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Cardiovascular Research 2005 67(2):333-341; doi:10.1016/j.cardiores.2005.03.016
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Copyright © 2005, European Society of Cardiology

Simvastatin inhibits NOR-1 expression induced by hyperlipemia by interfering with CREB activation

Javier Crespo, José Martínez-González, Jordi Rius and Lina Badimon*

Centro de Investigación Cardiovascular, CSIC/ICCC, Hospital de la Santa Creu i Sant Pau, Sant Antoni Maria Claret # 167, 08025 Barcelona, Spain

* Corresponding author. Tel.: +34 93 5565880; fax: +34 93 5565559. Email address: lbadimon{at}csic-iccc.santpau.es

Received 19 January 2005; revised 16 March 2005; accepted 21 March 2005


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Acknowledgments
 References
 
Objective: Our aim was to investigate whether neuron-derived orphan receptor-1 (NOR-1), an early gene induced by low density lipoproteins (LDL) in vascular smooth muscle cells (VSMC), is regulated by statins.

Methods: NOR-1 expression was analyzed in human VSMC in culture and in vivo in the aorta of diet-induced hyperlipemic pigs by RT-PCR and real-time PCR. [3H]Thymidine incorporation was used as an index of DNA synthesis. NOR-1 promoter activity was analyzed using a luciferase reporter system. Cyclic AMP response element binding protein (CREB) binding was assessed by EMSA and ELISA and CREB activation (phosphorylation in Ser133) by Western blotting.

Results: Simvastatin inhibited NOR-1 expression induced by LDL in VSMC and by hypercholesterolemia in the abdominal aorta of hyperlipemic pigs. The inhibition of the isoprenylation of geranylgeranylated proteins by simvastatin was key in both NOR-1 up-regulation and DNA synthesis induced by LDL. Inhibitors of RhoA (toxin B and exotoxin C3) and ROCK (Y-27632) mimicked the effect of simvastatin on NOR-1. Similarly both simvastatin treatment and cells transfected with a RhoA dominant-negative (RhoAT19N) showed inhibition of LDL-induced NOR-1 promoter activity. These effects were associated to the interference of the activation of CREB, a key transcription factor involved in NOR-1 induction. Finally, simvastatin prevented LDL induction of a reporter construct containing four consensus CRE and inhibited the expression of SMemb (a marker for dedifferentiated VSMC) dependent on CREB.

Conclusions: NOR-1 is a target for simvastatin in the vascular wall. We identified NOR-1 and CREB as key transcription factors mediating the effect of statins on VSMC proliferation through a mechanism dependent on RhoA/ROCK.

KEYWORDS Atherosclerosis; Gene expression; Lipoproteins; Smooth muscle cells; Statins


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 Acknowledgments
 References
 
Vascular smooth muscle cells (VSMC) migration and proliferation play key roles in vascular remodeling associated to atherosclerotic diseases [1]. Recently, neuron-derived orphan receptor-1 (NOR-1) has been described as a key early gene involved in VSMC proliferation [2,3]. NOR-1 is up-regulated by coronary angioplasty [2] and is over-expressed in atherosclerotic lesions from patients with coronary artery disease (CAD) [2,4]. NOR-1 is induced by growth factors and other key molecules involved in atherogenesis such as low density lipoproteins (LDL) [2,3,5]. LDL promote growth-related events, including up-regulation of key transcription factors and DNA synthesis, in a LDL receptor (LDL-R) independent manner [6–10].

Statins are competitive inhibitors of 3-hydroxy-3-methylglutharyl coenzyme A (HMG-CoA) reductase, the rate-limiting step in cholesterol synthesis [11]. Statins have been shown to be efficacious in reducing cardiovascular morbidity and mortality in primary and secondary prevention clinical trials [12,13]. The main pharmacological effect of statins is the reduction of plasma LDL-cholesterol levels; however, results from both clinical trials and from experimental animal models [14–18] suggest that statins could directly modulate vascular function. These pleiotropic effects of statins seem to be a result of the ability of these drugs to interfere postranslational processing of isoprenylated proteins involved in cell signaling (i.e. Rho and Ras proteins) and to regulate key genes controlling VSMC function [15,16,19–22].

