Synergistic Inhibition of Vascular Smooth Muscle Cell Migration by Phosphodiesterase 3 and...

Synergistic Inhibition of Vascular Smooth Muscle CellMigration by Phosphodiesterase 3 and

Phosphodiesterase 4 InhibitorsDaniel Palmer, Keith Tsoi, Donald H. Maurice

Abstract—Cyclic nucleotide phosphodiesterases (PDEs) hydrolyze cAMP or cGMP and terminate their signaling. Twoimportant families of PDEs that regulate cAMP signaling in cardiovascular tissues are the cGMP-inhibited PDEs(PDE3) and the cAMP-specific PDEs (PDE4). In this study, we have used a combination of an in vitro motility assayand a sensitive method for the measurement of cAMP in order to determine the relative roles of PDE3 and of PDE4 inthe regulation of cAMP-mediated inhibition of VSMC migration. Our data demonstrate that forskolin, an activator ofadenylyl cyclases, causes concentration-dependent inhibition of platelet-derived growth factor–induced VSMCmigration. Incubation of cultured VSMCs with a PDE4-selective inhibitor, Ro 20-1724, markedly potentiated both theantimigratory effect and the increase in cAMP caused by forskolin. Cilostamide, a PDE3-selective compound, did notaffect either the antimigratory activity of forskolin or its ability to increase cAMP. Cilostamide and Ro 20-1724interacted synergistically to potentiate the inhibition of VSMC migration by forskolin and caused a supra-additiveincrease in cAMP. These data are consistent with an important role for both PDE3 and PDE4 in the regulation ofcAMP-mediated inhibition of VSMC migration.(Circ Res. 1998;82:852-861.)

Key Words: cAMP n cyclic nucleotide phosphodiesterasen vascular smooth musclen migrationn platelet-derivedgrowth factor

Cell migration is vital in the processes of embryogenesis,wound healing, and bone remodeling and also in pathol-

ogies underlying diseases such as cancer.1,2 As it pertains tovascular biology, VSMC migration, from the medial to theintimal layer, has been strongly implicated in the develop-ment of atherosclerotic plaques and in the neointimal thick-ening found in restenosing arteries after balloon angio-plasty.3–5 Migration of VSMCs can be induced by a number ofblood-borne or vascular cell–secreted factors, includingPDGF,6 angiotensin II,7 transforming growth factor-b,8 insu-lin-like growth factor-1,9 fibroblast growth factor-2 (basicfibroblast growth factor),10,11 vitronectin,12 fibronectin,13 andoxidized LDL.14

Recently, a growing body of evidence has emerged impli-cating cAMP in the inhibition of VSMC migration.7,12,15–17

Specifically, studies using lipophilic structural analogues ofcAMP7,12,15,17and activators of adenylyl cyclases7,12,15–17havedemonstrated that an increase in cAMP positively correlateswith the inhibition of VSMC migration. Furthermore, thedownregulation of the major effector of cAMP, cAMP-dependent protein kinase (PKA), abrogates inhibition ofVSMC by forskolin.16

cAMP signaling in mammalian cells is terminated bycyclic nucleotide PDEs, a multifamily class of enzymes thatcatalyze the hydrolysis of cyclic nucleotides to 59-nucleotide

monophosphates, which do not activate cAMP effector pro-teins.18,19 Seven distinct PDE families (PDE1 to PDE7) havebeen designated, with each discriminated on the basis ofseveral criteria, including kinetic and regulatory properties aswell as molecular sequence.19 To date,'30 PDE isoenzymeshave been identified.19 Of the PDE families identified inVSMCs, members of the PDE3 (cGMP-inhibited) and PDE4(cAMP-specific) families have been shown to contribute tothe regulation of cAMP signaling and its impact on VSMCfunction.20–25 More specifically, inhibitors of PDE3 or PDE4activities increase VSMC cAMP, and PDE3 inhibitors havemarked effects on VSMC contraction-relaxation coupling.Although some reports have identified calmodulin-stimulatedPDE activity (PDE1) in homogenates of blood vessels20,21,23

and PDE1 has been shown to hydrolyze cAMP when thiscyclic nucleotide is present at high concentration,19 vasculareffects of selective PDE1 inhibitors, such as vinpocetine,26 donot correlate positively with inhibition of PDE1 activity andmay relate to other effects attributable to this compound.27,28

Low level PDE2 activity has been isolated only once fromporcine VSMCs, and no functional significance has beenattributed to this activity in VSMCs.20

Several studies have correlated an inhibition of PDE3activity in VSMCs with relaxation of aortic strips.29–31 Inaddition, Maurice et al25 have demonstrated that the selective

Received August 29, 1997; accepted February 9, 1998.From the Departments of Pathology (D.H.M.) and Pharmacology and Toxicology (D.P., K.T., D.H.M.), Queen’s University, Kingston, Ontario,

Canada.Correspondence to Dr D.H. Maurice, PhD, A221 Botterell Hall, Queen’s University, Kingston, Ontario K7L 3N6, Canada.E-mail [email protected]© 1998 American Heart Association, Inc.

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PDE3 inhibitor cilostamide synergizes with theb-adrenergicreceptor agonist isoproterenol to increase relaxation of rataorta. In contrast, selective PDE4 inhibitors are ineffective ateliciting relaxation of vascular smooth muscle in the absenceof a functional endothelium despite the fact that they repre-sent a significant portion of cAMP-PDE activity in theaorta.25,32 Combinations of isoproterenol and selective PDE4inhibitors, however, relax vascular smooth muscle in asynergistic fashion.24,25 PDE3 and PDE4 inhibitors in combi-nation also synergize to relax VSMCs.29,32

Whereas PDE3 inhibitors have potent vasorelaxant prop-erties, they are relatively ineffective at attenuating VSMCproliferation.33,34 However, selective inhibition of PDE3isozymes has been reported to potentiate the antiproliferativeeffects of forskolin, a direct activator of adenylyl cyclases.34

