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RAMP* Addition-3: "Atherogenic Mutations," March 9, 2000

The Unified Model of Atherogenesis and Acute IHD Death:
Additional Evidence Related to Monoclonality, and to Acquired Mutations.

by John W. Gofman, M.D., Ph.D., and Egan O'Connor, Editor

* RAMP is a short name for the book, Radiation from Medical Procedures in the Pathogenesis of Cancer and Ischemic Heart Disease, by John W. Gofman. Nov. 1999.

        *   Part 1.   Purpose of This Communication, and List of Papers
        *   Part 2.   The Unified Model: Some Excerpts from Chapter 45
        *   Part 3.   MonoClonality Observed in Plaque, Intimal Thickening, Media
        *   Part 4.   Numerical and Structural Chromosome Aberrations in Plaque
        *   Part 5.   Specific Satellite-Mutations and LOH, Plaque vs. Non-Plaque
        *   Part 6.   A Receptor-Gene Affecting Proliferative & Apoptotic Signals

 *   Part 1.  Purpose of This Communication, and List of Papers

          After RAMP went to press, we found additional papers (additional to the papers mentioned in Chapters 44, 45, 46) which have some relevance to our Unified Model of Atherogenesis and Acute IHD Death. The key premise of the Unified Model is that acquired mutations have a causal role in the genesis and progression of atherosclerosis.

          Here, the purpose of our RAMP Addition-3 is to indicate how the additional papers relate to the Unified Model, not to describe all aspects of the papers. The papers are listed alphabetically by principal author. We look forward eagerly to seeing future work by these and other investigators on any aspect of the key premise.

 *   Part 2.  The Unified Model: Some Excerpts from Chapter 45

          The Unified Model (which is proposed in Chapter 45 of RAMP) unites the observed relationships between plasma lipoproteins and IHD mortality, with the observed relationships between accumulated xray exposure and IHD mortality.

          As we see it, all our arterial beds (coronary, cerebral, and other) are in a lifelong process of clearing plasma lipoproteins out of the intimal layer after their influx from the lumen (Chapter 44, Part 5). In the intima, plasma lipoproteins are "foreign bodies," since normal metabolism would not use them in that location. We think that massive quantities of lipoproteins are processed in a lifetime in innumerable arterial walls. And obviously, most of this is handled successfully, since we rarely (if ever) see massive atherosclerosis all over the body ... (RAMP p.338).

          When a failure occurs in the lifelong "Foreign-Body Wars," it is localized. Lipids start serious accumulation at a particular site in the intima --- not everywhere. Only particular patches become atherosclerotic plaques, surrounded by grossly normal tissue. Why does a plaque develop where it does? Is this totally random? We do not think so ... (RAMP p.339).

  2a.   A "Whole New Ballgame":
The Mutated Clonal and Dysfunctional SMC

          We consider that a "whole new ballgame" begins in a coronary artery wherever a mutagen produces a clone of dysfunctional smooth muscle cells ... (RAMP p.339). Smooth muscle cells (SMCs) have essential jobs to perform in the "Foreign-Body Wars" of the intima (RAMP p.340). As part of the inflammatory response, they synthesize and secrete collagen, elastic tissue, and proteoglycans, to create the connective tissue (extra-cellular matrix) of a plaque. Also, they can produce a growth factor which induces migration and proliferation of more SMCs, as needed. Perhaps of real importance, they produce enzymes which can help protect a plaque's connective tissue, and they can also do the opposite. SMCs can produce enzymes which cause degradation of a plaque's connective tissue.

          During a person's lifetime, the arterial SMCs accumulate exposure to mutagens (including ionizing radiation from natural sources and especially from medical xrays). The frequency rises of cells which have acquired one or more mutations. Of course, not all mutations are consequential. The specific consequences (if any) depend upon which segment of the genome is altered. A person's population of mutated, arterial SMCs will include some or all of the following categories (RAMP p.340):

           *   The Mutated Non-Clonal SMC. For this type of mutated cell (non-clonal), the accumulated mutations do not confer any proliferative advantage. Thus, any acquired dysfunctions in a single cell are innocuous, because the dysfunctions are not magnified by a burgeoning clone of descendant cells with the same dysfunctions.

