Mutation Research, 239 (1990) 149-162 Elsevier

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MUTREV 07288

INTERNATIONAL COMMISSION FOR PROTECTION AGAINST ENVIRONMENTAL MUTAGENS AND CARCINOGENS

ICPEMC Working Paper 7/1/1 Mutational events in the etiology of arteriosclerotic plaques Arthur Penn Institute of Environmental Medicine, New York University, 550 First Avenue, New York, N Y 10016 (U.S.A.) (Accepted 17 May 1990)

Keywords: Monoclonal; Arteriosclerosis; Transformation; Viruses; Smooth muscle cells, proliferation

Summary The arteriosclerotic plaque is the lesion most often associated with cardiovascular disease, which is the leading cause of death in North America and Western Europe. Plaques are composed of cells (mostly smooth muscle cells but also macrophages and some lymphocytes) and formed elements (cellular debris, collagen, elastin, glycosaminoglycans, lipid droplets, cholesterol crystals and sometimes calcium deposits). Proliferation of smooth muscle cells is essential to plaque formation and development. Most theories of plaque development have viewed this proliferation as a secondary event following an initiating stimulus (e.g., endothelial injury). According to this view, the proliferating smooth muscle cells are otherwise identical to the large number of non-proliferating smooth muscle cells in the artery wall. The 'monoclonal' hypothesis of plaque formation presents a fundamentally different view; namely, that the cell proliferation critical to plaque development follows the stable transformation of smooth muscle cells and that the plaques can therefore be viewed as benign smooth muscle cell tumors of the artery wall. Environmental agents, including viruses and chemicals that have been previously associated with cell transformation and tumorigenesis may therefore also contribute directly to plaque development. Data are provided from in vivo and in vitro studies in support of this proposition. Evidence is also presented that in standardized assays human and animal plaque DNAs elicit responses similar to those elicited by tumor DNAs. Thus, both plaque formation and tumorigenesis may share common mechanisms.

All' correspondence and reprint requests should be addressed to the secreatary 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.

In this review, evidence is provided in support of the 'monoclonal' hypothesis of arteriosclerotic plaque formation. Experimental and clinical data, collected over the last 16 years, are presented that are consistent with the view that environmental mutagens, including viruses and chemical carcinogens, play a key role in plaque etiology. Since the objective of this review is to examine in detail the

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150 ' monoclonal' hypothesis and its implications, other hypotheses of plaque formation, e.g., 'response to injury' are not dealt with extensively. However, the essential aspects of these competing theories and the implications which arise from them are presented. References are also provided which describe these theories in greater detail. The review is organized in 6 sections. First, there is a short section on the pathophysiology of arteriosclerosis. This is followed by a brief discussion of 'response to injury' and 'inflammation' hypotheses as explanations of the primary factors in plaque etiology. Then, the early clonal analysis data in support of the 'monoclonal' hypothesis are presented. In the next section in vivo and in vitro studies with viruses and carcinogens are discussed. Problems associated with each of these experimental approaches are noted briefly. This is followed by a summary of recent transfection/tumorigenesis experiments with plaque DNA from both human and animal models, which indicate that transforming events play a role in plaque formation. In the last section the effects of DNA damage and repair upon plaque development are noted briefly. This section concludes with a synthesis of elements from both the 'monoclonal' and 'injury' hypotheses that is consistent with the bulk of experimental and clinical data that have been obtained to date.

Pathology Cardiovascular disease (CVD) is the leading cause of death in the United States and Western Europe. The major cause of CVD is atheroarteriosclerosis, an occlusive condition that develops in large arteries. The principal lesion associated with this condition is the arteriosclerotic plaque which arises, partly as a result of cell proliferation, in the intimal region of the artery wall, between the single layer of lumenal facing endothelium and the smooth muscle (medial) layer. The predominant cell type in plaques is the smooth muscle cell (SMC). In the atherosclerotic plaque a fibrous cap composed of SMC and connective tissue matrix covers the atheroma, which contains large amounts of extracellular lipid deposits and some cells, including SMC, macrophages and lymphocytes. In advanced plaques a necrotic core and calcification

may also be present. Lipid-laden macrophages, which can contribute to the 'foamy' appearance of many plaques, are often present as well. The formed elements of plaques include collagen, elastin, glycosaminoglycans and lipid deposits. The accumulation of cholesterol and cholesterol esters in plaques has been documented extensively in both clinical and experimental studies. A role has been suggested for lipid accumulation in the early stages of plaque development (reviewed in McGill, 1984). An early lesion, the fatty streak, is composed largely of 'foamy' cells, mostly lipid-laden macrophages. However, it is not clear how or even whether plaque development in later life depends on fatty streak formation during infancy and childhood. Regardless of whether there is a primary role for lipid deposition in the early stages of plaque development, there is no doubt that SMC proliferation is critical to this process.