Like PDE3 inhibition, PDE4 inhibition could only signifi-cantly limit VSMC proliferation in the presence of activatorsof adenylyl cyclases.34,35It is noteworthy that PDE4 inhibitionpotentiated the effects of forskolin to a greater extent than didPDE3 inhibition, consistent with the relative contributionof PDE3 and PDE4 activities in cultured VSMCs.36 Inaddition, PDE3 and PDE4 inhibitors have been shown tointeract synergistically, as they do in the process of relax-ation, to attenuate VSMC proliferation in VSMCs32 and inA10 cells, an immortalized VSMC-like cell line.37

Although a significant number of studies have focused onelucidating the role of PDE3 and PDE4 isozymes in theregulation of VSMC contraction and proliferation, a paucityof information exists on the contribution of these enzymes inthe process of cAMP-mediated inhibition of VSMC migra-tion. Furthermore, the differential capacity for specific PDEfamilies to impact on VSMC function, as indicated by thedisparate effects of PDE3 and PDE4 inhibitors on contractionand proliferation, has similarly not been suitably addressed asit applies to VSMC migration. Consequently, in the presentstudy, PDGF-induced rat aortic VSMC migration was quan-tified in the presence or absence of combinations of cilosta-mide (a selective PDE3 inhibitor),38 Ro 20-1724 (a selectivePDE4 inhibitor),39 IBMX (a nonselective PDE inhibitor),40

and forskolin to explore the respective roles of PDE3 andPDE4 in cAMP-mediated inhibition of VSMC migration.Similar combinations of these agents were used to relate thelevels of cAMP in the VSMCs to observed modulations ofmigratory ability.

Materials and MethodsMaterialsRecombinant human PDGF-BB, DMEM, HBSS, trypsin-EDTAsolution, penicillin-streptomycin antibiotic mixture, bovine calf se-

rum, and FBS were purchased from GIBCO BRL. Forskolin,8-bromo-cAMP, and 1,9-dideoxyforskolin were obtained from Re-search Biochemicals International. Ro 20-1724, bovine brain cal-modulin, and vinpocetine were acquired from Calbiochem-No-vachem Corp. HEPES, IBMX, EDTA, EGTA, dithiothreitol,phenylmethylsulfonyl fluoride, benzamidine HCl monohydrate (ben-zamidine), Tris-HCl, sodium chloride, and Triton X-100 were fromICN Biomedicals, Inc. Thep-nitrophenyl phosphate tablets wereacquired from Sigma-Aldrich, Ltd. Cilostamide was generouslyprovided by Dr H. Hidaka (Nagoya University School of Medicine,Nagoya, Japan). EHNA was from Biomol. Transwell cell culturechamber inserts (polycarbonate, tissue culture–treated, 6.5-mm di-ameter, and 8.0-mm pore size) were from the Corning Costar Corp.Trypan blue and Giemsa stain were purchased from BDH Chemicals.Affi-gel 601, column supports, gelatin, Dowex 50 (200 to 400 mesh),and aluminum oxide (alumina) were obtained from Bio-Rad Labo-ratories. Leupeptin was obtained from Boehringer-Mannheim. TheBCA protein assay and bovine serum albumin were purchased fromPierce. [3H]Hypoxanthine (24.1 Ci/mmol), [3H]cAMP (27 Ci/mmol),and [14C]59-AMP (590.4 mCi/mmol) were from NEN Life ScienceProducts. [14C]cAMP (283 mCi/mmol) was obtained from Amer-sham Life Science. All other items and chemicals (reagent grade)were obtained from Fisher Scientific.

Cell CulturePrimary cultures of rat aortic VSMCs (after isolation from rat aortaas previously described41) were a generous gift from Dr S.C. Pang(Department of Anatomy and Cell Biology, Queen’s University,Kingston, Canada). The identity of the cells was confirmed byimmunohistochemical detection of smooth muscle–specifica-actin.VSMCs were routinely cultured in DMEM supplemented with 10%bovine calf serum, 100 U/mL penicillin, and 100mg/mL streptomy-cin in a 37°C, 95% air/5% CO2, humidified atmosphere. To disso-ciate the cells for subculturing, VSMCs were washed once incalcium- and magnesium-free HBSS, treated with 0.05% trypsin and0.53 mmol/L EDTA for 2 to 5 minutes, and resuspended in growthmedium. VSMCs were seeded in 75-cm2 flasks with 106 cells in 15mL of medium per flask. In all experiments, VSMCs of passages 7through 16 were used.

cAMP PDE Activity AssayThe cyclic nucleotide PDE activity in homogenates of cultured VSMCswas assayed using a modification of the method of Davis and Daly.42

Briefly, cultures of VSMCs were homogenized in ice-cold lysis buffer(50 mmol/L Tris-HCl [pH 7.4], 5 mmol/L magnesium chloride,150 mmol/L sodium chloride, 1 mmol/L EDTA, 5 mmol/L benzami-dine, 1 mmol/L dithiothreitol, 1mmol/L leupeptin, 100mmol/L phenyl-methylsulfonyl fluoride, and 1% [vol/vol] Triton X-100). The homog-enate was centrifuged at 10 000g for 3 minutes, and the supernatant wasused for activity determinations. A sample of the homogenate (contain-ing '5 mg of protein) was added to reaction buffer (50 mmol/LTris-HCl [pH 7.4], 5 mmol/L magnesium chloride, 100mmol/L EGTA,and 0.1 nmol ['100 000 dpm] [3H]cAMP) in the presence of calcium(50mmol/L) along with calmodulin (10 U), vinpocetine (2 to 200mmol/L),EHNA (10 mmol/L), cilostamide (1mmol/L), Ro 20-1724 (10mmol/L),IBMX (500 mmol/L), or vehicle (DMSO or water) such that thereactions were carried out in a total volume of 100mL. The sampleswere incubated at 37°C for 30 minutes, and the reactions were halted bythe addition of 50mL of ice-cold 0.5 mol/L EDTA (pH 8.0). To correctfor recovery, 50mL [14C]59-AMP ('1600 dpm) and 0.2 mL HEPES-NaCl buffer (0.1 mol/L sodium chloride and 0.1 mol/L HEPES [pH8.5]) were added to the samples before purification of the nucleoside59-monophosphate reaction product. Products were purified via chro-matography using a polyacrylamide-boronate gel column (Affi-gel 601Bio-Rad, 1-mL bed volume). After the columns were prewashed with 8mL of HEPES-NaCl buffer, the samples were applied. The columnswere washed four times with 2 mL of HEPES-NaCl buffer andequilibrated with 1 mL of 0.05 mol/L sodium acetate (pH 4.8). Theradiolabeled nucleoside 59-monophosphate was eluted with 4 mL of0.05 mol/L sodium acetate (pH 4.8) and quantified using liquidscintillation counting. The eluted [3H]59-AMP was corrected for recov-

