Mouse Models of Atherosclerosis

Jonathan D. Smith

In the beginning of this decade the Nobel laureate David A collection of these macrophage foam cells makes up the Baltimore predicted that the 1990s would be the decade of first grossly visible lesion, called the fatty streak. The next the mouse, and the enormous bounty of biological informa- stage of lesion development is called the intermediate fibrous tion gained through the use of genetically modified mice plaque, which involves the proliferation of arterial smooth has turned his prediction into reality. muscle cells forming a cap over the fatty streak. The cells of Coronary heart disease is the most common cause of the foam-cell core often die, leading to the deposition of ex- death in the United States. Most cases are associated with tracellular cholesterol crystals. The advanced complex le- atherosclerosis, often initiated by hypercholesterolemia. sion is one that involves a thrombotic event, often due to the Atherosclerosis is a complex disease with both genetic and rupture of the plaque surface, exposing the highly environmental factors. Family history is a significant risk thromobogenic subendothelial surface, an event that can lead factor for cardiovascular disease, although the specific ge- to acute myocardial infarction. This progression of an ath- netic factors that lead to atherosclerosis susceptibility have erosclerotic lesion is not fast but rather develops over many been difficult to ascertain, as the vast majority of cases are years, and can be initiated during childhood and adolescence. not associated with a monogenic disorder like familial hy- The following is a list of questions that can be used to judge percholesterolemia, which is caused by a mutation in the the usefulness of animal models of atherosclerosis: (1) What low-density lipoprotein (LDL) receptor gene. Most cases of is the nature of the experimental lesions and their similarity atherosclerosis are thought to be associated with polygenic to human lesions; (2) is the plasma lipoprotein profile and factors, with variations in several minor genes coming to- metabolism similar to metabolism in humans; (3) what is the gether and interacting with the environment to create sus- time frame necessary for lesions to form, and how long does it ceptibility to this condition. As Robert Wissler has described take to breed the animals for the studies; (4) what is the cost in the introduction, it is practically impossible to study this of acquiring and maintaining the animals; (5) what is the abil- disease well in humans, as it is difficult to sort out the ge- ity to perform in vivo manipulations and imaging; and (6) netic and environmental components. Long-term dietary what is the ability of the model to take advantage of classical studies are obviously impossible to perform in humans. and molecular genetic approaches? Atherosclerosis develops slowly with age, and is often as- The mouse as a model meets many of these criteria, but certained only after a myocardial infarction, which can first it is important to acknowledge many important dif- mask the assignment of “affected” versus “unaffected” in ferences between mice and humans. The average lifespan clinical studies. Due to these confounders, using the termi- of a mouse is about 2 years, compared to about 75 years in nology of genetics, any genes associated with atherosclero- humans. Mice weigh much less, about 30 grams for the sis will therefore display incomplete penetrance with the adult. The lipid profile in the mouse is very different from phenotype of atherosclerosis. Thus there is a great need to that in humans, who carry about 75% of their plasma cho- develop animal models to discover the root causes of, and lesterol on LDL. Mice carry most of their cholesterol on genes that are associated with, this disease. high-density lipoprotein (HDL), which we know in humans The modern theory of atherogenesis, championed by is protective against atherosclerosis. Thus, mice fed their Russell Ross, is the “response to injury hypothesis,” which normal low-fat chow diet do not get atherosclerosis, while states that atherosclerosis is an excessive fibroproliferative it is a common disease in humans. One difference, which is response to an insult upon the arterial wall (1). In a typical an advantage of all animal models, is the ability to control scenario of human atherogenesis, plasma-derived lipopro- the environment and diet in mouse studies, which is im- teins are retained in, and modified by, the arterial wall. possible for long-term human studies. Human genetic stud- These modified lipoproteins are inflammatory and, by a ies are limited in range to various types of association stud- mechanism not completely understood, lead to endothelial ies. With mice, on the other hand, many additional kinds of cell activation and expression of cell-surface adhesion mol- genetic