15
Afherosclerosis, 85 (1990) 15-23 Elsevier Scientific Publishers Ireland, Ltd.
ATHERO
04542
Review
article
Animal models of atherosclerosis Mark L. Armstrong Cardiovascular
and Donald
D. Heistad
Center, Departments of Internal Medicine and Pharmacology, University and VA Medical Center, Iowa City, IA 52242 (U.S.A.)
of Iowa College of Medrcrne,
(Received 3 April, 1990) (Revised, received 18 June, 1990) (Accepted 26 June, 1990)
Key words:
Atherosclerosis;
Animal
models
Early rabbit models of atherosclerosis, summarized by Bailey [l], were described shortly after Marchand [2] identified atherosclerosis as a distinct fibrolipid intimal arteriopathy among the collection of entities that had been termed arteriosclerosis from the time of Lobstein [3]. These rabbit models provided the first evidence that dietary variables could be used to create experimental simulations of important morphological features of human atherosclerosis. In subsequent years numerous animal models have been used to study atherosclerosis. The most comprehensive description of the potential roles of animals in the study of atherosclerosis appeared in the proceedings of an international conference on comparative atherosclerosis published in 1965, in a large, beautifully illustrated volume, Comparative A therosclerosis [4]. Perusal of “Comparative Atherosclerosis” today shows how much our views of animal models have changed in the past 25 years. The differences are worth noting, since they make explicit our current sense of the roles of animal models in the study of atherosclerosis. For example, animals that
Correspondence to; Mark L. Armstrong, M.D., Department of Internal Medicine, University of Iowa College of Medicine, Iowa City, IA 52242, U.S.A. Phone: (319) 356-2708.
would most readily come to mind today in considering potential models for study are shown in Table 1. The list is much shorter than we would predict from the discussions and presentations in Comparative Atherosclerosis, with its expansive taxonomic view of animal species of potential use in evaluating lesion formation. Part of Comparative Atherosclerosis dealt with the search for species that showed spontaneous arterial lesions that bore some resemblance to atherosclerosis. This search has not been continued for several reasons. One reason is that descriptive surveys of lesion-bearing animals in uncontrolled settings now seem of minimal value. Another reason is that we have little interest in lesions in exotic species, even if systematic study were possible under conditions that permit lesion induction and modification. Many of us feel that no new animal species is likely to deliver information that is not potentially available in the common models shown in Table 1. Instead, the current tendency is to discover or create new models that are genetic variants of common species. Examples of genetic variants that are already reported [5-111 show great promise. Another shift in viewpoint relates to morphology of lesions. There is less concern today about the extent to which the lesions under study resemble human lesions. This point has been made
16 TABLE
I
MOST COMMON ROSIS
ANIMAL
Type of animal Birds Pigeons Common mammals Rabbits Swine Primates Old World monkeys
MODELS
OF ATHEROSCLE-
Type of lesion a
spontaneous,
induced
spontaneous, induced
induced
induced
a Spontaneous lesions are lesions that occur in association with genetic hypercholesterolemia. Induced lesions are lesions that occur in response to atherogenic diet.
cogently by a leading morphologist [12], when he notes that mere simulation of human lesions is not very helpful when the mechanisms of lesion formation are unlikely to have been similar to those that occur in man. The morphologic details of lesion formation in animal models continue to be important, however. This is why we are not quite satisfied when we view the figures in Comparative Atherosclerosis. We appreciate the superb details of the gross and light microscopic observations, but note little histochemical or immunologic evaluation and no fine structure studies. By today’s standards, the lesions seem only partially characterized. Basic to these shifts is an increasing emphasis on the process of lesion formation. For all the limitations of our knowledge of atherosclerosis, we believe that we have crucial leads regarding how lesions are formed. In contrast to the uncertainties of 1965, probably a majority of us now accept the concept that the lipid hypothesis [13,14] explains much of the human disease, just as induced or genetic lipid disorders are necessary in order to have multifocal atherosclerosis in animal models. The conviction of a plausible central etiology for Marchand’s disease frees us to use hyperlipidemic models to look for details of mechanisms of lesion formation and for factors that contribute to worsening or improvement of lesions. We typically use hyperlipidemia in our models as we set up appropriate conditions to study the effects of lipoprotein changes, coagulation mechanisms, formed elements of the blood, humoral agents and drugs
on the atherogenic process or on the dynamics of wall action. This has led to a more selective use of animals as models, because some of these questions are investigated more readily with the use of simpler biological systems as the appropriate models. Organ and cell cultures and even simpler systems bulk large in current studies. It is common for studies to comprise an animal component and several of these simpler systems. Growth in information about atherosclerosis in the past 25 years has led to a major shift in expectation on the part of investigators using animal models. We now consider atherosclerotic lesions to be not only preventable but modifiable to an important degree. The possibility of regression of atherosclerosis was first portrayed in animal models [15], and now the evidence that regression may be induced in human lesions is very convincing [16,17]. The current role of animal models in regression studies is to evaluate ways in which this established ameliorative effect may be enhanced or compromised. Finally, an important, newly recognized aspect of experimental atherosclerosis requires description in this paper, since it affects the interpretation of experimental and human lesions. It is now recognized that a common presentation of experimental atherosclerosis is one of enlargement of atherosclerotic arteries as plaque development takes place, so that the lumen size is preserved despite extensive plaque formation. This surprising observation, first described by Bond [23], has been confirmed [24-261, and is now recognized as a major compensatory mechanism by which stenosis of the lumen is quite effectively prevented in experimental atherosclerosis. Thus, stenosis is not readily induced in experimental atherosclerosis despite induction of extensive atherosclerotic plaques. These new experimental findings have led to recognition of the same compensatory mechanism at work in the early stages of plaque formation in man: precise autopsy observations [27] and echocardiographic studies at open chest surgery [28,29] have shown that in the early phases of human coronary atherosclerosis, stenosis of the lumen may be profoundly attenuated by this effect. The subsequent sequence of events that leads to stenosis of the lumen in human atherosclerosis has no experimental counterpart that leads to hemodynamically important stenosis in most atherosclerotic animals.