Monoclonality During the past 15 years, 3 hypotheses that have been advanced to explain the SMC proliferation have gained popularity. The best known of these 3 is the 'response to injury' hypothesis, according to which injury to the endothelium (e.g., by blood-borne factors or hemodynamic agents) is the principal stimulus to the intimal SMC proliferation (Ross, 1986). Although these factors clearly play a role in plaque development there is no compelling evidence that they are directly responsible for arterial SMC proliferation, in vivo. The subject of injury to the artery wall and its role in subsequent plaque development has been dealt with at length elsewhere (e.g., see Ross, 1986). It is important to recognize that 'response to injury' implies a polyclonal process. That is, during wound healing, daughter cells from a number of different parents are recruited to repopulate the wound site. Further consideration of the role of injury in plaque formation is made in the Discussion section of this review. The second of the hypotheses views plaque development as a special form of inflammatory response (reviewed recently in Munro and Cotran, 1988). The apparent involvement of macrophages in fatty streak formation, in the uptake of mod-

151 ified low-density lipoproteins, in the release of mitogens and in the adhesion of leucocytes to endothelial cells has contributed to this view. In both the 'response to injury' and 'inflammation' hypotheses, SMC proliferation is a reactive process. Proliferation occurs in response to mitogens or other stimulatory factors released by platelets or inflammatory cells. The SMC involved in intimal proliferation are representative of all the SMC in the artery wall. Of the 3 hypotheses, the most controversial one is the 'monoclonal' hypothesis first presented in 1973 (Benditt and Benditt, 1973; and discussed further by Benditt, 1974, 1977; and Poole, 1978). The 'monoclonal' hypothesis is fundamentally different from the other theories of plaque formation. The SMC involved in intimal proliferation are viewed as being derived from a stably transformed and therefore permanently altered cell population. In order to fully consider the consequences that would derive from the 'monoclonal' hypothesis, were it proven to be correct, the experiments and observations that led up to the formulation of the hypothesis will be presented in some detail. At the end of this review I will address the apparent incompatibilities between the theories. In the introduction to their report, Benditt and Benditt (1973) summarize 3 observations about plaques and plaque cells that are at odds with the view that plaques represent a response of the artery wall to injury. First, plaque SMC are smaller than are artery wall SMC at a repair site. Second, the extracellular material in fibrous plaques tends to be collagen, whereas elastin is more prominent in the normal artery wall or at a repair site. Third, plaque cells lack intercellular junctions. The Benditts observed that the two most likely interpretations of these observations were (a) that plaque cells were derived from a population of cells different from those of the arterial wall or (b) that plaque cells were derived from arterial SMC that had been transformed. The experimental approach they selected was based on a series of studies first reported in 1965 (Linder and Gartler, 1965). The studies clearly showed that human leiomyomas, benign uterine smooth muscle tumors, were monoclonal. The patients studied were black American females who were heterozygous for the X-linked

enzyme, glucose-6-phosphate dehydrogenase (G-6PD). Due to the random inactivation of the Xchromosome during development, analysis of any normal tissue should reveal a mosaic pattern; that is, each cell with either one or the other of the two X-chromosomes being active. Thus, the isozyme pattern from a sample of normal tissue should show approximately equal representation of both isozyme types (A and B). Tissues that are monoclonal in origin, e.g., many solid tumors, would be expected to display either, but not both, of the isozyme patterns. In their studies, Linder and Gartler studied 185 tumors, all but one of which displayed either the A form or B form of G-6-PD, but not both. Statistical analysis demonstrated the overwhelming probability that each tumor arose from only a single cell, i.e. that the tumors were monoclonal. 17 of the patients had multiple tumors. In all but one of these patients some of the tumors had the A isozyme and some the B isozyme. As Poole (1978) has pointed out, finding tumors of both A and B types in the same organ of a patient effectively counters the argument that cells in which a particular X-chromosome is active have a selective advantage over cells in which the other X-chromosome is active. Obviously, analysis of the extent of monoclonality will be difficult if there are large portions of normal tissue mixed in with the tumor tissue and will be meaningless if the tumors are polyclonal (e.g., venereal warts or multiple neurofibromas of von Recklinghausen's disease). Benditt and Benditt (1973) applied this G-6-PD isozyme approach to the study of plaque samples and normal wall from 4 autopsy patients. In 24 of 30 samples, plaques (up to 0.5 cm in diameter) had only one, or predominantly one G-6-PD isozyme type. In 57 of 59 normal artery samples, some as small as 0.1 mm 3, both enzyme types were present. The Benditts noted that these results are compatible with a monoclonal origin for plaques, which they suggested can be viewed as benign SMC tumors of the artery wall. The authors concluded by suggesting that viruses and chemical mutagens may play roles in plaque formation. Experiments addressing this suggestion will be summarized later in this review. These findings were confirmed two years later by Pearson et al. (1975) who reported that human

