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(Circulation Research. 2007;100:807.)
© 2007 American Heart Association, Inc.

Molecular Medicine

An Essential Role for gp130 in Neointima Formation Following Arterial Injury

Dong Wang, Zhimin Liu, Quanyi Li, Manjula Karpurapu, Venkatesh Kundumani-Sridharan, Huiqing Cao, Nagadhara Dronadula, Farhan Rizvi, Arun K. Bajpai, Chunxiang Zhang, Gerhard Müller-Newen, Kevin W. Harris, Gadiparthi N. Rao

From the Departments of Physiology (D.W., Z.L., Q.L., M.K., V.K.-S., H.C., N.D., F.R., A.K.B., G.N.R.) and Surgery (C.Z.), University of Tennessee Health Science Center, Memphis; Institut fur Biochemie (G.M.-N.), Rheinisch-Westflische Technische Hochschule Aachen, Germany; and Division of Hematology (K.W.H.), Department of Medicine, University of Alabama at Birmingham.

Correspondence to Gadiparthi N. Rao, PhD, Department of Physiology, University of Tennessee Health Science Center, Memphis, TN 38163. E-mail grao{at}physio1.utmem.edu

	Abstract

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Interleukin (IL)-6 induced vascular smooth muscle cell (VSMC) motility in a dose-dependent manner. In addition, IL-6 stimulated tyrosine phosphorylation of gp130, resulting in the recruitment and activation of STAT-3. IL-6–induced VSMC motility was found to be dependent on activation of gp130/STAT-3 signaling. IL-6 also induced cyclin D1 expression in a time- and gp130/STAT-3–dependent manner in VSMCs. Suppression of cyclin D1 levels via the use of its small interfering RNA molecules inhibited IL-6–induced VSMC motility. Furthermore, balloon injury induced IL-6 expression both at mRNA and protein levels in rat carotid artery. Balloon injury also caused increased STAT-3 phosphorylation and cyclin D1 expression, leading to smooth muscle cell migration from the media to the intimal region. Blockade of gp130/STAT-3 signaling via adenovirus-mediated expression of dngp130 or dnSTAT-3 attenuated balloon injury–induced STAT-3 phosphorylation and cyclin D1 induction, resulting in reduced smooth muscle cell migration from media to intima and decreased neointima formation. Together, these observations for the first time suggest that IL-6/gp130/STAT-3 signaling plays an important role in vascular wall remodeling particularly in the settings of postangioplasty and thereby in neointima formation.

Key Words: cyclin D1 • cytokines • migration • smooth muscle cells • transcription factors

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Interleukin (IL)-6 levels have been reported in coronary circulation after coronary artery angioplasty, and it was considered as a risk factor for restenosis.¹ The possible role of IL-6 in vessel wall remodeling is supported by the findings that its production was increased in vascular smooth muscle cells (VSMCs) in response to a variety of vascular agonists such as platelet-derived growth factor-BB (PDGF-BB), thrombin, and angiotensin II.^2,3 In addition, IL-6 has been shown to stimulate VSMC motility.^2,4,5 The IL-6 family of cytokines mediates its cellular effects via the signal transducer gp130.^6,7 IL-6, on binding to its soluble receptor, leads to homodimerization and activation of gp130.⁷ Activated gp130 recruits nonreceptor tyrosine kinases such as Jaks.^8,9 Jaks, in turn, phosphorylate gp130, thereby creating docking sites for the downstream signal transduction molecules.¹⁰ STAT-3 has been reported to be among the preferentially activated downstream effector transcriptional factor of IL-6/IL-6R/gp130 signaling module. IL-6/IL-6R/gp130 signaling plays an important role in immune function and inflammation.^11,12 Recent studies have shown that a phenylalanine knock-in substitution of the cytoplasmic Tyr757 residue of gp130 that disrupts the binding site for the negative regulator suppressor of cytokine signaling-3 leads to hyperactivation of STAT-3 causing gastric hyperproliferation.¹³ Both gp130 and STAT-3 have also been reported to be involved in the development of numerous cancers.^14–19 The studies from our laboratory showed that STAT-3 mediates both receptor tyrosine kinase and G protein–coupled receptor agonist–induced VSMC growth and motility.^20–22 Although the studies from our laboratory, as well as others, have indicated that IL-6 plays a role in VSMC motility,^2,5 the underlying mechanisms of its actions were never studied in this system. Here, we present evidence that IL-6 induces VSMC motility via activation of gp130/STAT-3 signaling, leading to cyclin D1 expression. In addition, vascular injury induced IL-6 production and STAT-3 activation in a time-dependent manner. Furthermore, blockade of gp130-STAT-3 signaling suppressed balloon injury–induced STAT-3 phosphorylation, cyclin D1 expression, SMC migration, and neointima formation. Together, these findings demonstrate a key role for IL-6/gp130/STAT-3 signaling in vascular wall remodeling following injury.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
An expanded Materials and Methods section is available in the online data supplement at http://circres.ahajournals.org.

