Ibuprofen inhibits migration and proliferation of human coronary artery smooth muscle cells by...

Ibuprofen inhibits migration and proliferation of humancoronary artery smooth muscle cells by inducing adifferentiated phenotype: role of peroxisomeproliferator-activated receptor γAbeer Dannouraa,c, Alejandro Giraldob,c, Ines Pereiraa, Jonathan M. Gibbinsb,c, Phil R. Dashb,c,Katrina A. Bicknella,c and Gavin Brooksb,c

aSchool of Pharmacy, bSchool of Biological Sciences, cInstitute for Cardiovascular and Metabolic Research, University of Reading, Reading, UK

KeywordsCAD, coronary artery disease; DES,drug-eluting stent; NSAIDs, nonsteroidalantinflammatory drugs; PCI, percutaneouscoronary intervention; PPAR, peroxisomeproliferator-activated receptor

CorrespondenceGavin Brooks, University of Reading,Whiteknights, PO Box 217, Reading,Berkshire, RG6 6AH, UK.E-mail: [email protected]

Received August 28, 2013Accepted November 16, 2013

doi: 10.1111/jphp.12203

Authorship contributions: (AD, AG) Theseauthors contributed equally to this work.(AD, AG, PD, JMG, KAB, GB) Participatedin research design, (AD, AG, IP) conductedexperiments, (AD, AG) performed dataanalysis, (AD, AG, GB) wrote or contributedto the writing of the manuscript.

Abstract

Objectives The search for agents that are capable of preventing restenosis andreduce the risk of late thrombosis is of utmost importance. In this study we aim toevaluate the in vitro effects of ibuprofen on proliferation and migration of humancoronary artery smooth muscle cells and on endothelial cells.Methods Cell proliferation was evaluated by trypan blue exclusion. Cell migra-tion was assessed by wound-healing ‘scratch’ assay and time-lapse video micros-copy. Protein expression was assessed by immunoblotting, and morphology byimmunocytochemistry. The involvement of the PPARγ pathway was studied withthe agonist troglitazone, and the use of selective antagonists such as PGF2α andGW9662.Key findings We demonstrate that ibuprofen inhibits proliferation and migrationof HCASMCs and induces a switch in HCASMCs towards a differentiated andcontractile phenotype, and that these effects are mediated through the PPARγpathway. Importantly we also show that the effects of ibuprofen are cell type-specific as it does not affect migration and proliferation of endothelial cells.Conclusions Taken together, our results suggest that ibuprofen could be aneffective drug for the development of novel drug-eluting stents that could leadto reduced rates of restenosis and potentially other complications of DESimplantation.

Introduction

Cardiovascular disease (CVD) remains the leading cause ofmorbidity and mortality worldwide, accounting for thedeath of one in three people each year.[1,2] Of the 17.3million deaths per year due to CVD, coronary artery disease(CAD) was responsible for over 40% of cases.[2] Currenttreatment modalities for CAD include percutaneous coro-nary intervention (PCI) with balloon angioplasty and stentimplantation. However, despite advances in stent technol-ogy (e.g. drug-eluting stents (DES)) that reduced substan-tially the rates of restenosis to less than 10%,[3,4] the ratesof late in-stent thrombosis, a devastating complication in

terms of morbidity and mortality, are higher with DEScompared with bare metal stents.[5] Recent reportssuggest that the risk of late-stent thrombosis appears to besteadily present over time,[6,7] and, indeed, there have beenreports of these events occurring 4.5–5 years after DESimplantation.[8,9] Thus, late in-stent thrombosis remainsa major problem in clinical practice.[10,11] DES containantiproliferative agents (e.g. rapamycin or paclitaxel) thattarget vascular smooth muscle cells (VSMCs) and inhibittheir migration and proliferation to the site of injuryfollowing stent deployment, thus ultimately preventing

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And PharmacologyJournal of Pharmacy

Research Paper

© 2014 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, 66, pp. 779–792 779

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neointimal formation and restenosis. However, DES alsointerfere with re-endothelialisation of the stent struts,which currently is thought to play a central role in thepathogenesis of very late (>12 months) in-stent thrombosisobserved with DES.[12,13] With this in mind, the search forcompounds capable of selectively inhibiting migration andproliferation of VSMCs, but sparing endothelial cell (EC)migration and proliferation, for use in DES is of utmostimportance.

During restenosis following stent deployment, VSMCsundergo significant changes in morphology and functionexhibiting a ‘synthetic’ or ‘dedifferentiated’ phenotype,whereby these cells synthesise components of theextracellular matrix and exhibit characteristics similar toimmature cells found in blood vessels during develop-ment.[14,15] In the intact vessel wall, however, VSMCs exhibita ‘differentiated’ or contractile phenotype characterised by aspindle shape and increased expression of contractile pro-teins that correlates with increased contractile ability.[15]

Peroxisome proliferator-activated receptor γ (PPARγ) is amember of the nuclear hormone-receptor superfamily.[16]

PPARγ expression has been shown to increase in the intimain response to injury.[17] It has been proposed that increasedactivity of PPARγ in blood vessels is important for limitingneointimal formation by enhancing the differentiation ofVSMCs and thereby reducing their migratory ability.[18] Pre-vious studies from our laboratory and others’ have demon-strated that ibuprofen, as well as certain other nonsteroidalanti-inflammatory drugs (NSAIDs), reduces VSMC prolif-eration in a dose- and cell cycle-dependent manner.[19,20] Inaddition, several lines of evidence indicate that NSAIDs,including ibuprofen, have the ability to activate PPARγreceptors in several cell types and tissues.[21–23] Here, weconfirm that ibuprofen inhibits proliferation and report forthe first time that ibuprofen inhibits migration of humancoronary artery smooth muscle cells (HCASMCs), and thatthis correlates with the induction of a differentiated pheno-type in these cells. We also report that at least some of theeffects of ibuprofen on HCASMCs are mediated throughPPARγ. More importantly, we also show for the first timethat the effect of ibuprofen is cell type-specific as it does notaffect migration and proliferation of endothelial cells. Takentogether, our results suggest that ibuprofen could be apotential molecule for incorporation into DES, which mayprevent restenosis without interfering with the process ofre-endothelialisation, thus having the potential of prevent-ing other complications of stent implantation.

Materials and Methods

Antibodies and reagents

Antibodies raised against SMα-actin (ab5694), SM22α(ab14106), GAPDH (ab8245), β-actin (ab6276) and Lamin

A (ab8980) were from Abcam (Cambridge, UK); antibodyraised against PPARγ (sc-7273) was from Santa Cruz Bio-technology (Dallas, TX, USA), antibody raised againstATF-2 (9226) was from Cell Signalling Technology(Danvers, MA, USA). Alexa Fluor 488 goat anti-mouse(A-11001), Alexa Fluor 594 phalloidin (A12381) andprolong Gold anti-fade reagent with DAPI (P36935) werefrom Life Technologies (Carlsbad, CA, USA). Most chemi-cals, including ibuprofen and troglitazone were obtainedfrom Sigma (Dorset, UK); Mitomycin C was fromCalbiochem (Feltham, UK); GW9662 was obtained fromBiomol (Exeter, UK); PGF2α was from Merck Chemicals(Nottingham, UK). Tissue culture plates and dishes wereNunc from Thermo Fisher (Waltham, MA, USA).

