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Original Paper
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Acta Biochim Biophys
Sin 2007, 39: 366-376 |
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doi:10.1111/j.1745-7270.2007.00291.x |
Ligands of Peroxisome
Proliferator-activated Receptor inhibit
Homocysteine-induced DNA Methylation of Inducible Nitric Oxide Synthase
yideng Jiang1*, jianzhong Zhang1, Jiantuan Xiong1, jun Cao1,
Guizhong Li1, and
Shuren Wang2
1 Department of Pathology, Ningxia Medical
College, Yinchuan 750004, China;
2
Department of Pathophysiology, West China College of Preclinical and Forensic Medical
Sciences, Sichuan University, Chengdu 610041, China
Received: January
22, 2007
Accepted: March 12,
2007
This work was
supported by a grant form the Specialized Research Fund for the Doctoral
Program of Higher Education (No. 20050610050)
*Corresponding
Author: Tel, 86-951-4083421; Fax, 86-951-4081245; E-mail, jwcjyd@163.com
Abstract Homocysteine (Hcy) is a risk factor for atherosclerosis. It is
generally accepted that inducible nitric oxide synthase (iNOS) is a key enzyme
in the regulation of vascular disease. The aim of the present study is to investigate the effects of peroxisome proliferator-activated
receptor ligands on iNOS in the presence of Hcy in human monocytes. Foam cells,
induced by oxidize low density lipoprotein (ox-LDL) and phorbol myristate
acetate (PMA) in the presence of different concentrations of Hcy, clofibrate
and pioglitazone in human monocytes for 4 d, were examined by oil red O
staining. The activity of iNOS was detected by real-time quantitative reverse
transcription-polymerase chain reaction and Western blot analysis. The
capability of DNA methylation was measured by assaying endogenous C5 DNA
methyltransferase (C5MTase) activity, and the iNOS promoter methylation level
was determined by quantitative MethyLight assays. The results indicated that
Hcy increased the activity of C5MTase and the level of iNOS gene DNA
methylation, resulting in a decrease of iNOS expression. Clofibrate and
pioglitazone could antagonize the hcy
effect on iNOS expression through DNA methylation, resulting in attenuation of
iNOS transcription. These findings suggested that Hcy decreased the expression
of iNOS by elevating iNOS DNA methylation levels, which can repress the
transcription of some genes. Peroxisome proliferator-activated receptor a/g ligands can
down-regulate iNOS DNA methylation, and could be useful for preventing
Hcy-induced atherosclerosis by repressing iNOS expression.
Key words homocysteine; DNA methyltransferase; PPARa/g ligand; iNOS DNA
methylation
Homocysteine (Hcy) is a sulfur-containing amino acid form during the metabolism of methionine. Hyperhomocysteine (HHcy) is found in 30% of patients with premature atherosclerosis of carotid and peripheral arteries [1]. Elevated plasma Hcy levels have been implicated as an independent risk factor for coronary heart diseases [2]. Evidences have also indicated that inducible nitric oxide synthase (iNOS) is the key mechanism in the regulation of vascular diseases. However, strong associations among HHcy, iNOS and atherosclerosis have been observed in many studies [3].
The precise mechanisms of Hcy-induced
atherosclerosis and iNOS anti-atherosclerosis are still to be fully
investigated. Much attention has been focused on the impacts of HHcy on dysfunction
and injury of vascular cells. Increasing evidence, however, indicates that HHcy
might also be involved in the regulation of disturbing the expression of
atherosclerosis-related genes through the interference of DNA methylation [4,5].
DNA methylation is a form of epigenetic
gene regulation that leads to the suppression of gene expression when occurring
in a regulatory region [6]. Some reports showed that it might be important for
atherogenesis, which is at least partially regulated by DNA methylation. ApoE
knockout mice and the neointima of balloon-denuded New Zealand White rabbit
aortas [6] showed that significant genomic hypomethylation developed during the
first replication of aortic smooth muscle cells (SMCs) in vivo, and that
hypomethylation occurred in some specific genes, such as 15-lipoxygenase and
extracellular superoxide dismutase, which have been indicated to be deeply
involved in atherosclerosis [7,8]. This might result from a direct regulatory
effect of hypomethylation on gene expression or from a secondary effect by
affecting DNA integrity and function.