17 TABLE
2
SUMMARY
OF ANIMAL
MODELS
Kind of animal Aoian models Chicken
IN STUDIES
OF LESION
FORMATION
Model status
Dietary induction of lesions; regression of diet-induced lesions; role of herpesvirus in lesion localization Marked interstrain difference in susceptibility to lesion formation: diet-aggravated atherosclerosis
Pigeon Other Japanese quail Turkey Ducks, geese Mammalian nonprimate models Rabbits and hares New Zealand white rabbits Special strains Watanabe (WHHL) St. Thomas Jackson Laboratory AX/JU strain Jackson Laboratory IIIVO/JU strain Houston RT rabbit Hybrid
USED
hares
Other small mammals Rodents Rats Mice Other rodents Cats Dogs Swine
Nonhuman primate models New World monkeys Squirrel monkey
Howler monkey Cebus monkey Marmosets Old World monkeys Macques Baboons Other monkeys Old World apes Gorillas and chimpanzees
Used mainly in drug screening Study of dissecting aneurysms Lesions may regress in response
Widely used for diet-induced Genetic
hyperlipidemia
(LDL) Genetic hyperlipidemia lipoproteins (VLDL, AX/JU
to diet
lesions
with elevation
of low density
with elevation IDL, LDL)
of several lower density
strain is hyperresponsive
to dietary
lipoprotein
cholesterol
IIIVO/JU strain is hyporesponsive to dietary cholesterol Recently characterized colony is hyporesponsive to dietary cholesterol Used to study monotypic versus monoclonal character of cells in lesions
Not a favored model for studies of atherosclerotic lesions Special strains show arterial lesions in response to very high cholesterol diet Not used for studies of atherosclerotic lesions Apparently no systematic use at present Lesions are difficult to produce and often are medial as well as intimal One of the best models. Lesions occur in both aorta and branch vessels; size of heart and vessels is sufficient for studies of cardiovascular function
Feral New World monkeys are not readily available; the advantage of this group of primates for studies of lesion formation may be too minimal to justify their use
One of the best models, but reduced availability may limit future use profoundly Future use will be increasingly restricted to evaluation lesions in genetically defined animals African green, patas, and other Old World species will similarly have limited future use
of
Lesion formation occurs in the great apes in response to diet, but these uniquely valuable animals are not available
18 With these points in mind, let us review the list of potential models shown in Table 2. The table includes most of the animals that have been evaluated in a systematic way for atherosclerotic lesions. Somewhat similar listings have appeared in previous reviews of potential animals for atherosclerosis research [18-221, which may be consulted for modest differences in descriptions and emphasis.
Birds
Birds are relatively hypercholesterolemic animals, with some tendency to develop atherosclerotic lesions in the absence of dietary induction. There is an increase in lesions with high-cholesterol diets, however, and important experimental evidence of regression of arterial lesions was shown with dietary manipulation in chickens [30]. More recently, chickens have been used to show the role of herpesvirus (Marak virus) [31] in lesion formation. Pigeons have been studied in a very informative way. Pigeon strains that differ in relation to the presence of lesions in response to basic avian hypercholesterolemia, have been further characterized in relation to responsiveness to dietary challenge with cholesterol [32,33]. There is a recently developed strain of Japanese quail that is particularly susceptible to cholesterol feeding. This feature has been used in screening drugs for effectiveness in lipid-lowering and lesion-suppressing properties [34,35]. The Japanese quail does not seem to have been used except for screening. The bronze-breasted turkey is famous for the development of aortic aneurysms that coexist with atherosclerotic plaques [36], and similar aneurysms have been found in white turkeys [37]. The turkey is rarely studied today. Similarly, ducks and geese were observed to develop fatty lesions that regress in response to diet [38], but the observation remains unexplored. In summary, pigeons are currently the main bird species used in studies of atherosclerotic lesions, because of contrasting responses to atherogenie challenge in the hyporesponive and hyperresponsive strains and the low cost of studies. The myocardial infarctions that develop in pigeons have been evaluated and found to be the result of
atheromatous embolism [39], in contrast to the usual atherothrombotic pathogenesis in humans. Rabbits and hares
The rabbit is so widely used that it is nearly a symbol of experimental atherosclerosis. The most common strain, the New Zealand white rabbit, has been severely criticized [40] with considerable justification for its limitations as an animal model. Generations of pathologists have despaired at the readiness of investigators to accept the state of massive lipid overload in the cholesterol-fed rabbit as an adequate representation of the atherosclerotic state in man. The rabbit is gaining new respect, however, for several reasons. One reason is that there is a growing tendency for investigators to use shortened induction times or lower doses of cholesterol, so that features of lesion structure other than huge populations of lipid-filled macrophages are more readily appreciated. Perhaps a more important reason is the recognition that the behavior of lesions in hyperlipidemia in rabbits tends to parallel that of lesions in other species in important ways [41]. The most important reason for the renewed interest in the New Zealand white rabbit, however, is that there now exist several genetic variants that offer expanded possibilites in exploring the relations of lipids to lesion behavior. The best known is the Watanabe strain [5], whose lipoprotein pattern on a standard chow diet is a fairly close approximation of monoclonal human familial hypercholesterolemia [6]. The lesions in the Watanabe rabbit are also considered close approximations of their human counterpart [7]. The St. Thomas strain [8], also recently characterized, has a broader spectrum of lower-density lipoprotein elevation and also develops atherosclerotic lesions on a standard chow diet. The Jackson Laboratories have established 2 strains that show contrasting responses to diet: the AX/JU strain is hyperresponsive to dietary cholesterol [9], and the IIIVO/JU strain is almost unresponsive to dietary cholesterol [lo]. Overturf and associates [ll] recently have partially characterized animals in a colony of New Zealand rabbits that are notably unresponsive to dietary cholesterol. Some other strains are occasionally used, but New Zealand rabbits, either the original strain or one of the emerging genetic variants, are the animal of choice for a multitude of studies. One criticism
