Correlation of lung surface area to apoptosis and proliferation in human emphysema

Transcription

1 Eur Repir J 2005; 25: DOI: / CopyrightßERS Joural Ltd 2005 Correlatio of lug urface area to apoptoi ad proliferatio i huma emphyema K. Imai*,#,e, B.A. Mercer #,",e, L.L. Schulma +, J.R. Soett 1 ad J.M. D Armieto #," ABSTRACT: Pulmoary emphyema i aociated with alteratio i matrix protei ad proteae activity. Thee alteratio may be liked to programmed cell death by apoptoi, potetially ifluecig lug architecture ad lug fuctio. To evaluate apoptoi i emphyema, lug tiue wa aalyed from 10 emphyema patiet ad ix idividual without emphyema (ormal). Morphological aalyi revealed alveolar cell i emphyematou lug with covoluted uclei characteritic of apoptoi. DNA fragmetatio wa detected uig termial deoxyucleotide traferae-mediated dutp ick-ed labellig (TUNEL) ad gel electrophorei. TUNEL revealed higher apoptoi i emphyematou tha ormal lug. Marker of apoptoi, icludig active capae-3, proteolytic fragmet of poly (ADP-riboe) polymerae, Bax ad Bad, were detected i emphyematou lug. Liear regreio howed that apoptoi wa iverely correlated with urface area. Emphyematou lug demotrated lower urface area ad icreaed cell proliferatio. There wa o correlatio betwee apoptoi ad proliferatio, uggetig that, although both evet icreae durig emphyema, they are ot i equilibrium, potetially cotributig to reduced lug urface area. I ummary, cell-baed mechaim aociated with emphyematou parechymal damage iclude icreaed apoptoi ad cell proliferatio. Apoptoi correlated with airpace elargemet, upportig epidemiological evidece of the progreive ature of emphyema. Thee data exted the udertadig of cell dyamic ad tructural chage withi the lug durig emphyema pathogeei. KEYWORDS: Capae, cell death, cell proliferatio, morphometry, termial deoxyucleotide traferae-mediated dutp ick-ed labellig A primary hypothei for the pathogeei of pulmoary emphyema i proteae atiproteae imbalace [1, 2]. Thi model ugget that alteratio i level of extracellular matrix (ECM)-degradig ezyme ad their ihibitor lead to a lo of matrix compoet of the lug. The hypothei i upported by tudie demotratig elevated expreio of proteae ad lo of ECM i the emphyematou lug compared with ormal [3 6]. However, the hypothei doe ot elucidate all apect of emphyema pathogeei, uch a the role of latet adeoviral ifectio [7]. It alo doe ot explai the accelerated, detructive ature of emphyema, eve after patiet top mokig [8, 9]. To clarify whether alterative cellular mechaim cotribute to emphyema pathogeei, ivetigator have focued o the role of programmed cell death by apoptoi [10 12]. YOKOHORI et al. [12] detected icreaed apoptoi of alveolar wall epithelial cell (although ot edothelial cell i emphyema lug). KASAHARA ad MATSUSHIMA [11] demotrated apoptoi of alveolar eptal ad edothelial cell accompaied by decreaed expreio of vacular edothelial growth factor (VEGF) i emphyematou lug tiue. Buildig o earlier work, which howed that blockig VEGF receptor igallig-iduced apoptoi ad ubequet emphyema i rat [10], cietit idetified igificatly reduced level of VEGF i the iduced putum of emphyema patiet compared with that of ormal idividual ad patiet with athma [13]. Thee tudie ugget that lo of edothelial cell maiteace factor cotribute to the pecific pathogeei of emphyema. I additio, the role of apoptoi i emphyema ha recetly bee foud to be ot merely correlative, but potetially cauative [14]. Direct itratracheal delivery of active capae-3 led to lo of alveolar epithelia ad emphyema-like tructural chage. Apoptoi AFFILIATIONS *Dept of Biochemitry, Nippo Detal Uiverity, Chiyoda-ku, Tokyo, Japa. # Dept of Medicie, Diviio of Molecular Medicie, " Ititute of Huma Nutritio, + Pulmoary Medicie, ad 1 Dept of Surgery, College of Phyicia ad Surgeo of Columbia Uiverity, New York, NY, USA. e Author cotributed equally to the tudy. CORRESPONDENCE J.M. D Armieto Columbia Uiverity College of Phyicia ad Surgeo 630 Wet 168th Street P&S 9-449, New York NY USA Fax: jmd12@columbia.edu Received: February Accepted after reviio: October SUPPORT STATEMENT Thi tudy wa upported by a Burrough Welcome Fud Career Award (J.M. D Armieto), the Alpha 1 Atitrypi Foudatio (J.M. D Armieto) ad a NIH Graduate Traiig Grat (B.A. Mercer). Europea Repiratory Joural Prit ISSN Olie ISSN VOLUME 25 NUMBER 2 EUROPEAN RESPIRATORY JOURNAL