Selected Abbreviations and Acronyms

DMSO 5 dimethyl sulfoxideEHNA 5 erythro-9-(2-hydroxy-3-nonyl)adenineIBMX 5 3-isobutyl-1-methylxanthineMAP 5 mitogen-activated proteinPDE 5 phosphodiesterase

PDGF 5 platelet-derived growth factorPKA, PKG 5 protein kinase A and G

VSMC 5 vascular smooth muscle cell

Palmer et al May 4, 1998 853

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ery of [14C]59-AMP and normalized for the total protein used in theassay, and the total cAMP-hydrolyzing activity in the sample wasexpressed as picomoles per minute per milligram protein. The BCAprotein assay (Pierce) was used (according to the manufacturer’sprotocol with bovine serum albumin as the standard) to determine thetotal protein concentration of each sample. The activities are represen-tative of at least three determinations for each agent(s).

Migration AssayVSMC migration assays were performed using a modified Boyden’schamber.7,43 Briefly, a confluent monolayer of VSMCs was washedwith calcium- and magnesium-free HBSS and treated with 0.05%trypsin and 0.53 mmol/L EDTA for 2 to 5 minutes to detach the cellsfrom the substratum. VSMCs were sequentially washed with growthmedium and with DMEM supplemented with 0.5% FBS. Isolatedcells were resuspended in DMEM/0.5% FBS to a concentration of6.73105 cells/mL, as determined using a hemocytometer. Viabilityof the cells used in the assay was determined by trypan blueexclusion, and viability was always.90%. VSMCs were allowed toequilibrate in DMEM/0.5% FBS for 1 hour before use. Transwellinserts (6.5-mm diameter, 8-mm pores) were immersed in a DMEM/0.25% gelatin solution for 1 hour before use without allowing thegelatin to dry. Approximately 23105 VSMCs, in a 300mL aliquot ofthe DMEM/0.5% FBS suspension, were added to the upper chamberof the Transwell inserts, and DMEM/0.5% FBS (500mL) was addedto the lower chamber (beneath the insert). After a 1-hour incubationat 37°C, under tissue-culturing conditions, individual inserts weretransferred to separate wells in a 24-well cluster plate in which 500mL of DMEM/0.5% FBS containing the chemotactic factorPDGF-BB (10 ng/mL) or vehicle (0.1 mol/L acetic acid) was present.When tested, forskolin (1 to 100mmol/L), 1,9-dideoxyforskolin(10 mmol/L), cilostamide (1mmol/L), Ro 20-1724 (10mmol/L),IBMX (500 mmol/L), 8-bromo-cAMP (1 mmol/L), or combinationsof these agents were added to the lower chamber with PDGF. Thevehicle (DMSO), at 0.2% of the total volume, was added to the lowerchamber in all experiments. Transwell apparatuses were incubatedfor 6 hours in a 37°C, 95% air/5% CO2, humidified atmosphere.After which time, cells remaining on the upper face of the membranewere removed by scraping with cotton swabs. VSMCs that hadmigrated to the lower face of the membrane were fixed for 12 to 16hours in 10% (wt/vol) paraformaldehyde in PBS at 4°C and stainedwith Giemsa stain for 1 hour. Membranes were washed in PBS andremoved from their support, and the number of migrating cells wasmeasured by light microscopy. Stained cells possessing a distinctnucleus and multiple projections that had clearly exited the pores ofthe filter were counted in eight random fields of view (magnification3200), such that the VSMC migrational activity was determined ascells per field of view. To ensure that effects on VSMC migrationwere not due to toxicity, VSMC suspensions were seeded in wells ofa 24-well cluster plate, which was precoated with a 0.25% gelatin/DMEM solution, and treated with forskolin (1 to 100mmol/L) andIBMX (500 mmol/L) or with vehicle (DMSO) for 1 or 6 hours, andviability was determined by trypan blue exclusion. Cell survival aftertreatment with the various pharmacological agents was also assessedusing a colorimetric acid phosphatase assay.44,45 Briefly, '10 000cells were added per well to a 96-well tissue culture cluster plate.Cells were treated for 6 hours with PDGF (10 ng/mL) and withvarying combinations of forskolin (1 to 100mmol/L), cilostamide(1 mmol/L), Ro 20-1724 (10mmol/L), and IBMX (500mmol/L) in100 mL/well of growth medium. After this incubation period, themedium was removed, and the cells were washed with 200mL/wellof PBS. Lysosomal acid phosphatase activity was assessed byincubation of the cells, at 37°C, with 100mL/well of a reaction buffercontaining 0.1 mol/L sodium acetate (pH 5.5), 0.1% Triton X-100,and 10 mmol/Lp-nitrophenyl phosphate (Sigma 104 phosphatasesubstrate). The reaction was stopped after 1 hour by the addition of10 mL/well of 1 mol/L sodium hydroxide. Enzyme activity wasdetermined by colorimetric measurement, at 405 nm, of the sampleswith a microplate reader. A linear relationship exists between cellnumber and acid phosphatase activity. Effects of individual drugtreatments were tested at least three times unless otherwise indicated.