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aortic wall, grossly free of plaque, had evidence of both G-6-PD isozyme types whereas plaques had isozyme patterns similar to those exhibited by leiomyomas. In the same study, the authors first reported on a clonal analysis of fatty streaks. This analysis was particularly important because, as noted earlier, many investigators believe(d) that the fatty streak is the precursor lesion to the fibrous capped atheromatous plaque, the most prevalent of the clinically significant plaques. Most investigators agree that not all fatty streaks progress to either experimentally or clinically significant plaques and the clonal analysis of fatty streaks by Pearson and co-workers confirms this (Pearson et al., 1975, 1978a,b). Of the 66 human aortic fatty streaks that they analyzed, 11 had G-6-PD isozyme patterns intermediate between those of polyclonal normal aortic wall and monoclonal leiomyomas. Subsequently, they reported that a subset of fatty streaks, from a larger group that they analyzed, had monoclonal characteristics (Pearson et al., 1983a). This finding and the absence of evidence for clonal selection as plaques increase in size (Pearson et al., 1975), are consistent with the conclusion that the monoclonal appearance of plaques is a result of their development from a group of monoclonal fatty streaks. The impossibility of carrying out a rigorous longitudinal study of the development of plaques from a subset of fatty streaks in humans has led predictably to a search for a suitable animal model in which to study this process. This in itself presents a problem because it is difficult to find species with X-linked electrophoretically separable isozymes of a given enzyme. One animal model that fulfills this requirement is the hybrid hare, produced by crossing the European hare with the Scandinavian snowshoe hare. Pearson and his colleagues have investigated whether the atherosclerotic plaques which appear in hybrid hares fed cholesterol are monoclonal (Pearson et al., 1983b). Although these plaques had some histologic features similar to those of human plaques, 92 of 93 plaques that were assayed were clearly polyclonal. The authors concluded that the hybrid hare is not a good model for studying the monoclonal nature of arteriosclerotic plaques via X-linked isozyme patterns. A similar conclusion was recently reached by a second group of investigators (Murray et al., 1988).

However, their results also suggested that (a) even in these hares there may at least be subpopulations of SMC that are monoclonal and that (b) within individual plaques there may be subpopulations of cells with monoclonal character. Experiments with the same hybrid hares studied by Pearson and co-workers showed no evidence of enzyme homozygosity in plaques of animals fed cholesterol. However, animals fed 25-hydroxycholesterol, a cholesterol oxidation product, showed evidence of plaque G-6-PD homozygosity. 10 years previously, Benditt had suggested that another cholesterol oxidation product, cholesterol a-oxide, might play a role in the etiology of clinically significant plaques (Benditt, 1977). Cholesterol a-oxide is a mutagen and inducer of tumors in laboratory animals. The presence in the circulation and consequent high availability to the artery wall of naturally occurring mutagens is a simple and relatively reliable way of insuring that cells at risk 'see' these agents. Murray et al. (1988) have noted that when portions of large plaques were grown in culture, the highest percentage of G-6-PD homozygosity was in the cells from the region closest to the arterial lumen. Cells in this region are most likely to make contact with circulating mutagens, in vivo. Viruses

In fight of the suggestion that viruses may be etiologic agents in atherosclerosis (Benditt and Benditt, 1973; Benditt, 1974), it was logical to ask whether viruses could induce atherosclerosis in one or more animal models. For reasons that will be made clear shortly most of the virus studies have been carried out with members of the herpes virus family. The most popular animal model for these virus studies has been the cockerel. An early indirect indication that herpes viruses might be related to cardiovascular disease was the observation that feline kidney cells infected with a feline herpes virus displayed accumulations of cholesterol crystals (Fabricant et al., 1973). The first clear demonstration that viruses could play an etiologic role in atherosclerosis came from the laboratory of the Fabricants and their co-workers. The first of a series of papers on this topic appeared in 1978

153 (Fabricant et al., 1978). Cockerels, free from pathogens and viruses, were injected at 2 days of age with Marek disease virus (MDV), an oncogenic herpes virus that causes malignant T cell lymphomas in chickens (Payne, 1972). When they were sacrificed 30 weeks later, 7 of 42 injected birds, but none of the 47 controls, had grossly visible atherosclerotic lesions. Microscopically, proliferative and fatty proliferative lesions were present in the thoracic aorta and coronary arteries in 50% of the injected birds but only in 5% of the uninjected controls. In another injected group, fed a diet containing 2% cholesterol from 15 to 30 weeks of age, atherosclerotic plaques appeared in 90% of the birds. Fewer than 20% of cholesterol-fed controls developed similar lesions. The histologic appearance of the virus-induced plaques was very similar to that of human atherosclerotic plaques. These studies were expanded and presented in greater detail the following year (Minick et al., 1979). Qualitatively similar results to those just noted had been presented 30 years earlier (Paterson et al., 1948, 1949; Paterson and Cottral, 1950). They reported that chickens suffering from neurolymphomatosis developed coronary artery lesions in which lipid accumulated. These findings and others led them to conclude that the arterial damage resulted from an infectious agent that was associated with the neurolymphomatosis. These conclusions were reached before MDV, the agent responsible for neurolymphomatosis, was isolated. Minick et al. (1979, p. 689) list a number of studies which report positive associations between virus infection and the development of arterial disease in both humans and experimental animals. Fabricant et al. also reported that turkey herpes virus (HVT), which is routinely used commercially to immunize chickens and thereby prevent MDVassociated tumors, was also partially effective at inhibiting MDV-associated atherosclerosis (Fabricant et al., 1983). Gross lesions appeared only in the MDV-injected birds. Microscopic lesions appeared in both MDV-injected and HVT-injected birds but there were many more birds with plaques in the MDV group than in the HVT group. One problem common to all these cockerel studies, is that no direct evidence was ever presented for MDV infection of plaque cells, although the pres-

