Sirtuin 1 Mediates the Actions of Peroxisome Proliferator ...

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1521-0111/90/5/522529$25.00 http://dx.doi.org/10.1124/mol.116.104679 MOLECULAR PHARMACOLOGY Mol Pharmacol 90:522529, November 2016 Copyright ª 2016 by The American Society for Pharmacology and Experimental Therapeutics Sirtuin 1 Mediates the Actions of Peroxisome Proliferator-Activated Receptor d on the Oxidized Low-Density LipoproteinTriggered Migration and Proliferation of Vascular Smooth Muscle Cells s Jung Seok Hwang, Sun Ah Ham, Taesik Yoo, Won Jin Lee, Kyung Shin Paek, Chi-Ho Lee, and Han Geuk Seo College of Animal Bioscience and Technology, Konkuk University, Seoul (J.S.H., S.A.H., T.Y., W.J.L., C.-H.L., H.G.S.); Department of Nursing, Semyung University, Jecheon (K.S.P.), South Korea Received April 11, 2016; accepted August 26, 2016 ABSTRACT Peroxisome proliferator-activated receptor d (PPARd) has been implicated in vascular pathophysiology. However, its functions in atherogenic changes of the vascular wall have not been fully elucidated. PPARd activated by GW501516 (2-[2-methyl-4-[[4- methyl-2-[4-(trifluoromethyl)phenyl]-1,3-thiazol-5-yl]methylsulfanyl]- phenoxy]acetic acid) significantly inhibited the migration and proliferation of vascular smooth muscle cells (VSMCs) triggered by oxidized low-density lipoprotein (oxLDL). These GW501516- mediated effects were significantly reversed by PPARd-targeting small-interfering RNA (siRNA), indicating that PPARd is involved in the action of GW501516. The antiproliferative effect of GW501516 was directly linked to cell cycle arrest at the G 0 /G 1 to S phase transition, which was followed by the down-regulation of cyclin- dependent kinase 4 along with increased levels of p21 and p53. In VSMCs treated with GW501516, the expression of sirtuin 1 (SIRT1) mRNA and protein was time-dependently increased. This GW501516-mediated up-regulation of SIRT1 expression was also demonstrated even in the presence of oxLDL. In addition, GW501516-dependent inhibition of oxLDL-triggered migration and proliferation of VSMCs was almost completely abolished in the presence of SIRT1-targeting siRNA. These effects of GW501516 on oxLDL-triggered phenotypic changes of VSMCs were also demonstrated via activation or inhibition of SIRT1 activity by resveratrol or sirtinol, respectively. Finally, gain or loss of SIRT1 function imitated the action of PPARd on oxLDL- triggered migration and proliferation of VSMCs. Taken together, these observations indicate that PPARd-dependent up-regulation of SIRT1 contributes to the antiatherogenic activities of PPARd by suppressing the migration and proliferation of VSMCs linked to vascular diseases such as restenosis and atherosclerosis. Introduction Oxidized low-density lipoprotein (oxLDL) is a proathero- genic risk factor that promotes the migration and growth of vascular smooth muscle cells (VSMCs) and macrophages in atherosclerotic lesions (Chatterjee and Ghosh, 1996; Sakai et al., 1996; Mine et al., 2002; Liu et al., 2014). VSMC prolif- eration and migration triggered by oxLDL were recently demonstrated as an important phenotypic change in multiple human vascular disorders such as atherosclerosis, and thus facilitating the formation and vulnerability of atherosclerotic plaque (Witztum and Steinberg, 2001). VSMCs play major roles in the maintenance of plaque stability as an important structural component of atherosclerotic plaque cap (Clarke and Bennett, 2006). Several studies have demonstrated that intimal migration and excess growth of VSMCs is a critical risk factor leading to restenosis and atherosclerosis (Stintzing et al., 2009; Brown et al., 2010). In fact, these phenotypic changes of VSMCs are regulated by interactions and network- ing between local cells and humoral factors within the arterial wall (Jiang et al., 2013). Thus, fine-tuned modulation of the migration and proliferation of VSMCs may have therapeutic potential in atherosclerotic complications. Peroxisome proliferator-activated receptor d (PPARd) par- ticipates in multiple physiologic processes, including cellular energy homeostasis, cell growth and differentiation, and inflammatory responses as a nuclear transcription factor activated by specific ligands (Giordano Attianese and This work was supported in part by the Basic Science Research Program through the National Research Foundation of Korea (NRF), funded by the Ministry of Science, ICT, and Future Planning [Grants NRF-2014R1A2A2A01004847 and 2015R1A5A1009701], and by the Next- Generation BioGreen 21 Program [Grant PJ01122201], Rural Development Administration, Republic of Korea. dx.doi.org/10.1124/mol.116.104679. s This article has supplemental material available at molpharm. aspetjournals.org. ABBREVIATIONS: CDK, cyclin-dependent kinase; DMEM, Dulbeccos modified Eagles medium; FBS, fetal bovine serum; GSK0660, methyl 3-[(4- anilino-2-methoxyphenyl)sulfamoyl]thiophene-2-carboxylate; GW501516, 2-[2-methyl-4-[[4-methyl-2-[4-(trifluoromethyl)phenyl]-1,3-thiazol-5- yl]methylsulfanyl]phenoxy]acetic acid; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; nLDL, native low-density lipoprotein; oxLDL, oxidized low-density lipoprotein; PCR, polymerase chain reaction; PPARd, peroxisome proliferator-activated receptor d; PPRE, peroxisome proliferator-activated receptor response element; siRNA, small-interfering RNA; SIRT1, sirtuin 1; VSMC, vascular smooth muscle cell. 522 http://molpharm.aspetjournals.org/content/suppl/2016/08/29/mol.116.104679.DC1 Supplemental material to this article can be found at: at ASPET Journals on April 7, 2022 molpharm.aspetjournals.org Downloaded from at ASPET Journals on April 7, 2022 molpharm.aspetjournals.org Downloaded from at ASPET Journals on April 7, 2022 molpharm.aspetjournals.org Downloaded from at ASPET Journals on April 7, 2022 molpharm.aspetjournals.org Downloaded from at ASPET Journals on April 7, 2022 molpharm.aspetjournals.org Downloaded from at ASPET Journals on April 7, 2022 molpharm.aspetjournals.org Downloaded from at ASPET Journals on April 7, 2022 molpharm.aspetjournals.org Downloaded from at ASPET Journals on April 7, 2022 molpharm.aspetjournals.org Downloaded from at ASPET Journals on April 7, 2022 molpharm.aspetjournals.org Downloaded from