Measurement of cAMP in Cultured VSMCsA confluent 75-cm2 flask of VSMCs was incubated with freshgrowth medium supplemented with 20 mCi/L [3H]hypoxanthine for16 hours. As previously described,46 the incubation of culturedVSMCs with [3H]hypoxanthine allows for the homogeneous labelingof both the ATP and GTP metabolic pools. Labeled VSMCs werewashed with calcium- and magnesium-free HBSS and subsequentlytreated with 0.05% trypsin and 0.53 mmol/L EDTA for 2 to 5minutes to detach the VSMCs from the flask. Dissociated cells werewashed, resuspended in DMEM/0.5% FBS, and incubated in a 37°C,95% air/5% CO2, humidified atmosphere for an hour. LabeledVSMCs (500mL, 23106 dpm) were seeded in 24-well cluster platesthat had been precoated with DMEM supplemented with 0.25%gelatin (wt/vol) and incubated for 1 hour at 37°C and 95% air/5%CO2. After the last equilibration period, cells were treated withforskolin (1 to 100mmol/L), cilostamide (1mmol/L), Ro 20-1724(10 mmol/L), or IBMX (500 mmol/L), alone or in combination, for30-minute, 1-hour, and 6-hour intervals. The drug vehicle (DMSO)was added to each well and represented no more than 0.2% of thetotal volume. Incubations were terminated by the addition of 0.5 mLof ice-cold 10% trichloroacetic acid, and'1000 dpm of [14C]cAMPwas added to each sample as an internal standard. cAMP wasisolated and purified via sequential column chromatography usingneutral alumina and Dowex 50 resin columns. [3H]cAMP and[14C]cAMP amounts were determined using liquid scintillation asdescribed previously.46 After correction for recovery, the [3H]cAMPpresent in the individual wells was expressed as a percentage of thetotal 3H in each well. Individual treatments were assayed in triplicatein at least three independent experiments unless otherwise indicated.

Statistical AnalysisData are presented as mean6SEM of at least three independentexperiments unless otherwise indicated. Statistical differences be-tween results were determined using unpaired ANOVA, with Dun-nett or Tukey-Kramer multiple comparison post hoc tests or unpairedStudent t tests as indicated. A value ofP,.05 was consideredstatistically significant.

ResultsEffects of Selective PDE Inhibitors on VSMCcAMP-Hydrolyzing PDE ActivityTo determine which isozymes represented major componentsof the total cAMP-hydrolyzing PDE (cAMP-PDE) activityand to permit for a rational interpretation as to which enzymeswould likely be of significant importance in the regulation ofcAMP-mediated inhibition of VSMC migration, cAMP-PDEactivity from VSMC homogenates was assayed in the pres-ence and absence of various PDE inhibitors (Table). Vinpo-cetine, a putative selective inhibitor of PDE1 isozymes, hadno significant effect on total PDE activity over the range ofconcentrations used (2 to 200mmol/L, Table). This result wasconsistent with the absence of calcium (50mmol/L)/calmod-ulin (10 U)–stimulated activity in these homogenates (Table).Similarly, EHNA (10 mmol/L), a selective inhibitor ofPDE2,47 was ineffective at inhibiting VSMC cAMP-PDEactivity, validating our previous work.36 Also consistent withour previous work,36 cilostamide (1mmol/L) and Ro 20-1724(10 mmol/L), selective PDE3 and PDE4 inhibitors, respec-tively, inhibited VSMC cAMP-PDE activity by'16% and40% individually and were strictly additive when combined(Table). The addition of vinpocetine (2 to 200mmol/L) to thiscombination of cilostamide and Ro 20-1724 did not furtherinhibit the cAMP-PDE activity, further attesting to theabsence of PDE1 in these cells (Table). The broad-spectrum

854 PDE3 and PDE4 Regulation of VSMC Migration



PDE inhibitor IBMX (500mmol/L) inhibited virtually all ofthe cAMP-PDE activity in these homogenates (Table). Thus,only PDE3 and PDE4 isozymes contribute significantly to thehydrolysis of cAMP in our VSMCs.

PDE3 and PDE4 Inhibitors PotentiateForskolin-Mediated Inhibition of PDGF-InducedVSMC MigrationConsistent with earlier reports,15–17 PDGF-BB caused a con-centration-dependent increase in the migration of cultured rataortic VSMCs when assayed using a modification of Boy-den’s chamber method.7 For our experiments, 10 ng/mLPDGF-BB was chosen, since this concentration gave an'50% maximal stimulation. In our studies, this concentrationof PDGF-BB stimulated VSMC migration by'4-fold from abasal migration of 32.9613.4 to 121.9614.1 cells per field ofview. Forskolin, an activator of adenylyl cyclases, inhibitedPDGF-induced migration of cultured rat aortic VSMCs (Fig-ure 1) and caused marked changes in the morphology of thesecells (see below). VSMC migration to 10 ng/mL PDGF wasnot inhibited by incubation with the lowest concentration offorskolin used in our studies (1mmol/L). However, higherconcentrations of forskolin (10 or 100mmol/L) did inhibitPDGF-induced migration by'21% and 58%, respectively(Figure 1). In both experiments in which it was measured,8-bromo-cAMP (1 mmol/L) also inhibited PDGF-inducedmigration, whereas 1, 9-dideoxyforskolin (10mmol/L), astructural analogue of forskolin that does not activate adeny-lyl cyclases, had no effect on PDGF-induced migration (not

shown). These data are consistent with prior reports offorskolin-mediated inhibition of PDGF-induced VSMC mi-gration and support the hypothesis that cAMP mediates theseeffects.

To ascertain the role(s) of cAMP-PDEs in regulating thisforskolin-mediated effect, inhibition of migration was alsomeasured in the presence of inhibitors of the major cAMP-PDE activities expressed in these cells. Whether used alone orin combination, neither the selective PDE3 inhibitor cilosta-mide (1 mmol/L), the selective PDE4 inhibitor Ro 20-1724(10 mmol/L), nor the broad-spectrum PDE inhibitor IBMX(500 mmol/L) inhibited PDGF-induced migration (notshown). In contrast to their effects alone, addition of some ofthese agents with forskolin markedly augmented the ability offorskolin to inhibit PDGF-induced migration (Figure 1). Inour experiments, a combination of 1mmol/L forskolin and10 mmol/L Ro 20-1724, two agents that when used alone hadno effect on PDGF-induced migration, caused a 30% de-crease in migration in response to PDGF-BB. Although theaddition of cilostamide did not increase the antimigratoryeffect of forskolin at this concentration, it potentiated theeffects of a combination of forskolin and Ro 20-1724 by afurther 38%, leading to a 68% total inhibition. Notably, thissynergistic potentiation of the effects of forskolin by cilosta-mide and Ro 20-1724 was not significantly different fromthat caused by a combination of IBMX and 1mmol/Lforskolin. In fact, IBMX and 1mmol/L forskolin inhibitedmigration in our studies by'76%.

