Nifedipine inhibits vascular smooth muscle cell proliferation and reactive oxygen species production through AMP-activated protein kinase signaling pathway

Vascular Pharmacology 56 (2012) 1–8

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Vascular Pharmacology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / v p h

Nifedipine inhibits vascular smooth muscle cell proliferation and reactive oxygen species production through AMP-activated protein kinase signaling pathway Jin Young Sung, Hyoung Chul Choi ⁎ Department of Pharmacology, Aging-associated Vascular Disease Research Center, College of Medicine, Yeungnam University, Daegu 705-717, Republic of Korea

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Article history: Received 22 March 2011 Received in revised form 24 May 2011 Accepted 13 June 2011 Keywords: AMP-activated protein kinase (AMPK) Nifedipine Vascular smooth muscle cells (VSMCs) Proliferation Reactive oxygen species

a b s t r a c t The dihydropyridine calcium channel blocker nifedipine induces specific pharmacological effects by binding to L-type calcium channels, which results in a reduced calcium influx in vascular smooth muscle cells (VSMCs) and is currently employed in antihypertensive drug. Dihydropyridine calcium channel blocker is reported to reduce oxidative stress and exhibits anti-proliferative effect in VSMCs. VSMCs are useful in the study of atherosclerosis because they show cell proliferation and reactive oxygen species (ROS) production with growth factor. To determine the mechanisms involved in these effects, we investigated the influence of nifedipine-induced AMP-activated protein kinase (AMPK) activation on VSMC proliferation and ROS production by using rat aortic VSMCs in vitro and in vivo. Nifedipine induced phosphorylation of AMPK in a dose-and time-dependent manner, and inhibited rat VSMC proliferation and ROS production following stimulation with 15% fetal bovine serum (FBS). Nifedipine also blocked the FBS-stimulated cell cycle progression through the G0/G1 arrest. Compound C, a specific inhibitor of AMPK, or AMPK siRNA reduced the nifedipine-mediated inhibition of VSMC proliferation. As an upstream kinase, LKB1 is required for nifedipineinduced AMPK activation in VSMCs. 7 days oral administration of 1 mg/kg nifedipine resulted in activation of LKB1 and AMPK in vivo. These data suggest that nifedipine suppress the VSMC proliferation and ROS production via activating LKB1-AMPK pathway. © 2011 Elsevier Inc. All rights reserved.

1. Introduction Structural changes of blood vessel are observed in many vascular diseases such as atherosclerosis. An abnormal proliferation of vascular smooth muscle cells (VSMCs) originating from the media can be considered a key event in atherogenesis (Ross, 1993; Tsaousi et al., 2011; Wang et al., 2010). Thus, compounds that interfere with the machinery of cell proliferation can be considered potential drugs of interest for the treatment of atherosclerosis. VSMCs express a large number of voltage-dependent calcium channels that actively participate in the responsiveness to various agonists through the control of intracellular Ca 2+ and Ca 2+ movements are undoubtedly involved in cell-cycle progression (Hughes, 1995; Machaca, 2010; Skelding et al., 2011). Therefore, VSMC proliferation is linked under the control of intracellular Ca 2+ concentration and the voltage-activated Ca2+ channels play an important role in intracellular Ca 2+ homeostasis (Short et al., 1993). There are several evidences that calcium channel blockers (CCBs) inhibit VSMC proliferation (Clunn et al., 2010; Jackson and Schwartz, 1992; Wu et al., 2009). Dihydropyridinetype CCBs are important drugs in the treatment of hypertension and ⁎ Corresponding author at: Department of Pharmacology, College of Medicine, Yeungnam University, 317-1 Daemyung5dong, Namgu, Daegu, Republic of Korea, 705717. Tel.: + 82 53 620 4353; fax: + 82 53 656 7995. E-mail address: [email protected] (H.C. Choi). 1537-1891/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.vph.2011.06.001