Reagents
For chemicals and biological reagents, see the online data supplement.

Construction of Adenoviral Vectors
For the detailed dngp130 cloning information, see the online data supplement. The construction of pAd-GFP and pAd-dnSTAT-3 were described previously.^23,24 The Ad-GFP, Ad-dngp130, and Ad-dnSTAT-3 virus were titrated using standard plaque assay.²⁵

Cell Culture
Rat VSMCs were isolated and subcultured as described previously.² VSMCs between 4 and 12 passages were quiesced by incubating in serum-free DMEM for 72 hours at 37°C and used to perform the experiments unless otherwise stated.

Cell Motility
VSMC motility was measured by cell wounding and Boyden chamber assays as described previously.^2,26 For the detailed procedures, see the online data supplement.

Chromatin Immunoprecipitation Assay
Chromatin immunoprecipitation (ChIP) assay was performed on VSMCs using a kit following the protocol of the supplier (Upstate Biotechnology, Lake Placid, NY). The primers designed for PCR amplification from the rat cyclin D1 promoter flanking the putative STAT-binding site located at –978²⁷ are as follows: forward, 5'-CAACGAAGCCAATCGGGAAGCTTC-3'; and reverse, 5'-CACCCTATACTTAAGCGGAGAGAA-3'. For the detailed procedure, see the online data supplement.

DNA Synthesis
VSMC DNA synthesis was measured by [³H]-thymidine incorporation as described previously.²⁴

Electrophoretic Mobility Shift Assay
Electrophoretic mobility shift assay was performed as described previously.²⁴

Western Blot Analysis
Western blot analysis was performed as described previously.²⁴

Rat Carotid Artery Balloon Injury
All of the animal protocols were performed in accordance with the relevant guidelines and regulations approved by the Internal Animal Care & Use Committee of the University of Tennessee Health Science Center. Balloon injury was performed essentially as described by us previously.²³ For morphometric analysis, carotid arteries were fixed in 10% formalin, dehydrated, and embedded in paraffin. Sections (5-µm thick) obtained at equally spaced intervals in the middle of injured and control common carotid artery segments were stained with hematoxylin/eosin. The intimal and medial areas were measured using NIH image 1.62 program and the intimal/medial ratios were calculated.

Delivery of Adenoviruses
After balloon injury, solutions of (100 µL) Ad-GFP (10¹⁰ plaque forming units [pfu]/mL), Ad-dngp130 (10¹⁰ pfu/mL), or Ad-dnSTAT-3 (10¹⁰ pfu/mL) were infused into the ligated segment of the common carotid artery for 30 minutes. The ligatures and catheter were then removed, the external carotid artery was ligated, and the incision was closed.

Double-Immunofluorescence Staining
For the detailed double immunofluorescence staining procedures, see the online data supplement.