experiments are possible, including breeding and ecules. Blood monocytes stick to these adhesion molecules genetic engineering. and then traverse the endothelium into the artery wall, There are many advantages of using mice for experimen- where they differentiate into macrophages. The macro- tal atherosclerosis research, including their relative ease pages then take up modified lipoproteins via scavenger re- and thriftiness to acquire and maintain. Their generation ceptors and thus become cholesterol-engorged foam cells. time is short, at about 9 weeks, 3 weeks for gestation and about 6 weeks until sexual maturity. It is easy to breed very large cohorts for experimental studies, and mice can The Rockefeller University, New York, New York

573 Vol 48, No 6 Laboratory Animal Science December 1998 develop atherosclerosis in a very short timeframe, as dis- diet contained 30% fat, 5% cholesterol, and 2% cholic acid, cussed below. Classical genetics in the mouse is very well and led to atherosclerosis in certain strains of inbred mice established and is aided immensely by the availability of such as C57BL6 (3). However, other strains were resistant hundreds of inbred strains. Many of these inbred strains to this diet. The F1 hybrids created by cross breeding ath- were created by lay people, mouse fanciers who bred mice erosclerosis-susceptible and -resistant strains had inter- for interesting coat colors. Thus, many genes have been iso- mediate levels of atherosclerosis, implying that genetic fac- lated in the homozygous state in these inbred strains. With tors were involved in determining atherosclerosis suscep- the coming of age of molecular genetics, it is now possible to tibility on this diet (4). However, this was still a very toxic add exogenous transgenes into mice, which can also be done diet on which the mice lost weight and often got sick with in many other species. However, uniquely in mice, it is also morbid respiratory infections. Beverly Paigen and her col- possible to knock out or replace endogenous genes; this is leagues modified the Thomas-Hartroft diet by blending it one of the main advantages of working in the mouse model. one part to three parts with a 10% fat diet to yield what is The major disadvantage of the mouse model is their small called by many scientists today the “Paigen diet,” which size, which makes it difficult but not impossible to perform consists of 15% fat, 1.25% cholesterol, and 0.5% cholic acid surgical manipulations and in vivo imaging. But there have (5). Paigen studied 10 different inbred mouse strains on been recent advances in these techniques that have over- this diet and found a large variation in atherosclerosis sus- come many of the size limitations, such as the ability to ceptibility; this variation did not correlate with the total perform imaging of abdominal atherosclerotic lesions in plasma cholesterol levels, implying that other factors were living mice, cardiac catheterization to determine cardio- involved in susceptibility to this diet. vascular function in free-ranging mice, and surgical liga- Paigen and her colleagues also developed assays that are ture of coronary arteries giving rise to myocardial ischemia. widely used to quantify atherosclerosis in the mouse model. What then are some of the uses for mouse models of ath- The most standard assay is the measurement of the cross- erosclerosis? First, this model is useful for the identifica- sectional lesion area in the aortic root (6). In this assay, tion of atherosclerosis susceptibility-modifying genes. Many freshly perfused and isolated hearts are fixed in formalin, humans who develop atherosclerosis have only moderate embedded in gelatin, frozen, and cut into thin sections at cholesterol and no other known risk factors. Thus, by using anatomically defined sites in the aortic sinus and valve various animal models it may be possible to identify genes region. These sections are stained for lipids, and the lesion that modify atherosclerosis susceptibility, and if this infor- area is measured microscopically. mation translates to humans, it will help in the identifica- Although this model has been widely employed and is tion of individuals with a genetic predilection toward ath- of significant use in the study of atherosclerosis, the pa- erosclerosis. These subjects can then be treated aggressively thology of the lesions are not ideally suited as a model for to reduce environmental and behavioral factors (e.g., diet, human atherosclerosis. This shortcoming led many inves- smoking, and physical inactivity) that play a role in ath- tigators to downplay the role of the mouse as a good model erosclerosis, and can receive pharmacological treatment to of atherosclerosis. Lesion formation in the diet-induced reduce known risk factors (e.g., hypercholesterolemia, low model is largely limited to the aortic root after feeding HDL, and