Gene Transfer of Redox Factor-1 Inhibits Neointimal...

Gene Transfer of Redox Factor-1 InhibitsNeointimal Formation

Involvement of Platelet-Derived Growth Factor-� Receptor Signaling viathe Inhibition of Reactive Oxygen Species–Mediated Syk Pathway

Hwan Myung Lee,* Byeong Hwa Jeon,* Kyung-Jong Won, Chang-Kwon Lee, Tae-Kyu Park,Wahn Soo Choi, Young Min Bae, Hyo Shin Kim, Sang Ki Lee, Seung Hwa Park,

Kaikobad Irani, Bokyung Kim

Abstract—The role of apurinic/apyrimidinic endonuclease-1/redox factor-1 (Ref-1) in vascular smooth muscle cells has yetto be clearly elucidated. Therefore, we attempted to determine the roles of Ref-1 in the migration induced byplatelet-derived growth factor (PDGF)-BB and in its signaling in rat aortic smooth muscle cells (RASMCs). Cellularmigration, superoxide (O��) production, Rac-1 activity, and neointima formation were determined in cells transfectedwith adenoviruses encoding for Ref-1 (AdRef-1) and small interference RNA of Ref-1. Overexpression of Ref-1 inducedby treatment with RASMCs coupled with AdRef-1 inhibited the migration induced by PDGF-BB. PDGF-BB alsoincreased the phosphorylation of the PDGF� receptor, spleen tyrosine kinase (Syk), mitogen-activated protein kinase,and heat shock protein 27, but these increases were significantly inhibited by AdRef-1 treatment. PDGF-BB increasedO�� production and Rac-1 activity, and these were diminished in cells transfected with AdRef-1. In contrast, RASMCmigration, phosphorylation of Syk and O�� production in response to PDGF-BB were increased by the knock down ofRef-1 with small interference RNA. The phosphorylation of PDGF� receptor in response to PDGF-BB was inhibitedcompletely by the Syk inhibitor and was partly attenuated by a NADPH oxidase inhibitor. PDGF-BB increased thesprout outgrowth of the aortic ring ex vivo, which was inhibited in the AdRef-1–infected RASMCs as compared withthe controls. Balloon injury–induced neointimal formation was significantly attenuated by the gene transfer of AdRef-1.These results indicate that Ref-1 inhibits the PDGF-mediated migration signal via the inhibition of reactive oxygenspecies–mediated Syk activity in RASMCs. (Circ Res. 2009;104:000-000.)

Key Words: apurinic/apyrimidinic endonuclease-1/redox factor-1 � vascular smooth muscle cells � migration� reactive oxygen species � spleen tyrosine kinase

Platelet-derived growth factor (PDGF) performs crucialfunctions in the regulation of vascular cell proliferation

and migration, resulting in circulatory disorders includingatherosclerosis. PDGF stimulates intracellular signal mole-cules with Src homology 2 (SH2) domains, including Src,phospholipase C, phosphatidylinositol 3-kinase (PI3K), andras/raf-1.1,2 Src, PI3K, and phospholipase C� perform crucialfunctions related to actin reorganization, growth, and migra-tion in response to PDGF in the vascular smooth musclecells,3 and these activities are mediated by the activation of afamily of serine/threonine-specific protein kinases, themitogen-activated protein kinases (MAPK), most notably p38MAPK.4 Recently, we determined that spleen tyrosine kinase(Syk), a 70-kDa non–receptor protein tyrosine kinase harbor-

ing 2 SH2 domains, binds to phosphorylated PDGF receptor(PDGF-R) and contributes to PDGF-mediated migration inthe vascular smooth muscle.5 Moreover, PDGF-R transmitsits signal into intracellular space with the production ofreactive oxygen species (ROS), particularly superoxide (O��)and hydrogen peroxide (H2O2).6,7 PDGF augments O�� levelsthrough NADPH oxidase (NOX), and the inhibition of NOXactivity reduces PDGF-induced chemotaxis in vascularsmooth muscle cells.8,9 Because ROS perform crucial func-tions in many aspects of cellular function, the redox systemcan be implicated in the regulation of vascular dysfunction.Furthermore, ROS stimulates PDGF-R phosphorylation.10

These results indicate that ROS may be involved in theactivation of PDGF-R via ROS-activated protein kinases.

Original received May 1, 2008; revision received October 16, 2008; accepted November 18, 2008.From the Institute of Medical Sciences (H.M.L., K.-J.W., C.-K.L., T.-K.P., W.S.C., Y.M.B., S.H.P., B.K.), School of Medicine, Konkuk University,

Danwol-dong, Chungju, South Korea; Infection Signaling Network Research Center (B.H.J., H.S.K., S.K.L.), Department of Physiology, ChungnamNational University, South Korea; and Cardiovascular Institute (K.I.), University of Pittsburgh Medical Center, Pa.

*Both authors contributed equally to this work.Correspondence to Bokyung Kim, Department of Physiology, Institute of Medical Sciences, School of Medicine, Konkuk University, Danwol-dong

322, Chungju 380-701, South Korea. E-mail [email protected]© 2008 American Heart Association, Inc.

Circulation Research is available at http://circres.ahajournals.org DOI: 10.1161/CIRCRESAHA.108.178699

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Despite these reports, the mechanisms underlying the rela-tionship between PDGF-R signaling and ROS remain to beclearly elucidated.