Cell culture

Primary human coronary artery endothelial cells(HCAECs) and primary HCASMCs were purchased fromPromoCell (GmbH, Heidelberg, Germany), and culturedaccording to manufacturer’s instructions using PromoCell’ssmooth muscle cell growth medium 2 (C-22062) orendothelial cell growth medium MV2 (C-22022), both ofwhich contains 5% serum without antibiotics or antifungalagents. Passaging was according to the manufacturer’sinstructions using DetachKit, also from PromoCell. Pri-mary human pulmonary microvascular endothelial cells(HPMECs) were purchased from PromoCell. Growth mediafor HPMECs was prepared to give a final concentrationof 20% (v/v) serum, and a concentration of 50 μg/mlendothelial cell growth supplement in M199 withGlutaMAX medium (Life Technologies).

For all experiments, cells were used between passages 3and 5 and were cultured, replacing with the same freshgrowth medium described earlier. For proliferation assays,HCASMCs (1 × 104/ml) and HPMECs (1 × 104/ml) wereseeded in 6-well plates. For wound-healing assays,HCASMCs (4 × 104/ml) and HCAECs (5 × 104/ml) wereseeded overnight in 12-well plates to form a confluentmonolayer. For time-lapse video microscopy experiments,both HCASMCs and HCAECs were seeded overnight at(1 × 104/ml) in 12-well plates. For immunoblotting,HCASMCs (2 × 105) and HCAECs (3 × 105) were platedin 60 mm tissue culture dishes. For immunostainingexperiments, glass cover slides were placed in 12-wellplates and coated with sterile 1% (v/v) gelatin in phos-phate buffered saline (PBS), then washed in PBS, andHCASMCs seeded at a density of 1 × 104/ml.

Proliferation analysis

Following an initial 24-h incubation, media were replacedwithout agonists (vehicle controls) or treated with different

Abeer Dannoura et al.Ibuprofen modulates human VSMC phenotype

© 2014 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, 66, pp. 779–792780

doses of ibuprofen. After a further 72 h, direct cell countingwas performed using trypan blue exclusion.[20]

Wound-healing assay

Migration was investigated using a wound-healing‘scratch’ assay as described earlier,[24,25] with minor modifi-cations. Briefly, 24 h after plating both HCASMCs andHCAECs at the desired cell density, medium was replacedin the absence (control) or presence of different concen-trations of ibuprofen or troglitazone, and in someinstances with the selective PPARγ inhibitors, GW9662 orPGF2α.[26–28] Mitomycin C, was added 1 h before scratch-ing at a concentration of 0.5 μg/ml. This concentra-tion of mitomycin C completely blocks proliferation ofHCASMCs within 1 h of administration (data not shown).A scratch in the cell monolayer was then generated with asterile 200 μl micropipette tip, and images of the gap werecaptured immediately (time 0 h) and repeated at 24 h(HCASMCs) or 10 h (HCAECs) using a phase contrastmicroscope with an attached ProgRes SpeedXT core 5digital camera with ProgRes CapturePro 2.8.8 (JenoptikSystems; GmbH). The extent of closure of the gap(HCAECs) or migrated cell number (HCASMCs) was ana-lysed using ImageJ (National Institutes of Health (NIH),Bethesda, MD, USA).

Time-lapse video microscopy

Time-lapse video microscopy was used for in-vitro real-time analysis of HCASMCs and HCAECs migration, asdescribed earlier,[29] with minor modifications. Cells werere-fed with fresh medium in the absence (control) or pres-ence of agonists such as ibuprofen or troglitazone, plus insome instances antagonists, such as GW9662. Followingtreatments for 30 min, cells were tracked for 48 h at15-min intervals. Over 90 cells were tracked in each of thethree separate wells per condition, with images capturedby a Nikon digital camera DXM 1200 Japan (NIS-elementsAR software) (Nikon Corporation, Kawasaki, Japan). Allmicroscopy stages were performed in a controlled environ-ment (37°C and 5% CO2). The distance that a particularcell had migrated within 48 h was analysed using ImageJsoftware (NIH).

Immunoblotting

Whole cell extracts were prepared as described previouslywith minor modifications.[20] Briefly, HCASMCs orHCAECs were washed gently in ice-cold PBS and scrapedinto 150 μl protein lysis buffer (50 mM Tris HCl pH 7.4,250 mM NaCl, 5 mM EDTA pH 8.0, with freshly addedIgepal (0.1% v/v)), supplemented with protease inhibitors(0.3 mM AEBSF, 10 μg/ml Leupeptin and 2 μg/ml

Aprotinin). Samples were incubated on ice for 10 min andthen centrifuged (10 000 × g, 5 min, 4°C). Supernatantswere transferred to fresh micro tubes and stored at −80°Cuntil use.

Nuclear extracts were prepared as described previously,with minor modifications.[30,31] Briefly, HCASMCs orHCAECs were washed gently in ice-cold PBS and scrapedinto 150 μl ice-cold nuclear buffer A (10 mM HEPESpH 7.9, 10 mM KCl, 1.5 mM MgCl2, 0.3 mM Na3VO4), sup-plemented with protease inhibitors (0.3 mM AEBSF,10 μg/ml Leupeptin and 2 μg/ml Aprotinin). Sampleswere incubated on ice for 5 min, then centrifuged(10 000 × g, 5 min, 4°C) and the pellets washed in 100 μlnuclear buffer A containing Igepal (0.1% v/v). Samples wereincubated for 5 min on ice, then centrifuged (10 000 × g,5 min, 4°C) and the pellets resuspended in 50 μl nuclearbuffer C (20 mM HEPES pH 7.9, 420 mM NaCl, 1.5 mMMgCl2, 0.3 mM Na3VO4, 0.2 mM EDTA pH 8.0, 25% (v/v)glycerol) supplemented with protease inhibitors as men-tioned earlier. Suspensions were incubated on ice for 1 hwith gentle vortexing every 15 min, then centrifuged(10 000 × g, 5 min, 4°C). Supernatants were transferred tofresh micro tubes and stored at −80°C until use. Proteinconcentrations were determined by the Bradford method.[32]

Samples were boiled with 0.33 vol of SDS-polyacrylamide gel electrophoresis sample buffer (10% SDS(w/v), 13% glycerol (v/v), 300 mM Tris HCl, pH 6.8,130 mM DTT, 0.2% bromophenol blue (w/v)). Proteins(20 μg) were separated by SDS-polyacrylamide gel electro-phoresis using 10% (w/v) resolving gels and 6% (w/v)stacking gels, and transferred to nitrocellulose as describedpreviously.[31] Nonspecific binding sites were blocked with5% (w/v) nonfat milk powder in TBS-T (20 mM Tris HClpH 7.5, 137 mM NaCl, 0.1% (v/v) Tween 20). Membraneswere incubated (overnight, 4°C) with primary antibodiesdiluted (1 : 1000 for PPARγ to 1 : 20000) in TBS-T contain-ing 5% nonfat milk powder for most antibodies, exceptPPARγ and ATF2 which were incubated in 5% (w/v) bovineserum albumin. Membranes were washed in TBS-T, incu-bated (60 min, room temperature) with horseradishperoxidase- (HRP-) conjugated secondary antibodiesfrom Dako (1/5000 to 1 : 25000 dilutions) in TBS-T con-taining 1% (w/v) nonfat milk powder, and were thenwashed in TBS-T. Bands were detected using enhancedchemiluminescence (ECL) Prime (GE Healthcare) and anImageQuant LAS4000 mini digital imager (GE Healthcare).ImageQuant 8.1 (GE Healthcare) software was used fordensitometric analysis.

