angiotensin-ii type 1 receptor interaction is a major regulator for

8/3/2019 Angiotensin-II Type 1 Receptor Interaction is a Major Regulator For

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Angiotensin-II Type 1 Receptor Interaction Is a Major Regulator forLiver Fibrosis Development in RatsHITOSHIYOSHfJI,SHIGEKIKURIYAMA, UNICHfYOSHII,YASUHIDE KENAKA,RYUICHINOGUCHI,

TOSHIYANAKATANI,HIROHISATSUJINOUE,AND HIROSHIEUKUI

T he r en i n -ang i o t ens i n sys t em (RAS ) i s f r equen t l y ac t i -va t ed i n pa t i en t s w i t h chron i c l i ve r d i seases. A ngi o t ens i n -I I(AT - II ) has bee n sugges t ed t o p l ay an i mpor t an t r o l e i n l i ve rf i b rogenes is . I t i nduces hepa t i c s t e l l a te ce l l (HS C) p ro l i f e r -a t i on and up- r egu l a t e s t he t r ans formi ng growt h f ac t o r 1~ 1(TGF-/31) express ion via AT-II type 1 receptor (AT1-R) invitro. T h e a i m o f th e p r e s e n t s t u d y w a s t o e x a m i n e t h e i n vivoe f f ec t o f candes a r t an (CA) , a c l i n i ca ll y used AT rR b l o cker(ARB) , and p e r i nd opr i l (P E ), an ang i o t ens i n - conver t i ng en-zyme (ACE ) i nh i b i t o r (ACE - I ) , on p i g se rum- i nduced l i ve rf i b ros is dev e l opm ent i n r a t s . T he c l i n i ca l l y ava i l ab le com pa-r ab l e doses o f CA and P E s i gn i fi can t l y a t t enua t ed t he f i b ro-s i s deve l opmen t . T hese i nh i b i t o ry e f f ec t s o f P E and CA werea l so found i n t he on-go i ng l i ve r fi b ros i s mode l . T he h epa t i chyd roxy pro l i n e and se r um f i b ros i s marke r s were s i gni f i-c a n t l y s u p p r e s s e d b y C A a n d P E t r e a t m e n t . F u r t h e r m o r e ,t he e~ smo ot h m usc l e ac t i n (~ -S M A) pos i t i ve ce l l s i n num berw e r e m a r k e d l y s u p p r e s s e d b y C A a n d P E t r e a t m e n t. S im i -l a r l y , t he hepa t i c T GF - ]~ p ro t e i n and messenger RNA(mRNA) l eve l s were s i gn i f i can t l y suppres sed . Our i n v i t r os t udy show ed t ha t AT - I I i nc r eased t he T GF-181 mRNA ex-pres s i on i n t he ac t i va t ed HS Cs , and t h i s e f f ec t was t o t a l l yb l ocke d by CA. T hese r e su l t s sug ges t ed t ha t t he RAS, e spe -cial ly AT-II and AT1-R interac t ion pla ys a pivota l role in l iverf i b ros is deve l op men t t h rough HS C ac t i va t ion . Because bo t hCA and P E a r e w i de l y u sed i n c l i n ica l p r ac t i ce w i t hou t s e r i-ous s i de e f f ec t s , t hese d rug s ma y prov i de an e f f ec t i ve newst rateg y for ant i - l ive r f ibrosis therap y. (HEPATOLOGY2001;34 : 745-750 . )

The renin -angio tensin system (RAS) i s repo r tedly act ivatedin pat ients wi th chron ic l iver diseases , such as c i r rhosis , l-3

Abbreviations: RAS, renin- angio tensi n system; A~f-II, angiotensin-II; ACE-I, angio-tensin-converting enzyme inhibitor; A TrR , angiotensin-II type 1 receptor; HSCs, he-patic stellate cells; TGF-/3> trans formi ng growt h factor/31; ARB, angio tensin -II type 1recept or blocker; PE, perindop ril; CA, candesartan; PBS, phos phate-b uffered saline;c~-SMA, R-smo oth muscle actin; ALT, alanine tra nsamin ase; T Bil, total bilirubin; P-Ill-P,procollagen III-N-peptide; mRNA, messen ger RNA; PCR, polymeras e chain reaction;VEGF, vascular endothelial grow th factor.Fro m the Third Depart ment of Internal Medicine, Nara Medical University, Kashi-hara, Nara, Japan.