           *   The Mutated Clonal but Otherwise Competent SMC. The accumulated mutations give this type of cell a proliferative advantage, so that its clone of descendants gradually replaces a small patch of non-clonal tissue. But because the mutated cells are still competent at doing their jobs, the clone is innocuous.

           *   The Mutated Clonal and Dysfunctional SMC. The accumulated mutations give such cells a proliferative advantage and cause them to be incompetent in some degree at performing one or more of their jobs. And because of their proliferative advantage, these dysfunctional cells gradually replace competent cells, at a localized patch of artery. These are the mutated cells which are central to the Unified Model.

  2b.  Multiple Paths to Dysfunction

          Within an arterial SMC, there are innumerable genes involved in completing all the wartime jobs assigned to the cell --- and innumerable types of mutation capable of rendering their products dysfunctional. A disabling mutation, of even one of these crucial genes in the progenitor of a clone (mini-tumor), can cause the clone to perform inadequately. Ionizing radiation inflicts mutations at random locations of a cell's genome, and there is no part of the genome which is inaccessible to radiation-induced mutation. Consequently, it is highly improbable that any two dysfunctional clones have identical disabling mutations (RAMP p.341).

          The Unified Model proposes that dysfunctional clones explain why a plaque grows at a particular site, explain why particular plaques have weak fibrous caps which rupture and kill the host, and explain why a spectacular dose-response is observed between exposure to medical xrays and age-adjusted mortality rates, by Census Divisions.

 *   Part 3.  MonoClonality Observed in Plaque, Intimal Thickening, Media

          An acquired mutation, confined to one or a few cells, is quite innocuous (Part 2a, above). Therefore, if acquired mutations have an important causal role in the genesis and progression of atherosclerosis, plaques should have at least one substantial population of clonal, dysfunctionally mutated cells in their history. Many technical difficulties remain, in establishing whether they do or do not.

  3a.  The Work of Earl Benditt and Others, 1973-1990

          In 1973, at the University of Washington Department of Pathology, Earl Benditt formulated a key question about atheromas: "What is the nature of the cellular proliferation involved in the `new formation,' the atherosclerotic plaque? ... Is it of multicellular or of monoclonal origin? The importance of this distinction lies in the fact that many neoplasms have been found to be of monoclonal origin. [A clone is a group of genetically identical cells descended from the same progenitor cell.] On the other hand, ordinary cell proliferations seen in embryogenesis, maintenance, and repair seem to be multicellular in origin."

          Benditt's method of seeking the answer to his question was adopted by Pearson and some others (details in RAMP, Chapter 44, Part 8). The answers from these investigators were quite variable, in the percent of the sampled atherosclerotic lesions judged to be monoclonal vs. the percent of samples from normal arterial tissue judged to be monoclonal. The variation may be due to the variable proportions of smooth muscle cells, endothelial cells, macrophages, and T-cells in the atheromas examined (Schwartz 1995, p.S131; Murry 1997, p.698).

          In other words, the particular method had its limitations. Moreover, the method cannot address whether a plaque's monoclonal cell-population is dysfunctionally mutated, or not. And furthermore, the method cannot rule out a multicellular origin even when plaque-tissue is judged to be monoclonal (Part 3b, below).

          We are sad to learn that Earl Benditt died in May 1996. We are very pleased that work related to his "monoclonal hypothesis" continues at the University of Washington --- where Schwartz, Murry, and Chung produced the papers discussed in Part 3b.

