In recent years, balloon angioplasty (BA) has beenperformed for coarctation of the aorta (CoA) and itseffectiveness has been reported.1–5 However, restenosisfollowing BA has also been reported and the long-termeffects of BA are not necessarily ideal. The restenosis ratefollowing BA is 50% or more and it is common in the nativeCoA; 3,4,6–8 it is thought that the tissues at the origin of theductus arteriosus (DA) play a part in restenosis.2,4,6,9

However, the exact mechanisms responsible for restenosisafter BA remain uncertain. In 1991, Kuro-o et al. clarifiedthe existence of three isoforms of the smooth muscle myosinheavy chain (MCH).10 These isoforms are detected in bloodvessel disorders, such as arteriosclerosis and post-percutaneous transluminal coronary angioplasty (PTCA)

changes,10–12 and they are pathologic markers of the smoothmuscle cell (SMC) activity. These isoforms are SM1, whichis a unique marker for SMC1, SM2, which is also themarker for a highly differentiated type of SMC, and SMemb,which is an undifferentiated non-muscle-type of MHC. TheSM2 appears in the DA and the umbilical artery during thefetal and noenatal period13–15 and may be a marker for tissuecellular organization of the DA. In the present study, wesurgically created a coarctation of the aorta (CoA) model inthe DA area surrounding the aorta in a newborn pig. Weexamined the transformation of SMC phenotypes usingimmunohistologic techniques and investigated the cause ofrestenosis following BA.

Methods

Animal preparation

We used a newborn pig model of CoA. Fourteen male andtwo female pigs were used (10–14-days-old; 2.4–4.1 kg).

Pediatrics International (2000) 42, 658–667

Original Article

Restenosis after balloon angioplasty of coarctation: Relationshipwith ductus arteriosus

KEN TAKAHASHI,1 TOSHIHIRO INO,2 MATAICHI OHKUBO,1 KATSUMI AKIMOTO1 ANDMASAHIKO KISHIROU1

Departments of Pediatrics, 1Juntendo University School of Medicine, Tokyo and 2Kameda MedicalCenter, Japan

Abstract BBaacckkggrroouunndd: Recently, balloon angioplasty (BA) has been used for the treatment of coarctation of the aorta(CoA) and the effectiveness of this treatment has been reported. However, the restenosis rate following BA innative CoA in the infant is high and the cause may be related to tissue properties at the origin of the ductusarteriosus (DA). However, the mechanisms responsible for restenosis remain uncertain.MMeetthhooddss//RReessuullttss: The present study was designed to examine transformation of the smooth muscle cell(SMC) phenotypes using immunohistologic studies and to investigate the cause of restenosis of CoA followingBA. A CoA model was surgically created in 15 newborn pigs (10–14-days-old; 2.4–4.1 kg). Balloon angio-plasty was performed 1 month after the initial operation. One or 3 months after BA, animals were killed andimmunohistologic studies on smooth muscle cell (SMC) antibodies against SM1, SM2 and SMemb of themyosin heavy chain (MHC) isoform were performed in the aorta at the CoA and DA. In the neointima, onlySMemb was positive. In the SMC layer of the DA, only SM2 was positive. One month after BA, the externallayer of the tunica media was strongly positive for SM2 only in the area around the origin of the DA.CCoonncclluussiioonnss: The first cause of restenosis is obstructive neointimal formation caused by the proliferation ofundifferentiated SMC into the subendothelial tissue. This proliferation seems to be triggered by BA. Thedistribution of SM2 1 month after balloon angioplasty showed a similar pattern of proliferation of SMC inthe external layer around the DA. This may represent a second mechanism of restenosis.

Key words balloon angioplasty, coarctation, ductus arteriosus, myosin heavy chain, smooth muscle cell.

Correspondence: Mataichi Ohkubo, Department of Pediatrics,Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo, Japan. Email: mohkubo@jane1.med.juntendo.ac.jp

Received 12 November 1999; revision 11 April 2000, accepted21 April 2000.

Piglets were anesthetized with the inhalation of isofluraneand underwent endotracheal intubation with assisted venti-lation using room air. Piglets then underwent left thoraco-tomy and the juxtaductal descending aorta was dissected freefor 3–5 cm. An absorbable suture of 4–0 Dexon or 5–0Maxon (American Cyanamid Co., Wayne, NJ, USA) waslooped around the DA side and a CoA was artificially formed.The pressure gradient was measured by direct puncture ofthe aorta above and below the CoA. We sought to producean intra-operative systolic gradient of 40–50 mmHg. Intra-muscular cefmetazon (500 mg/day) was given for 3 daysafter creation of the CoA model.