In the present study we show that statins prevent the up-regulation of NOR-1 induced by LDL in vitro in human VSMC and in vivo in the abdominal aorta of diet-induced hyperlipemic pigs. Simvastatin inhibited LDL-induced NOR-1 expression interfering RhoA/ROCK signaling and cyclic AMP response element binding protein (CREB) activation. Simvastatin could modulate other CREB-dependent genes such as SMemb, a marker for dedifferentiated VSMC. Thus, both inactivation of CREB and down-regulation of NOR-1 could be key events in switching off downstream genes involved in VSMC cell growth.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 Acknowledgments
 References
 
2.1. VSMC cultures
VSMC were obtained by a modification of the explant technique [19], from human non-atherosclerotic coronary arteries of hearts removed in transplant operations. The study was approved by the Reviewer Institutional Committee on Human Research of the Hospital of Santa Creu i Sant Pau that conforms to the Declaration of Helsinki. VSMC (from 3rd to 5th passages) were cultured in M199 (Gibco) supplemented with 20% fetal calf serum (FCS), 2% human serum, 2 mmol/L L-glutamine and antibiotics (100 U/mL penicillin and 0.1 mg/mL streptomycin). Cells were arrested in medium containing 0.4% FCS for 48 h and then were stimulated with LDL (30 mg protein/dL, for 1 h). Simvastatin (kindly provided by MSD), atorvastatin (kindly provided by Pfizer), mevalonate, geranylgeranyol and farnesol, were added 18 h before treatment. When other inhibitors were used, VSMC were pre-incubated with them for 30 min (unless otherwise state), these inhibitors were: toxin B (Tox B, an inhibitor of Rho proteins, Calbiochem), Y-27632 (a ROCK inhibitor; Tocris Coukson Ltd.,) and exotoxin C3 (a RhoA inhibitor; Calbiochem), the pre-incubation time with this compound was 24 h. The inhibitors did not produce any effect on cell morphology, cell apoptosis (assessed by staining with Hoechst 33258 colorant) or cell viability analyzed measuring the mitochondrial dehydrogenase activity by a commercial kit (XTT based assay for cell viabilityTM; Roche). In transfection experiments we used rat VSMC obtained by the explant technique [19]. Rat VSMC were cultured in DMEM supplemented with 10% FCS and antibiotics (100 U/mL penicillin and 0.1 mg/mL streptomycin).

2.2. Animals
Female pigs (Landrace/Largewhite [Piensos Victoria SA, Barcelona, Spain], mean body weight at initiation: 32 ± 4 kg) were randomized into two groups: normolipemic animals (n = 6) which were fed with a normal chow diet and hyperlipemic animals (n = 10) which were fed with a cholesterol-rich diet (2% cholesterol; 1% cholic acid; 20% beef tallow) for 100 days as described [22]. The hyperlipemic group was divided in two subgroups, one treated with simvastatin (2.5 mg/kg) (n = 5) and one treated with placebo (n = 5). Plasma cholesterol levels and hematological parameters were measured at baseline and at sacrifice. Because atherosclerotic lesions develop initially in the abdominal aorta, rings of this vessel were collected and frozen in liquid N2 to measure gene expression. All procedures were in accordance with institutional guidelines and followed the American Physiological Society and NIH guidelines for animal research.

2.3. Plasma biochemistry
Plasma total cholesterol was determined with an automatic analyzer (Kodak Ektachem DT System). Plasma lipoproteins were fractionated using the validated methods of the Lipid Research Clinic Program [23] and quantified spectrophotometrically (Kontron Instruments).

2.4. Lipoprotein isolation
Human LDL were isolated from pooled sera of healthy blood donors of the Barcelona area as previously described [20]. The content of protein (BCA protein assayTM; Pierce) and cholesterol (Cholesterol assay kitTM, RefLab) in the lipoproteins were determined by colorimetric assays. The absence of contamination by other lipoproteins was determined by electrophoresis on agarose gels (Paragon Electrophoresis kit, Beckman). Lipoproteins were endotoxin free, as determined by the Limulus Amebocyte Lysate pyrogen testing system (Biowhittaker Inc.), and did not contain any detectable levels of thiobarbituric-acid-reactive substances (TBARS).