19 of the atherosclerotic rabbit in the past was that the lesions did not regress. It is evident, however, that rabbit lesions are to some extent regressible [42,43], and it seems likely that emerging regimens will be increasingly effective in causing regression [44]. Thus, it seems possible that finding ways of enhancing regression in rabbit atherosclerosis might make the rabbit an important test animal for regimens most likely to have promise in regression of human lesions. The hybrid hare should be mentioned for its usefulness in studies of the clonality of cells in experimental atherosclerotic lesions [45]. In summary, the rabbit has undergone rehabilitation as a model of atherosclerosis, and rabbits seem likely to have continued major usefulness in future studies of atherosclerosis. Other small mammals
Rodents The possibility that small animals other than rabbits might be useful as animal models in atherosclerosis has been extensively explored in rodents. If suitable models existed among the many rodent species, it seems likely that they would be widely known and used. Although hyperlipidemic states are inducible in rats and mice, the resulting arterial lesions tend to be disappointingly meager. Other rodents are not superior in this respect. Papers appear from time to time advocating the use of rats or mice. Despite such advocacy, the attitude of most investigators in the field is probably illustrated by 2 leading groups of atherosclerosis research in the United States, who conducted initial evaluations in rodents [46,47], and quite promptly abandoned rodents in all subsequent research. Nonetheless, certain strains of mice seem to show improved lesion formation [21,48,49], and inbred strains of mice [49] are quite useful in examining the genetics of dyslipoproteinemia. We should consider the door ajar for future development of rodents as more satisfactory models of atherosclerotic lesions. Cats Cats have been used extensively as laboratory animals, but they have been almost skipped as
potential models of atherosclerosis. We know of only one paper in which cats were evaluated for lesions [50]. Lesions were indeed produced in response to atherogenic diet, but lesion characteristics and distribution were unlike those in humans, and the authors reverted to pigeon and primate models.
Dogs Dogs have been studied as models from time to time [51,523. The problems with dogs as models are that (1) atherogenesis cannot be produced in the absence of hypothyroidism or an unusual diet enriched in coconut oil, (2) the lesions are not very prominent in major arteries, and (3) medial lesions occur to a significant degree. Given the preemminence of dogs in cardiovascular studies, it is regrettable that these animals cannot be used more satisfactorily as atherosclerotic models. Study of the mechanisms of discrete lipid deposits in the media would seem to be one worthwhile topic for investigation, but this has not been evaluated. Swine Swine, in contrast to dogs, are superb models. Both full-sized and miniature swine have been extensively studied [53,54]. Lesions occur to some extent in free-living animals, and can be induced with atherogenic diet to an important degree. Lesions occur in both aorta and branch arteries. The vessels are large, facilitating physiologic and biochemical studies. Von Willebrand deficient strains [55,56] have been studied, permitting evaluation of the effect of coagulation pathways on atherogenic response. Nonhuman primates Primate models have been extensively characterized [57-591. Our group has used Old World primates for many years in correlated studies of arterial structure and function [60,61]. We have prized the advantages they offer in showing little lesion formation in controls, and in contrast extensive lesion formation in a multivessel distribution after dietary induction. We feel, nonetheless, that great caution should be taken in decisions about the future use of primates, because of their
20 decreasing availability and the expense and considerable knowledge base involved in providing proper maintenance and nutrition. Marked ecological change since publication of “Comparative Atherosclerosis” includes a shrinking pool of feral primates. Governmental regulations restrict availablity of some species. In the United States, studies funded through the National Institutes of Health must show that no nonprimate species is a reliable substitute. The National Institutes of Health has set up two centers for breeding and modeling of several Old World species. These centers are located at the Southwest Foundation for Research and Education, University of Texas, San Antonio; Texas, and at the Arteriosclerosis Research Center, Bowman Gray School of Medicine, Winston-Salem, North Carolina. The goal of these two centers is to provide experimental access and primatological expertise to scientists who have only occasional need of the unique features of animals belonging to the primate order. The comments that follow are made in the context of this background, which indicates that primates are a prized but limited resource.