Apurinic/apyrimidinic endonuclease/redox factor-1 (Ref-1) isa ubiquitous multifunctional protein that is involved in the baseexcision repair pathways of DNA subjected to oxidative dam-age.11 In addition, Ref-1 also promotes a variety of redox-mediated events regulating cell growth, differentiation,survival, and death, which are associated with activatorprotein-1, nuclear factor �B, p53, Egr-1, and cMyb.12,13

Ref-1 has been reported to inhibit the activation of Rac-1,an essential cofactor for the NOX complex. This results inthe inhibition of NOX, thereby reducing the level of ROS andendothelial cell growth and migration.14 Ref-1 has beendemonstrated to suppress intracellular oxidative stress andapoptosis via the modulation of Rac-1–regulated oxidation.15

We previously showed that tumor necrosis factor �–stimu-lated inflammation was inhibited by Ref-1 overexpression invascular endothelial cells.16 Moreover, Ref-1 expression wasupregulated as a consequence of ROS treatment and inatherosclerotic plaques.14,17 The finding that Ref-1 contrib-utes to the regulation of PDGF-stimulated proliferation sug-gests a possible role for Ref-1 in vascular smooth muscledysfunction.18 However, the roles of Ref-1 in smooth musclecell migration have yet to be fully determined. Therefore, inthis study, we attempted to determine the role of Ref-1 onPDGF-induced migration and balloon-injured carotid arteriesand its signaling associated with ROS production using rataortic smooth muscle cells (RASMCs) transfected with ad-enoviruses encoding for full-length Ref-1 (AdRef-1).

Materials and MethodsAn expanded Materials and Methods section is available in theonline data supplement at http://circres.ahajournals.org.

Cell Culture, Immunoblotting,and ImmunoprecipitationOur investigation conformed to the Guide for the Care and Use ofLaboratory Animals published by the (NIH Publication No. 85-23,revised 1996). All experiments and animal care were conducted inconformity with the institutional guidelines established by KonkukUniversity, South Korea. RASMCs (used at passages 5 to 9) wereisolated via treatment with enzymes from male Sprague–Dawley rats(6 weeks old; 190 g; n�6), which were purchased from Daehanbi-olink (Chungju, South Korea). Cell culture, immunoblotting, andimmunoprecipitation were performed as previously described.5

Adenoviral TransfectionAdenoviruses encoding for �-galactosidase (Ad�-gal) and full-length human AdRef-1 were generated via homologous recombina-tion in human embryonic kidney 293 cells, as previously described.19

RASMCs were infected for 2 hours in 2% FBS-DMEM at amultiplicity of infection (moi) (particle-forming units per cell) of 200of the specified adenovirus and subsequently incubated for 36 hoursin normal DMEM with the virus. The virus was then removed andthe cells were incubated for 18 hours in FBS free DMEM, followedby experimental treatments or assays. After adenoviral transfection,the efficiency of transfection obtained in the endothelial cells was�90%.

Cell Migration, Proliferation Assay, andSuperoxide ProductionCell migration was assessed with 48-well microchemotaxis Boy-den chambers (Neuro Probe, Gaithersburg, Md) as described

previously.5 Cell proliferation was determined via a 5-bromo-2�-deoxyuridine (BrdUrd) incorporation assay (Roche, Indianapolis,Ind). O�� production was measured via lucigenin-enhancedchemiluminescence.8

Rac-1 Pull-Down AssayRASMCs (2�105 cells) were treated with stimulants or inhibitorsand washed with ice-cold PBS, incubated for 5 minutes on ice in theextraction buffer, then centrifuged for 15 minutes at 16 000g at 4°C.Aliquots were obtained from the supernatant for comparisons ofprotein content. The supernatant proteins were incubated for 60minutes with the bacterially produced GST-PAK fusion protein(Cytoskeleton, Denver, Colo) harboring the Rac-1 binding region,bound to glutathione-coupled sepharose beads at 4°C. The beads andproteins bound to the fusion protein were then washed 3 times in anexcess of lysis buffer, eluted in Laemmli sample buffer (60 mmol/LTris, pH 6.8, 2% SDS, 10% glycerin, 0.1% bromphenol blue), andanalyzed for bound Rac-1 molecules via Western blotting.

Aortic Ring Assay, Balloon Injury, and AdenoviralGene TransferEx vivo RASMC migration was assessed via an aortic ring assayusing modified Matrigel.5 Male Sprague–Dawley rats (350 to 400 g)were anesthetized using a ketamine (80 mg/kg)/xylazine (12 mg/kg)mixture, and the left common and external carotid arteries wereexposed and isolated. A 2F Fogarty catheter (Edwards Lifesciences,Irvine, Calif) was introduced into the common carotid artery througharteriotomy in the external carotid artery and inflated to 1.0 to 1.5atmospheres, and a 10-mm injury was induced by withdrawn 4 times.After balloon removal, 100-�L aliquots of PBS containing Ad�-gal(1�109 particles; n�4) or AdRef-1 (1�109 particles; n�6) wereinjected through a cannula and allowed to incubate in the injuredsegment for 30 minutes. The external carotid was then tied and theblood flow was restored. Animals were euthanized after 2 weeks.Media and neointima areas of injured arteries were quantified byplanimetry (�m2) and intima-to-media ratios were calculated.