Peroxisome proliferator-activated receptors
(PPARs) have been implicated in macrophage biology, lipid homeostasis, and
atherogenesis. In addition, it is generally accepted that iNOS is a key factor
in the regulation of vascular disease [9,10]. Strong associations between PPARa/g
ligands and iNOS in atherosclerosis have been observed in many studies [10-12]. But the precise anti-atherosclerotic mechanisms of PPARa/g
ligands and the relationship between PPARa/g ligands
and iNOS remain obscure. Taking into account that Hcy interferes with the
earliest processes of lipid accumulation and metabolism [13], we examined the
capacity of C5 DNA methyltransferase (C5MTase) to transfer the methyl group to
DNA and iNOS DNA promoter methylation status and the effects of PPARa/g
ligands on iNOS DNA promoter methylation. The alteration of DNA methylation on
iNOS might be an important finding that could point to a mechanism against
atherosclerosis in which epigenetic gene silencing is a feature. The iNOS
uncovered aberrations of DNA methylation induced by Hcy as well as the effects
of PPARa/g
ligands and the PPAR ligands pathways could be a potential target for
anti-atherosclerosis therapy. These data for the first time indicate the effect
of DNA methylation on the iNOS gene in the context of atherosclerosis.
Materials and Methods
Cell cultures
Human blood from a healthy donor was drawn
into heparinized syringes. The whole blood
was separated into peripheral blood mononuclear
cells and neutrophils using the density gradient from Nycoprep 1.077 (Life Technologies, Chengdu,
China), then the monocytes were
isolated from peripheral blood mononuclear cells by adherence to a serum-coated culture flask for 2 h.
Adherent cells were then detached and
resuspended in RPMI-1640 medium containing 5% autologous plasma. Only cell preparations with a 95% viability or
greater were used. The cells were planted into 6-well plates and grown to 80%
confluence. Serum was deprived for 4 d cell synchronicity. The cells were then
cultured with 0.5 mg/ml phorbol
myristate acetate (PMA) (Sigma-Aldrich, St. Louis, USA) together with 100 mg/ml oxidize low density lipoprotein
(ox-LDL) and different concentrations of Hcy, and clofibrate (5 mM) and pioglitazone (10 mM).
Oil red O stained foam cells
The cultured monocytes were washed with
phosphate-buffered saline (PBS) three times and fixed in 2.5% glutardialdehyde
for 3 h, dipped in 2.5% potassium dichromate for 16 h, and stained in 1% oil
red O (Sigma-Aldrich) for 20 min to identify lipid droplets in cytoplasm. Cell
nuclei were then stained in hematoxylin for 15 s. All products were washed with
distilled H2O. monocyte-derived
foam cells were observed. Semi-quantitative analysis of foam cells was
evaluated by the percentage of positive oil red O staining cells.
Endogenous C5MTase activity
A modification of the assay developed by
Hattori et al. [14] was used to determine DNA MTase activity. A total of
1107 cells was scraped from plates, pooled into
ice-cold PBS and collected by centrifugation. The cells were suspended in 500 ml lysis buffer, then lysed by four cycles
of freezing at -70 șC and thawing at 37 șC. Protein concentration was determined by the Bradford
assay. Cell lysates containing 5 mg protein
were mixed with 0.5 mg poly[dI-dC]∙poly[dI-dC] and 1.5 mM S-adenosyl-L-[methyl-3H] methionine in a total volume of 20 ml and incubated at 37 șC for 2 h. The reactions were terminated by
adding 300 ml of stop solution (1% sodium dodecyl
sulfate, 2 mM EDTA, 5% 2-propanol, 125 mM NaCl, 1 mg/ml Proteinase K, 0.25
mg/ml carrier DNA) for 1 h at 37 șC.