Immunocytochemistry

HCASMCs were exposed to 500 μm ibuprofen for 72 h, thenwashed with PBS and fixed in 4% (v/v) formaldehyde

Abeer Dannoura et al. Ibuprofen modulates human VSMC phenotype


(10 min at room temperature). Following permeabilizationwith 0.1% Triton X-100 in PBS (10 min at room tempera-ture) and incubation with 1% (w/v) BSA in PBS containing0.1% Triton X-100 (20 min at room temperature) to blocknonspecific antibody binding, immunocytochemistry wasperformed as described previously with minor modifica-tions.[31] Briefly, cover slides were incubated with mousemonoclonal primary antibodies against β-actin (1 : 1000dilution for 60 min at 37°C), followed by Alexa Fluor 488anti-mouse secondary antibodies (1/200 dilution for 60 min,37°C), and then incubated with Alexa Fluor 594 phalloidin(1 : 200 dilution for 20 min at 37°C). Washes (×3) were per-formed with PBS between incubations. After the final wash,cover slides were mounted with ProLong Gold antifadereagent with DAPI (Life Technologies) and visualised using aZeiss AX10 Axioskop fluorescence microscope (Carl ZeissMicroImaging, GmbH). Images were captured using a ZeissAxioCam MRm digital camera with Software AxioVision Rel4.8.2 (Carl Zeiss MicroImaging, GmbH). Colour images wereconverted to greyscale using Adobe Photoshop (AdobeSystems, San Jose, CA, USA).

Statistical analysis

Statistical analysis was performed with Graphpad Prism 5.0(GraphPad Software, Inc., La Jolla, CA, USA). The data arepresented as mean ± standard error of the mean, and sig-nificance of results was determined by one-way analysis ofvariance with Dunnet’s or Tukey’s post test, and, whereappropriate, a paired two-tailed Student’s t-test. The resultswere considered significant when P < 0.05.

Results

Proliferation and migration of HCASMCsis inhibited by ibuprofen in adose-dependent manner

Proliferation and migration of VSMCs are key elements inthe pathophysiology of restenosis post stent implanta-tion.[33] Previous studies from our laboratory demonstratedthat A10 rat VSMC proliferation was inhibited by severalNSAIDs in a dose-dependent manner.[20] Here, we firstevaluated whether proliferation of primary HCASMCs issimilarly affected by ibuprofen. Treatment with differentconcentrations of ibuprofen did, indeed, cause an inhibi-tory effect on the proliferation of HCASMCs in a dose-dependent manner. Thus, whilst 100 μm ibuprofen had nosignificant effect, concentrations above 250 μm progres-sively inhibited proliferation of HCASMCs, with an IC50 of680 ± 120 μm (Figure 1a).

We then performed in-vitro studies to evaluate the effectsof ibuprofen on the migration of HCASMCs. Using awound-healing ‘scratch’ assay (a well-validated method for

evaluating migration of cells in vitro),[25] we first observedthat HCASMCs migrate individually rather than as a popu-lation of cells. Subsequent observations indicated that ibu-profen inhibits the number of cells that migrate to thewound in a dose-dependent manner. Thus, doses up to2000 μm ibuprofen were able to inhibit the migrated cellnumber with an IC50 of 410 ± 60 μm (Figure 1b and 1c),without significant cytotoxicity for the cells (as determinedby trypan blue exclusion assay; data not shown).

We then used time-lapse video microscopy to evaluate,in real time, the effects of ibuprofen on migration ofHCASMCs. Ibuprofen (500 μm) caused a significant reduc-tion in the average mean displacement (AMD) byHCASMCs over a period of 48 h (Figure 2), further con-firming the ability of ibuprofen to inhibit migration ofHCASMCs cells in vitro.

Ibuprofen induces a phenotypic switchin HCASMCs

Previous studies have shown that, during culture in thepresence of serum, VSMCs become de-differentiated,adopting an amoeboid shape and exhibiting increased syn-thesis of components of the extracellular matrix, thus theterm ‘synthetic’ that is commonly used to describe thisphenotype.[34] Associated with this change in phenotype,de-differentiated VSMCs also show a reduced expressionof contractile proteins, including SMα-actin, SM22α andF-actin.[33,34] In contrast, serum starvation or treatment withrapamycin induces differentiation of VSMCs with a charac-teristic spindle shape and increased expression of contrac-tile proteins, giving rise to the commonly used term,‘contractile’ phenotype[33–35] Importantly, during vasculardamage (e.g. stent deployment), VSMCs migrate and prolif-erate to the site of injury, and this correlates with a changefrom a contractile to a synthetic phenotype.[15]

Given the effects of ibuprofen on the proliferation andmigration of HCASMCs, we tested the possibility that theseeffects correlate with a change in the de-differentiation stateof these cells. Therefore, we investigated the morphology ofHCASMCs using immunocytochemistry. Treatment with500 μm ibuprofen for 72 h induced a change in morphol-ogy, from an amoeboid into a spindle shape (Figure 3a).Characteristically, the observed change in cell surface areainduced by ibuprofen mainly affects the width of the treatedcell whereas the length of the cell remains unchanged(Figure 3b–3d).

We next investigated whether the changes in morphologyinduced by ibuprofen correlated with changes in expres-sion of markers of the differentiated state.[34] HCASMCscultured in the presence of serum were either untreated(vehicle controls) or exposed to ibuprofen 500 μm, and theprotein levels of SMα-actin and SM22α were then assessed



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Figure 1 Ibuprofen inhibits migration and proliferation of human coronary artery smooth muscle cells (HCASMCs). (a) HCASMCs were eitheruntreated (vehicle control) or treated with increasing concentrations of ibuprofen (100–1000 μM). Trypan blue exclusion was used to determine theinhibitory effect of ibuprofen on HCASMC proliferation 72 h after incubation. Ibuprofen causes inhibition of proliferation in a dose-dependentmanner with an IC50 of 680 ± 120 μM. (b) HCASMCs were either unstimulated (vehicle controls) or stimulated with increasing concentrations ofibuprofen for 24 h. Mitomycn C was added at 23 h (1 h before ‘scratch’ wound) to prevent unwanted proliferation. Wound-healing ‘scratch’ assaywas then performed as described in Materials and Methods. Ibuprofen causes inhibition of migration in a dose-dependent manner with an IC50 of410 ± 160 μM (100–1000 μM). Data shown in (a) and (b) are representative of 3–4 independent experiments. (c) Representative images of theeffects of ibuprofen (500 and 750 μM) vs control on migration of HCASMCs. *P < 0.05 by one-way analysis of variance with Dunnett’s multiplecomparison test.



by immunoblotting analysis. As shown in (Figure 4a–4c),the protein levels of SMα-actin and SM22α wereupregulated following treatment with ibuprofen for 72 h.When we analysed the myofilament density of HCASMC, itrevealed that treatment with ibuprofen increased thephalloidin staining intensity of F-actin, another marker ofdifferentiation of VSMCs,[34] as compared with untreatedvehicle controls (Figure 4d), further supporting the pos-sibility that ibuprofen induces a switch from a synthetic to acontractile phenotype in HCASMCs.