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1521-0111/90/5/522–529$25.00 http://dx.doi.org/10.1124/mol.116.104679MOLECULAR PHARMACOLOGY Mol Pharmacol 90:522–529, November 2016Copyright ª 2016 by The American Society for Pharmacology and Experimental Therapeutics

Sirtuin 1 Mediates the Actions of PeroxisomeProliferator-Activated Receptor d on the OxidizedLow-Density Lipoprotein–Triggered Migration andProliferation of Vascular Smooth Muscle Cells s

Jung Seok Hwang, Sun Ah Ham, Taesik Yoo, Won Jin Lee, Kyung Shin Paek, Chi-Ho Lee,and Han Geuk SeoCollege of Animal Bioscience and Technology, Konkuk University, Seoul (J.S.H., S.A.H., T.Y., W.J.L., C.-H.L., H.G.S.);Department of Nursing, Semyung University, Jecheon (K.S.P.), South Korea

Received April 11, 2016; accepted August 26, 2016

ABSTRACTPeroxisome proliferator-activated receptor d (PPARd) has beenimplicated in vascular pathophysiology. However, its functions inatherogenic changes of the vascular wall have not been fullyelucidated. PPARd activated by GW501516 (2-[2-methyl-4-[[4-methyl-2-[4-(trifluoromethyl)phenyl]-1,3-thiazol-5-yl]methylsulfanyl]-phenoxy]acetic acid) significantly inhibited the migration andproliferation of vascular smooth muscle cells (VSMCs) triggeredby oxidized low-density lipoprotein (oxLDL). These GW501516-mediated effects were significantly reversed by PPARd-targetingsmall-interfering RNA (siRNA), indicating that PPARd is involved inthe action of GW501516. The antiproliferative effect of GW501516was directly linked to cell cycle arrest at the G0/G1 to S phasetransition, which was followed by the down-regulation of cyclin-dependent kinase 4 along with increased levels of p21 and p53.In VSMCs treated with GW501516, the expression of sirtuin

1 (SIRT1) mRNA and protein was time-dependently increased.ThisGW501516-mediated up-regulation of SIRT1 expressionwasalso demonstrated even in the presence of oxLDL. In addition,GW501516-dependent inhibition of oxLDL-triggered migrationand proliferation of VSMCs was almost completely abolishedin the presence of SIRT1-targeting siRNA. These effects ofGW501516 on oxLDL-triggered phenotypic changes of VSMCswere also demonstrated via activation or inhibition of SIRT1activity by resveratrol or sirtinol, respectively. Finally, gain orloss of SIRT1 function imitated the action of PPARd on oxLDL-triggered migration and proliferation of VSMCs. Taken together,these observations indicate that PPARd-dependent up-regulationof SIRT1 contributes to the antiatherogenic activities of PPARd bysuppressing the migration and proliferation of VSMCs linked tovascular diseases such as restenosis and atherosclerosis.

IntroductionOxidized low-density lipoprotein (oxLDL) is a proathero-

genic risk factor that promotes the migration and growth ofvascular smooth muscle cells (VSMCs) and macrophages inatherosclerotic lesions (Chatterjee and Ghosh, 1996; Sakaiet al., 1996; Mine et al., 2002; Liu et al., 2014). VSMC prolif-eration and migration triggered by oxLDL were recentlydemonstrated as an important phenotypic change in multiplehuman vascular disorders such as atherosclerosis, and thus

facilitating the formation and vulnerability of atheroscleroticplaque (Witztum and Steinberg, 2001). VSMCs play majorroles in the maintenance of plaque stability as an importantstructural component of atherosclerotic plaque cap (Clarkeand Bennett, 2006). Several studies have demonstrated thatintimal migration and excess growth of VSMCs is a criticalrisk factor leading to restenosis and atherosclerosis (Stintzinget al., 2009; Brown et al., 2010). In fact, these phenotypicchanges of VSMCs are regulated by interactions and network-ing between local cells and humoral factors within the arterialwall (Jiang et al., 2013). Thus, fine-tuned modulation of themigration and proliferation of VSMCs may have therapeuticpotential in atherosclerotic complications.Peroxisome proliferator-activated receptor d (PPARd) par-

ticipates in multiple physiologic processes, including cellularenergy homeostasis, cell growth and differentiation, andinflammatory responses as a nuclear transcription factoractivated by specific ligands (Giordano Attianese and

This work was supported in part by the Basic Science ResearchProgram through the National Research Foundation of Korea (NRF),funded by the Ministry of Science, ICT, and Future Planning [GrantsNRF-2014R1A2A2A01004847 and 2015R1A5A1009701], and by the Next-Generation BioGreen 21 Program [Grant PJ01122201], Rural DevelopmentAdministration, Republic of Korea.

dx.doi.org/10.1124/mol.116.104679.s This article has supplemental material available at molpharm.

aspetjournals.org.

ABBREVIATIONS: CDK, cyclin-dependent kinase; DMEM, Dulbecco’s modified Eagle’s medium; FBS, fetal bovine serum; GSK0660, methyl 3-[(4-anilino-2-methoxyphenyl)sulfamoyl]thiophene-2-carboxylate; GW501516, 2-[2-methyl-4-[[4-methyl-2-[4-(trifluoromethyl)phenyl]-1,3-thiazol-5-yl]methylsulfanyl]phenoxy]acetic acid; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; nLDL, native low-density lipoprotein;oxLDL, oxidized low-density lipoprotein; PCR, polymerase chain reaction; PPARd, peroxisome proliferator-activated receptor d; PPRE, peroxisomeproliferator-activated receptor response element; siRNA, small-interfering RNA; SIRT1, sirtuin 1; VSMC, vascular smooth muscle cell.