Incubation of VSMCs with a combination of 10mmol/Lforskolin and cilostamide did not result in a greater inhibition

Figure 1. Potentiation of forskolin-mediated inhibition of cul-tured rat aortic VSMC migration by PDE inhibitors. VSMC migra-tion in response to PDGF-BB (10 ng/mL) was determined usinga modification of Boyden’s chamber method7,43 as described in“Materials and Methods.” Effects of forskolin (1 to 100 mmol/L)in the absence or presence of cilostamide (1 mmol/L), Ro20-1724 (10 mmol/L), cilostamide and Ro 20-1724, and IBMX(500 mmol/L) on migrational activity were determined at 6 hours.Values are expressed as the mean6SEM of the number of cellspresent on the lower face of the Transwell (Corning CostarCorp) membrane in a 200-fold magnified field of view under alight microscope (cells per field of view) as determined from atleast three independent measurements. *P,.05 compared withPDGF-BB alone. **P,.05 compared with 1 or 10 mmol/L forsko-lin alone. #P,.05 compared with the combination of forskolinand Ro 20-1724. Unpaired one-way ANOVA with Tukey-Kramerpost hoc test was used for analyses.

Modulation of cAMP-Hydrolyzing PDE Activity inVSMC Homogenates

Treatment

cAMP-PDE Activity, pmol z

min21 z mg protein21

Experiment 1 Experiment 2

Vehicle 7467 7965

Vinpocetine

2 mmol/L 6763 z z z

20 mmol/L 6064 z z z

200 mmol/L 6266 z z z

Calcium (50 mmol/L) and calmodulin (10 U) z z z 9065

EHNA (10 mmol/L) 7563 z z z

Cilostamide (1 mmol/L) 5667* z z z

Ro 20-1724 (10 mmol/L) 4462* z z z

Cilostamide (1 mmol/L) and Ro 20-1724(10 mmol/L) 2965* z z z

Cilostamide (1 mmol/L), Ro 20-1724(10 mmol/L, and vinpocetine (2 mmol/L) 3265* z z z

Cilostamide (1 mmol/L), Ro 20-1724(10 mmol/L), and vinpocetine (20 mmol/L) 3262* z z z

Cilostamide (1 mmol/L), Ro 20-1724(10 mmol/L), and vinpocetine (200 mmol/L) 3164* z z z

IBMX (500 mmol/L) 663* z z z

Depicted are representative experiments for the determinations of PDEactivity, as described in “Materials and Methods.” Values are mean6SEM.

*P,.05 compared with vehicle-treated VSMC cAMP-PDE activity as deter-mined by unpaired one-way ANOVA and Dunnett multiple comparisons test.




of migration than was achieved with this dose of forskolinalone (Figure 1). Potentiation of the inhibitory effect onVSMC migration by 10mmol/L forskolin was, however,observed when Ro 20-1724 was added. Thus, whereas10 mmol/L forskolin inhibited PDGF-induced migration by21%, the combination of Ro 20-1724 and this concentrationof forskolin resulted in a 63% inhibition of the effect ofPDGF-BB. This represented a 3-fold potentiation of the effectof forskolin alone. Together, cilostamide and Ro 20-1724synergistically enhanced the effects of 10mmol/L forskolinsuch that this combination further reduced VSMC migrationto 80%, a 1.3-fold potentiation. It is notable that the extent towhich cilostamide was able to increase the inhibitory effectsof Ro 20-1724 and forskolin was diminished at the higherdose of forskolin (38% increase with 1mmol/L and 17% with10 mmol/L forskolin). As with the lower dose of forskolin,IBMX substantially augmented the inhibition mediated by10 mmol/L forskolin (from 21% to 76%), an enhancementequivalent to that mediated by the addition of both cilosta-mide and Ro 20-1724 to this concentration of forskolin. Toensure that nonspecific toxicological effects were not respon-sible for differences in VSMC migration observed after theaddition of cAMP-elevating agents, VSMC survival wasassessed using two separate tests. Under no circumstanceswere differences observed in VSMC survival after incuba-tions with the tested compounds, either alone or in combina-tion, using either trypan blue exclusion or lysosomal acidphosphatase activity assays (not shown).

Potentiation of Forskolin-Induced Increases inVSMC cAMP by PDE InhibitorscAMP levels in VSMCs treated with the combinations of theagents used to inhibit PDGF-induced migration were measuredat 30 minutes, 1 hour, and 6 hours (Figures 2 and 3). Under ourconditions, neither cilostamide (1mmol/L) nor Ro 20-1724(10mmol/L) alone caused a significant increase in cAMP levelsin VSMCs at any time point, whereas IBMX (500mmol/L)incubation of VSMC caused a doubling of cAMP (Figure 3).Forskolin (1 mmol/L) caused time-dependent increases incAMP, which reached a plateau between 30 minutes and 1 hour(Figures 2 and 3). Ro 20-1724, the PDE4 inhibitor, augmentedthe forskolin-induced increase in cAMP by 5.1-, 7.8-, and5.6-fold at the 30-, 60-, and 360-minute incubations, respec-tively. Although cilostamide did not potentiate the increases incAMP caused by any concentration of forskolin, it caused afurther increase in cAMP when combined with forskolin and Ro20-1724 (Figure 3). Thus, over the 30-minute, 1-hour, and6-hour time intervals, the combination of PDE3 and PDE4inhibitors potentiated the forskolin-induced increase in cAMP by'7.0-, 7.9-, and 6.6-fold, respectively. Similarly, the inclusionof IBMX resulted in a marked potentiation of the forskolin-mediated increase in cAMP such that the effects of forskolinwere 6.8-, 10-, and 9.9-fold larger than those caused by forskolinalone at the three time points. Incubation with 10mmol/L or100mmol/L forskolin increased VSMC cAMP levels by 50- and300-fold, respectively (not shown). In addition, IBMX aug-mented this increase in cAMP levels mediated by 10mmol/Lforskolin by 2.2-fold after 30 minutes (not shown).

