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Cardiovascular Research 2004 64(1):154-164; doi:10.1016/j.cardiores.2004.06.014
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Copyright © 2004, European Society of Cardiology

Inflammatory signaling pathway containing TRAF6 contributes to neointimal formation via diverse mechanisms

Takuya Miyaharaa,b,c, Hiroyuki Koyama*,a,b,c, Tetsuro Miyatab, Hiroshi Shigematsub, Jun-Ichiro Inoued, Tsuyoshi Takatoc and Hirokazu Nagawab

aDepartment of Vascular Regeneration, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo, Tokyo 113-8655, Japan
bDivision of Vascular Surgery, Department of Surgery, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo, Tokyo 113-8655, Japan
cDivision of Tissue Engineering, The University of Tokyo Hospital, 7-3-1 Hongo, Bunkyo, Tokyo 113-8655, Japan
dDepartment of Oncology, The Institute of Medical Science, The University of Tokyo, 4-6-1, Shiroganedai, Minato, Tokyo 108-0071, Japan

* Corresponding author. 7-3-1 Hongo, Bunkyo, Tokyo 113-8655, Japan. Tel.: +81-3-5800-8653; fax: +81-3-3811-6822. E-mail address: hkoyama-tky{at}umin.ac.jp

Received 15 March 2004; revised 31 May 2004; accepted 15 June 2004


   Abstract
Top
 Abstract
1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 References
 
Objective: The purpose of this study was to investigate the contribution of inflammatory signaling containing tumor necrosis factor receptor-associated factor 6 (TRAF6) to neointimal formation in a balloon injury model of rabbit carotid artery. Methods: Male Japanese white rabbits fed a normal diet were used. We transferred the dominant negative (DN) form of TRAF6 to a rabbit carotid artery that was subjected to balloon injury by in vivo electroporation method, and then evaluated its effect on intimal lesion formation after balloon injury. Results: An expression plasmid vector containing the TRAF6 DN sequence was successfully transferred to arterial wall cells, and its inhibitory effect on inflammatory signaling was confirmed by the marked suppression of nuclear factor-{kappa}B (NF{kappa}B) activity after injury. Morphometric analyses revealed significant inhibition of intimal lesion formation at 7 days after injury. Cell replication and accumulation of macrophages in the media were significantly decreased, and apoptosis was enhanced on day 2. Cell migration to the intima was suppressed on day 4. Extracellular signal-regulated kinase1/2 (ERK1/2) activity at 2 h after injury was also down-regulated. Interestingly, intimal cell replication was significantly blocked when TRAF6 DN was transfected at 7 days after injury. Conclusion: TRAF6 plays important roles in cell replication and migration, besides promotion of inflammatory cell infiltration and suppression of apoptosis.

KEYWORDS Intimal hyperplasia; Inflammatory signaling; Tumor necrosis factor receptor-associated factor 6; Nuclear factor-{kappa}B; Electroporation


   1. Introduction
Top
 Abstract
 1. Introduction
2. Materials and methods
 3. Results
 4. Discussion
 References
 
Multiple lines of evidence support the view that inflammatory reaction is one of the key factors in atherosclerotic lesion formation [1,2]. Previous studies have documented overexpression of the potent immune mediator CD40 and its counterpart CD40 ligand (CD40L) in experimental and human atherosclerotic lesions [3,4]. Moreover, prevention of CD40/CD40L interaction not only diminished the formation and progression of mouse atheroma, but also changed the content of lipid and collagen in established atherosclerotic plaques [4–6]. The cytoplasmic tail of CD40 bears two major signaling domains, namely NH2- and COOH-terminal domains, and CD40 signaling requires adaptor proteins termed tumor necrosis factor receptor-associated factors (TRAFs) [7,8] which bind to these domains. TRAF6 binds to the NH2-terminal cytoplasmic tail of CD40 [9], and then activates nuclear factor-{kappa}B (NF{kappa}B) and extracellular signal-regulated kinase1/2 (ERK1/2) [10]. NF{kappa}B plays an important role in the activation of cytokine and adhesion molecule genes involved in atherosclerosis and lesion development after vascular injury [11]. Recent studies demonstrated that inhibition of NF{kappa}B activity resulted in the prevention of neointimal formation [12,13]. Further, ERK1/2 is activated within minutes after injury of rat carotid artery, and the ERK cascade is also thought to be associated with cell replication in the arterial wall [14].

