|
|
|
Original Paper
|
|
|||
|
Acta Biochim Biophys
Sin 2007, 39: 19-26 |
||||
|
doi:10.1111/j.1745-7270.2007.00251.x |
Different Effects of
Homocysteine and Oxidized Low Density Lipoprotein on Methylation Status in the
Promoter Region of the Estrogen Receptor a
Gene
Yushan Huang1, Kejun Peng2, Juan Su1, Yuping Huang3, Yizhou Xu1, and Shuren Wang1*
1 Department of
Pathophysiology, West China School of Preclinic Medical Sciences & Forensic
Medicine, Sichuan University, Chengdu 610041, China;
2 Department of Laboratory
Medicine, Chengdu Medical College, Chengdu 610500, China;
3 Department of
Biochemistry and Molecular Biology, Gannan Medical College, Ganzhou 341000,
China
Received: October
10, 2006
Accepted: November
27, 2006
This work was supported
by the grants form the Specialized Research Fund for the Doctoral Program of
Higher Education (No. 20050610050) and the Science and Technology plan project
of the Department of Health of Jiangxi Province (No. 20062033)
*Corresponding
author: Tel, 86-28-85501268; Fax, 86-28-85503204; E-mail,
wangshuren1945@yahoo.com.cn
Abstract We investigated the effects of homocysteine (Hcy) and
oxidized low density lipoprotein (ox-LDL) on DNA methylation in the promoter
region of the estrogen receptor a (ERa) gene, and its potential mechanism in
the pathogenesis of atherosclerosis. Cultured smooth muscle cells (SMCs) of
humans were treated by Hcy and ox-LDL with different concentrations and for
different periods of time. The DNA methylation status was assayed by nested
methylation-specific polymerase chain reaction, the lipids that accumulated in
the SMCs and foam cell formations were examined with Oil red O staining. The
proliferation of SMCs was assayed by the
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method. The
results showed that ox-LDL in moderate concentrations (10-40 mg/L) induced de novo
methylation in the promoter region of the ERa
gene of SMCs; however, high concentrations (50 mg/L) of ox-LDL, resulted in
demethylation of ERa. The Hcy treatment resulted
in de novo methylation in the promoter region of the ERa gene with a concentration- and treating
time-dependent manner, and a dose-dependent promoting effect on SMC
proliferation. These data indicated that the two risk factors for
atherosclerosis had the function of inducing de novo methylation in the
promoter region of the ERa gene of SMCs. However, high
concentrations (50 mg/L) of ox-LDL induced demethylation, which indicates that
different risk factors of atherosclerosis with different potency might cause
different aberrant methylation patterns in the promotion region of the ERa gene. The atherogenic mechanism of Hcy
might involve the hypermethylation of the ERa
gene, which leads the proliferation of SMCs in atherosclerotic lesions.
Key words homocysteine; oxidized low density lipoprotein; estrogen
receptor a; DNA methylation;
atherosclerosis
DNA methylation is an epigenetic process
leading to the chemical modification of a genome [1]. The addition of the methyl
group to cytosine, mainly located in CpG nucleotides pairs, represents one of
the mechanisms by which the genome can behave as a 搑esponsive organ to environmental factors. Accumulating
evidence has shown aberrant DNA methylation patterns in various diseases,
including cancer, certain X-linked genetic diseases [2-3], autoimmune diseases [4], aging [5], etc. Until now, the estrogen receptor (ERa)
gene is the only gene known to have aberrant hypermethylation in its promotor
region in atherosclerosis development [6]. Homocysteine (Hcy) and oxidized low
density lipoprotein (ox-LDL) are two established risk factors for
atherosclerosis. So what are the effects of the two risk factors on the
methylation pattern of ERa gene?
ERa
gene is a potential growth suppressor gene [7]. In vivo and in vitro
models of vascular disease have found estrogen to be protective against smooth
muscle cell (SMC) proliferation and neointima formation [8]. Clinical evidence
has also strongly suggested that estrogen replacement therapy in postmenopausal
women might help prevent cardiovascular disease [9]. If the two important risk
factors for atherosclerosis could exert a profound influence on the methylation
pattern of the ERa gene, it would be
most helpful in our understanding of the mechanisms of atherosclerosis
development, and might even reveal some missing knowledge links between risk
factors and atherosclerosis development. We therefore designed the current
study to investigate the potential effects of Hcy and ox-LDL with various
concentrations and treating times on the methylation pattern of the ERa gene, and their possible correlation with
foam cell formation and SMC proliferation.
