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Cardiovascular Research Advance Access originally published online on June 7, 2008
Cardiovascular Research 2008 80(1):151-158; doi:10.1093/cvr/cvn157
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Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2008. For permissions please email: journals.permissions@oxfordjournals.org

Homocysteine modulates the effect of simvastatin on expression of ApoA-I and NF-{kappa}B/iNOS

Leonie G. Mikael1,2 and Rima Rozen1,2,*

1 Department of Human Genetics, McGill University, Montreal Children's Hospital Research Institute, 4060 Ste. Catherine West, Suite 241, Montréal, Quebec, Canada H3Z 2Z3
2 Department of Pediatrics, McGill University, Montreal Children's Hospital Research Institute, 4060 Ste. Catherine West, Suite 241, Montréal, Quebec, Canada H3Z 2Z3

* Corresponding author. Tel: +1 514 412 4358; fax: +1 514 412 4331. E-mail address: rima.rozen{at}mcgill.ca

Received 18 March 2008; revised 30 May 2008; accepted 5 June 2008

Time for primary review: 27 days


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 5. Conclusions
 Funding
 References
 
Aims: Statins can ameliorate atherosclerosis by inhibition of cholesterol biosynthesis or by modulation of inflammation. In earlier work, we showed that homocysteine (Hcy) reduced synthesis of apolipoprotein A-I (ApoA-I). Our goal in this study was to determine whether Hcy could interfere with the ability of simvastatin to increase ApoA-I synthesis or to modify statin-dependent regulation of inflammatory factors.

Methods and results: Human HepG2 hepatocarcinoma cells and murine RAW264.7 macrophages were treated with simvastatin, with and without Hcy, to examine the expression of ApoA-I and nuclear factor-{kappa}B (NF-{kappa}B) or the NF-{kappa}B target, inducible nitric-oxide synthase (iNOS), respectively. Mice with methylenetetrahydrofolate reductase (Mthfr) deficiency, an animal model of hyperhomocysteinemia, were administered simvastatin (in diets or by injection) for in vivo assessment of these interactions. In HepG2 cells, Hcy reduced the statin-dependent increases in ApoA-I protein, mRNA, and ApoA-I promoter activity. In RAW264.7 macrophages, simvastatin decreased, whereas Hcy increased, the expression of pro-inflammatory NF-{kappa}B protein; in the presence of both Hcy and simvastatin, the pro-inflammatory effect of Hcy prevailed. Hcy increased mRNA levels of iNOS in the macrophage line; the combination of Hcy and simvastatin resulted in a trend towards greater induction. In mouse studies, simvastatin decreased cholesterol levels, but levels of ApoA-I in Mthfr-deficient mice remained lower than those in Mthfr+/+ mice. Simvastatin injection increased iNOS protein and mRNA levels in peripheral blood of hyperhomocysteinemic Mthfr-deficient mice, but not in Mthfr+/+ mice. The drug also increased MTHFR protein in cells and mouse liver, an effect that was modified by Hcy.

Conclusion: These findings provide a link between statins and folate-dependent Hcy metabolism, and suggest that Hcy interferes with some anti-atherogenic and anti-inflammatory properties of simvastatin. Our work may have clinical relevance for hyperhomocysteinemic individuals on statin therapy.

KEYWORDS Homocysteine; Statin; Apolipoprotein AI; MTHFR; Inducible nitric-oxide synthase; Nuclear factor-{kappa}B


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 5. Conclusions
 Funding
 References
 
Statins, inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, are the first-line therapy in the treatment of cholesterol-induced cardiovascular disease (CVD).1 They inhibit endogenous cholesterol synthesis by blocking the rate-limiting step and are highly effective in reducing levels of low-density lipoprotein (LDL) and very-low-density lipoprotein (VLDL). Statins are also modestly effective in raising levels of high-density lipoprotein (HDL) and lowering triglycerides.2,3 Recent findings indicate that, in addition to modulating plasma lipids, statins exert pleiotropic anti-inflammatory effects which might contribute to their therapeutic role in CVD.4 The protective effects of statins on the vascular system include improvement of endothelial function, stabilization of fibrous plaques, decreased vascular inflammation, and reduction of C-reactive protein.5 In the cerebral circulation, statins up-regulate endothelial nitric-oxide synthase (eNOS) and inhibit inducible nitric-oxide synthase (iNOS), actions that may have neuroprotective potential.6

