and myocardial infarction. The studies with mice suggest that this apparent discrepancy is probably not explained by the possibility that Lp(a) is an acute-phase reactant that is induced by the clinical event.
The role of hepatic lipase The physiological functions of hepatic lipase in lipoprotein metabolism have been poorly understood. Recent studies of hepatic lipase knockout mice revealed, surprisingly, little or no impairment in the clearance of triacylglycerol-rich lipoprotein particles'-'. However, relative to normal mice, the knockout mice exhibited an accumulation
of a subclass of HDL particles, indicating that hepatic lipase is important in the remodeling and clearance of HDL. It should be noted that whereas hepatic lipase in humans is bound to the sinusoidal endothelium of liver, in mice it is largely present in the circulation. Thus, the specific role of hepatic lipase may differ between mice and humans.
ApoB and vitamin transport An unexpected result of studies with apoB knockout mice was the finding that homozygous knockout mice exhibited embryonic lethality, possibly owing to a failure of vitamin E transport~J. Human disorders resulting in very low levels of apoB (abetalipoproteinemia and some forms of hypobetalipoproteinemia) are associated with defective vitamin transport but are not lethal. Genetic dissection of complex traits related to atherosclerosis
Lipoprotein metabolism In mice, as in humans, most variations of lipoprotein metabolism and other traits associated with atherosclerosis are complex, m
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to humans. Seven separate loci contributing to multifactorial obesity (which may be more similar to human obesity) were revealed recently using the QTL mapping approach '-~'~4 (Fig. 5). Interestingly, three of these QTI.z overlap mendelian mutations (chromosomes 2, 6 and 7), suggesting that different alleles of the mendelian genes may contribute to common multifactorial obesity. The effects of four of the loci (chromosomes 6, 7, 12 and 15) on the percentage of body fat in a cross between strains C57BL/6J and Mus sprems are depicted in Fig. 6. The loci exhibit nonadditive interactions with respect to body fat. Certain loci (chromosomes 6 and 7) influence plasma cholesterol levels, whereas others (chromosomes 12 and 15) do not. The loci also exhibit striking differences with respect to the regional distribution of body fat '-'.
Future directions Identification of new genes
The identification of chromosomal loci contributing to complex traits relevant to Genotype atherosclerosis is only beginning and numerous new loci will doubtless be identified in the next decade. But will it be feasible to Figure 6. Interactions of four obesity loci in a mouse model for multifactorial obesity. Mincefrom a identify many of the underlying genes and backcross of (C57BI_]6J × Mus spretus) F1 × C57BI.J6J exhibit varying levels of obesity, ranging pathways? The identification of new genes from less than a few percent to more than 50 percent body fat. Median body fat (%) of mice with on the basis of position is currently very each of the 16 possiblecombinationsof alleles at four obesityloci (chromosomes 7, 6, 12 and 15) are indicated by red bars. Mice heterozygous for Mus spretus and C57BI_/IM alleles are represented laborious, and only about 40 human genes, by white boxes; mice homozygous for C57BL/6J alleles are represented by blue boxes. Each nearly all for mendelian disorders, have been genotypic group included between 13 and 29 mice. Data are taken from Ref. 25. successfully identified by positional cloning (and the majority of these studies were aided by chromosomal disruptions). However, as involving multiple genetic factors as well as environmental influ- increasingly dense expression maps (which identify chromosomal ences. The chromosomal regions involved can be identified by analysis locations of expressed genetic sequences) are constructed in humans of genetic crosses using a complete linkage map approach (also and mice, a positional candidate-gene approach will largely replace termed quantitative trait locus or QTL mapping)-". Such studies have a pure positional cloning strategy. Using this approach, QTLs and revealed a number of chromosomal regions (QTLs) that are impor- other linked loci are scanned for relevant candidate genes; the canditant in determining lipoprotein levels (Fig. 5), as well as the changes date genes can be tested directly, accelerating the identification of the in lipoprotein levels in response to high-fat diets ~''-'. Some, but not all, genes underlying the quantitative traits. A current map of QTLs and of these QTLs correspond to the locations of known candidate genes, candidate genes for atherosclerosis-related traits is shown in Fig. 5. indicating that new genes affecting lipoprotein metabolism remain to The complementation of mouse and human genetic studies in the disbe discovered. Recent studies have revealed that genetic differences section of complex genetic traits such as atherosclerosis is illustrated in the absorption of cholesterol, or the conversion of cholesterol into in Fig. 7. bile acids, are responsible, in part, for differences in dietary responsiveness among inbred strains of mice -'7. Disease models and gene therapy The ability