Local Overexpression of Toll-Like Receptors at the Vessel Wall Induces Atherosclerotic Lesion Formation
Synergism of TLR2 and TLR4
Objective— Atherosclerosis is now considered as a chronic inflammatory disease, and inflammation is closely related to immune systems, which consist of innate-immunity and adaptive-immunity. Recently, toll-like receptors (TLRs) have been identified as key components of innate-immunity. We examined the role of local expressions of TLRs at the vessel wall in atherosclerosis.
Methods and Results— We transfected cDNA encoding human TLR2 and TLR4 into the carotid arterial vessel wall of rabbits fed high-cholesterol diets with the use of HVJ-liposome. The rabbits were transfected with (1) pCMV-β-gal, (2) empty vector, (3) TLR2, (4) TLR4, (5) TLR2+4. X-gal staining and immunohistochemical analysis showed that the transfected plasmids were mainly expressed in the media. Neither TLR2 nor TLR4 transfection induced significant augmentation of atherosclerosis. Transfection of TLR2- and TLR4-containing HVJ synergistically accelerated atherosclerosis and increased expressions of vascular cell adhesion molecule 1, intercellular adhesion molecule 1, and MCP-1. Moreover, transfection of TLR2 and TLR4 resulted in synergistic activation of NF-κB at the vessel wall in vivo, and in vascular smooth muscle cells in vitro.
Conclusions— Expressions of both TLR2 and TLR4 at the vessel wall synergistically accelerated atherosclerosis. The present study revealed the role of TLRs expressed locally at the vessel wall in the early stage of atherosclerosis.
Atherosclerosis is now considered as a chronic inflammatory disease, and inflammation is closely related to immune systems.1,2 The immune systems contributing to atherosclerosis consist of the innate-immunity and the adaptive-immunity. The innate-immunity is the first line of the immune defense system which is based on detections of pathogen-associated molecular patterns. Recently, a family of Toll-like receptors (TLRs) has been identified as a key recognition component of innate-immunity.3,4 TLRs are originally identified as receptors that activate host defenses in response to microbial-derived ligands such as Gram-negative bacterial lipopolysaccharide (LPS).5
There are growing evidences showing the contribution of the TLR-signaling pathway to initiation and progression of atherosclerosis.6,7 In the advanced atherosclerotic lesions in human, TLR1, TLR2, and TLR4 were detected in not only infiltrating macrophages but also vascular cells such as endothelial cells and vascular smooth muscle cells, and they might contribute to plaque activation.8,9 The role of TLRs and their common downstream signaling adaptor molecule myeloid differentiation factor 88 (MyD88) in atherosclerosis have been demonstrated by use of gene-engineering mice.6 Deficiency of either TLR4 gene or MyD88 gene was shown to reduce atherosclerotic lesion formation in mice model of atherosclerosis.7 Recently Mullick et al demonstrated the importance of TLR2 expressions in non–bone marrow–origin cells such as endothelial cells on lesion formation by use of bone marrow transplantation in low-density lipoprotein receptor knock-out mice deficient in TLR2.10 It is, however, still not well clarified yet regarding the role of TLRs expressed locally at the vessel wall in atherosclerosis. Both TLR2 and TLR4 are expressed in atherosclerotic lesion, and TLRs use common downstream signaling pathways via MyD88. Therefore it is likely that the presence of two TLRs, TLR2 and TLR4, may synergistically be involved in the initiation and progression of atherosclerosis.
In this study, to clarify the contribution of the local expressions of TLR2 and TLR4 at the vessel wall to the initiation of atherosclerosis, we transfected plasmids containing TLR2 and TLR4 to the vessel wall of rabbits treated with the high-cholesterol diets. Interestingly, we demonstrated the synergistic effect of TLR2 and TLR4 overexpression on the initiation of atherosclerosis.
Plasmid Vector and Preparation of HVJ-Liposomes Containing Plasmids
Human TLR2 and TLR4 cDNA were generous gifts from Dr Shizuo Akira (Department of Host Defense, Research Institute for Microbial Diseases, Osaka University, Japan). The plasmids were grown in Escherichia coli and prepared with a Plasmid Mega Kit (Qiagen). In the present study, pFLAG-CMV without any expression insert was used as the control vector, and pCMV-β-gal plasmid (Promega) was used to confirm the expression of the injected plasmids. We used a high-efficiency transfection method with hemagglutinating virus of Japan (HVJ)-coated liposomes (Ishihara Industry, Japan). We prepared the HVJ-liposome complexes, containing pCMV-β-gal (200 μg), human TLR2 (200 μg), human TLR4 (200 μg), human TLR2+TLR4 (200 μg each), or the control vector.