Increased plasma homocysteine (Hcy) levels have become widely accepted as an independent risk factor for vascular disease.7,8 Hyperhomocysteinemia (HHcy), a condition that arises from disrupted Hcy metabolism, is caused by genetic and dietary deficiencies and has been implicated in the pathophysiology of atherosclerosis. A polymorphism (677C->T) in the enzyme methylenetetrahydrofolate reductase (MTHFR) is the most common genetic cause of mild HHcy.9 In a previous study, we showed that HHcy is associated with decreased Apolipoprotein A-I (ApoA-I) expression in humans and hyperhomocysteinemic Mthfr-deficient mice. We suggested that HHcy could increase the risk of atherosclerosis by decreasing ApoA-I through modulation of peroxisome-proliferator-activated receptor-alpha (PPAR{alpha}).10 The literature is mixed regarding the use of mice as an animal model to study the lipid-lowering effect of statins.11 Some studies have reported a decrease in ApoA-I levels following statin treatment in rodents, whereas others have seen an increase or no effect.12–14

Atherosclerosis is characterized by infiltration of monocytes into the arterial wall. The monocytes differentiate into macrophages and subsequently produce factors that promote atherosclerosis.15 One factor is nitric oxide (NO), which is regulated by one of three isoforms of NO synthase: eNOS, iNOS, and neuronal NOS (nNOS). eNOS-derived NO, at low concentrations, may modulate vasodilation and act as a haemodynamic regulator.16 iNOS, expressed mainly in macrophages, is responsible for the release of high NO levels. Excessive NO production due to elevated iNOS may exert cytotoxic effects on vascular cells. The iNOS gene promoter contains multiple cytokine-responsive elements including several nuclear factor-{kappa}B (NF-{kappa}B) binding sites17 and its expression is controlled by the activation of NF-{kappa}B.16

Hcy is known to activate NF-{kappa}B via oxidative stress in endothelial cells18 and potentially through protein kinase C in smooth muscle cells.15 iNOS expression in vascular smooth muscle cells is believed to promote atherosclerosis by increasing local oxidative stress. In rat aortic smooth muscle cells, Hcy treatment was shown to increase iNOS mRNA, activate NF-{kappa}B, and potentiate NF-{kappa}B cytokine activation.19 In human endothelial and vascular smooth muscle cells, HMG-CoA reductase inhibitors down-regulate the activation of NF-{kappa}B and activator protein-1 (AP-1),20 and in rabbit peripheral blood mononuclear cells (PBMCs), simvastatin is also believed to reduce NF-{kappa}B activity.21 In murine RAW264.7 macrophages, statins inhibit expression of lipopolysaccharide- (LPS) induced iNOS22 and up-regulate haem-oxygenase-1 expression.23 However, the iNOS-blocking effect of statins is cell-type dependent.24

In the present study, we tested the effect of simvastatin in vivo in Mthfr-deficient mice, a well-characterized animal model for mild HHcy and MTHFR deficiency in human populations due to polymorphism at bp677. We designed experiments for both long- and short-term simvastatin administrations to study some cholesterol-dependent and cholesterol-independent effects of statin and to investigate the potential interactions with Hcy. Human HepG2 and murine RAW264.7 macrophages were used for in vitro examination of statin and Hcy interactions.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 5. Conclusions
 Funding
 References
 
2.1 Materials
DL-Hcy was purchased from Sigma, Apo-Simvastatin from Apotex Inc., simvastatin from Calbiochem. Cell lines were obtained from American Type Culture Collection. Diets were purchased from Harlan Teklad. Materials for cell culture, RNA extraction and RT–PCR were purchased from Invitrogen and Qiagen.

2.2 Experimental model
Male and female Mthfr+/+, Mthfr+/– and Mthfr–/– mice on a C57BL/6 background were bred and housed as previously described.10 All animal experimentation was approved by the Animal Care Committee of the Montréal Children's Hospital (A5006-01) which conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). At 5 months, mice were placed on a control rodent diet (TD.75020) or the same diet supplemented with 0.1% w/w Apo-Simvastatin (TD.06597) which was ground to a powder prior to incorporation into rodent chow, as others have reported.12 After 6 weeks on diets, mice were sacrificed, blood was obtained through cardiac puncture and livers were frozen. Plasma Hcy, total and HDL-cholesterol were measured as previously described.25

For the short-term simvastatin experiment, 6-month-old Mthfr+/+ and Mthfr+/– mice were injected with simvastatin which was dissolved in ethanol, diluted in saline and administered via a single intraperitoneal injection at a dose of 50 mg/kg body weight. The control group received ethanol diluted in saline as control vehicle. There were 7 mice per genotype per treatment group. 16 h after simvastatin administration, blood was collected into EDTA tubes for PBMC isolation. Blood was diluted in phosphate-buffered saline (PBS 1X pH 7.4) enriched with dextrose (PBS-Dextrose) in a 1:1 ratio and the cells were separated on Ficoll gradient (Ficoll-Paque PREMIUM, GE Healthcare) by centrifugation at 2000 g for 4 min. PBMCs were collected and washed twice with cold PBS-Dextrose for use in western blotting or RNA isolation.

