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M i n i R e v ie w o n t h e C e ll u la r M e c h a n i s m s o f D i s e a s e

Fibroblasts M yof ibroblasts an d W oun d C ontra ct ionF r e d e r i c k G r i n n e l lDepartment of Cell Biology and Neuroscience, University of Texas Southwestern Medical Center, D allas, Texas 75235

c l in ica l case recons t ruc ted by Guido Majno (1975)f rom the Hippocra t i c records desc r ibes a wres t l e rwho v i s i t s the iatreion (out-pat ient c l inic) to betreated for a shou lder dis locat ion . With less invas ive proce-dures no longe r work ing to t igh ten the d i s loca t ion , the c l in icadopts the d ras t i c m easure o f induc ing wound con t rac t ion bypoking a ho t need le th rough the sk in o f the a rm pi t , and inth i s way the cav ity , in to w hich the hum erous i s m os t ly d i s -p laced , i s bes t s ca r red ove r and cu t o f f In Greek m edic inec i rca 400 B . C . E . , fam i l i a r i ty wi th w ound con t rac t ion a f t e rburn in ju ry a l ready was com m onplace .C losure o f cu taneous wounds invo lves th ree p roces ses :epi thel izat ion, connect ive t issue deposi t ion, and contrac-t ion . The con t r ibu t ion of each proces s va r ies accord ing tothe type o f wound . In gene ra l , ep i the l iza t ion re su l t s in re sur -fac ing of the wound ; connec t ive t i s sue depos i t ion re su l t s inrep lacem ent o f dam aged de rm is ; and con t rac t ion b r ings them arg ins o f open wounds toge the r (P eacock , 1984; C la rk ,1988; M as t , 1992) . In m am m als wi th loose sk in (m eaningloose ly a t t ached to the u nder ly ing t i s sue l aye r ), wou nd con-t rac t ion l eads to woun d c losure wi th l i t tl e s ca r r ing o r los sof func t ion . In hum an s , whose sk in is m ore f i rm ly a t t achedto un der ly ing t i ssues , the consequences o f con t rac t ion a rele ss bene f ic ia l , rang ing f rom m in im a l cosm et ic s ca r in som ecases to los s o f jo in t m ot ion or m a jor bo dy de form a t ion ino the rs . Consequent ly , a d i s t inc t ion has been m ade be tweencont rac t ion a s a norm a l p roces s o f wound c losure , and con-t rac tu re a s the abnorm al re su l t o f the con t rac t ion p roces swhere s ign i fcan t s ca r r ing o r los s o f func t ion occurs (Huntand Dunphy , 1979) . The pa tho log ic consequences o f t i s suecont rac t ion inc lude a va r ie ty o f condi t ions rang ing f ro m con-t rac tu re o f the f ib rous capsu le sur rounding breas t im plan t sto cons t r i c tu re o f ho l low organs (e . g . , the e sophagus ) a f t e rinjury (Skal l i and Gabbiani , 1988; Rudolph et a l . , 1992).In con tem pora ry ce l l b io logy , re sea rch on wou nd con t rac -t ion focuses on the wound fibroblas t . Skin f ibroblas ts nor-m a l ly a re s e s s i l e and qu iescen t , bu t shor t ly a f t e r cu taneouswounding , they b ecom e ac t iva ted . Ac t iva ted f ib rob las ts m i -gra te to the f ib ronec t in - f ib r in wou nd in te r face , p ro l i fe ra te ,and synthes ize a new col lagen-containing matrix cal led gran-u la t ion ti s sue . Aroun d the s am e t im e , wound con t rac t ion be -g ins . Once the woun d de fec t i s rep laced , the expanded f ib ro-blas t populat ion s tops dividing and regresses and extracel lularmatrix remodeling commences (Peacock, 1984; Clark, 1993).Desp i te the im por tance o f wound con t rac t ion fo r woundAddress all correspondence to E Grinnell, Department of Cell Biology andNeuroscience, University of Texas-Southwestern Medical Center, Dallas,TX 75235.

