Mutation Research, 239 (1990) 143-148
143
Elsevier MUTREV 07287
INTERNATIONAL COMMISSION FOR PROTECTION AGAINST ENVIRONMENTAL MUTAGENS AND CARCINOGENS
The possible involvement of somatic mutations in the development of atherosclerotic plaques Report of ICPEMC
Subcommittee
7/1
Conclusions and recommendations Membership of Subcommittee 7/1 B.A. Bridges (Brighton, Great Britain, Chairman), D.E. Bowyer (Cambridge, Great Britain), E.S. Hansen (Odense, Denmark), A. Penn (New York, U.S.A.) and K. Wakabayashi (Tokyo, Japan) (Accepted 17 May 1990)
Keywords: Atherosclerosis and carcinogens; Atherosclerosis and somatic mutations; ICPEMC Report
Introduction
Cardiovascular diseases are a major cause of human morbidity and mortality in developed industrialised countries. Epidemiological studies have revealed that major risk factors are dyslipoproteinemia, especially increased concentrations of serum low-density lipoproteins and decreased concentrations of serum high-density lipoproteins, smoking and the presence of other diseases such as hypertension and diabetes. Such risk factors may be associated with the formation of arterial lesions, or with the precipitation of clinically evident events such as thrombosis and myocardial
All correspondence and reprint requests should be addressed to the secretary of ICPEMC: Dr. J.D. Jansen, Medical Biological Laboratory TNO, P.O. Box 45, 2280 AA Rijswijk (The Netherlands). ICPEMC is affiliated with the International Association of Environmental Mutagen Societies (IAEMS) and the Institut de la Vie.
infarction or both. Risk factors do not necessarily indicate a causal relationship but may simply correlate with causal factors. There is, however, much evidence to suggest that dyslipoproteinemia and smoking reflect the causation or exacerbation of arterial lesions per se. Establishing the aetiology and even mechanisms of action of known risk factors in atherogenesis is complicated by the fact that atherosclerotic lesions are not homogeneous and a variety of morphologically distinct features are found within the same individual and often within the same artery. The commonly recognised lesions are (a) fibromuscular intimal thickenings, the cells of which are predominantly smooth muscle cells without lipid inclusions, (b) fatty dots and streaks, in which lipid accumulates intracellularly within so-called foam cells, which are mainly monocytederived macrophages, (c) fibro-fatty plaques, which are characterised by fat-filled cells, extracellular lipid accumulation and substantial accumulation of fibrous tissue which forms a fibrous cap, (d) atheromas, in which a large pool of ex-
0165-1110/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
144 tracellular lipid and necrotic gruel is seen at the base of a fibro-fatty lesion, (e) ulcerated, thrombosed and calcified lesions. It is plain from the morphological diversity of the lesions that a complex series of cellular events and cellular interactions occurs during lesion production and the balance between them determines the ultimate morphology of the lesion. The dominant phenomena to be considered are: (1) Infiltration of the arterial intima with plasma lipoproteins. (2) Migration of smooth muscle cells from the media into the intima where they proliferate and synthesise connective tissue, especially collagen, which contributes to the bulk of the growing lesion and constitutes the fibrous cap. (3) Involvement of blood monocytes which become fat-filled foam cells through the uptake of plasma iipoproteins, especially low-density and very-low-density lipoproteins that have been modified by oxidative and proteolytic reactions. (4) Involvement of T lymphocytes. This seems to occur mainly in the larger fibro-fatty lesions, where activated T cells may be found. Such an immunological reaction is likely to be a response to the formation of the lesion but could be involved in the initiation of the lesion. (5) Adhesion of platelets. This is thought to occur primarily in more advanced lesions where the integrity of the endothelium is compromised rather than in the earlier lesions with apparently intact endothelium. Platelets may well play a part in stimulating the growth of lesions. Attempts to explain the mechanism of lesion formation must take into account not only the accumulation of lipids and of blood cells within the intima, but also the participation of the smooth muscle cells from the media. The discovery by Ross that platelet-derived growth factor ( P D G F ) released from platelets at sites of endothelial injury, is mitogenic for smooth muscle cells, revived the concept, first proposed by Virchow a century ago, that atherogenesis is a reaction to injury (Ross and Glomset, 1973; Ross, 1986). The fibrogenic response of smooth muscle cells, apparently stimulated by mitogens such as P D G F shows interesting parallels with the fibrous repair reaction associated with chronic injury and chronic inflammation, in other sites in the body, for example in lung, in tuberculosis or in the pneumoconio-
ses. However, in 1973 Benditt and Benditt had proposed an alternative, although not necessarily mutually exclusive hypothesis, namely that the proliferation of the smooth muscle cells in the lesion is the result of their clonal growth in the manner of a benign tumour. This hypothesis was tested by analysis of the isoenzyme pattern in lesions from individuals (necessarily women) heterozygous for an X-linked marker enzyme, glucose-6-phosphate dehydrogenase (G6PD). Clear cut evidence for monotypism of fibro-fatty lesions, although not early fatty streaks was obtained. Such a result might have arisen because of the selective growth of cells containing one isoform of the enzyme, but this was ruled out by the fact that although individual lesions contained only one isoform, this was not the same type in every lesion, even within the same artery. Subsequent experiments by others confirmed the initial observations. Benditt and Benditt suggested that plaque formation was a response to a somatic mutational event which provided certain smooth muscle cells with a proliferative advantage over all their sister cells. They proposed that environmental mutagens could play a role in the aetiology of plaque formation. The observed lack of monotypism in fatty streaks may come about because any monotypism of smooth muscle cells in the lesions is masked by the heterogeneity of invading blood monocytes. From the outset, Benditt and Benditt's hypothesis elicited strong and mostly negative criticism. Although no convincing a priori reasons existed to discount their suggestion, it did not initially inspire experiments to try to test its validity. However, in 1986, Penn et al. reported studies of the D N A extracted from atherosclerotic lesions of human coronary arteries and showed that it possessed transforming ability when transfected into 3T3 cells and that such transformed cells elicited the appearance of tumours in nude mice. This result strongly suggested that human atherosclerotic plaques contain altered D N A capable of causing the cellular transformation and proliferation predicted by Benditt and Benditt's hypothesis. This result does not, of course, deny the possible role of arterial injury, inflammation and lipid metabolism in the development of plaques, but strengthens the possibility that a somatic mutation accompanies lesion formation.