The purpose of the present study, therefore, was to determine the role of TRAF6 in neointimal formation after balloon injury. We utilized a dominant negative form of TRAF6 (TRAF6 DN) [15] to inhibit the TRAF6-mediated signaling pathway. TRAF6 DN was transferred to the arterial wall by plasmid-based in vivo electroporation. Our data suggested that TRAF6 contributed to neointimal lesion formation after balloon injury through various mechanisms.


   2. Materials and methods
Top
 Abstract
 1. Introduction
 2. Materials and methods
3. Results
 4. Discussion
 References
 
2.1. Plasmid construction
The full coding region of the TRAF6 DN sequence was isolated from pMX-T6mut-puro by EcoRI and NotI digestion and then inserted into the expression plasmid vector containing FLAG tag (pME-FLAG) between the EcoRI and XhoI sites to make pME-FLAG-T6{Delta}RZ5 (TRAF6 DN plasmid), in which the FLAG epitope was tagged in the NH2-terminal of the TRAF6 DN sequence. A deletion mutant that lacks the RING finger and zinc fingers of TRAF6 acts as a dominant negative mutant, since TRAF6 stimulates both ERK and NF{kappa}B activity through its NH2-terminal region containing RING finger and zinc fingers [15]. The plasmid vector, pME-FLAG, which did not contain the TRAF6 DN gene, was used as a control plasmid. All these plasmids were grown in E. coli JM109, and purified using Qiagen Plasmid Mega Kits (Qiagen). In this study, TRAF6 DN plasmid-treated vessels (TRAF6 DN group) were compared with control plasmid-treated vessels (control group) to examine the role of TRAF6 after balloon injury.

2.2. Animals and gene transfer by in vivo electroporation
Male Japanese white rabbits (2.5–3.0 kg, Saitama Rabbitry, Saitama, Japan) fed a normal diet were used. This investigation conformed to the Guide for Care and Use of Laboratory Animals by the US National Institutes of Health (NIH publication No. 85-23, revised 1996). Rabbits were anesthetized by intramuscular injection of xylazine (2.5 mg/kg) and ketamine (50 mg/kg). Plasmid-based gene transfer by in vivo electroporation was performed as described by Matsumoto et al. [16]. Briefly, a midline neck incision was made to expose the carotid artery. After clamping the proximal site of the common carotid artery and the internal carotid artery, plasmid DNA solution at a concentration of 400 µg/ml was instilled into the luminal space of the carotid artery. Electronic pulses were delivered through an electronic pulse generator (Electro Square Porator ECM830, BTX, San Diego, USA) using electrodes at 30 voltage (pulse length: 20 ms, pulse interval: 80 ms, number of pulses: 10). The second pulse was stimulated with opposite polarity. Two segments taken 15–25 and 28–38 mm from the carotid bifurcation were stimulated in the same manner. After declamping, arterial circulation was restored.

To confirm in vivo gene transfer, the TRAF6 DN or control plasmid was transferred to the arterial wall and then identified at 2 days after transfer using a monoclonal antibody against FLAG tag (1:100, ANTI-FLAG M2 Monoclonal Antibody, Sigma). The rabbits were killed at 2 days after gene transfer by injection of an overdose of sodium pentobarbital. After perfusion with lactated Ringer's solution (Otsuka Pharmaceutical) and fixation with 4% phosphate-buffered paraformaldehyde (0.1 mol/l PO4buffer, pH 7.3), the carotid artery was excised and immersed in the same fixative for an additional 1 h. The resected arteries were embedded in paraffin and 4-µm cross-sections were cut. A monoclonal antibody against FLAG tag was applied after enzyme digestion with 0.125% trypsin and blocking with 1% normal goat serum. Subsequent incubation with biotinylated goat anti-mouse IgG (1:100 Vector Laboratories) and an ABC Elite kit (Vector) was performed.

