ISSN: 1524-4636 Copyright © 2007 American Heart Association. All rights reserved. Print ISSN: 1079-5642. Online

7272 Greenville Avenue, Dallas, TX 72514Arteriosclerosis, Thrombosis, and Vascular Biology is published by the American Heart Association.

DOI: 10.1161/01.ATV.0000257148.01384.7d 2007;

2007;27;540-547; originally published online Jan 4,Arterioscler. Thromb. Vasc. Biol.Wen-Jin Cherng

Shin-Yi Wang, Yu-Chih Liu, William L. Stanford, Richard D. Weisel, Ren-Ke Li and Chao-Hung Wang, Subodh Verma, I-Chang Hsieh, Agnes Hung, Ting-Tzu Cheng,

Intimal Hyperplasia After Vascular InjuryStem Cell Factor Attenuates Vascular Smooth Muscle Apoptosis and Increases

http://atvb.ahajournals.org/cgi/content/full/27/3/540located on the World Wide Web at:

The online version of this article, along with updated information and services, is

http://www.lww.com/reprintsReprints: Information about reprints can be found online at  

journalpermissions@lww.com410-528-8550. E-mail:

Fax:Kluwer Health, 351 West Camden Street, Baltimore, MD 21202-2436. Phone: 410-528-4050. Permissions: Permissions & Rights Desk, Lippincott Williams & Wilkins, a division of Wolters 

http://atvb.ahajournals.org/subscriptions/Biology is online at Subscriptions: Information about subscribing to Arteriosclerosis, Thrombosis, and Vascular

at University of Toronto on April 30, 2007 atvb.ahajournals.orgDownloaded from

Stem Cell Factor Attenuates Vascular Smooth MuscleApoptosis and Increases Intimal Hyperplasia After

Vascular InjuryChao-Hung Wang, Subodh Verma, I-Chang Hsieh, Agnes Hung, Ting-Tzu Cheng, Shin-Yi Wang,

Yu-Chih Liu, William L. Stanford, Richard D. Weisel, Ren-Ke Li, Wen-Jin Cherng

Objective—Stem cell factor (SCF) through its cognate receptor, the tyrosine kinase c-kit, promotes survival and biologicalfunctions of hematopoietic stem cells and progenitors. However, whether SCF/c-kit interactions exacerbate intimalhyperplasia through attenuating VSMC apoptosis induced by vascular injury has not been thoroughly investigated.

Methods and Results—VSMCs were stimulated with serum deprivation and H2O2 to induce apoptosis. The transcriptionof c-kit mRNA and the expression of the c-kit protein by VSMCs were estimated by Q-polymerase chain reaction andWestern blotting, respectively. The interactions of SCF and c-kit were investigated by in vitro and in vivo experiments.In vitro, H2O2 stimulation significantly induced apoptosis of VSMCs as evidenced by the 3- and 3.2-fold increases ofcleaved caspase-3 compared with those in the control group by Western blot and flow cytometric analyses, respectively(P�0.01). Stimulation of apoptosis also caused 3.5- and 9-fold increases in c-kit mRNA transcription and proteinexpression, respectively, by VSMCs compared with those in the control group. Administration of SCF (10 to 1000ng/mL) significantly lowered the amount of cleaved caspase-3 in H2O2-treated VSMCs (P�0.01). Specifically, SCFexerted this effect through activating Akt, followed by increasing Bcl-2 and then inhibiting the release of cytochrome-cfrom the mitochondria to the cytosol. In vivo, the mouse femoral artery was injured with a wire in SCF mutant (Sl/Sld),c-kit mutant (W/Wv), and colony control mice. In colony control mice, confocal microscopy demonstrated that thewire-injury generated a remarkable activation of caspase-3 on medial VSMCs, coinciding with upregulation of c-kitexpression. The wire-injury also caused an increase in the expression of SCF on surviving medial VSMCs and cells inthe adventitia. The upregulated c-kit expression in the vessel wall also facilitated homing by circulating SCF� cells.Compared with colony control mice, vascular injury in SCF mutant and c-kit mutant mice caused a higher number ofapoptotic VSMCs on day 14 and a lower number of proliferating cells, and resulted in significantly less neointimalformation (P�0.01) on day 28.

Conclusions—The interactions between SCF and the c-kit receptor play an important role in protecting VSMCs againstapoptosis and in maintaining intimal hyperplasia after vascular injury. (Arterioscler Thromb Vasc Biol. 2007;27:540-547.)