hyperhomocysteinemia). The identification of the Paigen-diet for periods of 14 weeks to 9 months. The these genes can be achieved by the candidate gene approach, lesions are quite small, only several hundred to a few thou- whereby candidate genes are bred into a specific mouse sand square micrometers, and they consist almost entirely model of atherosclerosis and then tested for their effects. of macrophage foam cells with little evidence for smooth Another way to identify these genes is through gene map- muscle cell involvement. Thus, this model is largely lim- ping and positional cloning methods, described below. The ited to the fatty streak stage and does not progress to re- second use of mouse models is the identification of the role semble human intermediate lesions. of various cell types in atherogenesis. Third, the mouse Despite this limitation of the diet-induced model, it has model can be used to characterize environmental and di- been available for the longest amount of time, and it has etary effects on atherosclerosis and, importantly, to test been used to map the genetic location of atherosclerosis- therapies that might block or reverse atherogenesis or le- susceptibility genes. The best characterized of these genetic sion progression. loci was named Ath-1, for the presumptive gene at that The earliest mouse model of atherosclerosis is the diet- genetic position that controls atherosclerosis susceptibil- induced model that was first characterized during the 1960s ity. Paigen, LeBoeuff, and their colleagues mapped this in Robert Wissler’s laboratory. Vesselinovitch and Wissler susceptibility locus to the short arm of chromosome-1 near tried five different diets in an attempt to induce athero- but separable from the apoa2 gene (7). They performed this sclerosis in mice. Most of these diets, which were devel- mapping by using classical mouse genetic techniques, em- oped for use in rats, led to weight loss and death of the ploying a set of recombinant inbred (RI) strains derived mice. On one of the diets, the animals were able to survive from a pair of inbred parental strains, one susceptible until they did develop some atherosclerotic lesions, which (C57BL6) and one resistant (C3H) to diet-induced athero- were then characterized (2). Shortly thereafter, Thompson sclerosis. Each RI strain is an inbred (homozygous) strain achieved further success in mouse studies using a diet that produced by Ϸ10 vertical brother-sister matings starting was developed by Thomas and Hartroft for use in rats. This with the F2 generation between a susceptible and a resis-

574 Mouse Models of Atherosclerosis

tant strain. Thus, each RI strain represents both parental amyloid A5 (19). Therefore, the genes that are mapped us- genotypes but has a unique combination of parental strain ing this model may be more indicative of the response to inheritance along the genetic map. the inflammatory diet rather than factors that are more The Ath-1 locus was also reported to regulate the Paigen- relevant to human atherosclerosis. diet effect on HDL cholesterol levels, such that diet-induced It has been a longstanding goal of many investigators decreases in HDL levels were observed in the atheroscle- around the world to create better mouse models for lipo- rosis-susceptible parental and RI strains. However, the protein disorders and atherosclerosis and to identify genes veracity of the Ath-1 locus as an atherosclerosis-suscepti- that may modify atherogenesis and lesion progression. In bility gene has been recently challenged. The initial map- 1992, Andrew Plump, in Jan Breslow’s lab, knocked out the ping of this locus used a qualitative ranking of lesion se- mouse apolipoprotein E (apoE) gene using the embryonic verity. Lusis and his colleagues repeated the atherosclero- stem cells and homologous recombination (20). This was sis analysis, using the quantitative assay described above, independently done at the same time by Nobuo Maeda, and on the same set of RI strains as Paigen used, and found was later also done by Janine VanRee and colleagues (21, that two of the lines were initially characterized incorrectly 22). The three mice had identical phenotypes. The first (i.e., atherosclerosis-susceptible versus -resistant). Upon phenotype observed was a lipoprotein disorder. Wild-type performing a statistical genetic analysis called quantita- mice, depending on the background strain, have total cho- tive trait locus mapping (QTL), the Ath-1 locus could no lesterol levels of about 60 to 100 mg/dl, mostly in HDL, longer be detected (9). Lusis also used another method to whereas apoE-deficient mice on a chow diet have total cho- search for the Ath-1 locus, employing the QTL analysis on lesterol levels of Ϸ500 mg/dl, which is composed mostly of a large F2 cohort generated from the same pair of athero- ␤VLDL, a cholesterol ester-enriched VLDL particle. These sclerosis-susceptible