Chondrocyte apoptosis plays an important role in cartilage degeneration in osteoarthritis (OA), and mechanical injury to cartilage induces chondrocyte apoptosis. In response to DNA damage, p53 expression is up-regulated, transcription activity is increased, and apoptosis signals are initiated. The p53-regulated apoptosis-inducing protein 1 (p53AIP-1) is one of the p53-regulated genes, and is activated in response to DNA damage. This study was undertaken to analyze p53 function after induction of apoptosis by shear strain in chondrocytes.
OA cartilage samples were obtained from subjects undergoing total knee replacement surgery, and normal cartilage samples were obtained from subjects undergoing surgery for femoral neck fracture. Chondrocytes were isolated from human cartilage and cultured. Expression of p53 and p53AIP in chondrocytes was detected by reverse transcriptase–polymerase chain reaction and Western blotting. Shear strain was introduced in normal human knee chondrocytes. To explore p53 function, normal human knee chondrocytes were pretreated with pifithrin-α or p53 small interfering RNA (siRNA) before induction of shear strain. Chondrocyte apoptosis was detected by expression of cleaved caspase 9 with Western blotting and TUNEL staining. Expression of p53 and p53AIP-1 was analyzed by Western blotting.
OA and normal chondrocytes expressed p53. OA chondrocytes showed much higher expression of p53 and p53AIP-1 than did normal chondrocytes. TUNEL-positive cells and expression of p53, p53AIP-1, and cleaved caspase 9 were increased by shear strain, but chondrocyte apoptosis was suppressed after pretreatment with pifithrin-α or p53 siRNA.
Our findings indicate that p53 and p53AIP-1 play important roles in human chondrocyte apoptosis. Down-regulation of p53 expression prevents cartilage from undergoing apoptosis introduced by shear strain.
Osteoarthritis (OA) is the most common degenerative disease of human articular cartilage, especially in the population older than 65 years (1). It is characterized by extracellular matrix damage and an important loss in tissue cellularity (2). Apoptosis, or programmed cell death, is a physiologic process for maintaining homeostasis in both embryogenic and adult tissue, including articular cartilage. Apoptosis may also play a role in diseases involving articular cartilage degeneration, such as OA (3).
It has been reported that chondrocytes undergo apoptosis in response to wounding or injurious compression (4). Chondrocyte apoptosis and necrosis have been reported in response to mature and immature bovine cartilage wounding, after injurious loading of calf cartilage explants, and after a transient injury in adult bovine cartilage (5). In analysis of human cartilage explants, it has been shown that mechanical injury induces chondrocyte apoptosis and the release of glycosaminoglycan from the matrix (6).
In the joint, chondrocytes are exposed to either dilatational (compressive hydrostatic pressure) or deviatoric stresses such as shear strain (7). Intermittent hydrostatic pressure increased aggrecan and type II collagen gene expression in normal chondrocytes, and induced changes in cell-associated protein in normal and OA chondrocytes (8). Intermittent hydrostatic pressure has been shown to induce apoptosis in vitro in a load- and time-dependent manner and is characterized by a loss of chondrocyte viability, internucleosomal DNA fragmentation, and activation of caspases (9). Shear strain in OA chondrocytes decreased aggrecan and type II collagen expression (7). Such molecular changes induced apoptosis with increasing membranous phosphatidylserine and nucleosomal degradation (7, 10).
Meanwhile, p53 has been identified as a 53-kd cellular protein and ultimate tumor suppressor gene (11). It is recognized as a pivotal regulatory protein that responds to a variety of signals and recruits an array of biochemical activities to trigger diverse biologic responses, most notably cell cycle arrest and apoptosis (12). Apoptosis in human chondrocytes from OA cartilage induced by hydrostatic pressure was accompanied by expression of p53 (9).
A newly identified molecule, p53-regulated apoptosis-inducing protein 1 (p53AIP-1), is a potential mediator of p53-dependent apoptosis and is induced by Ser-46-phosphorylated p53 (13). The molecule p53AIP-1 is expressed only when p53-dependent apoptosis is induced (14). Overexpression of p53AIP-1, a novel p53 target that mediates p53-dependent apoptosis, can result in p53 gene activation. In addition, p53AIP-1 promotes the release of cytochrome c, which forms an essential part of the apoptosome composed of Apaf-1 and procaspase 9 (15). In the early stage of p53 activation after DNA damage, phosphorylation of Ser-15 and some other sites occurs, which in turn promotes binding of p53 to promoters of cell cycle arrest genes, DNA repair genes, and other genes. However, if DNA damage is severe and repair is impossible, Ser-46 kinase is activated, leading to phosphorylation of Ser-46, a subtle change of p53 conformation, and a stronger affinity for p53AIP-1 (13). The presence of p53AIP-1 therefore represents actual cell apoptosis in response to severe DNA damage, such as DNA fragmentation.