2.5. RT-PCR
Total RNA was isolated using UltraspecTM (Biotex Laboratories) according to the manufacturer's recommendations and was reverse-transcribed. NOR-1 mRNA levels were analyzed by polymerase chain reaction (PCR) using the PCR DIG Labeling Mix (Roche) as described [2]. The specific NOR-1 oligonucleotides used were: 5'-AGGGCTGCAAGGGCTTTTTCAAGAGA-3' and 5'-TGCTTTCTACAGGAGCTGCT-3'. The specific c-fos oligonucleotides used were: 5'CATCATCCAGGCCCAGTG-3' and 5'-CTGAGCGAGTCAGAGGAAGG-3'. For both genes amplification was carried out by 24 cycles: denaturation 94 °C for 30 s; annealing, 61 °C for 1 min and polymerization, 72 °C for 1 min 30 s. PCR products were resolved by electrophoresis in agarose gels and transferred onto nylon membranes (NytranTM plus; Schleicher and Shuell) by a standard capillary technique. Blots were UV cross-linked. Detection of digoxigenin-labeled nucleic acids was performed with an anti-digoxigenin antibody linked to alkaline phosphatase and disodium 3-(4-methoxyspiro{1,2-dioxetano-3,2'-(5'-chloro)trycyclo[3.3.1.13,7]Decan}-4-yl) phenylphosphate (CSPD) was used as substrate. Levels of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were used to normalize results [2,3].

2.6. Real-time PCR
The inhibitory effect of simvastatin on NOR-1 mRNA levels was quantified using real-time PCR. Total RNA was obtained as described above. Assays-on-DemandTM (Applied Biosystems) of TaqManTM fluorescent real-time PCR primers and probes were used for NOR-1 (Hs00175077_m1) and for GAPDH (4326317E) and 18 S ribosomal RNA (4319413E) as endogenous controls.

2.7. Determination of DNA synthesis
Arrested human VSMC were stimulated with LDL (30 mg protein/dL) in medium containing 0.5 µCi/mL of [3H]thymidine (Amersham) in the presence or absence of simvastatin, mevalonate, geranylgeranyol or farnesol, and [3H]thymidine incorporation was determined as described [2,3].

2.8. Western blot analysis
Human VSMC were cultured and stimulated with lipoproteins as indicated above in presence or absence of simvastatin. Cell monolayers were washed with PBS and lysed with lysis buffer [1% SDS in 10 mmol/L Tris–HCl (pH 7.4), 1 mmol/L ortovanadate] or with Subcellular Proteome Extraction kit (Calbiochem) to obtain membrane and citoplasmatic protein. Proteins were analyzed by Western blot analysis as described previously [16]. Blots were incubated with an antibody against human CREB phosphorylated in Ser133 (C9102; Sigma); human CREB (C-21; Santa Cruz Biotechnology); human RhoA (26C4, Santa Cruz Biotechnology); human myosin heavy chain isoforms (SMemb, SM1 and SM2) [24] kindly provided by Dr. M. Aikawa (Brigham and Women's Hospital, Harvard Medical School, Boston, USA). Detection was performed using a horseradish peroxidase-labeled anti-rabbit IgG or horseradish peroxidase-labeled anti-mouse IgG, and the SupersignalTM detection system (Pierce). Equal loading of protein in each lane was verified staining filters with Pounceau.

2.9. Electrophoretic mobility shift assay (EMSA)
Nuclear extracts (3 µg) from VSMC and a doubled stranded probe corresponding to the sequence of human NOR-1 promoter (from –84 to –41; Nor/3CRE) containing three putative CRE motifs were used in an EMSA as described [2]. Super-shift experiments were performed with antibodies against human CREB (C-21, Santa Cruz Biotechnology).