hea l ing , the m echanism by which wound f ib rob las t s exe r tfo rce on the sur rounding ex t race l lu lar m a t r ix i s on ly beg in-n ing to be unders tood . Moreover , a l though i t has becom eeviden t tha t gene expres s ion and pro l i fe ra t ion o f these ce l l si s regu la ted by m echanica l fo rce , the m echanot ransduce rsand signaling m echan isms involved remain high ly speculative.Fibroblas ts , Myof ibroblas ts , and Wound Contrac t ionResearch carrie d out in the mid-1950's implica ted connect ivet i s sue ce l ls in the gene ra t ion o f the t ens i l e fo rce re spons ib lefor contract ion (Abercrom bie e t a l . , 1956). The ne wly formedgranula t ion t i s sue was sugges ted a s the o rgan of con t rac -ture (Bil l ingham and Russel l , 1956). Other s tudies showedtha t the fo rce -gene ra t ing ce l l s were loca l i zed a t the w oundm arg ins ra the r than in the cen te r o f the g ranu la t ion t i s sue(Watts e t a l . , 1958). Subsequently, Gabb iani e t a l . (1972) d i-rec t ly dem on s t ra ted tha t i so la ted s tr ips o f g ranu la t ion t i s suewere ab le to undergo a sm oo th m usc le - l ike con t rac t ion in v i -t ro and found tha t ce l l s wi th in the g ranu la t ion t i s sue exh ib i tsom e fea tu res o f sm ooth m usc le ce l l s such a s ac t in f i l am entbund les (s t ress f ibers) . The se myo-fibroblas ts were pro-posed to be re spons ib le fo r fo rce gene ra t ion , and the i r p res -ence has tu rned o u t to be a gene ra l fea tu re o f t is sues under -going contract ion (Skal li an d Gabbiani , 1987; Rudo lph et a l . ,1992).Ana lys i s o f cy toske le ta l m arke rs showed tha t a l thoughmyofibroblas ts express c~-smooth muscle act in, these cel lsa re de r ived f rom f ib rob las t s no t sm o oth m usc le ce l l s (Eddyet a l . , 1988; Darby et a l . , 1990). D uring gran ulat ion tissueform a t ion , m igra t ing f ib rob las t s d i f fe ren t i a te in to m yof ib ro-blas ts (Darby et a l . , 1990; W elch e t a l . , 1990). Ju dging fromthe presence o f f ib ronexus junc t ions , m yof ib rob las t s fo rmvery t ight adhes ions to the surrou nding f ibronect in-rich gran-u la t ion ti s sue (S inge r e t a l . , 1984; Tom asek and H aaksm a ,1991). The t im ing of the switch from fibro blas t to myofibro-b la s t phenotype appea rs to depend a t l eas t in pa r t on the ex-ten t to which the wound re s i s t s con t rac t ion (Darby e t a l . ,1990; Welch e t a l . , 1990). I t now seems l ikely that migrat ingf ib rob las t s a t the w ound m arg ins g ene ra te suf f ic ien t fo rce toin i t ia t e wound con t rac t ion . A s con t rac t ion p roceeds and re -s is tance increases , m igrat ing f ibrob las ts different ia te intomyofibroblasts and the act in cy toskeleton becom es organizedalong the l ines of greates t res is tance (Petrol l e t a l . , 1993).

I n V i t ro M o d e l s o f W o u n d C o n t ra c t i o nS everal in v i t ro m ode l s o f wound con t rac t ion have been de -ve loped us ing f ib rob last s cu l tu red in co l l agen or f ib r in m a-

