© 1999 American Heart Association, Inc.
Atherosclerosis and Lipoproteins |
Local Hypomethylation in Atherosclerosis Found in Rabbit ec-sod Gene
From the A. I. Virtanen Institute (M.O.L., S.M., M.O.H., K.A., J.J., S.Y.-H.) and the Department of Medicine (S.Y.-H.), University of Kuopio, Kuopio, and the Institute of Medical Technology (S.A.), University of Tampere, Tampere, Finland.
Correspondence to Seppo Ylä-Herttuala, A. I. Virtanen Institute, University of Kuopio, PO BOX 1627, 70211 Kuopio, Finland. E-mail Seppo.Ylaherttuala{at}uku.fi
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Abstract |
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Abstract—Extracellular superoxide dismutase (EC-SOD) protects arteries against deleterious effects of superoxide anions and the development of atherosclerosis. In this study, we cloned and characterized rabbit ec-sod gene. We identified 6 rabbit C-elements and 5 CpG clusters in the cloned sequence. One of the CpG clusters is located on the coding sequence. Because CpG clusters are potential sites for methylation and may explain the occurrence of mutations, methylation status of each of the CpG dimers located in the coding sequence CpG cluster was characterized using direct genomic sequencing. Unexpectedly, a marked reduction in the amount of methylated CpG dinucleotides in ec-sod gene was detected in atherosclerotic aortas as compared with normal aortic intima-media. Although alterations in DNA methylation are well characterized in malignant tumors, the presence of methylation changes in atherosclerosis has not been studied even though both diseases are characterized by excess cellular proliferation and alterations in gene expression. Further analysis of the whole genomic methylation by high-pressure liquid chromatography in normal and atherosclerotic aortas revealed a tendency for a decreased 5-methylcytosine (5-mC) content in atherosclerotic aortas as compared with normal arteries. Hypomethylation in atherosclerotic aortas occurred at the same level as has been reported from malignant tumors. Although a causal relationship between the methylation level and expression of EC-SOD cannot be proven, our results show that ec-sod hypomethylation is associated with the development of atherosclerosis and suggest that it may affect structure and function of ec-sod and other genes possibly involved in the development of atherosclerotic lesions.
Key Words: EC-SOD • mutations • CpG island • atherosclerosis • DNA methylation
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Introduction |
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Extracellular superoxide dismutase (EC-SOD) is an antioxidative enzyme that catalyzes dismutation of superoxide anion (O2.-) to less reactive hydrogen peroxide.1 Biochemical and in situ hybridization studies have shown the presence of high amounts of ec-sod mRNA and activity in early atherosclerotic lesions2 3 where the enzyme is synthesized by smooth muscle cells (SMC) and macrophages.3 The high level of ec-sod expression in the arterial wall is physiologically important in maintaining low extracellular O2.- concentration, which prevents harmful effects of O2.-, such as oxidation of cellular proteins and LDL and inactivation of endothelium-derived relaxing factor (NO).4 5
DNA methylation, ie, formation of 5-methylcytosine (5-mC) from a cytosine residue by methyltransferase is an important factor regulating gene expression at different stages of development.6 DNA methylation, which is a form of epigenetic gene regulation leading to suppression of gene expression, is commonly concentrated to CpG clusters.7 8 5-mC levels are not maintained during DNA replication, but methyl groups need to be added after each cycle of cell replication. In malignant tumors, genomic hypomethylation is associated to increased cellular proliferation, and oncogene and growth factor expression.9 10 11 12 13 Alterations in genome-wide- and gene-specific DNA methylation have been convincingly linked to the development of human cancers,14 inactivation of X chromosome,15 and genomic imprinting.16 However, there are no previous studies about the presence of hypomethylation in atherosclerotic lesions.