2.10. CREB binding by ELISA
Binding of nuclear extracts from VSMC, obtained with Transfactor Extraction kit (Clontech), to CRE consensus oligonucleotides were analyzed by an ELISA based method using the BD MercuryTM Transfactor CREB-1 kit (Clontech) according to the manufacturer's protocol.

2.11. Transient transfection and luciferase assays
Rat VSMC were transfected with luciferase expression vectors using LipofectamineTM Reagent and PlusTM Reagent (Invitrogen) according to the manufacturer's protocol. The constructs used were: pNOR{alpha}/–1703 containing the human NOR-1 promoter (from –1703 to +264) [25], kindly provided by Dr. N. Ohkura (Growth Factor Division, National Cancer Center Research Institute, Tokyo, Japan), a RhoA dominant-negative (RhoAT19N) and p-CREB-Luc (Stratagene) a reporter vector which contains the luciferase reporter gene driven by a basic promoter element (TATA box) plus four CRE cis-enhancer elements. A reporter plasmid containing a minimal promoter corresponding to dihydrofolate reductase was generated cloning this promoter [26], kindly provided by Dr. CJ. Ciudad (Facultad de Farmacia, Universidad de Barcelona, Barcelona, Spain), into PGL-3 basic vector (Promega). The resulting plasmid (p410-DHFR-Luc) was used to assess that simvastatin did not affect the overall VSMC transcription rate. Transfected cells were arrested for 48 h and then were stimulated with LDL for 4 h. Luciferase activity was measured in cell lysates using Luciferase assay kit (Promega). pSVβ-gal (Promega) was used as an internal control.

2.12. Statistical analysis
Results are expressed as mean ± SEM. A Stat View II (Abacus Concepts) statistical package for the Macintosh computer system was used for all analysis: multiple groups were compared by one-factor ANOVA, followed by Fisher PLSD to assess specific group differences.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 Acknowledgments
 References
 
3.1. Statins inhibit NOR-1 up-regulation in VSMC in culture and in vivo in the vessel wall
To analyze the effect of statins on NOR-1 mRNA levels induced by LDL, arrested VSMC were pre-incubated with simvastatin (increasing concentrations) or atorvastatin (10 µM) and then were induced with LDL (30 mg/dL, for 1 h). Statins inhibited LDL-induced NOR-1 expression in a dose-dependent manner (Fig. 1A).


Figure 1
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  		Fig. 1 Simvastatin inhibits NOR-1 expression in LDL-induced human VSMC and in abdominal aorta of hyperlipemic pigs. A. RT-PCR showing NOR-1 expression in human coronary SMC treated with simvastatin (SIM 1, 10 or 20 µmol/L) or atorvastatin (ATOR, 10 µmol/L) and stimulated with LDL (30 mg/dL, 1 h). Blots are representative of three independent experiments. B. Real time PCR showing NOR-1 expression in abdominal aorta samples from normolipemic pigs (NORMO) and from animals feed with a hyperlipemic diet (HYPER) treated or not with simvastatin (HYPER+SIM). P<0.05: *vs. NORMO; {dagger}vs. HYPER.

 
To determine whether the in vitro results were indeed showing a potential regulation of NOR-1 by statins in the vascular wall, NOR-1 mRNA levels were analyzed in abdominal aorta samples from normolipemic pigs and from animals fed with a hypercholesterolemic diet either treated or untreated with simvastatin. Plasma cholesterol levels were higher in hypercholesterolemic animals as a result of an increase in LDL levels, and they were not significantly modified by simvastatin (Table 1). NOR-1 mRNA levels were highly increased in abdominal aorta of hypercholesterolemic animals and they were significantly decreased by simvastatin treatment (Fig. 1B).