Key Words: apoptosis � c-kit tyrosine kinase � intimal hyperplasia � restenosis � stem cell factor

Neointimal formation, with resultant vascular remodeling,is a unifying pathological event complicating chronic

atherosclerosis, restenosis, and transplant arteriopathy, andremains the major limiting factor for the long-term efficacy ofvascular interventions, such as angioplasty and coronaryartery bypass graft surgery.1,2 VSMC proliferation coincideswith apoptosis in vessels undergoing angioplasty.3 The con-sequences of early-onset apoptosis in medial VSMCs aftervascular injury and late apoptosis in the neointima have not

been fully investigated. Although it plays an important role inlimiting neointimal growth and the subsequent intimal hyper-plasia, it may also exacerbate neointima formation at latertime points by provoking a greater wound-healing response inan effort to overcome the cellular deficit.4,5

Stem cell factor (SCF, Steel Factor) through its cognatereceptor, the tyrosine kinase c-kit,6 promotes survival,7 pro-liferation,8 mobilization,9 and adhesion10 of hematopoieticstem cells and their progenitors. Recently, the existence of

Original received April 15, 2006; final version accepted November 27, 2006.From the Division of Cardiology, Department of Internal Medicine (C.-H.W., I.-C.H., A.H., T.-T.C., S.-Y.W., Y.-C.L., W.-J.C.), Chang Gung

Memorial Hospital, Keelung; Chang Gung University College of Medicine, Taiwan; the Division of Cardiac Surgery (S.V.), St. Michael’s Hospital,Toronto, Canada; the Division of Cardiac Surgery (C.-H.W., S.V., R.D.W., R.-K.L.), Toronto General Hospital, Toronto, Canada; the Institute ofBioscience and Biotechnology (T.-T.C.), National Taiwan Ocean University; and the Institute of Biomaterials and Biomedical Engineering (W.L.S.),University of Toronto; Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Canada.

This work was presented at the annual meeting of the American Heart Association Scientific Sessions 2005, in Dallas, Texas, November 13–16, 2005Correspondence to Chao-Hung Wang, MD, Division of Cardiology, Department of Internal Medicine, Chang Gung Memorial Hospital, 222 Mai Chin

Road, Keelung, Taiwan. E-mail bearty@adm.cgmh.org.tw© 2007 American Heart Association, Inc.

Arterioscler Thromb Vasc Biol. is available at http://www.atvbaha.org DOI: 10.1161/01.ATV.0000257148.01384.7d

540 at University of Toronto on April 30, 2007 atvb.ahajournals.orgDownloaded from

this system has also been demonstrated in the vasculature.11,12

However, the role of its function in atherosclerosis is poorlyunderstood. In this study, we hypothesized that early-onsetapoptosis in medial VSMCs after vascular injury activates theSCF/c-kit system, which protects VSMCs from apoptosis andcontributes to over-growth of the neointima.

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

Animals StudiesWild-type C57BL/6, W/Wv(WBB6F1 hybrid strain, c-kit mutantmice), colony control WBB6F1 (�/�), Sl/Sld (Steel-Dickie;WCB6F1 hybrid strain, SCF mutant mice), and colony controlWCB6F1 (�/�) mice were purchased from Jackson Labs (BarHarbor, Me) and were used for the vascular injury studies. Allprocedures involving experimental animals were performed in ac-cordance with protocols approved by the institutional committees foranimal research of Toronto General Hospital, Mount Sinai Hospital,and Chang Gung Memorial Hospital and were conducted accordingto guidelines of the American Physiological Society.

Mouse Femoral Artery Wire-Injury ModelFemoral arterial injury was induced by inserting a straight springwire (0.38 mm in diameter, No. C-SF-15-15, Cook) for more than5 mm toward the iliac artery.13

Cell CultureHuman aortic smooth muscle cells were purchased from SmartecScientific (Cascade Biologics) and grown in 231 medium withsmooth muscle cell growth supplement, plus 50 U/mL penicillin and50 �g/mL streptomycin in a humidified atmosphere of 5% CO2.More than 97% of the cultured cells were VSMCs as confirmed byimmunostaining with a monoclonal �-smooth muscle actin (�SMA)antibody.14 Cells used for the experiments were in the third to fifthpassages and were 80% confluent. To produce apoptosis by serumdeprivation and H2O2, cells were washed with PBS, the medium wasreplaced with serum-free medium with H2O2 (800 �mol/L), and thecells were incubated and harvested at the indicated time points.Smooth muscle progenitors,15 endothelial progenitors,16 and late-outgrowth endothelial cells (OECs)16 were also cultured using thestandard methods mentioned previously.

Bone Marrow Transplantation ModelRecipient FVB mice at 8 weeks of age were lethally irradiated witha total dose of 9.0 Gy. eGFP transgenic mice (FVB background) thatubiquitously expressed enhanced GFP were used as the donors(Level Biotechnology Inc., Taipei, Taiwan).17 After irradiation, therecipient mice received unfractionated bone marrow cells (5�106)from eGFP mice by tail vein injection. At 8 weeks after the injection,these mice received wire injury to the femoral artery. Repopulationby eGFP-positive bone marrow cells was measured by flow cytom-etry to be 95%.