Materials and Methods
Materials
Dulbecco's modified Eagle medium (DMEM)/F12
medium and RPMI 1640 were purchased from Gibco Life Technology (Burlington,
Canada). Maleic dialdehyde (MDA) assay kit was purchased from the Jiancheng
Bioengineering Institute (Nanjing, China). Homocysteine, oil red O, sodium
bisulfite, hydroquinone and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT) were all obtained from Sigma-Aldrich (St. Louis, USA). Neonatal
bovine serum (NBS) was from Sijiqing Biotechnologn Company (Hangzhou, China).
Mouse anti-a-actin antibody was from Beijing Zhongshan
Golden Bridge Biotechnogn Company (Beijing, China). Total DNA extraction kit,
agarose gel DNA fragment recovery kit, Boracker reverse
transcription-polymerase chain reaction (RT-PCR) kit, and BIOZOL reagent all
were from Tianggen Biotech Company (Beijing, China). DNA mate and methylase
aluI were from TaKaRa Biotechnology Company, Ltd. (Dalian, China).
Cell culture and treatment
Fresh human umbilical cords were obtained
with informed consent from women with normal pregnancies undergoing abdominal delivery
in the West China Second Hospital (Chengdu, China). Approval was granted by the
sichuan province ethics
committee. Sampling of umbilical cord vessels was processed on the day of
delivery.
Umbilical vein smooth muscle cells were
prepared by the explant technique. After removal of the endothelium and
adventitia, the remaining tissue was cut into small pieces, planted on a tissue
culture flask using Pasteur-pipette, and then bathed in DMEM/F12 medium
supplemented with 20% NBS, 2 mM glutamine, 100 U/ml penicillin, and 100 mg/ml streptomycin. The cultures were
maintained at 37 ºC in a 5% CO2 humid atmosphere. Monolayer confluent vein
smooth muscle cells between 3-5
passages were used for all experiments. Cells were identified as vein smooth muscle
cells by positive staining with SMC-specific a-actin antibody, a marker of SMCs.
Confluent (85%-95%) human umbilical smooth muscle cells (HUSMCs) were
washed twice with phosphate-buffered saline (PBS) before the experiments. Then
the SMCs were treated with 50, 100, 200, 500, and 1000 mm Hcy in serum-free DMEM/F12 medium for 24, 48, and 72
h, or with 10, 20, 30, 40, and 50 mg/L ox-LDL for 48 h in DMEM/F12 medium,
respectively.
Preparation of nLDL and ox-LDL
Human native low density lipoprotein (nLDL)
was prepared by a density gradient ultracentrifugation of plasma from a healthy blood donor with a density
adjustment by sodium bromide [10], followed by dialysis against PBS at 4 ºC for 36 h to remove EDTA.
The nLDL was exposed to 10 mm CuSO4 for 20 h at 37 ºC [11]. The resultant ox-LDL was then dialyzed
against PBS with 100 mM EDTA at
4 ºC for 24 h, followed by concentration with
macrogol 2000 (Sigma-Aldrich). Ox-LDL was then passed through a 0.22 mm millipore filter for sterilization.
Identification of purity was carried out by 5% polyacrylamide gel
electrophoresis.
The protein content of nLDL and ox-LDL was
determined using the Coomassie Brilliant Blue G-250 method with albumin as the
standard.
The oxidative extent of ox-LDL was
monitored by measuring the thiobarbituric acid-reactive substance (TBARS) using
the MDA assay kit. TBARS was calculated as micromole malondialdehyde per gram
protein. The content of TBARS in the ox-LDL was 21 mmol/g.
Oil red O staining for foam
cells
HUSMCs were planted in a coverslip
pretreated by 0.1% polylysine in six-well plates. After 48 h of treatment by
ox-LDL, the cells were washed three times with PBS, fixed in 2.5%
glutaraldehyde for 3 h, dipped in 2.5% potassium dichromate for 16 h, and
stained in 1% oil red O for 20 min to identify lipid droplets in the cytoplasm
under a microscope. Cell nuclei were then re-stained in hematoxylin for 5 min.