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  		Table 1 Plasma lipid profile in normolipemic, hyperlipemic and hyperlipemic/simvastatin pigs

 
3.2. Geranylgeranylated proteins are key in DNA synthesis and NOR-1 expression induced by LDL
LDL-induced VSMC DNA synthesis inhibited by simvastatin was completely restored by mevalonate o geranylgeranyol but not by farnesol (Fig. 2A). Interestingly, simvastatin-inhibited NOR-1 expression was also specifically prevented by geranylgeranyol (Fig. 2B). In contrast, farnesol but not geranylgeranyol restored simvastatin-inhibited c-fos expression levels. Mevalonate, geranylgeranyol and farnesol alone did not produce any effect on basal NOR-1 expression (data not shown). Using real-time PCR we determined that the inhibitory effect of simvastatin on NOR-1 expression levels was completely abrogated by geranylgeranyol while farnesol did not produced a significant effect (Fig. 2C).


Figure 2
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  		Fig. 2 Simvastatin inhibition of DNA synthesis and NOR-1 expression induced by LDL is dependent on geranylgeranylated proteins. A. DNA synthesis ([3H]thymidine incorporation) in VSMC stimulated with LDL in the presence or absence of simvastatin (SIM, 20 µmol/L). The effect of mevalonate (MVA, 100 µmol/L), farnesol (FAR, 15 µmol/L) and geranylgeranyol (GER, 15 µmol/L) on SIM inhibition of DNA synthesis is shown. Results represent the mean ± SEM of three independent experiments performed in triplicate. B. NOR-1 and c-fos mRNA levels in VSMC treated as indicated in A. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a control. Blots are representative of three independent experiments. C. Quantitative real-time PCR data showing the inhibition by simvastatin (20 µmol/L) on LDL-induced NOR-1 mRNA levels and the reversion by GER. Results represent the mean ± SEM of three independent experiments performed in triplicate. P<0.05: *vs. LDL-treated cells; {dagger}vs. LDL/SIM and LDL/SIM/FAR. CT, control (non-induced cells).

 
3.3. RhoA/ROCK pathway is involved in NOR-1 up-regulation by LDL
Simvastatin interfered isoprenylation of RhoA (Fig. 3A), a geranylgeranylated protein involved in cell signaling [27,28] that could be implicated in NOR-1 up-regulation. In agreement with this result several inhibitors of the RhoA/ROCK pathway (tox B [an inhibitor of Rho family of small GTPases], exotoxin C3 [an inhibitor of RhoA] and Y-27632 [an inhibitor of ROCK]) mimicked the inhibitory effect of simvastatin on NOR-1 expression (Fig. 3B,C).


Figure 3
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  		Fig. 3 RhoA is involved in NOR-1 up-regulation by LDL. A. Western blot showing the inhibition of RhoA isoprenylation by simvastatin. RhoA protein levels in cytoplasm (cytop.) and membrane (memb.) of VSMC treated with simvastatin alone (SIM, 20 µmol/L) or simvastatin plus mevalonate (MVA, 100 µmol/L), geranylgeranyol (GER, 15 µmol/L) or farnesol (FAR, 15 µmol/L). Blots are representative of three independent experiments. B. NOR-1 mRNA levels analyzed by RT-PCR corresponding to human VSMC stimulated with LDL (30 mg/dL for 1 h) in the absence or presence of toxin B (Tox B, 2 or 5 ng/mL), Y-27632 (10 or 20 µmol/L) or exotoxin C3 (25 µmol/L). Blots are representative of three independent experiments. C. Graph showing the densitometric results of blots shown in B. Data are mean ± SEM. P<0.05: *vs Control cells; {dagger}vs cells treated with LDL alone. CT, control (non-induced cells).

 
In cells transfected with a construct containing NOR-1 promoter (pNOR{alpha}/–1703) simvastatin was also able to inhibit NOR-1 promoter activity, effect that was reverted by mevalonate and geranylgeranyol, but not by farnesol (Fig. 4A). In addition, in co-transfection experiments RhoAT19N (a RhoA dominant-negative) prevented LDL-induced NOR-1 promoter activity. In contrast, neither simvastatin nor RhoAT19N significantly modified the transcriptional activity of a minimal promoter (p410-DHFR-Luc) [26] (Fig. 4B). These results suggest that the inhibitory effect produced by both simvastatin and RhoAT19N on NOR-1 transcriptional activity was not due to a general (unspecific) inhibitory effect on cell transcription rate.