IL-6 ELISA
IL-6 in the tissue extracts was measured using an ELISA kit following the instructions of the manufacturer (Pierce, Rockford, Ill).

RNA Isolation, cDNA Synthesis, and RT-PCR
RNA was isolated from either cells or arteries using TRIzol reagent as per the guidelines of the manufacturer. For the detailed RT-PCR procedures and primer information, see the online data supplement.

In Vivo SMC Migration Assay
Although migration is difficult to quantify in vivo, the accumulation of cells in the intima early (4 days) after injury is considered to be mostly attributable to the migration of VSMCs from the injured media.²⁸ Therefore, the in vivo SMC migration was determined as described by Bendeck et al.²⁹ For the detailed procedure, see the online data supplement.

Statistics
All of the experiments were repeated at least 3 times, with similar patterns of results. Data are presented as mean±SD, and the treatment effects were analyzed by Student’s t test.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
IL-6 Stimulates gp130/STAT-3 Signaling in VSMCs
To understand the role of IL-6 in vascular diseases, we have studied its effects on VSMC motility using wounding and Boyden chamber assays. IL-6 induced VSMC motility in a dose-dependent manner, with a maximum 2- to 5-fold increase at 20 ng/mL (Figure 1A and 1B). To find whether IL-6 also modulates VSMC growth, we next tested its effect on VSMC DNA synthesis by [³H]-thymidine incorporation. As shown in Figure 1C, IL-6 up to 30 ng/mL had no significant effect on VSMC DNA synthesis. On the other hand, PDGF-BB (20 ng/mL) increased DNA synthesis by 4-fold as compared with control. To understand the mechanisms of IL-6–induced VSMC motility, we tested the role of STAT-3. IL-6 stimulated tyrosine phosphorylation of STAT-3 in a time-dependent manner, with a near maximum 2-fold increase at 10 minutes, and this effect was sustained, at least, for 1 hour (Figure 2A). To test whether IL-6–induced STAT-3 phosphorylation leads to an increase in its transcriptional transactivation capacity, its DNA binding activity was measured. IL-6 stimulated STAT-3 DNA binding activity in a time-dependent manner, with a maximum 3-fold increase at 2 hours (Figure 2B). The IL-6 family of cytokines uses gp130 as an integral part of their cognate receptor complex in mediating its cellular effects.^6,7 To test whether IL-6 activates gp130 in VSMCs, quiescent cells were treated with and without IL-6 (20 ng/mL) for the indicated times and an equal amount of protein from control and each treatment was analyzed for gp130 tyrosine phosphorylation via immunoprecipitation using anti-gp130 antibodies followed by immunoblotting with anti-PY20 antibodies. IL-6 stimulated tyrosine phosphorylation of gp130 in a time-dependent manner in VSMCs (Figure 3A, top). To find whether gp130 on its tyrosine phosphorylation recruits STAT-3, the anti-gp130 immunoprecipitates of control and IL-6–treated VSMCs were analyzed by Western blotting for STAT-3 using its specific antibodies. Anti-gp130 antibodies coprecipitated STAT-3 in a manner that is dependent on the state of gp130 tyrosine phosphorylation (Figure 3A, middle). To confirm these results, we next used the dominant negative mutant approach. VSMCs were transduced with Ad-GFP, Ad-dngp130, or Ad-dnSTAT-3 with a multiplicity of infection (moi) of 80, quiesced, and treated with or without IL-6 (20 ng/mL) for 60 minutes; and cell extracts prepared and analyzed for STAT-3 phosphorylation. Adenovirus-mediated expression of either dngp130 or dnSTAT-3 strongly inhibited IL-6–induced STAT-3 phosphorylation (Figure 3B and 3C). In support of these findings, we also studied the effect of dngp130 and dnSTAT-3 on IL-6–induced STAT-3 DNA binding activity. Adenovirus-mediated expression of dngp130 or dnSTAT-3 inhibited IL-6–induced STAT-3/DNA binding activity (Figure 3D).