Groups

Piglets with successful creation of a CoA were separatedinto two groups. There were six piglets in each group andthey were killed either 1 or 3 months after BA. As a controlgroup, a stenosis was formed in three piglets using the sameprocedure to the aorta across the DA but BA was notperformed; two of these pigs were killed 1 month after BAand the other one was killed 3 months after BA. In anothercontrol study, a stenosis was formed to the abdominal aortausing the same procedure and the pig was killed 1 monthafter BA.

Dilation technique

One month after the creation of the CoA model, BA wasperformed under anesthesia (inhaled isoflurane). In brief, theright femoral artery was exposed and ligated distally and an8 Fr sheath was inserted. Pigs were anticoagulated with100 U/kg heparin. At each catheterization, single-planecutfilm aortograms were performed in the lateral positionusing an 8 Fr Berman angiocatheter (Goodtec Inc.,Huntington Beach, CA, USA) to inject contrast medium at a dose of 1–1.5 mL/kg over 1–2 s. After determining thelateral angiographic diameter of the CoA, a NuMED Ballooncatheter (Canada Inc., Ontario, Canada) was used for BA.The balloon diameter used was selected on the basis of thediameter of the aorta immediately after the branch of a leftcarotid artery. When an insufficient result was seen, anotherballoon sized 1 mm larger was chosen. We used 6–8 atminflation pressure. Repeat aortography was recorded in thesame way as before BA. Experimental animals were killedeither 1 or 3 months after BA. Angiography was performedin order to determine the existence of restenosis and afterthis dissection was done.

Sample preparation and histologic study

Pigs were killed by a lethal dose of sodium pentobarbital(120 mg/kg) 1 or 3 months after BA. Aortic specimens

consisted of the CoA site, 3 cm of aorta both proximally anddistally and the renal artery. Specimens were fixed inmethanol–Carnoy’s fixative (60% methanol, 30% chloroformand 10% glacial acetic acid), embedded in paraffin andsectioned in the routine manner for immunohistochemistry11

and light-microscopic examination, and were then stained bythe hematoxylin–eosin and elastica van Gieson methods.

Transmission electron microscopy

The cross-sections for transmission electron microscopy(EM) were fixed in 5% glutaraldehyde in 0.1 mol/Lphosphatate buffer (pH 7.4). Sections were cut at 0.5 mmand were stained with toluidine blue to isolate selected areasfor ultrathin sectioning. Sections were mounted on coppergrids and stained with uranyl acetate and lead cirate.Photomicroraphs were taken with a H7100 transmissionelectron microscope (Hitachi, Tokyo, Japan) at 75 keV.

Immunohistochemistry

For identification of SMC differentiation, MHC isoforms(SM1, SM2 and SMemb) were immunostained on paraffin-embedded sections. Fixed specimens were embedded withparaffin and sectioned in 4 µm slices. After deparaffinizationand rehydration, immunoenzymatic staining was performedaccording to the ABC method with the Dako ABC kit(Vector Laboratories Inc., Burlingame, CT, USA). Sectionswere pre-incubated with 3% hydrogen peroxide and normalhorse serum to reduce non-specific reactions. Monoclonalantibodies against human SM1, SM2 and SMemb (YamasaCorp., Tokyo, Japan) were diluted 1:500, 1:200 and 1:1500,respectively, and then applied and incubated for 60 min atroom temperature. Sections were incubated with biotinylatedantimouse goat immunoglobulin for 30 min and thenincubated with ABC reagent for 30 min. Slides were rinsedin Tris-buffered saline. Peroxidase activity was revealed withthe use of 3,3′-diaminobenzidine tetrahydrochloride withhydrogen peroxide (0.014%). Sections were counterstainedwith methylene blue, dehydrated and mounted. In order toevaluate the distribution of each phenotype in the aortictunica media, the rate of SM1-, SM2- and SMemb-positivereactions were evaluated using immunohistologic studies.Because the distribution of SM2 differed markedly in theinternal and the external layers of the tunica media,evaluation was divided into the internal and external layersand was conducted at the level of the CoA area andabdominal aorta. The run of the tunica media SMC wasassumed to be concentric. Each specimen was judgedpositive or negative under ×200 magnification at the crossingpoint of the smooth muscle fiber and a straight line that runsthrough the center of each field to become the diameter of

Restenosis after balloon angioplasty 659

the aorta. The calculation of the positive rate was made bydividing the number of muscular fibers that were positive forthe immune staining by the total number of muscularfibers/100. An arbitrary number of ten views of eachspecimen were examined.