ResultsApoptosis Stimulation Upregulates c-kit mRNATranscription and Protein ExpressionAfter H2O2 stimulation, although there was no significantchange in the total caspase-3 amount, caspase-3 was tran-siently activated as indicated by the amount of cleavedcaspase-3 (Figure 1A). The activation of caspase-3 peaked at0.5 hour and then gradually returned back to the baseline.Apoptosis stimulation also induced a 25-fold increase in thenumber of apoptotic cells as indicated by Annexin-V�PI�

cells (Figure 1B). By RT-PCR and Q-PCR (Figure 1C and

1D, respectively), VSMCs had a low baseline level of c-kitmRNA transcription. In response to apoptosis stimulation, thetranscription of c-kit mRNA increased by approximately5.5-fold (1 hour after H2O2 administration). By Westernblotting, c-kit protein expression was present at the baselineand significantly increased after apoptosis stimulation inhuman VSMCs (Figure 1E) Immunofluorescent staining alsodemonstrated substantial c-kit expression on Annexin-V�

apoptotic VSMCs (supplemental Figure I). In the in vivoanimal model, wire injury activated caspase-3 throughout theentire vessel and stimulated c-kit and cleaved caspase-3coexpression on medial VSMCs at different time points(Figure 1F). Because the anti-c-kit antibody used in this study(sc-168) maps the cytoplasmic domain of the c-Kit protein(amino acids 925 to 975),18,19 the cytoplasmic staining patternwas observed in the majority of our immunofluorescenceimages.

SCF Attenuates the Activation of ApoptosisIn vitro, SCF (10 to 1000 ng/mL) was administered inVSMCs treated with H2O2. SCF significantly attenuated theamount of cleaved caspase-3 in a dose-dependent manner(Figure 2A). As estimated by flow cytometry, SCF (100ng/mL) caused a 3-fold decrease in the number of VSMCswith activated caspase-3 (Figure 2B). Using annexin-V toestimate the number of VSMCs undergoing apoptosis, SCF(1000 ng/mL) also significantly lowered the number ofapoptotic VSMCs from 24.7%�3.5% to 11.8%�2.7%(P�0.01) (Figure 2C). To clarify whether SCF also haseffects on proliferation in VSMCs with apoptotic stimulation,a proliferation assay was performed and showed that SCFsignificantly increased VSMC proliferation only at a highconcentration (1000 ng/mL) (Figure 2D). The in vivo exper-iments revealed that after the femoral artery had been injuredby the wire, SCF expression was greatly upregulated in theadventitia and in surviving medial VSMCs, providing a directsource of SCF for rescuing injured VSMCs (Figure 2E). Theupregulated transcription of SCF mRNA at injured sites wasalso quantified by Q-PCR, which demonstrated a 3.5-foldincrease in the SCF mRNA amount on injured vessels 4 daysafter wire-injury, compared with the baseline.

C-kit-Positive Cells Help SCF-Positive Cell HomingMouse femoral arteries were injured with a wire and thensubjected to immunostaining at indicated time points. Ondays 1 and 3, although remarkable SCF expression was notedin cells in the adventitia, no cells had adhered to the surfaceof the injured vessel wall (Figure 3A). On day 7 to 9 aftervascular injury, many SCF� cells had accumulated on thesurface of the injured vascular wall, close to the sites withc-kit protein expression (Figure 3A and 3B; supplementalFigure II). In the in vitro experiments, SCF expression wasinvestigated in a variety of bone marrow–derived progenitorcells, including smooth muscle progenitors, endothelial pro-genitors, OECs, and human aortic endothelial cells (supple-mental Figure IIIA through IIID). All these cells stronglyexpressed SCF, as demonstrated by immunofluorescent stain-ing and Western blot analysis (supplemental Figure IIIE).

Wang et al SCF Attenuates VSMC Apoptosis 541

at University of Toronto on April 30, 2007 atvb.ahajournals.orgDownloaded from

Progenitor cell homing involves cellular adhesion. Asmentioned above, OECs strongly expressed SCF. In theadhesion assay, apoptotic VSMCs greatly increased theadhesion of OECs, an interaction that was blocked byadministration of the anti-SCF antibody (Figure 3C). Further-more, a modified Boyden chamber was used to assess thechemoattractive potential of c-kit� cells to SCF� OECs.H2O2-stimulated VSMCs, compared with unstimulatedVSMCs, induced significantly more OEC migration (Figure3D). A significant abolishment of this migratory effect by anSCF blockade further supported the chemoattractive ability ofc-kit� cells to SCF� cells. Similar findings were also repeatedwith SCF� smooth muscle progenitors (data not shown). Toclarify the related mechanisms, our data demonstrated thatH2O2 stimulation upregulated the expression of SCF byVSMCs (supplemental Figure IVA). In a dose-dependentmanner, SCF significantly increased the production of VEGFand stroma-derived factor-1� (SDF-1�) by VSMCs undergo-ing apoptotic stimulation (supplemental Figure IVB andIVC). Through the expression of the receptors of VEGF

(VEGF-R2) and SDF-1� (CXCR4) on OECs20 and smoothmuscle progenitor cells,21 respectively, the migratory abilityof these cells toward VSMCs undergoing apoptotic stresssubstantially increased.