Received January 22, 2001; accepted July 30, 2001.S.K.'s present address is at the T hird D epartment of Internal Medicine, Kagawa Med-ical University, Kagawa, Japan.Address reprint requests to: Hitoshi Yoshiji, M.D., Ph.D., Third Department of Inter-

nal Medicine, Nara Medical University, Shijo-cho 840, Kashihara, Nara 634-8522,Japan.E-marl: [email protected]; fax: (81)-744-24-7122.

Copyright 2001 by the American Association for the Study o f Liver Diseases.0270-9139/01/3404-0018535.0010doi:10.1053/jhep.2001.28231

Angiotensin-I I (AT-II ) , whi ch i s an octapep t ide produ cedmain ly by proteoly t ic c leavage of it s precurso r AT-I by angio-t ens i n - conver t i ng enzyme (ACE ) , has many phys i o l og i c e f -fects , including vascular hormonal secret ion, t i ssue growth,and neu rona l act ivit ies. 4,5 AT-II i s a l so co nsidere d a poten t ia lmedi a t o r o f i n tr ahepa t i c por t a l hyper t ens i on , because i tsplasma level was increased in pat ients wi th c i r rhosis , and i t sadmin is t ra t ion indu ced elevat ion of the por tal pressure . 6 ,r Todate , several types of AT-II receptors have been ident i f ied. 5The AT-II type 1 recepto r (AT1-R) mediates m ost o f the bio-logical effects of AT-II , including increase in the int racel lularCa 2+ concent rat io n, cel l cont rac t ion, and prol i ferat ion. 5 Re-cent ly, losar tan, whic h i s used cl inically as an AT1-R antago-n i s t, has been shown t o r educe t he por t a l p r es sure i n hepa t i cci r rhosis, s I t has bee n revealed tha t AT-II indu ced cont rac t ionand proliferation of hepatic stellate ceils (HSCs).9 AT-II alsoincreased the transforming growth factor t3x (TGF-/31) andcol lagen-I gene express ion in the f ibroblas ts in vi tr o} -~2T hese b i o l og i ca l ac t i ons were medi a t ed p r edomi nan t l ythrough AT1-R. Accordingly, AT-II and AT1-R interact ionseems to have a pivotal role in l iver f ibrosis development .E v i dence t ha t t hese mol ecu l es a r e i mpor t an t in vivo in thedev elop men t of hepat ic f ibrosis , however , i s not avai lable .In the presen t s tudy, we exa mine d the ef fect of inhibi t ingthe RAS, especially the AT-II and AT1-R interaction, on l iverf ib rosi s i nduced by admi n i s t r a t ion o f p ig se rum. W e usedcl inically avai lable compo und s: an ACE in hibi tor (ACE-I) andan AT rR b l ocker (ARB) a t t he r apeu t i c concen t r a t i ons . W ealso examin ed the ef fect of these agents on HSC act ivat ion andthe hepa t ic TGF-/31 level dur ing l iver f ibrosis developm ent .

MATERIALS AND METHODSAnimals. Male Fischer 344 rats, aged 6 weeks, were purchasedfrom Japa n SLC Inc. (Hamam atsu, Shizuoka, Japan). The y werehoused in stainless steel, mesh cages und er co ntrol conditions oftemperature (23C _+ 3) and relative hu midity (50 % + 20%), with l0to 15 air changes per h our a nd light illumination for 12 hours a day.After a 1-week acclimatization period, the rats were divided into 4experimental groups. The animals were allowed access to food andtap water ad libitum throug hou t the acclimatization and experimen -

tal periods.Compounds and Animal Treatment. The ACE-I, perindopril (PE),and ARB, and candesartan (CA) were supplied by Daiichi Pharma-ceutical Co. (Tokyo,Japan) a nd Takeda Pharmaceutical Co. (Tokyo,Japan), respectively. The pig serum was purchased from Cosmo Bio.Co. (Tokyo , Japan). The same lo t of serum was used in all experi-ments. The rats were randomly divided into 6 groups. Groups 1 to 5(G1-5) received 0.5 mL of pig serum intraperitoneally twice weeklyfor 8 weeks. G1 was given the basal diet througho ut the experimen t,and was designed as a con trol group. One we ek after the pig seruminjection, the exp erime ntal animals in G2 and 3 started to receive PE745