All specimens were reviewed and the rates of immuno-staining were graded by two independent observers whowere blinded to other interpretations.

Immunoblotting for MHC isoforms

Abdominal arterial samples were frozen in liquid nitrogenand stored at –80°C until studied for western blotting. TheMHC polypeptides were separated by 7.5% sodium dodecylsulfate (SDS)–polyacrylamide gel electrophoresis as describedby Fukumoto et al.16 Immunoblotting was performed usingantibodies to human MHC isoforms SM1, SM2 and SMemb.The gel was incubated with a horseradish peroxidase-labeledanti-Ig secondary antibody at a 1:1500 dilution for 1 h,washed five times and developed for 10 s in an enhancedchemiluminescence (ECL) kit from Amersham (USA).

Data analysis

Results are presented as the mean~SD for normallydistributed variables. Comparisons between data were madeby paired t-test and comparisons between groups by analysisof variance. The level of statistical significance was set atP<0.05.

All experimentation and animal handling was conductedin a manner designed to minimize stress and discomfort tothe animals and conformed to the guidelines of the AnimalResearch Committee at Juntendo University.

Results

Histologic study 1 month after BA

Under optical microscopic examination, the fissure was seento reach the internal elastic membrane and the tunica media.The SMC proliferated from the tunica media into theendothelial cells. Then they formed the neointima. Undertransmission EM examination, cells in the neointima hadlarge nuclei; cell organelles, such as rough-surfaced endo-plasmic reticulum, Golgi apparatus and mitochondria, wereabundant and the number of myofilaments was decreased.These cells corresponded to a synthetic-type of SMC andexisted in large numbers. In the external layer of the tunicamedia, many cells contained few cell organelles andabundant myofilaments with a small nucleus, and thiscorresponded to a contractile-type of cell (Fig. 1).

Three months after BA

Under optical microscopy, the fissure was not seen and therewas a slight thickening of the residual neointima. Undertransmission EM, cells with abundant mitochondria andrough-surfaced endoplasmic reticulum were seen in theneointima, but they were not as abundant as they were 1month after BA. In the external layer of the tunica media,the nucleus of cells was small, an abundance of myofila-ments was seen and there were many cells with few cellorganelles. This was almost the same as 1 month after BA(Fig. 1).

Immunostaining for SMC MHC isoforms

In the neointima 1 month after BA, SMemb was positive,but SM1 and SM2 were negative. In the tunica media, inboth the internal and external layers, approximately half thecells were positive for SM1 and most cells were positive forSMemb. A similar pattern was seen in all cases in the areanear the DA and the abdominal aorta. There was a strongpositive tendency for SM2 in the external layer of the tunicamedia, but only in the area around the DA in specimens inwhich BA had been performed. In all specimens, some SMCwere positive for SM2 in the abdominal aorta and, in thecontrol specimens, the area around the DA was positive(Figs 2,3).

Three months after BA, SMemb was strongly positiveand SM1 and SM2 were negative in the newborn intima.Both SM1 and SMemb in the smooth muscle layers of thetunica media (external and internal layers) showed similarresults to those seen 1 month after BA. Most cells werepositive for SM2 in both layers after BA as well as in thearea around the DA and the abdominal aorta. In controlcases, similar results were noted (Figs 2,3). The distributionof MHC isoforms did not show the same localization as after1 month. It appeared that changes due to aging were thereason for this. In the DA 1 and 3 months after BA, onlySM2 was positive and SM1 and SMemb were negative(Fig. 4).