ence of MDV antigen in the artery wall was reported (Fabricant et al., 1981). Subsequent experiments by Fabricant et al. (1981) and by Hajjar et al. (1986) sought to correlate MDV infection with alterations in lipid accumulation and metabolism. The former showed that MDV-injected cockerels exhibited increased lipid deposits in arterial SMC. Hajjar and his co-workers studied patterns of lipid metabolism and accumulation in MDV-infected cockerels that were fed a normo-cholesterolemic diet. MDV-infected animals showed marked increases of aortic cholesterol esters (CE), triglycerides (TG), and phospholipids, compared to controls. These increases did not appear in MDV-injected cockerels that had been immunized previously with HVT. The increases in aortic CE deposition correlated well with decreases in acid and neutral cholesterol hydrolase activity and with increases in acyl cholesterol acyl transferase (ACAT; 2.3.1.26) activity. ACAT plays a key role in cholesterol esterification. These results were achieved without any significant increases in serum-cholesterol levels. Since aortic wall cells do not usually accumulate much CE, these authors suggest that CE sequestration in the aortic wall of MDV-treated cockerels may precede the appearance of the foam cell lesions thought by many to be characteristic of early stages of plaque development. The effects of MDV infection upon lipid disposition and metabolism in cockerel arteries were used as the basis for studies of the effects of herpes simplex virus (HSV) infection of human fetal arterial SMC (Hajjar et al., 1987). Fetal cells were chosen because they have less chance of harboring latent HSV than do adult cells. Following a 2-h exposure to HSV and 3 days of incubation at 37 o C, the infected cells exhibited > 40 x increase in CE and > 7 x increase in TG. Most of the CE fatty acids were saturated in HSV-infected cells whereas in control cells the saturated:unsaturated fatty acid ratio in CE----1. As was the case with cockerels, CE hydrolase activity was decreased in HSV-infected cells. When these cells were stimulated with arachidonic acid, there was decreased production of prostacyclin, an agonist of intracellular CE hydrolase. The authors concluded that the patterns of lipid deposition and metabolism in HSV-infected human arterial SMC

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are similar to those that have been described in human atherosclerosis, in vivo. The induction of plaques in MDV-injected cockerels encouraged investigators to look for evidence of herpes virus infections in human artery walls. Benditt et al. performed in situ hybridizations of HSV m R N A to wall samples from the anterior ascending thoracic aorta of patients undergoing coronary artery bypass surgery (Benditt et al., 1983). Specimens with thickened intima were generally positive whereas medial cells in these sections were negative. There was no hybridization to cytomegalovirus (CMV) or EpsteinBarr Virus (EBV) probes. Of the herpes viruses that affect man, EBV is most similar to MDV (Roizman et al., 1981). One problem with in situ hybridization studies of HSV probes and artery wall cells of surgery or autopsy patients is that infection with herpes virus is so common in people that it can be difficult to relate the presence of virus to the pathology of specific diseases in the tissues being sampled. In 1984, Gyorkey et al. reported results of an electron microscopic study of punch biopsy samples from plaque-free areas of the thoracic aortas from 60 atherosclerotic patients who were undergoing cardiovascular surgery (Gyorkey et al., 1984). Herpes virus virions were found sporadically in samples from 10 of 60 patients. The virus was present in SMC and more rarely in endothelial cells. The authors indicated that even in the 10 patients who tested positive, it was difficult to find evidence of the virus. Of the 1360 grids that were examined, only 35 displayed any evidence of the virions. No attempt was made to identify which of the 5 types of human herpes virus was present. The inability to find more extensive evidence of herpes infection in artery wall cells of bypass patients is striking and somewhat unexpected. In 1983, Melnick et al. published results of a study on tissue samples taken from 132 patients undergoing vascular surgery (Melnick et al., 1983). There were 126 samples from plaques and 6 from punch-biopsy samples of normal artery wall. Cell cultures were maintained from all of the punch biopsy samples and from 26 of the 126 plaque samples. 7 of the 26 plaque cultures were positive by indirect immunofluorescence for CMV anti-

gens as were 4 of 6 normal wall cultures. In both sets of cultures antigens were localized to the cytoplasm exclusively, and 10-30% of all cells in these cultures contained antigens to CMV. A recent report from the same group described high levels of antibodies to CMV in the sera of atherosclerotic patients undergoing vascular surgery (Adam et al., 1987). 90% of these patients displayed the CMV antibodies; and antibody titers were 2 × higher in patients than in controls. 74% of the controls also had antibodies to CMV. However, the percentage difference between patients and controls was reported to be highly significant ( p < 0.001). There was no correlation between antibody levels and either serum-cholesterol or serum T G levels. No significant differences in antibody titers of HSV1 and HSV2 were seen between surgical patients and controls. Finally, Yamashiroya et al. detected nucleic acids and antigens from HSV and CMV, but not EBV, in coronary arteries and thoracic aortas obtained at autopsy (Yamashiroya et al., 1988). All samples were taken from young trauma victims between 4.5 and 36 h after death. Although virus was not found in plaques it was identified in intimal layer 'foamy' cells. Even though the evidence is strong for an association of viruses of the herpes family with the artery wall, proof is still lacking for an etiologic role for these viruses in atherosclerosis. What follows is a brief summary of some of the mechanisms whereby these viruses could play such a role. A possible role for members of the herpes family in altering lipid metabolism has already been mentioned (Hajjar et al., 1986, 1987). At least 3 other possibilities have also been considered. Minick and co-workers discussed a role for these viruses in mediating cell injury (Minick et al., 1979). They noted that in MDV-infected cockerels, necrosis of the SMC layer (media) of the arteries was common and even preceded intimal thickening. If this observation were borne out in subsequent investigations, the appropriateness of the c o c k e r e l / M D V model to studies of plaque etiology might have to be re-examined since medial cell necrosis followed by intimal thickening is the reverse of what usually occurs during plaque development.