2.3. Balloon injury model
Under the same anesthesia, a 2Fr Fogarty balloon catheter (Baxter Healthcare) was introduced through the external carotid artery. The balloon was inflated and passed through the common carotid artery three times with constant rotation as described previously [14].

2.4. Experimental protocol
The rabbits were killed according to the experimental protocol (Fig. 1A and B). At 2 days after TRAF6 DN plasmid transfer, the carotid artery was subjected to balloon injury, and then the rabbits were killed to measure ERK1/2 activity at 2 h and NF{kappa}B activity at 6 h after balloon injury (Fig. 1A). Medial cell replication, macrophage infiltration, and apoptosis were evaluated at 2 days after balloon injury, SMC migration was observed by scanning electron microscopy (SEM) at 4 days after balloon injury, and neointimal hyperplasia was examined at 7 days after injury (Fig. 1A). To analyze intimal cell replication, gene transfer was performed at 7 days after injury and then the rabbits were killed at 2 days after gene transfer (Fig. 1B).


Figure 1
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  		Fig. 1 Experimental protocol. n, number of animals.

 
After perfusion and fixation as described above, four segments taken 17–19, 22–24, 29–31, and 34–36 mm from the carotid bifurcation were used for each histological study, and six rabbits were used for each experimental group.

2.5. Nuclear extract and NF{kappa}B gel-mobility shift assay
Nuclear extracts were prepared from two rabbits for each sample as described previously [12]. Gel-mobility shift assay was performed using Gel Shift Assay Systems (Promega) according to the manufacturer's instructions. NF{kappa}B consensus oligonucleotides were radiolabeled with [32P]ATP. For each reaction, 10 µg of nuclear extract was incubated with 32P-labeled consensus oligonucleotides for 20 min, and then electrophoresed on 4% acrylamide gel. The gel was dried and exposed to X-ray film.

2.6. Morphometric analysis of intimal hyperplasia
Fixed segments of the resected artery were embedded in paraffin, and 4-µm cross-sections were cut and stained with hematoxylin/eosin. The area of neointima and of media in each section were measured using Image J software (version 1.29) [17], and the ratio of neointimal area to medial area (NI/M ratio) was calculated for evaluation of neointimal formation.

2.7. Cell replication and macrophage infiltration
At 1, 9, and 17 h before the rabbits were killed, each rabbit was injected subcutaneously with 5-bromo-2'-deoxyuridine (BrdU) (25 mg/kg, Boehringer Mannheim). Replicating cells were identified with a monoclonal antibody against BrdU (Bu 20a, DAKO) and counterstained with hematoxylin. The percentage of BrdU-positive cell number to total cell number (BrdU labeling index) in the media or intima was calculated in each section [17].

Macrophage infiltration in the media was evaluated using a monoclonal antibody against rabbit macrophages (1:100, RAM11, DAKO) and counterstaining with hematoxylin. The percentage of immunostained cell number to total cell number (macrophage index) in the media was calculated in each section.

2.8. Smooth muscle cell (SMC) migration
The resected arteries were fixed overnight with phosphate-buffered 2.5% glutaraldehyde/2% paraformaldehyde solution. The opened vessels were prepared as described previously [17] and examined with a Hitachi S-430 scanning electron microscope at 20 kV and a magnification of x 90. The SMC migration from the media to the intima was evaluated by counting cells appeared on luminal surface at 4 days after injury as described previously [18]. Briefly, scanning electron micrographs of arterial luminal surface were taken, and a sheet with a ruled grid was placed over the micrograph. Each square of the grid was 81 mm2. The total area of the specimen and the area occupied by intimal SMCs were determined by counting squares. SMC migration was calculated from the proportion of the intimal area covered by SMCs to total intimal area (SMC migration index). For each section, 12 photomicrographs of fields were taken at random. The average value from 12 fields per section was used for statistical analysis.

