Download DNA Methylation, Smooth Muscle Cells, and Atherogenesis

ATVB in Focus
Smooth Muscle Cells
Series Editor:
Giulio Gabbiani
Previous Brief Reviews in this Series:
• Hillebrands J-L, Klatter FA, Rozing J. Origin of vascular smooth muscle cells and the role of circulating stem cells
in transplant arteriosclerosis. 2003;23:380 –387.
• Iso T, Hamamori Y, Kedes L. Notch signaling in vascular development. 2003;23:543–553.
• Kumar MS, Owens GK. Combinatorial control of smooth muscle-specific gene expression. 2003;23:737–747.
• Schaper W, Scholz D. Factors regulating arteriogenesis. 2003;23:1143–1151.
• Hao H, Gabbiani G, Bochaton-Piallat M-L. Arterial smooth muscle cell heterogeneity: Implications for atherosclerosis and restenosis development 2003;23:1510 –1520.
Downloaded from http://atvb.ahajournals.org/ by guest on April 28, 2017
DNA Methylation, Smooth Muscle Cells, and Atherogenesis
Mikko O. Hiltunen, Seppo Ylä-Herttuala
Abstract—DNA methylation is a form of epigenetic modification of the genome that can regulate gene expression.
Hypermethylation of CpG islands in the promoter areas leads to decreased gene expression, whereas promoters of
actively transcribed genes remain nonmethylated. Because of cellular proliferation and monoclonality of at least some
of the lesion cells, atherosclerotic lesions have been compared with benign vascular tumors.1,2 However, although
genetic and epigenetic background favors neoplastic transformation, atherosclerotic plaques never develop to malignant
tumors. Among cancer cells, common features are genome-wide hypomethylation, which correlates with transformation
and tumor progression. Recent studies have shown that DNA methylation changes occur also during atherogenesis and
may contribute to the lesion development. (Arterioscler Thromb Vasc Biol. 2003;23:1750-1753.)
Key Words: atherogenesis 䡲 DNA methylation 䡲 5-methylcytosine 䡲 epigenetic gene regulation 䡲 gene expression
N
studies, phenotypic transition is initiated by various factors
or injuries, eg, balloon denudation. The proliferative state
of the SMC requires profound changes in gene expression
and protein synthesis. We have shown that only a few
rounds of replication of contractile medial SMCs are
required to develop a significant hypomethylation of the
SMC genome.6 Consequences of this phenomenon on gene
expression are discussed later in this review. It has been
shown that some intimal synthetic SMCs are of monoclonal origin,1 which implicates that some clones of medial
SMCs have developed at least a transient growth advantage. The situation is somewhat similar to carcinogenesis,
where the tumor has gained a growth advantage.
Injury to the arterial wall, such as angioplasty, induces
endothelial dysfunction and stimulates SMC migration and
proliferation. The highest proliferative activity of SMCs
umerous factors in plasma (eg, lipoproteins, growth
factors, and monocytes) and arterial wall (eg, smooth
muscle cells [SMCs], matrix, and endothelium) contribute to
atherogenesis. The pathogenesis of atherosclerosis involves
mainly changes in the expression and function of genes rather
than mutations. Atherosclerosis begins with eccentric thickening of the intima, leading to complex lesions over several
decades.3 Intimal thickening forms in response to almost any
imaginable injury, including circulating factors and mechanical injury. Neointima is composed of SMCs, mesenchymal
intimal cells, and inflammatory cells.4
Before SMCs can migrate into intima, a transition in
their phenotype is required.5 Medial nonproliferating
SMCs have a contractile phenotype that enables them to
regulate vascular tone. When SMCs proliferate, they acquire a synthetic phenotype. As learned from animal
Received August 11, 2003; accepted August 11, 2003.
From the A.I. Virtanen Institute (M.O.H., S.Y.-H.) and Department of Medicine (S.Y.-H.), University of Kuopio, and Gene Therapy Unit (S.Y.-H.),
Kuopio University Hospital, Kuopio, Finland.