In the in vivo model of wire-induced femoral artery injuryin wild-type mice reconstituted with BM cells expressingeGFP (BMTGfp3Wild mice), results showed that smoothmuscle progenitor cells expressed SCF in the early phaseafter attaching to the injured vessel wall but became SCF-negative when they were mature in the neointima (supple-mental Figure IIIF and IIIG). Although it is not fullyunderstood how these cells adapt to this environment andhow differentiation is coordinated, these cells had highpotential to contribute to intimal hyperplasia.

SCF Attenuates Apoptosis Through theAkt-Bcl-2 PathwayAlthough there was no significant change in the total Aktamount, treatment with SCF transiently activated Akt, whichpeaked at 0.5 hour, followed by a significant increase in the

Figure 1. Apoptosis stimulation upregulates c-kit mRNA transcription and protein expression. A, Total and cleaved caspase-3 levelswere quantified by Western blotting at different time points after H2O2 stimulation. B, As estimated by flow cytometry, the number ofcells undergoing apoptosis (annexin-V�PI�) remarkably increased in response to H2O2 provocation. C and D, RT-PCR and Q-PCR wereperformed to estimate and quantify, respectively, the transcription of c-kit mRNA by human vascular smooth muscle cells (VSMCs) inresponse to H2O2 stimulation. E, Expression of the c-kit protein by human VSMCs was estimated by Western blotting after apoptosisstimulation. F, Coexpression of cleaved caspase-3 (red) and c-kit (green) was estimated by confocal microscopy before (control) andafter wire injury to a mouse femoral artery (C57BL/6; 630�). Yellow indicates coexpression (blue, nuclei). Arrowheads indicate internalelastic lamina. **P�0.01, compared with the control group (n�6 for each group). D1 and D2 indicate days 1 and 2, respectively. Scalebar�50 �m.

542 Arterioscler Thromb Vasc Biol. March 2007

at University of Toronto on April 30, 2007 atvb.ahajournals.orgDownloaded from

amount of Bcl-2 (Figure 4A). The control group revealed asignificant increase in the amount of cytosolic cytochrome-cwhich was maintained at a higher level in response toapoptosis stimulation compared with the baseline (Figure4B). However, in the SCF treatment group, cytosoliccytochrome-c levels significantly decreased and were main-tained at a very low level compared with the control group.

Apoptosis in SCF and c-kit Mutant MiceW/Wv mice are compound heterozygotes of a null c-kitmutation (W), and the W-viable (Wv) allele exhibits reducedkinase activity and represents the severest c-kit mutants thatsurvive gestation. Similarly, Sl/Sld mice are compound het-erozygotes of a null SCF mutation (Sl) and the Steel-Dickie(Sld) mutation, which lacks mSCF, and represent the severestSCF mutants that survive gestation. Thus, W/Wv mice have a

relative deficiency of c-kit kinase activity, although theaffected cells express normal to elevated levels of the c-kitreceptor and Steel-Dickie mice have a complete deficiency ofmembrane-bound SCF.

Intimal hyperplasia was significantly decreased in bothSCF mutant and c-kit mutant mice compared with colonycontrol mice (Figure 5A). As estimated by terminal deoxy-nucleotidyl transferase-mediated dUTP nick end-labeling(TUNEL) staining, the number of apoptotic cells tended to behigher on the vessel wall in both SCF and c-kit mutant micecompared with the media and adventitia of colony controlmice on day 7 after vascular injury (Figure 5C). On day 14,the number of apoptotic cells on the entire vessel wall wassignificantly higher in both SCF mutant and c-kit mutant micecompared with colony control mice (Figure 5B and 5C). Onthe other hand, the number of proliferating cells on the vessel

4D1D

LL

lortnoC

L

0

01

02

03

04

05

h1h5.0noc

noitavirped mureS

clea

ved

casp

ase

3 (%

)

FCS on

FCS

A B

E

0

5

01

51

02

52

03

Ann

exin

-V+

PI-

(%

)

†** **

‡ ‡‡

H2O2)lm/gn( FCS

+ + + + –– – 0001 001 01

C

PI

V-nixennA V-nixennA

%8.11%7.42

)lm/gn 0001( FCSFCS oN

0

5.0

1

5.1

2

5.2

3

5.3C

leav

ed C

aspa

se-3

+ + + + – FCS )lm/gn( 0001 001 01 – –

***†

‡ ‡

H2O2

**

**

H2O2 noitalumits

** **

FCS

HDPAG

D

H2O2

)lm/gn( FCS

)lm/gn 01( FGDP

+ + + + + + 0001 0001 001 01 — —

+ — — — — —

**

**

bA FCS — — — — — +

0

5.0

1

5.1

2

5.2

3

5.3

4

5.4

4yad1yadh21h0

yrujni eriw retfa

norm

aliz

ed t

o co

ntro

l

0

5

01

51

02

52

03

53

pro

lifer

atin

g ce

lls (

%)