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trices. E lsdale and B ard (1972) ~howed that fibroblasts cul-tured in col lagen matr ices acquire t i ssue- l ike phenotypiccharac te r is t ics not typica l ly observed in ce l ls in monolayercul ture . They reported tha t w i th matr ices f loa ting in the cul-ture medium , ce l l mot i l ity resul ted in col lapse of the matr ixin to a dense , opaque body less than one- tenth of the or ig ina lsize A simila r reorganiza tion process was observ ed whenfibroblasts were cul tured in f ibr in (Niewiarowski and Gold-stein, 1973). Subsequently, Bell et al . (1979) began studyingcol lagen-matr ix reorganiza t ion as an in v i t ro mode l of woundcontrac t ion .Fig . 1 i l lustra tes three co mm on var ia t ions o f the in v i t rocol lagen matr ix contrac t ion model . These a re : ( I ) f loa t ingmatr ix contrac t ion (reduc t ion in d iameter) ; II) anchoredmatr ix contrac t ion (reduc t ion in he ight) ; and III) st ressre laxat ion . T hese m odels d i f fe r marked ly in the i r mechani-ca l fea tures. In / , tension is d is t r ibuted iso t ropica l ly . In II,tension is distributed anisotropically. In III, mechanica lst ress deve lops during the per iod when the matr ix i s an-chored and then st ress d issipa tes a f te r the matr ix i s re leased.(In engineer ing ja rgon th is process would be ca l led st ra inrecovery ra ther than st ress re laxa t ion .) T he next two sec t ionswil l focus on contrac t ion of f loa t ing vs. anchored matr ices.Subsequent ly , s t ress re laxa t ion wi l l be d iscussed.Contrac t ion o f F loat ing vs. An cho redCol lagen M atr icesCurrent evidence suggests tha t contrac t ion o f floa t ing vs. an-chored col lagen matr ices i s s imi la r wi th regard to t rac t iona lremodel ing, in tegr ins, and extrace l lu la r fac tors (see be low).The m ajor d i f fe rence is in the end resul t . Con trac t ion o f af loa ting col lagen matr ix resul ts in a mechanica l ly re laxed t i s-sue , whi le contrac t ion of an anchored col lagen matr ix resul tsin a stressed tissue.Harr is e t al . (1981) show ed that contrac t ion o ccurs as aconsequ ence of mot i le ac t iv i ty by ce l ls t ry ing to migra tethrough the matr ix . This process was ca l led t rac t iona lremodel ing to d ist inguish i t f rom a smooth musc le- l ike con-t rac t ion . That i s , contrac t ion occurs as f ibroblasts spread andelongate , not as a l ready e longated ce l ls re t rac t the i r exten-sions (Grinne l l and Lamke, 1984) . Cel ls reorganize prox-imal ly loca ted col lagen f ibr i l s f i rs t and subseq uent ly the restof the matr ix as t rac t iona l forces a re propagated througho utthe cont inuous, in te r twined col lagen f ibr i l ne twork (Guidryand Grinnell , 1987).

0) (H)

l - t t _ tFigure 1.

(m)