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74 6 YO SH IJI ET AL HEPATOLOGYOctober 2001a n d C A b y g a v a g e o n c e a d a y a t a d o s e o f 8 m g / k g , w h i c h i s a c l i n i c a ll yc o m p a r a b l e d o s e f o r b o t h c o m p o u n d s . 1 3,14 W e a l s o e x a m i n e d t h ee f f ec t o f P E a n d C A o n t h e o n - g o i n g f i b ro s i s m o d e l . F o u r w e e k s a f t ert h e p i g s e r u m i n j e c t io n , t h e a n i m a l s i n G 4 a n d G 5 s t a r t e d t o r e c e iv eP E a n d C A i n t h e s a m e w a y a s G 2 a n d G 3 . T h e r a t s i n G 6 r e c e i v e d ap h o s p h a t e - b u f f e r e d s a l i n e ( P B S) i n j e c t i o n i n s t e a d o f t h e p i g s e r u ma n d w e r e g i v e n t h e b a s a l d i e t. A t t h e e n d o f a l l t h e e x p e r i m e n t s , a l lr a t s w e r e k i l le d u n d e r e t h e r a n e s t h e s ia , a n d e x a m i n e d f o r t h e s t u d yi t em s . A l l a n i m a l p r o c e d u r e s w e r e p e r f o r m e d a c c o r d i n g t o a p p r o v e dp r o t o c o l s a n d i n a c c o r d a n c e w i t h t h e r e c o m m e n d a t i o n s f o r t h ep r o p e r c a r e a n d u s e o f l a b o r a t o r y a n i m a l s.HistopathologicandlmmunohistochemicalExaminations. F i v e - m i c r o m -e t e r - t h i c k s e c t io n s o f f o r m a l i n - f i x e d a n d p a r a f f i n - e m b e d d e d l i v e rsw e r e p r o c e s s e d r o u t i n e l y f o r h e m a t o x y l i n - e o s i n a n d S i r i u s - r e d s t a i n -i n g . ~5 I m m u n o h i s t o c h e m i c a l s t a i n i n g o f c r s m o o t h m u s c l e a n t i g e n( a - S M A ) w a s p e r f o r m e d a s p r e v i o u s l y d e s c r i b e d w i t h p a r a f f i n - e m -b e d d e d se c t i o n s . 1 6,1 7 B r i e f ly , fo rm a l i n - f i x e d t i s su e s e c t i o n s we red e p a r a f f i n i z e d u s i n g x y l e n e a n d r e h y d r a t e d b y p a s s i n g t h r o u g h 9 9 % ,9 5 % , 7 0 % , 5 0 % , a n d 3 0 % e t h a n o l . N o n s p e c i f ic a n t i b o d y b i n d i n g w a sb l o c k e d w i t h s e r u m d i l u t e d 1 : 5 0 in P B S , a n d t h e e n d o g e n o u s b i o t i nw a s b l o c k e d w i t h 0 . 1 % a v i d i n f o r 1 5 m i n u t e s , f o l l o w e d b y 0 . 0 1 %b i o t i n f o r 1 5 m i n u t e s . T h e s e s e c t i o n s w e r e w a s h e d o n c e w i t h P B Sa f te r e a c h 1 5 - m i n u t e i n c u b a t i o n p e r i o d , t h e n r e a c t e d w i t h a p r i m a r ya n t i - ~ - S M A a n t i b o d y ( D A K O , K y o t o , J a p a n ; 1 : 1 0 0 ) f o r 1 h o u r a tr o o m t e m p e r a t u r e . A f t e r w a s h i n g t w i c e w i t h P B S , th e s e c t i o n s w e r ei n c u b a t e d w i t h a s e c o n d b i o t i n - l a b e l e d r a b b i t a n t i - r a t I g G ( N o v o c a s -t r a , B u r l i n g a m e , C A ) . T h e e n d o g e n o u s p e r o x i d a s e w a s b l o c k e d w i t h0 . 3 % h y d r o g e n p e r o x i d e i n P B S f o r 3 0 m i n u t e s , f o l l o w e d b y5 - m i n u t e w a s h i n g w i t h P B S. T h e s e c t i o n s w e r e t h e n i n c u b a t e d w i t hc o n j u g a t e d s t r e p t a v i d i n f o r 3 0 m i n u t e s , r i n s e d a g a i n i n P B S , a n df i n a ll y i n c u b a t e d w i t h d i a m i n o b e n z i d i n e f o r 3 m i n u t e s . S e m i - q u a n -t i ta t i v e a n al y s e s o f f ib r o s i s d e v e l o p m e n t a n d i m m u n o p o s i t i v e c e l la r e a w e r e c a r r i e d o u t w i t h F u j i - B A S 2 0 0 0 i m a g e a n a l y z i n g s y s t e m( F u j i , T o k y o , J