Transfection Small Interference RNASmall interference (si)RNA was designed to target the sequences ofrat Ref-1 (5�-CAAACUUCUGGUUUCCUUU-3�; accession no.NM_024148). The RNA of 19 nucleotides followed by TT weredesigned and chemically synthesized, purified, and annealed. Non-silencing siRNA (5�-CCUACGCCACCAAUUUCGU-3�) was pro-vided by Bioneer (Korea).

Statistical AnalysisData were expressed as the means�SE of the mean. The datawere statistically evaluated using Student’s t tests for compari-sons between pairs of groups and by ANOVA for multiplecomparisons. A probability value of �0.05 was considered bestatistically significant.

ResultsRef-1 on PDGF-BB–Induced Migrationin RASMCsWe first assessed the levels of adenoviral-mediated Ref-1overexpression in the RASMCs via Western blotting withanti–Ref-1 antibody. As is shown in Figure 1A, cellstransfected with AdRef-1 at 100 and 200 mois evidencedelevated Ref-1 expression in an moi-dependent manner.The level of Ref-1 expression in the AdRef-1–transfectedcells was greater than in those infected with Ad�-gal at200 mois, in a manner similar to that observed withendogenous Ref-1 expression in the nontransfected cells.The effects of Ref-1 overexpression were then tested on thePDGF-BB–induced migration of RASMC, via a Boyden

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chamber assay. RASMCs infected with Ad�-gal (100 and200 mois) did not affect cellular migration response to 10ng/mL PDGF-BB (Figure 1B). By way of contrast, theoverexpression of RASMCs with AdRef-1 in the range of 20to 200 mois inhibited PDGF-BB (10 ng/mL)–induced migra-tion in an moi-dependent manner, which reached a maximumat 200 mois of AdRef-1 (Figure 1B). Similar results of Ref-1were obtained in a linear wound-healing assay (data notshown).

Ref-1 on the Phosphorylation of Syk and theBinding of PDGF�-R to SykWe recently showed that Syk regulates PDGF-BB–inducedmigration via the p38 MAPK/heat shock protein (Hsp)27pathway in RASMCs.5 Thus, we attempted to determinewhether Ref-1 influences the activation of Syk in response toPDGF. The RASMCs were immunoprecipitated with anti-Syk antibody and were immunoblotted with anti–phosphoty-rosine antibody (pY; 4G10). The transfection of cells withAdRef-1 (200 mois) inhibited the increase of Syk phosphor-ylation induced by 10 ng/mL PDGF-BB, but transfection withAd�-gal (200 mois) did not (Figure 2A). The binding ofPDGF�-R to the signal molecules Src and Syk withintracellular SH2 domain has been shown to result in theactivation of the molecules.2 Therefore, we attempted to

determine the effects of Ref-1 on the binding of Sky toPDGF�-R in response to PDGF-BB. AdRef-1 (200 mois)inhibited the PDGF-BB (10 ng/mL)– enhanced binding ofPDGF�-R to Syk in RASMCs, but Ad�-gal (200 mois) didnot (Figure 2C).

Thus, we assessed the effects of Ref-1 on the phosphory-lation of p38 MAPK and Hsp27 in RASMCs. RASMCsinfected with AdRef-1 (200 mois) were shown to signifi-cantly inhibit the phosphorylation of p38 MAPK and Hsp27in response to PDGF-BB (10 ng/mL, 10 minutes), but cellstreated with Ad�-gal (200 mois) evidenced no such effect(Figure 3). PDGF-BB (10 ng/mL) induced an increase inextracellular signal-regulated protein kinase (ERK)1/2 phos-phorylation, which was inhibited in the AdRef-1 (200 mois)–transfected cells but not in the Ad�-gal (200 mois)–trans-fected cells (Figure 3A and 3C).

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Figure 1. Ref-1 overexpression on PDGF-BB–induced migrationin RASMCs. A, Overexpression of Ref-1 using AdRef-1. Thelevel of adenoviral-mediated Ref-1 overexpression in RASMCswas determined via Western blotting with an anti–Ref-1 anti-body. B, Effects of AdRef-1 and Ad�-gal on PDGF-BB–stimu-lated RASMC migration. Cells were preincubated with AdRef-1(20 to 200 mois) and Ad�-gal (100 and 200 mois) and thentreated with PDGF-BB (10 ng/mL) for 90 minutes (n�8). Cellmigration was evaluated via Boyden chamber assays. *P�0.05,significant difference between AdRef-1– and Ad�-gal–incubatedcells treated with PDGF-BB. Con indicates control; Ref-1,AdRef-1; �-gal, Ad�-gal.