Foam cells were counted under a microscope. Commonly in foam cells the area of
lipid droplets exceeded the area of the nucleus. Manual quantitative analysis
of foam cells under a microscope was carried out by randomly pre-selected view
fields to count the percentages of positive oil red O staining cells.
MTT assay for cell viability
Cell viability was determined by a
colorimetric assay based on the ability of viable cells to metabolize MTT. MTT
is a yellow tetrazolium salt that forms a blue formazan dye precipitate that
can be extracted using an organic solvent when it is reduced by the
mitochondria of metabolically active cells [12]. The HUSMCs were plated in 96
well plates at a density of 2105
cells/well in DMEM/F12 medium with 20% NBS. After 48 h, the HUSMCs were washed
twice with PBS, and then were treated with 50, 100, 200, 500 and 1000 mM Hcy in serum-free DMEM/F12 medium for 24
h, respectively. Three hours before the end of the cell incubation periods, the
culture medium was added with 20 ml of MTT
(5 g/L) to each culture well. After an additional 3 h of incubation at 37 ºC, the medium was removed and formazan
crystals were dissolved in 0.2 ml dimethyl sulfoxide for 30 min at 37 ºC. The optical density (OD) of each well was
measured at 560 nm using a microplate reader (Molecular Devices, Seattle, USA).
The data are expressed as percentages of the control viability measured in
untreated cells.
DNA extraction and sodium
bisulfite treatment
DNA extraction and sodium bisulfite
treatment were carried out as previously described [13]. Briefly, the cultured
HUSMCs were collected by scraping with a cell scraper, and washed three times
with PBS. Total DNA was extracted from cells using a total DNA extraction kit,
according to the manufacturer's protocols.
Ten micrograms of DNA in 50 ml of Tris-EDTA Buffer were denatured with
5.5 ml of 3 M NaOH at 37 ºC for 10 min, followed by a 16 h treatment
at 50 ºC after adding 30 ml of freshly prepared 10 mM hydroquinone and 520 ml of freshly prepared 3.6 M sodium
bisulfite (pH 5.0). The DNA was desalted using a home dialysis system with 1%
agarose, then incubated at 37 ºC
for 15 min with 5.5 ml of 3 M NaOH,
followed by ethanol precipitation with 33 ml of 3 M NaAC (pH 5.2), 4 ml of DNA mate and 300 ml of ethanol. After washing with 70% ethanol, the
gently dried DNA pellet was dissolved with 30 ml of Tris-EDTA Buffer at 65 ºC for 10 min. The DNA sample was immediately stored at -20 ºC until further use.
Nested
methylation-specific-polymerase chain reaction
Nested methylation-specific-polymerase
chain reaction (nMS-PCR) was used for the detection of methylation in the
promoter regions of the ERa gene.
nMS-PCR consists of two-step PCR amplifications after a standard sodium
bisulfite DNA modification in which unmethylated cytosine residues are
converted to thymine. Methylated cytosine residues are retained as cytosine at
CpG sites, and are then used to specifically amplify either methylated or
unmethylated DNA. The first step of nMSP uses an outer primer pair set that
does not contain any CpG. The second-step PCR was carried out with the
conventional PCR primers. Primers for the promoter region of the ERa gene were designed to include eight CPG
dinucleotides that have been linked to the regulation of the ERa gene expression. The summary of the
primers and product sizes of the nMS-PCR assays are shown in Table 1.
PCR products were gel purified with an
agarose gel DNA fragment recovery kit according to the manufacturer's
instructions and were sequenced by Invitrogen (Carlsbad, USA). To reduce
mispriming and to increase efficiency, touchdown (TD) PCR was used in the
amplification. Following hot start, samples were subjected to 20 cycles in a TD
program (94 ºC for 45 s, annealing
temperature for 45 s and 72 ºC
for 45 s for 20 cycles, followed by a 1 ºC decrease of the annealing temperature every second
cycle). After completion of the TD program, twenty cycles were subsequently run
(94 ºC for 45 s, 45 ºC for 45 s and 72 ºC for 45 s), ending with a 5 min extension at 72 ºC.