Male Japanese white rabbits were purchased from a breeder (SLC, Japan) and kept under conventional conditions in our animal facilities. Rabbits were divided into the following 5 groups: (1) those transfected with pCMV-β-gal, (2) those transfected with pFLAG-CMV without any expression insert, (3) those transfected with TLR2-HVJ, (4) those transfected with TLR4-HVJ, (5) those transfected with TLR2- and TLR4-HVJ. High cholesterol diets (containing 1% cholesterol) were started 2 weeks before the plasmids transfections and continued for the following 1 or 2 weeks until rabbits were euthanized. All animal experiments were conducted according to the Guidelines for Animal Experiments at Kobe University Graduated School of Medicine.
Surgical Procedure and Gene Transfer to Vessel Wall
The transfections were performed with the animals anesthetized by intravenous administration of 0.05 mg/g sodium pentobarbital. The left carotid artery was displayed widely by the median section of the neck. Blood flow was clamped transiently with artery clips. The common carotid artery was filled with HVJ-liposomes through a 24G catheter (TERUMO, Japan) inserted into the external carotid artery and incubated for 15 minutes, as previously described.11 After the incubation, the external carotid artery was ligated at the orifice. The blood flow to the common and internal carotid arteries was restored by releasing the clips, and the wound was closed.
Plasma Lipid Analysis
Two weeks after the plasmid transfection, venous blood samplings for measurements of plasma lipid level were performed. Plasma total cholesterol, HDL cholesterol, and triglyceride levels were measured by the enzymatic methods.
We examined the expressions of transfected β-gal at the vessel wall 2 weeks after the transfection according to the method described previously.12 Briefly, the sections were incubated at 37°C for 12 hours in PBS supplemented with 1 mg/mL X-gal (Sigma), 5 mmol/L potassium ferricyanide, 5 mmol/L potassium ferrocyanide, and 2 mmol/L MgCl2.
Histological and Immunohistochemical Analysis
Rabbits were euthanized 1 or 2 weeks after the plasmids injection, and the whole length of the left common carotid artery was removed. The middle portion of the common carotid artery was excised, embedded in OCT compound (Sakura, Japan), frozen in liquid nitrogen (LN2), and stored in −80°C. Serial 5-μm-thick cryosections of the carotid artery were provided for histological and immunohistochemical analysis. The neointimal areas of 3 sections were measured by the image processing software (Image-J) with a blinded manner, and the average data were used for n=1. Anti-human TLR2 (H-175) and TLR4 (M-16) antibodies were commercially obtained (Santa Cruz). An anti-rabbit monocyte/macrophage antibody (RAM-11) and an anti-human smooth muscle actin antibody (1A4) were also commercially obtained (DAKO). For the immunohistochemical staining with RAM-11 or 1A4 antibodies, the carotid arteries were immersion-fixed with the Bouin fixative. Anti-rabbit intracellular adhesion molecule-1 (ICAM-1) and VCAM-1 (VCAM-1) monoclonal antibodies (Rb2/3 and Rb1/9, respectively) were kindly provided by Dr M. Cybulsky (University of Toronto, Canada). The anti-rabbit MCP-1 monoclonal antibody13 was used for detection of rabbit MCP-1. Biotinylated anti-mouse and anti-goat antibodies (DAKO) were used for the secondary antibodies. The incubation with streptavidin-peroxidase was followed by the addition of the substrate 3′3′-diaminobenzidine. Collagen content in the sections was stained with Masson trichome.
NF-κB Electrophoretic Mobility Shift Assay
Rabbits were euthanized 1 week after the injection of plasmids, with or without intravenous injection of LPS (Escherichia coli O111:B4 LPS, 50 μg/kg, Sigma). In LPS-injected animals, we obtained carotid arteries 6 hours after the LPS injection. Nuclear proteins were isolated from the carotid arteries by use of ProteoExtract Subcellular Proteome Extraction Kit according to the manufacture’s instructions (Calbiochem).