Nitric oxide (NO), a gaseous free radical, is an important inducer of apoptosis and is synthesized by inducible nitric oxide synthase (iNOS). NO has been shown to induce chondrocyte apoptosis with the activation of p53 and caspase 3 in rabbit articular cartilage (16). Sodium nitroprusside, one of the NO donors, induced chondrocyte apoptosis with DNA fragmentation and production of reactive oxygen species (ROS) (17).
Lee et al (18) reported that cilostazol, which inhibits iNOS synthesis, prevented chondrocyte apoptosis and cartilage destruction with the inhibition of p53 phosphorylation in an experimental model of OA. Interleukin-1β (IL-1β), a proinflammatory cytokine, induced production of ROS and apoptosis in chondrocytes with the accumulation of p53 in vitro. Resveratrol, an anti-p53 activation agent, has been shown to inhibit IL-1β–induced apoptosis through inhibiting ROS production and release (19).
Therefore, we speculated that p53 and p53AIP-1 might play an important role in DNA damage–induced apoptosis of articular cartilage, and that decreased expression of p53 and p53AIP-1 might contribute to the pathogenesis of OA by down-regulating apoptosis in OA chondrocytes. In this study, we investigated p53 and p53AIP-1 expression in OA cartilage, and analyzed the function of p53 and p53AIP-1 in chondrocyte apoptosis induced by shear strain.
MATERIALS AND METHODS
Isolation and culture of chondrocytes.
Cartilage tissue samples were obtained from 6 patients with OA undergoing total knee replacement surgery. Diagnosis of OA was based on clinical, laboratory, and radiographic evaluations. Normal cartilage tissue samples were obtained from 5 age-matched patients undergoing surgery for femoral neck fracture. These 5 patients had no history of joint disease and had macroscopically normal cartilage. All patients were older than 65 years. All samples were obtained in accordance with the World Medical Association Declaration of Helsinki Ethical Principles for Medical Research Involving Human Subjects. Chondrocytes were isolated from cartilage tissue and cultured. Tissue samples were minced and digested in Dulbecco's modified Eagle's medium (DMEM; Gibco BRL, Grand Island, NY) containing 0.2% collagenase (Sigma, St. Louis, MO) at 37°C for 2 hours. Dissociated cells were cultured in DMEM supplemented with 10% fetal bovine serum (FBS; BioWhittaker, Walkersville, MD) and 100 units/ml of penicillin–streptomycin. After overnight culture, nonadherent cells were removed, and adherent cells were further incubated in fresh medium. All experiments were conducted using first-passage cells. The results of reverse transcriptase–polymerase chain reaction (RT-PCR) demonstrated that all samples produced type II collagen, and that OA chondrocytes synthesized type X collagen.
RT-PCR analysis of chondrocytes.
Chondrocytes were cultured in 6-well plates with various stimulants, and RNA was extracted with QIAshredder and RNeasy Mini kit, according to the recommendations of the manufacturer (Qiagen, Hilden, Germany). One microgram of total RNA was reverse-transcribed to first-strand complementary DNA with 1.25 μM oligo(dT) primer in 40 μl of PCR buffer II containing 2.5 mM MgC12, 0.5 mM dNTP mixture, 0.5 units RNase inhibitor, and 1.25 units murine leukemia virus RT (PerkinElmer, Foster City, CA) at 42°C for 60 minutes. The PCR buffer contained 1.5 mM MgC12, 0.2 mM dNTP mixture, 0.5 μM sense and antisense primers, 1.5 units of AmpliTaq Gold DNA polymerase, and 1 μl of RT reaction mixture in 20 μl of PCR buffer II (PerkinElmer). Thermal cycling conditions for p53 consisted of 35 cycles of denaturation at 95°C for 30 seconds, annealing at 65°C for 60 seconds, and extension at 72°C for 60 seconds. The primers used were 5′-ATGACTGCCATGGAGGAGTCACAG-3′ (sense) and 5′-CTAGCAGTTTGGGTTTCCTCCTTG-3′ (antisense).