145 In the light of the result by Penn et al., ICPEMC convened a subcommittee and charged it with accumulating and presenting in a coherent manner evidence bearing upon the monoclonal theory of atherogenesis. The following report and working papers are the outcome of the Committee's deliberations.
Clonality Conclusions There is evidence that a substantial fraction of human fibrous plaques is monotypic when subjected to G6PD isozyme analysis. There does not appear to be evidence that selection is an important factor in determining the isoenzyme composition of plaques so that it is reasonable to conclude that the majority of cells in such plaques are of monoclonal origin. This conclusion falls short of definitive proof, however, and it depends on the absence of smooth muscle cell islands of sufficient size in normal artery to have been the source of a monotypic but polyclonal plaque. This has been addressed experimentally twice and the results supported this contention. In humans the evidence regarding the monoclonality of fatty streaks is unclear. Some fatty streaks had monoclonal characteristics. These might be lesions with a predominant population of smooth muscle cells. It has to be recognized, however, that a monotypic character of all fatty streaks is hardly to be expected given the substantial presence of monocytes in such lesions, a fact that was not widely appreciated when the study was done. The hybrid hare is in principle a good experimental system in so far as the determination of isoenzyme types is concerned, but in the work carried out so far the material sampled appears to have comprised fatty lesions rather than fibrous plaques. Fatty lesions in the hare, like fatty streaks in humans, contain a substantial proportion of monocytes and so would not be expected to be monoclonal, an expectation confirmed experimentally. Recommendations A better experimental system is needed for the study of monoclonality of atherosclerotic plaques.
The hybrid hare has the advantage of easy isoenzyme typing and it might be worth examining whether fibrous plaques of this species are monotypic. If the plaques prove to be monotypic, the hybrid hare may present a useful model for studying the atherogenic effects of various mutagenic agents. Rodent systems also should not be discounted. Isoenzyme typing is feasible and one may study both the proliferation of smooth muscle cells and the development of fibrous plaques. Alternatively, the possibility of examining monoclonality using DNA-based methods should be considered since these could in principle be applied to a wide variety of systems including human material.
Involvement of somatic mutation Conclusions Only one laboratory has so far reported evidence of a heritable character in the D N A from human plaques that is not present in normal arterial wall. The character conferred via transfection a transformed phenotype on N I H 3T3 cells which were then able to induce tumours when injected into nude mice. A similar change, that might be likened to an activated oncogene, was found in D N A from plaques found in chickens treated with 7,12-dimethylbenz[a]anthracene (DMBA). It therefore seems likely that the plaques contain mutated DNA, but it should not be assumed in the latter case that the mutation was induced by the DMBA (see below). There is also the (perhaps remote) possibility that the mutation was present in the D N A not of smooth muscle cells but of some other cell type present in plaques but largely absent in normal arterial wall (e.g. monocytes/macrophages). The possible involvement of platelet-derived growth factor ( P D G F ) has been suggested as a basis for the transfectable phenotype but studies with the sis gene, which specifies the B chain of P D G F , have not supported this suggestion. The identity of the transfected gene is therefore at present unknown. There are reports that human smooth muscle cells can be transformed by pSV3neo to a form that expresses T antigen and accumulates numerous lipid droplets after treatment with fl-migrating very-low-density lipoprotein. This would suggest that the cells are expressing the scavenger recep-
146
tor. Lipogenesis at non-permissive temperature has also been reported in cultured smooth muscle cells transformed with T-24Ha-ras and temperaturesensitive SV40 large T antigen. The significance of such observations is still unclear although they do at least demonstrate that oncogene expression may induce in smooth muscle cells properties similar to those found in plaque cells. We conclude that there is accumulating evidence for a mutation in D N A from atherosclerotic plaques that may be of fundamental importance for understanding the mechanism of plaque formation. Recommendations It is important that the work demonstrating a transfectable mutant gene in h u m a n and chicken atherosclerotic plaque be independently confirmed and extended. The presence of the mutation in D N A of smooth muscle cells rather than some other component of plaques needs to be unambiguously determined. The use of cultured plaque cells may be of value in such studies.