2 K. IMAI ET AL. APOPTOSIS IN PULMONARY EMPHYSEMA occurred i the abece of iflammatio, demotratig the pecific role of programmed cell death i thi dieae [14]. The preet tudy wa udertake to ae the relatiohip betwee lug cell apoptoi, proliferatio, ad tiue urface area i patiet with pulmoary emphyema. MATERIALS AND METHODS Huma lug ample Huma lug tiue were collected from 16 patiet at New York Columbia Prebyteria Medical Ceter, New York, NY, USA, uder ititutioal guidelie. Te ample were from patiet with pulmoary emphyema: five obtaied from lug traplatatio ad five from lug volume reductio. Patiet had topped mokig o6 moth prior to urgery. Six ormal ample were from accidetal death victim or uued door lug harveted for traplat. For compario, lug tiue were obtaied from patiet with opecific itertitial peumoiti (56), eoiophilic grauloma (53), hypereitivity peumoiti (53) ad lymphagioleiomyomatoi (52). Immuohitochemical taiig for apoptoi ad cell proliferatio Surgically excied tiue were fixed i formali ad paraffi embedded. Cell proliferatio wa aeed by immuotaiig ectio with a atibody to huma proliferatig cell uclear atige (PCNA); cloe PC10; Sigma, St. Loui, MO, USA). Biotiylated ecodary atibody wa detected uig a avidibioti complex (Vector Laboratorie, Burligame, CA, USA). The ratio of PCNA-reactive cell over total cell (proliferatio idex) wa meaured at 640 magificatio. Immuotaiig wa alo performed uig atibodie agait huma Bad ad Bcl-2 (Traductio Laboratorie, Lexigto, KY, USA), ad Bax (PharMige, Sa Diego, CA, USA). Sectio were coutertaied with haematoxyli. Ultratructural aalyi of lug tiue Tramiio electro microcopy wa performed o explated lug tiue fixed i 2.5% glutaraldehyde. Tiue were ried i PBS ad pot-fixed cold i 2% omium tetroxide. Ultra-thi ectio were viewed with a tramiio electro microcope (1200 EX II, 80 KV; Jeol, Sudbyberg, Swede). Morphometric aalyi of airpace elargemet Mea liear itercept, iteral urface area ad fractioal lug volume were calculated i haematoxyli- ad eoi-taied lug ectio (5 mm), accordig to well-etablihed tereology method [15 18]. Digital image were captured uig a Macitoh computer ad Image Pro imagig oftware (Media Cyberetic, Sa Diego, CA, USA). A emitraparet grid of horizotal lie paced at 1-mm iterval wa overlaid oto 10 differet area of each 640 erial lug ectio. Cout of iterectio of thee lie with alveolar wall were ued to determie the iteral urface area. Cout of the umber of time the edpoit of the lie touched a alveolar wall were ued to calculate alveolar fractioal lug volume. Data were collected from 10 radom field per tiue ectio. I itu labellig of DNA cleavage DNA cleavage wa determied uig termial deoxyucleotide traferae (TdT)-mediated dutp ick-ed labellig (TUNEL) aay. Serial lug ectio were ubjected to bioti-treptavidi-horeradih peroxidae or fluorecei labellig of DNA trad break by TUNEL, uig the Dead Ed Colorimetric Apoptoi Detectio Sytem (Promega, Madio, WI, USA), ad the I Situ Cell Death Detectio Kit (Boehriger Maheim, Idiaapoli, IN, USA). A a poitive cotrol, lug ectio were treated with RNAe-free DNAe I (Boehriger Maheim). A a egative cotrol, the TdT ezyme wa omitted i parallel reactio. The ratio of TUNEL-poitive cell to total cell (apoptotic idex) wa meaured from.3,000 parechymal cell of each lug ample at 640 magificatio. Aemet of DNA fragmetatio To aay DNA ladderig, four emphyematou ad three ormal lug ample (100 mg wet weight) were digeted i 1.2 ml digetio buffer (0.1 mg?ml -1 proteiae K, 10 mm Tri-HCl, 0.1 M NaCl, 25 mm ethylee diamie tetra-acetic acid (EDTA), 0.5% odium dodecyl ulphate, ph 8.0). After protei extractio with pheol-chloroform/ioamyl alcohol ad dialyi agait 10 mm Tri-HCl, 1 mm EDTA, ph 8.0, ample were icubated with 1 mg?ml -1 of RNAe A for 1 h at 37 C, ad dialyi wa repeated. DNA (30 