New World monkeys
The New World primates have been described in depth [62], and their characteristics make it unlikely that the problems associated with their acquisition justify their future use in most types of study of atherosclerosis. Lesion formation in response to diet differs greatly among species, lesion distribution is unlike that seen in man, the health surveillance necessary is a major consideration, and there is probably little unique advantage to the use of any New World monkey.
Old World monkeys
Old World monkeys have been extensively studied [57-59,63,64]. At least seven species of the macaque genus have been used in atherosclerosis research. Currently, the cynomolgus is the most widely studied macaque. Rhesus and stumptails are also used to a considerable degree [64], however, and use of the Japanese macaque is reported periodically [59]. Lesion formation in response to atherogenic diet occurs at a greater rate in cynomolgus than rhesus [65]. Regression of lesions
occurs more readily in rhesus than in cynomolgus monkeys [65-671. There are no comparisons of other species of Old World monkeys under identical experimental conditions, but lesions in African green monkeys are probably characterized by more fibrosis [64] than is seen in macaques. Patas monkeys (Erythrocebus patus) also have relatively fibrotic lesions [68]. One macaque species, the Celebes ape (Macucu nigru), has shown a tendency to diabetes and the effect of hypercholesterolemia has been studied in that setting [69]. Baboons have less hypercholesterolemia in response to diet, and lesions are correspondingly smaller [70].
The great apes
Old World apes have been observed in captivity for lesions [71], and there are small systematic studies of lesion formation. The studies showed that a high fat, high-cholesterol diet produces marked hypercholesterolemia with formation of atherosclerotic lesions [72-741. Given the strategic pre-hominid position in primate evolution occupied by the great apes, this information, has the greatest qualitative significance. It is also unlikely to be supplemented significantly in the future, since the great apes are an endangered species. Conclusions
Animal models continue to have important roles in atherosclerosis research. In the eight decades in which animals have been used to study aspects of lesion formation in atherosclerosis, the potential choice of animal has expanded from rabbits, used in the earliest studies, to include many species of mammals and birds. During the past 25 years the actual choices of animals for study of atherosclerosis have narrowed. This reflects several developments, including greater acceptance of the lipid hypothesis as an important explanation of the human disease. Thus, there is an immediate advantage to the study of lesion formation and subsequent lesion change in several well-characterized hyperlipidemic models (pigeon, rabbit, swine and some primates). There is a growing sense that animals now uncharacterized are unlikely to be superior models. Interest is marked in genetic variants of common laboratory species that
21 show abnormal lipid metabolic states or changed responsiveness to atherogenic challenge. Both dogs and rodents are of quite limited value as models for study of lesion formation. Genetic variants, however, have already improved the potential choices in rabbits and may do so in rodents. Among larger mammals, swine and Old World primates are excellent models, but decreasing availability of primates raises a question of their future use.
13
14
15
16
Acknowledgements This work was supported 14230 and a VA Medical (D.D.H.)
by NIH Grant HL Investigator Award
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M.L., Effects of atherosclerosis and regression of lesions, Coronary Circ. Physiol. Pathol., (1990) in press. Clarkson, T.B., Lehner, N.D.M., Bullock, B.C. and Wagner, W.D., Atherosclerosis in New World monkeys. In Strong, J.P, (Ed.), Atherosclerosis in Primates, Primat Med., 9 (1976) 90. McNulty, W.P. and Malinow, M.R., The cardiovascular system, In T-W-Fiennes, R.N. (Ed.), Pathology of simian primates, Part I, Karger, New York, 1972, pp. 779-792. Clarkson, T.B., Hamm, T.E., Bullock, B.C. and Lehner, N.D.M., Atherosclerosis in Old World monkeys, In Strong, J.P, (Ed.), Atherosclerosis in primates, Primat Med., 9 (1976) 66. Armstrong, M.L., Atherosclerosis in rhesus and cynomolgus monkeys. In: Strong, J.P. (Ed.), Atherosclerosis in Primates, Primat Med., 9 (1976) 16. Armstrong, M.L. and Megan, M.B., Responses of two macaque species to atherogenic diet and its withdrawal, In Schettler, G. and Wiezel, A. (Eds.), Atherosclerosis III, Springer, New York, 1974, p. 336. Small, D.M., Bond, M.G., Waugh, D., Prack, M. and Sawyer, J., Physicochemical and histological changes in the arterial wall of nonhuman primates during progression and regression of atherosclerosis, J. Clin. Invest., 73 (1984) 1590. Armstrong, M.L., Trillo, A. and Pritchard, R.W., Naturally occurring and experimentally induced atherosclerosis in