Chondrocytes were washed 3 times with phosphate buffered saline and lysed in MOPS buffer (25 mM Tris, 1% Nonidet P40, 150 mM NaCl, 1.5 mM EGTA) supplemented with protease and phosphatase inhibitor mixture (Roche Diagnostics, Basel, Switzerland) on ice for 20 minutes. The lysates were centrifuged at 15,000 revolutions per minute for 20 minutes to remove cellular debris, and the supernatants were collected, and 3× electrophoresis sample buffer (Bio-Rad, Hercules, CA) was added (20).
Western blot analysis.
The concentration of proteins was quantified by the Bradford method with protein assay reagent (Bio-Rad), and diluted to an equal concentration with hypotonic buffer. Each sample was mixed with 3× electrophoresis sample buffer and electrophoresed on 7.5–15% polyacrylamide gradient gel (Biocraft, Tokyo, Japan) and transblotted electrically onto the blotting membrane (GE Healthcare, Buckinghamshire, UK). The expression of p53 protein was detected using mouse anti-human p53 monoclonal antibody (mAb; Santa Cruz Biotechnology, Santa Cruz, CA) and conjugated sheep anti-mouse IgG antibody (GE Healthcare). Phosphorylation of p53 at Ser-15 and Ser-46 (Cell Signaling Technology, Beverly, MA) was detected using rabbit anti-human monoclonal phospho-p53 and horseradish peroxidase (HRP)–conjugated goat anti-rabbit IgG antibody (GE Healthcare). The p53AIP-1 protein was detected using rabbit polyclonal antibody (Santa Cruz Biotechnology) and HRP-conjugated goat anti-rabbit IgG antibody (GE Healthcare) and visualized with ECL Plus reagent (GE Healthcare) with Chemilumino analyzer LAS-3000 mini (Fujifilm, Tokyo, Japan). Ataxia-telangiectasia mutated kinase (ATM) is a serine/threonine kinase that is phosphorylated when cells are exposed to DNA double-strand breaks, which are potent triggers of the DNA damage response (21). ATM phosphorylation indicates that DNA damage occurs following shear strain. The expression of phosphorylated ATM was detected using mouse anti-human phospho-ATM (Ser-1981) mAb (Cell Signaling Technology) and conjugated sheep anti-mouse IgG antibody.
Apoptosis is known to be associated downstream with the sequential activation of caspases. Apoptosis via the p53 pathway is related to the mitochondrial pathway. Apoptotic signals were confirmed by detection of cleaved caspase 9. Cytochrome c released from mitochondria associates with procaspase 9/Apaf-1, and this complex processes procaspase 9 into large and small fragments by self-cleavage under apoptotic stimulation (22). The expression of cleaved caspase 9 indicates the activation of the mitochondrial apoptotic pathway. The expression of cleaved caspase 9 was detected using mouse anti-human caspase 9 mAb (Upstate Biotechnology, Temecula, CA) and conjugated sheep anti-mouse IgG antibody. The expression of p53, p53AIP-1, and cleaved caspase 9 proteins was determined by semiquantitative analysis of a digitally captured image using the public domain NIH Image program (National Institutes of Health, Bethesda, MD; online at: http://rsb.info.nih.gov/nih-image/). Values were normalized to α-tubulin expression.
Culture of normal human knee chondrocytes and exposure to shear strain.
Normal human knee chondrocytes (Cambrex, Charles City, IA) were cultured in a humidified atmosphere of 5% CO2 and 95% air at 37°C in a Bullet kit. Before performing the experiments, we confirmed that normal human knee chondrocytes expressed type II collagen and sulfated proteoglycans but not type X collagen. Cells were grown to a subconfluent state and were then plated onto type I collagen–coated BioFlex plates with a deformable silicone rubber bottom surface at a density of 3 × 105 per well in DMEM/F-12 supplemented with 10% FBS and 100 units/ml of penicillin–streptomycin. Cyclic stretch experiments were performed using an FX-2000 Flexercell system (Flexcell International, McKeesport, PA). Shear strain was enforced at 2%, 5%, and 10% elongation for 6 hours (0.25 Hz), and at 10% elongation for 2, 6, and 12 hours (0.25 Hz). Chondrocytes were maintained in culture for 4 hours during the postloading period to provide time for metabolic changes to occur.