  3b.   Schwartz, Murry, Chung:
Monoclonal vs. Multicellular Origins

          The University of Washington group uses X-linked markers to judge whether a tissue-sample is monoclonal or not. The method is derived from the Lyon Hypothesis that early in human gestation, female embryos randomly and permanently inactivate either the paternal or maternal X-chromosome in each cell. If a female is heterozygous for a particular X-linked marker, the heterozygosity makes it possible to establish which cells are descended from a progenitor cell in which the paternal X-chromosome was permanently inactivated, and to establish which cells are descended from a progenitor cell in which the maternal X-chromosome was permanently inactivated.

          In their 1997 paper, Murry et al (1997, p.701) report as follows: "The principal findings of this study are 1) the majority of atherosclerotic plaques have a monoclonal population of cells, 2) the monoclonal population is composed of smooth muscle cells, 3) monoclonality is present both in aortic and coronary plaques, 4) although most samples of medial smooth muscle are polyclonal, a significant minority also show monoclonal characteristics, and 5) smooth muscle cells from diffuse intimal thickening also can show monoclonal characteristics." And in their abstract, Murry et al (1997, p.697) state:

          "The finding that normal arteries may have large X-inactivation patches raises the possibility that plaque monoclonality may arise by expanding a pre-existing clone of cells rather than generating a new clone by mutation or selection." In an earlier paper, Schwartz et al (1995, p.S133) also state: "If clonal expansion takes place during development and growth of the media or intima, prior to plaque formation, then lesions arising within a pre-existing clone would, by necessity, be clonal as well." Thus, this method by itself will not resolve Benditt's question (Part 3a): Do the SMCs of a plaque arise from a single progenitor cell, or from multiple cells?

          Using X-inactivation analysis, the Chung 1998 paper explores the approximate size of monoclonal patches in aortic samples of normal media and diffuse intimal thickening (DIT). Chung et al (1998, p.914) report that "patch size is surprisingly large" (often up to 4 mm in length) and: "These findings suggest that clonal expansion of smooth muscle cells occurs as part of normal aortic development." They also report, with caveats, (Chung 1997, p.920) that samples of normal media and DIT from persons ages 33 to 38 exhibit a significantly lower frequency of monoclonality than the samples from patients older than age 40.

  3c.  How the Findings Relate to Our Unified Model

          How do these findings relate to our Unified Model? Schwartz et al (1995, p.S136) write in their summary, "Any competent hypothesis of atherosclerosis must account for [the observation of] monoclonality. As noted above, it is possible that monoclonality of the intima is a normal part of development of the intima. This is a critical hypothesis, since the alternatives, i.e., existence of a proliferative subset or benign transformation of plaque smooth muscle cells, both imply unique properties of the plaque smooth muscle cell that would become prime targets in understanding the ontogeny of this most important vascular disease."

          The discovery, by X-inactivation analysis, that normal media and areas of DIT exhibit monoclonal patches, is fully consistent with the Unified Model. As we pointed out in Part 3a, X-inactivation analysis does not address the issue of clonal, dysfunctionally mutated smooth muscle cells at all. And the Seattle group also recognizes this. In their discussion, Chung et al (1997, p.922) point out:

          "It should be stressed, however, that this [the finding of large monoclonal patches in normal media and DIT] is not evidence against either mutation or clonal selection in atherogenesis." And they explicitly point out "the possibility that, irrespective of the underlying clonal architecture of the vessel wall, plaques may arise by a rare or mutagenic event."

 *   Part 4.  Numerical and Structural Chromosome Aberrations in Plaque

          Vanni 1990 and Casalone 1991 both use banding techniques to examine chromosomes, in cells cultured from atherosclerotic plaques.

  4a.  Vanni et al 1990

          Roberta Vanni and co-workers made "short-term cultures of SMC" obtained from three carotid plaques removed from different patients. They do not mention isolation of the SMCs from other types of cells in the plaques. "Only one case yielded a sufficient number of metaphases suitable for detailed analysis by G-banding: five of 11 metaphases had a 47,XX, +7 karyotype; of the remaining metaphases, three had a normal karyotype and three had random chromosome losses. The constitutional chromosome complement of the patient was 46,XX. Trisomy-7 was characteristic of the SMC of the atherosclerotic plaque of the patient" (Vanni 1990, p.273). Trisomy-7 refers to the presence of three copies of Chromosome-7 instead of two copies. Vanni et al note that Chromosome-7 includes the gene for the A-chain of Platelet-Derived Growth Factor (PDGF), the proto-oncogenes ra-1, met, A-raf-2, and receptor genes for "growth rate controlling factor and epidermal growth factor."