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nonhuman primates, In Kalter, S.S. (Ed.), The Use of Nonhuman Primates in Cardiovascular Diseases, Univ. Texas, Austin, TX, pp. 58-101. Howard, C.F. Jr., Vesselinovitch, D. and Wissler, R.W.. Correlation of aortic histology with gross aortic atherosclerosis and metabolic measurements in diabetic and nondiabetic Macaca nigra, Atherosclerosis, 52 (1974) 85. Strong, J.P. and McGill, H.C., Diet and experimental atherosclerosis in baboons, Am. J. Pathol., 50 (1967) 669. Stout, C. and Lemon, W.B.. Predominant coronary and cerebral atherosclerosis in captive nonhuman primates, Exp. Mol. Pathol., 10 (1969) 312. Andrus, S.B., Portman, O.W. and Riopelle, A.J., Comparative studies of spontaneous and experimental atherosclerosis in primates. II. Lesions in chimpanzees including myocardial infarction and cerebral aneurysms, Prog. Biothem. Pharmacol., 4 (1968) 393. Blaton, V., Vandamme, D., Declercq, B., Vastesaeger, M,, Mortelmans, J. and Peeters, H., Dietary induced hyperbetalipoproteinemia in chimpanzees: comparison to human hyperlipoproteinemia, Exp. Mol. Pathol., 20 (1974) 132. Blaton, V. and Peeters, H., The nonhuman primates as models for studying human atherosclerosis: studies on the chimpanzee, the baboon and rhesus macacus, Adv. Exp. Med. Biol., 67 (1975) 33.
Afherosclerosis, 85 (1990) 15-23 Elsevier Scientific Publishers Ireland, Ltd.
ATHERO
04542
Review
article
Animal models of atherosclerosis Mark L. Armstrong Cardiovascular
and Donald
D. Heistad
Center, Departments of Internal Medicine and Pharmacology, University and VA Medical Center, Iowa City, IA 52242 (U.S.A.)
of Iowa College of Medrcrne,
(Received 3 April, 1990) (Revised, received 18 June, 1990) (Accepted 26 June, 1990)
Key words:
Atherosclerosis;
Animal
models
Early rabbit models of atherosclerosis, summarized by Bailey [l], were described shortly after Marchand [2] identified atherosclerosis as a distinct fibrolipid intimal arteriopathy among the collection of entities that had been termed arteriosclerosis from the time of Lobstein [3]. These rabbit models provided the first evidence that dietary variables could be used to create experimental simulations of important morphological features of human atherosclerosis. In subsequent years numerous animal models have been used to study atherosclerosis. The most comprehensive description of the potential roles of animals in the study of atherosclerosis appeared in the proceedings of an international conference on comparative atherosclerosis published in 1965, in a large, beautifully illustrated volume, Comparative A therosclerosis [4]. Perusal of “Comparative Atherosclerosis” today shows how much our views of animal models have changed in the past 25 years. The differences are worth noting, since they make explicit our current sense of the roles of animal models in the study of atherosclerosis. For example, animals that
Correspondence to; Mark L. Armstrong, M.D., Department of Internal Medicine, University of Iowa College of Medicine, Iowa City, IA 52242, U.S.A. Phone: (319) 356-2708.
would most readily come to mind today in considering potential models for study are shown in Table 1. The list is much shorter than we would predict from the discussions and presentations in Comparative Atherosclerosis, with its expansive taxonomic view of animal species of potential use in evaluating lesion formation. Part of Comparative Atherosclerosis dealt with the search for species that showed spontaneous arterial lesions that bore some resemblance to atherosclerosis. This search has not been continued for several reasons. One reason is that descriptive surveys of lesion-bearing animals in uncontrolled settings now seem of minimal value. Another reason is that we have little interest in lesions in exotic species, even if systematic study were possible under conditions that permit lesion induction and modification. Many of us feel that no new animal species is likely to deliver information that is not potentially available in the common models shown in Table 1. Instead, the current tendency is to discover or create new models that are genetic variants of common species. Examples of genetic variants that are already reported [5-111 show great promise. Another shift in viewpoint relates to morphology of lesions. There is less concern today about the extent to which the lesions under study resemble human lesions. This point has been made
16 TABLE
I
MOST COMMON ROSIS
ANIMAL
Type of animal Birds Pigeons Common mammals Rabbits Swine Primates Old World monkeys
MODELS
OF ATHEROSCLE-
Type of lesion a
spontaneous,
induced
spontaneous, induced
induced
induced
a Spontaneous lesions are lesions that occur in association with genetic hypercholesterolemia. Induced lesions are lesions that occur in response to atherogenic diet.