The DNA was extracted with phenol-chloroform and ethanol precipitated. The
recovered DNA was resuspended in 30 ml of 0.3 M NaOH and incubated for 30 min
at 37 șC. DNA was spotted on GF/C filter discs
(Whatman, Shanghai, China) and dried. Filters were placed in scintillation
vials and incubated for 1 h at 60 șC with 500 ml of 0.5 M perchloric acid. Then 5 ml of scintillation
cocktail was added and radioactive 3H
incorporation into DNA was assessed using a liquid scintillation counter
(Beckman Coulter, Shanghai, China).
Real-time quantitative reverse
transcription-polymerase chain reaction (RT-PCR)
Genomic RNA was isolated from the cultured
cells by the E.Z.N.A Tissue RNA Kit (Omega Bio-tek). RNA was reversely
transcribed by the RevertAid First Strand cDNA Synthesis Kit (MBI) in a final
volume of 20 ml containing
5 ml RNA, 3 ml oligo dT primer, and 12 ml DEPC-treated water. The mixture was incubated at 70 șC for 10 min, then put on ice. Four
microliters 5 reaction buffer, 1 ml
ribonuclease inhibitor (recombinant; 20 u/ml), 2 ml dNTP mix, and 1 ml RevertAid M-MuLV Reverse Transcriptase (200 u/ml) were added and incubated at 20 șC for 10 min, then incubated at 48 șC for 60 min. cDNA was used for PCR.
Primers of iNOS (GenBank accession No.
AF440783) were designed with Primer Premier 5.0 software. The forward primer
was 5'-CTATGTAGCCCTAGTTTGTCC-3', the reverse primer was 5'-GTGGTAGATTGGCGTGAC-3'
and the probe primer was 5'-6-FAM-AGTCGTGTCGGGATGGGTGA-TAMRA-3'.
Real-time PCR was carried out using an iCycler iQ real-time PCR detection
system (Bio-Rad, Hercules, USA) with the program running for 40 cycles at
95 șC for 45 s, 60 șC for 60 s and 72 șC for 120 s. The melting curve analysis was carried out
at the range 55 șC
-95 șC by monitoring 6-FAM fluorescence with increasing
temperature (0.5 șC increments at 10 s
intervals). PCR-specific products were determined by clear single peaks at the
melting curves above 80 șC.
Real-time PCR was duplicated for each cDNA sample. Each gene RNA level was
acquired from the value of the threshold cycle (Ct) of the real-time PCR
related to that of glyceraldehyde-3-phosphate dehydrogenase (GAPDH; calibrator)
through the formula DCt [DCt=CtGAPDH (calibrator)-Ctsample].
Final results, expressed as N-fold differences in target gene expression
relative to the calibrator, termed "Ntarget", were determined as follows: NiNOS=2DCt(sample)-DCt(calibrator).
Western blot analysis of iNOS
Total protein was extracted from the
cultured monocytes and analyzed by Western blotting [15,16]. Briefly, cultured
cells were harvested by scraping with a plastic scraper. Extracts of whole
cells (5106) were isolated from cell culture by lysis
buffer. Protein concentrations were determined by Coomassie Protein Assay.
Polyacrylamide gel electrophoresis (7%-19%
polyacrylamide gradient gels) was
followed by electrophoretic transfer
of proteins from the gel to a nitrocellulose membrane. The membrane was
incubated in 10 ml of blocking solution (0.1 ml/cm2) for 2 h at room temperature with gentle agitation on
a platform shaker, and washed three times for 5 min each in TBST solution (50 mm Tris-HCl, pH 7.6, 150 mm NaCl, 0.05% Tween-20). The membrane
was then incubated with a monoclonal anti-iNOS antibody (at 1:250 dilution) in
10 ml primary antibody dilution buffer with gentle agitation at 4 șC, then washed three times with TBST
solution, and incubated with secondary antibody goat anti-rabbit horseradish
peroxidase-conjugated immunoglobulin G (Jackson ImmunoResearch, West Grove,
USA) in PBS at 1:2000 dilution containing 1% bovine serum albumin for 1 h at
room temperature. After being washed again three times with TBST solution, the membrane was incubated with 10
ml LumiGLO solution (New England Biolabs, Beverly, USA) with gentle agitation for
1 min at room temperature. Excess developing solution was drained out, but the
membrane was not dried. It was wrapped in plastic and exposed to X-ray film.