and -resistant parental strains. This remnant particles are thought to be primarily of intestinal study also failed to find the Ath-1 gene (10). origin, derived from chylomicrons that have undergone li- Nonetheless, this model has been used successfully to polysis so that the triglyceride core is depleted and replaced test for candidate genes’ effects on atherosclerosis. This is with cholesterol ester. Tony Hayek developed a more physi- a simple experiment. A candidate gene, mutation, ological “western-type” diet for mouse studies, which is simi- transgene, or knockout is bred onto the C57BL6 genetic lar in composition to an average American diet of several background, and the resulting mice are fed the Paigen diet. years ago, consisting of 21% fat by weight, 0.15% choles- The lesion area is compared with that of the parental terol, and no cholic acid. When fed this diet, wild-type mice C57BL6 mice in the same diet. By this method it has been have a two-fold elevation in plasma cholesterol, while apoE- shown that over-expression of ApoA-I, the major protein in deficient mice have over a three-fold elevation, to Ϸ2,000 HDL, leads to increased levels of HDL and decreased ath- mg/dl, again, mostly in ␤VLDL, but there is also an increase erosclerosis lesion area (11). Other examples of this use of in LDL (20). The post-prandial clearance of intestinally the candidate-gene method include the apoE transgene, derived lipoproteins is dramatically impaired in apoE-de- expressed either in the vascular wall or in the liver, which ficient mice (20). leads to decreased lesion area (12, 13). The tumor necrosis Atherosclerosis in apoE-deficient mice has been studied factor receptor knockout leads to increased lesion area (14). extensively by many labs around the world. Russell Ross’s The MHC class II deficiency leads to no change in lesion area, laboratory noticed monocyte adherence to endothelial cells showing that the immune response may not be a very large in lesion-prone areas of the aorta of chow-fed apoE defi- determinant of atherosclerosis area within this model (15). cient mice at 6 to 8 weeks of age (23). By 12 weeks of age This diet-induced model of atherosclerosis has also been fatty-streak lesions are apparent (23). By 20 weeks of age, used to test for environmental and drug effects in athero- many lesions progress beyond the fatty-steak stage and sclerosis. Alcohol feeding has been shown to decrease le- develop into fibro-proliferative intermediate lesions, which sions in this model (16). Psychosocial stress due to higher contain abundant extracellular matrix and smooth muscle cage density is associated with an increased lesion area as cells forming a cap over the foam-cell core (23). These le- well as total plasma cholesterol levels (17). The induction sions often have large necrotic areas in the core. of diabetes by injecting strepozotocin also leads to increased The apoE-deficient mouse was the first mouse model in lesion area (18). which atherosclerotic lesions progress beyond the foam-cell Although there are many uses of this model, there are also stage to form intermediate lesions, and thus quickly at- many disadvantages. The lesions are very small in mice at 4 tracted the attention of many investigators. Figure 1 dem- to 5 months of age, in the order of 200 to 1,000 square mi- onstrates the aortic root lesions of an apoE-deficient mouse crons in the aortic root. The lesions are largely confined to the after staining an unfixed frozen section with oil red-O and aortic root, and they usually do not develop beyond the early hematoxylin. Oil red-O stains the lipids in the lesions that foam-cell, fatty-streak stage. The diet is also unphysiological are primarily free and esterified cholesterol. The early fatty with regard to its extremely high cholesterol content, 1.25%, streak is a raised lesion composed mostly of monocyte-de- and the presence of cholic acid. In addition, Lusis and his rived macrophages and some CD4-positive T lymphocytes collaborators have shown that this diet is in itself inflamma- (24). The T cells in these early lesions are distributed tory, as it leads to the induction of hepatic NF-kB activation throughout the lesion and might make up 5 to 15% of the and the expression of acute phase reactants, such as serum lesion area. The more advanced lesion shows evidence of a

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Table 1. Comparison of atherosclerosis lesion pathology ApoE-deficient mouse Human Fatty streaks with macrophages and T cells yes yes Fibrous plaques with smooth muscle cells yes yes Lesion calcification yes yes Oxidized epitopes present yes yes Lesions form preferentially at branches and other sites with disturbed flow yes yes Diet responsive yes yes Aneurysms observed yes yes Plaque rupture observed no yes