Figure 4
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  		Fig. 4 Simvastatin inhibits LDL-induced NOR-1 promoter activity. A. Rat VSMC were transfected with pNOR{alpha}/-1703 (white bars). Arrested cells were induced with LDL (30 mg/dL for 4 h) in the presence of simvastatin alone (SIM, 20 µmol/L) or simvastatin plus mevalonate (MVA, 100 µmol/L), geranylgeranyol (GER, 15 µmol/L) or farnesol (FAR, 15 µmol/L). Black bars shown the results corresponding to the cotransfection with pNOR{alpha}/-1703 and RhoAT19N. CT, control (non-induced cells). Results represent the mean ± SEM of three independent experiments performed in triplicate. P<0.05: *vs cells treated with LDL alone; {dagger}vs cells treated with LDL+SIM; {ddagger}vs cells treated with LDL and transfected with pNOR{alpha}/-1703 alone. B. Luciferase activity corresponding to cells transfected with a reporter plasmid containing a minimal promoter (p410-DHFR-Luc). Experimental conditions are indicated in A. CT, control (non-induced cells). Results represent the mean ± SEM of three independent experiments performed in triplicate.

 
3.4. Simvastatin inhibits CREB phosphorylation and activity
Since CREB is a key transcription factor involved in LDL-induced NOR-1 expression [3], we analyzed the effect of simvastatin on CREB. To analyze the effect of simvastatin on CREB binding, nuclear extracts from VSMC preincubated with simvastatin and induced with LDL were analyzed by EMSA and ELISA. Simvastatin did not influence the binding of CREB either to specific sequences containing CRE sites present in NOR-1 promoter (analyzed by EMSA) (Fig. 5A) or to consensus CRE sequences (analyzed by ELISA) (Fig. 5B). However, simvastatin significantly inhibited early CREB phophorylation induced by LDL by 64 ± 5.8% (Fig. 6) (CREB phosphorylated levels: 100 ± 5% in LDL-treated cells versus 36 ± 5.8% in LDL+simvastatin-treated cells). This inhibitory effect was reverted by mevalonate and geranylgeranyol but not by farnesol. Exotoxin C3 and Y-27632 mimicked the inhibitory effect produced by simvastatin.


Figure 5
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  		Fig. 5 Simvastatin does not inhibit CREB–CRE binding. A. EMSA showing the ability of nuclear extracts from human VSMC to bind to the probe Nor/3CRE. Neither LDL nor simvastatin affected CREB binding to CRE motifs present in NOR-1 promoter. The super-shift effect of anti-CREB antibodies is shown. Blots are representative of three independent experiments. B. Binding of CREB to consensus CRE sequences analyzed by ELISA (see Methods). The results corresponding to nuclear extracts (20 µg) from control cells and from cells induced with LDL (30 mg/mL, 10 min) in the presence or absence of SIM (20 µmol/L) is shown. Control: non-induced cells; Comp: 20 µg of competitor oligodeoxynucleotides+nuclear protein of cells induced with LDL; CRE+: Mutant DNA-Coated well control+nuclear protein of cells induced with LDL. Results represent the mean ± SEM (n = 3 independent experiments performed in triplicate). P<0.05: *vs Control cells or cells treated with LDL alone or LDL+SIM.

 

Figure 6
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  		Fig. 6 Simvastatin inhibits CREB activation. A. Western blot from human VSMC showing CREB activation (phosphorylation in Ser133) by LDL (30 mg/dL, 10 min) and its inhibition by simvastatin (SIM, 20 µmol/L), and Y-27632 (20 µmol/L) and exotoxin C3 (C3, 25 µmol/L). The effect of mevalonate (MVA, 100 µmol/L), geranylgeranyol (GER, 15 µmol/L) and farnesol (FAR, 15 µmol/L) on SIM effect is shown. Blots are representative of three independent experiments. B. Graph showing the densitometric results of the experiments shown in A. Data are mean ± SEM. P<0.05: *vs Control cells; {dagger}vs cells treated with LDL alone, with LDL+SIM+MVA or with LDL+SIM+GER. CT, control (non-induced cells).

