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Department of Clinical Biochemistry, Rigshospitalet, 2100 Copenhagen, Denmark;
* Experimental Cardiovascular Research, Department of Medicine, Lund University, UMAS, 205 02 Malmö, Sweden; and
Institute of Plant Biology, Department of Plant Biochemistry, Royal Veterinary and Agricultural College, 1871 Frederiksberg, Denmark
3To whom correspondence should be addressed. E-mail: Silvio.Zaina{at}rh.dk.
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ABSTRACT |
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 TOP ABSTRACT LITERATURE CITED |
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KEY WORDS: • DNA methylation • homocysteine • atherosclerosis
Gene expression can be regulated at a genetic level by changes in DNA sequence and at an epigenetic level by mechanisms that are mutation independent. Epigenetic control of gene expression involves modifications of chromatin such as DNA methylation and several types of histone post-translational modifications such as acetylation, methylation, phosphorylation, and ubiquitination. Generally, conformationally relaxed chromatin (euchromatin) is a hallmark of potentially active genic regions and is associated with hypomethylated DNA and acetylated histones, whereas compact chromatin (heterochromatin) DNA is transcriptionally silent, hypermethylated, and bound to nonacetylated histones (1). Epigenetic mechanisms of gene regulation are crucial determinants of cellular behavior. For example, DNA methylation patterns are more or less stably inherited upon mitosis in adult cells, but deviations from normal DNA methylation patterns are associated with diseases such as cancer and atherosclerosis (2–4). The epigenetics of disease has been investigated intensely in recent years, particularly in cancer, because aberrant DNA methylation profiles are associated with advanced stages of disease and may represent markers of predisposition (5). Generally, tumor cells show two concomitant opposing changes in DNA methylation. As a whole, the genome is hypomethylated but selected CpG island-rich 5' regions of genes become densely hypermethylated (6). CpG island hypermethylation has been linked to tumor suppressor gene silencing, whereas DNA hypomethylation may cause oncogenic mutations by promoting chromosomal instability. Furthermore, because DNA methylation and histone acetylation are in principle reversible modifications, they have recently emerged as promising molecular targets in cancer therapy (7–9).
In contrast to cancer research, the involvement of epigenetic mechanisms in the context of atherosclerosis has been the subject of relatively little experimental investigation. Although the effects of hypercholesterolemia on chromatin structure had been documented by the early 1980s (10), the hypothesis that aberrant DNA methylation patterns drive atherogenesis was first formulated by P. E. Newman in the late 1990s (11). The author’s reasoning was that homocysteine effectively inhibits the folate- and vitamin B-12–dependent cycle converting methionine to S-adenosyl methionine (SAM),4 the main methyl group donor in DNA methylation reactions, and that hyperhomocysteinemia is an independent atherosclerosis risk factor (12). Therefore, abnormally low circulating levels of vitamin B-12 and folic acid were predicted to cause an accumulation of homocysteine, a parallel reduction in SAM, and consequently an insufficient DNA methylation capacity. Hypomethylated DNA would then be prone to mutations or aberrant gene expression patterns leading to the transition from a normal cellular phenotype to one that favors the development of vascular fibrocellular lesions (11). Subsequent experimental work consistently confirmed at least part of that predicted scenario (Table 1). In these studies, DNA methylation was assessed by methods that can reveal the net DNA methylation status (e.g., direct quantitation of methylcytosine residues or radioactive labeling of ends generated by methylation-sensitive restriction enzymes) but do not allow measurement of the extent of both de novo hyper- and hypomethylation. Global DNA hypomethylation at levels similar to those found in some cancer types was demonstrated in human, rabbit, and murine advanced atherosclerosis, in both vascular tissue and peripheral blood cells (13–15). In at least one study, circulating homocysteine levels were significantly correlated with the extent of DNA methyl-group loss in advanced atherosclerosis (15). Another study demonstrated DNA hypomethylation and hyperhomocysteinemia in peripheral blood cells from patients affected by end-stage renal disease, a condition associated with a high risk of cardiovascular disease (16). Furthermore, the latter work showed that folate dietary supplement restores normal DNA methylation levels (16). These results in humans were mirrored by the phenotype of mutant mice lacking methylenetetrahydrofolate reductase (MTHFR), an essential enzyme in the pathways regenerating SAM. MTHFR-null mutants show concomitant hyperhomocysteinemia, DNA hypomethylation, and aortic lipid deposits, possibly resembling fatty streaks that are observed in early atherosclerosis (17).