to perform planned genetic modifications in mice has Obesity and diabetes allowed the creation of a number of mendelian disease models relObesity and diabetes are strong risk factors for cardiovascular dis- evant to atherosclerosis". These include models for familial hypercholease. Six mendelian mutations resulting in obesity and diabetes in esterolemia (FH)9 and homocyst(e)inemia35. Such mice offer attractive mice have been identified over the years (Fig. 5). Three of the under- models for the study of genetic and environmental interactions conlying genes, obese (ob), agouti (a) and [at have now been cloned~% tributing to atherosclerosis and for the development of suitable The product of the ob gene is particularly interesting as it has been delivery vehicles and expression vectors for gene therapy. Although shown to influence appetite, food intake and energy balance by inter- gene therapy trials for FH have been initiated 3~, it is clear that imporaction with the diabetes (db) gene 3'-33. Clinical trials with the product tant basic problems relating to gene delivery and expression remain. of the ob gene, leptin, are ongoing. Discovery of the ob gene pro- Transplantation of bone marrow from normal mice into apoE knockvides an example of how studies with mouse models can be applied out mice demonstrated that bone-marrow-derived cells (presumably chr. 15
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The suitability of particular mouse models for atherosclerosis continues to stir debate. It human , c h r o m o s o m a l synteny= I mouse loci [ has been argued that the diet-induced lesions that occur in certain naturally occurring susceptible strains are poor models for atheropositional cloning or breeding~ sclerosis since the required diet is artificially positional candidate high in cholesterol and the lesions are at an positional gene congenic strains early stage of development. This reasoning has candidate led to the widespread use of certain models, gene particularly the apoE knockout mouse, as a positional cloning or background for physiological and genetic positional candidate gene studies. Although feasible, the expense of breeding mice onto the background of genetically engineered models is considerable. In adhuman gene sequence_ conservation= mouse gene dition, although the lesions in naturally occurring susceptible strains (such as C57BL/6J) are J human or mouse much smaller and less advanced, these strains biochemistry transgenics, provide a means for studying the early stages gene targeting, of atherogenesis. It will be interesting to biochemistry determine whether the same physiological and genetic factors influence lesion development in the various models. In mice, as in rabbits, the aorta has been used as a convenient large artery for mutation ~ clinical analysis "X~ls J screening drug examining atherogenesis. However, a recent study revealed that susceptibility to coronary lesions in mice is determined, in part, by genetic factors distinct from those for aortic [diagnostics] therapeutics[ lesions 5. Since coronary lesion development is of primary clinical interest, one may Figure7. Schematicdiagram showing how mouse models can accelerateidentificationof genes question whether scoring of aortic lesions contributing to complex human genetic traits. The left side of the diagram indicatesthe steps provides the most relevant information for involved in cloning a gene for a complex trait directly in humans and the right side indicatesthe human disease. steps in the mouse model.The searchesin miceand humans are bridged by chromosomalsynteny It is important to note that the mouse has (a conservationof linkagegroups in the two species)and sequenceconservation(allowingidentification of the homologous gene by cross-hybridization). A 'congenic strain' contains a small significant limitations for atherosclerosis chromosomal region derived from one strain that has been placed onto the genetic background research. There are, for example, several of a second strain by breeding. The use of congenic strains allows the isolation of individual fundamental differences in lipoprotein genescontributing to complextraits. metabolism between mice and humans, such as the absence in mice of cholesteryl ester transfer protein and Lp(a). Also, the small size of the mouse complicates studies of artery wall metabolism. atherosclerosis. It is interesting to note that, whereas the mouse Clearly, it will be important to continue studies with other estab- hardly deserved mention in atherosclerosis research a decade ago, it lished animal models such as non-human primates, pigs, rats and rab- was the subject of nearly half of the presentations at the 1995 Gordon bits; and results from studies with mice should be extrapolated to Research Conference on Atherosclerosis. What will the next ten years bring? Most of the significant candidate genes will be knocked humans with caution. out or overexpressed and new genes underlying complex traits related to atherosclerosis will be mapped and cloned. These studies Concludingremarks This review highlights only a small fraction of the recent obser- will reveal new pathways and generate new hypotheses that can then vations made in the mouse that are relevant to the problem of be tested in human subjects.