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Desvergne, 2015). This receptor heterodimerizes with retinoidX receptor in the consensus sequences named PPAR responseelements (PPRE) to modulate the gene expression of targetgenes (Tugwood et al., 1992). It was recently observed thatspecific ligand-mediated activation of PPARd in vascular cellsexerts antiatherosclerotic effects through antisenescent oranti-inflammatorymechanisms (Lee et al., 2003; Takata et al.,2008; Jandeleit-Dahm et al., 2009). Activation of PPARd alsosuppresses proliferation and inflammation of VSMCs byup-regulating transforming growth factor-b1 (Kim et al.,2008). We have also demonstrated that ligand-activatedPPARdmay stabilize atherosclerotic plaques by up-regulatingextracellular matrix proteins in VSMCs (Kim et al., 2009).Furthermore, activation of PPARd counteracts oxLDL-triggered apoptosis of VSMCs through transforming growthfactor-b and focal adhesion kinase signaling (Hwang et al.,2015). Therefore, it is especially important to examinewhether PPARd modulates the phenotype of VSMCs inducedby oxLDL.Sirtuin 1 (SIRT1) functions as a protein deacetylase to remove

the acetyl groups of diverse proteins in a NAD1-dependentmanner (Michan and Sinclair, 2007). SIRT1 participates inmultiple cellular processes such as modulation of the cellcycle, metabolism, and aging through interaction with diversesubstrates including forkhead transcription factor, histones,MyoD, PPARg coactivator-1a, and p53 (Vaziri et al., 2001;Langley et al., 2002; Brunet et al., 2004). Inhibitory effects ofSIRT1 on the migration and growth of VSMCs were reportedin multiple studies (Miyazaki et al., 2008; Li et al., 2011;Gorenne et al., 2013). Recently, a study reported that ligand-activated PPARd enhances transcription of SIRT1 throughSp1 but not PPRE in HuH7 cells (Okazaki et al., 2010).Therefore, we hypothesized that the adverse effects of PPARdon VSMC migration and growth may be in part stemmedby the up-regulation of SIRT1. Our findings indicate thatPPARd-induced up-regulation of SIRT1 mediates the abilityof this transcription factor to inhibit the phenotypic changesof VSMCs induced by oxLDL.

Materials and MethodsMaterials. GW501516 (GW501516 (2-[2-methyl-4-[[4-methyl-2-[4-

(trifluoromethyl)phenyl]-1,3-thiazol-5-yl]methylsulfanyl]phenoxy]-acetic acid) and low-density lipoprotein were purchased from EnzoLife Science (Farmingdale, NY) and Calbiochem (La Jolla, CA),respectively. Anti-b-actin antibody, 4ʹ,6-diamidino-2-phenylindole,mithramycin A, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT), and sirtinol were purchased from Sigma-Aldrich(St. Louis, MO). Resveratrol and GSK0660 (methyl 3-[(4-anilino-2-methoxyphenyl)sulfamoyl]thiophene-2-carboxylate) were obtainedfrom Calbiochem and Tocris Bioscience (Bristol, United Kingdom),respectively. Polyclonal antibodies specific for horseradish peroxidase-conjugated IgG, c-Myc, PPARd, and SIRT1 as well as monoclonalantibodies specific for cyclin D1, cyclin-dependent kinase (CDK) 2,p21, and p53 were from Santa Cruz Biotechnology (Dallas, TX). Amonoclonal anti-CDK4 antibody was purchased from Cell Signaling(Beverly, MA).

Cell Culture. Aortic VSMCs were isolated and incubated inDulbecco’s modified Eagle’s medium (DMEM) containing 1% antibi-otic and 20% fetal bovine serum (FBS) at 37°C under a condition of 5%CO2 and 95% air, as essentially described by Hwang et al. (2015).Briefly, the aortic explants dissected from rats were longitudinallycut, and the endothelial cells were removed. The isolated membranewas chopped into small pieces and incubated for a few days in DMEM

supplemented with 20% FBS. When VSMCs had fully migratedinto the medium, the cells were maintained in the same culturemedium.

Preparation of oxLDL. We prepared oxLDL as essentially de-scribed by Hwang et al. (2015). Briefly, native low-density lipoprotein(nLDL) was reacted with CuSO4 (final 5 mmol) at 37°C for 6 hours, andthen 1 mM EDTA was added to stop the reaction. After thoroughdialysis in phosphate-buffered saline, the extent of low-densitylipoprotein oxidation was determined by measuring thiobarbituricreactive substances (Siow et al., 1999).

Small-Interfering RNA Study. Gene-specific small-interferingRNA (siRNA)–mediated gene silencing was performed as essentiallydescribed by Hwang et al. (2015). Briefly, VSMCs were transfectedwith nonspecific sequence-targeting siRNA (Ambion, Austin, TX),SIRT1-targeting siRNA (Bioneer, Daejeon, South Korea), and PPARd-targeting siRNA (Ambion) using Welfect-Q (WelGENE, Daegu, SouthKorea) in serum-free medium. The effects of siRNA were analyzedafter incubation for 24 to 48 hours in fresh medium.

Assay of Cell Proliferation. VSMCs were synchronized toquiescence by incubating in DMEM containing 0.1% FBS for 24 hours.The cells were then treated with the indicated reagent for theindicated time in fresh DMEM containing 5% FBS, and exposed tonLDL or oxLDL. After incubation for 72 hours, the cells weremaintained for an additional 4 hours in the culture medium contain-ing MTT solution (final 0.1 mg/ml). After removal of the culturemedium, the resultant formazan crystals were dissolved in acidifiedisopropanol, and the optical density was measured using a microplatereader at 570 nm as described elsewhere (Ham et al., 2013).

Cell Cycle Determination. VSMCs were synchronized to quies-cence via incubation in 0.1% FBS-containing DMEM. After an over-night incubation, VSMCs were pretreated with GW501516 for24 hours in fresh DMEM. Finally, the cells were incubated in DMEMcontaining oxLDL for an additional 72 hours, and then centrifuged at1000 rpm for 5minutes to collect the cells. The cells were then fixed byincubating with 70% (v/v) ethanol at 270°C for 1 hour. After washingwith phosphate-buffered saline, a staining solution [10 mM Tris (pH7.0), 0.1% NP-40, 1 mM NaCl, 0.7 mg/ml ribonuclease A, and 5 mg/mlpropidium iodide] was added to the cells. The cellular DNA contentwas determined after incubation for 30 minutes in the dark, and cellcycle distributions were estimated with a FACSCalibur system (BDBiosciences, Franklin Lakes, NJ) and CellQuest Pro software (BDBiosciences).