2.3 Cell culture
Human HepG2 hepatocarcinoma cells and murine RAW264.7 macrophages were propagated at 37°C in 5% CO2 in DMEM-F12, and DMEM, respectively, containing 10% FBS, 100 µg/mL penicillin and streptomycin. For transfections and experiments involving treatment with compounds, cells were cultured in 24-well or 6-well plates.

2.4 Transient transfection studies
HepG2 cells were seeded at approximately 2.5 x 105 into 24-well plates and medium was changed regularly. At 90% confluence, cells were transfected with a human ApoA-I promoter construct either wild-type or mutated in the PPAR{alpha} response element (PPRE) that had been generated previously.10 Cells were contransfected with pCMV-β-galactosidase vector using LipofectamineTM 2000 Reagent in OPTI-MEM medium (Invitrogen) following the manufacturer's recommendations. Six hours after transfection, medium was replaced with fresh medium with or without Hcy and simvastatin, and the cells were harvested 16 h after treatment. Firefly luciferase activity was detected by a Luciferase Assay Kit (Promega) and transfection efficiencies were controlled by normalizing to β-galactosidase activity (AP-Biosystems) as previously described.10 Transfections were performed at least four times.

2.5 Western blotting
Cell lysates and mouse liver extracts were prepared and processed for electrophoresis and immunoblotting as described previously.10 Primary antibodies were anti-human ApoA-I (Calbiochem), anti-mouse ApoA-I (Biodesign International), anti-β-actin (Sigma), anti-albumin (Bethyl Laboratories), anti-NF-{kappa}B p65 (Santa Cruz Biotechnology), anti-iNOS (BD Biosciences), and anti-MTHFR.9 All primary antibodies were made in rabbit. Secondary antibody was peroxidase-coupled anti-rabbit IgG (Amersham). Signal detection was achieved with ECL Plus chemiluminescence system (Amersham) and exposure to X-ray films. Signals were quantified relative to control with Quantity One 4.1.0 software.

2.6 Quantitative real-time reverse transcription–PCR
RNA was isolated from liver, HepG2, RAW264.7 cells, and PBMC using RNeasy Mini kit (Qiagen) following the manufacturer's protocols. RNA was DNaseI-treated and reverse-transcribed using random primers. Real-time PCR was performed with the SyberGreen kit in Mx4000P QPCR system (Stratagene) or Biometra thermocycler. Primers for amplifying mouse and human ApoA-I, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), MTHFR, iNOS, and NF-{kappa}B are listed in Table 1. Samples were run in duplicate and the analysis of RT–PCR data was performed by normalizing against GAPDH.


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  		Table 1 Primer sequences used in q-RT–PCR

 
2.7 Statistical analysis
ANOVA and t-tests were used for statistical analyses with SPSS software. One-sample t-tests were used for comparison with untreated cells, and independent-sample t-tests were used to compare between treatments. P < 0.05 was considered significant.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 5. Conclusions
 Funding
 References
 
3.1 Effect of simvastatin and Hcy on ApoA-I and MTHFR protein levels in HepG2 cells
In the previous work, we showed that Hcy decreased ApoA-I expression in HepG2 cells.10 Here we investigated whether Hcy could interfere with the ability of simvastatin to induce ApoA-I. HepG2 cells treated with Hcy showed a significant decrease in ApoA-I protein compared with untreated cells after 6 h (Figure 1A and B). Simvastatin significantly increased ApoA-I levels 1.6-fold. When the cells were grown in the presence of both simvastatin and Hcy, levels of ApoA-I remained 30% lower compared to untreated cells and 57% lower compared to cells treated with simvastatin alone. Similar results were observed whether the cells were treated first with Hcy for 2 h followed by 4 h simvastatin treatment (Figure 1A, lane 5) or treated first with simvastatin for 4 h, followed by 2 h Hcy treatment (Figure 1A, lane 6) (P < 0.01 for all conditions compared to simvastatin alone). Hcy, therefore, counteracts ApoA-I induction by simvastatin. Methionine at the same concentration as Hcy did not affect simvastatin-dependent induction of ApoA-I (data not shown), consistent with a lack of effect of methionine alone on ApoA-I levels in our earlier report.10