Binding reactions were performed with 8 μg nuclear protein and 32P-labeled consensus oligonucleotide in a buffer containing 10 mmol/L HEPES (pH 7.9), 50 mmol/L KCl, 0.1 mmol/L EDTA, 2.5 mmol/L DTT, 10% glycerol, 0.05% NP40, 0.05 mg/mL poly (dI-dC) (Amersham) with or without a 25-fold excess of cold competitor oligonucleotide. The DNA complex was separated on a 4% polyacrylamide gel. The consensus oligonucleotide contained the NF-κB binding site was commercially obtained (Promega).
NF-κB-Luc Reporter Gene Assay
The NF-κB–dependent luciferase reporter plasmid, 3 NF-κB sites in tandem ligated into XhoI site of the pGL3pro plasmid, was kindly provided by Dr F. Kambe (Nagoya University, Japan).14 Rat vascular smooth muscle cells (VSMCs) were cultured in 60-mm dishes with 10% fetal bovine serum (FBS) containing Dulbecco’s modified eagle’s medium (DMEM, Sigma). We changed the culture medium to 2% FBS containing DMEM 2 hours before the transfection. VSMCs were cotransfected with the indicated plasmids using a FuGENE 6 reagent (Roche). We made 6 experimental groups: (1) VSMCs transfected with 1.0 μg of NF-κB-Luc plasmids alone (without any TLRs); (2) VSMCs transfected with 1.0 μg of NF-κB-Luc and 0.5 μg of TLR2 plasmids; (3) VSMCs transfected with 1.0 μg of NF-κB-Luc and 1.0 μg of TLR2 plasmids; (4) VSMCs transfected with 1.0 μg of NF-κB-Luc and 0.5 μg of TLR4 plasmids; (5)VSMCs transfected with 1.0 μg of NF-κB-Luc and 1.0 μg of TLR4 plasmids; (6) VSMCs transfected with 1.0 μg of NF-κB-Luc and 0.5 μg of TLR2 and 0.5 μg of TLR4 plasmids. β-galactosidase–expressing plasmids (0.5 μg, pCMV-β-gal plasmid, Promega) were also transfected to monitor the efficiency of transfection. Variable amounts of pFLAG-CMV were used to adjust total DNA amounts. Twenty-four hours after the transfection, the culture medium was changed to 10% FBS containing DMEM, 1 μg/mL human recombinant HSP60 (Sigma) containing DMEM, 1 μg/mL LPS (Escherichia coli O111:B4 LPS, Sigma) containing DMEM, or both 0.1 μg/mL K-235 LPS (Escherichia coli K-235 LPS, Sigma) and 0.1 μg/mL P. gingivalis LPS (Porphyromonas gingivalis LPS, InvivoGen) containing DMEM. The stimulation period was set to 12 hours for K235 LPS and P. gingivalis containing DMEM, and 30 hours for the others. After the stimulation, cells were harvested with a reporter lysis buffer (Promega). The luciferase activity in the cell lysates was assayed with the PicaGene Luciferase assay system (TOYO Inc, Japan), and the β-galactosidase activities were assayed with the β-galactosidase enzyme assay system (Promega).
Data were expressed as mean±SE. The significance of the difference between group means was analyzed by 1-way ANOVA followed by post hoc tests (PRISM 4.0, GraphPad). Values of P<0.05 were considered statistically significant.
Administration of the high-cholesterol diet to rabbits increased plasma total cholesterol levels in group 2, 3, 4, and 5, and there were no differences in lipid profiles among 4 groups (supplemental Table I, available online at http://atvb.ahajournals.org).