Correspondence to Seppo Ylä-Herttuala, MD, PhD, Department of Molecular Medicine, A.I. Virtanen Institute, University of Kuopio, PO Box 1627,
FIN-70211, Kuopio, Finland. E-mail [email protected]
© 2003 American Heart Association, Inc.
Arterioscler Thromb Vasc Biol. is available at http://www.atvbaha.org
1750
DOI: 10.1161/01.ATV.0000092871.30563.41
Hiltunen and Ylä-Herttuala
DNA Methylation, SMCs, and Atherogenesis
1751
Examples of Genes Implicated in Atherogenesis That Are at
Least Partially Regulated by DNA Methylation
Gene
IFN-␥38
PDGF-A39
MMP-240
MMP-740
MMP-940
TIMP-341
ICAM-142
Estrogen receptor-␣24
EC-SOD21
p5343
IFN indicates interferon; PDGF, platelet-derived growth factor; MMP, matrix
metalloproteinase; TIMP, tissue inhibitor of metalloproteinase; ICAM, intracellular adhesion molecule; EC-SOD, extracellular superoxide dismutase.
Downloaded from http://atvb.ahajournals.org/ by guest on April 28, 2017
Figure 1. Hypomethylation in atherosclerotic lesions. A,
5-methylcytosine content of genomic DNA extracted from
human arteries at different stages of atherosclerosis. Statistically
significant (*P⬍0.05) difference was observed when normal aortas (N; n⫽3) and fatty streaks (FS; n⫽23) were compared with
advanced lesions (AL; n⫽29) (ANOVA and modified t test). B,
5-methylcytosine content of genomic DNA extracted from atherosclerotic lesions induced by Western diet in ApoE knockout
mice. Normal control arteries were from ApoE⫺/⫺ mice fed with
a regular chow (*P⬍0.05; t test). C, Genomic 5-methylcytosine
contents of media and intima after denudation in normocholesterolemic NZW rabbits (media, n⫽2; intima, n⫽2) and in cholesterol-fed NZW rabbits (media, n⫽6; intima, n⫽6). Asterisks indicate statistical differences between intima and media (*P⬍0.01;
**P⬍0.005; t test). 5-methylcytosine analyses were done by
HPLC. Reprinted with permission from Reference 6.
occurs a few days after the injury, followed by matrix
formation from 1 week onwards, and may continue for
months, occluding the lumen and compromising the blood
flow.7
DNA Methylation Changes in SMCs
DNA methylation (ie, formation of 5-methylcytosines from
cytosine residues within CpG doublet by methyltransferases)
has a major role as a regulator of gene expression in
embryogenesis, X-chromosome inactivation in females,
genomic imprinting, and carcinogenesis.8 –10 DNA methylation is a form of epigenetic gene regulation that together
with altered binding profile of transcription factors commonly leads to suppression of gene expression when occurring in a regulatory region.11 According to current knowledge, 3 methyltransferases are responsible for genomic
methylation. Dnmt3a and Dnmt3b are responsible for de novo
methylation patterns, which are then maintained by Dnmt1.12
Although elusive, there must also be some demethylase
activity, because in the fertilized mouse eggs, the paternal
genome is rapidly demethylated before the replication of the
paternal pronucleus.13
During early development, certain chromosomal regions
become methylated (de novo methylation), controlling expression of genes that regulate cell differentiation. During
early stages of human carcinogenesis, genomic hypomethylation is a common phenomenon that is linked to transformation, tumor progression, and oncogene expression.14 –17 In
addition, it has been recently shown that hypomethylation
plays a causal role in tumor formation, possibly by inducing
chromosomal instability.18 The opposite situation, regional
DNA hypermethylation, is present in later stages of carcinogenesis and may lead to inactivation of tumor suppressor
genes.8,11,14 –17 Thus, changes in genomic methylation status
can lead to a selective growth advantage.