155 The herpes viruses might also mediate artery wall injury indirectly, via immunologic mechanisms. This possibility was suggested 15 years ago (Burch et al., 1973; Burch, 1974). At approximately the same time, a report on the presence of viral antigen-antibody complexes in the thoracic aortas of autopsy cases suggested that such complexes may play a role in the development of arterial disease (Smith et al., 1974). Minick et al. (1979) also list a series of earlier references which consider various immunologic mechanisms as contributing factors to the development of cardiovascular disease. A recent report from Russia supports the view that herpes virus interference with T-lymphocyte activity may play a role in plaque development (Gandzha et al., 1988). The summary, which is the only part of the article presented in English, claims that HSV-infected, cholesterol-fed rabbits displayed disorders of suppressor T cells. Aortic atherosclerosis in these rabbits was more severe than in uninfected cholesterol-fed controls. Of the 4 mechanisms that have been proposed to explain how herpes viruses might act as etiologic agents in the development of arteriosclerosis, the final one, and the one most germane to this overview, is that the viruses act as transforming agents. At present there are no published reports showing that SMC can be transformed by herpes viruses. In fact, a recent report from Nachtigal's laboratory is one of the few that describes the transformation of SMC by any virus (Nachtigal et al., 1987). They describe the transformation of rabbit arterial SMC by SV-40. The cells appeared to be transformed by both morphological and biochemical criteria, displayed lipid accumulation in the cytoplasm, grew as permanent lines in cell culture and retained SMC-specific antigens. A meeting abstract in the same year also reported marked changes in lipid metabolism and accumulation in SMC that had been transformed by SV-40 (Ruley et al., 1987). It is reasonable to expect that a number of laboratories presently are engaged in trying to transform SMC with specific members of the herpes family. However, even if we assume that HSV transformation of human SMC will be demonstrated, there will still be a major problem regarding an etiologic role for HSV in arteriosclerosis. That is the difficulty of directly demonstrating the presence of

HSV or viral products in cells following infection. This is true even in cases where cells are clearly transformed by morphological a n d / o r biochemical criteria, or where cells are tumorigenic in test systems. This problem has existed since the original demonstrations of a direct role of herpes viruses in cell transformation (Duff and Rapp, 1971, 1973). Hamster-embryo fibroblasts (HEF) were transformed by UV-irradiated HSV1 and HSV2. These transformed fibroblasts gave rise to tumors following injection into newborn Syrian hamsters. Although viral antigens were detected in the cells and HSV2 antibody was present in sera from tumor-bearing animals, no infectious virus was recovered from the transformed cells. Subsequent studies demonstrated that the transformed HEFs gradually lost the HSV DNA sequences with time (Minson et al., 1976). The inability to detect infectious virus or even viral nucleic acids or gene products in HSV-transformed mouse cells that were tumorigenic was also noted by Hampar et al. (1980). One explanation that fits these observations (reviewed in Hampar, 1981) is that of a 'hit and run' mechanism wherein cellular retention of herpes virus D N A would not be necessary, once the critical transforming event(s) have taken place. A second related explanation is that the viral D N A need not be the transforming agent. The latter could be a viral gene product present in an infected cell. The gene product could diffuse to a target cell which could already be either partially or fully transformed. This diffusing product could stimulate the target cell(s) to divide. Thus, the virally infected cells and the transformed cells need not be the same. A similar explanation was suggested recently to explain the apparent induction of Kaposi's sarcoma in transgenic mice carrying the HIV t a t gene (Vogel et al., 1988). There was no detectable HIV in Kaposi-type cells and a diffusible product of the t a t gene was invoked as the agent of transformation. Although these explanations are consistent with many observations and may even be correct, they remain problematical. Given the controversial nature of the 'monoclonal' hypothesis it will not be easy to argue for acceptance of a viral etiology for arteriosclerosis in the absence of direct evidence for the presence of virus in the affected tissue.

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Chemicals

The initial observation of the monoclonality of human plaques (Benditt and Benditt, 1973), and the ensuing suggestion that transforming agents might be involved in arteriosclerosis led to experiments in a small number of laboratories to determine whether carcinogenic chemicals could play a role in plaque initiation or development. The first major confirmation of this suggestion was provided by Albert and co-workers who reported that cockerels injected weekly with either of the two polycyclic aromatic hydrocarbon (PAH) carcinogens 7,12-dimethylbenz[ a ] a n t h r a c e n e (DMBA) or benzo[a]pyrene (B(a)P), displayed large proliferating arteriosclerotic plaques in their abdominal aortas (Albert et al., 1977). Since only large plaques were scored in this study it was not clear whether the carcinogens were actually inducing new plaques. Subsequent dose-response and temporal-response studies demonstrated that administration of a wide variety of PAHs to cockerels resulted in the accelerated development of pre-existing aortic plaques. Increasing doses of DMBA caused increases in cross-sectional area and mean plaque volume of abdominal aorta plaques (Penn et al., 1981b). Cockerels injected with DMBA from 4 to 8 weeks of age exhibited plaques as large as those appearing in 20-week-old control cockerels (Penn et al., 1981a). Both the carcinogen-associated and the spontaneous plaques appeared in the same regions of the artery. The numbers of plaques were the same in treated and control groups, and plaques in both groups were indistinguishable histologically and ultrastructurally (Batastini and Penn, 1984). These results strongly suggested that in this in vivo model, PAH carcinogens were acting more analogously to 'promoters' than to 'initiators'. This conclusion was strengthened by a recent demonstration that a number of PAHs, including 3 generally considered to be very weakly carcinogenic or non-carcinogenic, markedly accelerated plaque development in cockerels (Penn and Snyder, 1988). Regardless of whether PAH metabolites are behaving as 'initiators' or 'promoters' in artery wall cells, it is clear that these cells contain the requisite enzymes for bioactivation of PAHs.