coronary heart disease. Among dihydropyridine-based CCBs, the L-type Ca 2+ channel antagonist nifedipine inhibited VSMC proliferation in vitro and suppressed thickening caused by balloon angioplasty-induced injury in rats in vivo (Hirata et al., 2000). In this view where metabolic requirements for cell proliferation are largely identical in all normal and tumor cells, conserved low-energy checkpoint such as the AMP-activated protein kinase (AMPK) system functions as a suppressor of cell proliferation (Kim and Choi, 2010; Shackelford and Shaw, 2009). AMPK is a serine–theronine kinase involved in the regulation of cell metabolism (Hardie, 2000; Wang et al., 2009) and suppresses VSMC proliferation through inhibiting cell cycle progression and regulation of mitosis (Igata et al., 2005; Lizcano et al., 2004). Activation of AMPK required the phosphorylation at Thr172 in the activation loop of the α subunit by upstream kinase of AMPK (Hawley et al., 1995). Two upstream of AMPK got out through various studies. LKB1 and Ca 2+/calmodulin-dependent protein kinase kinase β (CaMKKβ) are the most characterized upstream activating AMPK. LKB1 is constantly active within cells. Phosphorylation and activation of AMPK by LKB1 requires an increase in AMP or AMP mimetic in response to drugs in cultured cells (Sakamoto et al., 2004). By contrast, CaMKKβ activates AMPK in an AMP independent manner, which is instead by a rise in the intracellular Ca 2+ concentration in response to nutrients or physiological stimulation (Hurley et al., 2005). Therefore, many of the cardiovascular benefits and the VSMC protection conferred by nifedipine are similar to those elicited by

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2.3. Western blot analysis

activation of AMPK. We hypothesized that nifedipine may exert beneficial effects on the cardiovascular system through activation of AMPK. Hence, the aim of this study was to investigate whether nifedipine affected VSMC proliferation and reactive oxygen species (ROS) production through activation of AMPK, and to identify the underlying signaling pathways in VSMCs.

Whole cell extracts were prepared by lysing the cells in pro-prep protein extract buffer. The protein concentration was quantified with protein assay reagent from Bio-Rad (Hercules, CA, U.S.A.). Equal amounts of protein were mixed with Laemmli Sample Buffer (BioRad) and heated for 5 min at 100 °C before loading. Total protein samples (30 μg) were subjected to 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) for 1 h 30 min at 100–120 V. The separated proteins were electrophoretically transferred onto a PVDF membrane for 1 h 20 min at 100 V using SD Semi-dry Transfer Cell. The membranes were blocked with 5% non-fat milk in Tris buffered saline (TBS) containing 0.05% Tween 20 (TBS-T) for 2 h at room temperature. The membranes were then incubated with the primary antibodies at a dilution of 1:1000 by overnight at 4 °C in TBST. The membranes were then washed with four changes of wash buffer (0.05% Tween 20 in TBS) and incubated for 1 h at room temperature in TBS containing anti-rabbit (Stressgen, Ann Arbor, MI, U.S.A.) or anti-mouse IgG (Santa Cruz, CA, U.S.A.) antibodies. Finally, after three more rinses with wash buffer, the membranes were exposed to ECL and ECL Plus western blot analysis detection reagents.

2. Materials and methods 2.1. Materials Nifedipine was purchased from Sigma-Aldrich (St. Louis, MO). Dulbecco's modified eagle medium (DMEM) and fetal bovine serum (FBS) were purchased from Thermo Scientific (Logan, UT, U.S.A.). Proprep protein extract buffer was purchased from Intron Biotechnology (Sungnam, Korea). Antibodies against AMPK, phospho-AMPK (Thr172), Acetyl CoA Carboxylase (ACC), phospho-ACC (Ser79), and phospho-LKB1 (Ser428) were obtained from Cell Signaling Technology (Beverly, MA, U.S.A.). A monoclonal antibody against β-actin was purchased from Sigma-Aldrich (St. Louis, MO). Compound C, AMPK inhibitor, was provided by Calbiochem (La Jolla, CA, U.S.A.). AMPK siRNA and LKB1 siRNA were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, U.S.A.). STO-609-acetic acid and ethylene glycol-bis (2-aminoethylether)N,N,N′,N′-tetraacetic acid (EGTA) were purchased from Sigma-Aldrich (St. Louis, MO).