Figure 2. SCF attenuates the activation of apoptosis. A, As estimated by Western blotting, SCF attenuated the amount of cleavedcaspase-3 in a dose-dependent manner. B, Estimated by flow cytometry, SCF significantly decreased the number of VSMCs withcleaved caspase-3 induced by apoptotic stimulation. C, As estimated by flow cytometry, SCF significantly decreased the number ofcells undergoing apoptosis (annexin-V�PI�) in response to H2O2 provocation. D, Effect of SCF on proliferation in VSMCs with H2O2 stim-ulation. **P�0.01, compared with the group with no SCF or PDGF treatment. E, The expression of SCF (green) on the injured vesselwall was remarkably upregulated after wire injury (red, nuclei). Arrowheads indicate internal and external elastic lamina. L indicateslumen (630�). Q-PCR was performed to quantify the upregulated transcription of SCF mRNA on injured vessel walls. *P�0.05,**P�0.01, compared with the control group. †P�0.01, ‡P�0.001, compared with the group with H2O2 stimulation, with no SCF treat-ment (n�6 for each group). Scale bar�50 �m.

Wang et al SCF Attenuates VSMC Apoptosis 543

at University of Toronto on April 30, 2007 atvb.ahajournals.orgDownloaded from

wall 7 days after wire injury was significantly lower in SCFmutant and c-kit mutant mice compared with colony controlmice (Figure 5D).

DiscussionThis study demonstrated that the SCF/c-kit system plays acritical role in the mechanisms of vascular remodeling,namely intimal hyperplasia, in response to the early-onsetapoptosis in medial VSMCs after injury. The transcription ofc-kit mRNA and the expression of c-kit protein by VSMCswere significantly upregulated in response to apoptotic stim-ulation. The increased c-kit expression on VSMCs not onlyhelps SCF exert its effects through protecting VSMCs fromapoptosis and increasing VSMC proliferation but also facil-itates the homing process of SCF-positive cells, whichcontribute to intimal hyperplasia. Furthermore, SCF attenu-ated the apoptosis of VSMCs through the Akt–Bcl-2 path-

way. The SCF/c-kit system orchestrated exacerbation ofneointimal formation after vascular injury at least in part byattenuating VSMC apoptosis and provoking a greater woundhealing response to overcome the cellular deficit.

Stem cell factor (SCF, Steel Factor) through its cognatereceptor, the tyrosine kinase c-kit,6 has been shown topromote survival,7 proliferation,8 mobilization,9 and adhe-sion10 of a variety of hematopoietic progenitors. Recently,this system was also demonstrated in the vascular system,although its role is still not fully understood. Matsui et alfound that SCF improves a variety of biological functions andsurvival of human umbilical vein endothelial cells.12 Hollen-beck et al showed that this system is expressed by and mayaffect VSMCs through an autocrine pathway.11 In the presentstudy, we demonstrated that this system also exerts its effecton the exacerbation of intimal hyperplasia through upregu-lating c-kit expression on apoptotic VSMCs, protecting

Figure 3. Upregulated expression of c-kit on VSMCs facilitates the homing process of SCF-positive cells. A, Confocal immunofluores-cent images show that 7 days after wire injury (using C57BL/6 mice), SCF-positive cells (red, arrows) had gradually accumulated on theinjured vessels (blue, nuclei; green, CD45) (630�). B, SCF-positive cells (red) homed in on c-kit-expressing sites (green) on the injuredvessel wall 7 to 9 days after wire injury. Coexpression of SCF and c-kit (yellow) was also noted on some cells in the vessel wall. Arrow-heads indicate the internal elastic lamina. L, M, and A indicate lumen, media, and adventitia, respectively. C, Progenitor cell hominginvolves cellular adhesion. Late-outgrowth of endothelial cells (OECs) strongly expressed SCF. In the adhesion assay, apoptotic VSMCsgreatly increased the adhesion of OECs (green), an interaction that was blocked by administration of an anti-SCF antibody. D,Increased migration of SCF� OECs toward c-kit� VSMCs (c-kit upregulated by apoptotic stress). Scale bar�50 �m.

544 Arterioscler Thromb Vasc Biol. March 2007

at University of Toronto on April 30, 2007 atvb.ahajournals.orgDownloaded from

VSMCs from apoptosis, and increasing VSMC proliferationand homing SCF� cells. In addition, associated mechanismsand the signaling pathway are also provided.

The consequences of early-onset apoptosis in medialVSMCs after vascular injury have not been fully investigated.VSMC apoptosis has been demonstrated in atherosclerosisand in restenotic lesions after angioplasty. In animal modelsof balloon vascular injury, medial VSMC apoptosis andsubsequent cell loss were observed soon after the injury.13