DNA damage was also detected and quantified by comet assay (23), after induction of shear strain at 10% elongation for 12 hours, with Trevigen's CometAssay, according to the recommendations of the manufacturer (Trevigen, Gaithersburg, MD). Briefly, cells were mixed with 1% (volume/volume) low-melting agarose and were immediately placed on a CometSlide (Trevigen). After the gels solidified, the slides with cells were subjected to electrophoresis and stained with 0.1% SYBR Green I. DNA damage was quantified using comet tail analysis. Seventy-five cells were randomly selected for the assay.
Internucleosomal DNA fragmentation.
After induction of shear strain at 10% elongation for 12 hours, internucleosomal DNA fragmentation was determined using a Quick Apoptotic DNA Ladder Detection kit, according to the recommendations of the manufacturer (BioVision, Palo Alto, CA). Briefly, the extracted DNA was concentrated, washed with ethanol, and subjected to 1.2% agarose gel electrophoresis.
Pretreatment of chondrocytes with pifithrin-α prior to shear strain induction.
Pifithrin-α is a chemical compound isolated for its ability to suppress p53-mediated transactivation and its ability to protect cells against p53-mediated apoptosis (24). It has been shown that pifithrin-α blocks p53-mediated apoptosis by suppressing the phosphorylation of p53 at Ser-15 (25). Chondrocytes were pretreated with 50 μM pifithrin-α (Sigma) for 24 hours in DMEM/F-12 medium supplemented with 10% FBS before the induction of loading.
Small interfering RNA (siRNA) transfection.
Lipofectamine 2000 was used to transfect p53 siRNA and nonspecific siRNA control into normal human knee chondrocyte monolayers, according to the recommendations of the manufacturer (Invitrogen, San Diego, CA). Briefly, 1 day before transfection, cells were plated on a 6-well plate in growth medium without antibiotics so that they would be 30–50% confluent at the time of transfection. Then, 100 pmoles of siRNA and Lipofectamine 2000 complexes were prepared and added to each well. After 6 hours of transfection, the complexes were removed and fresh medium containing 10% FBS was added. After 24 hours of transfection, the cells were replaced onto type I collagen–coated BioFlex plates with deformable silicone rubber bottom surfaces at a density of 3 × 105 per well, and shear strain was enforced. Transfection efficiency was determined by RT-PCR and Western blotting.
Before induction of shear strain, 3 × 105 chondrocytes were cultured in a type I collagen–coated BioFlex plate with a deformable silicon rubber surface. After exposure to shear strain, the chondrocytes cultured on the plates were fixed with 4% neutral buffered formalin for 10 minutes, and apoptotic cells were determined using a TUNEL assay kit, according to the recommendations of the manufacturer (Wako, Osaka, Japan).
Data are expressed as the mean ± SD unless otherwise indicated. For normally distributed data, Student's 2-tailed t-test was used for comparisons between the 2 groups.
Expression of p53 messenger RNA (mRNA) and protein in OA and normal chondrocytes.
RT-PCR demonstrated that p53 mRNA was expressed in all individual samples of OA and normal chondrocytes (Figure 1A). Western blotting confirmed that p53 protein was expressed in OA and normal chondrocytes (Figure 1B). Expression of p53 was significantly higher in OA chondrocytes than in normal chondrocytes.
Expression of p53AIP-1 in OA and normal chondrocytes.
Western blotting demonstrated that p53AIP-1 was expressed in all individual samples of OA and normal chondrocytes. Expression of p53AIP-1 was significantly higher in OA chondrocytes than in normal chondrocytes (Figure 1B).
DNA damage following 10% shear strain for 12 hours.
The length of the comet tail was significantly increased in chondrocytes after exposure to 10% shear strain for 12 hours, compared with that in nonloaded chondrocytes (P < 0.05) (Figure 2A). DNA fragmentation was not detected in nonloaded chondrocytes. In contrast, it was detected after loading shear strain (Figure 2A). Furthermore, phosphorylated ATM was not expressed in nonloaded chondrocytes but was expressed after loading shear strain (Figure 2A). These results indicated that DNA damage was induced by shear strain.
Chondrocyte apoptosis after exposure to 2%, 5%, and 10% shear strain.