Figure 1
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  		Figure 1 Effect of simvastatin (10 µmol/L) and Hcy (5 mmol/L) on ApoA-I and 70 kDa MTHFR protein in HepG2 cells after 6 h of treatment. (A) Representative western blot. Quantification of protein levels for (B) ApoA-I and (C) MTHFR relative to β-Actin (control). Cells treated with Hcy had decreased levels of ApoA-I and increased non-phosphorylated MTHFR (non-P, unfilled bars in C) compared to untreated cells. Cells treated with simvastatin had higher levels of ApoA-I and P-MTHFR (-P, filled bars). Hcy inhibits the simvastatin effect on ApoA-I and MTHFR whether the cells are treated with Hcy simultaneously with simvastatin (lane 4), preceding (lane 5), or following (lane 6) simvastatin treatment. Mean ± SEM of four independent experiments in duplicate. *P < 0.01 and **P < 0.05 compared to untreated, #P < 0.01 and •P < 0.05 compared to simvastatin-treated cells, t-tests.

 
We also examined the potential impact of simvastatin on MTHFR expression. MTHFR has two protein isoforms, 70 and 77 kDa, differing in the length of their coding sequence. The shorter 70 kDa isoform can be post-translationally modified by phosphorylation and, therefore, exists as phosphorylated (P-MTHFR) and non-phosphorylated (non-P-MTHFR) species. In HepG2 cells, only the 70 kDa isoform is present but can be observed as both P-MTHFR and non-P-MTHFR. Compared with untreated cells, treatment with Hcy significantly induced expression of non-P-MTHFR, whereas simvastatin induced P-MTHFR 2.4-fold (Figure 1A and C). In the presence of both the simvastatin and Hcy, the effect of Hcy was predominant, i.e. reduced levels of P-MTHFR (P < 0.05) compared to simvastatin treatment alone (Figure 1A, lanes 4–6).

Quantitative RT–PCR confirmed the effects of Hcy and simvastatin on ApoA-I mRNA expression, but no change was observed for MTHFR mRNA. Compared to untreated cells, Hcy reduced ApoA-I mRNA by 30%, simvastatin induced it by 40%, and in the presence of both compounds, ApoA-I reduction was 43% compared to simvastatin treatment alone and 20% lower compared to untreated cells (data not shown).

3.2 Effect of simvastatin and Hcy on ApoA-I promoter activity in HepG2 cells
To determine whether the simvastatin and Hcy interactions on ApoA-I expression were at the promoter level, HepG2 cells were transiently transfected with a human ApoA-I promoter construct, either wild-type (wt-ApoA-I) or mutated in a PPRE10 site (mut-ApoA-I). Addition of simvastatin increased wt-ApoA-I activity by 2.6-fold (Figure 2, P < 0.01). One mmol/L Hcy had no effect but 5 mmol/L Hcy decreased activity by 70% (P < 0.01) compared to transfections without treatment. This effect of Hcy on the ApoA-I promoter was consistent with our previous report.10 Compared to simvastatin treatment alone, activity of the wt-ApoA-I promoter in the presence of both simvastatin and Hcy was reduced by 54% (with Hcy at 1 mmol/L) and 85% (with Hcy at 5 mmol/L) demonstrating that Hcy inhibits statin-dependent ApoA-I up-regulation at the promoter level. The basal activity of the mutated promoter mut-ApoA-I was 50% that of wt-ApoA-I (P < 0.01); simvastatin increased its activity 1.6-fold and Hcy at 5 mmol/L decreased it by 80% compared to untreated cells. Compared to simvastatin treatment alone, mut-ApoA-I activity was reduced by 40% (with Hcy at 1 mmol/L) and 90% (with Hcy at 5 mmol/L) in the presence of both compounds. Mut-ApoA-I activity was significantly lower than that of wt-ApoA-I in all conditions. While 1 mmol/L Hcy alone had no effect, it was still able to significantly reverse simvastatin-induced activity of both wild-type and mutated ApoA-I promoters. The reduced, but not totally abolished, activity of mut-ApoA-I suggests that simvastatin may regulate ApoA-I through PPAR{alpha} and non-PPAR{alpha} mechanisms. Similar conclusions can be drawn for Hcy in these experiments, as reported in our earlier study.10