The Transfected Plasmids Were Expressed Mainly in the Media
In the group 1, we transfected the pCMV-β-gal containing HVJ to the common carotid arteries of rabbits. Histological studies of the transfected side of carotid arteries were performed two weeks after the transfection. As shown in supplemental Figure I, the serial sections were investigated with HE-staining (left), and anti-alpha smooth actin (1A4) immunohistochemistry (middle). To detect where the transfected plasmids were expressed, we performed X-gal staining (right). We confirmed that the injected pCMV-β-gal was still expressed 2 weeks after the transfection. Figure 1A shows TLR2 expressions and Figure 1B demonstrates TLR4 expressions in the carotid arteries of controls (without any HVJ injection), control-HVJ injected models, TLR2-HVJ injected models, TLR4-HVJ injected models, and TLR2+4-HVJ injected models. These samples were obtained 1 week after the each HVJ injection. The control carotid arteries (high-cholesterol–fed, without any HVJ injection) did not express TLR2, whereas expressed TLR4 in the endothelium. Immunohistochemical analysis showed the expressions of TLR2 proteins in the endothelium, media, and adventitia in TLR2-HVJ–injected models. The expressions of TLR4 proteins in the endothelium and the media were augmented in TLR4-HVJ–injected models. In TLR2+4-HVJ–injected models, the expressions of TLR2 and TLR4 were very vicinal in the media (arrowheads in Figure 1A and 1B).
Local Expression of TLR2 and TLR4 Synergistically Accelerated Atherosclerotic Lesion Formation
As shown in Figure 2, at 2 weeks after the plasmid transfection, the transfection of TLR2-HVJ or TLR4-HVJ alone induced a trend toward acceleration of atherosclerotic lesion formation, but it was statistically insignificant (0.03±0.01 mm2 in empty vector-treated, 0.06±0.02 mm2 in TLR2-treated, and 0.08±0.02 mm2 in TLR4-treated, not significant). On the other hand, we found that the transfection of TLR2- and TLR4-containing HVJ synergistically induced marked acceleration of atherosclerotic lesion formation (0.25±0.05 mm2 in TLR2 and 4-treated: P<0.05 compared with other groups). Either HVJ containing 400 μg of TLR2 alone or those containing 400 μg of TLR4 alone did not induce statistically significant acceleration of atherosclerotic process (data not shown). To reveal the main components of atherosclerotic lesions, histological investigations were performed in the arteries obtained 2 weeks after the transfection. As shown in supplemental Figure II, the atherosclerotic lesions were widely stained by anti-human smooth muscle actin antibody (1A4) and anti-rabbit macrophage antibody (RAM-11). Masson trichome staining showed broad collagen deposition in the atherosclerotic lesions. Therefore the atherosclerotic lesions were mainly consisted of vascular smooth muscle cells, macrophages, and collagen. These contents of atherosclerotic lesions were not different among these groups.
TLR2 and TLR4 Transfection Synergistically Accelerates the Expression of Adhesion Molecules in the Arteries
Leukocyte-endothelial cell adhesion is one of the key early events in the process of atherosclerosis. Therefore, immunohistochemical analysis of adhesion molecules was performed in the arteries obtained 1 week after the plasmids transfection. As shown in Figure 3A and 3B, the transfection of TLR2-HVJ alone or TLR4-HVJ alone slightly augmented the expression of ICAM-1 in the endothelium and that of VCAM-1 in the media, respectively. We found that the transfection of TLR2- and TLR4-containing HVJ further augmented the expression of ICAM-1 and VCAM-1. Then we examined immunohistochemical analysis of MCP-1, a key molecule for macrophage recruitment to the lesion. As shown in Figure 3C, the transfection of TLR2-HVJ alone or TLR4-HVJ alone had little effect on the expression of MCP-1. The transfection of TLR2- and TLR4-containing HVJ, however, synergistically accelerated the expression of MCP-1 in the arterial wall.
Transfection of TLR2 and TLR4 Synergistically Activated the Response of NF-κB in Arterial Wall
To address mechanisms of the augmented expressions of atherosclerosis-related molecules induced by the transfection of TLR2- and TLR4-containig HVJ, we investigated the activation of NF-κB in the vascular tissue. At first, the nuclear proteins were obtained from the plasmid-injected side of the carotid artery without LPS injection, but we could not detect any NF-κB shift bands. Therefore, we obtained carotid arteries 6 hours after the O111:B4 LPS injection. As shown in Figure 4, the NF-κB activation induced by the O111:B4 LPS stimulation was negligible in either vascular tissue from the TLR2-HVJ transfected rabbits (lane 2) or that from the TLR4-HVJ transfected rabbits (lane 3). However, as shown in the lane 4, transfection of TLR2- and TLR4-containing HVJ induced a marked activation of NF-κB in the arterial wall.