View larger version (16K): [in this window] [in a new window]   Figure 1. IL-6 induces VSMC motility but not DNA synthesis. A, A cell-free gap was made in a monolayer of quiescent VSMCs, treated with and without various doses of IL-6 for 24 hours, and cell motility was measured using wounding assay. B, Quiescent VSMCs were added to the upper chamber of the cell culture inserts that were placed in a 24-well plate. IL-6 at various doses was added to the lower chamber. After 8 hours of incubation at 37°C, the inserts were lifted out from the 24-well plate and the nonmigrated cells were removed from the upper side of the membrane with cotton swab. The migrated cells on the lower side of the membrane were fixed in methanol, stained with Giemsa–Wright, and counted under light microscope. C, Quiescent VSMCs were incubated with and without the indicated concentrations of IL-6 or 20 ng/mL PDGF-BB for 24 hours, and DNA synthesis was measured by [3H]-thymidine incorporation. *P<0.01 vs control.

View larger version (33K): [in this window] [in a new window]   Figure 2. IL-6 activates STAT-3 in VSMCs. Quiescent VSMCs were treated with and without IL-6 (20 ng/mL) for the indicated times and either cell or nuclear extracts were prepared. A, Cell extracts containing an equal amount of protein from control and each treatment were analyzed by Western blotting (IB) for pSTAT-3 using its phospho-specific antibodies. The blot was reprobed with anti–STAT-3 antibodies for normalization. B, Nuclear extracts containing an equal amount of protein from control and each treatment were assayed for STAT-3 DNA binding activity using its [32P]-labeled consensus double-stranded oligonucleotides as a probe. The bar graph shows the quantitative analysis of 3 experiments on STAT-3 phosphorylation. *P<0.01 vs control.

View larger version (25K): [in this window] [in a new window]   Figure 3. GP130 mediates IL-6 activation of STAT-3 in VSMCs. A, Quiescent VSMCs were treated with and without IL-6 (20 ng/mL) for the indicated times, and cell extracts were prepared. Protein (500 µg) from control and each treatment was immunoprecipitated with 2 µg of anti-gp130 antibodies, and the immunocomplexes were analyzed by Western blotting using anti-PY20 antibodies. The blot was reprobed sequentially with anti–STAT-3 and anti-gp130 antibodies. B through D, VSMCs were transduced with Ad-GFP (control), Ad-dngp130, or Ad-dnSTAT-3 (80 moi), quiesced, and treated with and without IL-6 (20 ng/mL) for 60 minutes and either cell or nuclear extracts were prepared. Cell extracts containing an equal amount of protein from control and each treatment were analyzed by Western blotting for pSTAT-3 using its phospho-specific antibodies (B and C). The blots were reprobed with anti–STAT-3 and/or anti-gp130 antibodies to show their endogenous or overexpressed levels. D, Nuclear extracts containing an equal amount of protein from control and each treatment were assayed for STAT-3 DNA binding using its [32P]-labeled consensus double-stranded oligonucleotides as a probe.