Measurement of Cell Migration. A wound healing assay wasadopted to measure VSMC migration as described elsewhere (Hamet al., 2014). To pause proliferation, VSMCs grown to confluency wereincubated in culture medium containing mitomycin C (final 8 mg/ml).After treatment with the indicated reagents for the indicated timeperiods, the VSMC monolayers were scratched with a sterile scraperand then treated with nLDL or oxLDL for an additional 72 hours infresh DMEM containing 5% FBS. The cells were fixed by incubatingwith 70% (v/v) ethanol at 270°C for 1 hour, and then stained in 0.2%crystal violet solution. Cell migration was determined by counting thecells migrated across the wound edge under a microscope.

Real-Time Polymerase Chain Reaction. Expression of SIRT1mRNAwas analyzed by real-time polymerase chain reaction (PCR) asdescribed elsewhere (Kim et al., 2013). Briefly, total RNAwas isolatedand reverse-transcribed into cDNA using TRIzol reagent (Invitrogen,Carlsbad, CA) and TOPscript RT DryMIX kit (Enzynomics, Seoul,South Korea), respectively. An aliquot of cDNA was amplified in a10-ml reaction solution containing 10 pM primers and 2 � Real-TimePCRmix (Solgent, Daejeon, SouthKorea), followed by 40 cycles of PCRamplification (10 seconds at 95°C, 10 seconds at 56°C, and 10 secondsat 72°C). The primer sequences used were SIRT1, 59-AGA ACC ACCAAAGCGGAAA-39 and 59-TCCCACAGGAGACAGAAACC-39; andGAPDH, 59-CAT GGC CTT CCG TGT TCC TA-39 and 59-CCT GCTTCA CCA CCT TCT TGA T-39. The cDNA levels of the target genewere determined as the fold ratio relative to GAPDH by the ΔΔCT

method (Hwang et al., 2014).

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Western Blot Analysis. The levels of protein were analyzed byimmunoblotting as described elsewhere (Hwang et al., 2015). In brief,whole cell lysates containing 50 mg of protein per lane were fraction-ated on a 10% SDS-polyacrylamide gel and blotted onto a polyvinyl-idene difluoride membrane (Hybond-P1, 0.45 mm pore; AmershamBiosciences, Buckinghamshire, United Kingdom). After blocking with5% skim milk, the membranes were probed with specific antibodies(anti-PPARd diluted to 1:500; anti-SIRT1 diluted to 1: 1000; anti-p53diluted to 1:500; anti-p21 diluted to 1:500; anti-CDK4 diluted to 1:1000; anti-CDK2 diluted to 1:1000; anti-cyclin D diluted to 1:500; anti-b-actin diluted to 1:3000) overnight at 4°C. After incubation with aperoxidase-conjugated secondary antibody, the immunoreactive bandwas visualized by conjunction with West-ZOL Plus (iNtRON Bio-technology, Seongnam, South Korea).

Vector Constructs. Recombinant adenoviral vectors were con-structed for lacZ and Myc-SIRT1 (GenBank: AF083106) expression.All cDNAs were ligated into the pAd/CMV/V5-DEST vector (Invitro-gen) using LR Clonase II and Gateway system (Invitrogen). Recombi-nant adenoviral vector was linearized with PacI and transferred into293A cells, and then cultured for 2 weeks in complete DMEM. ThepAd/CMV/V5-GW/lacZ vector (Invitrogen) as a control was alsointroduced to produce a lacZ-bearing adenovirus. The KIST virusfacility (Seoul, Korea) was used to produce adenoviruses. Themammalian expression vector pcDNA3.1-Flag-Sp1 was presented byProf. Jae-Hwan Kim (CHA University, Pangyo, South Korea).

Reporter Gene Assay. The PPRE3-tk-luc luciferase reportervector was a gift from Prof. Frank J. Gonzalez (National CancerInstitute, National Institutes of Health, Bethesda, MD). VSMCstransfected with 0.5 mg pcDNA3.1–PPARd, 0.5 mg pTK-PPRE-luc,0.5 mg pSV b-gal, and/or 0.5 mg pcDNA3.1–Myc-SIRT1 for 48 hourswere incubated with or without GW501516 and/or resveratrol for theindicated times. The cells were then lysed, and the reporter geneactivity was measured as described elsewhere (Hwang et al., 2015).

Statistical Analysis. One-way analysis of variance followed bythe Tukey-Kramer test was used to determine statistical significance.

ResultsActivation of PPARd Inhibits oxLDL-Induced

Proliferation of VSMCs. Because oxLDL increases the pro-liferation of cultured VSMCs (Chatterjee and Ghosh, 1996), weassessed whether activation of PPARd by GW501516 affectsthe VSMC growth stimulated by oxLDL. A concentration-dependent increase in proliferation was demonstrated inVSMCs treated with oxLDL relative to nLDL-treated cells, asdetermined by the MTT assay (Fig. 1A). By contrast, theoxLDL-stimulated VSMC proliferation was dose-dependentlyinhibited in cells treated with GW501516, a specific ligand forPPARd (Fig. 1B).To investigate the involvement of PPARd in the inhibition of

VSMC proliferation triggered by oxLDL, VSMCs were treatedwith PPARd-targeting siRNA or GSK0660, a PPARd-specificantagonist (Shearer et al., 2008). The expression of PPARdprotein was reduced in VSMCs transfected with PPARd-targeting siRNA, whereas nonspecific control siRNA did not(Supplemental Fig. 1). The addition of PPARd-targetingsiRNA or GSK0660 significantly reversed the inhibitory effectof GW501516 on VSMC proliferation triggered by oxLDL (Fig.1, C and D).To further characterize the antiproliferative action of

PPARd in oxLDL-treated VSMCs, the cell cycle distributionwas analyzed in VSMCs treated with or without oxLDL in thepresence or absence of GW501516. The cells were synchro-nized by incubating in medium containing 0.1% FBS for24 hours, and then exposed to vehicle (dimethylsulfoxide) or