Involvement of viruses Conclusions Although it has been unambiguously demonstrated that viruses of the herpes family can cause atherosclerotic plaques in experimental systems and such viruses have been reported to be associated with human plaques, we do not find any convincing evidence that they are responsible for the genesis of human plaques. We recognize the difficulty of establishing such a point, given the difficulty of detecting the presence of such infections or evidence for previous but not current infection. At present it seems likely that if viruses are involved they may operate as ' h i t and run' mutagens. It would be prudent, however, to be prepared for further evidence on this point.
Involvement of exogenous mutagens Conclusions The ability of large doses of ionizing radiation to give rise to atherosclerotic plaques may be regarded as established both in animal systems and in humans. Ionizing radiation is, of course, a powerful mutagen and the levels of gene muta-
tions and chromosome aberrations to be expected in cells surviving doses in excess of 10 G y are likely to be sufficiently great to explain the induction of plaques on a mutational model. However, such doses will also be expected to cause the death of the majority of cells in the irradiated field. During the course of a regime of radiotherapy, for example, this would result in surviving cells undergoing a large number of cell divisions in order to repopulate the arterial wall. This would in turn allow the clonal expansion of a mutated cell to a degree that would allow it to become established as a precurser of a plaque. Thus ionizing radiation could act as a mutagen or a promoter * or both. In animals the atherogenic action of radiation has been found in combination with other treatments such as a high cholesterol diet. The other area where studies in humans and animals appear to link up is smoking and polycyclic aromatic hydrocarbons (PAHs). Although epidemiological studies clearly show an association between cigarette smoking and atherosclerosis, the nature of the active agent(s) in the smoke is unknown and the data are unhelpful in distinguishing between a mutagenic and a ' p r o m o t e r / a c c e l e r a t o r ' mechanism. The ability of PAHs such as D M B A to cause the appearance of plaques in experimental animals is established, but the data are conflicting as to whether the number of plaques are increased, or merely their rate of progression. One case may be mentioned where a stochastic initiating event such as a mutation seems possible, namely the effect of two or three treatments of chickens with D M B A followed by prolonged exposure to methoxamine as a ' p r o m o t e r / accelerator' giving rise to thoracic lesions. The lesions were focal proliferations of intimal smooth muscle cells. Even the D M B A treatment cannot be assumed to have been acting as a mutagen since associated cytotoxicity may have been suffi* We have used the term promoter to refer to an agent that promotes the development of plaques whose initiation has already occurred. The use of the term has operational similarities with its use in connection with carcinogenesis. Although no mechanistic similarity is implied, the specific concept of clonal expansion suggested here for atherogenesis has previously been considered in the context of carcinogenesis. As with carcinogenesis, promotion of atherosclerotic lesions m a y well occur by more than one mechanism.
147 cient to allow clonal expansion of a preexisting spontaneously mutated cell. Elevated levels of gene mutations and chromosome aberrations are found in the peripheral lymphocytes of smokers but they are modest and one could not account for the greater severity of atherosclerotic disease in smokers on a mutational model unless the mutation frequency of smooth muscle cells were very much higher than that in circulating lymphocytes. This could occur if there were a special delivery mechanism for transporting the mutagen to the smooth muscle cell, or if metabolic activation of the mutagen were much greater in smooth muscle cells than lymphocytes. An example of a specific delivery mechanism might be the transport of PAHs in lipoproteins and their associated uptake by smooth muscle cells in a fatty streak. It is known that smooth muscle cells can activate PAHs but the levels of activation are not high. However, the activating ability of cells with fatty granules may be different. If PAHs have been transported to a cell by low-density lipoproteins and taken up they would be expected to induce the microsomal oxidative pathways so that metabolic activation would be increased. A combination of specific delivery of PAH plus induced microsomal oxidation pathways might be sufficient to raise the mutation rate to an extent consistent with the extent of atherosclerogenesis. We also wish to draw attention to vinyl chloride monomer and arsenic. These two human carcinogens both induce hemangiosarcomas which are endothelial tumours of blood vessels, usually in the liver. They also have been found to cause other vascular diseases in humans including atherosclerosis, although whether they initiate or accelerate the development of atherosclerotic plaques is not known. Dose-response analyses, however, indicate that the effect of arsenic on cardiovascular mortality is related to (past) peak exposure whereas the effect on mortality due to respiratory cancer seems to be more closely related to the time-weighted average exposure. It may be asked whether xeroderma pigmentosum (XP) individuals are also hypersensitive for the development of atherosclerotic plaques. Patients with XP are defective in the ability to repair D N A damage due to bulky D N A lesions and their