2.9. Apoptosis
Both common carotid arteries were harvested from their origin to the carotid bifurcation and cleared of adventitia. DNA was then extracted and analyzed for degradation into oligonucleosomes using the method of Rösl [19]. Briefly, DNA was extracted by overnight incubation of arterial tissues in DNA lysis buffer (20 mmol/l Tris–HCl [pH 7.4], 1% SDS, 5 mmol/l EDTA, and 100 µg/ml proteinase K) at 50 °C, followed by phenol/chloroform extraction. Then, 5 µg of arterial DNA was incubated with 10 µCi [32P]dCTP, cold dNTP except for dCTP, and 10 U Klenow polymerase (Stratagene) for 15 min at 30 °C. Unincorporated nucleotides were removed using a Wizard DNA cleanup system (Promega) according to the manufacturer's instructions. Radiolabeled DNA was electrophoresed on 1.8% agarose gel. The gel was dried and exposed to X-ray film.

Frozen sections were used for in situ end labeling of nicked DNA (four segments per animal). Terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) was used to analyze apoptotic cells according to the manufacturer's specifications (Apoptag, Serologicals). Nuclei were counterstained with hematoxylin. The percentage of TUNEL-positive nuclei to total nuclei in the media was calculated in each section.

2.10. ERK1/2 in-gel kinase assay
A cell lysate of carotid artery was prepared from one rabbit for each sample as described previously [14]. Equal amounts (20 µg of total protein) of each lysate were boiled for 5 min with sample buffer (50 mmol/l Tris [pH 6.8], 10% glycerol, 0.01% bromophenol blue, 1% SDS, and 360 mmol/l 2-mercaptoethanol [final concentration]) and then separated on SDS-PAGE gel containing myelin basic protein (MBP, Sigma) and incubated with [32P]ATP. Gel preparation, kinase reaction, and detection by autoradiography were performed as described by Duff et al. [20].

2.11. Statistical analysis
All values are shown as mean±S.E.M. Differences in the ratio of neointimal area to medial area (NI/M ratio), BrdU labeling index, macrophage index, SMC migration index, and the proportion of TUNEL-positive nuclei in a section in each group were analyzed by unpaired Student's t-test. Data were considered significant at P<0.05. For each histological study, the average value from four segments was used for statistical analysis.


   3. Results
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
4. Discussion
 References
 
3.1. Gene transfer by in vivo electroporation
Immunostaining using anti-FLAG tag antibody showed intense brown staining in the medial and intimal layers of the artery to which the TRAF6 DN or control plasmid had been transferred by in vivo electroporation method (Fig. 2A, B).


Figure 2
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  		Fig. 2 Photomicrographs of (A) TRAF6 DN plasmid-treated vessel and (B) control plasmid-treated vessel stained with anti-FLAG tag antibody. Intense brown staining is detected in the medial and intimal layers of each plasmid-treated vessel. L, lumen; I, intima; M (bar with arrows), media. Bar=100 µm. (C) NF{kappa}B activity determined by gel-mobility shift assay. Nuclear extract was incubated with [32P]ATP end-labeled consensus oligonucleotides. NF{kappa}B activity was inhibited in TRAF6 DN plasmid-treated vessels. NC, negative control (nuclease-free water without HeLa nuclear extract); PC, positive control (nuclease-free water with HeLa nuclear extract); N, normal carotid artery.

 
3.2. In vivo effect after transfer of TRAF6 DN
Activity of NF{kappa}B was detected at 6 h after balloon injury using gel-mobility shift assay. No activity was detected in normal carotid artery, but a marked increase in activity was detected in the control group. In comparison with the control group, NF{kappa}B activity was inhibited in the TRAF6 DN group (Fig. 2C).

3.3. TRAF6 DN prevents neointimal hyperplasia
Neointimal hyperplasia was evaluated at 7 days after balloon injury. A significant decrease in the ratio of neointimal area to medial area (NI/M ratio) was observed in the TRAF6 DN group, as compared with that in the control group (Fig. 3A, B, and C).


Figure 3
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  		Fig. 3 Effect of TRAF6 DN gene transfer on neointimal formation of rabbit carotid arteries after balloon injury. (A) Neointimal area/medial area ratio (NI/M) of TRAF6 DN plasmid-treated vessel was significantly reduced at 7 days after balloon injury (*P<0.01). Values are shown as mean±S.E.M. Photomicrographs of (B) control plasmid-treated vessel and (C) TRAF6 DN plasmid-treated vessel at 7 days after balloon injury. Sections were stained with hematoxylin/eosin. L, lumen; I, intima; M (bar with arrows), media; arrowhead, internal elastic lamina. Bar=100 µm.