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Figure 2. Effects of Ref-1 overexpression on the Syk activities inresponse to PDGF-BB. A and B, Effect of Ref-1 on PDGF-BB–induced Syk activation in RASMCs. Cells were incubated withAdRef-1 (200 mois) or Ad�-gal (200 mois) and then treated withor without PDGF-BB (10 ng/mL) for 10 minutes. The lysateswere immunoprecipitated with anti-Syk antibody and immuno-blotted with anti–phosphotyrosine(4G10) antibody. The lowerpanel shows the total expressions of Syk measured using anti–non-phospho-Syk antibody. The phosphorylation of Syk in thequiescent state of normal cells is deemed to be 100% (n�4). C,The interaction between PDGF�-R and Syk kinase in responseto PDGF-BB in RASMCs. Cells were incubated with AdRef-1(200 mois) or Ad�-gal (200 mois) and then treated with or with-out PDGF-BB (10 ng/mL) for 10 minutes (n�4). *P�0.05, signifi-cant difference from PDGF-BB–stimulated state. Con indicatescontrol; IB, immunoblotting; IP, immunoprecipitation; Ref-1,AdRef-1; �-gal, Ad�-gal; p-Y, phosphotyrosine.

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Ref-1 on Rac-1 Activation and O�� ProductionTo clarify the roles of Ref-1 in the production of O��, theeffects of AdRef-1 transfection were determined using lu-cigenin assays in RASMCs. PDGF-BB (10 ng/mL) effectedan increase in the production of O��. The level of O��

increased by PDGF-BB was inhibited by treatment with 2NOX inhibitors, diphenylene iodonium chloride (DPI)(10 �mol/L) and apocynin (1 mmol/L), and a ROS scavenger,N-acetyl-cysteine (NAC) (10 mmol/L) (Figure 4A). Piceat-annol, a Syk inhibitor, induced the complete abolition of thePDGF-BB–induced O�� production (Figure 4A). The level ofO�� in response to PDGF-BB was inhibited significantly incells transfected with AdRef-1 (200 mois) as compared withAd�-gal (200 mois) (Figure 4B). Because Rac-1 activation isan important step in the elevation of NOX activity in responseto receptor agonists or growth factors,14 the effects ofPDGF-BB and Ref-1 on the activity of Rac-1 were deter-mined. PDGF-BB (10 ng/mL) induced an increase in the levelof GTP-Rac-1 in both the cells transfected with AdRef-1 andthose with Ad�-gal. Rac-1 activation was inhibited signifi-cantly in the cells transfected with AdRef-1 (200 mois) ascompared with the cells transfected with Ad�-gal (200 mois)(Figure 4C and 4D).

Ref-1 and NOX Inhibitor on the Phosphorylationof PDGF�-RIt was reported that PDGF-induced ROS production mediatedby NOX, and PDGF-BB—induced migration was reduced by

the inhibition of NOX in vascular smooth muscle cells.8,9 Wedetermined whether treatment with an antioxidant and NOXinhibitors would affect the PDGF-BB–induced RASMCphosphorylation of PDGF�-R. As is shown in Figure 5A and5C, PDGF-BB (10 ng/mL) induced an increase in PDGF�-Rphosphorylation, which was inhibited by treatment with DPI(10 �mol/L), apocynin (1 mmol/L), and NAC (10 mmol/L).We also assessed the influence of Syk inhibitor on PDGF�-Ractivation. PDGF-BB (10 ng/mL) enhanced PDGF�-R activ-ity in RASMCs, and this was abolished completely as theresult of treatment with 30 �mol/L piceatannol (Figure 5Band 5D). We then attempted to determine the effects of Ref-1on PDGF�-R activation. As is shown in Figure 5E, transfec-tion with AdRef-1 (200 mois) partially inhibited the increasein PDGF�-R phosphorylation induced by PDGF-BB (10ng/mL), but transfection with Ad�-gal (200 mois) did not.

Effects of Ref-1 Knockdown inPDGF-Induced ResponsesTo further evaluate the roles of Ref-1 on the PDGF-BB–induced responses, we examined the effects of Ref-1 knock-down using a siRNA–Ref-1 technique. Cells transfected withRef-1 siRNA showed the diminution of Ref-1 expressioncompared with cells transfected with nonsilencing siRNA(Figure 6A). The migration stimulated by PDGF-BB (10ng/mL) was significantly increased in cells transfected withRef-1 siRNA (Figure 6B). Moreover, the transfection of cells


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Figure 3. Effects of Ref-1 overexpression on PDGF-BB–induced MAPK activities in RASMCs. A, Effects of Ref-1 on the activity of p38MAPK, Hsp27, and ERK1/2 in RASMCs induced by PDGF-BB. RASMCs were treated with AdRef-1 (200 mois) or Ad�-gal (200 mois)and then stimulated for 10 minutes with or without PDGF-BB (10 ng/mL). p38 MAPK, ERK1/2, and Hsp27 phosphorylations wereexamined using phospho-specific antibodies. Total expressions of MAPK and Hsp27 were determined using anti–non–phospho-specificand anti–�-actin antibodies. B through D, The statistical analysis of data in A. The ratio of phosphorylated/nonphosphorylated MAPKand phosphorylated Hsp27/�-actin in the basal state are expressed as 100% (n�4). *P�0.05, significant difference from PDGF-BB–stimulated state. Con indicates control; Ref-1, AdRef-1; �-gal, Ad�-gal.




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with Ref-1 siRNA increased the phosphorylation of Syk andthe binding of Syk to PDGF�-R induced by 10 ng/mL ofPDGF-BB (Figure 6C). In addition, the production of O��

was greater in Ref-1 siRNA–transfected cells compared withnonsilencing siRNA (Figure 6D). In contrast to the resultsobtained from Ref-1 siRNA, cells transfected with nonsilenc-ing siRNA showed no alteration, which was similar to cellmigration levels obtained from nontransfected controls.