mg) wa electrophoreed o 1.4% agaroe gel cotaiig 0.1 mg?ml -1 of ethidium bromide. Oly eve ample were aeed due to tiue lo durig proceig ad DNA purificatio ad/or iadequate tartig material from the remaiig ample. Immuoblottig of lug homogeate Itact ad cleaved capae-3 expreio wa determied i ix emphyematou ad five ormal lug ample homogeied i protei extractio buffer (20 mm Tri-HCl, ph 7.4, 0.15 M NaCl, 0.02% NaN 3, 1% NP-40, 1 mm pheylmethaeulfoyl fluoride, 2 mm N-ethylmaleimide, 10 mg?ml -1 leupepti, 1 mg?ml -1 aprotii, 10 mg?ml -1 peptati A, 1 mm EDTA). Weter blot wa performed o protei (120 mg) uig atibodie to huma capae-3 (cloe C31720; Traductio Laboratorie), capae-3 (cloe 67341A; PharMige), ad proteolytic fragmet of poly (ADP-riboe) polymerae (PARP, cloe G734; Promega), a ubtrate of capae-3. Statitical aalyi Reult of morphometry were aalyed uig a upaired t- tet or oe-way ANOVA. Liear regreio ad Pearo correlatio were ued to ae the relatiohip betwee urface area ad apoptotic ad proliferatio idice. Value for the Pearo correlatio (r) approach a value of +1.0 or -1.0, depedig o whether the factor are directly or iverely related, repectively. Liear regreio value (R 2 ) approach a optimal value of +1.0 ad idicate how well the data fit the regreio curve. Data were preeted a mea SD. A p-value,0.05 wa coidered igificat. RESULTS Cliical feature of patiet Lug tiue wa obtaied from 16 urelated adult. Relevat ad available cliical data ad mokig hitorie of thee idividual are provided i table 1. No differece i age wa detected betwee the two group. c EUROPEAN RESPIRATORY JOURNAL VOLUME 25 NUMBER 2 251

3 APOPTOSIS IN PULMONARY EMPHYSEMA K. IMAI ET AL. TABLE 1 Patiet characteritic Normal Emphyema Subject 6 10 Sex Male 2 5 Female 4 5 Age yr Smokig pack-yr NA FEV1 % pred NA FEV1/FVC % NA DL,CO % NA Data preeted a or mea SD. FEV1: forced expiratory volume i oe ecod; % pred: % predicted; FVC: forced vital capacity; DL,CO: diffuig capacity of the lug for carbo mooxide; NA: iformatio wa ot available for all ormal ample. Lug urface area, mea liear itercept ad fractioal volume fidig Surface area meauremet were made i all lug pecime uig tiue morphometry, ad the quatitative reult are how i table 2. A expected, lug tiue from patiet with emphyema demotrated igificatly lower urface area compared with ormal lug. Lug ectio from emphyema patiet demotrated icreaed mea liear itercept, reduced urface area ad fractioal lug volume compared with ormal lug ectio. Morphological detectio of apoptoi Haematoxyli- ad eoi-taied lug tiue ectio from ormal ad emphyematou lug are demotrated i figure 1. Scattered throughout the emphyematou lug tiue were cell exhibitig covolutio of uclear outlie morphologically characteritic of cell udergoig apoptoi (fig. 1b). Thee uclear chage were ot clutered i focal regio, a i typically ee i tiue ecroi. Apoptotic cell icluded edothelial cell, epithelial cell ad fibroblat. Neutrophil ifiltratio ito the alveolar pace or alveolar epta wa egligible. Biochemical characteriatio of apoptoi I itu detectio of DNA cleavage TUNEL reactio from ormal ad emphyema lug ectio are demotrated i figure 1d h. Both fluorecei TABLE 2 Surface area, mea liear itercept of all alveolar pace (MLI) ad volume of huma lug ample Characteritic Normal Emphyema p-value Surface area mm Volume fractio MLI mm Data preeted a mea SD. Group were compared uig the upaired t-tet. iothiocyaate (FITC) TUNEL for eitivity ad pecificity, ad biotiylated TUNEL reactio for aalyi of apoptotic cell i the cotext of the lug tiue were performed. TUNEL reactio uig FITC-cojugated ucleotide (fig. 1d ad e) exhibited little or o labellig i the ormal lug ( %), while more cell with itee labellig were preet throughout emphyematou tiue ( %; p ). TUNEL aay uig biotiylated ucleotide (fig. 1f, g ad h) howed o labellig i the ormal lug, while emphyema ectio were TUNEL poitive ( %; p,0.01). The higher apoptotic value obtaied with the bioti protocol are likely to be due to a icreaed backgroud, reultig from iflammatory cell peroxidae activity. Throughout the emphyema pecime, both