However, the molecular mechanisms of vascular cell apopto-sis remain to be elucidated, and the role of VSMC apoptosisin vascular remodeling is still a matter of controversy.22–25 Ithas been proposed that VSMC apoptosis prevents prolifera-tive vascular disease,22–25 because forced induction of VSMCapoptosis by gene modification results in a reduction ofvascular lesions. In contrast, it has also been postulated thatvascular cell apoptosis plays a role in the development ofvascular lesions, because exuberant balloon-induced apopto-sis results in enhanced neointimal formation.4 As proposed bythe current study, this phenomenon can be attributed, at leastin part, to the SCF/c-kit system. Our results demonstrated thatan apoptosis-stimulating stress substantially upregulated thetranscription of c-kit mRNA and the synthesis of c-kit protein.The c-kit receptor is a member of the type III receptortyrosine kinase family.26 This family of cytokine receptorsalso encompasses the c-fms receptor, the platelet-derivedgrowth factor receptors, and the flk-2/flt-3 receptor. Specifi-cally, our findings suggest that SCF exerts its antiapoptoticeffect through c-kit tyrosine kinase and then Akt, followed bya remarkable increase in the amount of intracellular Bcl-2,leading to a substantial inhibition of the release ofcytochrome-c from the mitochondria to the cytosol (Figure4C). Through this pathway, SCF significantly attenuates theactivation of caspase-3 and eventual cellular apoptosis.

On the other hand, the upregulated expression of c-kitreceptors on VSMCs undergoing apoptosis not only activatedthis SCF/c-kit system but also helped SCF-positive cellshome in on injured vascular sites. These SCF-positive cells

provided a substantial amount of SCF to rescue VSMCs fromapoptosis and also substantially contributed to the formationof neointima. The concentration of SCF in normal humanserum is, on average, 3.3 ng/mL.26 Our data showed that SCFalready exerts its effects at similar concentrations in adose-dependent manner at higher concentrations. Local SCFconcentrations at injured vascular sites, as provided by locallyaccumulated SCF-expressing cells or bone marrow–derivedprogenitor cells, are expected to be much higher than serumlevels. Consistent with this notion, our findings revealed thatsmooth muscle progenitor cells, late-outgrowth of endothelialcells, endothelial progenitor cells, and aortic endothelial cellsall express SCF. These findings are in line with the newparadigm that bone marrow–derived progenitor cells contrib-ute to intimal hyperplasia after vascular injury, as shownextensively in the literature.16,27,28 However, how the fate ofthese SCF-positive cells is decided in this microenvironmentstill remains to be elucidated.

The mouse femoral artery wire-injury model adopted in thepresent study represents a severe vascular injury model.Although the extent of vascular injury is considerably lesssevere in human angioplasty, in the era of extensive vascularstent intervention, similar stresses on vessel walls may existas atherosclerotic plaques are pushed outward. In this study,we also took advantage of SCF-mutant and c-kit mutant miceto gain further support for our hypothesis. In SCF-mutantmice, deficiencies of both mSCF and sSCF disrupted theability of the SCF/c-kit system to rescue injured VSMCs asindicated by extensive apoptotic events throughout all threelayers of the injured vessel wall. Their deficiencies in SCFalso substantially attenuated the contribution of SCF-positivecells to the formation of neointima. Although SCF is notdeficient in c-kit mutant mice, the lack of appropriate c-kittyrosine kinase signaling also led to remarkable apoptoticprocesses on the injured vessel wall, especially on thevascular media and adventitia. Through these mechanisms,these mutant mice ended up with significantly less intimalhyperplasia compared with colony control mice. The effect of

0

5

10

15

20

25

0h 0.5h 1h 2h 4h

tnemtaert FCS retfA

Nor

mal

ized

to c

ontr

ol Total AktAkt-pBcl-2

*

*

*

*

*

+ + + +-lm/gn 001 FCS h4 h2 h1 h5.0 h0

tkA latoT

p-tkA

2-lcB

tkAP-tkA

2-lcB

CtyC

xaB

9esapsaC

3esapsaC

FCS

tik-C sisotpopA

lairdnohcotiM

A

CnoitavratS

lm/gn 001 FCS+ + +- + ++-+ ++ +----

Bc-emorhcotyC

h6 h4 h2 h0 h6 h4 h2 h0

0

0.5

1

1.5

2

2.5

0h 2h 4h 6h

Control SCF

*†

*†

*

*†

lortnoC tnemtaert FCS

Nor

mal

ized

to

con

trol

s (0

h)

Figure 4. SCF attenuates VSMC apoptosisthrough the Akt–Bcl-2 pathway. A, Westernblot shows changes in different intracellularsignals in response to SCF treatment. B,Changes in cytosolic cytochrome-c levels inVSMCs in response to apoptosis stimulationwith or without SCF treatment. *P�0.01, com-pared with the baseline. †P�0.001, comparedwith the controls. C, The diagram depicts thepathway through which SCF exerts its effect(n�6 for each group).

Wang et al SCF Attenuates VSMC Apoptosis 545

at University of Toronto on April 30, 2007 atvb.ahajournals.orgDownloaded from

SCF in stimulating VSMC proliferation may also contributeto growth of the neointima. However, SCF exerts this effectonly at high concentrations.

In summary, we herein demonstrate a novel way by whichthe SCF/c-kit system works on vascular remodeling pro-cesses. These findings illustrate that the SCF/c-kit interactionis a very complicated process. To attenuate intimal hyperpla-sia or atherosclerotic processes, our work provides a rationalefor testing directed therapies aimed at interrupting the SCF/c-kit pathway in patients undergoing vascular interventionssuch as a bypass graft and angioplasty.