Effect of Ref-1 on the Sprout Growth ofAortic RingsThe effect of Ref-1 overexpression was evaluated in thecontext of the PDGF-BB–induced proliferation of RASMCs,via a BrdUrd assay. PDGF-BB (10 ng/mL) induced anincrease in RASMCs proliferation. RASMCs infected withAd�-gal (200 mois) exerted no significant effects on cellularproliferation responses to 10 ng/mL PDGF-BB (Figure I, A,in the online data supplement). The infection of RASMCswith AdRef-1 200 mois inhibited PDGF-BB (10 ng/mL)–induced proliferation (data not shown). To determine the roleof Ref-1 ex vivo, aortic ring assays were conducted usingMatrigel. The rings were embedded in Matrigel, and sproutoutgrowth was assessed in terms of cell migration andproliferation. As is shown in Figure 7A and supplementalFigure I, B, PDGF-BB (10 ng/mL) effected an increase in thesprout growth of the aortic rings, and this effect was attenu-ated by treatment with AdRef-1 (200 mois), but not bytreatment with Ad�-gal (200 mois).

Effect of Gene Transfer of AdRef-1 onNeointimal FormationTo investigate the effect of Ref-1 on the neointimal formationin carotid arteries, we used a model of balloon-injuredneointimal formation. Immunohistochemistry for PDGF�-Rindicated that only the neointima of balloon-injured carotidartery showed strongly positive to anti–PDGF�-R antibody(supplemental Figure II). Compared with Ad�-gal–treatedrats, gene transfer of AdRef-1 markedly decreased neointimalformation. The intimal/media ratio was also significantlyreduced in the AdRef-1–treated carotid arteries but not inredox mutant of Ref-1 (Ref-1/RMR) (Figure 7B and 7C).

DiscussionIn this study, we have demonstrated that Ref-1 overexpres-sion inhibited the migration and wound closure induced byPDGF-BB in the vascular smooth muscle cells. Moreover,PDGF-BB was shown to augment the activities of Rac-1 andNOX, and these activities were decreased in the Ref-1–overexpressed cells as compared with the �-gal–transfectedcells. Moreover, these results were verified by the results ofex vivo analysis with the outgrowth of vessel sprouts from theaortic strips and in vivo analysis with the neointima formationin balloon-injured rat carotid artery. These findings indicatethat the inhibition of PDGF-induced migration by Ref-1 isderived from the inhibition of Rac-1 activity that regulatesROS production. Although Ref-1 has been reported to inhibitNOX activity via a direct inhibition of Rac-1,14 this is, to thebest of our knowledge, the first report to clarify the role of

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Figure 4. Alteration of ROS production and Rac-1 activity in response to PDGF-BB in RASMCs. A, RASMCs were incubated with inhib-itors for 60 minutes and then treated with PDGF-BB (10 ng/mL) for 10 minutes. B, The supernatant from cells transfected with AdRef-1(200 mois) or Ad�-gal (200 mois) was added along with lucigenin and NADPH, and its chemiluminescence was repeatedly measured.The production in the quiescent state of normal cells is deemed to be 100% (n�4). C and D, RASMCs were treated with AdRef-1 (200mois) or Ad�-gal (200 mois) and then stimulated with or without PDGF-BB (10 ng/mL) for 10 minutes. Rac-1 GTP levels were deter-mined via a pull-down assay. GTP-Rac-1 in the quiescent state of normal cells is considered to be 100% (n�4). *P�0.05, significantdifference from PDGF-BB–stimulated state. Con indicates control; Ref-1, AdRef-1; �-gal, Ad�-gal.




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Ref-1 in vascular smooth muscle migration. In this study, thetransfection of Ref-1 in PDGF-BB–stimulated Syk kinasephosphorylation exhibited an effect similar to result seen withNOX inhibitor (apocynin, DPI) or ROS scavenger (NAC)treatment on the inhibition of PDGF-BB–mediated PDGF-Rand Syk kinase phosphorylations. Although the NOX inhib-itors used in this study have been widely used for NOXinhibition, they are reported to be an antioxidant and aninhibitor of flavin-containing enzyme.20,21 Therefore, furtherstudy will be needed for clarifying the action mechanism ofthese inhibitors in RASMCs. Ref-1 has been implicated in theinhibition of cellular responses, including apoptosis andapurinic/apyrimidinic repair.15,22 Moreover, vascular inflam-mation is attenuated in Ref-1–overexpressed cells.16 ROS isimportant in vascular cell responses and also contributes tothe elevation of migration in vascular smooth muscle cells.These results indicate that Ref-1 performs a crucial functionin the regulation of cellular ROS level and in the vasculartissue remodeling in circulatory disorders includingatherosclerosis.