GW501516, followed by stimulation with oxLDL. A significantdecrease in the G0/G1 population was observed in VSMCsexposed to oxLDL for 72 hours (Fig. 2, A and B). By contrast,the population of cells proceeding the S phase was signifi-cantly increased. GW501516, however, significantly reversedthe oxLDL-induced reduction in cell populations in the G0/G1

phase. Especially, the increased transition of oxLDL-stimulated cells into the S phase was also significantly sup-pressed by GW501516. These findings suggest that activationof PPARd by GW501516 causes the cell cycle arrest of VSMCtreated with oxLDL.Because a dynamic network of cell cycle–related molecules

such as CDKs, cyclins, and CDK inhibitors is associated withregulation of the cell cycle (Pavletich, 1999), we examined theexpression levels of key regulatory proteins in VSMCs treatedwith oxLDL in the presence or absence of GW501516. Wefound that oxLDL markedly suppressed the levels of p21 andp53 (Fig. 2, C and D), key regulators of the G1 to S phasetransition checkpoint (Toyoshima and Hunter, 1994;Pavletich, 1999). This oxLDL-dependent decrease in bothprotein levels was significantly reversed in the presence ofGW501516. In addition, up-regulation of CDK4, but notCDK2, induced by oxLDL was also significantly down-regulated by GW501516. These observations provide evidencethat GW501516-activated PPARd arrests oxLDL-induced cellcycle progression through expressional modulation of cellcycle–related proteins.Activation of PPARd Inhibits oxLDL-Stimulated

Migration of VSMCs. Next, we investigated the role ofGW501516 on oxLDL-triggered migration of VSMCs by anin vitro wound healing assay. With the proliferation index as areference basis, described previously elsewhere (Kim et al.,2010), we found that oxLDL significantly enhanced themigration of VSMCs whereas this effect of oxLDL on cellmigration was significantly suppressed by GW501516

Fig. 1. GW501516-activated PPARd attenuates oxLDL-triggered prolif-eration of VSMCs. (A) VSMCs were stimulated with either nLDL oroxLDL for 72 hours. (B) VSMCs treated with indicated concentration ofGW501516 for 24 hours were stimulated with either nLDL or oxLDL for72 hours. (C) GSK0660-pretreated cells were incubated for 30minutes, andthen exposed to GW501516. After incubation for 24 hours, the cells werestimulated with oxLDL for 72 hours. (D) PPARd siRNA-transfected cellswere incubated for 24 hours, and then pretreated with GW501516. Afterincubation for 24 hours, the transfectants were exposed to oxLDL for72 hours. AnMTT assay was performed to determine the cell proliferation.The results are expressed as mean 6 S.E. (n = 4 or 5). **P , 0.01 versusuntreated cells; #P, 0.05, ##P, 0.01 versus oxLDL-treated cells; †P, 0.05versus oxLDL plus GW501516-treated cells.

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treatment (Fig. 3, A and B). GW501516-mediated reduction inVSMC migration was not observed in cells transfected withPPARd-targeting siRNA (Fig. 3, C and D). These observationssuggest that the action of GW501516 is dependent on PPARdin the suppression of VSMC migration triggered by oxLDL.Activation of PPARd Induces SIRT1 Expression in

VSMCs. Because SIRT1 is implicated in neointima formationthrough modulation of VSMC migration and proliferation (Liet al., 2011), the expression level of SIRT1 was determined in

VSMCs treated with GW501516. The levels of both SIRT1mRNA and protein in VSMCs were significantly increased byGW501516 treatment in a time-dependent manner (Fig. 4, Aand B).To confirm whether this effect of GW501516 on the

up-regulation of SIRT1 is PPARd dependent, the effects ofPPARd-targeting siRNA and GSK0660 were examined.PPARd-targeting siRNA, but not nonspecific control siRNA,markedly inhibited GW501516-induced up-regulation of

Fig. 2. GW501516-activated PPARd reg-ulates cell cycle progression of VSMCstriggered by oxLDL. (A, B) Cells synchro-nized to quiescence by incubating inDMEM containing 0.1% FBS were pre-treated with GW501516 for 24 hours. Af-ter incubation for 72 hours in DMEMcontaining 5% FBS and oxLDL, the DNAcontent in cells stained with propidiumiodide was analyzed by flow cytometry.Representative histograms (A) and thecell number of each cell cycle (B) areplotted as mean 6 S.E. (n = 3). (C, D)The expression of cell cycle–regulatoryproteins was detected by Western blotanalysis. Representative blots (C) andthe fold changes in each protein to b-actinratio relative to untreated control (D) areshown as mean 6 S.E. (n = 4). *P , 0.05,**P , 0.01 versus 5% serum-treated cells;#P , 0.01, ##P , 0.05 versus oxLDL-treated cells.

Fig. 3. GW501516-activated PPARd attenuates oxLDL-triggered migration of VSMCs. (A, B) Mitomycin C(8 mg/ml)–treated cell monolayers were incubated for2 hours, and then washed with phosphate-buffered saline.After pretreatment with or without GW501516 for 24 hours,the cells were wounded by scraping with a razor blade, andthen stimulated with either nLDL or oxLDL in freshmedium containing 5% FBS. After incubation for 72 hours,a wound healing assay was performed to count the cells thatmigrated across the wound. (C, D) Cells transfected withPPARd-targeting siRNA were treated as described earlier,and a wound healing assay was performed as describedherein. Representative photographs (A, C) andmigrated cellnumbers (B, D) are shown as mean 6 S.E. (n = 6). Bars:100 mm. *P , 0.05, **P , 0.01 versus untreated cells; #P ,0.05 versus oxLDL-treated cells; †P , 0.05 versus oxLDLplus GW501516-treated cells.

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SIRT1 in VSMCs (Fig. 4C). In addition, the effect of GW501516on SIRT1 expression was also suppressed by GSK0660 pre-treatment (Fig. 4D), indicating the involvement of PPARd inthe up-regulation of SIRT1.Because Sp1 is implicated in PPARd-mediated SIRT1

expression (Okazaki et al., 2010), the effect of mithramycinA, a specific Sp1 inhibitor, was examined in PPARd-mediatedSIRT1 expression. When VSMCs were incubated in the pres-ence of mithramycin A, GW501516-induced up-regulation ofSIRT1 was markedly inhibited (Fig. 4E). This effect ofmithramycin A was also observed in GW501516-mediatedsuppression of VSMC proliferation triggered by oxLDL (Sup-plemental Fig. 2A). In addition, ectopic expression of Sp1potentiated the suppressive effect of GW501516 on the VSMCproliferation triggered by oxLDL (Supplemental Fig. 2B).These findings indicate that GW501516-regulated SIRT1 isan important factor in the modulation of VSMC proliferationstimulated by oxLDL.On the other hand, SIRT1 has been reported to inhibit the

transcriptional activity of PPARg (Picard et al., 2004), anothermember of PPAR nuclear family, so we assessed the effects ofSIRT1 overexpression and activation on the transcrip-tional activity of PPARd. Although GW501516 significantly

increased the transcriptional activity of PPARd, the ectopicexpression of SIRT1 or addition of resveratrol did not signif-icantly affect the GW501516-activated increase in the tran-scriptional activity of PPARd, which suggests that SIRT1 didnot directly influence the transcriptional activity of PPARd inVSMCs (Supplemental Fig. 3).Down-Regulation of SIRT1 Abrogates the Inhibitory