skin is hypersensitive to the carcinogenic effect of ultraviolet light and psoralens plus UVA. Their cells in culture are hypersensitive to the mutagenic action of a wide variety of agents including UV and PAHs, but not ionizing radiation and active oxygen species. However, recent evidence indicates that factors other than or in addition to somatic mutation are responsible for the high incidence of skin cancers in XP individuals (Bridges, 1990) and the excess of internal tumours in XPs is small and confined to certain sites (Kraemer et al., 1984). Despite fairly detailed attention, there does not appear to be any report of an association of XP with cardiovascular disease in general or atherosclerosis in particular, although it is doubtful whether the specific question has ever been addressed. It is interesting to note that one may induce tumours of the medial layer (leiomyomas and leiomyosarcomas) by treatment of mice with p-hydrazinobenzoic acid. Although these tumours are different in their location and morphology from the intimal lesions of atherosclerosis, the experimental results have demonstrated that environmental mutagens may reach the smooth muscle cells of the arterial media. The view has been advanced that atherosclerotic plaques may be viewed simply as a benign type of tumour but we would urge caution in this regard. Plaques may (and probably do) contain cells with a 'transformed' phenotype but they are more complex systems than tumours and there is unlikely to be a single etiological cause. Overall, we conclude that some agents with mutagenic potential certainly accelerate and possibly initiate the development of atherosclerotic plaques but a link between the observed effect and the mutagenic mechanism cannot be made with any confidence at the present time. Recommendations There are, of course, many areas deserving of attention concerning the action of chemicals in enhancing atherosclerotic plaque formation, but three would seem to have particular relevance to the possible involvement of a mutagenic component. Firstly, with all agents, greater attention needs to be given to the problem of distinguishing be-
148
tween effects which are the result of single step events and those which require the continuous presence of an agent for a period. These are formally analogous to the 'initiation' and 'promotion' steps postulated for carcinogenesis, indeed there may be common mechanistic similarities. It should not be forgotten, however, that a mutational step need not be the first step in the genesis of a plaque. Studies to distinguish between mutational and 'promotion/acceleration' effects need to be tied in with oncogene expression. Dependence on dose-rate and time-course modelling must also be considered. Secondly, sensitive modern techniques such as D N A post-labelling, monoclonal antibodies to D N A adducts, and two-dimensional N M R need to be applied for the detection of D N A damage. With smoking, or exposure to PAHs, for example, one would like to know if there is any evidence for a specifically and significantly elevated amount of damage in the cells of fatty streaks as compared with cells in normal arterial wall, and with circulating lymphocytes. It is necessary to demonstrate such a difference if a mutagenic mechanism for the action of cigarette smoke is to be regarded seriously. Thirdly, more sensitive epidemiological methods need to be employed in studying populations exposed to suspected atherogenic agents. It is also recommended that studies on populations exposed to mutagens and carcinogens should bear in mind the possibility of atherosclerotic lesions as an endpoint in addition to cancers.
Involvement of endogenous mutagens
Conclusions Even if it is accepted that somatic mutation plays a role in the development of atherosclerotic plaques, it does not follow that the mutations are induced or are inducible by exogenous agents (environmental mutagens). The presence of mutagens generated by normal metabolism within the body is now well established, e.g. active oxygen species, nitrosamines, aldehydes, fecapentenes. There is, however, little evidence bearing on the extent to which such agents might reach the myocytes of the arterial media and be involved in the induction of mutations in smooth muscle cells. A parallel worth noting, however, is between active oxygen species and ionizing radiation. The latter exerts much of
its mutagenic effect by means of the generation of active oxygen species following ionization. Two recent papers (Carew et al., 1987; Kita et al., 1987) have drawn attention to the ability of the antioxidant probucol (4,4'-(isopropylidenedithio)bis(2,6-di-tert.-butylphenol)) to prevent the progression of atherosclerosis in the Watanabe heritable hyperlipidemic rabbit. This has been interpreted in terms of a restriction of the oxidation of LDLP, and there is evidence to support this view. Nevertheless it would seem wise also to consider the possibility that the probucol might be scavenging active oxygen species that otherwise would be mutagenic.
Recommendations The possibility of D N A damage by endogenous mutagens is in principle open to investigation using the same sensitive assays outlined above for exogenous mutagens. Active oxygen species are certainly amenable to investigation in this way, as one can readily alter their concentration in vivo, e.g. by treatment with scavengers such as probucol.