          The finding, that only 3 out of 11 metaphases had the normal number of chromosomes, is very unusual --- except for cancer cells. Vanni et al comment, "Although our cytogenetic evidence must be confirmed by further investigations, the finding of a chromosome change in SMC of an atherosclerotic plaque would support the hypothesis of the plaque as a benign tumor and trisomy-7 in particular as a common denominator of different neoproliferative processes." We are unaware that Vanni et al have published "further investigation."

          We note that Trisomy-7 was observed in 45% of the metaphases. If there had been more than 11 metaphases, the percentage might have been either higher or lower. On the basis of such very limited observations, not much should be said, especially when we do not know what percentage of the cultured cells were really SMCs.

  4b.  Casalone et al 1991

          Casalone et al (1991, p.139) performed cytogenetic analysis (by chromosome QFQ banding) "on primary cell cultures obtained from human fibrous plaques which had been separated from the uninvolved arterial wall." The donors were 16 male and 2 female patients. Using a variety of antibodies, on cells from the same cultures used for cytogenetic analysis, they determined that the cells were predominantly SMCs (Casalone 1991, p.142).

          Casalone et al report on the observed frequency of aneuploidy (wrong number of chromosomes), the type of aneuploidy, and detectable deletions and translocations in the plaque samples. They report (Casalone 1991, pp.140-141) that "All but one of the control cultures failed to grow at the same rate as the plaque cultures, so that chromosome analysis was not usually possible. The control of case 3 was an exception, giving 35 metaphases at first passage, all 46,XY." They comment (p.142): "The conspicuous lack of success with control cultures from the normal intima indicates an enhanced proliferative capacity of the cells from the plaques."

          It is the view of Casalone et al (1991, p.142) that "The presence in five cases of two or more separated clones, and the predominance of normal metaphases in all cases, cast doubt on the monoclonal theory."

          We disagree. Banding techniques are helpful in detecting aneuploidy and some very gross structural chromosomal aberrations. There is no reason at all to assume that atherogenesis must be tied to the particular mutations detectable by banding. Cells with the proper number of chromosomes can belong to a dysfunctionally mutated clone of SMCs, whose shared mutations would not be detectable by banding techniques (e.g., Parts 5 and 6 below).

 *   Part 5.  Specific Satellite-Mutations and LOH, Plaque vs. Non-Plaque

          The frequency of specific genetic mutations, in DNA extracted from atherosclerotic plaques vs. DNA extracted from adjacent normal tissue, was reported in a set of three papers: Kiaris 1996, Spandidos 1996, and Hatzistamou 1996. The papers focus on mutations located in selected satellite-regions of the DNA. Satellites are DNA segments characterized by short, repetitive sequences of base-pairs.

          In these studies, the extracted DNA was not solely (perhaps not even predominantly) from smooth muscle cells. All thirty plaque specimens were obtained from myocardial infarction autopsy cases (17 males, 13 females), but the specimens were neither the "culprit" plaques nor from the coronary arteries. Twenty specimens were taken from the aorta and ten from the basilar cerebral artery. Kiaris et al state (p.48): "The plaques were selected to be not calcified, and measured around 0.5 cm in diameter. Histologically, all specimens contained foam cells as the main component. Calcified specimens and the specimens with significant fibrous components were excluded from the study." The same specimens were apparently used in Spandidos 1996 (p.137) and Hatzistamou 1996 (p.187).