The PCR products were separated by
electrophoresis through a 1% agarose gel containing ethidium bromide. DNA bands
were visualized by ultraviolet light.
A HUSMC DNA sample without any treatment was
used as the unmethylated control, and the methylated control DNA target was
prepared through treating DNA with methylase AluI that methylates cytosine
residues within all CpG dinucleotides in vitro.
Isolation of total RNA
Total RNA was extracted from the HUSMC
treated with 200 mm Hcy in serum-free DMEM/F12 medium for 24,
48 and 72 h with BIOZOL reagent, according to the manufacturer's protocols. The
purity of RNA samples was checked by measuring the absorbance of RNA at 260 and
280 nm and calculating A260/A280. RNA samples with A260/A280=1.8 were used for further studies. For checking the
integrity of RNA samples, agarose formaldehyde gel was used. Samples with
intact bands of 28S and 18S RNA were used for the semiquantitative RT-PCR.
Semiquantitative RT-PCR
Semiquantitative RT-PCR was carried out
using a Boracker RT-PCR kit. Briefly, for the reverse transcription reaction, 3
ml of total RNA together with 1 ml of Oligo (dT)18 (50 pmol/ml) was denatured at 75 ºC for 5 min, then put on ice for at least 1
min. It was then added to 4 ml of 5´RT buffer, 2 ml
of dNTP mixture (each 10 mM), 1 U of RNase inhibitor, and 1 ml of ReverTra Ace at a final volume of 20 ml. The reaction was allowed to proceed for
1 h at 42 ºC. After completion
of the reaction, the enzyme was inactivated at 99 ºC for 5 min, then stored at -20 ºC
until further use. Two and a half microliters of the total cDNA sample were
amplified in a single-stage PCR using primers designed for the ERa and the housekeeping genes, human
glyceraldehyde-3-phosphate dehydrogenase (G3PDH), with primers as follows: ERa forward, 5'-CCCTTGCTATGTTACTAAGCGTGAG-3'
(2387-2412); ERa reverse, 5'-TGCCATAGGAATACAAGAGGGTGCT-3' (2652-2627); G3PDH forward, 5'-ACCACAGTCCATGCCATCAC-3';
G3PDH reverse, 5'-TCCACCACCCTGTTGCTGTA-3'.
Amplification of cDNA specific for the ERa gene was carried out using primers
resulting in a PCR product of 266 bp, and amplification for G3PDH resulted in a
PCR product of 450 bp. Each reaction mixture contained 0.25 mM dNTPs, 1 ml of primers (20 mM), 1.25 U Taq DNA polymerase and PCR buffer at a
total volume of 25 ml. The following
parameters were used for amplification: denaturation at 94 ºC for 45 s, annealing at 61 ºC for 45 s and extension at 72 ºC for 45 s. At the end of the desired
cycles, the final incubation was carried out at 72 ºC for 5 min. After amplification of all samples for 30
cycles, 10 ml of PCR products were electrophoretically
separated on 1% agarose gel. The quantification of RT-PCR products was carried
out by measuring the densitometric analysis of agarose gels using an image
analyzer (NIH image), then, the ratios (ERa/G3PDH) were calculated.
Data analysis
The data were analyzed using the software SPSS
12.0 for Windows. Data were presented as mean±SD. For comparison between multiple groups,
quantitative data were analyzed using one-way ANOVA and least significant
difference test, and qualitative data were analyzed using a c2 test. Values less than 0.05 were
considered significant.
Results
The effect of ox-LDL on foam
cell formation
The SMC treated by ox-LDL for 48 h
displayed a remarkable and dose-dependent lipid accumulation as shown in figs. 1 and 2. The foam cells
increased linearly with the increment of ox-LDL concentration, which was in
accordance with the knowledge that ox-LDL is a risk factor for atherosclerosis.
The effect of Hcy on cell
viability
fig. 3 displayed an increasing proliferation of SMC along
with the increment of Hcy concentration, which indicated a dose-dependent
promoting effect of Hcy on SMC proliferation.
Alterations of methylation
status of the ERa gene in HUSMCs treated by
ox-LDL and Hcy with different concentrations and different treating times
Fig. 4 displayed the results of the methylation status of
the ERa gene with different treatments. Fig.