We divided the tunica media smooth muscle layer intothe internal and external layers according to the methodsdescribed earlier and made a comparative study of theimmunohistochemistry in these two layers. Differences inSM1 were not seen between the area around the DA and the abdominal aorta or in the external and internal layers ofthe tunica media in the same period. The positive rate wasmuch higher after 1 month compared with 3 months at everylevel. Each control specimen was within the range of theaverage of ~1SD. The external layer of the area around the DA showed higher ~positive rates of SM2 than that ofthe internal layer in comparison with the organization after 1 month (73.6~18.8 vs 25.8~19.5, respectively;

660 K Takahashi et al.

P<0.005). Moreover, the positive rate of the external layerwas higher in the area around the DA than it was in theabdominal aorta (73.6~18.8 vs 18.3~12.2, respectively;P<0.005). In control specimens, the positive rates of SM2were almost the same in the external as in the internallayers, as well as in the area around the DA and theabdominal aorta. They were within the range of the average~1SD (Fig. 5). The effects of BA stimulation on the areaaround the DA was thought to be the cause of the positiverates to SM2 in this area.

Three months after BA, the positive rates of SM2increased overall and no significant difference betweenregions was seen. An increase in the positive rate was

thought to be due to aging. The SMemb showed a highpositive rate in all areas 1 month after BA. Statistically, therewas a significant difference in the internal layer in the areaaround the DA and the abdomen (97.4~1.7 vs 95.8~1.5,respectively; P<0.05), but both cases were almost 100%positive and this difference was thought to be clinicallyunimportant. The positive rate after 3 months decreasedmore compared with that after 1 month and this was thoughtto be due to aging. A significant difference was notedbetween the external and internal layers in the abdominalaorta (84.5~8.7 vs 79.9~8.3, respectively; P<0.05).However, this difference in MHC isoform transformationwas thought to be clinically insignificant (Fig. 5).

Restenosis after balloon angioplasty 661

Fig. 1 Transmission electron microscopy of the (a,c) neointima and (b,d) external layer of the tunica media. In the neointima, both(a) 1 and (b) 3 months after balloon angioplasty (BA), the cells had large nuclei; in addition, rough-surfaced endoplasmic reticulum,Golgi apparatus and mitochondria were abundant. However, the number of myofilaments was decreased. These characteristicscorrespond to a synthetic-type of smooth muscle cell and existed in large numbers. In the external layer of tunica, there were manycells with a small nucleus that contained few cell organelles both (b) 1 and (d) 3 months after BA. These charcteristics correspond to acontractile-type of cell. This was almost the same after BA.

Immunoblotting

To confirm the antigen–antibody reaction results, westernblotting was performed on the abdominal aorta. The SM1appeared at 204 kDa and SM2 and SMemb appeared at200 kDa. The amount of SM1 and SMemb was almost thesame after 1 and 3 months, but the amount of SM2 appearedgreater after 3 months compared with 1 month. These resultswere the same as the immunohistochemistry results and werethought to be caused by aging (Fig. 6).

Discussion

Stenosis from neointima proliferation

Currently, BA is actively performed for the CoA of the aortaafter surgical operation to treat restenosis.5 There has beenmuch research into the mechanism of restenosis followingPTCA following BA. In 1991, Kuro-o et al. clarified theexistence of three types of isoforms of the smooth muscleMHC, namely SM1, SM2 and SMemb.10–12 These MHCisoforms are pathologic markers of SMC activity. They are

classified into SM1, which is a unique marker for SMC,SM2, which is also the marker for a highly differentiatedtype of SMC, and SMemb, which is an undifferentiated non-muscle type of MHC.

In the present study, SMemb was found to be positive inthe neointima 1 and 3 months after BA. In EM examina-tions, cell organelles were noted to be abundant. Thissuggests that the SMC in the neointima were in an activesynthetic state. This is similar to the results reported forrestenosis lesions following PTCA11,17 or in animal experi-mental models of the coronary arteries.18 It is thought thatSM1 is transformed into an undifferentiated type andproliferates to the subendothelium following stimulating byBA and that this is the cause of restenosis lesions of CoA.