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The outstanding questions • Mice differ from humans in several aspects of lipoprotein metabolism and probably other pathways contributing to atheroselerosis. How important will such differences be in extrapolating results in the mouse to humans? • Which mouse models (e.g. naturally occurring strains or genetically engineered animals) will be most useful in examining mechanisms contributing to atherosclerosis? • What strategies will be most useful in the identification of new genes contributing to atherosclerosis? • What is the nature of the genes determining differences in atherosclerosis susceptibility among inbred strains of mice? Will these genes be important in determining susceptibility variations in human populations? • How do risk factors such as hyperlipidemia, diabetes, hypertension and high homocysteine levels interact to promote atherogenesis?
Acknowledgements. Work in the authors' laboratory was supported, in part, by NIH grants HL30568, HIA2488 and HL28481. D.M.S. was supported, in part, by NIH training grant 5T32DK07688-02. We are grateful to C. Hedrick,A. Fyfe and J-H. Qiao for help in the preparationof the manuscript;and J. Berliner and A. Watson for preparation of Fig. 3.
References 1 Paigen, B. et al. (1990) Atherosclerosis susceptibility differences among progenitors of recombinant inbred strains of mice, Arteriosclerosis I0, 316-323 2 Lusts, AJ. (1993) The mouse model for atherosclerosis, Trends Cardiovasc. MetL 3, 135-143 3 Paigen, B., Plump, A.S. and Rubin, EM. (1994) The mouse as a model for human cardiovascular disease and hyperlipidemia, Curr. Opm. Lipidol. 5, 258-264 4 Breslow, J.L. (1994) Lipoprotein metabolism and atherosclerosis susceptibility in transgenic mice, Curr. Opin. LtpidoL 5, 175-184 5 Qiao, J-H. et aL (1994) Pathology of atheromatous lesions in inbred and genetically engineered mice: genetic determination of arterial calcification, Arterioscler. Tbromb. 14, 1480-1497 6 Warden, C.H. et aL (1993) Atherosclerosls in trunsgenic mice overexpressing apolipoprotein All, Science 261,469--472 7 Qiao, J-H. et aL (1993) Involvement of the tyrosiuase gone in deposition of cardiac lipofuscin in mice: association with aortic fatty streak development, J. Clin. Invest. 92, 2386-2393 8 Nakashima, Y. et al. (1993) ApoE-deficient mice develop lesions of all phases of atherosclerosis throughout the arterial tree, Arterwscler. Thromb. 14, 133-140 9 lshibashi,S. et aL (1994) Massive xanthomatosis and atherosclerosis in cholesteralfed low density lipoprotein receptor-negative mice, J. Clin. hwest. 93, 1885-1893 10 PurcelI-Huynh, D.A. et al. (1995) Transgeuic mice expressing high levels of human apolipoproteiu B develop severe atherosclerotic lesions in response to a high-fat diet, J. Clin. Invest. 95, 2246-2257 11 Paszty, C., Maeda, N., Verstuyfl, J. and Rubin, E.M. (1994) Apolipoprotein AI transgene corrects apolipoprotein E deficiency-induced atheroosclerosis in mice, J. Clin. Invest. 94, 899-903 12 Plump, A.S., Scott, C. and Breslow, J.L. (1994) Human apoAl gone expression increases high density lipoprotein and suppresses atherosclerosis in the apolipoprotein E-deficient mouse, Proc. NatlAcad. Sci. USA 91, 9607-9611 13 Li, H., Reddick, R.L. and Maeda, N. (1993) Lack of apoAl is not associated with increased susceptibilityto atheresderosis in mice, Arterioscler. Thromb. 13, 1814-1821 14 Linton, M.E et al. (1993) Transgenic mice expressing high plasma concentrations of human apolipoprotein B100 and lipoprotein(a), J. Clin. Invest. 92, 3029-3037