In this study, we have cloned rabbit ec-sod gene and identified 6 repeat elements and 5 CpG clusters in the cloned sequence. A marked hypomethylation of the ec-sod gene was found in atherosclerotic aortas. At the whole genomic level, a tendency for hypomethylation was detected in atherosclerotic lesions. The results suggest that hypomethylation may play a role in the proliferation of SMC and development of atherosclerotic lesions.
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Methods |
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Library Screening
Approximately 500 000 plaques from a rabbit heart genomic library (Stratagene) were screened twice using random primed 32P-labeled ec-sod probe (386 bp).17 Positive clones were isolated and subcloned into pBluescript SK vector (Stratagene). Sequencing of the clone SA1 and SA2 (Genbank accession No. Y13339 and AJ007044) was carried out using automated laser fluorescence sequencer (A.L.F., Pharmacia). Sequence analysis was done by Genetics Computer Group program package, Center for Scientific Calculation, Webgene launcher program package, Institute of Advanced Biomedical Technologies and Censor program.18
Experimental Animals
Tissue samples were isolated from a total of 38, 6- to
8-month-old New Zealand White (NZW) rabbits and 7, 1- to 3-year-old
Watanabe heritable hyperlipidemic (WHHL)
rabbits.19 Isolated samples were aortic intima-media,
heart, liver, spleen, and sperm. WHHL rabbit lesions were used as a
model of atherosclerosis because the composition of
their lesions resembles human atherosclerotic lesions.20
Because it is difficult to find normal arterial wall in
WHHL rabbits, normal NZW aortas were used as controls in the
methylation analysis.
DNA Preparation
DNA was extracted (500 mg tissue wet weight) in 5 mL of
digestion buffer containing 10 mmol/L Tris, 1 mmol/L EDTA,
0.3 mol/L NaAc, 1% SDS, and 100 µg proteinase K (pH 8.0). Tissue
preparations were incubated overnight at +37°C, extracted once with
phenol-chloroform and twice with chloroform. Isolated DNA was
precipitated with ethanol and dissolved in Tris-EDTA-buffer
(10 mmol/L Tris-HCL, 1 mmol/L EDTA, pH 8.0).
Screening of the Mutations
Mutations in the coding sequence were screened using
polymerase chain reaction (PCR) techniques. The coding sequence
was amplified in 2 segments. The first sequence was amplified using a
primer pair 5'-GGGCCTGTTGAATTTCCTGAGAT-3' and
5'-GCACGGCCAGCGGGTTGTAGT-3' and sequenced by primers
5'-GGATGTTGCAAGTGACCAGGC-3' and 5'-GCCAGCGGGTTGTAGTGG-3'. The second
segment was amplified by a primer pair 5'-GCGCCCACTACAACCCGCTG-3' and
5'-CCGGGGCGCGCGGACTTTTC-3' and sequenced by primers
5'-ACTACAACCCGCTGGCCGTGC-3' and 5'-CTTTCGCGCCGTCGCTTCTTG-3. The length
of the amplified fragments were 595 and 331 bp, respectively. Tissue
samples from the isolated tissues of 5 NZW rabbits were amplified in 5
duplicates, each of which was sequenced on both strands. The PCR
product was sequenced by cycle sequencing employing automated
sequencer.
Bisulphite Modifications
Reaction was done as described21 to convert
nonmethylated cytosines to uracils. Two micrograms of DNA was
dissolved in 30 µL of H2O, and 3 µL of 2
mol/L NaOH was added. Samples were incubated 10 minutes at room
temperature, and 0.4 mL of a modification mixture was added to each
sample. The modification mixture was done as follows: 1.9 g sodium
metabisulfite (Sigma) was dissolved in 2 mL of
H2O. NaOH (0.7 mL of 2 mol/L) and 0.5 mL of 1
mol/L hydroquinone (Fluka) were added. pH was adjusted to 5.0, and the
volume was filled to 4 mL with H2O. Samples were
then incubated at 50°C for 4 hours. DNA was purified with Wizard DNA
Clean-Up System (Promega) according to the protocol of manufacturer.