 
3.4. TRAF6 DN suppresses cell replication and macrophage infiltration
Given the inhibitory effect of TRAF6 DN on neointimal hyperplasia, we investigated how TRAF6 DN influenced neointimal formation after balloon injury. At 2 days after balloon injury, medial cell replication was evaluated by the BrdU labeling index. The medial BrdU index was significantly lower in the TRAF6 DN group than in the control group (Fig. 4A, B, and C). Intimal cell replication was measured at 9 days after balloon injury. The intimal BrdU index was also significantly lower in the TRAF6 DN group than in the control group (Fig. 4D, E, and F).


Figure 4
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  		Fig. 4 Effect of TRAF6 DN gene transfer on medial (A, B, and C) and intimal cell replication (D, E, and F). (A) Medial BrdU labeling index of TRAF6 DN plasmid-treated vessel was significantly reduced. (D) Intimal BrdU labeling index of TRAF6 DN plasmid-treated vessel was also significantly reduced. Values are shown as mean±S.E.M. (*P<0.01). Photomicrographs of (B) and (E) control plasmid-treated vessels and (C) and (F) TRAF6 DN plasmid-treated vessels. Sections were immunostained with anti-BrdU antibody and counterstained with hematoxylin. BrdU-positive cells stain brown. L, lumen; I, intima; M (bar with arrows), media; arrowhead, internal elastic lamina. Bar=100 µm.

 
Macrophage infiltration was examined at 2 days after balloon injury using anti-macrophage antibody, and was significantly suppressed in the TRAF6 DN group as compared with that in the control group (Fig. 5A, B, and C).


Figure 5
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  		Fig. 5 Effect of TRAF6 DN gene transfer on macrophage infiltration at 2 days after balloon injury. Macrophage infiltration was quantified by the ratio of macrophage number to total cell number (macrophage index). (A) Macrophage index of TRAF6 DN plasmid-treated vessel was significantly reduced (*P<0.01). Values are shown as mean±S.E.M. Photomicrographs of (B) control plasmid-treated vessel and (C) TRAF6 DN plasmid-treated vessel. Sections were immunostained with RAM11 and counterstained with hematoxylin. RAM11-positive cells stain brown. L, lumen; M (bar with arrows), media. Bar=100 µm.

 
3.5. TRAF6 DN inhibits SMC migration
At 4 days after balloon injury, SMC migration was observed by SEM. SMCs were generally arranged in a longitudinal orientation parallel to the direction of blood flow. SMC migration was significantly inhibited in the TRAF6 DN group (Fig. 6A, B, and C).


Figure 6
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  		Fig. 6 Effect of TRAF6 DN gene transfer on SMC migration into intima at 4 days after balloon injury. SMC migration was quantified by scanning electron microscopic survey of the luminal surface. (A) SMC migration of TRAF6 DN plasmid-treated vessel was significantly reduced (*P<0.01). Values are shown as mean±S.E.M. Electron micrographs of (B) control plasmid-treated vessel and (C) TRAF6 DN plasmid-treated vessel. Bar=50 µm.

 
3.6. Effect on apoptosis after balloon injury
To evaluate apoptosis, rabbits were killed at 2 days after balloon injury. The percentage of TUNEL-positive cell number to total medial cell number was significantly increased in the TRAF6 DN group, as compared with that in the control group (Fig. 7A, B, and C). We evaluated apoptosis by another method using detection of DNA fragmentation. In the normal carotid artery, DNA degradation was not detected. In the control group, the same band pattern was observed, while DNA extracted from TRAF6 DN plasmid-treated vessels revealed a smeared pattern, a hallmark of apoptosis (Fig. 7D).