4(A) illustrates the effect of ox-LDL on the methylation status of the ERa gene. DNA from normal HUSMC (without any
treatment) amplified only with the unmethylated primers, which suggested that
the promotion region of the ERa gene of
SMC was unmethylation under normal conditions. The ox-LDL induced de novo
methylation of the promotion region of the ERa gene in mild-moderate concentrations. All of the
methylated primers and unmethylated primers produced evident PCR products in 10-40 mg/L of ox-LDL. Ox-LDL in high
concentrations (50 mg/L), however, showed a demethylation effect on the ERa gene. The PCR product with methylated
primer was totally lost, possibly indicating that the effect of ox-LDL on de
novo methylation of the ERa gene is
not a function that is associated with the increase of ox-LDL concentration.
Fig. 4(B-D)
illustrated the effect of Hcy with different concentrations and different
treating times. The results exhibited significant dose-dependent and treating
time-dependent de novo methylation in the promotion region of the ERa gene. Along with the prolongation of
treating time and increased Hcy concentration, the methylated bands became more
and more strong, while the unmethylated bands became weaker and weaker. In the
72-h treating group the unmethylated bands were totally lost, leaving only methylated
bands.
The above results indicate that the two
important risk factors of atherosclerosis could exert their effects on
atherosclerosis development by inducing aberrant methylation in the promotion
region of the ERa gene. This generally
occurred in hypermethylation, which might interfere with expression, but the
exerting behavior might vary with potency.
Alterations of ERa mRNA expression in HUSMCs with a
different methylation status of the ERa gene
To determine whether the ERa promoter methylation status was associated
with ERa mRNA expression levels, we used
semiquantitative RT-PCR to examine ERa
mRNA expression in HUSMCs treated with 200 mM Hcy for 24, 48 and 72 h (Fig. 5). From the
above results, we found that along with the prolongation of treating time, the
methylation degree was increased. We also found that along with the
prolongation of treating time, the ERa
mRNA expression was decreased.
Discussion
Atherosclerosis is a disease of large and medium-sized
arteries and is characterized by lipid accumulation and SMC migration and
proliferation [14-15]. Multiple
mechanisms have been implicated in its pathogenesis. The earliest recognized
gross lesion in atherogenesis is the fatty streak, characterized by an
accumulation of cells loaded with cholesteryl esters (foam cells) just beneath
the endothelium. Oxidized LDL has been implicated for playing a critical role
in foam cell formation. The uptake of ox-LDL by monocyte/macrophage through
scavenger receptors is the earliest and essential process in foam cell
generation. Our data also show an obvious and dose-dependent promoting effect
of ox-LDL on foam cell formation [16].
Ox-LDL is believed to exert diverse
biological effects on atherosclerosis genesis. Sukhanov et al. used two
cDNA microarray systems that contain a total of 35,932 unique genes to identify
the genes differentially regulated by ox-LDL in human aortic smooth muscle
cells (HASMC). They observed significant increases in RNA levels for 180 named
genes and significant decreases for 192 named genes. They demonstrated that
ox-LDL predominantly elevates the expression of genes involved in cell-cell
interactions, membrane transport, oncogenesis, apoptosis, and transcription and
decreases the expression of genes responsible for protein and nucleic acid
biosynthesis, lipid metabolism, and humoral responses [17].
Zaina et al. [18] recently reviewed
the evidences proposing that lipids and lipoproteins can act as nuclear factors
regulating chromatin structure and gene expression. The interaction between
chromatin and small lipid molecules, such as cholesterol and lipid peroxidative
products, have also been proved by increasing experimental evidences. And the
association of hyperlipidemic lipoprotein profiles and aberrant DNA methylation
patterns at early stages of atherosclerosis was also been proved in mice and in
cultured human macrophages
ERa,
upon activation by estrogen, regulates a variety of cellular activities,
including the inhibition of cell proliferation. Such an anti-proliferative
effect also involves the proliferation of SMCs [19]. To date, ERa is the only gene known to have
differential CpG island methylation in its promotion region in atherosclerosis.
In atheromas, ERa methylation was significantly
increased when compared with normal proximal aortas [20]. Since the ox-LDL
played an important role in foam cell formation and could modify chromatin
structure to exert epigenetic regulations on gene expression, could the ox-LDL
also play an atherogenic role by way of inducing aberrant methylation in the
promoter sequence of the ERa gene?