The percentage of TUNEL-positive apoptotic cells significantly increased in a force-dependent manner after loading shear strain for 6 hours (P < 0.05) (Figure 2B). Expression of p53 increased in a force-dependent manner after loading 2%, 5%, and 10% elongation shear strain for 6 hours (Figure 2C). Phosphorylation of p53 at both Ser-15 and Ser-46 was increased after loading shear strain compared with nonloaded samples (Figure 2C). In nonloaded chondrocytes, p53AIP-1 was not detected; however, it was detected after loading shear strain. Cleaved caspase 9 was increased in a force-dependent manner (Figure 2C). Relative expression of cleaved caspase 9 increased in a force-dependent manner, as shown by analysis with NIH Image software (Figure 2D).
Chondrocyte apoptosis after exposure to 10% shear strain for 2, 6, and 12 hours.
The percentage of TUNEL-positive apoptotic cells significantly increased in a time-dependent manner after loading 10% elongation strain (P < 0.05) (Figure 3A). Western blotting revealed that expression of p53 increased after loading 10% elongation shear strain for 2, 6, and 12 hours (Figure 3B). Phosphorylation of p53 at Ser-15 and Ser-46 increased in samples after loading shear strain compared with nonloaded samples (Figure 3B). In nonloaded chondrocytes, p53AIP-1 was not detected; however, it was detected after loading shear strain. Expression of cleaved caspase 9 increased in a time-dependent manner after exposure to 10% elongation strain (Figure 3B). Relative expression of cleaved caspase 9 also increased in a time-dependent manner, as shown by analysis using NIH Image software (Figure 3C).
Chondrocyte apoptosis after exposure to 10% shear strain following treatment with a p53 inhibitor.
The percentage of TUNEL-positive apoptotic cells was decreased in chondrocytes pretreated with pifithrin-α (50 μM) for 24 hours before loading 10% shear strain, compared with untreated samples (Figure 4A). Expression of p53 and p53AIP-1 was still detected by Western blotting when chondrocytes were pretreated with 50 μM pifithrin-α. Pifithrin-α inhibited the phosphorylation of p53 at Ser-15 in treated chondrocytes compared with untreated samples (the latter shown in Figure 3). However, pifithrin-α did not inhibit the phosphorylation of p53 at Ser-46 compared with untreated samples. Expression of cleaved caspase 9 was decreased compared with that in untreated samples (Figure 4B). Relative expression of cleaved caspase 9, analyzed using NIH Image software, was also decreased compared with that in untreated samples (Figure 4C).
Effect of p53 siRNA.
RT-PCR showed that expression of p53 mRNA was significantly inhibited after 48 hours of transfection with p53 siRNA (Figure 5A). Western blotting confirmed that the expression of p53 protein also decreased (Figure 5B). The p53 expression was inhibited to ∼14.9% of control by siRNA transfection.
Chondrocyte apoptosis after p53 siRNA transfection and exposure to 10% shear strain.
The cyclic stretch experiments demonstrated that the percentage of TUNEL-positive apoptotic cells induced by shear strain was significantly decreased when p53 siRNA was transfected (Figure 6A). Western blotting showed that p53, phosphorylation of p53 at Ser-15 and Ser-46, and p53AIP-1 expression decreased when p53 siRNA was transfected. Cleaved caspase 9 expression was also decreased compared with that in samples transfected with nonspecific control siRNA (Figure 6B). Relative expression of cleaved caspase 9, analyzed using NIH Image software, significantly decreased (Figure 6C).
In recent years, many authors have shown that the rate of chondrocyte apoptosis is increased in OA cartilage and speculated on the role of apoptosis in the pathogenesis of OA (26), suggesting possible targets for new therapeutic strategies. Excessive mechanical stress in articular cartilage has been implicated as a contributing factor in the pathogenesis of OA (27). Thus, modulation of the mechanism mediated by substances inducing apoptosis is being considered as a novel strategy for the treatment of OA.
Meanwhile, several studies have analyzed the expression of p53 in cartilage, and its correlation with the presence of apoptotic cells (28–30). In this study, we confirmed that p53 is expressed in OA and normal chondrocytes, and demonstrated for the first time that expression of p53AIP-1 in OA chondrocytes was much higher than in normal chondrocytes. These results suggest that p53 and p53AIP-1 may play important roles in the onset or progression of OA.