alveolar ad meechymal cell exhibited poitive TUNEL taiig. I additio, everal macrophage-like cell with TUNELpoitive cytoplamic bodie ca be ee, characteritic of phagocytoi of apoptotic cell (fig. 1h). TUNEL aay performed o lug ectio from patiet with diffue itertitial lug dieae were imilar to ormal ample. Aay performed without TdT ezyme howed o reactivity (data ot how). Ultratructural aalyi of emphyema lug cofirmed morphological chage of apoptoi i everal cell type. The mot promiet feature were cytoplamic codeatio ad vacuoliatio, chromati codeatio ad coective tiue degradatio. Electro microcopy (fig. 2a) of emphyema lug tiue how two apoptotic cell i cloe appoitio to a healthy cell. Thee cell demotrate cytoplamic ad uclear codeatio with irregularitie i cell hape, characteritic of apoptoi. The preece of apoptoi i the emphyema lug ample wa ext examied uig biochemical aalyi of DNA ladderig (fig. 2b). Agaroe gel electrophorei of DNA from ormal ample revealed itact high molecular weight pecie. I cotrat, ample E2, from a patiet with the mot cliically ad morphometrically evere emphyema, demotrated the mot exteive ladderig patter with fragmet of,180 baepair. Sample E1, E3, ad E4 came from patiet with le evere emphyema tha E2. Thee ample did ot how ladderig, but oethele did reveal a mear o the gel. Expreio of activated capae-3 ad cleaved PARP i lug homogeate Capae are apartate-directed cyteie proteae with a pivotal role i the executio of apoptoi, ot ecroi. Thu, capae-3 Weter blot aalye of ormal ad emphyematou lug tiue were performed (fig. 3). Pro-capae-3 (32 kda) wa detected with imilar frequecy i ormal ad emphyematou lug homogeate (fig. 3a). Thee fidig were cofirmed with a rabbit polycloal atibody agait capae-3 (data ot how). However, the activated ubuit of capae-3 (p17 ad p12) were detected oly i emphyematou lug homogeate (fig. 3b). I additio, a atibody that pecifically react to the proteolytic fragmet of PARP, a ubtrate of active capae-3, demotrated reactivity i the emphyema lug homogeate but ot i ormal lug (fig. 3c). The additioal bad ruig at 80 kda may repreet further degradatio of PARP. 252 VOLUME 25 NUMBER 2 EUROPEAN RESPIRATORY JOURNAL

4 K. IMAI ET AL. APOPTOSIS IN PULMONARY EMPHYSEMA a) b) c) d) e) f) g) h) FIGURE 1. Morphological examiatio ad termial deoxyucleotide traferae-mediated dutp ick-ed labellig (TUNEL) taiig of huma lug tiue. Haematoxyli ad eoi taiig of ormal (a) ad emphyema (b) lug ectio. The arrow i b) ad c) idetifie uclear pykoi ad covolutio. d, e) Fluorecei iothiocyaate-labelled TUNEL reactio from ormal ad emphyematou lug tiue. f, g) Biotiylated TUNEL reactio from ormal ad emphyematou lug ectio. h) Idetificatio of macrophage-like cell cotaiig TUNEL-poitive material i their cytoplam. Scale bar510 mm. Expreio of apoptoi-aociated protei Bcl-2, Bad, ad Bax i the huma lug The iitiatio ad progreio of apoptoi occur through everal protei of the Bcl-2 family, icludig Bcl-2, Bad, ad Bax [19]. Immuohitochemical taiig of thee protei i ormal ad emphyema lug tiue i demotrated i figure 4. Bcl-2 wa ot detected i either ormal or emphyematou lug tiue (fig. 4a ad b). I additio, Bax ad Bad expreio were ot detected i ormal lug tiue (fig. 4c ad f). However, expreio of both protei wa detected i emphyematou lug (fig. 4d ad 4g). I emphyema ample, Bax taiig wa localied to alveolar epithelial cell (fig. 4d, arrow), wherea Bad taiig wa ditributed amog what appeared to be both epithelial ad meechymal cell (fig. 4g, arrow). Iteretigly, everal macrophage-like cell demotrated icluio of Bax-poitive material i their cytoplam (fig. 4g). Idividual value of urface area, apoptoi, ad cell proliferatio Value of urface area, apoptoi, ad proliferatio i the idividual ample are preeted uig catter plot (fig. 5). The ditributio of urface area ad apoptoi wa broad amog emphyema ample, but remaied igificatly c EUROPEAN RESPIRATORY JOURNAL VOLUME 25 NUMBER 2 253