Distribution of SM2 and specificity of DA

Because the DA shrinks and closes after birth, its cellularfeatures are different from those of other blood vessels. Theappearance of the MHC isoform of SMC is significant. Asfor other large blood vessels, SM2 is negative in the neonatalperiod and becomes positive with age.16 However, the DA

662 K Takahashi et al.

Fig. 2 Immunohistochemistry for smooth muscle cell antibodies against (a,d) SM1, (b,c) SM2 and (c,f) SMemb in the aorta at thecoarctation of the aorta (CoA) 1 (a–c) and 3 months (d–f) after balloon angioplasty (BA). (a–c) One month after BA, the fissurereached the tunica media. Smooth muscle cells proliferated from the tunica media into the endothelial cells and formed the neointima.In the neointima, only SMemb was positive (c). In the tunica media, approximately half of all cells were positive for SM1 (a) and mostcells were positive for SMemb (c). There was a strong positive tendency for SM2 in the external layer of the tunica media (b). (d–f) Three months after BA, the fissure was not seen and the neointima remained. In the neointima, only SMemb was positive (f),similar to results obtained 1 month after BA. In the tunica media, approximately half of all cells were positive for SM1 (d) and mostcells were positive for SMemb (f). Staining for SM2 showed the same positivity both in the internal and the external layers of thetunica media (e).

SMC is already SM2 positive in the fetal period13–15 and thisis regarded as an indicator of the DA in the fetal period orthe neonatal period. Similarly, in the present study, SM2 wasonly positive in the SMC of the DA. The DA and umbilicalartery share common features that distinguish them fromother vessels. They both close at birth, contract whenexposed to oxygen, contract sensitively to endothelin-1 andare positive for SM2.13,19 Therefore, it is thought possible

that the SM2 is related to the mechanisms of contraction andclosure of these vessels at birth.11,19 Moreover, the SM2-positive cells appear significantly in the early stages ofdevelopment of organs such as the bladder and intestines,which receive stimulation more than other organs regardingsmooth muscle other than blood vessels.14 It is presumedthat SM2-positive cells are related to strong contractility, asmentioned above.

Restenosis after balloon angioplasty 663

Fig. 3 Immunohistochemistry for smooth muscle cell antibodies against SM2 in the aorta at (a,d) the coarctation of the aorta (CoA)and (c,f) the abdominal aorta 1 (a–c) and 3 months (d–f) after balloon angioplasty (BA) and in control cases (b,e). There was strongpositivity for SM2 in the external layer of the tunica media at the CoA 1 month after BA (a). However, in control cases (b,e), SM2positivity was not different between the internal and external layers. Results for SM2 were more positive at 3 months (d–f) than1 month (a–c) after BA and there was no difference between the internal and the external layers in all cases. This was thought to be theresult of aging.

Fig. 4 Immunohistochemistry for smooth muscle (SM) cell antibodies against (a) SM1, (b) SM2 and (c) SMemb in a cross-section ofthe ductus arteriosus 3 months after balloon angioplasty (BA). Only staining for SM2 was positive, while results for SM1 and SMembwere almost negative.

Restenosis in native coarctation following BA in infants

In contrast, restenosis in typical CoA following BAspecifically in infants was expected as the cause, differingfrom that seen after PTCA. To explain the cause of CoA,many theories concerning DA cellular organization havebeen advocated. For example, there is Hutchin’s Branchpointtheory.20 Other theories, such as the theory of Monma et al.on hypoplasia in the isthmus area and shrinkage of the DA,21

and the theory of the existence of a DA cellular organization(seen at aorta rebuilding operations)22,23 have also beenproposed. Moreover, the diameter of the stenosis before theprocedure,24 the size of the hypoplasia in the narrowregion,2,9,25 balloon size6,26,27 and the degree of cystic medialnecrosis8 are all risk factors for restenosis following BA forCoA. However, age has a great influence on restenosis rates.For the neonatal period, the restenosis rate reaches80%,2,3,4,9,28 by 3 months of age it is 50%4 and at less than

664 K Takahashi et al.

Fig. 5 The positive rate ofsmooth muscle (SM) cell anti-bodies against (a,b) SM1, (c,d)SM2 and (e,f) SM3 1 (a,c,e) and3 months (b,d,f) after balloonangioplasty (BA). The SM1 wasmore positive 1 month after BA(a) compared with 3 monthsafter BA (b) in all areas; SM2was more positive in the externallayer in the media at thecoarctation of the aorta (CoA),for which BA was performed,than in the internal layer or atabdominal aorta 1 month afterBA (c). Three months after BA,the positive rates of SM2increased overall (d) comparedwith 1 month after BA. TheSMemb showed high positiverates in all areas 1 (e) and 3months (f) after BA and wasmore positive at 3 monthscompared with 1 month afterBA. The reason for thedifferences at 1 and 3 monthsafter BA in SM1 and SMembwas thought to be the effect ofaging. (d), BA was performed;(m) control (stenosis wasformed to the area around theductus arteriosus, but BA wasnot performed); (×) control(stenosis was formed to theabdominal aorta and BA wasperformed).