Figure 2
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  		Figure 2 Effect of simvastatin (10 µmol/L) and Hcy (1 or 5 mmol/L) on ApoA-I promoter activity in HepG2 cells. Simvastatin increased activity of the wild-type ApoA-I promoter construct (wt-ApoA-I) and an ApoA-I promoter construct mutated for PPRE (mut-ApoA-I). Five millimolar per litre Hcy reduced both basal and simvastatin-induced promoter activity, while 1 mmol/L Hcy reduced the simvastatin-induced activity. Mean ± SEM of four independent experiments in duplicate. *P < 0.01 and **P < 0.05 compared to untreated, #P < 0.05 compared to simvastatin-treated, •P < 0.01 compared to wt-ApoA-I for each treatment, t-tests.

 
3.3 Effect of simvastatin on cholesterol, ApoA-I, Hcy, and MTHFR levels in Mthfr-deficient mice
Wild-type and Mthfr-deficient mice were placed on control and simvastatin-containing diets for 6 weeks. On the control diet, Mthfr+/– and Mthfr–/– mice had significantly lower levels of plasma and liver ApoA-I protein as well as liver ApoA-I mRNA compared to Mthfr+/+ mice (data not shown). Simvastatin reduced levels of ApoA-I protein in plasma and liver without altering ApoA-I mRNA, and also lowered levels of total and HDL-cholesterol. However, levels of ApoA-I protein and HDL remained lower in Mthfr-deficient mice compared to Mthfr+/+ mice on both diets. The genotype differences for ApoA-I were observed in our previous study of Mthfr-deficient mice on a BALB/c background.10

Plasma Hcy levels were significantly higher in Mthfr+/– and Mthfr–/– compared to Mthfr+/+ mice, but there were no differences due to simvastatin. A negative correlation (r = –0.52, P < 0.01) was observed between total plasma Hcy and ApoA-I levels in mice on both diets. We previously observed a similar correlation in BALB/c mice.10

Liver MTHFR protein levels were also analysed in the afore-mentioned experiments. Figure 3A shows both the phosphorylated (P-MTHFR) and non-phosphorylated (non-P-MTHFR) forms of the 70 kDa MTHFR isoform from Mthfr+/+ mice on control (lanes 1–2) and simvastatin (lanes 3–4) diets. Mthfr+/– mice had lower MTHFR levels, as expected, in both dietary groups (Figure 3B, P < 0.001). Simvastatin increased MTHFR levels (P < 0.01), predominantly the phosphorylated isoform (Figure 3A); this pattern was similar to that observed for MTHFR in HepG2 cells (Figure 1A). Simvastatin did not alter Mthfr mRNA levels (data not shown).


Figure 3
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  		Figure 3 Effect of simvastatin on liver MTHFR protein levels in mice. (A) Representative western blot showing both phosphorylated (-P) and non-phosphorylated (non-P) 70 kDa MTHFR isoform from Mthfr+/+ mice on control (lanes 1–2) and simvastatin (lanes 3–4) diets. (B) Quantification of total MTHFR protein relative to β-Actin in Mthfr+/+ and Mthfr+/– mice, n = 5–11 animals per group. *P < 0.001 for genotype, #P < 0.01 for diet, ANOVA.

 
3.4 Effect of simvastatin and Hcy on NF-{kappa}B p65, MTHFR, and iNOS in RAW264.7 cells
To determine whether Hcy could interfere with some of the anti-inflammatory activities of simvastatin, we measured protein levels of the NF-{kappa}B p65 subunit, since Hcy can increase NF-{kappa}B activity15,18 and statins decrease expression of iNOS, a down-stream effector of NF-{kappa}B, in some cell types.22 In murine RAW264.7 macrophages treated with simvastatin (Figure 4A), there was a ~25% reduction in NF-{kappa}B p65 (P < 0.05) after 8 h, compared to untreated cells. Hcy treatment increased NF-{kappa}B p65 levels 1.5-fold (P < 0.05) compared to untreated cells. In the presence of both simvastatin and Hcy, there was a 2-fold increase in NF-{kappa}B p65 compared to simvastatin alone (P < 0.05) and no change compared to Hcy alone. This increase was not observed for NF-{kappa}B p65 mRNA, as assessed by semi-quantitative RT–PCR (data not shown).