Transfection of TLR2 and TLR4 Synergistically Activated the NF-κB Activation in Vascular Smooth Muscle Cells Under Stimulation With Serum and LPS
To further confirm the synergistic activation of NF-κB by TLR2 and TLR4 observed in vivo, we transfected rat vascular smooth muscle cells (VSMCs) with the NF-κB-Luc reporter plasmid and TLR2 or TLR4 plasmids. As shown in Figure 5A, serum significantly activated the NF-κB through TLR2 and TLR4. Cotransfection of TLR2 (0.5 μg) and TLR4 (0.5 μg) to VSMCs induced significantly higher serum-stimulated NF-κB activity compared with TLR2 (1.0 μg)-transfected or TLR4 (1.0 μg)-transfected VSMCs. HSP60 (Figure 5B) also activated NF-κB through TLR2 and TLR4. We found the dose-dependent relations between the NF-κB activity stimulated by HSP60 and the transfected doses of TLR2 and TLR4. O111:B4 LPS (Figure 5C) significantly activated NF-κB via TLR2 and TLR4. Under O111:B4 LPS stimulation, co-transfection of TLR2 (0.5 μg) and TLR4 (0.5 μg) to VSMCs induced significant higher NF-κB activity compared with TLR2 (1.0 μg)-transfected or TLR4 (1.0 μg)-transfected VSMCs. Neither stimulation of TLR4-transfected VSMCs with P. gingivalis LPS nor TLR2-transfected VSMCs with K-235 LPS induced noticeable changes in NF-κB activity. Stimulation with K-235 LPS and P. gingivalis LPS (Figure 5D) significantly activated NF-κB via TLR2 and TLR4, respectively. Under costimulation with K-235 LPS and P. gingivalis LPS, TLR2 and TLR4 cotransfected VSMCs showed significantly higher NF-κB activity compared with that in single TLR2 or TLR4 transfected VSMCs. Therefore, cotransfection of TLR2 and TLR4 induced synergistic, not additive, activation of NF-κB in VSMCs when stimulated by serum, O111:B4 LPS, or both of K-235 LPS and P. gingivalis LPS.
In the present study, we investigated the role of TLR2 or TLR4 expressed locally at the vessel wall in atherosclerotic lesion formation. Single transfection of TLR2- or TLR4-HVJ into the vessel wall under hypercholesterolemia had minimum effects on atherosclerotic lesion formation. Surprisingly, the transfection of TLR2- and TLR4-containing HVJ synergistically accelerated atherosclerotic lesion formation in association with augmented expression of adhesion molecules. The present study revealed the role of locally-expressed TLRs in the early stage of atherosclerosis.
In this rabbit model of atherosclerosis, only minimum atherosclerotic changes develop in carotid arteries by the 4-week treatment with the high cholesterol diet.11 Single transfection of either TLR2 or TLR4 into the vessel wall failed to show significant changes in atherosclerotic process, although they induced a trend toward acceleration of lesion formation. This may partly be attributable to an insufficient dose of transfected TLRs, or to the insufficiency of some adapter molecules of TLRs such as MD2 or CD14. The most important finding in the present study was that the cotransfection of TLR2 and TLR4 genes markedly augmented atherosclerotic process. Because TLRs are known to share common downstream signal pathways, it seems possible that individual TLR synergistically augments the downstream outcomes. Indeed, the synergistic effects of TLRs on cytokine production from macrophages and on contraction of air-way smooth muscle cells have been reported.15,16 Our study demonstrated the synergistic effects of TLR2 and TLR4 on atherosclerotic lesion formation.
There is growing evidence supporting the proatherosclerotic effects of TLR4. Vink et al showed that application of a LPS-containing cuff around the femoral artery resulted in augmentation of neointima formation in mice.17 This effect was likely mediated via TLR4, because the neointima was markedly thinner in mice deficient in TLR4 than in wild-type mice. Therefore TLR4 was involved in the neointima formation, which was mainly composed of VSMCs. In our model, the atherosclerotic lesion was also mainly composed of VSMCs.
On the other hand, there has been only limited information on the role of TLR2 in the process of atherosclerosis. Although not all studies support the expression of TLR2 in atherosclerotic vessels, we found that not only TLR4 but also TLR2 was expressed in VSMCs of atherosclerotic samples obtained by directional coronary angiotomy (DCA).18 From the present results, it is intriguing to assume that signals mediated by locally-expressed TLR2 may serve to augment the proatherosclerotic effects mediated by TLR4 at the vessel wall.