The present result shows that the promotion region of the ERa gene in HUSMCs displays de novo
methylation upon the treatment of mild-moderate concentrations of ox-LDL.
However, high concentrations of ox-LDL caused demethylation in the promotion
region of the ERa gene. There is no
dose-effect relationship between the ox-LDL concentration and the methylation
level, but our data showed a clear dose-dependent promoting effect of ox-LDL on
foam cell formation. The findings of this study can bring forward a tentative
idea that the ox-LDL might not primarily play its pathogenic role in foam cell
formation and atherogenesis by resulting in aberrant methylation in the promoter
of the ERa gene.
Homocysteine, when its concentration is
elevated in plasma, has been considered an independent risk factor for
cardiovascular disease. The atherogenic mechanism of hyperhomocysteinemia can
involve a variety of effects, including vascular endothelial
dysfunction/injury, attenuation of
NO-mediated vasodilatation, disturbance in the antithrombotic activities of the endothelium, oxidative
stress, and activating nuclear factor-kB
leading to recruitment of leukocytes and monocytes etc. [21,22]. Homocysteine is also
intimately associated with S-adenosylmethionine, the methyl donor for more
than 100 different transmethylation reactions, including DNA methylation
[23,24]. So hyperhomocysteine can interfere with the epigenetic modification of
a genome. Hiltunen et al. [25] have reported that a genomic
hypomethylation occurred during atherogenesis in human, mouse and rabbit
lesions and MTase was expressed in atherosclerotic lesions . Altered gene expression and cell proliferation
in atherosclerotic lesions have some similar characteristics with certain solid
tumors, which have shown genomic hypomethylation and hypermethylation in some
tumor suppression genes. Our research group has recently also found that Hcy
could increase the activity of methyltransferase (data to be published). So
perhaps Hcy could exert its atherogenic effect by inducing abberant methylation
patterns in the ERa gene.
In fact, the data
in the present study demonstrate a dose-dependent de novo methylation in
the promotion region of the ERa gene along with the
increasing homocysteine concentration and treating time. We found that
hypermethylation status of promoters could inhibit the ERa expression.
Meanwhile the stimulated cell viability assay showed a dose-dependent promoting
effect of Hcy on SMC proliferation. This result potentially suggests that the
de novo methylation in the promotion region of the ERa gene, induced by
Hcy might involve the mechanism of SMC proliferation in atherogenesis.
In summary, our
experimental results indicate that both ox-LDL and Hcy could cause aberrant
methylation in the promotion region of the ERa gene, but the
ox-LDL-induced aberrant methylation did not exhibit any clear correlation with
its effect of inducing foam cell formation. However, the Hcy-induced
hypermethylation in the promotion region of the ERa gene showed
similar dose-dependent patterns with the promoting effect on SMC proliferation,
which might suggest a novel mechanism involved in atherogenesis by hyperhomocystine.
References
1 Bird A. The essentials of DNA methylation.
Cell 1992, 70: 5-8
2 Bell MV, Hirst MC, Nakahori Y, MacKinnon RN,
Roche A, Flint TJ, Jacobs PA et al. Physical mapping across the fragile
X: hypermethylation and clinical expression
of the fragile X syndrome. Cell. 1991, 64: 861-866
3 Sado T, Fenner MH, Tan SS, Tam P, Shioda T,
Li E. X inactivation in the mouse embryo deficient for Dnmt1: distinct effect of hypomethylation on
imprinted and random X inactivation. Dev Biol 2000, 225: 294-303
4 Richardson B. DNA methylation and autoimmune
disease. Clin Immunol 2003, 109: 72-79
5 Richardson B. Impact of aging on DNA
methylation. Ageing Res Rev 2003, 2: 245-261
6 Dong C, Yoon W, Goldschmidt-Clermont PJ. DNA
methylation and atherosclerosis. J Nutr 2002, 132: 2406-2409
7 Ross R. The pathogenesis of atherosclerosis: a perspective for the 1990s. Nature
1993, 362: 801-809
8 Schwartz SM, Majesky MW, Murry CE. The
intima: development and
monoclonal responses to injury. Atherosclerosis 1995, 118: 125-140
9 Hulley S, Grady D, Bush T, Furberg C,
Herrington D, Riggs B, Vittinghoff E. Randomized trial of estrogen plus
progestin for secondary prevention of coronary heart disease in postmenopausal
women. Heart and Estrogen/progestin Replacement Study (HERS) Research Group.