iNOS promoter methylation
analysis
Bisulfite conversion and DNA recovery Bisulfite modification was carried out using the EZ DNA Methylation Kit (Zymo Research, Orange, USA) following the manufacturer's instructions. The bisulfite-treated DNA was isolated using the Wizard DNA Clean-Up System (Promega, Madison, USA). The DNA was eluted by 50 ml of warm water and 5.5 ml of 3 M NaOH was added and allowed to stand for 5 min. The DNA was ethanol precipitated with glycogen as a carrier and resuspended in 20 ml of water.
Standard curve Peripheral blood leukocyte (PBL) DNA was used as a substrate for M.SssI treatment as described by the manufacturer [17]. PBL DNA (0.05 mg/ml) was incubated with M.SssI at a concentration of 1 U/mg DNA (0.05 U/ml) and 0.16 mM AdoMet (Sigma-Aldrich) overnight at 37 șC. Then extra AdoMet (to 0.20 mM) and M.SssI (to 0.065 U/ml) were added followed by a second overnight incubation at 37 șC. The sample was stored at 4 șC. To generate unmethylated human DNA as control samples for testing the MethyLight reactions, sperm and PBL DNA were amplified using a WGA kit (Molecular Staging, New Haven, USA) as described by the manufacturer. The unmethylated and methylated DNA were then treated with bisulfite and recovered as described above. The following ratios were prepared (methylated/unmethylated): 0/100, 10/90, 25/75, 50/50, 75/25, 90/10 and 100/0. each sample was examined by real-time PCR analysis in duplicate. We correlated the DCt values with the predefined prevalence of methylated alleles. The curve had a sigmoid shape with a linear part in the range of 10%-90% of methylated DNA. From this we deduced an algorithm to calculate the methylation ratio of an unknown sample from its DCt value.
MethyLight reactions and methylation calculations We carried out a novel quantitative analysis of methylated alleles that is essentially a major improvement over a previous method based on real-time PCR (MethyLight) [18]. We used a VIC-labeled probe that specifically hybridizes to the sequence derived from the methylated allele, and a FAM-labeled probe that binds to the sequence generated from the unmethylated allele. The amount of fluorescent dye released during PCR is measured by a real-time PCR system and is directly proportional to the amount of PCR product generated. The binding site of the probes covers three differently methylated CpG dinucleotides. Their improved sequence specificity facilitates relative quantification of methylated and unmethylated alleles that are simultaneously amplified in a single tube.
PCR primers were designed to amplify the
bisulfite-converted antisense strand of the iNOS sequence. The PCR primers and
probes and the strategy for designing the MethyLight reaction are listed in Fig.
1. The PCR was carried out using a 96-well optical tray with caps at a
final reaction volume of 20 ml.
Samples contained 10 ml of
TaqMan Universal PCR Master Mix (Bio-Rad), 2 ml of bisulfite-treated DNA, 2.5 mM each of the iNOS forward primer and PPARa/g
reverse primer and 150 nM each of the fluorescently labeled probes iNOS met and
unmet. Initial denaturation at 95 șC for 5 min to activate the AmpliTaq Gold DNA polymerase was followed by
40 cycles of denaturation at 95 șC
for 15 sec and annealing and extension at 60 șC for 1 min.
The MethyLight data specific for the
methylated iNOS gene were expressed as the percentage of methylated reference
values and the percentage of unmethylated reference values, and calculated
similarly to a recent report [19], but with the following changes. The
percentage of methylated DNA molecules in a real-time PCR experiment is given
by c, DCt=Ct-FAM-Ct-HEX=Log2[c/(1-c)]. To account for the differential efficacy of the
PCR (methylated/unmethylated) and probe activity, we restate the model as DCt=a+bLog2[c/(1-c)], with a and b representing the additional
effects. The following equation was deduced from the results generated by the
standard curve, c=100/[1+2[(2-DCt)/-0.68], a=2 and b=0.68. Each MethyLight reaction was
carried out between three and six times, and the data shown are the mean percentage
of methylated reference values or the mean percentage of unmethylated reference
values of the three measurements.