2.4. Cell proliferation assay We used 2 different methods, MTT assay and bromodeoxyuridine incorporation (BrdU). VSMCs were seeded on 24-well plates at 1 × 104 cells per well in DMEM supplemented with 10% FBS. After different treatments, 50 μl of 1 mg/ml MTT solution was added to each well (0.1 mg/well) and incubated for 4 h. The supernatants were aspirated, and the formazan crystals in each well were solubilized with 200 μl dimethyl sulfoxide (DMSO). An aliquot of this solution (100 μl) was placed in 96-well plates. Cell proliferation was assessed by measuring the absorbance at 570 nm using a microplate reader. We examined another cell proliferation, as a synthetic nucleotide 5′-bromodeoxyuridine (BU colorimetric, 1 μM) incorporation belonged to the improved generation of kits for measuring DNA synthesis (Roche Diagnostics GmbH, Mannheim, Germany). This assay is based on the detection of BrdU

2.2. Cell culture Sprague–Dawley rats were anesthetized by pentobarbital (50 mg/kg). VSMCs were isolated from thoracic aorta and the connective tissue was removed. Tissue was processed using a 1 mm chop setting in a 10 cm culture dish, and cultured with 50% FBS-DMEM with 1% antibiotics and incubated in a CO2 incubator (5% CO2, 37 °C). Aortic VSMCs were grown in DMEM with 10% FBS and 1% antibiotic (penicillin 10,000 U/ml). We used VSMCs from 6 to 8 passages at 70–90% confluence in 10 cm dishes and cell growth was arrested by incubation of the cells in serum-free DMEM for 24 h prior to use.

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Fig. 1. Effect of nifedipine on phosphorylation of AMPK and ACC in VSMCs. VSMCs were incubated with different concentrations of nifedipine (1, 10, and 100 nM) for 1 h (A and C) or with 100 nM nifedipine for various durations (B and D). AMPK phosphorylation at Thr172 (p-AMPK) and ACC phosphorylation at Ser79 (p-ACC) were determined by western blot analysis. Representative blots (A and B) and densitometric analyses (C and D) are shown. Values are mean ± S.E.M from 3 independent measurements. *p b 0.05 versus the control (0 nM nifedipine or time 0).

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incorporated into the genomic DNA of proliferating cells. Cells grown in 96-well tissue-culture microplates are labeled by the addition of BrdU for 4 h. During this labeling period, BrdU is incorporated in place of thymidine into the DNA of cycling cells. After removing the labeling medium, the cells are fixed, and the DNA is denatured in one step by adding this kit. The complexes are detected by the subsequent substrate reaction. The reaction product is quantified by measuring the absorbance at 547 nm using a microplate reader. 2.5. Flow cytometric analysis for cell cycle VSMCs were treated with or without nifedipine for 48 h. Cells (1 × 10 4) were trypsinized and fixed in 95% ethanol overnight. Fixed cells were stained with propidium iodide (PI) (50 μg/ml) for 30 min at 37 °C. PI enters the cells and stains the nucleus. PI stained cells were filtered using a 5 ml polystyrene round bottom tube with a cellstrainer cap prior to flow cytometry. All flow cytometry measurements were done using a FACSCalibur (Becton Dickinson, San Jose, CA, U.S.A.). Cell cycle analysis was performed using Cell-Quest prosoftware.