cogently by a leading morphologist [12], when he notes that mere simulation of human lesions is not very helpful when the mechanisms of lesion formation are unlikely to have been similar to those that occur in man. The morphologic details of lesion formation in animal models continue to be important, however. This is why we are not quite satisfied when we view the figures in Comparative Atherosclerosis. We appreciate the superb details of the gross and light microscopic observations, but note little histochemical or immunologic evaluation and no fine structure studies. By today’s standards, the lesions seem only partially characterized. Basic to these shifts is an increasing emphasis on the process of lesion formation. For all the limitations of our knowledge of atherosclerosis, we believe that we have crucial leads regarding how lesions are formed. In contrast to the uncertainties of 1965, probably a majority of us now accept the concept that the lipid hypothesis [13,14] explains much of the human disease, just as induced or genetic lipid disorders are necessary in order to have multifocal atherosclerosis in animal models. The conviction of a plausible central etiology for Marchand’s disease frees us to use hyperlipidemic models to look for details of mechanisms of lesion formation and for factors that contribute to worsening or improvement of lesions. We typically use hyperlipidemia in our models as we set up appropriate conditions to study the effects of lipoprotein changes, coagulation mechanisms, formed elements of the blood, humoral agents and drugs
on the atherogenic process or on the dynamics of wall action. This has led to a more selective use of animals as models, because some of these questions are investigated more readily with the use of simpler biological systems as the appropriate models. Organ and cell cultures and even simpler systems bulk large in current studies. It is common for studies to comprise an animal component and several of these simpler systems. Growth in information about atherosclerosis in the past 25 years has led to a major shift in expectation on the part of investigators using animal models. We now consider atherosclerotic lesions to be not only preventable but modifiable to an important degree. The possibility of regression of atherosclerosis was first portrayed in animal models [15], and now the evidence that regression may be induced in human lesions is very convincing [16,17]. The current role of animal models in regression studies is to evaluate ways in which this established ameliorative effect may be enhanced or compromised. Finally, an important, newly recognized aspect of experimental atherosclerosis requires description in this paper, since it affects the interpretation of experimental and human lesions. It is now recognized that a common presentation of experimental atherosclerosis is one of enlargement of atherosclerotic arteries as plaque development takes place, so that the lumen size is preserved despite extensive plaque formation. This surprising observation, first described by Bond [23], has been confirmed [24-261, and is now recognized as a major compensatory mechanism by which stenosis of the lumen is quite effectively prevented in experimental atherosclerosis. Thus, stenosis is not readily induced in experimental atherosclerosis despite induction of extensive atherosclerotic plaques. These new experimental findings have led to recognition of the same compensatory mechanism at work in the early stages of plaque formation in man: precise autopsy observations [27] and echocardiographic studies at open chest surgery [28,29] have shown that in the early phases of human coronary atherosclerosis, stenosis of the lumen may be profoundly attenuated by this effect. The subsequent sequence of events that leads to stenosis of the lumen in human atherosclerosis has no experimental counterpart that leads to hemodynamically important stenosis in most atherosclerotic animals.
17 TABLE
2
SUMMARY
OF ANIMAL
MODELS
Kind of animal Aoian models Chicken
IN STUDIES
OF LESION
FORMATION
Model status
Dietary induction of lesions; regression of diet-induced lesions; role of herpesvirus in lesion localization Marked interstrain difference in susceptibility to lesion formation: diet-aggravated atherosclerosis
Pigeon Other Japanese quail Turkey Ducks, geese Mammalian nonprimate models Rabbits and hares New Zealand white rabbits Special strains Watanabe (WHHL) St. Thomas Jackson Laboratory AX/JU strain Jackson Laboratory IIIVO/JU strain Houston RT rabbit Hybrid
USED
hares
Other small mammals Rodents Rats Mice Other rodents Cats Dogs Swine
Nonhuman primate models New World monkeys Squirrel monkey
Howler monkey Cebus monkey Marmosets Old World monkeys Macques Baboons Other monkeys Old World apes Gorillas and chimpanzees
Used mainly in drug screening Study of dissecting aneurysms Lesions may regress in response
Widely used for diet-induced Genetic
hyperlipidemia
(LDL) Genetic hyperlipidemia lipoproteins (VLDL, AX/JU
to diet
lesions
with elevation
of low density
with elevation IDL, LDL)
of several lower density
strain is hyperresponsive
to dietary
lipoprotein
cholesterol
IIIVO/JU strain is hyporesponsive to dietary cholesterol Recently characterized colony is hyporesponsive to dietary cholesterol Used to study monotypic versus monoclonal character of cells in lesions
Not a favored model for studies of atherosclerotic lesions Special strains show arterial lesions in response to very high cholesterol diet Not used for studies of atherosclerotic lesions Apparently no systematic use at present Lesions are difficult to produce and often are medial as well as intimal One of the best models. Lesions occur in both aorta and branch vessels; size of heart and vessels is sufficient for studies of cardiovascular function
Feral New World monkeys are not readily available; the advantage of this group of primates for studies of lesion formation may be too minimal to justify their use
One of the best models, but reduced availability may limit future use profoundly Future use will be increasingly restricted to evaluation lesions in genetically defined animals African green, patas, and other Old World species will similarly have limited future use
of
Lesion formation occurs in the great apes in response to diet, but these uniquely valuable animals are not available
18 With these points in mind, let us review the list of potential models shown in Table 2. The table includes most of the animals that have been evaluated in a systematic way for atherosclerotic lesions. Somewhat similar listings have appeared in previous reviews of potential animals for atherosclerosis research [18-221, which may be consulted for modest differences in descriptions and emphasis.