Table 1 compares some of the salient features of atherosclerotic lesions that form in humans and apoE-deficient mice. Lesion progression and cell types are similar, as is the presence of oxidized lipoproteins. The major difference Figure 1. Histology of atherosclerotic lesion from an apoE-defi- of this mouse model, as is the case for most of the other cient mouse. Fibrous lesion in aortic root of 4-month-old apoE- models of experimental atherosclerosis, is that plaque rup- deficient mouse fed the western-type diet. Unfixed cryosection was stained for lipids with oil red-O, and counterstained with hema- ture is not observed, whereas plaque rupture is fairly com- toxylin. mon in humans and can lead to heart attacks. One poten- tial reason for the lack of plaque rupture in mice is that smooth muscle cell cap. These intermediate lesions still the diameter of the aorta is less than 1 mm, which is even have macrophages, especially at the shoulder of the le- smaller than the diameter of the major coronary arteries sion and around the necrotic core, but there are many in humans. As the vessel diameter decreases, the surface fewer T cells, which now have an exclusively subendo- tension increases exponentially; thus, in the mouse there thelial distribution (24). may be so much surface tension that plaque rupture would When apoE-deficient mice are fed the western-type diet, not be likely to occur. the lesions are larger, compared to those of mice fed the The apoE-deficient mouse model of atherosclerosis can chow diet. Average lesion areas in an aortic root section, as then be used to: (1) identify atherosclerosis susceptibil- measured by the Paigen method, are Ϸ400,000 ␮m2 and ity modifying genes, by the candidate-gene and gene-map- 100,000 ␮m2 for 16-week-old apoE-deficient mice fed the ping methods; (2) identify the role of various cell types western-type and chow diets, respectively, while C57BL/6 in atherogenesis; (3) identify environmental factors af- mice fed the Paigen diet have aortic root lesions of <1,000 fecting atherogenesis; and (4) assess therapies that might ␮m2 at the same age. Russell Ross looked carefully at the block atherogenesis or lesion progression. effect of diet on the time course of lesion formation and The candidate-gene approach is similar to what was de- progression. In mice on the western-type diet, all stages of scribed above for the diet-induced model. The relevant lesion progression occur at an earlier age, compared to le- mutation, transgene, or knockout is bred onto the apoE- sion progression in mice on the chow diet (23). Thus, the deficient background, and effects on atherosclerosis are apoE-deficient mouse is a very useful model for studying determined. There have been numerous examples of can- the mechanism of dietary regulation of lesion formation didate genes tested by breeding with apoE-deficient mice. and progression. And, the fact that the lesions develop For example, when Andrew Plump, and independently Ed- within a very short time is another advantage of this model ward Rubin, bred the human apoAI transgene onto the of atherosclerosis, compared to many other models. apoE-deficient mouse, there was a dramatic decrease in The lesions in the apoE-deficient mice grow so large upon lesion size. At 4 months of age, there was a 40-fold decrease aging that they can be observed macroscopically without in lesion area in the apoE-deficient mice that expressed any staining, in situ (23). Another way to assay atheroscle- high levels of the human apoAI transgene, leading to HDL rosis in this model is by the en face method, in which the levels of at least 105 mg/dl (26, 27). whole descending aorta is opened longitudinally, removed, Another candidate gene we tested was the macrophage and stained for lipids (25). When viewed this way, it is ap- colony-stimulating factor (MCSF). The Op, or osteopetrotic, parent that the lesions do not form in a diffuse pattern, mouse has a frame shift mutation in the MCSF gene lead- but rather at specific sites of lesion predilection where the ing to complete MCSF deficiency. We bred this mutation flow is turbulent, such as at the aortic arch and branch onto the apoE-deficient background. We observed a dra- points. With this assay the percentage of the surface area matic 4-to 10-fold decrease in the atherosclerotic lesion area, of the aorta covered by lesions can be measured; this per- despite a 2.5-fold increase in plasma cholesterol levels in centage is found to correlate with age and fat content of these double-mutant mice (28). These mice had decreased the diet. A third assay that can be used to determine the numbers of monocytes, supporting the central role of mono- extent of lesion formation in the aorta is the biochemical cyte-macrophages in atherogenesis, and suggesting that the measurement of free and, particularly, esterified cholesterol monocyte is a good target for the development of drugs to mass in the aorta. prevent atherosclerosis due to hyperlipidemia.