Figure 7
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  		Fig. 7 Effect of TRAF6 DN gene transfer on apoptosis at 2 days after balloon injury. Apoptosis was quantified by TUNEL staining as the ratio of TUNEL-positive nuclei to total nuclei. (A) TUNEL positivity of TRAF6 DN plasmid-treated vessels was significantly reduced (*P<0.01). Values are shown as mean±S.E.M. Photomicrographs of (B) control plasmid-treated vessel and (C) TRAF6 DN plasmid-treated vessel. Sections were immunostained with terminal deoxynucletidyl transferase (TdT) enzyme and counterstained with hematoxylin. TUNEL-positive nuclei stain brown. L, lumen; M (bar with arrows), media. Bar=100 µm. (D) Autoradiographs of DNA gel electrophoretogram revealed that fragmentation into oligonucleotides was more intense in DNA extracted from TRAF6 DN plasmid-treated vessels. DNA was end-labeled with [32P]dATP. L, 100 bp DNA ladder; N, normal carotid artery.

 
3.7. TRAF6 DN blocks ERK1/2 activity
The activity of ERK1/2 was measured at 2 h after balloon injury using an in-gel kinase assay with MBP as the substrate. Scarce activity was detected in normal carotid artery. ERK1/2 activity of two bands at molecular masses of 44 kDa (ERK1) and 42 kDa (ERK2) was higher in the control group, while the activity in the TRAF6 DN group was markedly lower (Fig. 8).


Figure 8
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  		Fig. 8 ERK1/2 activity determined by in-gel kinase assay. Cell lysate from carotid artery was separated on SDS-PAGE gel containing MBP and incubated with [32P]ATP. Phosphorylated MBP was observed at molecular masses of 44 kDa (ERK1) and 42 kDa (ERK2). ERK1/2 activity was inhibited in the TRAF6 DN plasmid-treated vessel. N, normal carotid artery.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
References
 
TRAF6 is a transducer of both NF{kappa}B and ERK1/2 activation [10]. NF{kappa}B activation is significantly involved in neointimal hyperplasia after balloon injury [11–13]. Further, we previously demonstrated that the ERK1/2 pathway regulates replication of medial SMCs after arterial injury [14]. TRAF6 is the only TRAF family member that participates in signal transduction of both the TNF receptor (TNFR) superfamily and the interleukin-1 (IL-1R)/Toll-like receptor (TLR) superfamily [21,22], and TLR4 has also been shown to be involved in neointimal formation [23]. Accordingly, these findings suggest that TRAF6 plays a crucial role in neointimal lesion formation through various mechanisms, and TRAF6-mediated signal transduction could be a new therapeutic target in the treatment of atherosclerotic lesions.

In the present study, we used a plasmid-based electroporation method to transfer TRAF6 DN to arterial wall cells. Immunostaining against FLAG tag, which was inserted into the TRAF6 DN and control plasmid, was performed to confirm that the transferred plasmid was expressed in the rabbit carotid artery. Then, NF{kappa}B activity was measured to confirm the function of the expressed TRAF6 DN. The previous study showed that the gene expression by the plasmid-based electroporation method in rabbit carotid artery was detected only during the first week with a maximum at 2 days after transfer, although the gene transfer efficiency was {approx}20 times as high as that by sole plasmid application [16]. The increase in NF{kappa}B activity after arterial balloon injury was transient (peak at 6 h after injury) [24]. Therefore, in the present study, balloon injury was performed at 2 days after electroporation, and then NF{kappa}B activity was examined at 6 h after balloon injury. We found that the TRAF6 DN plasmid was sufficiently transferred to the vascular wall by in vivo electroporation, and then activation of NF{kappa}B in injured vessels was successfully inhibited by TRAF6 DN, indicating that this strategy could be feasible for prevention of TRAF6-mediated NF{kappa}B activation.