IL-6–Induced VSMC Motility Requires gp130/STAT-3–Dependent Cyclin D1 Expression
To understand the role of gp130/STAT-3 signaling in IL-6–induced VSMC motility, cells were transduced with Ad-dngp130 or Ad-dnSTAT-3 (80 moi), quiesced, and subjected to IL-6–induced motility. As measured by wounding and Boyden chamber assays, adenovirus-mediated expression of either dngp130 or dnSTAT-3 completely inhibited VSMC motility induced by IL-6 (Figure 4A and 4B). It was demonstrated that STATs modulate the expression of cyclin D1 in some cell types.³⁰ In addition, a role for cyclin D1 in the regulation of cell migration has been reported.^31,32 To identify the potential target genes of gp130/STAT-3 signaling in the path of VSMC motility, we next studied the time course effect of IL-6 on cyclin D1 expression. IL-6 induced cyclin D1 expression both at mRNA and protein levels in a time-dependent manner, with a maximum 3-fold increase at 2 hours, and these levels were sustained, at least, for 16 hours (Figure 5A and 5B). Furthermore, suppression of gp130/STAT-3 signaling by adenovirus-mediated expression of dngp130 or dnSTAT-3 completely inhibited IL-6–induced cyclin D1 expression, both at mRNA and protein levels (Figure 5C through 5E). Transfac analysis of the cloned rat cyclin D1 promoter²⁷ revealed the presence of a putative STAT binding site spanning from –978 to –986 (5'-TTCCTGGAA-3'). To find whether STAT-3 binds to cyclin D1 promoter, we have performed both electrophoretic mobility shift and ChIP assays. A 2-fold increase in STAT-3/DNA binding activity was observed with rat cyclin D1 promoter sequence, 5'-TCTGGTTCCTGGAAGGGCAA-3', encompassing the putative STAT binding site as a [³²P]-labeled probe in response to IL-6 (Figure 5F). ChIP of control and various time periods of IL-6–treated (20 ng/mL) VSMCs with anti–STAT-3 antibodies followed by PCR amplification using primers spanning –1013 to –411 region of cyclin D1 promoter revealed increased binding of STAT-3 to this region in response to IL-6 treatment as compared with control (Figure 5G). These results clearly indicate that STAT-3 is indeed involved in IL-6–induced cyclin D1 expression in VSMCs. To find whether cyclin D1 plays a role in VSMC migration, we used a small interfering RNA (siRNA) approach. Compared with the effect of scrambled control siRNA, cyclin D1 siRNA inhibited IL-6–induced cyclin D1 expression (Figure 6A). IL-6 induced motility in VSMCs that were transfected with scrambled control siRNA. However, IL-6 failed to stimulate the motility of VSMCs that were transfected with cyclin D1 siRNA (Figure 6B and 6C).

View larger version (18K): [in this window] [in a new window]   Figure 4. IL-6–induced VSMC motility requires activation of gp130/STAT-3 signaling. VSMCs that were transduced with Ad-GFP (control), Ad-dngp130, or Ad-dnSTAT-3 with a moi of 80 were quiesced and subjected to IL-6–induced motility by wounding (A) or Boyden chamber (B) assays. *P<0.01 vs control; **P<0.01 vs IL-6 treatment alone.

View larger version (36K): [in this window] [in a new window]   Figure 5. IL-6 induced cyclin D1 expression in gp130/STAT-3–dependent manner in VSMCs. A and B, Quiescent VSMCs were treated with or without IL-6 (20 ng/mL) for the indicated times, and RNA or proteins were isolated. A, An equal amount of RNA from control and each treatment was analyzed by RT-PCR for cyclin D1 and ß-actin mRNA levels using their specific primers. B, An equal amount of protein from control and each treatment was analyzed by Western blotting for cyclin D1 and CDK4 levels using their specific antibodies. C and D, Conditions were the same as in A, except that cells were transduced with Ad-GFP, Ad-dngp130, or Ad-dnSTAT-3 with a moi of 80 and quiesced before they were subjected to IL-6 treatment and analysis of cyclin D1 and ß-actin mRNA levels. E, Conditions were the same as in B, except that cells were transduced with Ad-GFP, Ad-dngp130, or Ad-dnSTAT-3 with a moi of 80 and quiesced before they were subjected to IL-6 treatment and Western blot analysis of cyclin D1 and ß-tubulin levels. F, Nuclear extracts were prepared from quiescent VSMCs that were treated with IL-6 (20 ng/mL) for various times, and an equal amount of protein from each condition was analyzed for STAT DNA binding activity using a [32P]-labeled putative STAT binding sequence from rat cyclin D1 promoter as a probe. G, ChIP was performed by use of anti–STAT-3 antibodies in control VSMCs and VSMCs treated for various times with IL-6 (20 ng/mL), and the resulting DNA fragments were subjected to PCR amplification using primers spanning –1013 to –411 region of rat cyclin D1 promoter. The bar graphs in A, C, and D represent the quantitative analysis of 3 independent experiments. *P<0.01 vs control; **P<0.01 vs IL-6 treatment alone.