The effects of ibuprofen on HCASMCs arepartially mediated by PPARγ

Many effects of NSAIDs are mediated via cyclooxy-genase (COX)-independent mechanisms;[36] indeed, previ-ous studies have demonstrated that ibuprofen and otherNSAIDs are agonists of PPARγ.[21–23] On the other hand, themajor metabolite of arachidonic acid via COX in VSMCs isPGF2α.[37] Interestingly, PGF2α is a selective antagonist ofPPARγ.[26,27] To study the molecular mechanisms of theeffects of ibuprofen in HCASMCs, we evaluated whetherthe selective natural antagonist of PPARγ, PGF2α,[26,27] orthe selective synthetic antagonist, GW9662,[28] could preventthe effects of ibuprofen in HCASMCs.

Figure 5a shows that the inhibitory effect of ibuprofen onproliferation of HCASMCs was almost completely abro-gated by pre-incubation with either PGF2α or GW9662.Similarly, analysis of migration by wound-healing ‘scratch’assay and time-lapse video microscopy in the presence ofeither PGF2α or GW9662, in combination with ibuprofen,suggest that the PPARγ pathway is also involved in mediat-ing the effects of ibuprofen on HCASMC migration

(Figure 5b and 5c). Thus, whereas incubation with eitherantagonist alone does not significantly affect the migrationof HCASMCs compared with control conditions, incuba-tion of HCASMCs with ibuprofen in the presence of eitherPGF2α or GW9662 significantly but not completelyreverses the effects of ibuprofen (Figure 5b and 5c).

We then explored whether the effects of ibuprofen onmorphology and expression of markers of differentiation inHCASMCs were also mediated via PPARγ. As shown in(Figure 3a and 3b), cell surface area in HCASMCs treatedwith ibuprofen in combination with GW9662 (3 μm) wassimilar to that of medium alone-treated control cells, andwas significantly higher than those treated with ibuprofenalone, suggesting that PPARγ does, indeed, play a role in thephenotype switch induced by ibuprofen. However, whilstGW9662 significantly inhibits the effects on morphologyinduced by ibuprofen, they do not return to levels seen inuntreated control cells (Figure 3a and 3b), suggesting thatother pathways might also be involved in mediating thesemorphological effects. Indeed, troglitazone, a selectivePPARγ agonist, induces changes in the morphology ofHCASMCs, but these are qualitatively different from thoseinduced by ibuprofen (Figure 3a). Thus, whilst ibuprofeninduces a change in morphology resembling the character-istic spindle shape of differentiated VSMCs as describedpreviously,[34,35] troglitazone affects both cell width and celllength in HCASMCs as opposed to ibuprofen that mainlyaffects cell width without affecting cell length (Figure 3a–3d). Finally, we evaluated the expression of protein markersof VSMC differentiation. Significantly, whilst ibuprofeninduced expression of both SMα-actin and SM22α(Figures 4a–4c, 6a–6c), incubation with PGF2α did not sig-nificantly inhibit these effects. In fact, SM22α expressionlevels in the presence of PGF2α alone, or in combinationwith ibuprofen, did not differ; the effect of troglitazone wascomparable to that of ibuprofen (Figure 6a and 6c). In con-trast, whereas PGF2α had a trend to inhibit the effects ofibuprofen on SMα-actin expression, this was not signifi-cant, and troglitazone failed to induce expression of thisprotein above control levels (Figure 6a and 6b).

Ibuprofen does not affect migration andproliferation of ECs

Taken together, our results suggest that ibuprofen inhibitsproliferation and migration of HCASMCs and this is par-tially mediated by the PPARγ pathway. In clinical practice,the ideal DES would prevent restenosis and also late andvery late in-stent thrombosis. It has been suggested thatthe currently available DES target VSMC migration andproliferation, but in doing so they also preventre-endothelialisation of the stent struts, with the risk of verylate in-stent thrombosis steadily occurring over time,[6,7]

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Figure 2 Time-lapse video microscopy of the effects of ibuprofen(500 μM) compared with control. Ibuprofen inhibits migration ofhuman coronary artery smooth muscle cells (HCASMCs). Data are pre-sented as bar and whisker graphs, representative of three independentexperiments, and shown as mean ± standard error of the mean of theaverage migrated displacement (AMD). *P < 0.05 as compared withcontrols.



indicating that complete healing of the vessel wall is neverachieved. Novel DES that prevent migration and prolifera-tion of VSMCs whilst permitting the migration and prolif-eration of ECs, and thus allowing re-endothelialisation,could offer a significant advantage for DES therapy. There-fore, we performed ‘scratch’ wound assays and time-lapsevideo microscopy analysis to evaluate the effects of ibupro-fen on migration of HCAECs.

As shown in (Figure 7a), ibuprofen at all concentrationstested (100–1000 μm) does not significantly affect migra-tion of HCAECs as measured via a ‘scratch’ wound assay.

Similarly, ibuprofen at a concentration of 750 μm, whichpotently inhibits migration of HCASMCs (Figures 1b and2), fails to inhibit migration of HCAECs as demonstratedby time-lapse video microscopy analysis (Figure 7b). Incontrast, troglitazone does affect migration of HCAECs asmeasured by wound-healing assay and time-lapse videomicroscopy (Figure 7a and 7b). We also evaluated the effectsof ibuprofen on proliferation of HPMECs. Whilst doses of100–1000 μm had no significant effect on proliferation ofthese cells, there was significant inhibition of proliferationat doses of 2000 μm, with an estimated IC50 of 1500 ±

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Figure 3 Ibuprofen induces a phenotypic switch in human coronary artery smooth muscle cells (HCASMCs). (a–c) HCASMCs were untreated(control) or exposed to troglitazone (100 μM) or GW9662 (3 μM) alone or in combination with ibuprofen (500 μM) for 72 h. (a) Cells wereimmunostained; pictures are representative of three independent experiments. Scale bar = 50 μm. (b) Areas, (c) lengths, and (d) widths were meas-ured for at least 40 cells in each experiment. Results are mean ± standard error of the mean for three independent experiments. *P < 0.05,**P < 0.01, one-way analysis of variance with Tukey’s post-test.



50 μm (Figure 7c). Interestingly, this IC50 is up to threetimes higher than that required to inhibit proliferation ofHCASMCs. Taken together, these experiments suggest thatthe effect of ibuprofen on migration and proliferation atdoses below 2000 μm is cell type-specific, thus affecting onlyHCASMCs while sparing migration and proliferation ofECs.