 
Finally, in transfection experiments both simvastatin treatment and RhoAT19N-transfection significantly reduced the transcriptional activity of a reporter plasmid containing 4 CRE sites (p-CRE-Luc) (Fig. 7A). In addition, to evidence whether simvastatin could specifically modulate the expression of other CREB-depending genes, we analyzed the effect of simvastatin on non-muscle myosin heavy chain-B (also known as SMemb) [29]. Indeed, simvastatin inhibited LDL-induced protein expression levels of SMemb, without modification of other SMC markers non-regulated by CREB (SM1 and SM2) (Fig. 7B,C).


Figure 7
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  		Fig. 7 Simvastatin potentially regulates other CREB-dependent genes. A. Simvastatin and the RhoA dominant negative (RhoAT19N) inhibit the promoter activity induced by LDL (30 mg/dL for 4 h) of a construct composed only of CRE boxes (p-CREB-Luc). CT, control (non-induced cells); p-CREB-Luc alone (white bars); cotransfection with RhoAT19N (black bar). Results represent the mean ± SEM of three independent experiments performed in triplicate. P<0.05: *vs control cells (CT); {dagger}vs cells treated with LDL alone. B. Western blots showing levels of SMemb and other VSMC markers (SM1 and SM2) in cells exposed to LDL (30 mg/dL for 24 h) in the presence or absence of simvastatin (20 µmol/L) and geranylgeranyol (GER, 15 µmol/L). C. Graph showing the result corresponding to the quantification of SMemb protein levels. Data from two independent experiments performed in triplicate. P<0.05: *vs Control cells; {dagger}vs cells treated with LDL alone or with LDL+SIM+GER. CT, control (non-induced cells).

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
Acknowledgments
 References
 
NOR-1 is a member of the NGFI-B family of orphan nuclear receptors involved in cell proliferation in different cell types including VSMC [2,3,5,30], that is induced by coronary angioplasty in vivo [2] and is over-expressed in atherosclerotic lesions from CAD patients [2,4]. Recently, we have show that NOR-1 is involved in LDL-induced VSMC proliferation and that LDL induces NOR-1 expression by a CREB-dependent mechanism [3]. Here, we show that simvastatin, an HMG-CoA reductase inhibitor that inhibits VSMC proliferation [15,16], inhibits the NOR-1 up-regulation induced by hyperlipidemia in the vascular wall of hypercholesterolemic pigs and in LDL-induced VSMC, by interfering RhoA signaling and CREB activation.

Simvastatin, at concentrations that efficiently inhibit cell proliferation, inhibits NOR-1 expression induced by LDL in human VSMC. In addition, in the porcine model simvastatin prevented the up-regulation of NOR-1 observed in the abdominal aorta of animals fed with a hyperlipemic diet. Under our experimental conditions of prolonged administration of a cholesterol and saturated fat rich diet (100 days) to promote cholesterol intestinal absorption, simvastatin did not significantly reduced plasma LDL-cholesterol levels but strongly reduced NOR-1 expression in the porcine vessels (quantitatively assessed by real time PCR). These results suggest a direct effect of simvastatin on the vascular wall, which is in concordance with previous results showing vascular effect of these drugs unrelated with plasma cholesterol lowering [15–18].

The effect of simvastatin on both DNA synthesis and NOR-1 expression induced by LDL in VSMC was reverted by geranylgeranyol but not by farnesol. In contrast, the LDL-induced expression of c-fos was reverted by farnesol but not by geranylgeranyol, consistent with the well-known role of Ras (a farnesylated protein) in c-fos up-regulation [31]. Our present results suggest a prominent role of NOR-1 over other genes, such as the proto-oncogene c-fos, in the growth-related effects promoted by LDL in VSMC.