1--1

Adhesive in te rac t ions be tween ce l ls and col lagen requiredfor contrac t ion a re media ted by a2/31 in tegr ins (S chiro e t a l . ,1991; Klein et al ., 1991). Tensile force was found to dependon an in tac t ac t in cytoske le ton (Bel l e t a l . , 1979; Bel lows e ta l . , 1982; Guidry and Griune l l , 1985) and myosin lightcha in k inase (MLCK) ac t iv i ty (Ehrl ich and Griswold , 1984;Van Bockxmeer e t a l . , 1984; Ehrl ich e t a l . , 1991) .Col lagen matr ix contrac t ion requires se rum (Ste inberg e ta l . , 1980; Guidry and Grinne l l , 1985) , which indica tes tha tce l l contrac t i l i ty can be regula ted by extrace l lu la r fac tors .The ac t iv i ty of se rum can be replaced or enhanced by puri -f ied growth fac tors . For instance , t ransforming growth fac tor(TGF-/~) stimulates co ntractio n o f both floating and an-chored col lagen matr ices (Montesano and Orc i , 1988; Fine-smith e t a l . , 1990; Fukamizu and Grinne l l , 1990) . TGF-/~a lso has been reported to promote f ibroblast d i f fe rent ia t ioninto myofibroblasts (Ronnov-Jessen and Petersen, 1993; Des-moul i e t a l . , 1993) . Pla te le t der ived growth fac tor (PDG F)st imula tes matr ix contrac t ion (Clark e t a l. , 1989; Gul lberge t a l . , 1990) , but by a mechanism independent o f TGF-/3(Tingst rom e t a l ., 1992) . Fac tors tha t inhibi t matr ix contrac-t ion inc lude f ibroblast growth fac tor (Finesm ith e t a l . , 1990;Dubertret et al ., 1991) and interferon 3' (Gillery et al .,1992) . In genera l , the downstream signa l ing mechanisms bywhich growth fac tors regula te matr ix contrac t ion a re un-known, but prote in k inase C probably is involved (Danowskiand Harr is , 1988; Guidry , 1992) ,C o l la g e n M a t r i x C o n t ra c ti o n a n d t h e C e l l P h e n o ty p eThe f ibroblast phenotype tha t deve lops as a consequence ofcol lagen matr ix contrac t ion d i ffe rs dramat ica l ly dependingon whether the matr ices a re f loa t ing or anchored. Contrac-t ion of f loat ing col lagen matr ices g ives r ise to a m echanica l lyre laxed t i ssue whose ce l ls have morph ologica l an d prol i fe ra -t ive fea tures resembl ing dermis, whereas anchored matr icesdeve lop in to a s t ressed t i ssue resembl ing granu la t ion t i ssue .Stra in gauge measurem ents have shown tha t the force exer tedby f ibroblasts in anchored col lagen matr ices i s comparablewith tha t genera ted in contrac t ing skin wounds or dur ingtooth e rupt ion (K asugai e t a l . , 1990; Delvoye e t a l . , 1991;Ko lodney and Wysolmerski , 1992) .In f loa t ing col lagen matr ices, f ibroblasts deve lop ste l la temorpho logy w i th l ong p roc e sse s a nd a c y to ske l e t a l me sh -work (Bel l e t a l . , 1979; Bel lows e t a l. , 1981). In mark ed con-t rast , ce l ls in anchored matr ices become bipolar and or ienta long l ines of tension (Stopak and Harr is , 1982; Bel lows e ta l . , 1982) . Cel ls deve lop prominent s t ress f ibers and f ibro-nexus junc t ions and resem ble myofibroblasts (Farsi andAubin , 1984; Mochi ta te e t a l . , 1991; Tomasek e t a l . , 1992) .Therefore , f ibroblasts can contrac t a t i ssue matr ix in v i t rowithout d i f fe rent ia t ing in to myofibroblasts , and the appear-ance of myofibroblasts corre la tes wi th deve lopm ent of s t ressin the matr ix .Fibroblasts in f loa t ing vs. anchored col lagen matr icesshow profoun d differences in cell proliferation. After co ntrac-t ion of f loa ting col lagen matr ices, there i s a marked dec l inein ce l lu la r DNA synthesis (Sarber e t a l . , 1981; Nishiyamaet a l . , 1989; Nakagawa e t a l . , 1989a) . Th e ce l ls beco me ar-rested in Go (Kono e t a l . , 1990) , and ce l l regression begins(Nakagawa e t a l . , 1989b) . Cel ls in anchored m atr ices, on theother hand, cont inue to synthesize DN A and increase in ce l lnum ber (Nishiyama e t a l . , 1989; Nakagawa e t a l . , 1989a ,b) .

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A l s o , s u b je c t i n g f i b r o b l a s t s i n f l o a t i n g c o l l a g e n m a tr i c e s t oe x te r n a l s t r e s s r e su l t s in i n c r e a s e d c e l l g r o w th J a in e t a l . ,1 9 9 0 ) .T h e l o w p r o l i f e r a t i v e c a p a c i ty o f f i b r o b la s t s i n f lo a t i n g c o l -l a g e n m a tr i c e s a p p e ar s to r e f l e c t d e c r e a s e d r e s p o n s i v e n e s s o fth e c e l l s t o g r o w th fa c to r s N a k a g a w a e t a l . , 1 9 8 9 a ; N i s h i -y a m a e t a l . , 1 9 9 0 , 1 9 9 1 ). R e c e n t s tu d i e s h a v e s h o w n th a tP D G F r e c e p to r s o n f i b r o b l a s t s i n f l o a t i n g c o l l a g e n m a tr i c e sl o s e th e i r c a p a c i ty to a u to p h o s p h o r y l a t e i n r e s p o n s e toP D G F L i n a n d G r i n n e l l, 1 9 9 3 ) .