AcknowledgmentsWe thank Rei-Chang Chen and Hsiu-Fu Mei for technical assistancewith performing the real-time PCR and confocal microscope.

Sources of FundingThis work was supported in part by the National Science Council ofTaiwan (NSC 94-2134-B-182A-191 and NSC 94-2134-B-182A-192)

and in part by Heart and Stroke Foundation of Canada (to S.V. andR.D.W.).

DisclosuresNone.

References1. Babapulle MN, Eisenberg MJ. Coated stents for the prevention of reste-

nosis: Part I. Circulation. 2002;106:2734–2740.2. Savage MP, Douglas JS, Jr., Fischman DL, Pepine CJ, King SB, III,

Werner JA, Bailey SR, Overlie PA, Fenton SH, Brinker JA, Leon MB,Goldberg S. Stent placement compared with balloon angioplasty forobstructed coronary bypass grafts. Saphenous Vein De Novo Trial Inves-tigators. N Engl J Med. 1997;337:740–747.

3. Han DK, Haudenschild CC, Hong MK, Tinkle BT, Leon MB, Liau G.Evidence for apoptosis in human atherogenesis and in a rat vascularinjury model. Am J Pathol. 1995;147:267–277.

4. Rivard A, Luo Z, Perlman H, Fabre JE, Nguyen T, Maillard L, Walsh K.Early cell loss after angioplasty results in a disproportionate decrease inpercutaneous gene transfer to the vessel wall. Hum Gene Ther. 1999;10:711–721.

0

2

4

6

8

41D7D3D1D

dliW

dlS/lSvW/W

02468012141

41D7D3D1D

0246

80121

41D7D3D1D

**

* ** *

**

0

5.0

1

5.1

2

5.2

3

5.3

I/M r

atio

(%

)

****

ynoloC lS/lS d W/W v

slortnoc

C

aititnevdAaideMamitnioeN

No.

ofa p

opto

tic

c ells

-ctnatum FCSslortnoc ynoloC tik tnatum

82D

41D

A

B

D

76-iK

slortnoc ynoloC tnatum FCS -c tik tnatum

0

5

01

51

02

52

03N

o.

of

Ki-

67+

cel

ls(H

PF

)

***

ynoloC lS/lS d W/W v

slortnoc

41D

Figure 5. Apoptosis in SCF and c-kit mutant mice. A, Intimal hyperplasia 28 days after femoral artery wire injury is shown by H&Estaining in colony control, SCF mutant, and c-kit mutant mice (n�6 to 7 in each group). I/M indicates the intima/media ratio. B and C,Apoptotic cells (brown, arrows) are enumerated by TUNEL staining through all three layers of the femoral artery 1, 3, 7, and 14 daysafter wire injury. D, Cell proliferation (indicated by Ki67) was estimated in vessels 14 days after wire injury (red, �-SMA; green, Ki67;blue, nuclei; cyan, Ki67� nucleus, indicated by arrows). *P�0.05, **P�0.01, compared with colony control mice (n�6 for each group).Arrowheads indicate internal and external elastic laminae. Scale bar�50 �m.

546 Arterioscler Thromb Vasc Biol. March 2007

at University of Toronto on April 30, 2007 atvb.ahajournals.orgDownloaded from

5. Walsh K, Smith RC, Kim HS. Vascular cell apoptosis in remodeling,restenosis, and plaque rupture. Circ Res. 2000;87:184–188.

6. Chabot B, Stephenson DA, Chapman VM, Besmer P, Bernstein A. Theproto-oncogene c-kit encoding a transmembrane tyrosine kinase receptormaps to the mouse W locus. Nature. 1988;335:88–89.

7. Domen J, Weissman IL. Hematopoietic stem cells need two signals toprevent apoptosis; BCL-2 can provide one of these, Kitl/c-Kit signalingthe other. J Exp Med. 2000;192:1707–1718.

8. Leary AG, Zeng HQ, Clark SC, Ogawa M. Growth factor requirementsfor survival in G0 and entry into the cell cycle of primitive humanhemopoietic progenitors. Proc Natl Acad Sci U S A. 1992;89:4013–4017.

9. Fleming WH, Alpern EJ, Uchida N, Ikuta K, Weissman IL. Steel factorinfluences the distribution and activity of murine hematopoietic stem cellsin vivo. Proc Natl Acad Sci U S A. 1993;90:3760–3764.

10. Levesque JP, Leavesley DI, Niutta S, Vadas M, Simmons PJ. Cytokinesincrease human hemopoietic cell adhesiveness by activation of very lateantigen (VLA)-4 and VLA-5 integrins. J Exp Med. 1995;181:1805–1815.

11. Hollenbeck ST, Sakakibara K, Faries PL, Workhu B, Liu B, Kent KC.Stem cell factor and c-kit are expressed by and may affect vascular SMCsthrough an autocrine pathway. J Surg Res. 2004;120:288–294.