Regarding geranylgeranylated proteins involved in these effects, RhoA seems to play a key role because simvastatin interfered with RhoA membrane translocation and inhibitors of RhoA (exotoxin C3), Rho proteins (toxin B) or ROCK (Y-27632) mimicked the simvastatin inhibitory effect on NOR-1 expression. The critical role of Rho in the simvastatin anti-proliferative effects on VSMC is in agreement with previous papers showing that VSMC proliferation inhibited by statins is highly dependent on geranylgeranylated proteins [19,32,33]. In fact, newly synthesized RhoA is isoprenylated and translocated to membranes coincident with progression of the G1 to S phase in growing cells [34], and the down-regulation of p27Kip1 by RhoA mediates the induction of DNA synthesis in VSMC [33,35]. Previously we showed that LDL-induced NOR-1 is mediated by GPCR, from our present data we propose that NOR-1 up-regulation by LDL would be modulated by GPCR-dependent pathways via RhoA/ROCK. In fact, the RhoA/ROCK pathway is a mediator of GPCR signaling [36] which play a crucial role regulating different processes including cell cycle and growth processes and that has been involved in neointimal formation in balloon-injured arteries [37].

Since LDL induced NOR-1 in a CREB-dependent manner [3], we analyze whether simvastatin could modulate NOR-1 preventing CREB function. Neither LDL nor simvastatin affected CREB binding to CRE boxes present in the NOR-1 promoter or to oligonucleotides containing consensus CRE sequences, according with the ability of CREB to constitutively bind to its response element [38]. However, simvastatin significantly reduced CREB activation (phosphorylation in Ser133) and NOR-1 promoter activity in cell transfected with a luciferase reporter system. In addition, specific inhibition of RhoA or ROCK also reduced CREB phosphorylation, and a RhoA dominant negative (RhoAT19N) completely prevented NOR-1 promoter activity. Therefore, our results show that in VSMC functional RhoA is required for LDL-induced cell signaling leading to CREB activation. The inhibition of CREB by statins could be relevant beyond NOR-1 down-regulation because recent papers argue for a main role of CREB in VSMC survival/proliferation and vascular remodeling processes [39–41]. In fact, our results using a reporter plasmid containing 4 CRE boxes (p-CRE-Luc) suggest that simvastatin could potentially regulate other genes containing functional CRE sites. Moreover, we show that LDL induced while simvastatin inhibited the protein levels of SMemb, a marker for dedifferentiated VSMC, that increases in intimal SMC of hypercholesterolemic animal models [42] and after balloon angioplasty [43]. Since SMemb is activated by CREB (through a functional CRE site present in its promoter) [29], we could consider that LDL may up-regulate its expression through a CREB-dependent mechanism while simvastatin reverts this effect on the basis of its interference in RhoA and CREB activation. However, further experiments are needed to demonstrate this relationship.

In summary, the vascular effects of LDL could be modulated by simvastatin treatment both by controlling plasma LDL levels and by directly regulating cell signaling pathways. Our results emphasize the role of RhoA in the growth promoting effects induced by LDL and suggest that NOR-1 and CREB could be key transcription factors mediating the in vivo effects of statins on the vascular wall. Further studies, including immunohistochemical analyses, are needed to confirm the involvement of NOR-1 in the vascular effects of statins.


   Acknowledgments
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Acknowledgments
References
 
This work has been possible thanks to funds provided by FIS-PI020361, FIS C03-01/Recava the Freedom to Discover Program of Bristol–Myers Squibb Foundation (USA) and Catalana-Occidente. We thank Dr. M. Aikawa, for providing the antibodies against human MHC isoforms and Dr. C.J. Ciudad for the p410-DHFR minimal promoter. We thank Guthrie cDNA Resource Center (www.cdna.org) for the gift of the RhoAT19N construction. The authors thank Dra. Sonia Sanchez from the animal facility of our center and the Heart Transplant Team of the Division of Cardiology and Cardiac Surgery of the Hospital de la Santa Creu i Sant Pau for their collaboration. Authors are indebted to the technical assistance provided by Silvia Aguiló. Javier Crespo and Jordi Rius have been recipients of a Research Fellowship from Fundación de Investigación Cardiovascular and DURSI, respectively.


   Notes
 
Time for primary review 25 days


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Acknowledgments
 References
 

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