  5a.  Kiaris 1996: One Site Assessed for Mutations

          Kiaris et al (1996, p.47) state that "The aim of the present study was to investigate whether instability, at a minisatellite region located downstream of the H-ras proto-oncogene possessing enhancer activity, is a detectable phenomenon in atherosclerotic plaques. Thirty specimens were analyzed by polymerase chain reaction (PCR) in order to reveal alterations of the repetition number and by restriction fragment length polymorphism (RFLP) with BstNI restriction endonuclease for the detection of point mutations within the 28 base-pair core repetitive element. No point mutations were found among the 30 cases tested; however, alterations of the repetition number of the core were detected in 5 (17%) cases. Our results suggest that instability at the H-ras minisatellite may be associated with development of the disease."

          And how did the plaque specimens compare with the controls? Kiaris et al (1996, p.49) explain that "Five (17%) among 30 cases exhibited the generation of novel VNTR alleles in the atherosclerotic tissue which were absent from the control normal tissue of the same patient and thus interpreted as positive for instability ..."

          At the end of their paper, Kiaris et al (1996, p.50) discuss how their finding might relate to a possibly monoclonal origin of atherosclerotic plaques: "The accumulation of somatic mutations during the development of the disease, due to an increased mutational rate [genomic instability], results in the generation of a heterogeneous population of cells comprising the atherosclerotic tissue." Please see our own comment in Part 5d.

  5b.   Spandidos 1996:
Seven Additional Sites Assessed for Mutations

          Spandidos et al (1996, p.137) state: "In the present study, we investigated whether an elevated mutational rate is detectable in human atheromatous plaques. Thirty specimens were assessed for microsatellite instability (MI) by 7 microsatellite markers, and MI in at least one marker was apparent in 6 (20%) cases. Our data suggest that decreased fidelity in DNA replication and repair may be associated with the development of the disease." Describing the method, the authors assure (p.138):

          "MI was scored by comparing the electrophoretic pattern of the microsatellite markers amplified from the paired DNA preparations that corresponded to the atherosclerotic plaque with adjacent normal tissue. The analysis in the MI positive cases was repeated at least twice and the results were highly reproducible." Blinding of the comparisons is not mentioned. In their discussion, Spandidos et al (1996, p.138) comment, appropriately:

          "Examining the specimens with additional markers might increase our figures. The precise significance of these findings remains obscure because the information as regards the genetic basis of the disease is limited. However, we may postulate that the relatively high mutational rate of the atherosclerotic lesions, as reflected in the instability of the microsatellite sequences, indicates a destabilization of the genome which may affect other genes resulting in the disregulation of the cells harbouring these mutations ... It would be of interest to screen DNA repair genes for mutations in atherosclerotic plaques exhibiting MI and investigate whether these mutations are also present in the germline of the patients ..."

  5c.   Hatzistamou 1996:
LOH: 18 Microsatellites Screened for Deletions

          Hatzistamou et al (1996, p.186) state: "The aim of the present investigation was to perform an allelotype analysis in 30 atherosclerotic lesions in order to reveal any deletions involved in the development of the disease. Eighteen chromosomal arms were tested by one microsatellite marker located on each arm, and allelic imbalance in at least one marker was observed in 7 (23%) of the cases. Futhermore, the analysis revealed the presence of microsatellite instability (MI) in 10 (33%) cases, suggesting that an increase in the mutation rate may be involved in the formation of the plaque. These results highlight the mutation concept for atherosclerosis and suggest that Loss of Heterozygosity (LOH) and MI may be involved in the development of the disease."

          Also, the authors report (p.187): "LOH was scored when a significant reduction in the intensity of one allele in the heterozygous specimens was observed in the plaque DNA. MI was scored when altered mobility or the generation of novel microsatellite alleles was observed in the plaque DNA. The analysis in the MI and LOH positive cases was repeated at least twice and the results were highly reproducible."