One of the earliest demonstrations of this was by Juchau and co-workers, who reported that aryl4-monooxygenase, a microsomal enzyme involved in B(a)P bioactivation, is present in aortas of rabbits, monkeys and man. Low concentrations of cytochrome P-450 were also detected in aortic wall microsomal fractions (Juchau et al., 1976). A few years later, investigators from the same laboratory demonstrated that cultures of human fetal aortic SMC can metabolize B(a)P and DMBA (Bond et al., 1979). The metabolites included both proximate and ultimate mutagenic compounds. Similar results with chick aortic homogenates were presented the following year by the same group (Bond et al., 1980). They also demonstrated that the PAH metabolites could bind covalently to DNA. In both sets of studies (Bond et al., 1979, 1980) pretreatment with inducing agents resulted in marked increases in enzymatic activity and/or PAH metabolism. Subsequently, two other reports from this group strongly suggested that interaction of PAH metabolites with the artery wall might directly precede plaque initiation. In 1983, evidence was presented that $9 fractions from aortic homogenates of atherosclerosis-susceptible pigeons both had higher levels of inducible monooxygenases and could bioactivate B(a)P better than $9 fractions from aortas of atherosclerosis resistant pigeons (Majesky et al., 1983). These resuits are particularly striking because the original studies of susceptibility and resistance to atherosclerosis in pigeons were carried out on 5 strains of pigeons, none of which had been exposed to PAHs (Clarkson et al., 1959). In 1985, there was a report that focal, proliferating plaques appeared in the thoracic aortas of cockerels which had received initiating injections of DMBA followed by twice weekly 'promotional' injections of the alpha adrenergic agonist, methoxamine (Majesky et al., 1985). Thus, an initiation-promotion protocol, comparable to that used in classic rodent carcinogenesis studies, can elicit plaque formation in cockerels. Two other sets of studies of PAH-associated arterial lesions have been reported recently. Two strains of mice were each fed an atherogenic diet and injected with the PAH carcinogen, methylcholanthrene (3MC) (Paigen et al., 1985, 1986).

157 The two strains differed in the inducibility of cytochrome P-450 enzymes and in their susceptibility to 3MC-induced tumorigenesis. Arterial lesions increased in size and number in both groups. However, the results were more pronounced in the strain in which P-450-induced tumorigenesis and PAH-associated arterial lesion formation cosegregated. In the other study, p-hydrazinobenzoic acid (HBA), a chemical present in mushrooms, was tested for its possible carcinogenic activity via lifetime administration (in the drinking water) to random-bred Swiss mice (McManus et al., 1987). The Swiss mouse is a well characterized animal model for studying carcinogenesis, but these animals rarely develop arteriosclerosis. 50% of the males that were fed HBA died of aortic rupture. Aortic tumors appeared in 40% of treated males but only in 4% of controls. The tumors, some of which were benign and others malignant, all retained biochemical and morphological markers characteristic of SMC. The authors also note that at early times in the experiments, HBA-treated mice displayed proliferative lesions in the intima-media that were similar to vascular changes characteristic of human arteriosclerosis. An interesting ancillary aspect of this work is that it arose from an interest in the carcinogenic properties of natural substances. No mention is made of the 'monoclonal' hypothesis, even though these results confirm the prediction that chemical carcinogens may play a role in plaque development. Possibly the strongest argument for a role of carcinogenic chemicals in clinically significant plaque formation comes from recently published epidemiological studies (Chen et al., 1988). The carcinogen in this case was an inorganic chemical, arsenic. Chen et al. reported on an isolated Taiwanese population that suffers from blackfoot disease. The association of this disease with arsenic in well water was first pointed out by Wu et al. nearly 30 years ago (Wu et al., 1961; referenced in Chen et al., 1988). The current report shows that a direct relationship exists between arsenic exposure and arteriosclerosis as well as between arsenic exposure and increased mortality from cardiovascular disease. There was also increased mortality from a variety of cancers in the population suffering from blackfoot disease. In light of