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2.6. Transfection of AMPK and LKB1 siRNA Transfection of VSMCs with siRNA was performed using lipofectamine 2000 reagent, according to the manufacturer's instructions. Aliquots of 1 × 10 4 cells were plated on 6 wells on the day before transfection and grown to about 70% confluence. The cells were then transfected with 10 μM AMPK and LKB1 siRNA (Santa Cruz Biotechnology Inc., Santa Cruz, CA, U.S.A.) plus 100 pmol of lipofectamine for 6 h in Opti-MEM® I reduced serum medium (Invitrogen, Carlsbad, CA, U.S.A.). Following an incubation period of 48 h, the AMPK and LKB1 protein levels were measured using western blot analysis, while the cell proliferation was analyzed using the MTT assay. 2.7. Measurement of intracellular ROS Intracellular ROS production was measured by 2′,7′-dichlorodihydrofluorescein fluorescence using live imaged laser scanning and fluorescence-activated cell sorting (FACS) analysis. Cells were pretreated with various agents (nifedipine, compound C, AMPK siRNA, and LKB1 siRNA) and incubated in the dark for 10 min in the presence of

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Fig. 2. Effect of nifedipine on cell proliferation and cell cycle progression. VSMC proliferation was determined by the MTT assay (A) and BrdU incorporation assay (B). Cells were stimulated with 15% FBS for 48 h and then treated with nifedipine (1, 10, and 100 nM) for 24 h. Data are represented as the mean ± S.E.M (n = 4). Nifedipine regulates VSMC proliferation through cell cycle arrest (C). Cells were seeded onto 6 well plates at a density of 1 × 104 cells/ml, treated with 15% FBS for 48 h and then treated with nifedipine (1, 10, and 100 nM) for 24 h. Cells were detached by trypsinization and studied with flow cytometry. *p b 0.05 versus the control, † p b 0.05 versus 15% FBS.

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10 μM DCF-DA. ROS generation was detected from the oxidation of DCF fluorescence using a fluorescein isothiocyanate (FITC) filter set. For FACS analysis, cells were treated with nifedipine and incubated with DCF-DA at a final concentration of 10 μM for 30 min at 37 °C. Then the treated cells were washed twice with PBS to remove the extracellular compounds and resuspended in 0.5 ml PBS for cytometry analysis. The change in fluorescence intensity was monitored by flow cytometry (FACScan, Becton-Dickinson, San Jose, CA, U.S.A.).

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2.8. Oral administration of nifedipine Adult male C57BL/6 mice (aged 12–14 weeks, weighing 20–23 g) were used in the present study. Nifedipine (1 mg/kg per day) was administered orally for 7 days (Inaba et al., 2009). The mice were anesthetized by pentobarbital (50 mg/kg). Aorta was isolated, immediately carried out western blot analysis.

Results are expressed as mean± SEM from at least three independent experiments. Differences between data sets were assessed by oneway analysis of variance (ANOVA) or Bonferroni's test. 3. Results 3.1. Nifedipine activates AMPK in VSMCs Incubation of VSMCs with nifedipine (1, 10, 100 nM for 1 h or 100 nM for 1–12 h) resulted in dose-and time-dependent phosphorylation of AMPK at Thr172 (Fig. 1). This phosphorylation was correlated with AMPK activity. At the same time, we also measured the phosphorylation of ACC at Ser79, phosphorylation of ACC is an indicator of AMPK activity. Consistent with AMPK activation, dose- and timedependent increases of ACC phosphorylation were observed by nifedipine treatment. The AMPK activation reached its maximum at 1–6 h after nifedipine treatment. 3.2. Nifedipine suppresses VSMC proliferation stimulated by 15% FBS and induces G0/G1 cell cycle arrest We examined the effect of nifedipine on VSMC proliferation and cell cycle arrest. Nifedipine decreased VSMC proliferation induced by 15% FBS in a dose-dependent manner. In MTT assay to assess the viability and the proliferation of cells, nifedipine gradually decreased VSMC proliferation (Fig. 2A). Similar result was obtained in BrdU incorporation of DNA synthesizing cells (Fig. 2B). To examine the cell cycle progression, we investigated cell cycle arrest using a flow cytometry. Compared with cells treated with 15% FBS, nifedipine significantly increased the cells in G0/G1 phase (Fig. 2C). 3.3. Inhibition of AMPK activation restores VSMC proliferation To further demonstrate the interaction of nifedipine and AMPK activation, we examined the effects of AMPK inhibitors on VSMC proliferation. Nifedipine decreased VSMC proliferation stimulated with 15% FBS (from 167.5 ± 1.6% to 109.9 ±5.8%). Compound C, a specific inhibitor of AMPK, reduced the nifedipine-mediated inhibition of VSMC proliferation (from 109.9 ± 5.8% to 145.2 ± 2.1%). Genetic inhibition of AMPK with siRNA also restored the nifedipineinduced anti-proliferative effect (from 109.9 ± 5.8% to 145.2 ± 5.1%) (Fig. 3A). The nifedipine-induced AMPK and ACC phosphorylation in VSMCs was significantly inhibited by pretreatment of compound C or AMPK siRNA (Fig. 3B). These results indicate that nifedipine-induced AMPK activation inhibits VSMC proliferation.