a p a n ) i n 6 o c u l a r f i el d s ( m a g n i f i c a ti o n 4 0 ) p e r s p e c -i m e n o f 7 r a t s. W e d i d n o t c o u n t t h e c ~ - S M A - p o s i ti v e v e s s e ls i n t h ep o r t a l a r e a , w h i c h w e r e a s s u m e d t o b e h e p a t i c a r t e r i e s . W e o n l yi n c l u d e d o ~ - S M A - p o si ti v e c e l ls i n t h e s i n u s o i d a l l i n i n g f o r i m a g ea n a l y s i s .Serum Markers. A t t h e e n d o f t h e e x p e r i m e n t , s e r u m s a m p l e s w e r eo b t a i n e d f r o m t h e a b d o m i n a l a o r t a . T h e a l a n i n e a m i n o t r a n s f e r a s e( A L T ) a n d t o t a l b i l i r u b i n ( T B i l ) w e r e a s s e s s e d u s i n g t h e r o u t i n el a b o r a t o r y m e t h o d . T h e s e r u m m a r k e r s f o r f ib r o s i s d e v e l o p m e n tn a m e l y , 7 S - c o l l a g e n , p r o c o l l a g e n I I I - N - p e p t i d e ( P - I I I -P ) , a n d h y a l -u r o n i c a c i d w e r e a l s o m e a s u r e d a s p r e v i o u s l y d e s c r i b e d . 18Hepatic Hydroxyproline Content and TGF-IJ1 Expression. T h e h e p a t i ch y d r o x y p r o l i n e c o n t e n t w a s d e t e r m i n e d a s p r e v i o u s l y d e s c r ib e d . 19B r ie f ly , l i v e r s p e c i m e n s w e r e w e i g h e d , a n d 2 0 m g o f f r o z e n s a m p l e sw e r e h y d r o l y z e d i n 6 m o l / L H C 1 i n a n a u t o c l a v e f o r 2 4 h o u r s . A f t e rc e n t r i fu g a t i o n , t h e s u p e r n a t a n t w a s m i x e d w i t h 1 % p h e n o l p h t h a l e i na n d 8 N - K O H t o o b t a i n a s o l u t i o n a t p H 7 t o 8 . T h i s s o l u t i o n w a ss t i r r e d w i t h K C 1 a n d b o r a t e b u f f e r ( p H 8 . 2 ) f o r 1 5 m i n u t e s a t r o o mt e m p e r a t u r e a n d f o r a n o t h e r 1 5 m i n u t e s a t 0 C, a f t er c h l o r a m i n e Ts o l u t i o n w a s a d d e d a n d s t i r r e d f o r 6 0 m i n u t e s a t 0 C. A f t e r a d d i t i o no f 3 .6 m o l / L s o d i u m t h i o s u l f at e , t h e s o l u t i o n w a s i n c u b a t e d f o r 3 0m i n u t e s a t 1 2 0 C a n d s t i r r e d w i t h t o l u e n e f o r 2 0 m i n u t e s . T h e n ,E h r l i c h ' s s o l u t i o n w a s a d d e d t o t h e s u p e r n a t a n t a f t e r c e n t r i f u g a t io na t 2 , 0 0 0 r p m a t 4 C a n d l e f t f o r 30 m i n u t e s a t r o o m t e m p e r a t u r e . T h ea b s o r b a n c e w a s m e a s u r e d a t 5 6 0 n m . T h e h y d r o x y p r o l i n e c o n te n twa s e x p re s se d a s / * g / g we t l i v e r . Th e TGF-J$ 1 e x p re s s i o n l e v e l i n t h el i v e r w a s m e a s u r e d b y t h e P R E D I C T A a s s a y k i t ( G e n z y m e C o . , C a m -b r i d g e , M A ) . L i v e r l y s a te s w e r e p r e p a r e d a s d e s c r i b e d p r e v i o u s l y ,2a n d t h e s a m p l e s w e r e e q u a l i z e d f o r p r o t e i n c o n c e n t r a t i o n p r i o r t ome a su r i n g t h e TGF-1 3 1 l e v e l . Al l TGF-1 3 1 p ro t e i n l e v e l s we re a s se s se da s t h e a c t i v e f o r m b y a d d i t i o n o f H C1 21 a n d w e r e e x p r e s s e d a s n g / m go f t h e t o t a l li v e r p r o t e i n .Isolation and Culture of HSCs. T h e l i v e r H S C s w e r e i s o l a t e d fr o m t h el i v e r o f F 3 4 4 r a t s a s d e s c r i b e d p r e v i o u s l y ,22 w i t h a m i n o r m o d i f i c a -t i o n . B r i e f ly , t h e l i v e r wa s p e r fu se d wi t h Kre b s ' -R i n g e r so l u t i o n fo l -l o w e d b y 0 . 1 % p r o n a s e E a n d 0 . 0 3 2 % c o l l a g e n a s e ( N a k a r a i , K y o t o ,