1 year it is 40%.3 Balloon angioplasty of the restenosis aftersurgical treatment gives similar results related to age and thefrequency of restenosis is high.6 The participation of the DAcellular organization is important in the restenosis processafter BA and in aortic remodeling.2,6,9 There is a possibilitythat the brisk activity of the DA cellular organization isrelated to the frequency of restenosis caused by aging.4 Inthe present experiments, SM2 became strongly positive inthe external layer of the tunica media of the area around theDA 1 month after BA. However, control animals showedalmost equal positive rates between the external and internallayers of the tunica media in the areas around the DA andabdominal aorta. Although SM2-positive cells did not enterin continuously from the DA juncture to the aortic tunicamedia smooth muscle layer in this experiment, these resultsshow that DA-type cellular oganization or similar SMC existin the external layer of the tunica media around the DA andthat there is a possibility that they have been transformed toSM2 by BA. The SM2-positive cells are possibly linked tostrong contractility, as described.14,19 When BA is performedon native CoA, the possible occurrence of restenosisfollowing BA may be due to the contractility of the SMCwhen transformed into SM2-positive cells. Three months

after BA, the positive rate of SM2 had increased uniformlyin all specimens, including control specimens. The positiverate showed no significant difference between the externaland internal layers of the tunica media or between theabdominal aorta and the area around the DA. The SM2-positive rate increases with aging in both humans andanimals.11,12 Pigs increased their weight to 30 kg (average) inthe 3 months after BA. With this weight gain, the systolicblood pressure increases from an average of 65 mmHg 1month after BA to 120 mmHg 3 months after BA. There arereports that SM2 is superior to SM1 in contractility14,19 andthat SM2 may be a cause of blood pressure elevation.

Distribution of SM1 and SMemb in the tunica media

The SM1 does not show a characteristic distribution likeSM2 and differences at 1 and 3 months in SM1 positivitywere not seen. However, the positive rate after 3 monthsdecreased overall compared with 1 month after BA and thischange was thought to be due to the transformation to SM2,which accompanies aging. The SMemb became stronglypositive (as mentioned above) in the neointima. In the tunicamedia smooth muscle layer, the positive rate was higher

Restenosis after balloon angioplasty 665

Fig. 6 Immunoblotting for smooth muscle (SM) cell antibodies against SM1, SM2 and SMemb. Compared with 1 month afterballoon angioplasty (BA), the amount of SM2 seen 3 months after BA was markedly increased, whereas the amount of SM1 andSMemb was unchanged.

overall after 1 month. Statistically, it was calculated that thepositive rate of the internal layer of the tunica media of thearea around the DA is significantly lower than the abdominalaorta, but both rates were close to 100%. Therefore, thisdifference was considered to be clinically insignificant. Thepositive rate 3 months after BA decreased overall comparedwith 1 month after BA. It is thought that the transformationto a mature-type of the SM2 phenotype with aging is similarin both humans and animals.11,12

Study limitations

In these experiments we created a coarctation model in thenewborn pig and conducted BA experiments. A restenosismodel was formed at the isthmus of the CoA of the aortaregion and involved the DA cellular organization. However,like clinical CoA, the involvement of the DA cellularorganization did not form the stenosis. Therefore, this didnot accurately reproduce the clinical reaction after BA toCoA. However, it was thought that because currently noCoA animal model exists, this was the best method. Becauseit was necessary to perform an operation and BA during theneonatal period, we selected pigs that would grow toapproximately 3 kg 2 weeks after birth. We used anti-humanMHC antibodies to perform histoimmunologic examinations.Because we were able to obtain consistent results fromimmunohistologic studies and immunoblots, these results areconsidered significant.

Acknowledgments

We thank Dr K Nishimoto (Juntendo University School ofMedicine) for cooperation in this study, Mrs Y Kojima, Mr A Furuhata and Mr Y Sueyoshi for the excellenttechnical assistance and Mr S Ichinose, Mr T Tsukada andMr T Kanai for animal care. Finally, we acknowledgeProfessor Y Yamashiro of Juntendo University School ofMedicine.

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