Statistical analysis
Results are expressed as mean±SEM. The data were analyzed using one-way anova and additional analysis used Student-Newman-Keuls test for
multiple comparisons within treatment
groups or Student's t-test for between two groups. P<0.05 was
considered significant.
Results
effect of Hcy on foam cells
derived from monocytes
Fig. 2 shows the effects when foam cells were induced after
4 d incubation with PMA and ox-LDL, 0, 50, 100, 200 or 500 mm Hcy, and clofibrate and pioglitazone in
monocytes. A great number of foam cells were found by oil red O staining. The
foam cells cultured with various concentrations of Hcy (50 to 500 mM) for 4 d were significantly elevated
compared with the control (P<0.05). Hcy at 100 mm produced the highest stimulation of foam
cells (P<0.001), indicating that Hcy is important for foam cell
formation. The role of the addition of clofibrate and pioglitazone were further
tested that they can decrease foam cell formation (P<0.05).
Endogenous C5MTase activity
To determine whether Hcy is able to induce changes in C5MTase in human monocytes, the monocytes were cultured with PMA and ox-LDL as well as different concentrations of Hcy, and clofibrate and pioglitazone. The endogenous C5MTase activity is shown in Fig. 3. Hcy up-regulated the activity of C5MTase, with maximum activity at a concentration of 100 mm Hcy (p<0.01, compared with the control group). The other experimental groups also showed significant elevation of C5MTase activities (p<0.05). The cells cultured with clofibrate and pioglitazone decreased the activity of C5MTase in cultured monocytes within 4 d (p<0.05, compared with the ox-LDL+PMA+Hcy group).
Effect of Hcy on iNOS RNA and
protein expressions
To determine whether Hcy and PPARa/g ligands modulate the expression of iNOS
mRNA, total RNA was isolated from the cultured monocytes treated with Hcy at
different concentrations, clofibrate and pioglitazone. After normalization
against GAPDH mRNA, the mRNA level of iNOS was down-expressed in all
Hcy-treated groups compared with the control group (P<0.05). The
decrease in the levels of iNOS mRNA after incubation with 100 mm Hcy reached the maximum when cultured
monocytes were incubated with Hcy (50500 mm) for 4 d. But the increasing concentrations of Hcy
did not result in dose-dependent decreasing effects on mRNA levels of iNOS.
There was no significant difference in mRNA expression of iNOS between the
experimental groups. PPARa/g
ligands also increased iNOS production (Fig. 4).
The effects of Hcy on the protein levels of
iNOS were measured by Western blot analysis, and showed a result similar to
that of iNOS mRNA expression. The lowest protein expression of iNOS was also at
a concentration of 100 mM Hcy,
and there was no significant difference in iNOS protein levels between various
dosages of Hcy. PPARa/g ligands increased iNOS protein expression.
These results show that PPARa/g ligands not only reduced the production of iNOS protein but also iNOS mRNA levels in cultured human
monocytes (Fig. 5).
Hcy-induced iNOS methylation
changes
To explore the possible role of the Hcy and
PPARa/g
ligands in iNOS methylation levels, the methylation status of the iNOS promoter
region was investigated in cultured monocytes. Using a quantitative TaqMan-based
real-time PCR, it was found that an increase in Hcy dose (50, 100, 200 and 500 mM) led to a significant increase in iNOS
DNA methylation by 31%, 87%, 32% and 50%, respectively (p<0.05), and decrease in
clofibrate and pioglitazone 32% and 64% compared with the control, and 26% and
61% compered to 100 mM Hcy (Fig. 6)
by 32%, 26% and 64%, 61% (p<0.01).
These findings suggested that DNA methylation played a role in iNOS expression
observed in human monocytes.