We next compared the expression levels of PPARγ in bothHCASMCs and HCAECs. First, we were able to determinethat PPARγ, as a nuclear receptor protein, was presentmainly in the nuclear fractions of both cell types(Figure 8a). To verify the loading of nuclear fractions, mem-branes were probed with ATF2 antibody, and, as shown in(Figure 8b and 8c), densitometric analysis showed thatPPARγ was more abundant in HCASMCs than in HCAECs

when levels were normalised to the loading control, ATF2(Figure 8b and 8c). Interestingly the protein expressed inboth cell types had a molecular mass of ≈53 kDa (seeDiscussion).

Discussion

Restenosis post stent implantation is a complexpathophysiological process, which involves proliferationand migration of VSMCs to the site of injury (e.g. followingballoon angioplasty and stent deployment) and a changein VSMCs towards a synthetic/proliferative phenotype.[33]

Indeed, the restenotic vessel wall is mostly composed ofextracellular matrix proteins synthetised by dedifferentiatedproliferative VSMCs.[14] Recent advances in stent technol-

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Figure 4 Ibuprofen increases the expression of protein markers of differentiation. (a) Human coronary artery smooth muscle cells (HCASMCs) wereunstimulated (control) or exposed to 500 μM ibuprofen for 72 h, and expression of SM α-actin and SM22α protein levels was assessed byimmunoblotting. Images are representative of three independent experiments. (b) and (c) Densitometric analyses of immunoblotts. Data are pre-sented as mean ± standard error of the mean for three independent experiments. (d) Fluorescence intensity analysis of F-actin expression onHCASMCs untreated (control) or exposed to 500 μM for 72 h. F-actin fibers were stained with Alexa Fluor 594 phalloidin. *P < 0.05, paired two-tailed Student’s t-test.



ogy, including DES containing antiproliferative agents (e.g.rapamycin and paclitaxel) have reduced significantly therate of restenosis to less than 10% over the past decade.[3,4]

However, despite the success of DES in reducing restenosis

rates, there is a constant threat of late and very late in-stentthrombosis, particularly following abrupt discontinuationof dual antiplatelet therapy.[5] Several factors appear to beimplicated in the biology of late in-stent thrombosis, butmost evidence suggest it is the lack of selectivity of DES tar-geting not only VSMCs but also endothelial cells, thus inter-fering with the process of re-endothelialisation and vesselwall repair.[12,13] Thus, novel compounds capable of selec-tively targeting VSMCs migration and proliferation withoutinterfering with the process of re-endothelialisation areurgently needed. Indeed, the ideal stent should be capableof maintaining low rates of restenosis, reducing the risk ofearly or late in-stent thrombosis, and promoting vessel wallhealing by modulating the inflammatory response withinthe vessel wall.[15,38] Several second-generation DES, contain-ing everolimus or zotarolimus, share the molecular mecha-

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Figure 5 The effects of ibuprofen on proliferation and migration ofhuman coronary artery smooth muscle cells (HCASMCs) are mediatedvia PPARγ. (a) HCASMCs were either untreated (vehicle controls) orexposed to either PGF2α (100 nM) or GW9662 (3 μM) in the presenceor absence of ibuprofen (500 μM). Trypan blue exclusion was used toevaluate HCASMC proliferation. (b) Cells were untreated (vehicle con-trols) or exposed to either PGF2a or GW9662 in the presence orabsence of ibuprofen (500 μM) for 72 h. Mitomycn C (0.5 μg/ml) wasadded at 1 h before the ‘scratch’ wound to prevent unwanted prolif-eration. Wound-healing ‘scratch’ assay was then performed. (c) Time-lapse video microscopy of the effects of ibuprofen (500 μM) onmigration of HCASMCs in the presence or absence of GW9662 com-pared with vehicle controls and troglitazone (100 μM). Data are pre-sented as bar and whisker graphs, representative of three independentexperiments, and shown as mean ± standard error of the mean of theaverage migrated displacement (AMD). *P < 0.05 one-way ANOVAwith Tukey’s post-test.

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Figure 6 Ibuprofen-mediated expression of protein markers of differ-entiation is not dependent on PPARγ. (a) Immunoblotting analysis ofmarkers of contractile proteins expressed in HCASMCs in response totroglitazone (100 μM), or ibuprofen 500 μM alone or in combinationwith PGF2α (100 nM) vs vehicle controls. (b) and (c) Densitometricanalyses of immunoblots relative to GAPDH. Data presented aremean ± standard error of the mean from three independent experi-ments. *P < 0.05, one-way analysis of variance with Tukey’s post-test.



nisms of their predecessor, sirolimus (rapamycin), andtherefore target VSMCs as well as endothelial cells. Whilstrecent clinical trials suggest improved outcomes comparedwith first generation DES up to 2 years of follow up,[39]

their performance in terms of late complications such aslate and very late (>12 months) stent thrombosis remain tobe determined.

We, and others, have previously shown that certainNSAIDs, including the propionic acid derivative, ibuprofen,are capable of reducing rat VSMCs proliferation in a dose-and cell cycle-dependent manner.[19,20] Here, we studied theability of ibuprofen to inhibit proliferation and migrationof HCASMCs. First, we demonstrated that ibuprofen was

able to inhibit both proliferation and migration ofHCASMCs in a dose-dependent manner. Of note, the con-centration required to inhibit proliferation of HCASMCs(IC50 680 ± 120 μm) is similar to the dose that inhibitedA10 rat VSMCs proliferation in our previous study (IC50646 μm).[20] A similar concentration range is required forinhibition of HCASMC migration (IC50 410 ± 60 μm) asdemonstrated by wound-healing ‘scratch’ assay and also bytime-lapse video microscopy. Interestingly, we also foundthat the effects of ibuprofen on HCASMC migration andproliferation correlated with the induction of a phenotypicswitch on HCASMCs towards a differentiated, contractilestate, characterised by a spindle shape morphology and

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Figure 7 Ibuprofen does not affect migration and proliferation of endothelial cells. (a) Human coronary artery endothelial cells (HCAECs) wereuntreated, exposed to different concentrations of ibuprofen (100 μM–1000 μM), or exposed to troglitazone (100 μM) for 24 h. Results shown aremean ± standard error fo the mean for three independent experiments. *P < 0.05, one-way analysis of variance with Tukey’s post-test. (b) Time-lapse video microscopy on HCAECs treated with troglitazone (100 μM) or GW9662 (3 μM) alone or in combination with ibuprofen (500 μM). Dataare presented as bar and whisker graphs, representative of three independent experiments, and shown as mean ± standard error of the mean of theaverage migrated displacement (AMD). (c) Human pulmonary microvascular endothelial cells (HPMECs) were either untreated (vehicle controls) ortreated with increasing concentrations of ibuprofen (100–2000 μM). Direct cell counting using Trypan blue exclusion was used to determine theinhibitory effect of ibuprofen on HPMEC proliferation 72 h after incubation. Ibuprofen causes inhibition of proliferation in a dose-dependentmanner with an IC50 of 1500 ± 50 μM. *P ≤ 0.05 by one-way analysis of variance with Dunnett’s multiple comparison test.