Morphological Characterization ofMigrated VSMCsPhotomicrographs depicting changes in the appearances ofVSMCs incubated with the various pharmacological agentsstudied are shown (Figure 4). Panels a and b show VSMCsthat had migrated in the absence and in the presence of 10

Figure 2. Effects of the PDE inhibitors cilostamide, Ro 20-1724,and IBMX on time-dependent forskolin-induced increase inVSMC cAMP. Intracellular cAMP was measured after incubationof [3H]hypoxanthine-prelabeled VSMCs with forskolin (1 mmol/L)in the presence or absence of cilostamide (1 mmol/L), Ro20-1724 (10 mmol/L), and IBMX (500 mmol/L). [3H]cAMP valuesare expressed as a percentage of the total 3H. Values aremean6SEM from triplicate determinations in a representativeexperiment. Similar results were obtained in at least five deter-minations. All values were significantly different (P,.05) fromforskolin alone, with the exception of the combination of forsko-lin and cilostamide. Values determined for incubations of forsko-lin plus Ro 20-1724 were only significantly different (P,.05) fromforskolin plus Ro 20-1724 plus cilostamide at the 30-minutetime point. Unpaired Student t test was used for analyses.

Figure 3. Effects of PDE inhibitors, in the presence of 1 mmol/Lforskolin, on VSMC cAMP levels. [3H]cAMP was determinedafter incubation of [3H]hypoxanthine-labeled VSMCs with forsko-lin (1 mmol/L), cilostamide (1 mmol/L), Ro 20-1724 (10 mmol/L), acombination of cilostamide and Ro 20-1724, or IBMX(500 mmol/L) for 30 minutes. [3H]cAMP values are expressed asa percentage of the total 3H. Values are the mean6SEM of atleast eight determinations in three independent experiments.*P,.05 compared with basal (DMSO). #P,.05 compared with1 mmol/L forskolin alone. **P,.05 compared with the combina-tion of forskolin and Ro 20-1724. Unpaired Student t test wasused for analyses.




ng/mL PDGF-BB, respectively. In both instances, cells arewell spread out over the membrane surface and displaydistinct lamellipodia and pseudopodia. Addition of 1mmol/Lforskolin (panel c) caused little change in cell morphology orin cell number. Although incubation of VSMCs with thecombination of forskolin (1mmol/L) and cilostamide (paneld) did not result in fewer cells migrating in response toPDGF-BB, some cells appeared to develop a more spindlyappearance characterized by multiple, thin, branching pro-cesses and a compact cell body. Addition of Ro 20-1724 withforskolin (1 mmol/L) resulted in a notable decrease in thenumber of cells that had migrated to the lower face of themembrane and also resulted in a large number exhibitingthe spindly appearance (panel e). In combination, cilostamideand Ro 20-1724, in the presence of forskolin (1mmol/L),substantially decreased the number of cells that had migrated,and of those that were present, all displayed the spindlymorphology (panel f). Furthermore, a substantial number ofVSMCs under these conditions were seen to remain withinthe pores of the filter. The spindly appearance was alsoobserved with the concurrent incubation of VSMCs withforskolin (1 mmol/L) and IBMX (panel g) or with higherconcentrations (10mmol/L) of forskolin (panel h). Sinceincubation of VSMCs with 1,9,-dideoxyforskolin, the inac-tive forskolin analogue, did not result in the appearance ofspindly cells or in inhibition of migration (not shown) andsince 1 mmol/L 8-bromo-cAMP mimicked both of theseeffects of forskolin (not shown), it is reasonable to proposethat both phenomenon were cAMP-mediated. Of further note,this morphology was also seen to be adopted by VSMCs onthe upper face of the Boyden’s chamber membrane under theconditions of the assay as well as by VSMCs under standardculturing conditions when treated with forskolin and 8-bro-mo-cAMP, suggesting that the morphology is not necessarilylimited to migrating cells.

DiscussionThe present study represents the first detailed investigationinto the roles played by specific PDE families in the regula-tion of VSMC migration. Although previous reports haveestablished that cAMP-elevating agents and structural ana-logues of cAMP are capable of inhibiting VSMC migration inresponse to various chemotactic agents,7,12,15–17,48cAMP-PDEinvolvement in the regulation of this process has receivedconsiderably less attention. For our studies, cultured rat aorticVSMCs were used. We have previously characterized thecAMP-PDE activities present in these cells.36 In addition tothis previous characterization, we have demonstrated herethat the cells used in the present study expressed no detect-able PDE1 activity as assessed by the inability of calcium-calmodulin to stimulate VSMC cAMP-PDE activity as wellas the inability of a selective PDE1 inhibitor, vinpocetine, toaffect the total cAMP-hydrolyzing activity when used aloneor in combination with cilostamide and Ro 20-1724, selectivePDE3 and PDE4 inhibitors, respectively. Although somereports have shown that vinpocetine can relax isolated bloodvessels,27 the fact that this agent does not potentiate nitrova-sodilator–or atrial natriuretic peptide–induced increases invascular cGMP28 may perhaps indicate that PDE1 inhibition

was not the molecular basis of its vasorelaxant properties.Consistent with our previous report,36 the lack of effectobtained using EHNA, a selective PDE2 inhibitor, attests tothe lack of this activity in these cells. Also in agreement withour previous report, PDE3 and PDE4 activities accounted for.60% of the total cAMP-hydrolyzing activity in the VSMCsused in the present study. Furthermore, the near total inhibi-tion of cAMP-PDE activity by IBMX was taken to mean thatour cultured VSMCs did not express PDE7, a novel cAMP-specific PDE that is insensitive to IBMX.49,50Because of theseconsiderations, we chose to determine the relative roles ofPDE3 and PDE4 in the regulation of VSMC migration bycAMP. A recent report has indicated that PDE4 isozymesexhibit differential sensitivity to classical PDE4 inhibitors,such as rolipram and Ro 20-1724.51 The rat aortic VSMCsused here predominantly express PDE4D (authors’ unpub-lished data, 1997), a PDE4 isozyme that is inhibited potentlyby Ro 20-1724 (IC50, '0.7 mmol/L),51 thus validating theselection and dose of our selective PDE4 inhibitor.