It has been suggested that the ROS generated by NOXactivation can stimulate growth factor receptors.23 The eleva-tion of intracellular ROS levels as the result of PDGF-Ractivation was found to be the cause of full receptor activationin certain cells, most notably vascular smooth musclecells.10,24 Moreover, the treatment of cells with NOX inhibitoror catalase resulted in the inhibition of PDGF-R phosphory-lation.8 In this study, we determined that the inhibition of

NOX activity and ROS scavenging inhibited, although notcompletely, the phosphorylation of PDGF�-R in response toPDGF-BB. These results indicate that initial levels of ROSreleased as the result of PDGF stimulation may participate inthe regulation of PDGF-R activity. Moreover, ROS directlystimulate intracellular signals activated by PDGF stimulation.It was recently determined that Src is dually regulated; firstly,by its direct stimulation of PDGF-R, and secondly, by theROS production induced by PI3K- and protein kinase C–me-diated NOX activation.8 Furthermore, in both the presentstudy and previous reports, PDGF was demonstrated toincrease the phosphorylation of Syk, and this effect wasinhibited by ROS inhibitors or Ref-1 transfection.5 By way ofcontrast, Syk kinase inhibitor effected the complete abolitionof PDGF-R activity in response to PDGF-BB. Syk is acti-vated by ROS in lymphoma cells.25 Moreover, ROS havebeen shown to regulate a protein tyrosine phosphatase (PTP),SHP-2, resulting in the activity of proteins that harbor theSH2 domain, including Syk.26 According to these results, itcan be surmised that ROS stimulates Syk activity via PTP invascular smooth muscle cells. In the present study, Sykactivity was partly inhibited in Ref-1–transfected cells. Theseresults indicate that Syk kinase is regulated by the activationof PDGF-R via NOX-mediated ROS production and PDGF-Ris also simultaneously regulated by the activation of Sykkinase. Src kinase is reportedly required for the full activationof PDGF-R and PDGF-associated signaling.27,28 In accor-

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Figure 5. Effects of ROS and Syk inhibitors on the PDGF-BB–induced PDGF�-R phosphorylation in RASMCs. A and B, RASMCs wereincubated with NAC (10 mmol/L), apocynin (Apo) (1 mmol/L), DPI (10 �mol/L), and piceatannol (PIC) (30 �mol/L) for 60 minutes, andwere then treated with PDGF-BB (10 ng/mL) for 10 minutes. C and D, The statistical analysis of data from A (n�4) and B (n�4),respectively. Phosphorylation in quiescent state is deemed to be 100%. *P�0.05, significant difference from PDGF-BB (10 ng/mL)–stimulated state. E, Effects of Ref-1 transfection on the PDGF�-R phosphorylation. PDGF�-R phosphorylation was measured in a simi-lar fashion to that in A and B (n�3). Con indicates control; IB, immunoblotting; IP, immunoprecipitation; p-Y, phosphotyrosine.




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dance with these results, we hypothesized that PDGF-BBinitially stimulates Syk activity via NOX activity and thatthese consequently allow for the full activation of PDGF-R.As described above, the results of this study show that ROS

scavenger and NOX inhibitors do not completely inhibit Sykphosphorylation in response to PDGF-BB. Our findings,therefore, suggest that Syk can be stimulated directly byPDGF-R without mediation via ROS-associated stimulation

Ref-1

ββ-Actin

Con Non Ref-1

A

DC

B

IB : PDGFβ-R

IP : Syk

PDGF-BB

Con Non siRNA Con Non siRNA

IB : Syk

IB : p-Y

0

100

200

300

400

PDGF-BB


PDGF-BB


37

49

37

(kDa)

18082

64

(kDa)82

64

0

30

60

90

Mig

rate

d C

ells

/Fie

ld

*

*

O2−

Prod

uctio

n (%

)Figure 6. Effect of Ref-1 knockdown on PDGF-BB–induced responsiveness in RASMCs. A, Ref-1 expression in Ref-1 knockdown cells.Cells were transfected with 1 nmol/L nonsilencing siRNA and Ref-1 siRNA or not (control). Ref-1 expression was confirmed by immu-noblotting using an anti–Ref-1 antibody (n�3). B, Effect of Ref-1 siRNA transfection (1 nmol/L) on PDGF-BB (10 ng/mL)–induced migra-tion in RASMCs. Cell migration was evaluated via Boyden chamber assays (n�8). C, Effect of Ref-1 siRNA on PDGF-BB–induced Sykactivation in RASMCs (n�3). D, Ref-1 siRNA on O�� production in response to PDGF-BB. O�� production was determined by lucigeninchemiluminescence in RASMCs. The production in the quiescent state of normal cells is deemed to be 100% (n�4). Con indicatescontrol; IB, immunoblotting; Non, nonsilencing; IP, immunoprecipitation; p-Y, phosphotyrosine.

Figure 7. Effects of gene trans-fer of AdRef-1 in neointimal for-mation. A, Effects of AdRef-1 onthe sprout growth of aortic ringsinduced by PDGF-BB. Micro-scopic images of the effects ofAdRef-1 or Ad�-gal on PDGF-BB–induced sprout outgrowthare shown (n�4). Aortic rings(1 mm) were cultured andembedded in Matrigel and werethen treated with Ad�-gal (200mois) or AdRef-1 (200 mois) inthe presence or absence ofPDGF-BB (10 ng/mL). Aorticrings were treated with Ad�-galor AdRef-1 without (top images)or with 10 ng/mL PDGF-BB(bottom images). B and C,Carotid arteries of rats treatedAd�-gal and AdRef-1 were ana-lyzed after 2 weeks of ballooninjury. Representative images ofballoon-injured carotid arterywith Ad�-gal and with AdRef-1(n�4 to 6). Intima–media ratio ofcross-sectional area (�m2) wasquantified by planimetry.*P�0.05. Con indicates control;Ref-1, AdRef-1; �-gal, Ad�-gal.