5 APOPTOSIS IN PULMONARY EMPHYSEMA K. IMAI ET AL. a) a) 32 kda b) 24 kda 17 kda 12 kda c) 85 kda 80 kda Normal Emphyema Jurkat Pro-capae-3 Active capae-3 PARP b) FIGURE 3. Capae-3 activity ad poly (ADP-riboe) polymerae (PARP) cleavage i huma lug. Weter blot aalyi of homogeate from ormal ad emphyematou huma lug (120 mg of protei per lae). a) Pro-capae-3 expreio (32 kda) i ee i all ample. The lat lae how reactivity i lyate from Jurkat cell timulated by ati-fa atibody a a poitive cotrol. b) Expreio of the pro-form of capae-3. Expreio of the 12- ad 17-kDa fragmet with a 24-kDa itermediate form detected by a rabbit polycloal atibody i the emphyema ample. c) Expreio of the proteolytic fragmet of PARP, a ubtrate of capae-3, i emphyematou lug tiue homogeate but ot i the ormal lug. The preece of the additioal bad ruig at 80 kda may repreet further degradatio of PARP. M N1 N2 N3 E1 E2 E3 E4 Normal Emphyema FIGURE 2. Biochemical aalye of apoptoi i huma lug. a) Electro micrograph howig the ultratructure of alveolar epta of a emphyema lug ectio. Apoptotic cell (arrow) adjacet to a ormal cell illutrate cytoplamic codeatio ad hrikage, with codeatio of uclear chromati. Lo of cotact with the extracellular matrix cotact i alo oberved. Scale bar52 mm. b) DNA wa iolated from three ormal ad four emphyematou lug ad aalyed by agaroe gel electrophorei (30 mg of DNA per lae). DNA from all emphyema ample howed a mear, with ample E2 from a patiet with evere emphyema revealig exteive ladderig. Lae 1 how a 1-kb DNA ladder. differet from ormal lug. The emphyema ample demotrated lower average lug urface area tha ormal lug (fig. 5a). All emphyema ample demotrated higher apoptoi level tha the ormal ample (fig. 5b). Data i figure 5b are from FITC TUNEL cout of apoptotic cell. Level of cellular proliferatio were alo examied uig immuohitochemitry for PCNA (fig. 5c). Iteretigly, emphyematou lug demotrated higher level of cell proliferatio tha ormal lug (p,0.05). Correlatio ad liear regreio aalye of tiue morphometry with apoptotic ad proliferatio idice The reult of cell morphology ad tiue morphometry tudie uggeted a potetial aociatio betwee cell lo ad lug architecture. Thu, the correlatio betwee lug urface area ad apoptoi (FITC TUNEL), a determied by TUNEL taiig i the idividual ample (fig. 6a), wa plotted. Higher level of apoptoi were detected a lug urface area decreaed (r5-0.75; p50.001). Next, the level of cell proliferatio wa compared relative to urface area. Although cell proliferatio (PCNA taiig) wa igificatly higher i emphyematou lug, o igificat correlatio betwee cell proliferatio ad urface area wa detected (fig. 6b). Further, o tatitical correlatio betwee cellular apoptoi ad proliferatio wa detected, although the poitive lope of the regreio lie idicate that higher proliferative rate may be occurrig i ample with higher rate of apoptoi (fig. 6c). Thee data ugget that the kietic of cell lo (apoptoi) ad cell replacemet (proliferatio) may be a critical feature of emphyema pathogeei. DISCUSSION The preet tudy demotrate exteive cell death by apoptoi, i combiatio with coective tiue degradatio, i the lug tiue of patiet with pulmoary emphyema. Apoptoi wa demotrated ad cofirmed by everal differet techique, icludig cell morphology, DNA fragmetatio ad apoptoi-related protei expreio. Thee reult cofirm ad exted obervatio made by other [10 12]. I additio, the data provide a correlatio of apoptoi with a morphometric meaure of the emphyematou proce. Each method of demotratig apoptoi i the preet tudy provide iight ito the potetial role of apoptoi i the pathogeei of pulmoary emphyema. By morphological aalyi, cattered apoptotic cell were viible i the abece of tiue iflammatio. Ultratructural aalyi cofirmed cytoplamic ad uclear codeatio i apoptotic cell. Thee feature ditiguih apoptoi from tiue ecroi [20]. I the latter, ecrotic cell exhibit cellular wellig ad rupture of the plama membrae, while the ucleu remai relatively itact. I additio, i tiue ecroi, there i a aociated iflammatory repoe. The morphological feature of the cell i the emphyema tiue i thi tudy were coitet with apoptoi. Cellular apoptoi wa cofirmed by DNA fragmetatio by i itu ed labellig ad gel electrophorei. However, o pecific cell type wa predomiately affected. Thee reult are imilar 254 VOLUME 25 NUMBER 2 EUROPEAN RESPIRATORY JOURNAL