Figure 4
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  		Figure 4 (A) Effect of simvastatin (50 µmol/L) and Hcy (5 mmol/L) on NF-{kappa}B p65 protein subunit and MTHFR protein, assessed by western blotting, in RAW264.7 macrophages after 8 h of treatment. Cells treated with simvastatin show a decrease in protein levels of NF-{kappa}B p65 relative to β-Actin and no significant change in MTHFR. Treatment with Hcy increases levels of NF-{kappa}B p65 and non-P-MTHFR. Simultaneous treatment with Hcy and simvastatin shows a similar increase in NF-{kappa}B to that seen with Hcy alone. Mean ± SEM of four experiments in duplicate. (B) Effect of simvastatin and Hcy on iNOS mRNA levels, assessed by semi-quantitative RT–PCR, in RAW264.7 macrophages after 6 h of treatment. Simvastatin treatment does not affect iNOS mRNA levels but Hcy treatment increases iNOS mRNA 1.8-fold compared to untreated cells. Treatment with both compounds induces iNOS over 2-fold compared to simvastatin treatment alone (P < 0.01) and 1.3-fold compared to Hcy treatment alone (P= 0.07). Values normalized to GAPDH. Mean ± SEM of three experiments in duplicate. *P < 0.05 compared to untreated cells, #P < 0.05 and **P < 0.01 compared to simvastatin treatment alone, t-tests.

 
Although treatment with simvastatin increased P-MTHFR in HepG2 cells (Figure 1), it had no significant effect on MTHFR protein in RAW264.7 cells (Figure 4A). Hcy increased non-P-MTHFR approximately 4-fold compared to untreated cells (Figure 4A), as it did in HepG2 cells (Figure 1A), and as we have reported elsewhere.26 This pattern for MTHFR did not change in the presence of both Hcy and simvastatin.

We also examined whether simvastatin and Hcy could modulate iNOS expression in RAW264.7 cells. Since endogenous levels of iNOS protein were barely detectable by immunoblotting, we measured iNOS mRNA by semi-quantitative RT–PCR. Simvastatin treatment did not affect iNOS mRNA. Hcy increased iNOS expression 1.8-fold compared to untreated cells (Figure 4B, P < 0.05). In the presence of both simvastatin and Hcy, this induction was significantly higher compared to simvastatin alone (2-fold increase, P < 0.01) and showed a borderline significant increase compared to Hcy alone (1.3-fold, P = 0.07). Total levels of PPAR{alpha} mRNA and protein remained unchanged in response to simvastatin or Hcy at the 8 h time point (data not shown).

3.5 Effect of simvastatin on iNOS in PBMCs of Mthfr-deficient mice
To examine the possible interaction between simvastatin and Hcy in the inflammatory response in vivo, we isolated mice PBMCs after an injection of simvastatin at a dose known to elicit an anti-inflammatory response.27 Because NF-{kappa}B could not be detected in these PBMCs, we measured iNOS again as a downstream effector of NF-{kappa}B. There were no differences in iNOS expression due to genotype in the control group (Figure 5A and B). However, simvastatin significantly increased iNOS levels in PBMCs (P < 0.001 for treatment, ANOVA). Since the ANOVA indicated that there was an interaction between simvastatin and genotype (P < 0.05), we performed t-tests which revealed that this treatment effect was significant only for the Mthfr+/– group (P < 0.001). The 2-fold increase in iNOS expression in hyperhomocysteinemic Mthfr+/– mice in the simvastatin group, compared to Mthfr+/– mice in the control group was confirmed at the mRNA level by semi-quantitative RT–PCR (data not shown).


Figure 5
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  		Figure 5 Effect of simvastatin on iNOS protein in PBMCs from mice, after a single intraperitoneal injection of either simvastatin (50 mg/kg body weight) or ethanol (control vehicle). n = 6–7 mice per group. iNOS levels from PBMCs were measured after 16 h. (A) Representative western blot showing iNOS from control and simvastatin-treated Mthfr+/+ and Mthfr+/– mice. (B) Quantification of iNOS relative to β-Actin. *P < 0.001 for treatment and P < 0.05 for interaction between genotype and treatment, ANOVA. #P < 0.001 for comparison of Mthfr+/– in simvastatin group to Mthfr+/– in control group, t-test.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
5. Conclusions
 Funding
 References
 
Statins have become an important first-line therapy in the prevention and treatment of CVD through an inhibition of cholesterol synthesis and modulation of the inflammatory response. With the recognition of HHcy as a risk factor for CVD, the interaction between Hcy and statins may have clinical relevance. We suggest that Hcy can interfere with some anti-atherogenic and anti-inflammatory properties of simvastatin based on experiments with Mthfr-deficient mice as an in vivo model of HHcy, and in vitro studies in HepG2 cells and RAW264.7 macrophages.

