Recent Advances in Nutritional Sciences

Nutrition and Aberrant DNA

Methylation Patterns in
Atherosclerosis: More than Just
Hyperhomocysteinemia?1,2

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
(79).
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 authors 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 hy-

Silvio Zaina,3 Marie Wickstrom Lindholm,* and Gertrud Lund

Department of Clinical Biochemistry, Rigshospitalet, 2100
Copenhagen, Denmark; *Experimental Cardiovascular Research,
Department of Medicine, Lund University, UMAS, 205 02 Malmo,
Sweden; and Institute of Plant Biology, Department of Plant
Biochemistry, Royal Veterinary and Agricultural College, 1871
Frederiksberg, Denmark

ABSTRACT Methylation is a reversible modification of

DNA participating in epigenetic regulation of gene expression. It is now clear that atherosclerosis is associated with
aberrant DNA methylation patterns in the vascular tissue
and peripheral blood cells, but the origin of this anomaly is
poorly understood. Based on evidence that global DNA
hypomethylation coexists with hyperhomocysteinemia in
advanced human atherosclerosis, it is widely assumed that
altered DNA methylation patterns in atherosclerosis are
mainly secondary to a decrease in factors essential for the
synthesis of S-adenosyl methionine (SAM, the main methyl
group donor in DNA methylation reactions), such as folate
and vitamin B-12, or to homocysteine-induced blocking of
SAM biosynthesis. Nonetheless, recent work expanded this
view by showing that both local DNA hyper- and hypomethylation occur in early atherosclerosis in normohomocysteinemic mice and that atherogenic lipoprotein profiles
promote DNA hypermethylation in cultured human macrophages. These findings suggest that during early atherosclerosis, nutritional factors affect DNA methylation patterns by mechanisms that are likely to be independent of
vitamin or homocysteine levels. These data have the potential to assist in the identification of preventive or therapeutic avenues for cardiovascular disease. J. Nutr. 135: 5 8,
2005.
KEY WORDS: DNA methylation

atherosclerosis

homocysteine

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 posttranslational modifications such as acetylation, methylation,
1
Supported by the Danish Research Council, Danish Heart Foundation
(Hjerteforeningen), Novo Nordisk Foundation, and the Lundbeck Foundation.
2
Manuscript received 15 September 2004.
3
To whom correspondence should be addressed. E-mail: Silvio.Zaina@rh.dk.

4
Abbreviations used: apoE, apolipoprotein E; MTHFR, methylenetetrahydrofolate reductase; SAM, S-adenosyl methionine; SMC, smooth muscle cells.

0022-3166/05 $8.00 2005 American Society for Nutritional Sciences.

ZAINA ET AL.

TABLE 1
Synopsis of studies documenting changes in DNA methylation at the level of the whole genome or individual genes
Sample
Human, rabbit aortae
ApoE-null mice aortae1
Injury-induced rabbit neointima
Human PBMC
Rabbit aortae
Human aortae
ApoE-null mice aortae
ApoE-null mice PBMC
ApoE-null mice PBMC
Human macrophage THP-1 cells

Disease stage or
treatment
Advanced
Advanced
Advanced
Advanced
Advanced
Preatherosclerotic and
advanced
Preatherosclerotic
Advanced
Atherogenic lipoprotein
profile

Genetic element

DNA status

Reference

Whole genome
Whole genome
Whole genome
Whole genome
ec-sod gene
Estrogen receptor gene
Whole genome

Hypomethylated
Hypomethylated
Hypomethylated
Hypomethylated
Hypomethylation at exon
Hypermethylation at promoter
Hypo- and hypermethylation with excess
of the former
Hypo- and hypermethylation
Hypo- and hypermethylation with excess
of the former
Hypermethylation

(13,14)
(14)
(14)
(15)
(13)
(21)
(20)

Whole genome
Whole genome
Whole genome

(20)
(20)
(20)

1 The study compared apoE-null mice fed a high-cholesterol or a normal diet. PBMC, peripheral blood mononuclear cells.

pomethylation. 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 (1315). 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).
The above data are consistent with the view that hyperhomocysteinemia-induced DNA hypomethylation is an important driving force in atherogenesis, implying that folate administration is a sensible therapeutic avenue to counteract
DNA hypomethylation, if not hyperhomocysteinemia per se
(18,19). However, recently published work expanded this
model by suggesting that aberrant DNA methylation patterns
in atherosclerosis are likely to have more complex causes than
simply hyperhomocysteinemia (20). The authors analyzed hyperlipidemic, atherosclerosis-prone apolipoprotein E (apoE)null mutant and control wild-type mice at the early stages of
the disease (at 4 wk of age, when hyperlipidemia is already
established but no histological sign of aortic lesions is detectable) and in the presence of relatively advanced fibrocellular
lesions (at the age of 6 mo). A methylation-sensitive DNA
fingerprinting technique was used, allowing measurement of
the relative contribution of hyper- and hypomethylation to
the net genome-wide DNA methylation profile. The study
demonstrated both DNA hyper- and hypomethylation, with a
slight but significant excess of the latter, in aortae of 4-wk old
apoE-null mice. The same pattern, but with a larger excess of
hypomethylated sites, was present in the aortae of affected
6-mo-old mutant mice. The peripheral blood cell DNA of
apoE-null mice showed aberrant DNA hypo- and hypermethylation in both age groups, but significant excess hypomethylation was observed only in 6-mo-old mice. Furthermore,

DNA methylation patterns were within the expected tissueto-tissue variation in all control tissues examined. These results show that the initial stages of atherogenesis are associated
with a rearrangement of genomic DNA methylation patterns
including both hyper- and hypomethylation, rather than a
unidirectional change toward global hypomethylation. The
authors went further, elucidating mechanisms underlying aberrant DNA methylation patterns in early atherosclerosis, by
stimulating the human macrophage THP-1 cell line with
lipoprotein mixes reproducing lipoprotein ratios of ApoE-null
mice (high VLDLLDL to HDL ratio) or wild type mice (low
VLDLLDL to HDL ratio). A relatively short stimulation
with this atherogenic lipid profile induced a significant DNA
hypermethylation compared with untreated cells or cells incubated with a lipoprotein mix reproducing the VLDLLDL
to HDL ratio of wild-type control mice (20).
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

DNA METHYLATION IN ATHEROSCLEROSIS

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 homocysteinefolate 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

FIGURE 1 Schematic representation of atherosclerosis progression from a normal blood vessel (left) to the development of a fatty
streak and an elevated fibrocellular lesion (far right). The DNA methylation status of vascular cells is indicated for stages of the disease at
which it was studied.

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).
ACKNOWLEDGMENT
We thank Angelo Santino for discussions on nutritional factors
and disease.

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