Cultured VSMCs are a preferred model for the presentstudy of VSMC migration since they represent VSMCs in asynthetic phenotype similar to that found for migrating cellsin vivo.4 VSMC migration, in the present study, was inresponse to the addition of the potent and pathophysiologi-cally relevant chemotactic factor PDGF-BB.4 Selective inhib-itors of PDE3 (cilostamide, 1mmol/L) and PDE4 (Ro20-1724, 10mmol/L) isozymes, when used at concentrationsthat were selective and additive in terms of their effects oncAMP-PDE activity, had no effect on PDGF-induced VSMCmigration when used alone or in combination. This result wasconsistent with a previous study by Tanaka et al,48 in which itwas reported that a selective PDE3 inhibitor, E-1020, had noeffect on serum-induced VSMC migration over concentra-tions ranging from 0.1 to 10mmol/L. Furthermore, in thepresent study, a broad-spectrum PDE inhibitor, IBMX(500 mmol/L), was also ineffective at negatively modulatingthe migratory ability of VSMCs. The significant role forPDE3 and PDE4 activities was shown when inhibitors ofthese activities were combined with forskolin. More specifi-cally, the present study has demonstrated that cAMP-medi-ated inhibition of VSMC migration is regulated by a complexinterplay between these two PDEs. Thus, whereas cilosta-mide had a relatively modest impact on the concentrationdependence of forskolin-induced inhibition of VSMC migra-tion, Ro 20-1724 markedly potentiated this inhibitory effectof forskolin. Evidence for a significant interaction betweenPDE3 and PDE4 activities in modulating this cell functionwas demonstrated when addition of both of these selectivePDE inhibitors caused a synergistic potentiation of forskolin-induced inhibition of VSMC migration. These data demon-strated that PDE3 and PDE4 inhibitors could interact syner-gistically to modulate cellular effects mediated by cAMP.Similar interactions have been reported in relation to theeffects of these compounds on regulating relaxation-contrac-tion coupling in VSMCs.29 In their study, synergism betweenPDE3 and PDE4 inhibitors was attributed to the supra-additive increases in cAMP in the tissues studied. In ourexperiments, a similar phenomenon was observed. Thus, Ro20-1724 potentiated the forskolin-induced increase in cAMP




Figure 4. Representative photomicrographs of migrated VSMCs. VSMC migration was in response to PDGF-BB (10 ng/mL) for 6 hoursusing a modification of the method of Boyden.7,43 Incubation conditions included 0.5% FBS (a), 10 ng/mL PDGF-BB (b), PDGF-BB and1 mmol/L forskolin (c), PDGF-BB, 1 mmol/L forskolin, and 1 mmol/L cilostamide (d), PDGF-BB, 1 mmol/L forskolin, and 10 mmol/L Ro20-1724 (e), PDGF-BB, 1 mmol/L forskolin, cilostamide, and Ro 20-1724 (f), PDGF-BB, 1 mmol/L forskolin, and 500 mmol/L IBMX (g),and PDGF-BB and 10 mmol/L forskolin (h). Original magnification 3200.




at all time points studied, and the addition of cilostamidefurther augmented these increases. Given that cilostamidewas unable to potentiate forskolin-induced increases incAMP, these data were consistent with an important role forPDE3 only when PDE4 was inhibited. These findings identifya significant role for PDE4 in modulating the antimigratorypotential of cAMP, consistent with the VSMC cAMP-PDEactivity attributable to PDE4 isozymes relative to othercAMP-hydrolyzing PDEs. This observation stands in markedcontrast to the role played by PDE4 in the regulation ofrelaxation-contraction coupling of VSMCs.29

Our data demonstrate that although forskolin, when usedalone or in the presence of selective PDE inhibitors, inhibitedPDGF-stimulated VSMC migration and increased VSMCcAMP in a concentration-dependent manner, no relationshipbetween the absolute level of cAMP generated by the variouscombinations of agents tested and their inhibitory potentialexisted. For example, although 100mmol/L forskolin in-creased cAMP to a level'10-fold that achieved by acombination of 1mmol/L forskolin and 500mmol/L IBMX,the resulting levels of inhibition of migration with thesetreatments were virtually identical. There exists at least twopotential explanations for these findings. First, a coordinatedregulation of PKA activity by adenylyl cyclase and cAMP-PDE activities could influence the steady-state concentrationof cAMP required for full activation of PKA in cells in amanner independent of absolute cAMP levels.52 In support ofthis model, Deeg et al53 demonstrated that parotid glandamylase secretion was stimulated by a coordinated increase inboth adenylyl cyclase and PDE activity such that the cellsseemed to respond to an increase in cAMP metabolism eventhough the levels of cAMP did not change. In addition,subcellular colocalization of selected PDE and PKA isoformsmay allow for a coordinated regulation of function.52 Second,the absence of a more significant inhibition of VSMCmigration with combinations of agents giving rise to verylarge increases in cAMP may be due to a significant non-cAMP–inhibitable component of PDGF-induced migration.Also, our data demonstrate that there exists a thresholdincrease of cAMP that is required in treated VSMCs tomediate this inhibition of migration. For example, although1 mmol/L forskolin significantly increased cAMP levels, thisconcentration did not result in any measurable change inmigration. In seeming contradiction with this cAMP-medi-ated mechanism of inhibition, findings of Mooradian et al54

suggest that inhibition of adenylyl cyclase, via activation of aGi heterotrimeric GTP-binding protein, could promote inhi-bition of VSMC migration. However, given that Gia canmodulate effectors other than adenylyl cyclases and that thebg subunits are also able to effect changes in cell function, itis possible that this correlation of activity with function is notmechanistic.55