References Benditt, E.P., and J.M. Benditt (1973) Evidence for a monoclonal origin of human atherosclerotic plaques, Proc. Natl. Acad. Sci. (U.S.A.), 70, 1753-1756. Bridges, B.A. (1990) Sunlight, DNA damage and skin cancer: a new perspective, Jpn. J. Cancer Res., 18, 105-107. Carew, T., D. Schwenke and D. Steinberg (1987) Antiatherogenic effect of probucol unrelated to its hypocholesterolemic effect: Evidence that antioxidants in vivo can selectively inhibit low density lipoprotein degradation in macrophage-rich fatty streaks and slow the progression of atherosclerosis in the Watanabe heritable hyper-lipidemic rabbit, Proc. Natl. Acad. Sci. (U.S.A.), 84, 7725-7729. Kita, T., Y. Nagano, M. Yokode, K. lshii, N. Kume, A. Ooshima, H. Yoshida and C. Kawai (1987) Probucol prevents the progression of atherosclerosis in Watanabe heritable hyperlipidemic rabbit, an animal model for familial hypercholesterolemia, Proc. Natl. Acad. Sci. (U.S.A.), 84, 5928-5931. Kraemer, K.H., M.M. Lee and K. Scotto (1984) DNA repair protects against cutaneous and internal neoplasia: Evidence from xeroderma pigmentosum. Carcinogenesis, 5, 511-514. Penn, A., S.J. Garte, L. Warren, D. Nesta and B. Mindich (1986) Transforming gene in human atherosclerotic plaque DNA, Proc. Natl. Acad. Sci. (U.S.A.), 83, 7951-7955. Ross, R. (1986) The pathogenesis of atherosclerosis - an update, N. Engl. J. Med., 314, 488-500. Ross, R., and J.A. Glomsett (1973) Atherosclerosis and the arterial smooth muscle cell, Science, 180, 1332-1339.
143
Elsevier MUTREV 07287
INTERNATIONAL COMMISSION FOR PROTECTION AGAINST ENVIRONMENTAL MUTAGENS AND CARCINOGENS
The possible involvement of somatic mutations in the development of atherosclerotic plaques Report of ICPEMC
Subcommittee
7/1
Conclusions and recommendations Membership of Subcommittee 7/1 B.A. Bridges (Brighton, Great Britain, Chairman), D.E. Bowyer (Cambridge, Great Britain), E.S. Hansen (Odense, Denmark), A. Penn (New York, U.S.A.) and K. Wakabayashi (Tokyo, Japan) (Accepted 17 May 1990)
Keywords: Atherosclerosis and carcinogens; Atherosclerosis and somatic mutations; ICPEMC Report
Introduction
Cardiovascular diseases are a major cause of human morbidity and mortality in developed industrialised countries. Epidemiological studies have revealed that major risk factors are dyslipoproteinemia, especially increased concentrations of serum low-density lipoproteins and decreased concentrations of serum high-density lipoproteins, smoking and the presence of other diseases such as hypertension and diabetes. Such risk factors may be associated with the formation of arterial lesions, or with the precipitation of clinically evident events such as thrombosis and myocardial
All correspondence and reprint requests should be addressed to the secretary of ICPEMC: Dr. J.D. Jansen, Medical Biological Laboratory TNO, P.O. Box 45, 2280 AA Rijswijk (The Netherlands). ICPEMC is affiliated with the International Association of Environmental Mutagen Societies (IAEMS) and the Institut de la Vie.
infarction or both. Risk factors do not necessarily indicate a causal relationship but may simply correlate with causal factors. There is, however, much evidence to suggest that dyslipoproteinemia and smoking reflect the causation or exacerbation of arterial lesions per se. Establishing the aetiology and even mechanisms of action of known risk factors in atherogenesis is complicated by the fact that atherosclerotic lesions are not homogeneous and a variety of morphologically distinct features are found within the same individual and often within the same artery. The commonly recognised lesions are (a) fibromuscular intimal thickenings, the cells of which are predominantly smooth muscle cells without lipid inclusions, (b) fatty dots and streaks, in which lipid accumulates intracellularly within so-called foam cells, which are mainly monocytederived macrophages, (c) fibro-fatty plaques, which are characterised by fat-filled cells, extracellular lipid accumulation and substantial accumulation of fibrous tissue which forms a fibrous cap, (d) atheromas, in which a large pool of ex-
0165-1110/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
144 tracellular lipid and necrotic gruel is seen at the base of a fibro-fatty lesion, (e) ulcerated, thrombosed and calcified lesions. It is plain from the morphological diversity of the lesions that a complex series of cellular events and cellular interactions occurs during lesion production and the balance between them determines the ultimate morphology of the lesion. The dominant phenomena to be considered are: (1) Infiltration of the arterial intima with plasma lipoproteins. (2) Migration of smooth muscle cells from the media into the intima where they proliferate and synthesise connective tissue, especially collagen, which contributes to the bulk of the growing lesion and constitutes the fibrous cap. (3) Involvement of blood monocytes which become fat-filled foam cells through the uptake of plasma iipoproteins, especially low-density and very-low-density lipoproteins that have been modified by oxidative and proteolytic reactions. (4) Involvement of T lymphocytes. This seems to occur mainly in the larger fibro-fatty lesions, where activated T cells may be found. Such an immunological reaction is likely to be a response to the formation of the lesion but could be involved in the initiation of the lesion. (5) Adhesion of platelets. This is thought to occur primarily in more advanced lesions where the integrity of the endothelium is compromised rather than in the earlier lesions with apparently intact endothelium. Platelets may well play a part in stimulating the growth of lesions. Attempts to explain the mechanism of lesion formation must take into account not only the accumulation of lipids and of blood cells within the intima, but also the participation of the smooth muscle cells from the media. The discovery by Ross that platelet-derived growth factor ( P D G F ) released from platelets at sites of endothelial injury, is mitogenic for smooth muscle cells, revived the concept, first proposed by Virchow a century ago, that atherogenesis is a reaction to injury (Ross and Glomset, 1973; Ross, 1986). The fibrogenic response of smooth muscle cells, apparently stimulated by mitogens such as P D G F shows interesting parallels with the fibrous repair reaction associated with chronic injury and chronic inflammation, in other sites in the body, for example in lung, in tuberculosis or in the pneumoconio-