The bisulphite reaction was completed by NaOH and DNA was precipitated
with 5 mol/L ammonium acetate and ethanol.
PCR and Genomic Sequencing
One hundred ng of freshly modified DNA was used for the
PCR amplification of the ec-sod gene CpG island. For the
first PCR, primers (5'-GATTGGATGTTGTAAGTGATT-3' and
5'-AACCAAAAAAAAACTTCAAC-3') covered a 871 nucleotide
region. PCR conditions were as follows: 96°C for 4 minutes, 80°C
for 3 minutes, followed by 96°C for 20 seconds, 52°C for 40
seconds, 72°C for 90 seconds, for 39 cycles. Two separate nested PCR
were carried out after the first PCR to amplify the sequence containing
all CpG sites within the ec-sod gene coding region (Figure 1
). For the upstream PCR, primers
(5'-GTAAAAC-GACGGCCAAGTGATTAGGTTGGGGG-3' and biotinylated
5'-CCAAAAAAAAACTTCAACA/GC-3')
covered a 857 nucleotide region, whereas the downstream
primers
(5'-GTAAAACGA-CGGCCATT/CGGGGATTTGAGTTAGG-3'
and the biotinylated primer shown above) covered a 446
nucleotide region. PCR conditions were as follows: 96°C
for 3 minutes, 80°C for 3 minutes, followed by 96°C for 20 seconds,
51°C for 15 seconds, 72°C for 90 seconds, for 33 cycles for the
upper 857 nucleotide region. The lower 446
nucleotide region was amplified with the same program
having annealing step at 52°C for 15 seconds. PCR products were
isolated with streptavidin-coated magnetic beads (Dynal AB) and
sequencing reactions were carried out using the AutoRead kit
(Pharmacia) with fluorescently labeled primers as described.
Reaction products were analyzed on automated sequencer and
the methylation status of each CpG doublet was categorized into the
following 5 levels: 0%, 25%, 50%, 75%, and
100%.22 23
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Total 5-mC Analysis
Perkin-Elmer high-pressure liquid chromatography
(HPLC) system was used to analyze cytosine
methylation at the whole genomic level as described.24 The
column was constructed from Beckman Ultrasphere 5-µm
particles.
Northern Blot Analysis
Tissue distribution of ec-sod mRNA was determined by
Northern blot analysis. Total RNA was isolated from rabbit
normal and atherosclerotic intima-media, heart, liver, and spleen by
guanidium isothiocyanate method and mRNA was isolated by Quick
Prep mRNA isolation kit (Pharmacia). One µg of mRNA was separated by
electrophoresis on a denaturing formaldehyde 1.5% agarose gel and
transferred to HYBOND N nylon membrane (Amersham). The membranes were
hybridized with a random-primed 32P-CTP-labeled
rabbit ec-sod (386 bp) and human ß-actin (1.1
kb) probes as described.17
Cell Culture Studies
The effect of ec-sod CpG cluster hypomethylation on
the ec-sod expression was analyzed by culturing
duplicate 10-cm plates (5 to 10x106 cells) of
rabbit aortic SMC in the presence of 3 µmol/L 5-azacytidine
(Sigma) for 1 month.25 As a control, we used SMC
grown in the absence of 5-azacytidine to obtain the basic level of
ec-sod expression. RNA and DNA were isolated from the
cultured cells and the methylation level and ec-sod mRNA
expression were determined as described above.
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Results |
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Rabbit ec-sod Gene
We have cloned and characterized rabbit ec-sod gene, which has a 735 bp nonintronic coding sequence in the third exon similar to human and mouse ec-sod genes26 27 (Figure 2
).