J a p a n ) s o l u t i o n a t 3 7 C . T h e d i g e s t e d l iv e r w a s e x c i s e d a n d m i n c e d ,a n d i n c u b a t e d i n K r e b s ' - R in g e r s o l u t i o n c o n t a i n i n g 0 . 0 8 % p r o n a s e E ,0 . 0 4% c o l l ag e n a s e , a n d 2 0 / ~ L / m L D N a s e f o r 3 0 m i n u t e s a t 3 7 C ( p H7 . 3 ). A f t e r p a s sa g e t h r o u g h a n y l o n m e s h , t h e c e l ls w e r e c e n t r i fu g e da t 4 5 0 g f o r 8 m i n u t e s . T h e H S C - e n r i c h e d f r a c t io n w a s o b t a i n e d b yc e n t r i f u g a t io n i n 8 .2 % N y c o d e n z ( N y c o m e d P h a r m a A S , O s lo , N o r -w a y ) s o l u t i o n a t 1 , 4 0 0 g f o r 2 0 m i n u t e s . T h e H S C s i n t h e u p p e r w h i t el a y e r w e r e w a s h e d b y c e n t r i f u g a t i o n a t 4 5 0 g f o r 8 m i n u t e s a n d s u s -p e n d e d i n D u l b e c c o ' s m o d i f i e d E a g l e m e d i u m c o n t a i n i n g 1 0 % f e t a lc a l f s e r u m , 1 0 0 U / m L p e n i c i l l i n , a n d 1 0 0 m U / m L s t r e p t o m y c i n . T h ec e l l v i a b i l i t y wa s o v e r 9 5 % a s d e t e rm i n e d b y t h e t ry -p a n b l u e e x c l u -s i o n t e s t . Th e c e l l s we re p l a t e d a t a d e n s i t y o f 5 1 05 c e l ls / mL o nu n c o a t e d 6 0 - r a m p l a s t ic d i s h es . A f t e r 5 d a y s i n c u l t u r e , H S C s b e c a m em y o f i b r o b l a s t - li k e w i t h r e d u c e d l i p i d v e si c le s a n d i n c r e a s e d i m m u -n o re a c t i v e c ~ -SM A,a n d 7 d a y s a f t e r p l a t i n g a l l o f t h e c e i l s we re w e l ls p r e a d a n d a - S M A p o s i t iv e .23 F r o m d a y 1 0 , t h e m e d i u m w i t h o rw i t h o u t t r e a t m e n t o f A T - I I ( 1 / , m o l / L ) ( N a k a l a i , K y o t o , J a p a n ) a n dC A ( 1 / ~ m o l / L ) w a s c h a n g e d e v e r y 2 4 h o u r s a n d c e l l c u l t u r e w a sc o n t i n u e d u n t i l d a y 1 2 .RNA Expression of TGF-lJl by Real-Time Polymerase Chain Reaction.T G F- 13 1 m e s s e n g e r R N A ( m R N A ) e x p r e s s i o n w a s e v a l u a t e d b y r e a l -t i m e p o l y m e r a s e c h a i n r e a c t i o n ( P C R ) a s d e s c r i b e d p r e v i o u s l y23-25f o r b o t h t h e in vitro a n d in vivo s t u d i e s. F o r t h e in vivo s t u d y , m R N Aw a s e x t r a c t e d f r o m t h e w h o l e l i v e r o f e a ch e x p e r i m e n t a l g r o u p ( n =5 ) . Fo r t h e in vitro s t u d y , m R N A w a s e x t r a c t e d f r o m H S C s o f t h eu n t r e a t e d c o n t r o l , A T - I I ( 1 / x m o l / L ) - t r e at e d a n d A T - I I p lu s C A ( 1/ x m o l / L ) - t re a t e d g r o u p s ( n = 4 e a c h ) . F o r c o m p l e m e n t a r y D N A s y n -t h e s is , T a q m a n r e v e r s e t r a n s c r i p t io n r e a g e n t s w e r e u s e d a s d e s c r i b e di n t h e m a n u f a c t u r e r ' s m a n u a l o f t h e A B I P r i s m 7 7 0 0 S e q u e n c e D e -t e c t i o n S y s t e m ( P E A p p l i e d B io s y s t e m s , F o s t e r C i t y , C A ) , w h i c h w a su s e d f o r r e a l - ti m e P C R a m p l i f i c a t i o n f o l l o w i n g t h e T a q m a n U n i v e r -s a l P C R M a s t e r M i x P r o t o c o l ( P E A p p l i e d B i o s y st e m s ) . R e la t iv eq u a n t i f i c a t io n o f g e n e e x p r e s s i o n w a s p e r f o r m e d a s d e s c r i b e d i n t h em a n u a l b y u s i n g g l y c e r a l d e h y d e - 3 -p h o s p h a t e d e h y d r o g e n a s e a s a ni n t e r n a l c o n t r o l. T h e t h r e s h o l d c y c l e a n d t h e s t a n d a r d c u r v e m e t h o dw e r e u s e d f o r c a l c u l a ti n g t h e r e l a ti v e a m o u n t o f t h e t a r g e t R N A a sd e s c r i b e d f o r P E. T h e f o l l o w i n g t e m p e r a t u r e s w e r e u s e d : h o l d 5 0 Cf o r 2 m i n u t e s , h o l d 6 0 C f o r 3 0 m i n u t e s , h o l d 9 4 C f o r 5 m i n u t e s ,c y c l e 4 5 re p e a t s 9 4 C fo r 1 mi n u t e , 5 5 C fo r 1 mi n u t e , 7 2 C fo r 1m i n u t e . T o p r e v e n t g e n o m i c D N A c o n t a m i n a t i o n , a ll R N A s a m p l e sw e r e s u b j e c t e d t o D N a s e I d i g e s t i o n a n d c h e c k e d b y 4 0 c y c l e s o f P C Rt o c o n f i r m t h e a b s e n c e o f a m p l i f i e d D N A .Statistical Analysis. To a s se s s t h e s t a t i s t i c a l s ig n i f i c a n c e o f i n t e r -g r o u p d i f f e re n c e s in t h e q u a n t i t a ti v e d a t a , B o n f e r r o n i ' s m u l t i p l ec o m p a r i s o n t e s t w a s p e r f o r m e d a f t e r o n e - w a y A N O V A , f o l l o w e d b yB a r l e tt ' s t e s t to d e t e r m i n e t h e h o m o l o g y o f v a r ia n c e .