ses. However, in 1973 Benditt and Benditt had proposed an alternative, although not necessarily mutually exclusive hypothesis, namely that the proliferation of the smooth muscle cells in the lesion is the result of their clonal growth in the manner of a benign tumour. This hypothesis was tested by analysis of the isoenzyme pattern in lesions from individuals (necessarily women) heterozygous for an X-linked marker enzyme, glucose-6-phosphate dehydrogenase (G6PD). Clear cut evidence for monotypism of fibro-fatty lesions, although not early fatty streaks was obtained. Such a result might have arisen because of the selective growth of cells containing one isoform of the enzyme, but this was ruled out by the fact that although individual lesions contained only one isoform, this was not the same type in every lesion, even within the same artery. Subsequent experiments by others confirmed the initial observations. Benditt and Benditt suggested that plaque formation was a response to a somatic mutational event which provided certain smooth muscle cells with a proliferative advantage over all their sister cells. They proposed that environmental mutagens could play a role in the aetiology of plaque formation. The observed lack of monotypism in fatty streaks may come about because any monotypism of smooth muscle cells in the lesions is masked by the heterogeneity of invading blood monocytes. From the outset, Benditt and Benditt's hypothesis elicited strong and mostly negative criticism. Although no convincing a priori reasons existed to discount their suggestion, it did not initially inspire experiments to try to test its validity. However, in 1986, Penn et al. reported studies of the D N A extracted from atherosclerotic lesions of human coronary arteries and showed that it possessed transforming ability when transfected into 3T3 cells and that such transformed cells elicited the appearance of tumours in nude mice. This result strongly suggested that human atherosclerotic plaques contain altered D N A capable of causing the cellular transformation and proliferation predicted by Benditt and Benditt's hypothesis. This result does not, of course, deny the possible role of arterial injury, inflammation and lipid metabolism in the development of plaques, but strengthens the possibility that a somatic mutation accompanies lesion formation.
145 In the light of the result by Penn et al., ICPEMC convened a subcommittee and charged it with accumulating and presenting in a coherent manner evidence bearing upon the monoclonal theory of atherogenesis. The following report and working papers are the outcome of the Committee's deliberations.
Clonality Conclusions There is evidence that a substantial fraction of human fibrous plaques is monotypic when subjected to G6PD isozyme analysis. There does not appear to be evidence that selection is an important factor in determining the isoenzyme composition of plaques so that it is reasonable to conclude that the majority of cells in such plaques are of monoclonal origin. This conclusion falls short of definitive proof, however, and it depends on the absence of smooth muscle cell islands of sufficient size in normal artery to have been the source of a monotypic but polyclonal plaque. This has been addressed experimentally twice and the results supported this contention. In humans the evidence regarding the monoclonality of fatty streaks is unclear. Some fatty streaks had monoclonal characteristics. These might be lesions with a predominant population of smooth muscle cells. It has to be recognized, however, that a monotypic character of all fatty streaks is hardly to be expected given the substantial presence of monocytes in such lesions, a fact that was not widely appreciated when the study was done. The hybrid hare is in principle a good experimental system in so far as the determination of isoenzyme types is concerned, but in the work carried out so far the material sampled appears to have comprised fatty lesions rather than fibrous plaques. Fatty lesions in the hare, like fatty streaks in humans, contain a substantial proportion of monocytes and so would not be expected to be monoclonal, an expectation confirmed experimentally. Recommendations A better experimental system is needed for the study of monoclonality of atherosclerotic plaques.
The hybrid hare has the advantage of easy isoenzyme typing and it might be worth examining whether fibrous plaques of this species are monotypic. If the plaques prove to be monotypic, the hybrid hare may present a useful model for studying the atherogenic effects of various mutagenic agents. Rodent systems also should not be discounted. Isoenzyme typing is feasible and one may study both the proliferation of smooth muscle cells and the development of fibrous plaques. Alternatively, the possibility of examining monoclonality using DNA-based methods should be considered since these could in principle be applied to a wide variety of systems including human material.