Database analysis revealed 6 regions of rabbit C-repetitive
sequences that represent typical short interspersed sequences
(SINE) elements.28 These repeats have originated
partly from tRNA genes29 and evolved by retrotransposition
similar to Alu repeats30 found in human ec-sod
gene26 or B repeats28 that are
present in mouse gene. Human gene also contains a LINE 2B
sequence in front of the coding sequence and 35-bp long poly-T after
the coding sequence. LINE sequences are "long period interspersed
sequences" in mammalian genomes that are retroposons generated from
RNA polymerase II transcripts.31
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According to sequence analysis, rabbit ec-sod
gene contains 5 CpG clusters, mainly located at the 5' region of the
cloned sequence. Similar CpG island patterns can also be seen on human
and mouse genes (Figure 2
). The first cluster is 365 bp long,
55% CG rich, with the observed/expected
(O/E) value of 0.57 for CpG
cluster. The second cluster is 254 bp long, 61% CG rich, and has the
O/E value of 0.57. The
length of the third cluster is 330 bp long, 65% GC rich, and has the
O/E value of 0.55. These
islands are separated by short sequences and may have arisen from a
single CpG cluster. The fourth cluster is 338 bp
long, 57% GC rich, with the
O/E value of 0.5. The fifth
and the largest (1067 bp) island is located on the coding sequence of
the gene (Figure 1). Sequence in this island is 72% GC rich
containing 121 CpG dinucleotides. The
O/E ratio of the last CpG
cluster (0.87) is well above the limit used to determine the presence
of these clusters.32
Comparison of the DNA and amino acid sequences between the species is
shown in Figure 1. Sequence similarity between rabbit and human,
rat, or mouse DNA is 80%, 67%, or 63%, respectively (Figure 1A).
CpG island comparison reveals a striking similarity among
the species. Most of the conserved dimers are located in the region
encoding for the active center. At nucleotide positions 74,
192, 324, 345, 450, 471, and 612, 3 of the CpG dimers are conserved,
whereas the fourth is a TpG dimer suggesting a possible deamination
process. Independent dimers are well conserved between rabbit and human
(69% similarity), whereas rabbit and rat or mouse show 33% and 31%
similarity, respectively. Amino acid sequence is also well conserved
between the species (Figure 1B). Sequence similarity between
rabbit and human, rat and mouse amino acids is 80%, 71%, and 68%,
respectively.
Mutation Screening
Previously the following mutations have been identified:
Arg213
Gly in the human cDNA region
encoding the carboxyterminal end of the protein,33 34
Asn21Asp in the amino-terminal portion of
mouse cDNA and a 10-bp deletion in 3' UTR of mouse cDNA35
(Figure 1A). Because CpG dimers are at high risk of mutations,
we screened mutations from rabbit lung, kidney, spleen, liver, brain,
and heart tissues. Three new mutations were identified in the coding
sequence: 1) 45 AC, 2) 76 GC, and 3) 157 CT (Figure 1A).
The first mutation is located in a putative signal peptide,
but it does not change the amino acid sequence, whereas the 2 latter
point mutations caused changes in the amino acid chains
Glu26Gln and
Arg52Trp, respectively (Figure 1B). The
first mutation created a new CpG dimer between dimers 6 and 7, whereas
the third mutation disrupted CpG dimer 19. The third mutation is
probably caused by deamination of the methylated CpG dimer. In 4 out of
5 animals, the new mutations caused allele variation. Mutations
were also present in germ line.