Effect of PPARd on VSMC Migration and ProliferationTriggered by oxLDL. Because the expression of SIRT1 wasup-regulated in VSMCs by PPARd activation, we examinedthe role of SIRT1 in PPARd-associated modulation of cellmigration and proliferation in VSMCs stimulated withoxLDL. The expression of SIRT1 was time-dependently de-creased in the presence of oxLDL, but this oxLDL-triggeredreduction of SIRT1 was dose-dependently reversed byGW501516 (Fig. 5, A and B).To further examine whether this GW501516-mediated re-

covery of SIRT1 expression directly affects oxLDL-stimulatedmigration and proliferation of VSMCs, we examined the effectof SIRT1-targeting siRNA in VSMCs (Supplemental Fig. 4).Although GW501516 significantly inhibited oxLDL-stimulatedmigration and proliferation of VSMCs, siRNA-mediated knock-down of SIRT1 in VSMCs almost completely abolished the

Fig. 4. GW501516-activated PPARd induces SIRT1 expres-sion. (A, B) VSMCs were treated with GW501516 for theindicated time periods. The total RNA or cell lysate wasanalyzed by real-time PCR (A) or immunoblot (B), respec-tively, as described in Materials and Methods. The foldchange in SIRT1 to GAPDH cDNA ratio relative to time0 was determined and is plotted as mean 6 S.E. (n = 4).**P , 0.01 versus untreated cells. (C) VSMCs transfectedwith PPARd-targeting siRNA or nonspecific siRNA for24 hours were stimulated with or without GW501516 for24 hours, and then were harvested and subjected toimmunoblot analysis. (D, E) VSMCs pretreated withGSK0660 (D) or mithramycin A (E) for 30 minutes werestimulated with or without GW501516 for 24 hours andwere then analyzed byWestern blotting as described herein.

Fig. 5. RNAi-mediated knockdown of SIRT1 abrogates theactions of GW501516 on the migration and proliferation ofVSMCs triggered by oxLDL. (A) Cells were treated withoxLDL for the time period indicated. (B) GW501516-pretreated cells were incubated for 24 hours, and then wereexposed to oxLDL for 72 hours. Immunoblot analysis wasperformed to detect the expression of SIRT1 protein usingspecific antibodies for SIRT1 and b-actin. (C) SIRT1 siRNA-transfected cells were incubated for 24 hours, and thenwerepretreated with or without GW501516. After incubation for24 hours, the transfectants were exposed to oxLDL for72 hours. An MTT assay was performed to determine cellproliferation. (D) SIRT1 siRNA-transfected cell monolayerswere incubated for 24 hours, and then were pretreatedconsecutively with or without mitomycin C (8 mg/ml) and/orGW501516 for 2 hours and 24 hours, respectively, beforeexposure to oxLDL for 72 hours. A wound healing assay wasperformed to count the cells that migrated across thewound, and is expressed as mean 6 S.E. (n = 4). *P ,0.05, **P , 0.01 versus untreated cells; #P , 0.05 versusoxLDL-treated cells; †P , 0.05 versus oxLDL plusGW501516-treated cells.

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effect of GW501516 on oxLDL-triggered migration and pro-liferation (Fig. 5, C and D). These observations suggest thatPPARd inhibits oxLDL-triggered cell migration and prolifera-tion through modulation of SIRT1 expression.Ectopic Overexpression of SIRT1 Mimics the

Suppressive Action of PPARd in VSMC Migration andProliferation Triggered by oxLDL. To further character-ize the critical role of SIRT1 in oxLDL-triggered cell migrationand proliferation, the effects of SIRT1 overexpression wereexamined in VSMCs. The ectopic expression of SIRT1 in-creased the level of SIRT1 in VSMCs (Supplemental Fig. 5). Inline with the levels of SIRT1 expression, the oxLDL-triggeredmigration and proliferation of VSMCs were significantlyattenuated, indicating that SIRT1 is an important protein inPPARd-mediated inhibition of VSMC migration and prolifer-ation triggered by oxLDL (Fig. 6).SIRT1 Is an Important Factor in PPARd-Mediated

Suppression of VSMC Migration and ProliferationTriggered by oxLDL. To confirm the functional significanceof PPARd-mediated induction of SIRT1, we investigated theimpact of SIRT1 activity on PPARd-mediated inhibition ofVSMC migration and proliferation in cells triggered byoxLDL. Blockade of SIRT1 activity by sirtinol, a specific SIRT1inhibitor, reversed the inhibitory actions of PPARd on theVSMCmigration and proliferation triggered by oxLDL (Fig. 7;Supplemental Fig. 6). By contrast, resveratrol, a SIRT1

activator, significantly inhibited both oxLDL-stimulated cellmigration and proliferation to a similar degree as GW501516did in VSMCs (Fig. 7). This inhibitory effect of GW501516 wasfurther enhanced in the presence of resveratrol, suggestingthat the actions of PPARd on the VSMC migration andproliferation are mediated through expressional regulationof SIRT1 (Fig. 7; Supplemental Fig. 6).

DiscussionTranscriptional activation of specific target genes by PPARd

elicits a broad range of cellular responses tomaintain vascularhomeostasis (Lee et al., 2003; Kim et al., 2008, 2009). Althoughdiverse target genes participate in the vascular actions ofPPARd, the effector molecules associated with the vascularactions of PPARd have not been fully elucidated. Here, weshowed that a specific ligand GW501516-activated PPARdinhibits oxLDL-stimulated migration and proliferation ofVSMCs, which trigger formation of a fibrous capsule in thepathogenesis of atherosclerosis (Dzau et al., 2002; Gorenneet al., 2013). This PPARd-mediated inhibition is dependent onSIRT1, a deacetylase that has a multitude of roles in themodulation of cardiovascular diseases (Ota et al., 2010). InVSMCs treated with GW501516, the mRNA and protein ofSIRT1 were up-regulated. Chemical- or siRNA-mediatedmodulation of SIRT1 activity or expression abrogated theinhibitory actions of PPARd on themigration and proliferationof VSMCs triggered by oxLDL. Furthermore, adenovirus-mediated ectopic expression of SIRT1 potentiated these effectsof GW501516 on VSMCs.The antiatherogenic properties of PPARd are, in part,

associated with its activity to induce the intracellular multi-functional gene SIRT1 in VSMCs. Our present observationsare consistent with a previous finding demonstrating thatactivation of PPARd suppresses premature senescence byup-regulating SIRT1 in human endothelial cells exposed toangiotensin II (Kim et al., 2012). In addition, SIRT1 trans-genic mice exhibited protection against atherosclerosis viainhibition of VSMC migration and proliferation, which un-derlie neointima formation (Li et al., 2011). By contrast,another study showed that SIRT1 promoted proliferation by