the 'monoclonal' hypothesis these results suggest that arsenic may act in all these cases by helping to transform cells in each of the target tissues. The actual mechanism(s) whereby arsenic initiates various cancers or arteriosclerosis remain(s) to be determined. The above experiments with cockerels and mice, treated with a wide variety of chemicals, including strong and weak carcinogens, have shown that these compounds can apparently induce and can certainly accelerate arteriosclerotic plaque development. However, the effective doses of these compounds in most of the experiments summarized above are many times higher than those to which humans are normally exposed. For example, in the PAH studies with chickens and mice the doses ranged from 0.1 to 40 m g / k g body weight. In the HBA studies, mice received lifetime exposures at a dose of 0.125%. Thus, the primary value of the P A H / c a r c i n o g e n experiments just summarized lies less in specifying which PAHs can contribute to plaque development clinically or even experimentally, than in suggesting a mechanism whereby certain environmental chemicals could 'initiate' or 'promote' plaque development. This mechanism is not merely a variant form of injury to the artery wall. Indeed, if these chemicals react in vascular tissue similarly to the way they act in other tissues and if oncogenic viruses play a direct role in plaque formation then a transforming mechanism has to be considered for involvement in plaque etiology and development. Transformation Since none of the experiments summarized to this point addressed directly the question of whether transforming events are associated with plaque development, either clinically or experimentally, an experimental approach was adopted (Penn et al., 1986) that had been popularized previously in oncogene studies. D N A extracted from surgically removed human coronary artery plaques was transfected into NIH3T3 cells. Individual foci were collected, expanded in culture and injected into nude mice. Tumors arose that were morphologically and ultrastructurally indistinguishable from those which arose in nude mice after injection of T24 DNA-transformed cells.

158 D N A of the T24 human bladder carcinoma cell line contains an activated Harvey r a s oncogene that is not present in human plaque DNA. Tumors appeared in 20-100% of mice injected with plaque DNA-transformed cells. The latent period for these tumors was >/7 weeks compared to 2-3 weeks for the T24-derived tumors. No tumors arose in mice that had been injected with 3T3 cells transfected with non-transforming DNA. The plaque DNAassociated tumors hybridized to a human D N A probe. Human artery D N A was negative in these assays. Comparable experiments were also carried out with plaque DNA from cockerels that had been injected weekly with DMBA from 4 to 20 weeks of age (Penn et al., 1989). The DNAs for transfection, which also included samples from artery walls grossly free of plaque and from artery walls that underlay the plaques, were co-transfected with pSV2neo, which confers resistance to gentamycin. Gentamycin-resistant colonies of 3T3 cells were collected after 18 days and injected into nude mice. All 5 mice injected with 3T3 cells that had been transfected with plaque DNA developed tumors. The presence of chicken D N A in the transfected cells was confirmed by hybridization to a chicken genomic probe. No tumors appeared in mice injected with cells that had been transfected with aortic DNA. These two sets of transfection data strongly suggest that plaque DNA from both clinical and experimental samples displays molecular alterations comparable to those that have been described for many tumor DNAs. Discussion

During the past two decades a large body of experimental evidence has been collected in support of the view that injury to the endothefial cells lining the lumenal surface of the artery wall results in the general stimulation of arterial SMC proliferation that is essential for arteriosclerotic plaque formation. The data summarized above support a different contention, namely, that transformation of one or more SMC is the critical event that must precede SMC proliferation. Although the 'injury' and 'monoclonal' hypotheses are generally regarded as being mutually exclusive, the available evidence points to possible

roles for both injury and transformation in plaque etiology. The 'synthesis' of the injury and monoclonal hypotheses can be summarized as follows: The primary events in plaque etiology involve D N A damage and its fixation. Elaboration of this damage in a clinically or experimentally significant way may require injury to the arterial wall. The argument in support of this view is analogous to that advanced for a multi-' hit' process in tumorigenesis. A similar argument was presented by Trosko and Chang who proposed that somatic cell mutations were involved not only in the etiology of cancer but in that of arteriosclerosis and diabetes, as well (Trosko and Chang, 1980). It is notable that most of the data which support a role for viruses, chemicals and transforming events in plaque etiology were not available when this argument was first presented. According to the 'multi-hit' argument, at least 3 distinct sets of requirements would have to be met in sequence for plaque development to begin. First, two or more independent molecular events must take place, involving damage to critical regions of D N A in one or more cells. Second, the DNA damage either must not be repaired or must be repaired with poor fidelity. Third, the cell(s) with D N A damage must be stimulated to divide. The D N A damage could take one or more of the following forms: an inherited gene defect, spontaneous mutation, or environmental insult. Included among the latter are viral infection and chemical mutagenesis, e.g., via mutagenic components of ingested food or inhaled cigarette smoke. Either of these sets of chemical mutagens might be solubilized in and transported to arterial SMC by serum lipoproteins. The second component of this triad, repair, is something of a 'black box', since there is very little that is known about the capacity of arterial SMC to repair D N A damage. However, indirect evidence suggests that if repair does occur in arterial SMC in vivo, then either it proceeds slowly or does not occur with high fidelity. Benditt and Benditt (1973) examined only aortas and iliac arteries from 3 patients and found an average of 10 plaques/patient. Since none of these plaques was occlusive and other arteries in which plaques often develop (e.g., carotid, coronary, femoral and popliteal) were not sampled, it follows that pa-