R E S U L T SHistologic Findings and Fibrosis Markers. A s s h o w n i n F ig . 1 A ,S i r i u s - r e d s t a i n i n g s h o w e d t h a t 8 - w e e k t r e a t m e n t w i t h t h e p i g

s e r u m r e s u l te d i n a m a r k e d l iv e r fi b r os i s d e v e l o p m e n t ( G 1 ) .I n c o n t r a s t, t r e a t m e n t w i t h P E ( G 2 ) a n d C A ( G 3 ) s i g n i f i c a n t l ya t t e n u a t e d t h e f i b r o si s d e v e l o p m e n t ( F i g . 1 B a n d C , r e s p e c -t iv e ly ) c o m p a r e d w i t h G 1 . D e n s i t o m e t r i c a n a ly s i s s h o w e dt h a t t h e f ib r o s i s a r e a s w e r e m a r k e d l y s u p p r e s s e d i n t h e P E -a n d C A - t r e a t e d r a ts (P < . 0 1) . W e n e x t e x a m i n e d w h e t h e rt h e s e i n h i b i t o r y e f f e ct s c o u l d a l s o b e f o u n d i n t h e o n g o i n gf i b r o s is e x p e r i m e n t a l m o d e l . I n t h i s e x p e r i m e n t , P E a n d C Aw e r e a d m i n i s t e r e d 4 w e e k s a f t e r th e p i g s e r u m i n j e c t i o n . A ss h o w n i n F i g . l D a n d E , b o t h P E ( G 4 , 1 D ) a n d C A ( G 5 , 1 E )e x e r t e d s i g n i f i c an t i n h i b i t o r y e f f ec t s o n l i v e r f i b r o s is d e v e l o p -m e n t e v e n i n t h e o n g o i n g f i b r o s i s m o d e l . T h e d e g r e e s o f in -h i b i t o r y e f f ec t s i n G 4 a n d G 5 w e r e s i m i l a r t o G 2 a n d G 3 ,r e s p e c t i v e l y ( P < . 0 1 v s . c o n t r o l ) ( F i g . 2 ) . N o f i b r o s i s d e v e l -o p m e n t w a s f o u n d i n t h e P B S - t r e a t e d g r o u p ( G 6 ) . T h e l i v e rh y d r o x y p r o l i n e c o n t e n t a n d t h e s e r u m c o n c e n t r a t i o n s o f 7 4c o l l a g e n , P - I I I- P , a n d h y a l u r o n i c a c i d w e r e a l l s i g n i f i c a n t l y