Most of our knowledge about the significance of DNA
methylation comes from developmental biology, cancer biology, and studies with targeted deletions of mouse Dnmt
genes.19 DNA hypomethylation in cancer cells occurs in
highly and moderately repeated DNA sequences. These
include heterochromatic DNA repeats, dispersed retrotransposons, and endogenous retroviral elements.20 In addition to
cancer, we have shown recently that genomic hypomethylation is present in advanced human atherosclerotic lesions,
lesions of apolipoprotein E (ApoE) knockout mice, and
neointima of balloon-denuded New Zealand White (NZW)
rabbit aortas (Figure 1).6 We have also shown that significant
genomic hypomethylation develops during the first replications of aortic SMCs in vitro and that hypomethylation occurs
in some specific genes, such as 15-lipoxygenase and extracellular superoxide dismutase.6,21 The Table shows examples
of genes that may be important for atherogenesis and are at
least partially regulated by DNA methylation. Because arterial SMCs are terminally differentiated, replication does not
normally occur and methylation machinery is probably minimally active. It is possible that DNA hypomethylation is
associated with changes in gene expression during the lesion
development. This may result from a direct regulatory effect
of the hypomethylation on the gene expression or be a
1752
Arterioscler Thromb Vasc Biol.
October 2003
operative. It is possible that de novo methylation activity that
would be required to increase genomic methylation level is
not induced during SMC proliferation. Circumstantial evidence from in situ hybridization analysis of the expression of
Dnmt1 supports the conclusion that increased Dnmt1 expression is associated with increased cellular proliferation in
atherosclerotic lesions.6
Other Cancer Biomarkers and Atherosclerosis
Downloaded from http://atvb.ahajournals.org/ by guest on April 28, 2017
Figure 2. Schematic representation of the possible role of
genomic methylation in SMC proliferation.
secondary effect by affecting DNA integrity and function. It
has also been shown that regional hypermethylation occurs in
atherosclerosis. Estrogen receptor-␣ gene was found to have
an increased methylation level in atheromas compared with
normal aorta.22,23 Estrogen receptor-␣ gene was also shown to
be methylated in SMCs in vitro during the phenotypic
switch.24
It has been reported that overexpression of platelet-derived
growth factor, c-myc, and other growth regulatory genes
occurs during atherogenesis and that SMC proliferation can
be successfully inhibited by blocking the expression or
activity of these genes.25–27 Several studies have shown that
when culturing various cell lines in the presence of a
methylation inhibitor, 5-azadeoxycytidine, expression of
some inactive genes can be activated.28 Similar effects may
be caused by hypomethylation in vivo. The fact that overexpression of Dnmt in cancer cannot reverse genomic hypomethylation could be explained by the lack of de novo
methylation activity of the enzyme.8,29
A crucial question regarding hypomethylation is whether it
is a consequence of SMC proliferation or whether it contributes to the increased proliferative activity. However, as
learned from cancer cells, important consequences of DNA
hypomethylation may be a decreased karyotypic stability and
altered heterochromatic-euchromatic interactions favoring
oncogenesis.20 It has been reported from cancer tissue that
Dnmt activity is actually increased despite the genome-wide
hypomethylation.30,31 Dnmt1 activity can be seen as a compensatory mechanism to maintain genomic methylation pattern, because only 2 rounds of replications are required for
genomic hypomethylation if maintenance methylation activity provided by Dnmt or other methyl transferases is not
Genomic instability (ie, chromosomal alterations and alterations in microsatellite sequences) is characteristic for human
cancers, and it has been suggested that loss of mechanisms
that protect genome integrity contributes to tumorigenesis.32
In normal cells, microsatellite sequence repeats are accurately
maintained during replication, but in tumor cells they vary in
length (ie, microsatellite instability). Although phenotypically silent, microsatellite instability indicates defects in
DNA replication and maintenance machinery. These errors in
the replication may lead to activation of proto-oncogenes and
in turn to inactivation of tumor suppressor genes. In human
atherogenesis, microsatellite instability occurs in 20% to 33%
of cases and may be linked to the increased proliferation rate
of SMCs.33,34
A special mechanism is required for the termini of the
eukaryotic chromosomes (ie, telomere) to complete replication during cell division. To overcome the end-replication
problem that produces gaps at the 5⬘ ends of the newly
synthesized DNA, an enzyme called telomerase must add
sequence repeats onto the preexisting 3⬘ overhangs of the
chromosomes.35 Telomerase activity has been found in germ
line cells and in cancer cells but not in somatic cells.36 This
attrition of the telomere length leads eventually to the
cessation of cell proliferation. Therefore, the actual biological
age of the cells (ie, the number of cell divisions) may be
determined by a biological clock that resides in the telomeres.