Human vascular smooth muscle cells are reported to express functional TLR2 and TLR4 signal complexes linked to chemokine and proinflammatory cytokine release.19 The expressions of TLR2 and TLR4 in vascular smooth muscle cells were augmented in the atherosclerotic lesions.18 Therefore, in our study, the overexpression of TLR2 and TLR4 mainly in vascular smooth muscle cells contributed to the process of atherosclerosis.
As the mechanism responsible for the augmented atherosclerotic lesion formation by cotransfection of TLR2- and TLR4-containing HVJ, we spotlighted the NF-κB–mediated signaling pathway. The NF-κB–mediated signaling pathway is the main downstream signal transduction pathway from TLRs including both TLR2 and TLR4. NF-κB regulates gene expressions of a number of key molecules in atherogenesis. We therefore investigated the activation of NF-κB in vivo and in vitro. In the experiment in vivo, NF-κB was markedly activated by O111:B4 LPS injection in the carotid arteries transfected with TLR2- and TLR4-containig HVJ. O111:B4 LPS was reported to contain both TLR4 and TLR2 agonists.20,21 In our model, the transfection of neither TLR2-HVJ nor TLR4-HVJ alone was enough to induce a noticeable activation of NF-κB in the arterial samples. Thus, the local transfection of TLR2- and TLR4-containing HVJ synergistically augmented the NF-κB activation induced by TLR2 and TLR4 ligands. Then we demonstrated the synergistic augmentation of NF-κB activation in VSMCs when both of TLR2 and TLR4 were cotransfected and stimulated by serum, O111:B4 LPS, or both of K-235 LPS and P. gingivalis LPS. K-235 LPS was reported as TLR4-specific LPS, and P. gingivalis LPS as TLR2-specific LPS.22 It is possible that this synergistic NF-κB activation served to accelerate atherosclerosis in the carotid arteries transfected with TLR2- and TLR4-containing HVJ. Further, we demonstrated the synergistically-augmented expressions of ICAM-1, VCAM-1, and MCP-1, which are induced via the NF-κB–mediated signaling pathway and serve as the key molecules in the process of atherosclerosis.
As the limitation of our study, although we have shown the proatherogenic actions of locally-expressed TLRs at the vessel wall, the ligands for TLR activation in vivo were not identified in the present study. It has been revealed that not only exogenous but endogenous ligands can activate TLRs.23–25 These endogenous ligands are mostly produced during tissue damage or stress. As a candidate of such an endogenous factor, we examined the effect of HSP60 stimulation on NF-κB activation in TLR-transfected VSMCs, but the synergistic activation was not detected in the presence of TLR2 and TLR4. Therefore, some factors in serum other than HSP60 might act as endogenous ligands that synergistically stimulated both TLR2 and TLR4. Recently it was reported that hyaluronan fragments (low-molecular-weight hyaluronan) were produced by tissue damage, acted as an endogenous signal and provoked inflammation through the activation of both TLR2 and TLR4.26,27 Although we did not examine the effects of low-molecular-weight hyaluronan, it might be the candidate ligand for the synergistic activation of NF-κB in the presence of TLR2 and TLR4. In addition, it is possible that some mechanisms other than the synergistic activation of NF-κB–mediated signals were also involved in the acceleration of atherosclerotic lesion formation in the vessels cotransfected by TLR2 and TLR4. Further studies are needed.
In conclusion, our study demonstrated that the presence of TLR2 and TLR4 at the vessel wall, particularly in VSMCs, synergistically promotes atherosclerotic process under hypercholesterolemia. We also showed that costimulation of TLR2 and TLR4 induced the synergistic activation of NF-κB at the vessel wall and in VSMCs in vitro, which likely resulted in induction of key molecules of atherosclerosis such as ICAM-1, VCAM-1, and MCP-1. In human atherosclerotic vessels, however, TLR2 and TLR4 may not be expressed in the same cells, but independently expressed in different cells at the vessel wall. In such cases, it is possible that paracrine factors produced by either TLR2-mediated or TLR4-mediated signals act to promote another TLR-mediated signals related to atherosclerosis and lead to acceleration of atherosclerotic lesion formation.
Sources of Funding
This study was supported in part by Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology, Tokyo, Japan.
Original received December 30, 2006; final version accepted August 20, 2007.
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