Methylation of ec-sod CpG Cluster in Atherosclerotic
Aorta and Other Rabbit Tissues
Because CpG clusters are potential sites for methylation and
may explain the occurrence of mutations, the methylation status of 109
dimers (Figure 1A) of the ec-sod CpG
cluster covering the coding sequence was determined using direct
genomic sequencing. Methylation level of the dimers was
analyzed from normal aortic intima-media, atherosclerotic
intima-media, and from heart, liver, spleen, germ line cells (sperm),
and cultured rabbit aortic SMC. According to preliminary
analysis, no differences were found in the whole genomic
methylation level between NZW and WHHL rabbit tissues, eg, kidney
values in NZW versus WHHL are 3.85 versus 3.9, respectively
(Laukkanenet al, unpublished data, 1999). The sequencing
analysis of the CpG cluster showed higher 5-mC content in the
island in all tissues studied as compared with atherosclerotic aorta
and germ line, which was nonmethylated throughout the cluster (Figure 3). Based on the sequencing
analysis, the 3' end of the island was almost completely
methylated in normal and atherosclerotic aorta, as well as in all
tissues analyzed. A clear difference was seen in the 5' end of
the island between normal (n=4) and atherosclerotic aorta (n=4) in
which the 5' end of the island is almost completely nonmethylated.
Also, the boundary between the hypomethylated and methylated region is
well defined. The comparison of different rabbit tissues shows no
common methylation pattern, but the distribution of 5-mC varies between
tissues. The CpG island located on the coding sequence was completely
nonmethylated in all germ line samples analyzed (n=3).
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For the analysis of the whole genomic methylation, DNA was extracted, digested, and subjected for HPLC analysis from 14 normal and 9 atherosclerotic rabbit aortas. The analysis revealed a 3.48±0.11% average content of 5-mC in normal aortic intima-media as compared with 3.16±0.25% in atherosclerotic lesions, which corresponds to a 9% decrease in the 5-mC content in atherosclerotic aortas. However, the difference did not reach statistical significance.
Methylation of the CpG Island in Cultured SMC
To analyze the effect of hypomethylation on the
expression of ec-sod, primary rabbit aortic SMC were
cultured in the presence of 5-azacytidine, which is known to induce a
general hypomethylation. Sequencing analysis revealed that the
ec-sod CpG cluster was completely methylated in control
cells (Figure 3). This supports earlier observations that have
shown that in cell lines, genes associated with CpG clusters are
methylated if they are not necessary for cell
survival.36 It is interesting to note that the
methylation pattern of the CpG cluster showed only minor changes
(<10%) after induction of hypomethylation with 3 µmol/L
5-azacytidine (Figure 3). The decrease was random and did not
resemble the hypomethylation pattern observed in atherosclerotic aorta.
This implies that hypomethylation in ec-sod in vivo may have
arisen by a specific mechanism. HPLC analysis was used to
confirm the effect of 5-azacytidine treatment on the genome-wide
methylation. The induction of hypomethylation caused a marked 52%
reduction in the genome-wide methylation as compared with control cells
(data not shown).
Northern Blot Analysis
Northern blot analysis of tissue distribution of
ec-sod mRNA showed the highest expression in normal aortic
intima-media, whereas atherosclerotic intima-media had decreased
ec-sod mRNA synthesis, as shown in previous
experiments.3 Heart showed much lower expression and
liver and spleen very little, if any, expression (Figure 4). The distribution is comparable with
reported enzyme activity in the same tissues.37 Northern
blot analysis from cultured SMC (see above) showed that minor
hypomethylation of the coding region did not increase mRNA expression
in 5-azacytidine–treated SMC as compared with untreated control cells
(Figure 4).
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Discussion |
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Ec-sod gene has so far been cloned from human26 and mouse.27 Each of these genes contains a nonintronic coding sequence located in the third exon and species characteristic SINE elements that are known to be retroposons. Rabbit C-element is a characteristic SINE element with defined structural features.30 38 In human and mouse ec-sod genes, corresponding LINE elements can be found in the 5' region of the sequence. The importance of these elements for the function of ec-sod gene is unknown but may reflect the evolutionary origin of the gene.