JAMA 1998, 280: 605-613
10 Redgrave TG, Roberts DC, West CE. Separation
of plasma lipoproteins by density-gradient ultracentrifugation. Anal Biochem
1975, 65: 42-49
11 Itabe H, Yamamoto H, Suzuki M, Kawai Y,
Nakagawa Y, Suzuki A, Imanaka T et al. Oxidized phosphatidylcholines
that modify proteins. Analysis by monoclonal antibody against oxidized low
density lipoprotein. J Biol Chem 1996, 271: 33,208-33,217
12 Waterfield CJ, Westmoreland C, Asker DS,
Murdock JC, George E, Timbrell JA. Ethionine toxicity in vitro:
the correlation of data from rat
hepatocyte suspensions and monolayers with in vivo observations. Arch
Toxicol 1998, 72: 588-596
13 Yang ZH, Shang ZB, Yu J. The methylation
profiles of the promoter CPG islands of nine tumor associated gene correlate
with their expressions in three lung cancer cell line. Tumor 2004, 24: 216-222
14 Yla-Herttuala S, Nikkari T, Hirvonen J,
Laaksonen H, Mottonen M, Pesonen E, Raekallio J et al. Biochemical composition
of coronary arteries in Finnish children. Arteriosclerosis 1986, 6: 230-236
15 Benditt EP, Benditt JM. Evidence for a
monoclonal origin of human atherosclerotic plaques. Proc Natl Acad Sci USA
1973, 70: 1753-1756
16 Ylitalo R, Jaakkola O, Lehtolainen P,
Yla-Herttuala S. Metabolism of modified LDL and foam cell formation in murine
macrophage-like RAW 264 cells. Life Sci 1999, 64: 1955-1965
17 Sukhanov S, Hua Song Y, Delafontaine P. Global
analysis of differentially expressed genes in oxidized LDL-treated human aortic
smooth muscle cells. Biochem Biophys Res Commun 2003, 306: 443-449
18 Zaina S, Dossing KB, Lindholm MW, Lund G.
Chromatin modification by lipids and lipoprotein components: an initiating event in atherogenesis?
Curr Opin Lipidol 2005, 16: 549-553
19 Schwartz SM, Majesky MW, Murry CE. The intima:
development and monoclonal
responses to injury. Atherosclerosis 1995, 118: 125-140
20 Post WS, Goldschmidt-Clermont PJ, Wilhide CC,
Heldman AW, Sussman MS, Ouyang P, Milliken EE et al. Methylation of the
estrogen receptor gene is associated with aging and atherosclerosis in the
cardiovascular system. Cardiovasc Res 1999, 43: 985-991
21 Au-Yeung KK, Woo CW, Sung FL, Yip JC, Siow YL.
Hyperhomocysteinemia activates nuclear factor-kappaB in endothelial cells via
oxidative stress. Circ Res 2004, 94: 28-36
22 Dayal S, Bottiglieri T, Arning E, Maeda N,
Malinow MR, Sigmund CD, Heistad DD et al. Endothelial dysfunction and
elevation of S-adenosylhomocysteine in cystathionine beta-synthase-deficient
mice. Circ Res 2001, 88: 1203-1209
23 Guilland JC, Favier A, Potier de Courcy G,
Galan P, Hercberg S. Hyperhomocysteinemia: an
independent risk factor or a simple marker of vascular disease? 1. Basic data.
Pathol Biol 2003, 51: 101-110
24 Lee ME, Wang H. Homocysteine and
hypomethylation. A novel link to vascular disease. Trends Cardiovasc Med 1999,
9: 49-54
25 Hiltunen MO, Turunen MP, Hakkinen TP, Rutanen
J, Hedman M, Makinen K, Turunen AM et al. DNA hypomethylation and
methyltransferase expression in atherosclerotic lesions. Vasc Med 2002, 7: 5-11