Involvement of somatic mutation Conclusions Only one laboratory has so far reported evidence of a heritable character in the D N A from human plaques that is not present in normal arterial wall. The character conferred via transfection a transformed phenotype on N I H 3T3 cells which were then able to induce tumours when injected into nude mice. A similar change, that might be likened to an activated oncogene, was found in D N A from plaques found in chickens treated with 7,12-dimethylbenz[a]anthracene (DMBA). It therefore seems likely that the plaques contain mutated DNA, but it should not be assumed in the latter case that the mutation was induced by the DMBA (see below). There is also the (perhaps remote) possibility that the mutation was present in the D N A not of smooth muscle cells but of some other cell type present in plaques but largely absent in normal arterial wall (e.g. monocytes/macrophages). The possible involvement of platelet-derived growth factor ( P D G F ) has been suggested as a basis for the transfectable phenotype but studies with the sis gene, which specifies the B chain of P D G F , have not supported this suggestion. The identity of the transfected gene is therefore at present unknown. There are reports that human smooth muscle cells can be transformed by pSV3neo to a form that expresses T antigen and accumulates numerous lipid droplets after treatment with fl-migrating very-low-density lipoprotein. This would suggest that the cells are expressing the scavenger recep-
146
tor. Lipogenesis at non-permissive temperature has also been reported in cultured smooth muscle cells transformed with T-24Ha-ras and temperaturesensitive SV40 large T antigen. The significance of such observations is still unclear although they do at least demonstrate that oncogene expression may induce in smooth muscle cells properties similar to those found in plaque cells. We conclude that there is accumulating evidence for a mutation in D N A from atherosclerotic plaques that may be of fundamental importance for understanding the mechanism of plaque formation. Recommendations It is important that the work demonstrating a transfectable mutant gene in h u m a n and chicken atherosclerotic plaque be independently confirmed and extended. The presence of the mutation in D N A of smooth muscle cells rather than some other component of plaques needs to be unambiguously determined. The use of cultured plaque cells may be of value in such studies.
Involvement of viruses Conclusions Although it has been unambiguously demonstrated that viruses of the herpes family can cause atherosclerotic plaques in experimental systems and such viruses have been reported to be associated with human plaques, we do not find any convincing evidence that they are responsible for the genesis of human plaques. We recognize the difficulty of establishing such a point, given the difficulty of detecting the presence of such infections or evidence for previous but not current infection. At present it seems likely that if viruses are involved they may operate as ' h i t and run' mutagens. It would be prudent, however, to be prepared for further evidence on this point.
Involvement of exogenous mutagens Conclusions The ability of large doses of ionizing radiation to give rise to atherosclerotic plaques may be regarded as established both in animal systems and in humans. Ionizing radiation is, of course, a powerful mutagen and the levels of gene muta-
tions and chromosome aberrations to be expected in cells surviving doses in excess of 10 G y are likely to be sufficiently great to explain the induction of plaques on a mutational model. However, such doses will also be expected to cause the death of the majority of cells in the irradiated field. During the course of a regime of radiotherapy, for example, this would result in surviving cells undergoing a large number of cell divisions in order to repopulate the arterial wall. This would in turn allow the clonal expansion of a mutated cell to a degree that would allow it to become established as a precurser of a plaque. Thus ionizing radiation could act as a mutagen or a promoter * or both. In animals the atherogenic action of radiation has been found in combination with other treatments such as a high cholesterol diet. The other area where studies in humans and animals appear to link up is smoking and polycyclic aromatic hydrocarbons (PAHs). Although epidemiological studies clearly show an association between cigarette smoking and atherosclerosis, the nature of the active agent(s) in the smoke is unknown and the data are unhelpful in distinguishing between a mutagenic and a ' p r o m o t e r / a c c e l e r a t o r ' mechanism. The ability of PAHs such as D M B A to cause the appearance of plaques in experimental animals is established, but the data are conflicting as to whether the number of plaques are increased, or merely their rate of progression. One case may be mentioned where a stochastic initiating event such as a mutation seems possible, namely the effect of two or three treatments of chickens with D M B A followed by prolonged exposure to methoxamine as a ' p r o m o t e r / accelerator' giving rise to thoracic lesions. The lesions were focal proliferations of intimal smooth muscle cells. Even the D M B A treatment cannot be assumed to have been acting as a mutagen since associated cytotoxicity may have been suffi* We have used the term promoter to refer to an agent that promotes the development of plaques whose initiation has already occurred. The use of the term has operational similarities with its use in connection with carcinogenesis. Although no mechanistic similarity is implied, the specific concept of clonal expansion suggested here for atherogenesis has previously been considered in the context of carcinogenesis. As with carcinogenesis, promotion of atherosclerotic lesions m a y well occur by more than one mechanism.