Although there is abundant information about gene expression, cellular activation, and proliferation of SMCs, relatively
little attention has been paid on the biological age of the
vascular cells. It is hypothesized that the age of the vascular
cells may have important effects on the response to injury and
various growth stimuli.37 These responses may also be
modulated by genomic methylation status of the vascular
cells. Figure 2 presents possible contribution of epigenetic
changes on SMC proliferation during atherogenesis.
Summary
It is concluded that hypomethylation occurs in atherosclerotic
lesions and that it seems to be associated with an increased
transcriptional activity. It is possible that alterations in DNA
methylation may play an important role in atherogenesis and
that interventions directed toward methylation machinery
may be useful for the treatment of vascular disorders.
Acknowledgments
The Academy of Finland, Sigrid Juselius Foundation, and Finnish
Foundation for Cardiovascular Research supported this study. The
authors would like to thank Marja Poikolainen for preparing the
manuscript.
Hiltunen and Ylä-Herttuala
DNA Methylation, SMCs, and Atherogenesis
References
Downloaded from http://atvb.ahajournals.org/ by guest on April 28, 2017
1. Benditt EP, Benditt JM. Evidence for a monoclonal origin of human
atherosclerotic plaques. Proc Natl Acad Sci U S A. 1973;70:1753–1756.
2. Penn A, Garte SJ, Warren L, Nesta D, Mindich B. Transforming gene in
human atherosclerotic plaque DNA. Proc Natl Acad Sci U S A. 1986;83:
7951–7955.
3. Ylä-Herttuala S, Nikkari T, Hirvonen J, Laaksonen H, Möttönen M,
Pesonen E, Raekallio J, Åkerblom HK. Biochemical composition of
coronary arteries in Finnish children. Arteriosclerosis. 1986;6:230 –236.
4. Haust MD. The natural history of human atherosclerotic lesions. In:
Moore S, ed. Vascular Injury and Atherosclerosis. New York: Dekker;
1981:1–23.
5. Campbell GR, Chamley-Campbell JH. Smooth muscle phenotypic modulation: role in atherogenesis. Med Hypotheses. 1981;7:729 –735.
6. Hiltunen MO, Turunen MP, Hakkinen TP, Rutanen J, Hedman M,
Makinen K, Turunen AM, Aalto-Setala K, Yla-Herttuala S. DNA hypomethylation and methyltransferase expression in atherosclerotic lesions.
Vasc Med. 2002;7:5–11.
7. Nikol S, Huehns TY, Hofling B. Molecular biology and post-angioplasty
restenosis. Atherosclerosis. 1996;123:17–31.
8. Bestor TH, Tycko B. Creation of genomic methylation patterns. Nat
Genet. 1996;12:363–367.
9. Ramsahoye BH, Davies CS, Mills KI. DNA methylation: biology and
significance. Blood Rev. 1996;10:249 –261.
10. Razin A, Shemer R. DNA methylation in early development. Hum Mol
Genet. 1995;4:1751–1755.
11. Greger V, Passarge E, Hopping W, Messmer E, Horsthemke B. Epigenetic changes may contribute to the formation and spontaneous
regression of retinoblastoma. Hum Genet. 1989;83:155–158.
12. Jones PA, Baylin SB. The fundamental role of epigenetic events in
cancer. Nat Rev Genet. 2002;3:415– 428.
13. Mayer W, Niveleau A, Walter J, Fundele R, Haaf T. Demethylation of the
zygotic paternal genome. Nature. 2000;403:501–502.
14. Goelz SE, Vogelstein B, Hamilton SR, Feinberg AP. Hypomethylation of
DNA from benign and malignant human colon neoplasms. Science. 1985;
228:187–190.