View larger version (17K): [in this window] [in a new window]   Figure 6. Cyclin D1 mediates IL-6–induced VSMC motility. A, VSMCs were transfected with control or cyclin D1 siRNA, quiesced, and treated with or without IL-6 (20 ng/mL) for 16 hours; and cell extracts were prepared and analyzed by Western blotting for cyclin D1 and ß-tubulin levels using their specific antibodies. B and C, Conditions were the same as in A, except that cells were treated with or without IL-6 (20 ng/mL) for 24 and 8 hours to measure cell motility by wounding (B) and Boyden chamber (C) assays, respectively. *P<0.01 vs control; **P<0.01 vs IL-6 treatment alone.

gp130/STAT-3 Signaling Mediates Balloon Injury–Induced Cyclin D1 Expression, SMC Motility, and Neointima Formation
To relate the role of IL-6/gp130/STAT-3 signaling to vascular wall remodeling, we first studied the time course effect of balloon injury on IL-6 expression in rat carotid artery. As shown in Figure 7A and 7B, a 7-fold increase of IL-6 production both at mRNA and protein levels was observed 3 days after balloon injury. Increased STAT-3 tyrosine phosphorylation was observed in injured arteries compared with uninjured arteries and adenovirus-mediated transduction of dngp130 or dnSTAT-3 into injured arteries significantly decreased this effect (Figure 7C). Similarly, increased cyclin D1 expression was observed at both mRNA and protein levels at day 3 in balloon-injured arteries compared with uninjured arteries, and these responses were suppressed by both dngp130 and dnSTAT-3 (Figure 7D and 7E). In addition, coimmunofluorescence staining revealed that balloon injury–induced cyclin D1 expression is colocalized with SMC -actin but not with CD45, and dngp130 and dnSTAT-3 significantly diminished this colocalization (Figure 7F). This result indicates that balloon injury–induced and gp130/STAT-3–mediated cyclin D1 expression occurs in SMCs rather than in inflammatory cells. Peak aortic SMC proliferation occurs in the medium between the second and third days after balloon injury^33,34 and is followed by the migration of SMCs to intima. Intimal SMC proliferation takes place between the fourth and seventh days after balloon injury.^33,34 Because SMC migration from media to intima is an important factor in the formation of neointima, we next studied the effect of adenovirus-mediated expression of green fluorescent protein (GFP), dngp130, and dnSTAT-3 on balloon injury–induced SMC migration. Compared with the effect of GFP, adenovirus-mediated expression of either dngp130 or dnSTAT-3 inhibited balloon injury–induced SMC migration (Figure 8A). To understand the role of gp130/STAT-3 signaling in restenosis, balloon injury was performed in rat carotid artery and adenovirus expressing GFP, dngp130, or dnSTAT-3 was transduced into injured arteries. Two weeks after the surgery, arteries were isolated and analyzed for either dngp130 and dnSTAT-3 expression or neointima formation. Adenovirus-mediated transduction of dngp130 or dnSTAT-3 into injured arteries resulted in substantial expression of these proteins even 2 weeks after injury (Figure 8B). Adenovirus-mediated expression of either dngp130 or dnSTAT-3 reduced balloon injury–induced neointima formation by 40% (Figure 8C).