Discussion
Atherosclerosis has been viewed as bland
deposits of excess lipid in the vascular wall, but mounting evidence has
redefined them as dynamic sites of chronic inflammation [20,21]. Considerable
epidemiological evidence has identified Hcy as a risk factor for
arteriosclerosis, but the mechanisms of Hcy-induced atherosclerosis have been
linked to inflammation, oxidative stress, and apoptosis. Increasing evidence,
however, indicates that HHcy might also be involved in disturbing the
expression of atherosclerosis-related genes through the interference of DNA
methylation [22,23].
Previous studies have reported the effects
of Hcy-induced DNA methylation in vascular SMCs (VSMCs) [24,25]. However, our
experiments showed that Hcy not only promoted C5MTase activity but also induced
DNA hypermethylation of the iNOS gene promoter and the downgrade of iNOS
expression in foam cells derived from monocytes. We also showed that PPARa/g
ligands decreased activity of DNA MTase and down-regulated iNOS DNA
methylation, which repressed iNOS expression, resulting in
anti-atherosclerosis. This finding has important clinical implications, and is
supported by the following evidence. First, the plasma concentration of Hcy in
patients suffering from homocystinuria is as high as 500 mm [26,27]. Patients with plasma Hcy levels
>50 mm are at increased risk for vascular
disorders [28,29]. This is in the range used in the present study. Second, in
the present study, we have also produced insights regarding the mechanisms
responsible for Hcy-induced iNOS promoter region DNA methylation in foam cells
derived from monocytes.
Although the molecular mechanism underlying
Hcy-induced atherosclerosis has been the subject of intensive investigation,
most previous studies have focused on the influence of Hcy on endothelial cells
and VSMCs [30,31]. Recent studies have shown that Hcy might also act directly
on the immune cells to initiate and promote the progression of atherosclerosis
[32,33]. Holven et al. [34] suggested that Hcy might exert its
atherogenic effect by enhancing the inflammatory response. Similarly, Hcy might
work through a new mechanism involving the up-regulation of iNOS DNA
methylation, especially in the development of atherosclerotic plaque in
patients with vascular disorders [35]. This should provide new insight to our
understanding of Hcy-induced atherosclerosis.
We tested C5MTase activity, which acted as
a mediator in Hcy-induced DNA methylation, and found that Hcy (=50 mm) elevated C5MTase activity. A great
difference between Hcy and cysteine is a one-carbon methyl group transfer
metabolism, which involves the Hcy, but not the cysteine. The transmethylation
modulation of genomic DNA (gDNA) is a very important epigenetic way for the
regulation of gene expression, and has been shown to involve the expression of
some atherosclerosis-related genes. In this cycle, the methyl-group for gDNA
modification is catalyzed by DNA methyltransferase. After the methyl-group is
transferred to DNA or other target compounds, the produced S-adenosylhomocysteine
is then hydrolyzed to Hcy. Hcy can be recycled to methionine, or be eliminated
by other metabolic ways. An abnormal increased Hcy concentration might
interfere with this cycle, and result in feedback impacts on DNA MTase and the
methylation status of gDNA. In the present study, we have found that C5MTase
activity can be triggered by the Hcy range of 50500 mm. It has been reported that MTase activity in cancer
tissue was actually increased in spite of the genome-wide hypomethylation [36].
MTase activity can be seen as a compensatory mechanism to maintain the genomic
methylation pattern. Our unpublished data suggest that the Hcy level
significantly increases MTase activity both in VSMCs and monocytes.
Second, we have distinguished the expression
of iNOS. The present study has provided evidence that iNOS is the major enzyme
responsible for Hcy-induced atherogenesis, our results indicating that iNOS
protein expression was induced by Hcy in foam cells derived from monocytes.
iNOS is believed to produce low amounts of NO to execute physiological and/or
anti-inflammatory functions. The inflammation-associated expression of iNOS and
the subsequent overproduction of NO are assumed to be responsible for the
cardiovascular failure [37]. The results also showed that iNOS promoter was
hypermethylated after Hcy treatment. A reasonable explanation is that
increasing concentrations of Hcy increases the intracellular ratio of
S-adenosylmethionine (SAM)/S-adenosylhomocysteine and stimulates DNMT activity
that the methyl group of SAM transfers to iNOS. Increased iNOS gene promoter
hypermethylation is silenced in association with the CpG-island methylation. for this reason, lowered iNOS
expression is a reasonable, and easy, explanation [38,39]. But the result of decreased
iNOS expression levels contradicts the report by Woo et al. [40]. A
potential, yet plausible explanation is that there are differences in iNOS
expression between foam cells and macrophages. In many mechanisms involved in
the synthesis of iNOS in the process of foam cell development, in which iNOS
promoter DNA methylation might be predominant, iNOS replication was repressed
by promoter hypermethylation.