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15 Callow. M.J.. Slolzfus, L.I.. I~lwn. R.M. and Rubin, E.M (1t~94) Expression of human apolipoprotein B and assembly of lipoprotein(a) in transgenic mice. Proe. Natl Acad. Set. USA 91. 2130-2134 16 Berliner,J.A et at. (1995) Atherosclerosis: basic mechanisms, oxidation, inflammation, and genetics. Cwculatton 91, 2488-2496 17 Palinski,W. ct at. (1994) ApoE-deficient mice are a model of lipoprotein oxidation in atherogenesis: demonstration of oxidation specific epitopes in lesions and high titers of autoantibodies to malondialdehyde-lysine in serum, Artertosch, r. TIv'omb. 14, 605-616 18 Liao, E et al. (1994) Genetic evidence for a common pathway mediating oxidative stress, inflammatory, gene induction, and aortic fatty streak formation in mice, J. CIm. Invest. 94. 877-884 19 Fyfe,A.I., Qtan, J-H. and Lusts,A.J. (1994) Immune deficient mice develop typical atherosclerotic fatty streaks when fed an atherogenic diet, J. Clin. Invest. 94, 2516-2520 20 Willnow,T.E., Sheng, Z., lshibashi,S and Herz.J. (1994) Inhibition of hepatic chylomicron remnant uptake by gene transfer of a receptor agonlst,Science 264, 1471-1464 21 Frazer, K.A., Narla, G., Zhang, J.L. and Rubin, E.M (1994) The apolipoprotein(a) gene is regulated by sex hormones and acute-phase inducers in YAC transgenic mice, Nat. Genet. 9, 424--431 22 Homanics, G.E. et aL (1995) Mild dyslipidemia in mice following targeted inactivation of the hepatic lipase gone. J. Biol. Chem. 270, 2974-2980 23 Farese, R.V, Jr et al. (1995) Knockout of the mouse apolipoprotein B gone results in embryonic lethality in homozygutes and protection against diet-induced hypereholesterolemia in heterozygotes. Proc. NatlAcad. Set. USA 92, 1774-1778 24 Lander, E.S. and Schork, N.J. (1994) Genetic dissection of complex traits, Scieace 265, 2037-2048 25 Warden. C.H. et at. (1995) Identification of four chromosomal loci determining obesity in a multifactorial mouse model, J. Clin. Invest. 95, 1545-1552 26 PurcelI-Huynh, D.A. et al. Genetic factors in lipoprotein metabolism: analysis of a genetic cross between inbred mouse strains NZB/BINJ and SM/J using a complete linkage map approach, J. Chn. Invest. (ra press) 27 Kirk. E.A. et al. (1995) Hyper- and hypo-responsiveness to dietary fat and cholesterol among inbred mice: search for level and variability genes. J. Ltptd Res. 36, 1522-1532 28 Zhang, Y. et al. (1994) Positional cloning of the mouse obese gene and its human homologue, Nature 372, 425--432 29 Bultman, S.J., Michand, E.J. and Woychik R.P. (1992) Molecular characterization of the mouse agouti locus, Cell 71, 1195-1204 30 Naggert, J.K. et at. (1995) Hyperproinsulinaemia in obese fat/fat mice associated with a carboxypeptidase E mutation which reduces enzyme activity, Nat. Genet. 10, 135-142 31 Pelleymounter, M.A. el aL (1995) Effects of the obese gene product on body weight regulation in ob/ob mice, Science 269, 540-543 32 Hallas, J.L. et aL (1995) Weight-reducing effects ofthe plasma protein encoded by the obese gene, Science 269, 543-546 33 Campfield, L.A. et at. (1995) Recombinant mouse ob protein: evidence for a peripherul signal linking adiposity and central neural networks, Science 269, 546-549 34 West, D.B., Goudey-Lefevre,J., York, B. and Truett, G. (1994) Dietary obesity linked to genetic loci on chromosomes 9 and 15 in a polygenic mouse model, J. Clia. hn,est. 94, 1410-1416 35 Watanabe,M. et al. (1995) Mice deficient in cystathionine I~-synthase:animal models for mild and severe homocyst(e)inemia, Proc. NadAcad. Sci. USA 92, 1585-1589 36 Grossman, M. et al. (1994) Successful ex vivo gene therapy directed to liver in a patient with familial hypereholesterolemia, Nat. Genet. 6, 335-341 37 Linton, M.E, Atkinson, J.B. and Fazio, S. (1995) Prevention of atherosclerosis in apolipoprotein E-deficient mice by hone marrow transplantation, Science 267, 1034-1037 38 Silver, L.M., Nadeau, J.H. and Goodfellow, P.N., eds (1994) Mammalian Genome 5 (Suppl.), Springer-Verlag