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Fig. 3. Inhibitory effect of compound C and AMPK siRNA on the anti-proliferative action of nifedipine. Cells were stimulated with 15% FBS for 48 h and then treated with compound C (10 μM) or transfected with AMPK siRNA in the presence of nifedipine. Cell proliferation was determined by the MTT assay (A). Data are represented as the mean ± S.E.M (n= 4). Protein expressions of p-AMPK, AMPK, and p-ACC were determined by western blot analysis (B). Representative blots from three independent experiments were shown. *p b 0.05 versus the control, †p b 0.05 versus 15% FBS, #p b 0.05 versus 15% FBS + nifedipine.

3.4. Inhibition of CaMKKβ does not alter nifedipine-induced AMPK activation Recent studies have suggested that CaMKKβ functions as AMPK upstream kinase under conditions in which intracellular Ca2+ increases (Hawley et al., 2005). To determine whether Ca2+ or CaMKKβ is required for nifedipine-induced AMPK activation, we first investigated the effect of STO-609, a CaMKKβ inhibitor, on nifedipine-induced AMPK activation in VSMCs. STO-609 doesn't affect nifedipine-enhanced phosphorylation of AMPK, ACC, and LKB1 (Fig. 4A). These results suggest that CaMKKβ was not required of nifedipine-induced AMPK activation in VSMCs. To further establish that LKB1 is required for nifedipine-activated AMPK in VSMC, we suppressed LKB1 expression in VSMC by using LKB1 siRNA. LKB1 siRNA, but not con siRNA, suppressed the protein expression of LKB1. Corroborating previous studies suggested a requirement of LKB1 for phosphorylation of AMPK, we found that LKB1 siRNA inhibited nifedipine-induced phosphorylation of both AMPK and ACC (Fig. 4B). These experiments suggest that LKB1 is required for nifedipine-induced AMPK activation in VSMCs. We also investigated that nifedipine-induced AMPK activation in the absence of Ca2+. Fig. 4C showed that Ca2+ chelation with EGTA (10 mM) doesn't affect nifedipine-enhanced phosphorylation of AMPK, ACC, and LKB1.