15. Gama-Sosa MA, Slagel VA, Trewyn RW, Oxenhandler R, Kuo KC,
Gehrke CW, Ehrlich M. The 5-methylcytosine content of DNA from
human tumors. Nucleic Acids Res. 1983;11:6883– 6894.
16. Feinberg AP. Genomic imprinting and gene activation in cancer. Nat
Genet. 1993;4:110 –113.
17. Counts JL, Goodman JI. Alterations in DNA methylation may play a
variety of roles in carcinogenesis. Cell. 1995;83:13–15.
18. Gaudet F, Hodgson JG, Eden A, Jackson-Grusby L, Dausman J, Gray JW,
Leonhardt H, Jaenisch R. Induction of tumors in mice by genomic
hypomethylation. Science. 2003;300:489 – 492.
19. Laird PW. Mouse models in DNA-methylation research. Curr Top
Microbiol Immunol. 2000;249:119 –134.
20. Ehrlich M. DNA methylation in cancer: too much, but also too little.
Oncogene. 2002;21:5400 –5413.
21. Laukkanen MO, Mannermaa S, Hiltunen MO, Aittomaki S, Airenne K,
Janne J, Yla-Herttuala S. Local hypomethylation in atherosclerosis found
in rabbit ec-sod gene. Arterioscler Thromb Vasc Biol. 1999;19:
2171–2178.
22. Post WS, Goldschmidt-Clermont PJ, Wilhide CC, Heldman AW,
Sussman MS, Ouyang P, Milliken EE, Issa JP. Methylation of the
estrogen receptor gene is associated with aging and atherosclerosis in the
cardiovascular system. Cardiovasc Res. 1999;43:985–991.
23. Dong C, Yoon W, Goldschmidt-Clermont PJ. DNA methylation and
atherosclerosis. J Nutr. 2002;132:2406S–2409S.
24. Ying AK, Hassanain HH, Roos CM, Smiraglia DJ, Issa JJ, Michler RE,
Caligiuri M, Plass C, Goldschmidt-Clermont PJ. Methylation of the
25.
26.
27.
28.
29.
30.
31.
32.
33.
34.
35.
36.
37.
38.
39.
40.
41.
42.
43.
1753
estrogen receptor-alpha gene promoter is selectively increased in proliferating human aortic smooth muscle cells. Cardiovasc Res. 2000;46:
172–179.
Marin ML, Gordon RE, Veith FJ, Tulchin N, Panetta TF. Distribution of
c-myc oncoprotein in healthy and atherosclerotic human carotid arteries.
J Vasc Surg. 1993;18:170 –176.
Wilcox JN, Smith KM, Williams LT, Schwartz SM, Gordon D. Plateletderived growth factor mRNA detection in human atherosclerotic plaques
by in situ hybridization. J Clin Invest. 1988;82:1134 –1143.
Shi Y, Hutchinson HG, Hall DJ, Zalewski A. Downregulation of c-myc
expression by antisense oligonucleotides inhibits proliferation of human
smooth muscle cells. Circulation. 1993;88:1190 –1195.
Haaf T. The effects of 5-azacytidine and 5-azadeoxycytidine on chromosome structure and function: implications for methylation-associated
cellular processes. Pharmacol Ther. 1995;65:19 – 46.
Laird PW, Jaenisch R. DNA methylation and cancer. Hum Mol Genet.
1994;3:1487–1495.
Lee PJ, Washer LL, Law DJ, Boland CR, Horon IL, Feinberg AP.
Limited up-regulation of DNA methyltransferase in human colon cancer
reflecting increased cell proliferation. Proc Natl Acad Sci U S A. 1996;
93:10366 –10370.
Issa JP, Vertino PM, Wu J, Sazawal S, Celano P, Nelkin BD, Hamilton
SR, Baylin SB. Increased cytosine DNA-methyltransferase activity
during colon cancer progression. J Natl Cancer Inst. 1993;85:1235–1240.
Hartwell L. Defects in a cell cycle checkpoint may be responsible for the
genomic instability of cancer cells. Cell. 1992;71:543–546.