          In their discussion, Hatzistamou et al (1996, p.187) comment: "Originally, LOH was reported in the development of human tumours and represents a manifestation of the recessive behavior of the onco-suppressor genes. The present findings suggest that the deletion of onco-suppressor genes is also detectable in atherosclerotic plaques and is probably associated with the disease. Although the incidence of LOH reported in the present investigation is not very high, this is the first report to our knowledge which demonstrates LOH in atherosclerotic lesions. The use of more markers in the regions exhibiting LOH, may increase the figures and provide evidence for the precise location of the genes involved. This may also reveal the inactivation of specific `atherogenesis suppressor genes.' However, LOH in these chromosomal arms has already been reported in a variety of human tumours, providing evidence for the pleiotropic effects of the onco-suppressor genes in the human diseases."

  5d.  How All Three Papers Relate to Our Unified Model

          All three papers establish that, when DNA extracted from atherosclerotic tissue is compared with DNA from normal adjacent tissue, from 17% to 33% of the atherosclerotic specimens show a higher frequency of mutation at the examined DNA segments than do the corresponding non-plaque specimens. The DNA segments chosen for examination were segments known to be associated with one type of genomic instability (MI) and with certain cancers.

          It is natural to investigate such sites first, and we are excited that the exploration has started. We emphatically agree with Spandidos et al (1996) that, in further exploration, DNA sites for repair-genes deserve special attention. Our Unified Model proposes that innumerable other sites exist where acquired mutations in smooth muscle cells could produce a clone of dysfunctional cells, with the dysfunctions causing the genesis and progression of Ischemic Heart Disease.

          The set of three papers, above, cannot address the issue of monoclonality, as it relates to the Unified Model. And they could not do so, even if the examined DNA extracts had been exclusively from smooth muscle cells. The reason: These papers examine a few pre-selected segments of DNA --- eighteen in Hatzistamou 1996. If these selections do not happen, by chance, to include the particular mutated segments of a particular plaque, there is no test of the Unified Model.

          Moreover, the Unified Model predicts that the mutations, responsible for atherogenesis and acute IHD deaths, will differ from one plaque to another (RAMP, Chapters 45 and 46). The day will arrive, when enough will be known to say that particular genes and other particular segments of the genome cannot possibly have any causal role in the genesis and progression of atherosclerotic plaque. Meanwhile, very few segments of the genome can be ruled out.

 *   Part 6.  A Receptor-Gene Affecting Proliferative & Apoptotic Signals

          McCaffrey et al (1997, p.2182) report that in their study, "DNA from human atherosclerotic and restenotic lesions was used to test the hypothesis that microsatellite instability leads to specific loss of the Type II receptor for TGF-B1 (TBR-II), causing acquired resistance to TGF-BI." TGF is Transforming Growth Factor.

          They found tiny deletions (1 and 2 base-pairs) in the TBR-II gene, mostly (but not exclusively) "in the replication error-prone A-10 microsatellite region ... The mutations could be identified within specific patches of the lesion, while the surrounding tissue, or unaffected arteries, exhibited the wild-type [normal] genotype. This microsatellite deletion causes frameshift loss of receptor function, and thus, resistance to the anti-proliferative and apoptotic effects of TGF-B1. We propose that microsatellite instability in TBR-II disables growth inhibitory pathways, allowing monoclonal selection of a disease-prone cell type within some vascular lesions."

          McCaffry et al (1997, p.2183) note in their Introduction that "Because TGF-B1 can be a potent, autocrine growth inhibitor for a variety of cells, including smooth muscle cells, an acquired mutation in the Type II receptor would confer a relative growth advantage on the cells bearing the mutation, allowing monoclonal, or oligoclonal, selection in a given tissue."

          In the Discussion section about their findings, McCaffrey et al (1997, p.2187) comment: "This analysis probably underestimates the true frequency of the TGF-B receptor mutations for several reasons ..." which they state. Then they add, "By extension, it seems likely that other anti-proliferative or apoptotic genes could acquire mutations, thereby conferring resistance and monoclonal expansion."

          We certainly concur. The concept, that there are numerous mutational paths leading to clonal and dysfunctionally mutated SMCs, is a central concept in the Unified Model of Atherogenesis and Acute IHD Events.

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