12. Matsui J, Wakabayashi T, Asada M, Yoshimatsu K, Okada M. Stem cellfactor/c-kit signaling promotes the survival, migration, and capillary tubeformation of human umbilical vein endothelial cells. J Biol Chem. 2004;279:18600–18607.

13. Sata M, Maejima Y, Adachi F, Fukino K, Saiura A, Sugiura S, Aoyagi T,Imai Y, Kurihara H, Kimura K, Omata M, Makuuchi M, Hirata Y, NagaiR. A mouse model of vascular injury that induces rapid onset of medialcell apoptosis followed by reproducible neointimal hyperplasia. J MolCell Cardiol. 2000;32:2097–2104.

14. Ozawa T, Mickle DAG, Weisel RD, Koyama N, Ozawa S, Li RK.optimal biomaterial for creation of autologous cardiac grafts. Circulation.2002;106 (suppl I):176I–182I.

15. Fukuda D, Sata M, Tanaka K, Nagai R. potent inhibitory effect ofsirolimus on circulating vascular progenitor cells. Circulation. 2005;111:926–931.

16. Wang CH, Ciliberti N, Li SH, Szmitko PE, Weisel RD, Fedak PW, AlOmran M, Cherng WJ, Li RK, Stanford WL, Verma S. Rosiglitazonefacilitates angiogenic progenitor cell differentiation toward endotheliallineage: a new paradigm in glitazone pleiotropy. Circulation. 2004;109:1392–1400.

17. Hsiao YC, Chang HH, Tsai CY, Jong YJ, Horng LS, Lin SF, Tsai TF.Coat color-tagged green mouse with EGFP expressed from the RNApolymerase II promoter. Genesis. 2004;39:122–129.

18. Lucas DR, al-Abbadi M, Tabaczka P, Hamre MR, Weaver DW, Mott MJ.c-Kit expression in desmoid fibromatosis. Comparative immunohisto-chemical evaluation of two commercial antibodies. Am J Clin Pathol.2003;119:339–345.

19. Makhlouf HR, Remotti HE, Ishak KG. Expression of KIT (CD117) inangiomyolipoma. Am J Surg Pathol. 2002;26:493–497.

20. Yoon CH, Hur J, Park KW, Kim JH, Lee CS, Oh IY, Kim TY, Cho HJ,Kang HJ, Chae IH, Yang HK, Oh BH, Park YB, Kim HS. Synergisticneovascularization by mixed transplantation of early endothelial pro-genitor cells and late outgrowth endothelial cells: the role of angiogeniccytokines and matrix metalloproteinases. Circulation. 2005;112:1618–1627.

21. Zhang LN, Wilson DW, da Cunha V, Sullivan ME, Vergona R, RutledgeJC, Wang YX. Endothelial NO synthase deficiency promotes smoothmuscle progenitor cells in association with upregulation of stromal cell-derived factor-1alpha in a mouse model of carotid artery ligation. Arte-rioscler Thromb Vasc Biol. 2006;26:765–772.

22. Fukuo K, Inoue T, Morimoto S, Nakahashi T, Yasuda O, Kitano S,Sasada R, Ogihara T. Nitric oxide mediates cytotoxicity and basicfibroblast growth factor release in cultured vascular smooth muscle cells.A possible mechanism of neovascularization in atherosclerotic plaques.J Clin Invest. 1995;95:669–676.

23. Fukuo K, Nakahashi T, Nomura S, Hata S, Suhara T, Shimizu M,Tamatani M, Morimoto S, Kitamura Y, Ogihara T. Possible participationof Fas-mediated apoptosis in the mechanism of atherosclerosis. Geron-tology. 1997;43(Suppl 1):35–42.

24. Cai W, Devaux B, Schaper W, Schaper J. The role of Fas/APO 1 andapoptosis in the development of human atherosclerotic lesions. Athero-sclerosis. 1997;131:177–186.

25. Geng YJ, Henderson LE, Levesque EB, Muszynski M, Libby P. Fas isexpressed in human atherosclerotic intima and promotes apoptosis ofcytokine-primed human vascular smooth muscle cells. ArteriosclerThromb Vasc Biol. 1997;17:2200–2208.

26. Broudy VC. Stem cell factor and hematopoiesis. Blood. 1997;90:1345–1364.

27. Sata M, Saiura A, Kunisato A, Tojo A, Okada S, Tokuhisa T, Hirai H,Makuuchi M, Hirata Y, Nagai R. Hematopoietic stem cells differentiateinto vascular cells that participate in the pathogenesis of atherosclerosis.Nat Med. 2002;8:403–409.

28. Werner N, Kosiol S, Schiegl T, Ahlers P, Walenta K, Link A, Bohm M,Nickenig G. Circulating endothelial progenitor cells and cardiovascularoutcomes. N Engl J Med. 2005;353:999–1007.

Wang et al SCF Attenuates VSMC Apoptosis 547

at University of Toronto on April 30, 2007 atvb.ahajournals.orgDownloaded from