147 cient to allow clonal expansion of a preexisting spontaneously mutated cell. Elevated levels of gene mutations and chromosome aberrations are found in the peripheral lymphocytes of smokers but they are modest and one could not account for the greater severity of atherosclerotic disease in smokers on a mutational model unless the mutation frequency of smooth muscle cells were very much higher than that in circulating lymphocytes. This could occur if there were a special delivery mechanism for transporting the mutagen to the smooth muscle cell, or if metabolic activation of the mutagen were much greater in smooth muscle cells than lymphocytes. An example of a specific delivery mechanism might be the transport of PAHs in lipoproteins and their associated uptake by smooth muscle cells in a fatty streak. It is known that smooth muscle cells can activate PAHs but the levels of activation are not high. However, the activating ability of cells with fatty granules may be different. If PAHs have been transported to a cell by low-density lipoproteins and taken up they would be expected to induce the microsomal oxidative pathways so that metabolic activation would be increased. A combination of specific delivery of PAH plus induced microsomal oxidation pathways might be sufficient to raise the mutation rate to an extent consistent with the extent of atherosclerogenesis. We also wish to draw attention to vinyl chloride monomer and arsenic. These two human carcinogens both induce hemangiosarcomas which are endothelial tumours of blood vessels, usually in the liver. They also have been found to cause other vascular diseases in humans including atherosclerosis, although whether they initiate or accelerate the development of atherosclerotic plaques is not known. Dose-response analyses, however, indicate that the effect of arsenic on cardiovascular mortality is related to (past) peak exposure whereas the effect on mortality due to respiratory cancer seems to be more closely related to the time-weighted average exposure. It may be asked whether xeroderma pigmentosum (XP) individuals are also hypersensitive for the development of atherosclerotic plaques. Patients with XP are defective in the ability to repair D N A damage due to bulky D N A lesions and their
skin is hypersensitive to the carcinogenic effect of ultraviolet light and psoralens plus UVA. Their cells in culture are hypersensitive to the mutagenic action of a wide variety of agents including UV and PAHs, but not ionizing radiation and active oxygen species. However, recent evidence indicates that factors other than or in addition to somatic mutation are responsible for the high incidence of skin cancers in XP individuals (Bridges, 1990) and the excess of internal tumours in XPs is small and confined to certain sites (Kraemer et al., 1984). Despite fairly detailed attention, there does not appear to be any report of an association of XP with cardiovascular disease in general or atherosclerosis in particular, although it is doubtful whether the specific question has ever been addressed. It is interesting to note that one may induce tumours of the medial layer (leiomyomas and leiomyosarcomas) by treatment of mice with p-hydrazinobenzoic acid. Although these tumours are different in their location and morphology from the intimal lesions of atherosclerosis, the experimental results have demonstrated that environmental mutagens may reach the smooth muscle cells of the arterial media. The view has been advanced that atherosclerotic plaques may be viewed simply as a benign type of tumour but we would urge caution in this regard. Plaques may (and probably do) contain cells with a 'transformed' phenotype but they are more complex systems than tumours and there is unlikely to be a single etiological cause. Overall, we conclude that some agents with mutagenic potential certainly accelerate and possibly initiate the development of atherosclerotic plaques but a link between the observed effect and the mutagenic mechanism cannot be made with any confidence at the present time. Recommendations There are, of course, many areas deserving of attention concerning the action of chemicals in enhancing atherosclerotic plaque formation, but three would seem to have particular relevance to the possible involvement of a mutagenic component. Firstly, with all agents, greater attention needs to be given to the problem of distinguishing be-
148
tween effects which are the result of single step events and those which require the continuous presence of an agent for a period. These are formally analogous to the 'initiation' and 'promotion' steps postulated for carcinogenesis, indeed there may be common mechanistic similarities. It should not be forgotten, however, that a mutational step need not be the first step in the genesis of a plaque. Studies to distinguish between mutational and 'promotion/acceleration' effects need to be tied in with oncogene expression. Dependence on dose-rate and time-course modelling must also be considered. Secondly, sensitive modern techniques such as D N A post-labelling, monoclonal antibodies to D N A adducts, and two-dimensional N M R need to be applied for the detection of D N A damage. With smoking, or exposure to PAHs, for example, one would like to know if there is any evidence for a specifically and significantly elevated amount of damage in the cells of fatty streaks as compared with cells in normal arterial wall, and with circulating lymphocytes. It is necessary to demonstrate such a difference if a mutagenic mechanism for the action of cigarette smoke is to be regarded seriously. Thirdly, more sensitive epidemiological methods need to be employed in studying populations exposed to suspected atherogenic agents. It is also recommended that studies on populations exposed to mutagens and carcinogens should bear in mind the possibility of atherosclerotic lesions as an endpoint in addition to cancers.
Involvement of endogenous mutagens
Conclusions Even if it is accepted that somatic mutation plays a role in the development of atherosclerotic plaques, it does not follow that the mutations are induced or are inducible by exogenous agents (environmental mutagens). The presence of mutagens generated by normal metabolism within the body is now well established, e.g. active oxygen species, nitrosamines, aldehydes, fecapentenes. There is, however, little evidence bearing on the extent to which such agents might reach the myocytes of the arterial media and be involved in the induction of mutations in smooth muscle cells. A parallel worth noting, however, is between active oxygen species and ionizing radiation. The latter exerts much of
its mutagenic effect by means of the generation of active oxygen species following ionization. Two recent papers (Carew et al., 1987; Kita et al., 1987) have drawn attention to the ability of the antioxidant probucol (4,4'-(isopropylidenedithio)bis(2,6-di-tert.-butylphenol)) to prevent the progression of atherosclerosis in the Watanabe heritable hyperlipidemic rabbit. This has been interpreted in terms of a restriction of the oxidation of LDLP, and there is evidence to support this view. Nevertheless it would seem wise also to consider the possibility that the probucol might be scavenging active oxygen species that otherwise would be mutagenic.
Recommendations The possibility of D N A damage by endogenous mutagens is in principle open to investigation using the same sensitive assays outlined above for exogenous mutagens. Active oxygen species are certainly amenable to investigation in this way, as one can readily alter their concentration in vivo, e.g. by treatment with scavengers such as probucol.
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