J.Y. Sung, H.C. Choi / Vascular Pharmacology 56 (2012) 1–8

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Fig. 4. Nifedipine-induced AMPK activation in VSMCs is independent of CaMKKβ. Preincubation of CaMKKβ inhibitor STO-609 abolishes nifedipine-induced phosphorylation of AMPK, LKB1, and ACC in VSMCs (A). VSMCs were treated with or without STO-609 (10 μM) for 1 h and then treated with nifedipine (100 nM) for 1 h. The blot is a representative of three blots from three independent experiments. LKB1 is required for nifedipine-induced AMPK activation in VSMCs (B). VSMCs were transfected with control siRNA or LKB1 siRNA for 48 h. The transfected cells were treated with nifedipine (100 nM) for 1 h. The blot is a representative of three blots from three independent experiments. Effect of Ca2+ chelator EGTA on nifedipine-induced phosphorylation of AMPK, LKB1, and ACC in VSMCs (C). VSMCs were treated with nifedipine (100 nM) for 1 h in the presence or absence of Ca2+ chelator EGTA (10 mM, 1 h prior to nifedipine). Nifedipine-induced phosphorylations of AMPK, LKB1, and ACC were not altered by pretreatment with EGTA. Densitometric analyses are shown for Fig.4D. Values are mean ± S.E.M from 3 independent measurements. *p b 0.05 versus the control.

3.5. Nifedipine-induced AMPK activation attenuates intracellular reactive oxygen species production

levels. These findings indicate that nifedipine activates AMPK in aortic tissue in vivo, as observed in cultured VSMCs.

We tested whether nifedipine-induced AMPK activation decreased ROS production in VSMCs and observed ROS level by fluorescence microscope after treating with pharmacological inhibitor of AMPK, compound C, AMPK siRNA, and LKB1 siRNA. After exposure by 15% FBS for 24 h, cell showed morphological characteristics of ROS production. The 15% FBS-generated ROS was decreased by nifedipine, but compound C, AMPK siRNA, and LKB1 siRNA restored ROS production (Fig. 5A). Next, a FACS analysis presented in Fig. 5B shows 15% FBS increased ROS levels, whereas pre-treatment with nifedipine (100 nM) blocked 15% FBS-induced ROS production (FBS 161% versus nifedipine 104%). We then studied the effects of compound C, AMPK siRNA, and LKB1 siRNA on suppressed ROS production. Nifedipine-induced AMPK activation significantly decreased ROS production. These effects of nifedipine were abolished by compound C, AMPK siRNA, and LKB1 siRNA. These data showed that nifedipine inhibited 15% FBS-induced ROS production through AMPK activation.

4. Discussion

3.6. Nifedipine-induced AMPK activation in vivo We wanted to show in vivo administration of nifedipine activates AMPK in aorta. The nifedipine oral dose (1 mg/kg) in this study was used in cardiovascular experimental setting (Inaba et al., 2009). This paper reported that oral administration with 1 mg/kg nifedipine resulted in a distinct change in the size of neointimal formation of injured artery model. As observed in Fig. 6A and B, oral administration of nifedipine (1 mg/kg for 7 days) significantly increased the phosphorylation of AMPK, ACC, and LKB1 without any change in total AMPK protein

Ca 2+ channel blockers have been demonstrated to reduce the severity of experimentally induced atherosclerosis (Jackson et al., 1989). Also, Ca 2+ channel blockers inhibited the development of atherosclerosis due to their inhibition of VSMC proliferation (Stepien et al., 2002). Therefore, it is interesting to determine the mechanisms whereby these drugs exert their anti-atherosclerotic actions. Since VSMC proliferation plays a critical role in the development of atherosclerosis, we investigated the influence of nifedipine-induced AMPK activation on the proliferation of VSMC isolated from rat aorta, by using a model of cell culture. In the present study, we demonstrated for the first time that nifedipine activated AMPK by phosphorylating AMPK at Thr 172 and inhibited 15% FBS-induced proliferation and ROS production in VSMCs. The extent of AMPK phosphorylation at Thr 172 strongly reflects its activity (Hardie, 2004). Nifedipine also increased phosphorylation of ACC, an AMPK downstream substrate, at Ser 79. We think that Ca 2+ channel blockers, at least, dihydropyridine-based Ca 2+ channel blockers has the ability to activate AMPK. In our unpublished experiment, amlodipine also activates AMPK in VSMC. Nifedipineinduced AMPK activation has an attractive characteristic, because AMPK was reported to mediate beneficial and bio-protective effects of metformin and statin (Choi et al., 2008; Zhou et al., 2001). AMPK is a serine/threonine protein kinase, which serves as an energy sensor in eukaryotic cells. Several studies revealed that AMPK activation strongly suppressed cell proliferation in normal cells as well as in tumor cells (Motoshima et al., 2006). These effects of AMPK appear to be