Mechanical stimuli represent important regulators of chondrocyte function and induce mediators of inflammation (31–33). Certain types of mechanical injury can also induce chondrocyte death. In animal models, and in human cartilage injuries, compression causes chondrocyte death (4, 34). In a previous study, shear strain induced molecular changes associated with apoptosis whereas hydrostatic pressure increased matrix macromolecule expression (8).
In this study, to investigate DNA damage following shear strain, we performed comet assay, internucleosomal DNA fragmentation, and Western blot analysis of phosphorylated ATM. ATM is phosphorylated when cells are exposed to DNA double-strand breaks, which are potent triggers of the DNA damage response (21). ATM phosphorylates a number of proteins, including many tumor suppressors, such as p53, Brca1, and Chk2 (35), and is involved in cell cycle check point control, apoptotic responses, and DNA repair (36). Therefore, these results suggest that shear strain induced DNA damage, and enhanced expression of p53 and p53AIP-1.
Islam et al (9) demonstrated that hydrostatic pressure induces apoptosis and increases p53 mRNA and protein expression in cultured human chondrocytes. In addition, the frequency of ISNEL-positive cells has been shown to correlate with p53 expression in OA and RA cartilage (37). At the molecular level, induction of apoptosis by hydrostatic pressure is characterized by up-regulation of p53, c-Myc, and Bax (8).
In this study, the increase in the percentage of TUNEL-positive apoptotic chondrocytes seen after loading shear strain indicated that the percentages of cells undergoing apoptosis were increased in a force- and time-dependent manner. However, we also considered the possibility that TUNEL staining might be positive for necrotic cells in addition to apoptotic cells. Therefore, apoptosis was confirmed by detecting the cleavage of caspase 9.
Pifithrin-α has been isolated by screening of the chemical library in a cell-based readout system and selected for its ability to reduce p53-dependent transactivation. It has been shown that pifithrin-α can protect cells against p53-mediated apoptosis induced by various stimuli (38). Pifithrin-α analog was effective in protecting neuronal cells against a variety of fatal insults and reducing the side effects of anticancer drugs (39). In a rat model, inhibition of p53 with pifithrin-α effectively protected hepatocytes against endotoxin-induced apoptosis (40). In this study, we demonstrated that phosphorylation of p53 at Ser-15 was suppressed and chondrocyte apoptosis was decreased when chondrocytes were pretreated with pifithrin-α. However, p53, p53AIP-1, and phosphorylation of p53 at Ser-46 were not directly suppressed with pifithrin-α. These results suggest that pifithrin-α actually protects chondrocytes against shear strain–induced apoptosis by inhibition of the phosphorylation of p53 at Ser-46.
Zaman et al (41) demonstrated that systemic administration of proteasome inhibitors, a novel class of anticancer drugs, induced apoptosis of stem-like and proliferative chondrocytes in the growth plate in young mice, and that proteasome inhibition–induced apoptosis was associated with the up-regulation of p53 and apoptosis-inducing factor. In a rat chondrogenic cell line, suppression of p53 with siRNA partly rescued chondrocytes from proteasome inhibition–induced apoptosis (41).
In this study, we demonstrated that expression of p53 and p53AIP-1 increased with the activation of apoptotic processes in normal human chondrocytes following exposure to shear strain. However, pifithrin-α reduced chondrocyte apoptosis after loading shear strain. Furthermore, down-regulation of endogenous p53 by transfection of p53 siRNA inhibited chondrocyte apoptosis after loading shear strain. Our results suggest that shear strain induces apoptosis via p53 and the mitochondrial pathway.
In conclusion, we have demonstrated that p53 and p53AIP-1 are important molecules in chondrocyte apoptosis induced by elongation shear strain, and that down-regulation of exogenous or endogenous p53 expression inhibits chondrocyte apoptosis. Thus, down-regulation of p53 may be a therapeutic target for OA.
All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published. Dr. Nishiyama had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.
Study conception and design. Hashimoto, Nishiyama, Hayashi.
Acquisition of data. Hashimoto, Nishiyama, Hayashi, Takebe.
Analysis and interpretation of data. Hashimoto, Nishiyama, Hayashi, Fujishiro, Takebe, Kanzaki, Kuroda, Kurosaka.
We thank Ms Kyoko Tanaka, Ms Minako Nagata, Ms Masako Sakaguchi, and Ms Maya Yasuda for technical assistance and Janina Tubby for English language assistance.