Mutation screening of the coding sequence revealed 3 new mutations. Two of these point mutations also changed the amino acid sequence. In principle, mutations could be at least partially explained by methylation because methylated cytosines in CpG dimers are frequently deaminated and mutated to thymidines, which has lead to rarity of these nucleotides39 because the mutation pressure on methylated CpG is 40 times higher than on nonmethylated dinucleotides.40 In spite of the high mutation pressure, we found only 1 dimer that was deaminated and mutated to TpG. This suggests a strong protection of the methylated dimers to keep cytosines in the ec-sod sequence. This is also supported by the similarity of the CpG pattern on the coding sequence of the cloned genes. Deamination and mutation of dimers would change the amino acid composition, which could lead to disappearance of the functional gene from the genome.
We characterized the methylation level of the CpG island located
on the coding sequence of the gene. CpG islands are normally found in
nonmethylated motifs in germ line and in tissues where the gene
associated to the island is expressed.7 However,
ec-sod CpG island located on the coding sequence shows a
high degree of methylation in all adult tissues studied (Figure 3), whereas in germ line it was completely nonmethylated,
revealing that the island is not protected against methylation and that
the methylation occurs during embryogenesis. The distinct
hypomethylation in ec-sod 5' region in atherosclerotic aorta
clearly differs from other analyzed tissues. In SMC, where
artificial hypomethylation was produced by 5-azacytidine, the local
methylation status of the ec-sod CpG cluster remained almost
unchanged. This is indicative for independent regulation and
maintenance of the methylation pattern of the ec-sod
CpG cluster, which is also supported by the random decrease of
methylation in 5-azacytidine-treated SMC as compared with the well
defined decrease in atherosclerotic aorta.
Northern blot analysis of mRNA isolated from different tissues indicated that arterial wall is one of the major sites of ec-sod expression. Variation between tissues clearly demonstrates that ec-sod is not a housekeeping gene but is strongly induced in certain physiological environments. This is supported by our previous findings showing that the expression of ec-sod is increased in the early phase of atherogenesis and decreased in complicated and connective tissue-rich human and rabbit lesions.3
It is not known whether hypomethylation of ec-sod in atherosclerotic lesions contributes to lesion formation or whether it occurs as a consequence of the pathological process. Also, as has been reported earlier,7 our results do not indicate a causal relationship between methylation pattern of the CpG island and ec-sod expression. However, it is conceivable that hypomethylation has at least partly developed through increased cell proliferation, which is a typical feature in atherogenesis.41 Because ec-sod methylation level in normal arterial wall was already lower than in other tissues, we cannot exclude a possibility that specific conditions in the vessel wall, such as deficiency in cytosine methyl transferase activity, may contribute to the hypomethylation of the CpG island. It is also possible that, similar to cancer cells, hypermethylation of genes involved in the inhibition of cell growth or apoptosis may be involved.
To our knowledge this is the first demonstration of hypomethylation in atherosclerotic lesions. The reduction observed in the methylation status of atherosclerotic lesions as compared with normal aortic intima-media is similar to the hypomethylation level reported in malignant tumors.16 According to preliminary results, similar hypomethylation can be found in human atherosclerotic plaques and in SMC isolated from intima of balloon-denudated rabbit arteries (Hiltunen et al, unpublished observation, 1999). These findings also support the hypothesis that lesion SMC may differ from normal arterial SMC, which might be reflected as differences in gene expression and potential for gene regulation.42 43 44 Normally, a genome-wide hypomethylation in malignant tumors is associated with increased expression of oncogenes and growth factors that leads to excess cell proliferation. Because SMC proliferation is an important factor in the development of atherosclerotic lesions,41 it is possible that similar mechanisms may at least partially account for the progression of atherosclerotic lesions in rabbit arteries.
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Acknowledgments |
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The authors want to thank professor Markku Kulomaa for critical comments regarding the manuscript. The authors also want to thank Mervi Nieminen, Arja Korhonen, and Anne Karppinen for technical assistance and Marja Poikolainen for preparing the manuscript. This study was supported by Finnish Foundation for Cardiovascular Research, Finnish Academy, Sigrid Juselius Foundation, and Ida Montin Foundation.
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