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and imply that PDGF-BB stimulates Syk via both ROS-dependent and independent pathways.

The migration of vascular smooth muscle cells is com-monly known to be elevated by several factors, particularlyPDGF. Previously, we determined that Syk inhibitors inhibitthe PDGF-induced increase in vascular cell migration.5 Asdescribed above, Syk activity was inhibited in the Ref-1–transfected cells as compared with the control cells. More-over, the binding of Syk to PDGF-R as the result of PDGFtreatment was also inhibited by transfection with Ref-1. BothSrc and Syk have also been previously associated withPDGF-R, and this association appears to be a prerequisite forkinase activation.3 We previously showed that the Src familykinase Lyn phosphorylates Syk and functions as an upperstream of Syk in rat mast cells.29 Similarly, the Src inhibitorinhibited the PDGF-induced migration and phosphorylationof Syk. The activation of receptor tyrosine kinases, includingPDGF-R, can result in Src and Syk activation, and thesesignals stimulate the elevation of MAPK phosphorylation.30

PDGF is known to increase both MAPK activation andmigration in several cells, which suggests that MAPK may bea crucial pathway in PDGF-mediated migration. In thepresent study, PDGF increased the phosphorylation of p38MAPK and Hsp27 in RASMC. The results from previousreports demonstrated that p38 MAPK inhibitor and Hsp27-siRNA attenuate PDGF-induced migration in vascularsmooth muscle cells.5,31 Moreover, Ref-1 transfection andtreatment with Syk inhibitors were determined to inhibit theincreased phosphorylation of p38 MAPK and Hsp27 inducedby PDGF. These results indicate that Ref-1 contributes toPDGF-induced migration via the inhibition of ROS produc-tion, which consequently induces Syk activity, resulting inthe phosphorylation of p38 MAPK/Hsp27.

Moreover, ROS is involved in the proliferation, as well asthe migration, of vascular smooth muscle.32 This studyindicated that PDGF-induced proliferation was attenuated inRef-1–overexpressed cells. These results, therefore, indicatethat ROS induce an increase in proliferation in vascularsmooth muscle cells, which results from the inhibition of thereduction of ROS production in cells via Ref-1 treatment.ERK1/2 has been identified as a common pathway forexaminations of the mechanisms by which the proliferation ofvascular smooth muscle and ROS enhance the activity of thiskinase.33 Moreover, in this study, PDGF-induced ERK1/2activity was also inhibited in cells transfected with Ref-1.These results indicate that PDGF-induced proliferation ismediated by ROS production and that this is inhibited byRef-1 treatment.

In summary, we have demonstrated that Ref-1 transfectioninhibits the neointimal formation, RASMC migration, prolif-eration, and phosphorylation of Syk and p38 MAPK, both ofwhich were enhanced by PDGF-BB. Moreover, Ref-1 over-expression induced the diminution of the activity of Rac-1and treatment with NOX inhibitor or ROS scavenger partiallyinhibited the phosphorylation of PDGF�-R. RASMC migra-tion, phosphorylation of Syk, and O�� production in responseto PDGF-BB were increased by the knockdown of Ref-1.PDGF-BB enhanced the activity of Syk kinase, and theinhibition of Syk abolished the phosphorylation of PDGF-R.

These results indicate that Ref-1 inhibits the PDGF-mediatedmigration signal via the ROS-mediated Syk activity.

Sources of FundingThis work was supported by a grant from the Korea Research Founda-tion Grant, funded by the Korea Government (MOEHRD), the RegionalResearch Universities Program/Chungbuk BIT Research-Oriented Uni-versity Consortium (KRF-2005-015-E00021) and the Korea Science &Engineering Foundation through the Infection Signaling Network Re-search Center (grant R13-2007-020-01000-0).

DisclosuresNone.

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Irani and Bokyung KimWahn Soo Choi, Young Min Bae, Hyo Shin Kim, Sang Ki Lee, Seung Hwa Park, Kaikobad Hwan Myung Lee, Byeong Hwa Jeon, Kyung-Jong Won, Chang-Kwon Lee, Tae-Kyu Park,

Mediated Syk Pathway−Oxygen Species Receptor Signaling via the Inhibition of Reactive βPlatelet-Derived Growth Factor-

Gene Transfer of Redox Factor-1 Inhibits Neointimal Formation. Involvement of

Print ISSN: 0009-7330. Online ISSN: 1524-4571 Copyright © 2008 American Heart Association, Inc. All rights reserved.is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231Circulation Research

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Cir Res AHA-2008-178699/R2

Supplement Material

Materials

Piceatannol was purchased from Tocris Bioscience (Bristol, UK). N-acetyl-cysteine

(NAC) was acquired from Calbiochem (San Diego, CA). Diphenilene iodonium

chloride (DPI), apocynin, NADPH, and lucigenin were from Sigma (St Louis, MO).