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The implications of the latter study may be manyfold. First, it suggests that DNA hypermethylation is one of the early molecular "hits" in atherogenesis. Although only a very limited number of individual genes have been screened for DNA methylation aberrations in atherosclerosis to date, these findings are consistent with the observation that DNA hypermethylation at the human estrogen receptor gene is associated with atherosclerosis and, noticeably, with aging, a cardiovascular disease-predisposing factor (21). Second, lipids or lipoprotein components are likely candidate factors behind such changes. We anticipate that further studies will uncover additional atherogenic dietary factors that can modify DNA methylation and chromatin structure, including lipids, lipoprotein constituents, as well as other yet unidentified molecules, thus opening new perspectives to the prevention of atherosclerosis by the use of commonly available dietary constituents (22). Third, if these findings have any relevance to human atherosclerosis, it can be concluded that efforts to counteract DNA hypomethylation by folate supplementation or other strategies aimed at compensating the effects of hyperhomocysteinemia should be considered with caution because they may favor SAM overproduction and DNA hypermethylation at early stages of atherosclerosis, with potential proatherogenic consequences. Interestingly, a similar dilemma poses itself in cancer therapy because ongoing experimentations with DNA hypomethylation-promoting drugs, although based on sound molecular evidence, contrast with the oncogenic effects of loss of DNA methyltransferase activity in animal models (23,24). Important clues to this issue are bound to come from extensive clinical trials assessing the effect of vitamin supplements on the development of cardiovascular disease (25). Published studies in apoE-null mice suggest that vitamin supplements confer a homocysteine-independent protection, although the normocysteinemic status of this animal model complicates the extrapolation of the data to humans (26). Fourth, the data provide insights into causal relations between hyperhomocysteinemia and DNA hypomethylation. The latter could not be a consequence of homocysteine imbalance because apoE-null mice are not hyperhomocysteinemic (27). One alternative possibility is that DNA hypomethylation occurs passively as a consequence of defective maintenance of DNA methylation patterns in proliferating cells (28). Although this cannot be excluded in the case of intensely proliferating fibrocellular lesions, it is an unsatisfactory explanation for the preatherosclerotic aortae of apoE-null mice, in which the authors of the present review failed to detect any abnormal cell proliferation. Rather, the data discussed to date suggest that DNA hypomethylation in early atherosclerosis is mechanistically unrelated to the homocysteine-folate system and, in close similarity to the concomitant DNA hypermethylation, is the result of chromatin-modifying dietary or endogenous factors.
In light of the above data, what is the relevance of DNA hypomethylation in advanced atherosclerosis? We propose that the bulk of DNA hypomethylation detected in aortic advanced fibrocellular plaques is a passive phenomenon due to loss of methyl groups in highly proliferating smooth muscle cells (SMC), similar to the observed hypomethylated status of injury-induced neointimal tissue DNA (14). Furthermore, DNA hypomethylation in peripheral blood cells may reflect hyperproliferation of cell types involved in immune or inflammatory responses during atherosclerosis (29,30). This does not exclude the possibility that homocysteine may cause DNA hypomethylation indirectly through promoting SMC proliferation or activation of immune responses, thus explaining the observed correlations between homocysteine levels and the degree of DNA hypomethylation (31,32). Accordingly, Yi et al. (33) did not detect any significant correlation between plasma homocysteine and plasma SAM levels, or between the latter and the extent of peripheral blood cell DNA hypomethylation, thus giving weight to the view that homocysteine excess does not necessarily result in limiting methyl group donor levels.
In conclusion, recent findings suggest that early atherogenesis is associated with rearrangements of DNA methylation patterns involving both hypo- and hypermethylation and that nutritional factors are likely to play a central role in these alterations (Fig. 1). In advanced phases of the disease, cell hyperproliferation results in a predominant DNA hypomethylation. Furthermore, the molecular mechanisms underlying the significance of hyperhomocysteinemia as an independent risk factor for cardiovascular disease are probably not explained by a direct effect on DNA methylation, but rather are to be ascribed to at least some of the many known cellular functions of homocysteine (31).
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Manuscript received 15 September 2004.
4 Abbreviations used: apoE, apolipoprotein E; MTHFR, methylenetetrahydrofolate reductase; SAM, S-adenosyl methionine; SMC, smooth muscle cells.
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LITERATURE CITED |
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TOPABSTRACTLITERATURE CITED |
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