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In a similar study, we tested the role of the immune sys- transgenic mice created independently in Stephen Young’s tem in this hyperlipidemic model of atherosclerosis. Hayes and Edward Rubin’s labs (36, 37). These mice synthesize Dansky bred the apoE-deficient mice to the Rag-1-deficient human apoB only in their livers, as the intestinal enhancer mice. Rag-1 is a recombinase involved in antibody and T- is not present in these transgenic constructions. The hu- cell receptor gene rearrangement, and is absolutely essen- man apoB transgene leads to the production of both the tial in the maturation of T and B cells. Although the apoE- full-length apoB-100 protein and, via RNA editing, the trun- deficient/Rag-1-deficient mice totally lack cellular and hu- cated apoB-48 protein. These mice have mildly increased moral immune systems, there was only a 40% decrease in LDL and total cholesterol levels, with a lipoprotein profile lesion area on the chow diet at 16 weeks of age (24). These showing a distinct LDL peak, in contrast to wild-type mice, lesions at this age and on this diet are primarily foam-cell which have only a distinct HDL peak. The apoB transgenic fatty streaks, where T cells are common. These double knock- mouse is also a very diet-responsive model. Upon feeding out mice were also fed the western-type diet until 16 weeks of the Paigen diet, the cholesterol goes up to about 300 mg/dl. age, so that lesions progressed to the fibro-proliferative stage, Atherosclerosis is not observed when the mice are on fed a in which T cells are less prevalent. On this regimen, the lack chow diet, but it develops in response to feeding either the of an immune system had no effect on lesion area (24). Thus, Paigen diet or the western-type diet (38, 39). in contrast to the MCSF deficiency, which decreased mono- Recently the last two models have been bred with each cyte-macrophages and led to a 4- to 10-fold effect on lesion other to generate the human apoB transgenic/LDL recep- area, knocking out of the immune system played only a mi- tor-deficient model. These mice have an even larger increase nor role, and only on the early lesions. in LDL cholesterol and may prove to be an excellent model ApoE-deficient mice have also been used to look for envi- for atherosclerosis research. All these genetically engi- ronmental and drug effects on atherosclerosis and to test neered mouse models of atherosclerosis are available from novel therapies. For instance, certain antioxidant drugs The Jackson Laboratory, making them quite easy for in- have been shown to decrease lesion area in this model (29). vestigators to acquire and use. Estrogen therapy also decreases lesion area in this model In summary, the mouse is an excellent model for experi- (30). One novel therapy tested in this model is bone mar- mental atherosclerosis research. There are three quantita- row transplantation. Wild-type, apoE-competent bone mar- tive atherosclerosis assays in use: cross-sectional lesion area row, when transplanted into apoE-deficient hosts, abolishes at the aortic root, en face lesional surface area, and aortic atherosclerosis, as the macrophages that differentiate from cholesterol and cholesterol ester mass. The diet-induced the bone marrow precursors secrete enough apoE to nor- atherosclerosis model has been used effectively, but the le- malize the cholesterol levels (31). Gene therapy has also sions