Birds
Birds are relatively hypercholesterolemic animals, with some tendency to develop atherosclerotic lesions in the absence of dietary induction. There is an increase in lesions with high-cholesterol diets, however, and important experimental evidence of regression of arterial lesions was shown with dietary manipulation in chickens [30]. More recently, chickens have been used to show the role of herpesvirus (Marak virus) [31] in lesion formation. Pigeons have been studied in a very informative way. Pigeon strains that differ in relation to the presence of lesions in response to basic avian hypercholesterolemia, have been further characterized in relation to responsiveness to dietary challenge with cholesterol [32,33]. There is a recently developed strain of Japanese quail that is particularly susceptible to cholesterol feeding. This feature has been used in screening drugs for effectiveness in lipid-lowering and lesion-suppressing properties [34,35]. The Japanese quail does not seem to have been used except for screening. The bronze-breasted turkey is famous for the development of aortic aneurysms that coexist with atherosclerotic plaques [36], and similar aneurysms have been found in white turkeys [37]. The turkey is rarely studied today. Similarly, ducks and geese were observed to develop fatty lesions that regress in response to diet [38], but the observation remains unexplored. In summary, pigeons are currently the main bird species used in studies of atherosclerotic lesions, because of contrasting responses to atherogenie challenge in the hyporesponive and hyperresponsive strains and the low cost of studies. The myocardial infarctions that develop in pigeons have been evaluated and found to be the result of
atheromatous embolism [39], in contrast to the usual atherothrombotic pathogenesis in humans. Rabbits and hares
The rabbit is so widely used that it is nearly a symbol of experimental atherosclerosis. The most common strain, the New Zealand white rabbit, has been severely criticized [40] with considerable justification for its limitations as an animal model. Generations of pathologists have despaired at the readiness of investigators to accept the state of massive lipid overload in the cholesterol-fed rabbit as an adequate representation of the atherosclerotic state in man. The rabbit is gaining new respect, however, for several reasons. One reason is that there is a growing tendency for investigators to use shortened induction times or lower doses of cholesterol, so that features of lesion structure other than huge populations of lipid-filled macrophages are more readily appreciated. Perhaps a more important reason is the recognition that the behavior of lesions in hyperlipidemia in rabbits tends to parallel that of lesions in other species in important ways [41]. The most important reason for the renewed interest in the New Zealand white rabbit, however, is that there now exist several genetic variants that offer expanded possibilites in exploring the relations of lipids to lesion behavior. The best known is the Watanabe strain [5], whose lipoprotein pattern on a standard chow diet is a fairly close approximation of monoclonal human familial hypercholesterolemia [6]. The lesions in the Watanabe rabbit are also considered close approximations of their human counterpart [7]. The St. Thomas strain [8], also recently characterized, has a broader spectrum of lower-density lipoprotein elevation and also develops atherosclerotic lesions on a standard chow diet. The Jackson Laboratories have established 2 strains that show contrasting responses to diet: the AX/JU strain is hyperresponsive to dietary cholesterol [9], and the IIIVO/JU strain is almost unresponsive to dietary cholesterol [lo]. Overturf and associates [ll] recently have partially characterized animals in a colony of New Zealand rabbits that are notably unresponsive to dietary cholesterol. Some other strains are occasionally used, but New Zealand rabbits, either the original strain or one of the emerging genetic variants, are the animal of choice for a multitude of studies. One criticism
19 of the atherosclerotic rabbit in the past was that the lesions did not regress. It is evident, however, that rabbit lesions are to some extent regressible [42,43], and it seems likely that emerging regimens will be increasingly effective in causing regression [44]. Thus, it seems possible that finding ways of enhancing regression in rabbit atherosclerosis might make the rabbit an important test animal for regimens most likely to have promise in regression of human lesions. The hybrid hare should be mentioned for its usefulness in studies of the clonality of cells in experimental atherosclerotic lesions [45]. In summary, the rabbit has undergone rehabilitation as a model of atherosclerosis, and rabbits seem likely to have continued major usefulness in future studies of atherosclerosis. Other small mammals
Rodents The possibility that small animals other than rabbits might be useful as animal models in atherosclerosis has been extensively explored in rodents. If suitable models existed among the many rodent species, it seems likely that they would be widely known and used. Although hyperlipidemic states are inducible in rats and mice, the resulting arterial lesions tend to be disappointingly meager. Other rodents are not superior in this respect. Papers appear from time to time advocating the use of rats or mice. Despite such advocacy, the attitude of most investigators in the field is probably illustrated by 2 leading groups of atherosclerosis research in the United States, who conducted initial evaluations in rodents [46,47], and quite promptly abandoned rodents in all subsequent research. Nonetheless, certain strains of mice seem to show improved lesion formation [21,48,49], and inbred strains of mice [49] are quite useful in examining the genetics of dyslipoproteinemia. We should consider the door ajar for future development of rodents as more satisfactory models of atherosclerotic lesions. Cats Cats have been used extensively as laboratory animals, but they have been almost skipped as
potential models of atherosclerosis. We know of only one paper in which cats were evaluated for lesions [50]. Lesions were indeed produced in response to atherogenic diet, but lesion characteristics and distribution were unlike those in humans, and the authors reverted to pigeon and primate models.