View larger version (17K): [in this window] [in a new window]   Figure 7. Balloon injury induces the expression of IL-6, and blockade of gp130/STAT-3 signaling suppresses balloon injury–induced STAT-3 phosphorylation and cyclin D1 expression. At 72 hours or the indicated times after balloon injury, control uninjured and balloon-injured arteries were isolated and either RNA or proteins were extracted. A, One microgram of RNA from control and various times of balloon-injured arteries was analyzed by RT-PCR for IL-6 and GAPDH mRNA levels using their specific primers. B, By ELISA, the IL-6 levels in control and 72-hour balloon-injured aortic tissue extracts were measured. C and E, An equal amount of protein from control and 72-hour balloon-injured Ad-GFP, Ad-dngp130, or Ad-dnSTAT-3–transduced arteries was analyzed by Western blotting for pSTAT-3 (C) and cyclin D1 (E) levels using their specific antibodies. The blots were reprobed with anti–ß-tubulin antibodies for normalization. D, Conditions were the same as in C, except that an equal amount of RNA from each was analyzed by RT-PCR for cyclin D1 and ß-actin mRNA levels as described in the legend of Figure 5A. F, Double-immunofluorescence staining for the indicated molecules was performed 4 days after balloon injury following a standard protocol. *P<0.01 vs uninjured artery.

View larger version (29K): [in this window] [in a new window]   Figure 8. Blockade of gp130/STAT-3 signaling suppresses balloon injury (BI)-induced SMC migration and neointima formation in rat carotid arteries. Soon after balloon injury, the rats received adenovirus expressing GFP, dngp130, or dnSTAT-3 by infusion into the injured arteries. A, For the measurement of SMC migration, 4 days after balloon injury, rats were euthanized and the injured right common carotid arteries and uninjured left common carotid arteries were dissected out, fixed, opened longitudinally, and stained for nucleus using anti-histone antibodies; and the cells in the luminal region were counted. B and C, All of the conditions were the same as in A, except that, 2 weeks after balloon injury, either arteries were isolated and protein was extracted and analyzed by Western blotting for gp130 and STAT-3 levels using their specific antibodies or arteries were fixed, sectioned, and stained with hematoxylin/eosin stain and morphometric analysis performed and the intimal/medial (I/M) ratios were calculated. The bar graphs in A and C represent the quantitative analysis of SMC migration and neointima formation, respectively. *P<0.05 vs Ad-GFP-BI alone (n=6).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The important observations of this report are as follows. (1) IL-6 activated gp130/STAT-3 signaling in VSMCs. (2) Blockade of gp130/STAT-3 signaling inhibited IL-6–induced VSMC migration. (3) IL-6 induced cyclin D1 expression in a gp130/STAT-3–dependent manner in VSMCs. (4) Suppression of cyclin D1 levels by the use of its specific siRNA inhibited IL-6–induced VSMC migration. (5) Vascular injury induced IL-6 expression in a time-dependent manner. (6) Vascular injury also resulted in the activation of gp130/STAT-3 signaling and induction of cyclin D1 expression. (7) Downregulation of gp130/STAT-3 signaling via adenovirus-mediated expression of their respective dominant negative mutants inhibited balloon injury–induced STAT-3 activation, cyclin D1 induction, SMC migration and neointima formation. Earlier studies by us as well as others have suggested that IL-6 stimulates VSMC migration in vitro.^2,4,5 In addition, IL-6 was considered as a risk factor for restenosis because its levels increased in patients with coronary artery angioplasty.¹ Despite this, its relation to proliferative vascular wall diseases, particularly restenosis and the potential mechanisms of its involvement in vascular dysfunctions, were not well understood. In this direction, the present results show that IL-6 stimulates gp130/STAT-3 signaling in the mediation of VSMC migration in vitro. It is also intriguing to note that IL-6 induces cyclin D1 expression via gp130/STAT-3 signaling, resulting in VSMC migration. The other notable observation of the present study is that balloon injury induces the expression of IL-6 to substantial levels in rat carotid artery. This result along with the findings that IL-6 levels were also elevated in patients who have undergone coronary artery angioplasty reinforces the notion that IL-6 is indeed a risk factor for restenosis. A role for IL-6 in restenosis can be further supported by the following observations: (1) STAT-3 phosphorylation and cyclin D1 expression occurred following its production in balloon-injured arteries; and (2) downregulation of gp130/STAT-3 signaling by adenovirus-mediated expression of dngp130 or dnSTAT-3 attenuated balloon injury–induced cyclin D1 expression and neointima formation. Because the balloon injury–induced cyclin D1 expression was predominantly colocalized with SMC -actin, but not with CD45, and dngp130 and dnSTAT-3 diminished this colocalization, it is likely that gp130/STAT-3–mediated cyclin D1 expression occurs in SMC rather than in inflammatory cells in the artery in response to injury. The induction of expression of IL-6 in the artery by injury may also aid an inflammatory response, thereby causing the migration and infiltration of T lymphocytes into the lesion. The presence of T lymphocytes in both atherosclerotic and restenotic lesions has been observed.^35,36 Recent studies have also provided evidence for the role of IL-6/gp130/STAT-3 signaling in inflammation involving T-cell infiltration.¹²