increased expression of known contractile protein markersof differentiation.[33–35]

Accumulating evidence indicates that NSAIDs havepleiotropic effects in many cell types, and therefore mayhave a therapeutic role in several conditions including pre-vention and treatment of certain types of cancer,[36] and inprevention and treatment of neurodegenerative diseases,such as Alzheimer disease.[22] It has been demonstrated pre-viously that ibuprofen, as well as other NSAIDs, activatePPARγ in several cell types.[21,40] Indeed, ibuprofen at thedose range used in our studies, has been shown to activatethe PPARγ pathway.[23,41] In addition, ibuprofen reduces glialinflammation via a PPARγ-dependent mechanism, thusexplaining the beneficial effects of ibuprofen and otherNSAIDs in Alzheimer’s disease.[23,42] The report of Weberet al. suggests that the effects of ibuprofen on VSMCs pro-liferation are COX-independent.[19] Significantly, PPARγappears to be protective in the cardiovascular systemand has important regulatory roles in vascular biology.[43]

Our data indicate that ibuprofen-induced inhibition ofHCASMC proliferation appears to be mediated primarilyby the PPARγ pathway, since pre-incubation with the selec-tive PPARγ antagonists, PGF2α and GW9662, almost com-pletely restored the proliferative ability of HCASMCs to

control levels. To our knowledge, only few studies haveaimed to elucidate the mechanisms whereby NSAIDsinhibit VSMCs proliferation. One study found that severalNSAIDs, including ibuprofen, inhibit A10 VSMCs prolifera-tion via inhibition of store operated Ca2+ entry (SOCE)channels, an important Ca2+ entry pathway involved in cellproliferation.[44] Another study showed that aspirin inhib-ited VSMCs proliferation by inducing phosphorylation ofAMP-activated protein kinase.[45] The precise mechanismswhereby ibuprofen via activation of PPARγ inhibitproliferation in VSMCs remain to be elucidated; furtherstudies are needed to clarify this. Several agonists of PPARγhave been described including: thiazolidinediones (e.g.ciglitazone, troglitazone, pioglitazone, rosiglitazone) andnatural ligands, including prostanoids, such as 15d-PGJ2and n3-polyunsaturated fatty acids.[43] These PPARγagonists appear to inhibit VSMCs proliferation via down-stream mechanisms that include: inhibiting the expres-sion of the transcription factor c-fos,[46,47] blockade ofangiotensin-II-induced activation of extracellular signal-regulated kinase 1/2 (ERK1/2);[18,48,49] suppression of cellcycle signaling through decreased phosphorylation of theretinoblastoma protein (Rb),[50] a mediator of G1/S progres-sion; and prevention of mitogen-induced degradation ofp27(Kip1),[50] an inhibitor of cdk and Rb phosphorylation.

The ibuprofen-induced phenotypic switch in HCASMCsalso appears to be mediated by PPARγ, as pre-incubationwith GW9662 in the presence of ibuprofen induces ade-differentiated amoeboid morphology similar to that ofcontrol cells, whereas incubation with ibuprofen aloneinduces a differentiated spindle-shaped morphology. Simi-larly, PPARγ plays an important role in the migratory activ-ity of HACSMCs as suggested by the partial rescue of themigratory activity of these cells by incubation with thePPARγ antagonists, PGF2α and GW9662, in the presence ofibuprofen.

Since the effect of PPARγ inhibitors did not completelyabrogate the migratory response, other pathways could beinvolved in this response in addition to PPARγ. We specu-late that one such pathway could be COX2. Indeed, one ofthe main metabolites of arachidonic acid in VSMCs isPGF2α,[37] and this metabolite is a natural selective antago-nist of PPARγ.[26,27] Similarly, in-vivo studies have shownthat there is increased expression and activity of COX2 inthe intima following vascular injury.[17,51] Furthermore, thesynthesis of PGF2α via COX activity is increased byPDGF,[52,53] an important regulator of the differentiationstate of VSMCs, which induces dedifferentiation of VSMCstowards a synthetic phenotype and promotes their migra-tion and proliferation.[54–56] In agreement with these obser-vations, our preliminary data using the wound-healing‘scratch’ assay, indicate that celecoxib, a COX2 selectiveinhibitor, at doses that are nontoxic to the cells

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Figure 8 PPARγ is mainly a nuclear protein and is more abundantlyexpressed in human coronary artery smooth muscle cells (HCASMCs)relative to human coronary artery endothelial cells (HCAECs). (a) and(b) Immunoblotting analysis of PPARγ expression in untreatedHCASMCs vs HCAECs. (a) PPARγ is mainly expressed in the nucleus. (b)Representative images of immunoblotting for PPARγ in HCASMCs vsHCAECs. (c) Densitometric analysis showing relative abundance ofPPARγ normalised to ATF2 protein as loading control.



(6.25–25 μg), prevents migration of rat A10 VSMCs whilstsparing HCAECs migration (unpublished observations).

Of note, our data also indicate that migration and prolif-eration of ECs is not affected by ibuprofen, suggesting thatthe effect of ibuprofen is cell type-specific. This is an impor-tant novel finding from our study and, to our knowledge,this is the first evidence indicating that a molecular com-pound such as ibuprofen has the ability to selectively inhibitVSMCs proliferation and migration whilst sparing ECs pro-liferation and migration. The potential implications of thisobservation could translate into the development of novelDES that could have advantages over those DES currently inuse by not only preventing the development of restenosis,but also at the same time, by not interefering with migra-tion and proliferation of ECs, facilitating the process ofre-endothelialisation of the stent struts. The reasons for theselectivity of ibuprofen are not clear. PPARγ are ligand-activated transcription factors that belong to a nuclearreceptor superfamily of which two isoforms, PPARγ1(53 kDa) and PPARγ2 (57 kDa), are formed from the samegene by alternative mRNA splicing.[57,58] Our results indicatethat a single protein band of approximately 53 kDa isdetectable in nuclear extracts of both HCASMCs andHCAECs (Figure 8), likely corresponding to the PPARγ1isoform. This is in agreement with previous studies sug-gesting that only PPARγ1 is expressed in VSMCs andendothelial cells of different species and tissues.[59,60] We alsoobserved that the relative expression levels of PPARγ appearto be higher in HCASMCs compared with HCAECs, andthis could partly explain the sensitivity of HCASMCs to theeffects of ibuprofen.

Drugs currently used in DES, including sirolimus,paclitaxel and zotarolimus, inhibit proliferation of VSMCsin nanomolar concentrations.[61,62] Thus, a potential limita-tion of our study is that IC50 concentrations of ibuprofenfor migration and proliferation appear to be relatively highwhich may present a challenge to the feasibility of usingibuprofen in a DES.