Dogs Dogs have been studied as models from time to time [51,523. The problems with dogs as models are that (1) atherogenesis cannot be produced in the absence of hypothyroidism or an unusual diet enriched in coconut oil, (2) the lesions are not very prominent in major arteries, and (3) medial lesions occur to a significant degree. Given the preemminence of dogs in cardiovascular studies, it is regrettable that these animals cannot be used more satisfactorily as atherosclerotic models. Study of the mechanisms of discrete lipid deposits in the media would seem to be one worthwhile topic for investigation, but this has not been evaluated. Swine Swine, in contrast to dogs, are superb models. Both full-sized and miniature swine have been extensively studied [53,54]. Lesions occur to some extent in free-living animals, and can be induced with atherogenic diet to an important degree. Lesions occur in both aorta and branch arteries. The vessels are large, facilitating physiologic and biochemical studies. Von Willebrand deficient strains [55,56] have been studied, permitting evaluation of the effect of coagulation pathways on atherogenic response. Nonhuman primates Primate models have been extensively characterized [57-591. Our group has used Old World primates for many years in correlated studies of arterial structure and function [60,61]. We have prized the advantages they offer in showing little lesion formation in controls, and in contrast extensive lesion formation in a multivessel distribution after dietary induction. We feel, nonetheless, that great caution should be taken in decisions about the future use of primates, because of their
20 decreasing availability and the expense and considerable knowledge base involved in providing proper maintenance and nutrition. Marked ecological change since publication of “Comparative Atherosclerosis” includes a shrinking pool of feral primates. Governmental regulations restrict availablity of some species. In the United States, studies funded through the National Institutes of Health must show that no nonprimate species is a reliable substitute. The National Institutes of Health has set up two centers for breeding and modeling of several Old World species. These centers are located at the Southwest Foundation for Research and Education, University of Texas, San Antonio; Texas, and at the Arteriosclerosis Research Center, Bowman Gray School of Medicine, Winston-Salem, North Carolina. The goal of these two centers is to provide experimental access and primatological expertise to scientists who have only occasional need of the unique features of animals belonging to the primate order. The comments that follow are made in the context of this background, which indicates that primates are a prized but limited resource.
New World monkeys
The New World primates have been described in depth [62], and their characteristics make it unlikely that the problems associated with their acquisition justify their future use in most types of study of atherosclerosis. Lesion formation in response to diet differs greatly among species, lesion distribution is unlike that seen in man, the health surveillance necessary is a major consideration, and there is probably little unique advantage to the use of any New World monkey.
Old World monkeys
Old World monkeys have been extensively studied [57-59,63,64]. At least seven species of the macaque genus have been used in atherosclerosis research. Currently, the cynomolgus is the most widely studied macaque. Rhesus and stumptails are also used to a considerable degree [64], however, and use of the Japanese macaque is reported periodically [59]. Lesion formation in response to atherogenic diet occurs at a greater rate in cynomolgus than rhesus [65]. Regression of lesions
occurs more readily in rhesus than in cynomolgus monkeys [65-671. There are no comparisons of other species of Old World monkeys under identical experimental conditions, but lesions in African green monkeys are probably characterized by more fibrosis [64] than is seen in macaques. Patas monkeys (Erythrocebus patus) also have relatively fibrotic lesions [68]. One macaque species, the Celebes ape (Macucu nigru), has shown a tendency to diabetes and the effect of hypercholesterolemia has been studied in that setting [69]. Baboons have less hypercholesterolemia in response to diet, and lesions are correspondingly smaller [70].
The great apes
Old World apes have been observed in captivity for lesions [71], and there are small systematic studies of lesion formation. The studies showed that a high fat, high-cholesterol diet produces marked hypercholesterolemia with formation of atherosclerotic lesions [72-741. Given the strategic pre-hominid position in primate evolution occupied by the great apes, this information, has the greatest qualitative significance. It is also unlikely to be supplemented significantly in the future, since the great apes are an endangered species. Conclusions
Animal models continue to have important roles in atherosclerosis research. In the eight decades in which animals have been used to study aspects of lesion formation in atherosclerosis, the potential choice of animal has expanded from rabbits, used in the earliest studies, to include many species of mammals and birds. During the past 25 years the actual choices of animals for study of atherosclerosis have narrowed. This reflects several developments, including greater acceptance of the lipid hypothesis as an important explanation of the human disease. Thus, there is an immediate advantage to the study of lesion formation and subsequent lesion change in several well-characterized hyperlipidemic models (pigeon, rabbit, swine and some primates). There is a growing sense that animals now uncharacterized are unlikely to be superior models. Interest is marked in genetic variants of common laboratory species that
21 show abnormal lipid metabolic states or changed responsiveness to atherogenic challenge. Both dogs and rodents are of quite limited value as models for study of lesion formation. Genetic variants, however, have already improved the potential choices in rabbits and may do so in rodents. Among larger mammals, swine and Old World primates are excellent models, but decreasing availability of primates raises a question of their future use.
13
14
15
16
Acknowledgements This work was supported 14230 and a VA Medical (D.D.H.)
by NIH Grant HL Investigator Award
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