I n a d d i t i o n to c h a n g e s i n c e l l p r o l i f e r a t io n , f i b r o b l a s ts i nf l oa t in g c o l l a g e n m a t r i c e s a l s o s h o w d e c r e a s e d c o l l a g e n b i o -s y n t h e si s a n d i n c r e a s e d r e l e a s e o f c o l l a g e n a s e c o m p a r e dw i th c e l l s i n a n c h o r e d m a tr i c e s N u s g e n s e t a l . , 1 9 8 4 ;U n e m o r i a n d W e r b , 1 9 8 6 ; P a y e e t a l . , 1 9 8 7 ; F u k a m i z u a n dG r i n n e l l , 1 9 9 0 ) . C h a n g e s i n c o l l a g e n a n d c o U a g e n a s e b i o -s y n th e s i s b y f i b r o b l a s t s i n f lo a t i n g m a tr i c e s d e p e n d o n t r a n -scr ipt ional as we l l as posttranscr iptional m echa ni sm s Mauche t a l . , 1 9 8 8 ; L a m b e r t e t a l . , 1 9 9 2 ; E c k e s e t a l . , 1 9 9 3 ) . T h e s ef i n d i n g s s u g g e s t th a t m e c h a n i c a l o r g a n i z a t i o n o f th e t is s u ec a n r e g u l a te e x tr a c e l l u l a r m a tr i x b i o s y n th e s i s a n d r e m o d e l -i n g a s w e l l a s c e l l p r o l i f e r a t i o n .S t r e ss Re l a x a t i o nI f f lo a t in g c o l l a g e n m a t r i c e s r e s e m b l e d e r m i s a n d a n c h o r e dm a tr i c e s r e s e m b l e g r a n u l a t i o n t i s s u e , th e n s t r e s s r e l a x a t i o nr e p r e s e n t s th e t r a n s i t i o n f r o m g r a n u l a t i o n t i s s u e to d e r m i so r s c a r ) , a l b e i t o n a t i m e s c a l e th a t is m a r k e d l y s p e d u p . U n -l i k e th e s l o w h o u r s to d a y s ) c o n tr a c t i o n o f f lo a t i n g o r an -c h o r e d c o l l a g e n m a tr i c e s , c o n tr a c t i o n o f s t r e s s e d c o l l a g e nm a tr i c e s o c c u r s i n m i n u te s . T h e f i b r o b l a s t s th e m s e l v e s c o n -t r a c t a s i n d i c a te d b y th e r e t r a c t i o n o f c e l l p s e u d o p o d i a a n dc o l l a p s e o f a c t i n f i l a m e n t b u n d l e s M o c h i ta t e e t a l . , 1 9 9 1 ;T o m a s e k e t a l . , 1 9 9 2 ) . I n th i s c a s e , th e m e c h a n i s m a p p e a rsto i n v o l v e a s m o o th m u s c l e - l i k e c o n tr a c t i o n r a th e r th a n t r ac -t i o n a l r e m o d e l i n g . I n ta c t s t r e s s f i b e r s a r e r e q u i r e d a n d th ep r o c e s s is r e g u l a te d b y s e r u m fa c to r s T o m a s e k e t a l . , 1 9 9 2 ) .R e c e n t l y , th r o m b i n h a s b e e n fo u n d to s u b s t i tu te fo r s e r u ma n d s h o w n to s t i m u l a te m y o s i n l i g h t c h a i n p h o s p h o r y l a t i o nK o l o d n y a n d E l s o n , 1 9 9 3 ) th r o u g h a G -p r o te i n r e g u l a te dpathway Pi l cher , B . K . , and J . J . Tom asek . 1993. M o l B i olCel l 4 : 3 0 0 a ) .A c c o m p a n y i n g s t r e s s r e l a x a t i o n , f i b r o b l a s t s s h o w t r a n -s i e n t e c t o c y t o s i s o f a n n e x i n - c on t a i n in g v e s i c l e s L e e e t a l . ,1 9 9 3 ) , r e l e a s e o f c e l l s u r fa c e f i b r o n e c t i n M o c h i ta t e e t a l . ,1 9 9 1 ) , a n d i n a c t iv a t i o n o f P D G F r e c e p to r s L i n a n d G r i n -n e l l , 1 9 9 3 ) . C e l l p r o l i f e r a t io n a n d c o l l a g e n s y n th e s i s d e -c l i n e r a p i d l y a s th e c e l l s s w i t c h f r o m a n a c t iv a ted to r e s t i n gp h e n o ty p e I w i g e t a l . , 1 9 8 1 ; M o c h i ta t e e t a l . , 1 9 9 1 ) . T h es i g n a l i n g m e c h a n i s m s c o n tr o l l i n g th e s e d i f f e r e n t e v e n t s h a v ey e t t o b e d e te r m i n e d , b u t o n e o f th e e a r l i e s t f i b r o b l a s t r e -s p o n s e s to s t r e s s r e la x a t i o n 5 -1 0 m i n ) i s a c t iv a t i o n o f a cy -c l i c A M P / p r o te i n k i n a s e A s i g n a l in g p a th w a y H e , J . , a n d EG r i n n e l l . 1 9 9 3 . M o l B i ol Cel l 4 : 3 6 4 a ) .C h a n g e s i n c e l l p r o l i f e r a t i o n a n d b i o s y n th e t i c a c t i v i ty a f -t e r s t r e s s r e la x a t i o n p r o v i d e i n s i g h t i n to th e p o s s i b l e m e c h a -n i s m o f m y o f i b r o b l a s t d i s a p p e a r a n c e a t t h e e n d o f w o u n dh e a l i n g . A s l o n g a s th e t i s s u e i s u n d e r m e c h a n i c a l s t r e s s , c e l lp r o l i f e r a t io n a n d b io s y n th e t i c a c t i v i ty w i l l p e r s i s t . O n c e m e -c h a n i c a l s t r e s s i s r e l i e v e d , u s u a l l y b y a c o m b i n a t i o n o fw o u n d c o n tr a c t i o n a n d b i o s y n th e t i c a c t iv i ty , c e l l s w i l l s w i t c hto a n o n -p r o l i f e r a t i v e p h e n o ty p e a n d b e g i n to r e g r e s s e v e n