6 K. IMAI ET AL. APOPTOSIS IN PULMONARY EMPHYSEMA a) b) c) d) e) f) g) FIGURE 4. Immuohitochemical taiig for Bcl-2, Bax ad Bad i huma lug tiue ectio. Bcl-2 wa ot detected i either ormal (a) or emphyematou (b) lug tiue. Bax ad Bad expreio wa ot detected i ormal lug tiue (c, f), but both protei were preet i emphyematou lug ample (d, g). I emphyema ample, Bax taiig wa detected i alveolar epithelial cell (d, arrow), wherea Bad taiig wa ditributed amog epithelial ad meechymal cell (g, arrow). I additio, everal macrophage-like cell demotrated icluio of Bax-poitive material i their cytoplam (e). Scale bar520 mm (e) ad 60 mm (g). to obervatio of KASAHARA ad coworker [10, 11], who demotrated apoptoi of both alveolar eptal ad edothelial cell. Relative pecificity of apoptoi for pulmoary emphyema wa uggeted by the abece of TUNEL reactivity i patiet with diffue itertitial lug dieae. Thee reult are alo i agreemet with the obervatio of KASAHARA ad colleague [10, 11], who foud o apoptoi i the lug of patiet with primary pulmoary hyperteio. The fidig of TUNEL-poitive material withi alveolar macrophage ugget a role for macrophage a caveger of apoptotic cell, a how for other cell ytem [21]. The curret author have exteded previou fidig by demotratig biochemical evidece of apoptoi ad capae activatio i emphyematou lug tiue. The equece of capae activatio i a idipeable proce i the apoptoi pathway [22]. Capae-3 fuctio dowtream of cell damage i apoptoi, playig a pivotal role i targetig molecule for proteolyi [23]. Proteolyi of PARP ito a 85-kDa fragmet, a how i the emphyema ample i the curret tudy, i a pecific evet mediated by capae-3 [24]. c EUROPEAN RESPIRATORY JOURNAL VOLUME 25 NUMBER 2 255

7 APOPTOSIS IN PULMONARY EMPHYSEMA K. IMAI ET AL. a) 220 Surface area mm b) 5 Apoptoi idex % c) ** ** # a) Apoptoi idex % b) Proliferatio idex % c) Surface area mm Proliferatio idex % Normal Emphyema FIGURE 5. Scatter plot of idividual lug ample. Lie repreet the media value. Surface area meaure (a) ad apoptoi by fluorecei iothiocyaate termial deoxyucleotide traferae-mediated dutp ick-ed labellig (b) were igificatly differet from ormal ample. c) The average proliferatio rate i the emphyema ample wa higher tha that for ormal lug. # :p50.049; **: p,0.01. A importat fidig of the curret tudy wa the elevatio of both apoptoi ad cell proliferatio i the emphyema ample (fig. 5b ad c, repectively). Iteretigly, correlatio aalye revealed a igificat ivere relatiohip betwee urface area ad apoptoi (fig. 6a), with o correlatio betwee urface area ad cell proliferatio (fig. 6b). Together, thee data ugget that, although both cell parameter are Proliferatio idex % Apoptoi idex % icreaed i emphyema, apoptoi ad proliferatio may ot have the ame relatiohip to lug urface area. Thi mechaim could explai the accelerated, detructive ature of the emphyematou proce that exceed the rate predicted 3 FIGURE 6. Relatiohip betwee urface area, apoptoi (fluorecei iothiocyaate termial deoxyucleotide traferae-mediated dutp ick-ed labellig) ad cell proliferatio. Idividual patiet value are how. a) A ivere correlatio betwee urface area ad apoptotic idex i oberved by liear regreio (p50.001). There i o igificat correlatio betwee urface area ad proliferatio (b), or betwee apoptoi ad proliferatio (c), eve though emphyema ample had higher level of both of thee cellular idice. However, the poitive lope of the regreio lie ugget that apoptoi ad proliferatio icreae together VOLUME 25 NUMBER 2 EUROPEAN RESPIRATORY JOURNAL