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Fig. 5. Effect of nifedipine on ROS production induced by 15% FBS. ROS generated viable cells have a uniform bright green color in their cytoplasm and nuclei (A). For observation of intracellular ROS by fluorescence microscope, cells were treated with 15% FBS for 24 h in the presence of nifedipine and then treated with compound c (10 μM) for 2 h or transfected with AMPK siRNA, LKB1 siRNA. DCF-DA (10 μM) is applied for 10 min. ROS production was assessed by FACS analysis (B). *p b 0.05 versus the control, † p b 0.05 versus 15% FBS, #p b 0.05 versus 15% FBS + nifedipine.

mediated through multiple mechanisms including regulation of the cell cycle and inhibition of protein synthesis. In this study, nifedipine decreased VSMC proliferation induced by 15% FBS in a dose-dependent

manner and the anti-proliferative mechanism turned out to be a cell cycle arrest at G0/G1 phase. Although the precise mechanism of nifedipine remains to be clarified, the results of our study indicate that

J.Y. Sung, H.C. Choi / Vascular Pharmacology 56 (2012) 1–8

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Acknowledgments This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MEST) (20100007386) (2010).

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Fig. 6. Effect of nifedipine oral administration on the phosphorylation of AMPK, ACC, and LKB1 in aortic tissue. Sample was prepared from aortas of male C57BL/6 mice (aged 12–14 weeks, weighing 20–23 g) as described in Methods (A). Nifedipine (1 mg/kg) was administered orally for 7 days, n = 2 to 3 for each group. Densitometric analyses are shown (B). *p b 0.05 versus the control.

nifedipine can impede VSMC proliferation through activation of AMPK, because pharmacologic and genetic inhibition of AMPK restored the nifedipine-mediated inhibition of VSMC proliferation. We further investigated upstream kinase for AMPK activation because CaMKKβ has been reported to activate AMPK in response to increase of intracellular Ca 2+ levels (Hurley et al., 2005). Although STO-609, a CaMKKβ inhibitor, doesn't influence nifedipine-induced AMPK phosphorylation, LKB1 siRNA inhibited nifedipine-induced phosphorylation of both AMPK and ACC. These results indicate that LKB1 is required for nifedipine-induced AMPK activation in VSMCs. ROS have been proposed to be important signaling molecules in many biological events such as cell proliferation which is important in atherosclerosis (Park et al., 2004). Oxidative stress and the production of intracellular ROS have been implicated in the pathogenesis of cardiovascular disease, in part by promoting vascular smooth muscle proliferation (Jin and Berk 2004). We demonstrated that nifedipine was capable of inhibiting 15% FBSinduced ROS production. This effect was also reversed by pharmacologic and genetic inhibition of AMPK. Several Ca2+ channel blockers have been demonstrated to have antioxidant effect (Cominacini et al., 2003) and this study provides the evidence that nifedipine reduces intracellular ROS production by activating AMPK. Our data showed that the inhibitory effects of nifedipine on VSMC proliferation correlate with decreased ROS production. Modulation of AMPK activation leads to down regulation of ROS production that controls VSMC proliferation. The intrinsic link between VSMC proliferation and ROS production often relates directly to their regulatory actions. Furthermore, it has been reported that lercanidipine can suppress the proliferation of VSMCs via inhibiting cellular ROS (Wu et al., 2009). Based on the in vitro findings, we next examined nifedipineinduced AMPK activation in vivo. Similar to the in vitro findings, oral administration of nifedipine led to a significant activation of AMPK, ACC,

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