tend to be small and are limited to early fatty-streak been tested in this model. Recombinant defective adenovi- stage. This model is also criticized because of the toxicity rus carrying the apoE cDNA, when injected into apoE-defi- and inflammatory responses due to the diet. Of the geneti- cient mice, can cure these mice, albeit temporarily, of their cally engineered models, the apoE-deficient model is the apoE deficiency and lead to the normalization of their plasma only one that develops extensive atherosclerotic lesions on cholesterol levels and a decrease in atherosclerosis (32). a chow diet. It is also the model in which the lesions have There are two additional mouse models of atherosclero- been characterized most thoroughly. The lesions develop sis that have proven very useful. The first is the LDL re- into fibrous plaques; however, there is no evidence that ceptor-deficient mice made in 1993 in Joachim Herz’s lab plaque rupture occurs in this model. The LDL receptor-de- (33). These mice have a more modest lipoprotein abnormal- ficient and the human B transgenic models have elevated ity than the apoE-deficient mice, with increases in LDL LDL levels, but no lesions, or only very small lesions, form and VLDL cholesterol leading to a total plasma cholesterol on the chow diet. However, robust lesions do form on the of about 250 mg/dl on a chow diet. On this diet, and at that western-type diet. level of plasma cholesterol, LDL receptor-deficient mice do What will the future hold for atherosclerosis research in not get atherosclerosis (33). However, this is a very diet- mice? I predict that many new genes will be identified that responsive model. After these mice are fed the Paigen diet, play important roles in atherogenesis in mice, and some of their plasma cholesterol levels soar to about 1,500 mg/dl, these genes will also play a role in this disease in humans. and large atherosclerotic lesions form (34). It has also been There is going to be increased use of mice for testing vari- shown that feeding the less toxic western-type diet also ous diets, drugs, and other therapies for the treatment and leads to the development of large lesions, with plasma cho- prevention of atherosclerosis. I predict that we will see the lesterol levels of about 400 mg/dl (35). The lesion pathol- development of a plaque-rupture model. I forecast that we ogy in this model is not as well characterized as in the apoE- will also see a mouse model in which many of the mouse deficient model, but it does appear similar in that the le- genes for apolipoproteins, their receptors, and lipoprotein sions can progress beyond the foam-cell fatty-streak stage metabolic enzymes have been replaced with human genes, to the fibro-proliferative intermediate stage. This model is to recapitulate, as much as possible, human lipoprotein now widely used because the hyperlipidemia is less extreme metabolism in a mouse. I think that this will be a very than in the apoE-deficient model, and the lipoprotein pro- attractive model in which to study both lipoprotein metabo- file is more similar to the human lipoprotein profile. lism and atherosclerosis in the future. The second additional mouse model is the human apoB

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Dr. Wissler: I wondered as you were talking whether there is any way that you can convert the mouse so that n = 10 the LDL-to-HDL ratio is more like the human without any dietary intervention?