Taken together, our results suggest that the in-vitro effectsof ibuprofen on migration and proliferation, coupled withthe induction of a phenotype switch towards a differenti-ated state whilst sparing migration of HCAECs, could beexploited by the incorporation of ibuprofen into a DES toprevent restenosis post stent implantation and potentiallyreduce other complications of DES implantation by not

interfering with migration and proliferation of ECs.Whether our in vitro observations could be translated clini-cally into a DES of increased performance remains to bedemonstrated. Future studies will aim to evaluate the fea-sibility of ibuprofen-containing DES in in-vivo animalmodels of vascular injury and restenosis.

Conclusion

In conclusion, this study demonstrates for the first time thatibuprofen induces a phenotypic change in HCASMCs,which is consistent with the reduced proliferative andmigratory ability of these cells. The effects of ibuprofen onmorphology and proliferation of HCASMCs are mediatedmainly by the PPARγ pathway. Migration of HCASMCsappears to be mediated in part by PPARγ, since pre-incubation with PPARγ antagonists significantly, but notcompletely, reverses the effects of ibuprofen; other pathways(e.g. via COX 2) could be involved in this response. Impor-tantly, migration and proliferation of ECs is not affectedby ibuprofen at concentrations that potently inhibitHCASMCs proliferation and migration, indicating thatHCASMCs are targeted selectively by ibuprofen. Thereasons for the selectivity of ibuprofen affecting HCASMCswhereas it spares migration of HCAECs remain unclear,but the relatively higher expression levels of PPARγ inHCASMCs could be, at least in part, responsible for the sen-sitivity of HCASMCs to this drug. Our in-vitro observationscould translate into reduced rates of restenosis post angio-plasty with stent deployment of an ibuprofen-containingDES, providing ibuprofen-induced inhibition of prolifera-tion and migration of HCASMCs and could potentially alsolead to a reduced risk of other complications following stentimplantation, as ibuprofen spares the migration and prolif-eration of ECs.

Declarations

Conflict of interest

The Author(s) declare(s) that they have no conflicts ofinterest to disclose.

Funding

This work was supported by University of Reading, UK, andTishreen University, Syria.

References

1. Allender S et al. European Cardiovas-cular Disease Statistics. Brussels: Euro-pean Heart Network, 2008.

2. Mendis S et al. Global Atlas on Cardio-vascular Disease Prevention andControl. Geneva: World HealthOrganization in collaboration with theWorld Heart Federation and the

World Stroke Organization, 2011. vi,155 p.

3. Babapulle MN et al. A hierarchi-cal Bayesian meta-analysis of ran-domised clinical trials of drug-eluting



stents. Lancet 2004; 364: 583–591.

4. Windecker S et al. Sirolimus-elutingand paclitaxel-eluting stents for coro-nary revascularization. N Engl J Med2005; 353: 653–662.

5. Urban P, De Benedetti E. Thrombosis:the last frontier of coronary stenting?Lancet 2007; 369: 619–621.

6. Daemen J et al. Early and late coro-nary stent thrombosis of sirolimus-eluting and paclitaxel-eluting stents inroutine clinical practice: data from alarge two-institutional cohort study.Lancet 2007; 369: 667–678.

7. Shen ZJ et al. Five-year clinicaloutcomes after coronary stentingof chronic total occlusion usingsirolimus-eluting stents: insights fromthe rapamycin-eluting stent evaluatedat Rotterdam Cardiology Hospital-(Research) Registry. Catheter Cardio-vasc Interv 2009; 74: 979–986.

8. Layland J et al. Extremely late drug-eluting stent thrombosis: 2037 daysafter deployment. Cardiovasc RevascMed 2009; 10: 55–57.

9. Al-Dehneh A et al. Drug-eluting stentthrombosis 1659 days after stentdeployment: case report and literaturereview. Tex Heart Inst J 2010; 37: 343–346.

10. Garg S, Serruys PW. Coronary stents:looking forward. J Am Coll Cardiol2010; 56: S43–S78.

11. Wilson JM. Unintended consequences:avoiding restenosis and stent throm-bosis. Tex Heart Inst J 2010; 37: 341–342.

12. Lüscher TF et al. Drug-eluting stentand coronary thrombosis: biologicalmechanisms and clinical implica-tions. Circulation 2007; 115: 1051–1058.

13. Stähli BE et al. Drug-eluting stentthrombosis. Ther Adv Cardiovasc Dis2009; 3: 45–52.

14. Litvin J et al. Expression and functionof periostin-like factor in vascularsmooth muscle cells. Am J Physiol CellPhysiol 2007; 292: C1672–C1680.

15. Rzucidlo EM. Signaling pathwaysregulating vascular smooth musclecell differentiation. Vascular 2009;17(Suppl. 1): S15–S20.

16. Dreyer C et al. Control of theperoxisomal beta-oxidation pathwayby a novel family of nuclear hormonereceptors. Cell 1992; 68: 879–887.

17. Bishop-Bailey D et al. Intimal smoothmuscle cells as a target for peroxi-some proliferator-activated receptor-Œ ≥ ligand therapy. Circ Res 2002; 91:210–217.

18. Goetze S et al. Peroxisomeproliferator-activated receptor-gammaligands inhibit nuclear but notcytosolic extracellular signal-regulatedkinase/mitogen-activated proteinkinase-regulated steps in vascularsmooth muscle cell migration. JCardiovasc Pharmacol 2001; 38: 909–921.

19. Weber A-A et al. Cyclooxygenase-independent inhibition of smoothmuscle cell mitogenesis by ibuprofen.Eur J Pharmacol 2000; 389: 67–69.

20. Brooks G et al. Non-steroidal anti-inflammatory drugs (NSAIDs) inhibitvascular smooth muscle cell prolifera-tion via differential effects on the cellcycle. J Pharm Pharmacol 2003; 55:519–526.

21. Lehmann JM et al. Peroxisomeproliferator-activated receptors α andγ are activated by indomethacinand other non-steroidal anti-inflammatory drugs. J Biol Chem1997; 272: 3406–3410.

22. Bernardo A et al. Nuclear recep-tor peroxisome proliferator-activatedreceptor-γ is activated in rat microglialcells by the anti-inflammatory drugHCT1026, a derivative of flurbiprofen.J Neurochem 2005; 92: 895–903.

23. Heneka MT et al. Acute treatmentwith the PPARγ agonist pioglitazoneand ibuprofen reduces glial inflamma-tion and Aβ1–42 levels in APPV717Itransgenic mice. Brain 2005; 128:1442–1453.

24. Rodriguez LG et al. Wound-healingassay. Methods Mol Biol 2005; 294:23–29.

25. Liang C-C et al. In vitro scratch assay:a convenient and inexpensive methodfor analysis of cell migration in vitro.Nat Protocols 2007; 2: 329–333.

26. Marx N et al. Peroxisome proliferator-activated receptor gamma activators

inhibit gene expression and migrationin human vascular smooth musclecells. Circ Res 1998; 83: 1097–1103.

27. Reginato MJ et al. Prostaglandinspromote and block adipogenesisthrough opposing effects onperoxisome proliferator-activatedreceptor γ. J Biol Chem 1998; 273:1855–1858.

28. Leesnitzer LM et al. Functional conse-quences of cysteine modification inthe ligand binding sites of peroxisomeproliferator activated receptors byGW9662. Biochemistry 2002; 41:6640–6650.