4.1 Hcy inhibition of simvastatin-induced ApoA-I production in vitro
Several mechanisms have been proposed by which elevated levels of Hcy can lead to atherosclerosis including direct effects on lipid metabolism and transport, protein modification, oxidative stress, and/or endoplasmic reticulum stress.28 We explored another mechanism and demonstrated that HHcy reduced the expression of ApoA-I in men with coronary artery disease, Mthfr-deficient mice, and HepG2 cells.10 In this study, we show that simvastatin induces ApoA-I protein and mRNA, as well as ApoA-I promoter activity in HepG2 cells; these observations are consistent with earlier studies in HepG2 cells and hamster hepatocytes.29,30 However, Hcy inhibits the statin-dependent increases in ApoA-I protein, mRNA, and promoter activity in HepG2 cells. Another group has confirmed our initial findings on Hcy regulation of ApoA-I protein levels, although, in contrast to our results, they did not observe changes in ApoA-I mRNA levels.31

In the promoter studies, Hcy at concentrations of both 1 and 5 mmol/L antagonized the effect of simvastatin on the wild-type ApoA-I promoter and a promoter that was mutated at a PPRE site. Statins can upregulate ‘A-site’ activity of the human ApoA-I promoter through activation of PPAR{alpha}.29 Our data are consistent with this finding, but they also allude to activity in the promoter region independent of the PPRE site since simvastatin was able to enhance activity in the mutant as well as wild-type promoters.

4.2 Simvastatin effects in vivo
Levels of total and HDL-cholesterol and ApoA-I protein decreased in plasma and liver of mice following treatment with simvastatin-containing diets in all three MTHFR genotype groups. These decreases in ApoA-I due to simvastatin have been reported in some13 but not all rodent studies.12–14 The statin-dependent decrease in HDL in mice is distinct from the statin effects on HDL in human populations. In this study, we observed a statin-dependent increase in human ApoA-I promoter activity in HepG2 cells. Whether statin has the same effect on the murine ApoA-I promoter remains to be determined.

Despite the differences in statin action between mice and humans, MTHFR deficiency in our mice was associated with reduced plasma and liver ApoA-I levels, and reduced HDL-cholesterol and total cholesterol, in mice on control and statin-containing diets. Since we and others10,31 have demonstrated that HHcy in humans is also associated with decreased ApoA-I levels, it is possible that statins may not be able to completely reverse the Hcy-induced ApoA-I decrease.

Simvastatin treatment of mice did not significantly affect plasma Hcy levels; this observation has been reported in some but not all clinical reports on statins.32,33 Ridker et al.33 reported that both lovastatin and placebo were associated with Hcy lowering. Statins also reduced the recurrence of coronary events in individuals with high Hcy, suggesting that HHcy did not interfere with statin action. Our findings in mice are consistent with this observation, since statins effectively reduced cholesterol in all groups of mice, yet the differences between genotypes in HDL-cholesterol were still evident. In a smaller study that reported a reduction in Hcy after simvastatin therapy, decreased Hcy was not associated with a reduction in total cholesterol, triglycerides, LDL, and ApoB.34 In addition to the afore-mentioned types of studies that are centred on statin therapy, it would be useful to examine the interaction between Hcy and statin in the clinical trials that are currently underway to target Hcy lowering.

Simvastatin may exert some of its pleiotropic effects indirectly through an increase in MTHFR expression, which regulates Hcy levels, as observed in HepG2 cells and mouse liver. Although simvastatin increased MTHFR levels in liver, plasma Hcy was not influenced by simvastatin. This finding could be due to the fact that plasma levels reflect export of Hcy from many tissues. Nonetheless, it is possible that there are tissue-specific changes in Hcy or related metabolites, due to the drug's effect on MTHFR. MTHFR is also required for generation of S-adenosylmethionine which is utilized as a carbon donor in a wide variety of methylation reactions. We have recently suggested that MTHFR may be important in cell survival, since it is up-regulated during endoplasmic reticulum stress.26 The intriguing connection between simvastatin and MTHFR warrants further investigation, particularly in individuals with the MTHFR polymorphism.