8 K. IMAI ET AL. APOPTOSIS IN PULMONARY EMPHYSEMA by traditioal Fletcher curve, eve after patiet top mokig [8, 9]. Iteretigly, the icreae i cell proliferatio may explai why the elevated rate of apoptoi doe ot caue total lo of parechyma. Recetly, YOKOHORI et al. [12] alo aalyed apoptoi ad cell proliferatio i emphyema lug. They detected much higher level of PCNA taiig (media value were,2.5% for emphyema lug ad 0.1% for ormal) ad lower bioti TUNEL taiig (,0.9% for emphyema lug ad 0.1% for ormal) tha the curret author. The reao for the differece betwee the curret author reult ad thoe of YOKOHORI et al. [12] may ret i the ample populatio. Firt, the average age of the ample populatio i the curret tudy wa youger ad the emphyema ample came from patiet with a horter mokig hitory, averagig 40.9 pack-yr compared with a average of 95 pack-yr for the ample i the tudy by YOKOHORI et al. [12]. Sample i the curret tudy were collected from emphyema patiet who had quit mokig o6 moth prior to the collectio of the lug tiue. I additio, although o report wa made o the morphometric aemet of airpace, it i poible that the ample i the tudy by YOKOHORI et al. [12] had more evere emphyema tha thoe i the curret tudy. Mot importatly, the ormal ample i the tudy by YOKOHORI et al. [12] were from lug cacer patiet, which could have a higher proliferative capacity tha the ormal lug ample i the cotrol group i the curret tudy. The idetificatio of Bax expreio i emphyema, but ot i the ormal lug, provide cellular lik to apoptoi. Bax protei are pro-apoptotic molecule, whoe expreio i icreaed whe cell die by depletio of cell adheio to the ECM [25]. Bax protei couteract the protective effect of Bcl-2 protei ad trigger capae activatio [22]. Data from the curret tudy demotrate o Bcl-2 expreio, with elevated Bax ad Bad reactivity i emphyematou lug ample. I additio, there were macrophage-like cell cotaiig Baxpoitive particle withi their cytoplam. Thi i a itriguig fidig i a dieae ivolvig icreaed level of proteae that potetially dirupt the matrix to modify cellular adheio ad igallig. The curret tudy exted obervatio that cellular apoptoi occur i emphyema, but doe ot etirely clarify the mechaim of apoptoi. Ituitively, apoptoi may be related to the diruptio of the ECM. Importatly, the obervatio of cellular apoptoi doe ot egate the role of proteae i emphyema pathogeei. Ideed, it may repreet a cotiuum of the detructive proce, i which lo of matrix attachmet lead to icreaed cellular Bax ad capae-3 activity, drivig apoptoi of alveolar cell. It i poible that the icreaed proteae burde reult i lo of local matrix. Thu, it i coceivable that the cell death occurrig i emphyema may be aoiki, programmed cell death due to lo of matrix adheio [26]. Certaily, the icreaed proteae burde i emphyema could potetiate lo of matrix, cleavage of cell urface receptor, cell detachmet, ad ubequet lo of igallig eceary for cell urvival. I additio, other mechaim, uch a activatio of cell urface death receptor or ivolvemet of Fa ligad [27], may play a role i lo of pulmoary cell i emphyema. Alteratively, blockade of VEGF receptor igallig iduce cellular apoptoi ad emphyema, uggetig that ihibitio of edothelial growth factor play a role i the pathogeei of dieae [10, 11]. Regardle of the iitiatig cellular iult, the preet tudy demotrate that apoptoi occur i emphyema lug cocomitat with dieae progreio. Thi i a mechaim that may explai the detructio of the lug durig progreio of the dieae. Numerou tudie have demotrated a role for apoptoi i a variety of chroic huma dieae, icludig eurodegeerative dieae, heart failure, atherocleroi ad viral dieae [28]. I everal of thee dieae, ati-apoptotic aget are expected to treat patiet or low dieae progreio. May of thoe aget are uder evaluatio ad could potetially be applied to dirupt the progreive tiue lo characteritic of pulmoary emphyema. To the curret author kowledge, o previou report have examied the relatiohip betwee cellular mechaic ad lo of alveolar architecture, a reported here. The icreae i cell death a morphometric lug urface area fall ugget that lo of cell cotribute to reduced itegrity of alveolar epta, reultig i icreaed pulmoary compliace ad reduced elaticity. Thi hypothei i upported by tudie o emphyema iductio followig pulmoary capae delivery i mice [14]. However, coiderig the evet i revere equece, it i coceivable that the lo of matrix alog the alveolar wall iduce cell death without compeatory icreae i cell proliferatio. Noethele, much work remai to be carried out to