159 tients afflicted with severe atherosclerosis may have a dozen or more macroscopic plaques at various sites. If, as this study implies, D N A damage in intimal SMC precedes plaque development, it is reasonable to conclude that such damage was not repaired (well) wherever these plaques arose. Also, in the initiation-promotion studies in cockerels, a single 5-mm segment of thoracic aorta, immediately proximal to the coeliac artery bifurcation, was analyzed, in each of 8 birds (Majesky et al., 1985). 6 of 8 segments had plaques with an average of > 8 plaques/segment. No control group had more than 2 plaques/segment. Only 2 injections of DMBA, one week apart, were administered per cockerel. The 'promoter' used in these studies, methoxamine, is not thought to be genotoxic. Thus, any repair of D N A damage that occurred in intimal SMC was insufficient to interfere with development of a number of plaques. Inability to effectively remove D N A damage, regardless of the reason, would be important only if the third requirement for plaque formation were also met, i.e., the damaged SMC would have to be stimulated to divide. It is here that injury could play a critical role. One of the most popular descriptors of transformed cells is that they have a proliferative advantage over non-transformed cells of the same type. However, the value of a proliferative advantage will be moot if the cells in question are not stimulated to divide. Arterial SMC present special problems in terms of cell proliferation because they exist in a number of phenotypic states (Chamley-Campbell et al., 1979; Campbell and Campbell, 1985). At one extreme are 'contractile' cells, which are highly differentiated, non-dividing SMC of the type normally found in the medial layer of the undisturbed adult artery wall. At the other extreme are 'synthetic' cells, which can be actively dividing and usually display an increase in synthetic organelles (Golgi, free ribosomes, rough endoplasmic reticulum). 'Synthetic' cells are found at sites of injury. There is indirect evidence that SMC directly involved in plaque formation are in the 'synthetic' state. There is a report that 'synthetic' SMC are slightly smaller than are 'contractile' cells from the same tissue (Yokota et al., 1978). In their ultrastructural studies of plaque development in the cockerel, Moss

and Benditt (1970) described plaque SMC as being slightly smaller than SMC in the medial layer of the artery wall. Numerous studies (referenced in Campbell and Campbell, 1985) indicate that the arterial SMC which proliferate in response to injury have characteristics of the 'synthetic' phenotype. As the injury is repaired and the artery wall becomes reendothelialized, the phenotype modulates to the 'contractile' state. Two points about SMC proliferation should be recognized. First, 'synthetic' and 'contractile' refer to phenotypic alterations of cells regardless of their genotype, so both transformed and untransformed SMC would be expected to display these changes. Second, even when SMC are stimulated to divide, in vivo, there are bursts of proliferation followed by long quiescent periods, rather than continuous rounds of cell division (Penn et al., 1981a). Repeated cycles of limited endothelial damage and repair, might each stimulate one or only a few rounds of cell division. If there are SMC with a proliferative advantage (i.e., transformed) at a site where injury is likely to occur, this could ultimately yield a sizable population of SMC, derived from a single transformed cell, within each plaque. The observations and assumptions that follow about SMC transformation are consistent with what is currently known about plaque development. Partially or fully transformed SMC are expected to be distributed randomly throughout the arterial intima. Any exogenous agent that can either transform cells by itself or complete the transformation of cells that are already partially transformed would increase the number of potential plaque 'stem' cells. If circulating levels of the lipoproteins that can solubilize and transport certain mutagens, are kept elevated for an extended period of time, the chances increase of (fully) transforming intimal SMC. The same would be true if high levels of mutagens (e.g., cholesterol metabolites or cigarette smoke components) were maintained in the circulation for extended periods of time. Any permanent decrease in the supply of plaque-stimulating factors in the circulation should decrease the chances of developing clinically or experimentally significant plaques. This would help explain why long-time cigarette smokers can expect to derive some cardiovascular benefit from

160

smoking cessation even if they wait until after their first heart attack to do so (Hermanson et al., 1988). As reviewed earlier in this article, there is abundant evidence that injury to the arterial wall can stimulate SMC proliferation. However, gross injury to large portions of the arterial wall is not readily compatible with the focal nature of arteriosclerotic plaques. In fact, as has been discussed elsewhere, scanning electron microscope studies indicate that relatively persistent gross endothelial injury seems to occur only at later stages of plaque development (Schwartz and Reidy, 1987). If genetically altered SMC are the progenitor cells of plaques, if these cells are distributed randomly throughout the artery wall, as seems reasonable, and if injury plays a role in plaque formation, then the focal nature of plaques points to localized injury as a possible stimulus for the proliferation of the already transformed SMC. This raises the following question: Is there evidence that locally occurring and recurring injuries take place in the artery wall in vivo, at sites of plaque formation, even in the absence of exogenously administered agents? The answer is ' yes'. A number of years ago evidence was presented that hemodynamic stress at specific locations in the artery wall correlated well with sites of plaque localization (Fry, 1968; Caro et al., 1971) thus confirming an observation made some years earlier (Stebhens, 1960). Alterations in blood flow have been found at 'flow dividers' e.g., branching points of arteries, and at points just proximal to those branching points. In the initiation-promotion studies with DMBA and methoxamine the enhanced plaque development was found just proximal to the coeliac artery branch point in the aorta (Majesky et al., 1985). Recent results suggest that small gradients of shear stress, of the type that are thought to occur at flow dividers, can lead to turnover of a limited number of endothelial cells (Davies et al., 1986). However, as yet, there is no evidence that gradients such as these will lead to intimal SMC proliferation, in vivo. At least two points should be noted if endothefial injury is to be included along with transforming events as a necessary component of the early stages of clinically significant plaque development. First, recurring, plaque-relevant injury

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