Fig. 6. Adenovirus-mediated overexpression of SIRT1 potentiates theeffects of PPARd on the migration and proliferation of VSMCs triggered byoxLDL. (A) Ad-LacZ- or Ad-SIRT1-infected cells were incubated for48 hours, and then were pretreated with or without GW501516 for24 hours. After incubation for 72 hours with or without oxLDL, an MTTassay was performed to determine the cell proliferation. (B, C) Ad-LacZ- orAd-SIRT1-infected cells monolayers were incubated for 48 hours, and thenwere treated with mitomycin C (8 mg/ml) for 2 hours. After pretreatmentwith or without GW501516 for 24 hours, the cells were stimulated with orwithout oxLDL for 72 hours. A wound healing assay was performed (B),and the migrated cell numbers (C) are shown as mean6 S.E. (n = 4). Bars:100 mm. *P , 0.05.

Fig. 7. Activator- or inhibitor-mediated modulation of SIRT1 activitymimics the actions of PPARd on the migration and proliferation of VSMCstriggered by oxLDL. (A) GW501516-pretreated cells were incubated for24 hours, and then were exposed to sirtinol or resveratrol for 30 minutes.After stimulation with oxLDL for 72 hours, an MTT assay was performedto analyze the cell proliferation. (B) Mitomycin C (8 mg/ml)-treated cellmonolayers were incubated for 2 hours, and then were pretreated with orwithout GW501516 for 24 hours. After exposure to sirtinol or resveratrolfor 30 minutes, the cells were stimulated with oxLDL for 72 hours. Awound healing assay was performed to analyze cell migration, and isexpressed as mean 6 S.E. (n = 4). **P , 0.01 versus untreated cells; #P ,0.05, ##P , 0.01 versus oxLDL-treated cells; †P , 0.05 versus oxLDL plusGW501516-treated cells.

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preventing senescence of primary endothelial cells culturedfrom porcine aorta (Zu et al., 2010).Although the action of SIRT1 in cell proliferation is debat-

able, this study demonstrates that activation of PPARdinhibits oxLDL-stimulated VSMC migration and prolifera-tion. In human atherosclerotic plaques, the reduced expres-sion of SIRT1 has also been detected (Gorenne et al., 2013);therefore, it may be possible to control the stability of plaquesthrough SIRT1 up-regulation in a process mediated by PPARdactivation. Our current observations provide new clues in therole of PPARd as a potential novel therapeutic target incardiovascular diseases associated with VSMC migrationand proliferation such as atherosclerosis and restenosis.Reduced expression of SIRT1 was demonstrated in VSMCs

isolated from plaques and human atherosclerotic plaques(Gorenne et al., 2013). In accordance with results in athero-sclerotic plaques, our study has demonstrated that theexpression of SIRT1 in VSMCs was markedly suppressed byexposure to oxLDL, which is implicated in various aspectsof cardiovascular diseases, in particular atherosclerosis(Trpkovic et al., 2015). This oxLDL-induced suppression ofSIRT1 expression was dose-dependently reversed by theactivation of PPARd in VSMCs. In fact, the direct modulatoryproperties of SIRT1 in the migration and proliferation ofVSMCs induced by oxLDL were demonstrated by siRNA- oradenovirus-mediated modulation of SIRT1 expression as wellas chemical-mediated modulation of SIRT1 activity. Accord-ingly, oxLDL-induced down-regulation of SIRT1 seems to beinvolved in the pathogenesis of vascular disorders triggered byoxLDL.Our finding show that activation of PPARd significantly

enhances SIRT1 expression regardless of oxLDL in VSMCs.Although the role of PPARd in SIRT1 expression was notdirectly evaluated, the Sp1 inhibitor mithramycin A clearlyinhibited the GW501516-induced expression of SIRT1. Thisfinding was in line with reported results showing that ligand-activated PPARd transcriptionally activates the SIRT1 genethrough Sp1 in VSMCs and hepatocyte-derived Huh7(Okazaki et al., 2010; Kim et al., 2012). Our present resultsindicate that Sp1 is a critical factor in the PPARd-mediatedinduction of SIRT1 expression instead of PPRE.In addition, Sp1 also inhibits VSMC proliferation by

transcriptionally increasing mitofusin-2 or growth inhibitoryprotein p27 (Andrés et al., 2001; Sorianello et al., 2012). Inline with the previous reports, modulation of Sp1 by inhibitoror ectopic overexpression significantly affected to the actionsof GW501516 on the VSMC proliferation triggered by oxLDL.SIRT1 also inhibits vascular injury–induced cell proliferationin carotid artery-ligated mice (Li et al., 2011), which stressesthe importance of SIRT1 in restenosis. In fact, up-regulationof SIRT1 by PPARd inhibits platelet-derived growth factor– orinterleukin-1b–induced migration and proliferation ofVSMCs in a cell cycle–dependent manner (Lim et al., 2009;Kim et al., 2010). Furthermore, activation or inhibition ofSIRT1 activity by resveratrol or sirtinol, respectively, modu-lated VSMC migration and proliferation triggered by oxLDLin our present experimental conditions. These observationsindicate that PPARd inhibits oxLDL-triggered migration andproliferation of VSMCs through a mechanism linked toSIRT1.PPARd-dependent up-regulation of SIRT1 is a key event in

the inhibition of VSMC migration and proliferation triggered

by oxLDL. SIRT1, a NAD1-dependent histone deacetylase,has a variety of cellular functions in cell cycle regulation,organism longevity, and energy metabolism (Finkel et al.,2009). Our study showed that GW501516-activated PPARdsuppressed oxLDL-triggered migration and proliferation ofVSMCs. This effect of PPARd on VSMC migration andproliferation was linked to the levels of SIRT1, indicating thatSIRT1 is a critical factor in the modulation of atheroscleroticpathogenesis (Li et al., 2011; Gorenne et al., 2013). Thus,SIRT1 has emerged as an important modulator that promotesvascular functions (Li et al., 2011; Gorenne et al., 2013).Such findings provide a rationale for SIRT1 as a drug target