The aim of the present study was to determine whether neointimal hyperplasia after balloon injury could be inhibited by TRAF6 DN. We found that TRAF6 DN inhibited neointimal thickening at 7 days after balloon injury. Intimal lesion formation consists of three predominant components; increase in cell number, cell migration and accumulation of extracellular matrix [25,26]. SMC proliferation and migration across the line of injury have been quantitatively evaluated using tritiated thymidine incorporation and SEM in previous experimental studies [25–27]. After balloon injury, ERK1/2 activity in the media was increased within 1 h, and medial SMC replication was markedly increased with a maximum on day 2, which was followed by significant macrophage and lymphocyte infiltration to the intima. SMC migration from the media to the intima was observed from 4 days after injury, and following these responses SMCs arrived in the intima and replicated until 2 weeks after injury. The time course of apoptosis is considered to parallel the cell proliferation after vascular injury [28], and a previous study reported there was a 25% loss of medial SMCs in rat carotid arteries at 48 h after injury [29]. On the basis of these findings, we used the present protocol of experiments to evaluate each component of intimal lesion formation. Especially, to evaluate medial and intimal cell replication we used the different protocols in which the transferred gene expression effectively acted upon each phase of medial and intimal cell replication, since peak gene expression occurred at 2 days after in vivo electroporation-mediated gene transfer. The present study demonstrated that transfer of TRAF6 DN significantly inhibited medial cell replication, intimal cell replication, cell migration from the media to the intima, and recruitment of macrophages after balloon injury, and further showed that apoptosis in the media was enhanced by TRAF6 DN. Suppression of cell replication and an increase in apoptosis might decrease the cell number in the intima and media, possibly contributing to the inhibition of intimal lesion formation after balloon injury. We considered that inhibition of macrophage infiltration and enhancement of apoptosis were caused by the suppression of NF{kappa}B activation. Previous studies obtained similar findings after the suppression of NF{kappa}B activation, in which transfer of NF{kappa}B decoy or I{kappa}B{alpha} was utilized [12,13].

The findings of the present study indicated two possible mechanisms to explain the prevention of medial cell replication. One is suppression of ERK1/2 signaling, and the other is inhibition of NF{kappa}B activation. The ERK1/2 cascade is located downstream of CD40 signaling, and activation of the ERK1/2 cascade initiates medial cell replication after arterial injury. Indeed, our previous study showed marked activation of ERK1/2 immediately after balloon injury of rat carotid artery, and also demonstrated that PD98059, a selective inhibitor of ERK kinase-1, significantly blocked medial cell replication after injury [14]. Meanwhile, recent studies showed that NF{kappa}B activation was also linked to medial cell replication in the arterial wall. Marra et al. [30,31] suggested that inhibition of NF{kappa}B activity was associated with up-regulation of p21Cip1/Waf1and p27Kip1 levels in human SMCs stimulated with platelet-derived growth factor.

The most interesting finding of the present study is that intimal cell replication was also inhibited by transfer of TRAF6 DN. Surprisingly, there has been little evidence showing how intimal cell replication is promoted after arterial injury. Our previous study demonstrated that intimal cell replication was not controlled by basic fibroblast growth factor or ERK1/2 signaling, which are potent mitogen and mitogenic signal pathways for medial cells [17]. Recent studies, however, have provided a hint as to the mechanisms of intimal cell replication. A clinical trial using a sirolimus-coated stent showed marked prevention of in-stent lesion formation, indicating the possibility that the anti-immune effect of sirolimus decreased cell proliferation in the in-stent lesion [32]. Further, an experimental study showed the association of cell replication and leukocyte accumulation in the intimal lesion after stent implantation in an atherosclerotic artery [33]. These findings suggest a crucial role of the inflammatory reaction in intimal cell replication, and the data of TRAF6 DN in the present study support this speculation.

SMC migration was also inhibited by TRAF6 DN. This might be because inflammatory signaling was suppressed by TRAF6 DN. Inflammatory cells potentially secrete various proteases including matrix metalloproteinases (MMPs), and cell movement in the arterial wall is linked to activation of some proteases [34,35]. Macrophages stimulate other inflammatory cells to secrete proteases, and also potentially secrete proteases themselves [36,37].

In conclusion, the present study showed that the inflammatory signaling pathway containing TRAF6 markedly contributed to intimal lesion formation in an arterial injury model using rabbit carotid artery. Transfer of TRAF6 DN significantly inhibited medial cell replication, intimal cell replication, cell migration, and recruitment of macrophages, and further enhanced apoptosis after balloon injury, which possibly resulted in the significant suppression of the intimal lesion size after injury. These data suggest that the TRAF6-mediated signaling pathway could be a therapeutic target for the treatment of vascular occlusive disease.


   Acknowledgements
 
This study was supported by a Grant-in-Aid for Scientific Research (C) from the Ministry of Education, Science, and Culture of Japan (13671218).


   Notes
 
Time for primary review 14 days


   References
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 References
 

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