4.3 Hcy interference with anti-inflammatory properties of simvastatin
During the onset of atherosclerosis, NF-{kappa}B activation is associated with increased expression of inflammatory factors.18 Active NF-{kappa}B binds to {kappa}B-binding motifs in the promoters of several immunomodulator genes including iNOS. In RAW264.7 macrophages, expression of the p65 subunit of NF-{kappa}B decreased in response to simvastatin and increased in response to Hcy. In the presence of both compounds, NF-{kappa}B p65 levels were 2-fold higher compared to simvastatin treatment alone demonstrating that Hcy may counteract the effect of simvastatin. We also observed that Hcy induced mRNA levels of iNOS, and that simvastatin was unable to reverse this induction. To investigate the potential interaction between HHcy and statins in the inflammatory pathway in vivo, we examined a short-term exposure to simvastatin in wild-type and hyperhomocysteinemic mice. The increase in iNOS protein and mRNA in hyperhomocysteinemic mice following statin treatment, but not in normohomocysteinemic mice, provides evidence for an interaction.

The iNOS-modulating effect of statins is dependent on the cell type and on the specific statin. Statins can reduce LPS-induced iNOS production and NF-{kappa}B activation in RAW264.7 macrophages depending on the level of their hydrophobicity.22 Lovastatin inhibits iNOS expression in rat macrophages,35 whereas atorvastatin up-regulates iNOS expression in vascular smooth muscle cells. In rat aortic vascular smooth muscle cells, atorvastatin up-regulates iNOS expression and activity resulting in increased NO production, but this induction is not NF-{kappa}B-dependent.36 Interesting to note that in this same cell line, Hcy was shown to induce iNOS expression and NO production via NF-{kappa}B activation.19 Our results are consistent with this finding since Hcy increased the expression of the NF-{kappa}B p65 subunit and of iNOS in RAW264.7 macrophages. The increase in iNOS levels in PBMCs of Mthfr+/– mice in response to simvastatin is consistent with our in vitro data and suggests that simvastatin may have different effects on the inflammatory response depending on the levels of Hcy. The molecular mechanisms by which statins and Hcy interact in the inflammatory cascade require dissection, particularly since atherosclerosis is associated with chronic inflammation.

Peritoneal macrophages cultured from PPAR{alpha}+/+ mice injected with simvastatin respond in vitro by a reduction of LPS-induced iNOS, whereas PPAR{alpha}–/– mice do not respond, suggesting that simvastatin activity is through a PPAR site.27 Our in vitro and in vivo data suggest that the effect of Hcy and simvastatin on iNOS was greater than either compound alone. Whether these effects on iNOS occur through Hcy- and statin-dependent modulation of PPAR{alpha} remain to be determined. We have nonetheless reported a putative PPRE in the sequence upstream of the Mthfr promoter and shown that PPAR{alpha} up-regulates MTHFR.10

4.4 Limitations of the study
In order to increase intracellular Hcy levels in cultured cells, 5 mmol/L Hcy is required in vitro.10,26,37 Although supraphysiological, this concentration is necessary to attain a transient increase in intracellular Hcy and does not affect cell viability.37 We used DL-Hcy in our experiments, which are only 50% biologically active in cells, thereby necessitating higher concentrations of the compound. Nonetheless, in the promoter studies, Hcy at a concentration of 1 mmol/L was effective in inhibiting simvastatin-induced ApoA-I promoter activity. Furthermore, some of the interactions between statin and Hcy metabolism were reproduced in vivo. The interaction between Hcy and simvastatin on iNOS in RAW264.7 macrophages (Figure 4B) was also detected in mouse PBMCs, where simvastatin increased iNOS protein (Figure 5) in Mthfr-deficient mice that have a modest elevation in plasma Hcy.


   5. Conclusions
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 5. Conclusions
Funding
 References
 
We suggest that Hcy may influence the anti-atherogenic properties of simvastatin through regulation of ApoA-I and the anti-inflammatory properties of simvastatin through modulation of the NF-{kappa}B/iNOS pathway. Our observations are based on cultured human cells, cultured murine macrophages, and in vivo data from PBMCs of HHcy mice administered simvastatin. It would be important to determine whether similar phenomena occur in vivo in humans. Studies in human populations are, therefore, required to determine whether statin therapy is influenced by HHcy or by the presence of the common MTHFR polymorphism.


   Funding
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 5. Conclusions
 Funding
References
 
Canadian Institutes of Health Research (MOP-43232).


   Acknowledgements
 
We thank Qing Wu and Andrea Lawrance for technical assistance.

Conflict of interest: none declared.


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 5. Conclusions
 Funding
 References
 

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