Spandidos DA, Ergazaki M, Arvanitis D, Kiaris H. Microsatellite instability in human atherosclerotic plaques. Biochem Biophys Res Commun.
1996;220:137–140.
Hatzistamou J, Kiaris H, Ergazaki M, Spandidos DA. Loss of heterozygosity and microsatellite instability in human atherosclerotic plaques.
Biochem Biophys Res Commun. 1996;225:186 –190.
Watson JD. Origin of concatameric T7 DNA. Nat New Biol. 1972;239:
197–201.
Counter CM, Avilion AA, LeFeuvre CE, Stewart NG, Greider CW,
Harley CB, Bacchetti S. Telomere shortening associated with chromosome instability is arrested in immortal cells which express telomerase
activity. EMBO J. 1992;11:1921–1929.
de Bono DP. Olovnikov’s clock: telomeres and vascular biology. Heart.
1998;80:110 –111.
White GP, Watt PM, Holt BJ, Holt PG. Differential patterns of methylation of the IFN-gamma promoter at CpG and non-CpG sites underlie
differences in IFN-gamma gene expression between human neonatal and
adult CD45RO-T cells. J Immunol. 2002;168:2820 –2827.
Lin XH, Guo C, Gu LJ, Deuel TF. Site-specific methylation inhibits
transcriptional activity of platelet-derived growth factor A-chain
promoter. J Biol Chem. 1993;268:17334 –17340.
Sato N, Maehara N, Su GH, Goggins M. Effects of 5-aza-2⬘deoxycytidine on matrix metalloproteinase expression and pancreatic
cancer cell invasiveness. J Natl Cancer Inst. 2003;95:327–330.
Wild A, Ramaswamy A, Langer P, Celik I, Fendrich V, Chaloupka B,
Simon B, Bartsch DK. Frequent methylation-associated silencing of the
tissue inhibitor of metalloproteinase-3 gene in pancreatic endocrine
tumors. J Clin Endocrinol Metab. 2003;88:1367–1373.
Tanaka Y, Fukudome K, Hayashi M, Takagi S, Yoshie O. Induction of
ICAM-1 and LFA-3 by Tax1 of human T-cell leukemia virus type 1 and
mechanism of down-regulation of ICAM-1 or LFA-1 in adult-T-cellleukemia cell lines. Int J Cancer. 1995;60:554 –561.
Schroeder M, Mass MJ. CpG methylation inactivates the transcriptional
activity of the promoter of the human p53 tumor suppressor gene.
Biochem Biophys Res Commun. 1997;235:403– 406.
Downloaded from http://atvb.ahajournals.org/ by guest on April 28, 2017
DNA Methylation, Smooth Muscle Cells, and Atherogenesis
Mikko O. Hiltunen and Seppo Ylä-Herttuala
Arterioscler Thromb Vasc Biol. 2003;23:1750-1753; originally published online August 28,
2003;
doi: 10.1161/01.ATV.0000092871.30563.41
Arteriosclerosis, Thrombosis, and Vascular Biology is published by the American Heart Association, 7272
Greenville Avenue, Dallas, TX 75231
Copyright © 2003 American Heart Association, Inc. All rights reserved.
Print ISSN: 1079-5642. Online ISSN: 1524-4636
The online version of this article, along with updated information and services, is located on the
World Wide Web at:
http://atvb.ahajournals.org/content/23/10/1750
Permissions: Requests for permissions to reproduce figures, tables, or portions of articles originally published
in Arteriosclerosis, Thrombosis, and Vascular Biology can be obtained via RightsLink, a service of the
Copyright Clearance Center, not the Editorial Office. Once the online version of the published article for
which permission is being requested is located, click Request Permissions in the middle column of the Web
page under Services. Further information about this process is available in the Permissions and Rights
Question and Answer document.
Reprints: Information about reprints can be found online at:
http://www.lww.com/reprints
Subscriptions: Information about subscribing to Arteriosclerosis, Thrombosis, and Vascular Biology is online
at:
http://atvb.ahajournals.org//subscriptions/