PDGF-BB was purchased from R&D Systems (Minneapolis, MN). The following

antibodies were used in this study: monoclonal anti-Syk (Abcam, Cambridge, UK),

polyclonal anti-Syk, anti-Ref-1 (Santa Cruz, Santa Cruz, CA), anti-phospho

tyrosine(4G10) (Upstate, Lake Placid, NY), anti-p38 MAPK, anti-phospho p38 MAPK

(Cell Signaling, Beverly, MA), anti-heat shock protein 27 (Hsp27), anti-phospho Hsp27

(Affinity BioReagents, Golden, CO), anti-extracellular–regulated protein kinase 1/2

(ERK1/2), anti-phospho ERK1/2 (Promega, Madison, WI), anti-PDGFβ-R, and anti-β-

actin (Sigma) antibodies.

Immunoprecipitation

After cells were lysed in extraction buffer (20 mmol/L HEPES, pH 7.5, 1% Nonidet P-

40, 150 mmol/L NaCl, 10% glycerol, 10 mmol/L NaF, 1 mmol/L Na3VO4, 2.5 mmol/L

4-nitrophenylphosphate, 0.5 mmol/L PMSF, and 1 tablet of complete proteinase

inhibitor cocktail), lysates containing 500 µg of proteins were incubated for 5 hours

with 4 µg/mL of antibodies at room temperature. The immunocomplex was then

precipitated overnight using protein A-agarose beads (Sigma) at 4°C. The beads were

washed with PBS containing tween-20 and were re-suspended in SDS sample buffer

containing 40 mmol/L Tris-HCl (pH 6.8), 8 mmol/L EGTA, 4% 2-mercaptoethanol,

Cir Res AHA-2008-178699/R2

40% glycerol, 0.01% bromophenol blue, and 4% SDS, then boiled for 5 minutes. The

collected protein samples were immunoblotted with antibodies.

Cell Migration and Proliferation Assay

The RASMCs were seeded in 96-well microtiter plates at a density of 2×103 cells per

well. After various treatments for 24 hours, BrdU–labeling solution (10 µmol/L) was

added to the cells, followed by 12 hours of incubation. The BrdU antibody complexes

were detected using a Wallace 1420 VICTOR 3TM luminometer (PerkinElmer, Boston,

MA).

Superoxide Production

After various treatments, the RASMCs were lysed with tris buffer (50 mmol/L Tris, 150

mmol/L NaCl, 1 mmol/L EDTA, 1 mmol/L PMSF, 1 mmol/L Na3VO4, 5 mmol/L NaF,

1% triton X-100, and 10% Glycerol, pH 7.2). The supernatant was treated with 10

µmol/L lucigenin and 500 µmol/L NADPH. After 5 minutes, its chemiluminescence

was repeatedly measured using the luminometer over 20 minutes, at 2-minute intervals.

A single value was determined by the average of five readings at 20 minutes. Data

normalization was applied to the total protein contents.

Aortic Ring Assay

The endothelium and adventitium of aorta obtained from Sprague Dawley rats (aged

eight weeks, n = 8) were enzymatically removed, and the vessels were cut into rings (1

mm). The rings were then placed and embedded in 48-well plates coated with Matrigel

(BD Bioscience). The embedded rings were incubated for 3 days in 10% FBS-medium,

Cir Res AHA-2008-178699/R2

then transfected with AdRef-1 or Adβ-gal under transfection conditions. The rings were

stained with Diff-Quik and photographed on day 5.

Transfection Small Interference RNA

After 12 hours incubation of RASMCs in antibiotic-free DMEM containing 10% FBS,

the medium was replaced with fresh antibiotics and FBS-free DMEM. The cells were

subsequently incubated with the small interference RNA (siRNA) or non-silencing

control siRNA to a final concentration of 1 nmol/L siRNA using transfection reagent

(Welfect-QTM Gold, Welgene, Korea). After transfection for 48 hours, cells were

harvested with the extraction buffer. The relative expression of Ref-1 was determined

by immunoblotting analysis using an anti-Ref-1 antibody.

Cir Res AHA-2008-178699/R2

Supplement Figures

Online Figure I. Effects of Ref-1 on the sprout growth of aortic rings and the proliferation of RASMCs induced by PDGF-BB. (A) Effects of AdRef-1 on PDGF-BB–induced proliferation. Cells were treated with PDGF-BB (10 ng/mL) for 24 hours and cell proliferation was quantified using a BrdU method. (B) Aortic rings (1 mm) were cultured and embedded in Matrigel, and were then treated with Adβ-gal (200 MOI) or AdRef-1 (200 MOI) in the presence or absence of PDGF-BB (10 ng/mL). Proliferation (A, n = 5) and outgrowth (C, n = 4) in control is considered to be 100%. * Denotes a significant difference from PDGF-BB–stimulated state, with P<0.05.

Cir Res AHA-2008-178699/R2

Online Figure II. Immunohistochemistry for PDGFβ-R in the balloon-injured neointima of rat carotid artery. (A) Normal carotid artery. (B) Balloon-injured carotid artery (14 days). Only neointima of balloon-injured carotid artery showed strongly positive to anti-PDGFβ-R (red arrow), suggesting PDGFβ-R is upregulated in the balloon-injured carotid artery. Counterstained with hematoxyline, × 400.

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