29. Schmidt CE et al. Integrin cytoskeletalinteractions in migrating fibroblastsare dynamic, asymmetric, and regu-lated. J Cell Biol 1993; 123: 977–991.

30. Markou T et al. Glycogen synthasekinases 3alpha and 3beta in cardiacmyocytes: regulation and conse-quences of their inhibition. Cell Signal2008; 20: 206–218.

31. Giraldo A et al. Feedback regulationby Atf3 in the endothelin-1-responsivetranscriptome of cardiomyocytes:Egr1 is a principal Atf3 target. BiochemJ 2012; 444: 343–355.

32. Bradford MM. A rapid and sensitivemethod for the quantitation of micro-gram quantities of protein utilizingthe principle of protein-dye binding.Anal Biochem 1976; 72: 248–254.

33. Martin KA et al. Rapamycin promotesvascular smooth muscle cell differen-tiation through insulin receptorsubstrate-1/phosphatidylinositol 3-kinase/Akt2 feedback signaling. J BiolChem 2007; 282: 36112–36120.

34. Han M et al. Serum deprivationresults in redifferentiation of humanumbilical vascular smooth musclecells. Am J Physiol Cell Physiol 2006;291: C50–C58.

35. Cox LR, Ramos K. Allylamine-induced phenotypic modulation ofaortic smooth muscle cells. J ExpPathol (Oxford) 1990; 71: 11–18.

36. Thun MJ et al. Nonsteroidal anti-inflammatory drugs as anticanceragents: mechanistic, pharmacologic,and clinical issues. J Natl Cancer Inst2002; 94: 252–266.



37. Brinkman HJ et al. Involvement ofcyclooxygenase- and lipoxygenase-mediated conversion of arachidonicacid in controlling human vascularsmooth muscle cell proliferation.Thromb Haemost 1990; 63: 291–297.

38. Simons M. VEGF and restenosis: therest of the story. Arterioscler ThrombVasc Biol 2009; 29: 439–440.

39. Silber S et al. Unrestricted randomiseduse of two new generation drug-eluting coronary stents: 2-yearpatient-related vs stent-related out-comes from the RESOLUTE AllComers trial. Lancet 2011; 377: 1241–1247.

40. Jaradat MS et al. Activation ofperoxisome proliferator-activatedreceptor isoforms and inhibition ofprostaglandin H2 synthases by ibu-profen, naproxen, and indomethacin.Biochem Pharmacol 2001; 62: 1587–1595.

41. Shimada T et al. PPARgamma medi-ates NSAIDs-induced upregulation ofTFF2 expression in gastric epithelialcells. FEBS Lett 2004; 558: 33–38.

42. Sastre M et al. Nonsteroidal anti-inflammatory drugs repress β-secretase gene promoter activity by theactivation of PPARγ. Proc Natl AcadSci U S A 2006; 103: 443–448.

43. Hamblin M et al. PPARs and the car-diovascular system. Antioxid RedoxSignal 2009; 11: 1415–1452.

44. Munoz E et al. Nonsteroidal anti-inflammatory drugs inhibit vascularsmooth muscle cell proliferation byenabling the Ca2+-dependent inacti-vation of calcium release-activatedcalcium/orai channels normally pre-vented by mitochondria. J Biol Chem2011; 286: 16186–16196.

45. Sung JY, Choi HC. Aspirin-inducedAMP-activated protein kinase activa-tion regulates the proliferation ofvascular smooth muscle cells fromspontaneously hypertensive rats.

Biochem Biophys Res Commun 2011;408: 312–317.

46. Law RE et al. Troglitazone inhibitsvascular smooth muscle cell growthand intimal hyperplasia. J Clin Invest1996; 98: 1897–1905.

47. Benson S et al. Peroxisome proliferator-activated receptor (PPAR)-gammaexpression in human vascular smoothmuscle cells: inhibition of growth,migration, and c-fos expression bythe peroxisome proliferator-activatedreceptor (PPAR)-gamma activatortroglitazone. Am J Hypertens 2000; 13:74–82.

48. Graf K et al. Troglitazone inhibitsangiotensin II-induced DNA synthesisand migration in vascular smoothmuscle cells. FEBS Lett 1997; 400:119–121.

49. Goetze S et al. PPAR gamma-ligandsinhibit migration mediated by multi-ple chemoattractants in vascularsmooth muscle cells. J CardiovascPharmacol 1999; 33: 798–806.

50. Wakino S et al. Peroxisomeproliferator-activated receptor gammaligands inhibit retinoblastoma phos-phorylation and G1--> S transition invascular smooth muscle cells. J BiolChem 2000; 275: 22435–22441.

51. Ogawa M et al. A critical role ofCOX-2 in the progression ofneointimal formation after wire injuryin mice. Expert Opin Ther Targets2009; 13: 505–511.

52. Schror K, Weber AA. Roles of vaso-dilatory prostaglandins in mitogenesisof vascular smooth muscle cells.Agents Actions Suppl 1997; 48: 63–91.

53. Weber AA et al. Antimitogenic effectsof vasodilatory prostaglandins incoronary artery smooth muscle cells.Basic Res Cardiol 1998; 93(Suppl. 3):54–57.

54. Ferns GA et al. Inhibition ofneointimal smooth muscle accumula-tion after angioplasty by an antibody

to PDGF. Science 1991; 253: 1129–1132.

55. Pukac L et al. Platelet-derived growthfactor-BB, insulin-like growth factor-I,and phorbol ester activate differentsignaling pathways for stimulation ofvascular smooth muscle cell migra-tion. Exp Cell Res 1998; 242: 548–560.

56. Cospedal R et al. Platelet-derivedgrowth factor-BB (PDGF-BB) regula-tion of migration and focal adhesionkinase phosphorylation in rabbitaortic vascular smooth muscle cells:roles of phosphatidylinositol 3-kinaseand mitogen-activated proteinkinases. Cardiovasc Res 1999; 41: 708–721.

57. Kliewer SA et al. Differential expres-sion and activation of a family ofmurine peroxisome proliferator-activated receptors. PNAS 1994; 91:7355–7359.

58. Tontonoz P et al. Stimulation ofadipogenesis in fibroblasts by PPAR32,a lipid-activated transcription factor.Cell 1994; 79: 1147–1156.

59. Law RE et al. Expression and functionof PPARγ in rat and human vascularsmooth muscle cells. Circulation 2000;101: 1311–1318.

60. Zahradka P et al. Peroxisomeproliferator-activated receptor Œ ±and Œ ≥ ligands differentially affectsmooth muscle cell proliferation andmigration. J Pharmacol Exp Ther 2006;317: 651–659.

61. Parry TJ et al. Drug-eluting stents:sirolimus and paclitaxel differentiallyaffect cultured cells and injuredarteries. Eur J Pharmacol 2005; 524:19–29.

62. Chen YW et al. Zotarolimus, a novelsirolimus analogue with potent anti-proliferative activity on coronarysmooth muscle cells and reducedpotential for systemic immunosup-pression. J Cardiovasc Pharmacol 2007;

49: 228–235.



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