Dr. Smith: In the LDL receptor-deficient and apoB SEM) n = 9

Ϯ transgenic models, there are increases in LDL even on the 2

␮ chow diet. The combination of these two models leads to ( even higher LDL levels. Mean lesion area Dr. Wissler: But is the HDL still very high? Dr. Smith: The HDL levels in mice are strain depen- dent. In the wild-type mice, they can be anywhere from ICAM-1 +/+ ICAM-1 -/- about 50 to over 100 mg/dl. But when you start making the animals hyperlipidemic, either by diet or by genetic Figure 2. Aortic root lesion area in apoE-deficient mice with or engineering, the HDL levels decrease. Thus, in many of without ICAM-1 deficiency. Lesion area was determined in 16- week-old chow-fed male mice. Although the ICAM-1-deficient mice these models the mice are actually approaching a human- had a larger mean lesion area, this difference was not statisti- like lipoprotein profile where the LDL-to-HDL ratio is cally significant by a two-tailed t test (P = 0.32). similar to the human ratio. Dr. Butler: Has anyone tried antibodies to adhesion Dr. St. Clair: One of the things that has always struck molecules to block atherosclerosis in these mouse models? me about atherosclerosis in general is the great variability Dr. Smith: The effects of various adhesion-molecule within groups. I think that the dogma these days is that, in knockouts on atherosclerosis have been studied in mice. most animal models, you need 20 to 25 animals per group Denisa Wagner bred the P-selectin knockout mice with LDL in order to expect to see a significant difference in what receptor-deficient mice, and the resulting double knockout you are testing. I would have thought that in genetically mice displayed smaller early lesions than the singly LDL identical mice this variability would have been reduced. receptor-deficient mice, although this difference was not But I was struck by a slide that Allen Fogelman showed observed for intermediate fibrous lesions (40). Using the one time of recombinant inbred strains of C57BL6 mice diet-induced model, Arthur Beaudet tested several knock- still revealing significant variation in lesion size. Do you outs, including P-selectin, ICAM-1, and CD18, all of which have any explanation for that? led to smaller fatty streaks when compared to wild-type Dr. Smith: When we first developed the apoE-deficient mice, although the basal lesion area in this study was quite mice, it was on a mixed and outbred genetic background, small, <1,000 ␮m2 (41). We have done some work with some and lesion variation was present, but still, by using liter- of the ICAM-1 knockout mice developed by Gutierrez- mate controls, we were able to perform good statistical Ramos (42). By breeding we created double apoE-deficient/ analysis and show significant differences on groups with ICAM-1-deficient mice, and we found that lesion area was sizes of Ϸ10 per gender. Now that these mutations have no different in these 16-week-old chow-fed mice than in been bred back onto homozygote inbred strains like the the singly apoE-deficient mice (Figure 2). Thus, the results C57BL6 or FVBN, we have found about a two-fold decrease of the ICAM-1 knockout are different in these two mouse in lesion variation compared to the outbred mice, but there models of atherosclerosis. There continues to be a lot of still is quite a bit of lesion variation. We think that there interest in adhesion molecules and their role in athero- are stochastic events that play a role in atherogenesis. Thus, sclerosis. VCAM-1 is under study in several labs and is we find that with numbers of about 10 per group, one can thought to be expressed specifically at sites of lesion pre- statistically detect a lesion area decrease of about 40%, or dilection, such as branch points, and only in hypercholes- a two-fold increase. If you are looking for smaller differ- terolemic animals. But there are also new adhesion mol- ences, a larger sample size would be needed. ecules that are surfacing. Judy Berliner has some very el- egant work in the beginning stages showing that the CS1 domain of fibronectin, which can bind to activated integrin, References may be a major player in monocyte adhesion during athero- 1. Ross, R. 1993. The pathogenesis of atherosclerosis: a perspec- genesis in the mouse. tive for the 1990s. Nature 362:801–809. 2. Vesselinovitch, D., and R. W. Wissler. 1968. Experimental Dr. Wissler: As you pointed out, there are some real limi- production of atherosclerosis in mice. 2. Effects of atherogenic tations to the mouse model in terms of what you can do in and high-fat diets on vascular changes in chronically and terms of manual interventions. Are there any advances in acutely irradiated mice. J. Atheroscler. Res. 8:497–523. this area? 3. Thompson, J. S. 1969. Atheromata in an inbred strain of Dr. Smith: There are a few labs that have made remark- mice. J. Atheroscler. Res. 10:113–122. 4. Roberts, A., and J. S. Thompson. 1976. Inbred mice and able progress in microsurgery and in the study of cardiac their hybrids as an animal model for atherosclerosis research. function in catheterized mice that are mobile and even exer- Adv. Exp. Med. Biol. 67:313–327. cising (43). It is possible to make transgenics in other spe- 5. Paigen, B., A. Morrow, C. Brandon, et al. 1985. Variation cies, such as pigs, which are more amenable to surgical main susceptibility to atherosclerosis among inbred strains of mice. Atherosclerosis 57:65–73. nipulation, but the ability to make knockouts is still unique to the mice.

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