in vascular disorders, including restenosis and atherosclero-sis. In fact, many other studies have provided evidence thatthe level of SIRT1 expression is intimately linked to athero-sclerotic vascular disorders (Li et al., 2011; Kim et al., 2012;Gorenne et al., 2013). Because down-regulated level of SIRT1has been demonstrated in VSMCs of the atheroscleroticplaque region (Gorenne et al., 2013), it may be possible toinhibit the proatherogenic effect of decreased SIRT1 in thevascular wall by inducing SIRT1 expression through activa-tion of PPARd.In conclusion, SIRT1 was identified as a key molecular

target of PPARd-mediated antimigration and antiprolifera-tion actions in VSMCs stimulated with oxLDL. These obser-vations indicate that GW501516-activated PPARd inhibits themigration and proliferation of VSMCs triggered by oxLDL bymodulating SIRT1 expression, suggesting that reinforcementof SIRT1 expression by PPARd could be a novel therapeu-tic strategy for vascular disorders such as restenosis andatherosclerosis.

Authorship Contributions

Participated in research design: Hwang, Seo.Conducted experiments: Hwang, Ham, Yoo, W.J. Lee,Performed data analysis: Hwang, Paek, C.-H. Lee, Seo.Wrote or contributed to the writing of the manuscript: Hwang,

Paek, Seo.

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SIRT1 mediates the actions of PPARδ on the oxLDL-triggered

migration and proliferation of vascular smooth muscle cells

Jung Seok Hwang, Sun Ah Ham, Taesik Yoo, Won Jin Lee, Kyung Shin Paek, Chi-Ho

Lee, and Han Geuk Seo

Molecular Pharmacology

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Supplemental Data

Supplemental Fig. 1. Effects of siRNA on the expression of PPAR in VSMCs. Cells

were transfected with PPARδ-targeting siRNA or nonspecific control siRNA. After

incubation for 48 h, an aliquot of the cell lysate was analyzed by Western blotting. The

levels of PPARδ were markedly reduced upon transfection of PPARδ-targeting siRNA,

whereas control siRNA, consisting of a pool of nonspecific sequences, had no effect on

PPARδ levels.

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Supplemental Fig. 2. Effects of Sp1 on the proliferation of VSMCs induced by oxLDL.

(A) Cells pretreated with mithramycin A for 30 min were exposed to GW501516. After

incubation for 24 h, cell were stimulated with oxLDL for 72 h. (B) Cells transfected

with pcDNA3.1 vector or pcDNA3.1-Flag-Sp1 for 48 h were pretreated with or without

GW501516. Following incubation for 24 h, the cells were incubated in the presence or

absence of oxLDL for 72 h. Cell proliferation was determined by the MTT assay.

Inhibition of Sp1 by mithramycin A or ectopic expression of Sp1 affected to the actions

of GW501516 on the proliferation of VSMCs induced by oxLDL, suggesting that

ligand-activated PPARδ counteracts the actions of oxLDL by upregulating SIRT1 via

Sp1 in the VSMCs. Data are expressed as the means ± SE (n=4). **P<0.01, *P<0.05.

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Supplemental Fig. 3. Effect of SIRT1 overexpression or activation in the

transcriptional activity of PPAR in VSMCs. (A) Cells transfected with pcDNA3.1-

PPAR (0.5 g), pTK-PPRE-luc (0.5 µg), pSV β-gal (0.5 µg), pcDNA3.1 vector (0.5

g), and/or pcDNA3.1-Myc-SIRT1 (0.5 g) for 48 h were incubated in the presence or

absence of GW501516 (GW) for 24 h. (B) Cells transfected with pcDNA3.1-PPAR

(0.5 g), pTK-PPRE × 3-luc (0.5 µg), and pSV β-gal (0.5 µg) for 48 h were pretreated

with resveratrol (Res) for 30 min, and then incubated with GW501516 for 24 h. Cells

were lysed in a luciferase reporter lysis buffer and an aliquots of total lysates were used

to determine luciferase activity with Microlumat Plus LB96V (EG&G Berthold).

Variations in transfection efficiency were normalized to β-galactosidase activity as

determined by the enzyme assay system (Promega). Although GW501516 significantly

increased the transcriptional activity of PPAR, the ectopic expression of SIRT1 or

addition of resveratrol did not significantly affect to this GW501516-activated increase

in the transcriptional activity of PPAR. Data are expressed as the means ± SE (n=3).

NS, not significant.

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Supplemental Fig. 4. Effects of siRNA on the expression of SIRT1 in VSMCs. Cells

were transfected with SIRT1-targeting siRNA or control siRNA for 48 h and then

harvested. Western blot analysis was performed using the antibodies indicated. The

levels of SIRT1 were markedly reduced upon transfection of SIRT1-targeting siRNA

compare to control siRNA.

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Supplemental Fig. 5. Ectopic expression of SIRT1 by adenoviral infection. VSMCs

were infected with Ad-LacZ or Ad-SIRT1. Following incubation for 48 h, the cells were

lysed, and aliquots of whole-cell lysates were subjected to Western blot analysis with

anti-SIRT1 or anti-β-actin antibodies to detect adenovirus-mediated expression of

SIRT1 in VSMCs. The levels of SIRT1 were markedly increased upon infection with

Ad-SIRT1, whereas control Ad-LacZ had no effect on SIRT1 levels.

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Supplemental Fig. 6. Effects of a SIRT1 activator or inhibitor on oxLDL-induced

proliferation and migration of VSMCs. Cell monolayers were treated with mitomycin C

(8 μg/ml) for 2 h. After washing with PBS, the cells were pretreated with or without

GW501516. After incubation for 24 h, cells were exposed to sirtinol or resveratrol for

30 min, followed by exposure to oxLDL for 72 h. Cells were then scraped with a razor

blade and incubated in fresh medium containing 5% FBS. After incubation for 72 h, the

cells that migrated across the wound were photographed. Bars, 100 μm. Modulation of

SIRT1 activity by sirtinol or resveratrol influenced the actions of GW501516 on the

proliferation and migration of VSMCs induced by oxLDL.