In addition to characterizing the ability of forskolin and thePDE inhibitors to attenuate VSMC migration, we also exam-ined the effect of these agents on VSMC morphology (Figure4). As stated previously, PDGF-BB (10 ng/mL) caused aprofound increase in the number of cells found on the lowerface of the membrane (Figure 1). Consistent with a migratoryphenotype, cells on the lower face of the membrane possessed

prominent lamellipodia and pseudopodia (Figure 4b).1,2 Treat-ment with 1 mmol/L forskolin or with the PDE inhibitorsalone did not reduce the number of cells present on the lowerface of the membrane or migratory phenotype (Figure 4c).When combined, this concentration of forskolin and eithercilostamide or Ro 20-1724 gave rise to a spindly morphologyin the cells that had migrated (Figure 4d and 4e). These cellswere characterized as having a compact cell body withmultiple, thin, branching processes. Also of note was theabsence in these cells of the lamellipodia observed in cellsthat had migrated in the absence of these agents. A secondaryobservation was that under conditions in which the majorityof the cells observed had a spindly appearance, a significantpercentage of cells appeared to have remained within themembrane pores. Moreover, Bornfeldt et al16 have demon-strated a similar tendency for VSMCs to remain in the poresof a modified Boyden’s chamber apparatus when treated withsphingosine-1-phosphate, an agent that elevates cAMPthrough an as-yet-undetermined mechanism. The appearanceof this morphology correlated with increases in intracellularcAMP. Consistent with this hypothesis is the observation thatVSMCs treated with 10mmol/L forskolin almost exclusivelydisplayed this morphology (Figure 4h). Moreover, at a higherconcentration (100mmol/L), the same compound evoked notonly a substantial decrease in the cell number but also acomplete change in cell morphology to the spindly type forall VSMCs found on the lower face. As further support for arole for cAMP in mediating these changes in cell shape,1,9-dideoxyforskolin (10mmol/L) was unable to bring abouta similar change in the appearance of these cells, whereas1 mmol/L 8-bromo-cAMP did. Similarly, when potentiatedby IBMX or the combination of selective PDE inhibitors(Figure 4f and 4g), VSMCs exclusively exhibited the spindlymorphology. In relation to these findings, since recent reportshave indicated that elevations in cAMP correlated with actinfilament disassembly, this mechanism could, at least in part,explain the morphological changes in VSMCs observedunder these conditions.16,56 The functional consequence ofadopting this morphology is unclear, since treatments thatinduced this change in cell shape did not necessarily decreasethe migratory ability of VSMCs (Figures 1, 4c, and 4d).

Given that agents that increase cAMP or cGMP cansynergistically regulate VSMC function through effects onPDE3, the data presented in the present study have bothphysiological and pharmacological implications.25 Two endo-thelium-derived vasoactive agents that regulate VSMC func-tion through cAMP or cGMP are prostacyclin and EDRF.25

Indeed, loss of endothelial cell function, which results indecreased release of these endothelium-derived vasoactivesubstances, has been shown to be an early event in thedevelopment of atherosclerosis, a process in which VSMCmigration has a critical role.57 Of further note is the observa-tion that balloon catheterization of rat aorta results in aselective upregulation of PDE4 activity, consistent with thedata presented here, suggesting a prominent role for PDE4isoenzymes in the regulation of cAMP-mediated inhibition ofVSMC migration.58 The molecular basis for this cAMP-mediated inhibition of VSMC migration has not, however,been fully elucidated. Although some results16 would appear




to support a central role for PKA as the primary effector forcAMP in this mechanism, others7 suggest the involvement ofmultiple systems. In this regard, the recent observation thatcAMP can activate the cGMP-dependent protein kinase(PKG) represents one other possible avenue through whichcAMP could act.59,60 Since PDGF-induced VSMC migrationis ultimately dependent on increases in intracellular calci-um,1,2,9 cAMP-mediated decreases in cytosolic calcium maycontribute to the diminished migratory ability of VSMCstreated with cAMP-elevating agents.61 In addition, cAMP-mediated changes in cytoskeletal structures16,56 and alterationof gene expression via the cAMP-response element bindingprotein may also be important.62 In addition to these otherpossible mechanisms of inhibition, activation of PKA bycAMP elevating agents has been shown to attenuate VSMCproliferation, presumably by negatively modulating the sig-naling by the MAP kinase pathway.63 A role for the MAPkinase pathway may also be involved in the process of cellmotility as indicated by a recent study by Graf et al.64

Antisense oligodeoxynucleotides directed against mRNA forthe MAP kinase pathway components ERK-1 and ERK-2inhibited PDGF-BB–induced VSMC migration, suggestingan important role for the MAP kinase pathway in the signaltransduction events regulating VSMC migration and thepotential that cAMP could act to inhibit this process via thismechanism.

In conclusion, the present study demonstrates that selectivePDE3 and PDE4 inhibitors can potentiate the cAMP-medi-ated antimigratory effects of forskolin in VSMCs. Since aninhibitor of PDE4 markedly potentiated the effects of for-skolin in the present study, whereas a PDE3 inhibitor hadmore modest effects, and in light of the observation thatPDE4 is upregulated in the aorta in response to balloonangioplasty, PDE4 inhibitors may represent a class of agentsthat are useful in limiting the VSMC migration occurring inresponse to endothelial damage but have limited impact onoverall blood pressure. Further studies relating the generalityof our observations to other activators of adenylyl cyclase,such as prostaglandins andb-adrenergic receptor agonists,should address this potential.

AcknowledgmentsThis study was supported by the Heart and Stroke Foundation ofOntario. Dr Maurice is a Career Research Scientist in HealthSciences sponsored by the Pharmaceutical Manufacturers of Canada-Health Research Foundation/Medical Research Council of Canada,D. Palmer is the recipient of an Ontario Graduate Scholarship, and K.Tsoi is the recipient of a summer studentship from the Pharmaceu-tical Manufacturers of Canada-Health Research Foundation/MedicalResearch Council of Canada. The authors are grateful to Dr S.C.Pang (Department of Anatomy and Cell Biology, Queen’s Univer-sity, Kingston, Canada) for provision of rat aortic vascular smoothmuscle cells.

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Daniel Palmer, Keith Tsoi and Donald H. Mauriceand Phosphodiesterase 4 Inhibitors

Synergistic Inhibition of Vascular Smooth Muscle Cell Migration by Phosphodiesterase 3

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