in the cont inued presence of growth factors. This v iew isconsistent wi th commonplace surgical exper ience that in-creased skin tension cont r ibutes to increased scarr ing Aremet al . , 1976; Bu rgess et al . , 1990). Moreover, i t helps explainthe role of external pressure in reducing wound con t ractureLarson et al . , 1971; Rockw el l et al . , 1989).D r s . W i l l i a m S n el l a n d R i c h a rd A n d e r s o n m a d e h e l p f u l c o m m e n t s a n d s u g -g e s t i o n s r e g a r d i n g t h i s m a n u s c r i p t .

T h e a u t h o r ' s r e s ea r c h h a s b e e n s u p p o r t e d b y N a t i o n a l I n s t it u t e s o f H e a l t hg r a n t G M 3 1 3 2 1 .R e c e i v e d f o r p u b l ic a t io n 3 0 A u g u s t 1 9 9 3 a n d i n r e v is e d f o r m 1 1 N o v e m b e r1993.

R e f e r e n c e sA b e r c r o m b i e , M . , M . H . F l i n t , an d D . W . J a m e s . 1 9 56 . W o u n d c o n tr a c t io nin r e la t ion to co l lagen forma t ion in scorbut ic gu inea p igs . J. Embryol. Exp.Morphol. 4 : 1 6 7 - 1 7 5 .A r e m , A . J . , a n d J . W . M a d d e n . 1 9 76 . E f f e c ts o f s t r e s s o n h e a l i n g w o u n d s .I . In te rmi t ten t noncyc l ica l tens ion . J. Surg. Res. 2 0 : 9 3 - 1 0 2 .Be l l , E . , B . Iva r sson , and C. M er r i l l . 1979 . P rod uc t ion of a t i ssue - l ike s t ruc tureby con t r ac t ion of co l lagen la t t ic e s by h um an f ib robla s ts o f d i f f e ren t p ro l i f e ra -t ive po ten t ia l in v i t ro . Proc. Natl. Acad. Sci. USA. 7 6 : 1 2 7 4 - 1 2 7 8 .Be l lows, C. G . , A . H . M e lche r , and J. E . Aub in . 1981 . 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