defie the mechaim ivolved i apoptoi durig the progreio of pulmoary emphyema. ACKNOWLEDGEMENTS The author are grateful to M. Selma (Itituto Natioal de Efermedade Repiratoria, Mexico DF, Mexico) for the geerou gift of tiue ectio from patiet with itertitial lug dieae. REFERENCES 1 Sider GL, Lucey EC, Stoe PJ. Pitfall i atiproteae therapy of emphyema. Am J Repir Crit Care Med 1994; 150: S131 S Shapiro SD. The pathogeei of emphyema: the elatae: atielatae hypothei 30 year later. Proc Aoc Am Phyicia 1995; 107: Imai K, Dalal S, Che E, et al. Huma collagaae (matrix metalloproteiae-1). Expreio i the lug of patiet with emphyema. Am J Repir Crit Care Med 2001; 163: Filay GA, O Dricoll L, Ruell KJ, et al. Matrix metalloproteiae expreio ad productio by alveolar macrophage i emphyema. Am J Repir Crit Care Med 1997; 156: Stockley RA. Neutrophil ad proteae/atiproteae imbalace. Am J Repir Crit Care Med 1999; 160: S49 S52. 6 Ohihi K, Takagi M, Kurokawa Y, Satomi S, Kottie Y. Matrix metalloproteiae-mediated extracellular matrix protei degredatio i huma pulmoary emphyema. Lab Ivet 1998; 78: c EUROPEAN RESPIRATORY JOURNAL VOLUME 25 NUMBER 2 257

9 APOPTOSIS IN PULMONARY EMPHYSEMA K. IMAI ET AL. 7 Retamale I, Elliott WM, Mehi B, et al. Amplificatio of iflammatio i emphyema ad it aociatio with latet adeoviral ifectio. Am J Repir Crit Care Med 2001; 164: Fletcher CM, Peto R. The atural hitory of airflow obtructio. BMJ 1977; 1: Gelb AF, McKea RJ Jr, Breer M, Eptei JD, Zamel N. Expadig kowledge of lug volume reductio. Am J Repir Crit Care Med 2001; 163: Kaahara Y, Tuder R, Taraeviciee-Stewart L, et al. Ihibitio of VEGF receptor caue a lug cell apoptoi ad emphyema. J Cli Ivet 2000; 106: Kaahara T, Matuhima K. Edothelial cell death ad decreaed expreio of vacular growth factor ad vacular edothelial growth factor receptor 2 i emphyema. Tred Immuol 2001; 22: Yokohori N, Aohiba K, Nagai A. Icreaed level of cell death ad proliferatio i alveolar wall cell i patiet with pulmoary emphyema. Chet 2004; 125: Kaazawa H, Aai K, Hirata K, Yohikawa J. Poible effect of vacular edothelial growth factor i the pathogeei of chroic obtructive pulmoary dieae. Am J Med 2003; 114: Aohiba K, Yokohori N, Nagai A. Alveolar wall apoptoi caue lug ditructio ad emphyematou chage. Am J Repir Cell Mol Biol 2003; 28: Thurlbeck WM. Iteral urface area ad other meauremet i emphyema. Thorax 1967; 22: Duill MS. Quatitative method i the tudy of pulmoary pathology. Thorax 1962; 17: Weibel ER. Priciple ad method for the morphometric tudy of the lug ad other orga. Lab Ivet 1963; 12: Weibel ER. Morphometry of the huma lug: the tate of the art after two decade. Bull Eur Phyiopathol Repir 1979; 15: Yag J, Liu X, Bhalla K, et al. Prevetio of apoptoi by Bcl-2: releae of cytochrome c from mitochodria blocked. Sciece 1997; 275: Wyllie AH, Kerr JF, Currie AR. Cell death: the igificace of apoptoi. It Rev Cytol 1980; 68: Lorezi M, Gerhardiger C. Early cellular ad molecular chage iduced by diabete i the retia. Diabetologia 2001; 44: Graville DJ, Carthy CM, Hut DW, McMau BM. Apoptoi: molecular apect of cell death ad dieae. Lab Ivet 1998; 78: Wolf BB, Schuler M, Echeverri F, Gree DR. Suicidal tedecie: apoptotic cell death capae family proteiae. J Biol Chem 1999; 274: D Amour D, Germai M, Orth K, Dixit VM, Poirier GG. Proteolyi of poly(adp-riboe) polymerae by capae 3: kietic of cleavage of moo (ADP-riboyl)lated ad DNAboud ubtrate. Radiat Re 1998; 150: Merto GR, Cella N, Hye NE. Apoptoi i accompaied by chage i BcL-2 ad Bax expreio, iduced by lo of attachmet, ad ihibited by pecific extracelluar matrix protei i mammary epithelial cell. Cell Growth Differ 1997; 8: Michel JB. Aoiki i the cardiovacular ytem: kow ad ukow extracelluar mediator. Arteriocler Thromb Vac Biol 2003; 23: Yauda N, Gotoh K, Miatoguchi S, et al. A icreae of oluble Fa, a ihibitor of apoptoi, aociated with progreio of COPD. Repir Med 1998; 92: Rudi CM, Thompo CB. Apoptoi ad dieae: regulatio ad cliical relevace of programmed cell death. Au Rev Med 1997; 48: VOLUME 25 NUMBER 2 EUROPEAN RESPIRATORY JOURNAL