Furthermore, some evidence has indicated
that PPARa/g
ligands reduce inflammation and anti-atherosclerosis [3]. However, our data
have shown that PPARa/g ligands can partially block Hcy-induced
C5MTase activity and are involved in the mechanism of Hcy-induced DNA
methylation in cultured human monocytes. This is consistent with the fact that
PPARa/g ligands
have an anti-atherosclerotic effect in cultured VSMCs. PPARa/g
ligands play an important role in transferring the methyl group to DNA or other
target compounds [41]. we have
also examined the effects of PPARa/g ligands on Hcy-induced response to iNOS,
and showed that PPARa/g ligands clearly inhibited Hcy-induced iNOS
in cultured human monocytes. These results suggest that Hcy-induced iNOS can be
regulated by peroxisome proliferators [42], and the activation of PPARa/g
might have beneficial effects in patients with atherosclerotic disorders.
The dosage of Hcy used in the present study is clinically relevant, from moderate hyperhomocysteinemia (Hcy concentration of 100 mm, found in up to 40% of patients with myocardial infarction, stroke, or venous thrombosis) to severe hyperhomocysteinemia (Hcy concentration of 500 mm, found in patients with inherited homocystinuria). The impacts of various concentrations of Hcy on the one-carbon methyl group transfer metabolism, however, did not show a dose-effect relationship. The highest effects on aberrant methylation of iNOS and the factors involved in the pathway of DNA methylation and the SAM cycle were at the Hcy concentration of 100 mm. Increased Hcy concentrations, on the contrary, exert weaker effects on the aberrant methylation of iNOS and involved factors. This was unexpected, but might be reasonable. Hyperhomocysteinemia has been found to be associated with many deleterious effects, including pro-apoptosis on endothelial cells, promoting proliferation of VSMCs, activating some inflammatory pathways and coagulation cascades, even mediating cholesterol dysregulation. Hcy concentrations higher than 100 mm might exert more direct injurious effects, such as oxidative stress and apoptosis, whereas moderate hyperhomocysteinemia might have a milder impact on epigenetic modulation of gene expression [43]. There is a proliferation of SMCs at the level of 100 mm Hcy, but viable cell counts progressively reduce in Hcy concentrations of 200 mm and above. This phenomenon suggests that the varied detrimental effects of Hcy could be attributed to different concentrations by different mechanisms. In mild and moderate hyperhomocysteinemia, Hcy might primarily influence the epigenetic regulation of gene expression through the interference of methyl group transfer metabolism, whereas in higher Hcy concentrations, the essential impacts might be more directly injurious through oxidative stress, pro-apoptosis, and inflammation.
Our findings uncovered Hcy-induced hypermethylation in the iNOS promoter and increased activity of DNA MTase, similar to the fingding in cancer tissues. It is possible that alterations in iNOS promoter hypermethylation play an important role in atherogenesis. However, there is no proven direct relationship between iNOS promoter hypermethylation and atherogenesis, there might simply be an association between these two processes, but our findings also suggested that different concentrations of Hcy exert different effects through different mechanisms. Therefore, interventions directed towards HHcy should be more specific, taking into account the Hcy concentration in each case and related mechanisms, for the treatment of vascular disorders in the treatment of vascular disorders. PPARa/g ligands clearly inhibit Hcy-induced iNOS. The induction of iNOS promoter hypermethylation by HHcy and the anti-atherosclerotic effect of PPARa/g ligands is a new, hitherto unreported element of their mechanisms. These findings reveal a novel role for Hcy in the pathogenesis of human vascular disease.
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