It was shown that IL-6 plays an important role in both cell migration and proliferation.^{2,4,5,13,14,18,37} Although IL-6 via gp130 activates the Jak/STAT and SHP2/Ras/MAPK pathways, the former signaling was shown to be more important for regulation of cell migration.^12,18 The fact that IL-6 induces VSMC migration, and that this response was inhibited by both dngp130 and dnSTAT-3, further highlights the importance of gp130/STAT-3 signaling in the regulation of cell motility, particularly of VSMCs. The gp130/STAT-3 activation also appears to be critical in the migration of SMC from media to intima as blockade of this signaling attenuated SMC motility as well as reduced neointima formation. Recent studies have suggested that cyclin D1 plays a role in the regulation of cell motility.^31,32 In this respect, our results identify cyclin D1 as a target molecule of gp130/STAT-3 signaling in VSMCs mediating IL-6–induced motility. Even more interesting, balloon injury–induced cyclin D1 expression was also suppressed by the blockade of gp130/STAT-3 signaling. These findings, therefore, suggest that IL-6/gp130/STAT-3 signaling via targeting cyclin D1 expression plays a key role in VSMC migration and neointima formation. It has also been reported that targeting the downregulation of cyclin D1 by pharmacological agents inhibits neointima formation.³⁸ The capacity of IL-6 to stimulate cyclin D1 expression in VSMCs with its lack of effect on DNA synthesis further supports that cyclin D1 is involved in IL-6–induced VSMC motility. However, blockade of cyclin D1 levels suppressed PDGF-BB–induced VSMC migration and proliferation (V.K.-S. et al, unpublished observations). Taken together, these findings reveal that cyclin D1 plays a role in both VSMC migration and proliferation. Although the mechanism(s) by which cyclin D1 influences VSMC motility remains speculative, recent reports suggest that it binds to transcriptional-factor–interacting factors such as p300 and inhibits its histone deacetylase activity, facilitating enhanced gene expression.^32,39 Although future studies are required to prove this mechanism, our finding that depletion of cyclin D1 levels diminishes IL-6–induced VSMC motility strongly suggests an additional role for this molecule besides its involvement in cell-cycle regulation. IL-6 has also been reported to induce the expression of monocyte chemotactic protein-1 (MCP-1) in VSMCs involving Jak/STAT signaling.⁴⁰ Because MCP-1 is a potent chemoattractant for VSMCs,⁴¹ it is possible that besides cyclin D1, IL-6 via activation of gp130/STAT-3 signaling targets other genes such as MCP-1 in the stimulation of VSMC motility and neointima formation.

In summary, the present findings show for the first time that IL-6/gp130/STAT-3 signaling plays a crucial role in vascular wall remodeling in response to injury.

	Acknowledgments

 
Sources of Funding

This work was supported by NIH grant HL69908 (to G.N.R.).

Disclosures

None.

	Footnotes

 
Original received August 25, 2006; revision received January 10, 2007; accepted January 12, 2007.

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