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F1~. 1. Microphotographs of liversections from the pig serum-treatedrats. (A) Untreated control group(G1); (B and D) ACE-I (PE; 8 mg/kg/d) administration started 1 (G2)or 4 (G4) weeks after the pig seruminjection. (C and E) ARB (CA; 8 rag/kg/d) administration started 1 (G3)or 4 (GS) weeks after the pig seruminjection. The liver in G1 shows anextensive fibrosis development, butmild fibrosis in G2-5. (F) No fibrosisdevelopment was found in the PBS-treated group (G6) (Sirius-red stain-ing, original magnification )


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748 YOSHIJI ET AL HEPATOLOGY Oc to ber 2001TABLE 1. Effect of RAS Inhibi tor on Several Mar kers i n Animals Tr eated W ith Pig Serum

Hydroxypr oline Hyaluronic P-III-P T Bil(/~g/g) Acid (ng/mL) (ng/mL) 7S (ng/mL) ALT (U/L) (mg/mL)

Pig se ru m 396.4 2 104.1 107.4 _+ 27.7 31.2 _+ 6.2 133.2 + 23.4 66.2 2 12.1 0.2 + 0.1+P E 74.4 + 16.4" 30.8 + 10.7" 7.1 -+ 1.8" 32.2 -- 5.4* 56.6 -+ 12.1 0.2 -- 0.1+C A 62.2 + 4.1" 25.6 _+ 10.4" 6.6 -+ 1.4" 22.3 -+ 4.8* 63.5 -+ 15.4 0.1 -+ 0. iPBS 28.8 -+ 11.3" 3.7 -+ 0.7* ND ND 56.3 -+ 12.4 0.2 -+ 0. i

NOTE. Results are express ed as mean s + SD (n = 10). Abbreviation: ND, not determined.*Statistically significant difference as compared with the pig serum -tre ated gro up.

Effect ofRAS Inhibitors on TGF-~l Expression in the Liver. Sincei t has been show n that TGF-]3t acts on the act ivated HSCs inan au tocr ine man ner , 26 we e xam ined the ef fect of PE and CAtreatment on the TGF-t31 express ion level in the l iver . Weper formed enzyme- l i nked i mmunosorben t a s say t o eva l ua t ethe act ivated TGF-]3i protein express ion level . The TGF-t31protein concent rat ion in the whole l iver was s igni f icant lylower in the l iver of PE- t reated (G2 :28 .6 _+ 6.3 ng/mg wetl iver , n = 10) and CA -t reated (G3:24.2 + 4.1) ra ts than in thecont ro l ra t s (55.4 + 8.2) (P < .01, F ig. 4B). We also exam inedthe mR NA expres s ion level of TGF-t31 in the who le l iver . Asshow n in F ig. 5A, mRNA express ion of TGF-13I a lmost corre-spon ded to the p rotein level of TGF-]31 (F ig. 4B). The d egreeof TGF-]31 prote in a nd mRNA express ion level in the l iver ofeach group also corresponded to the number of c~-SMA-pos-Rive cells.

Effect of AT -II and AR B on TGF-[31 mRN A Expression in the Acti-vated HSCs In Vit ro. To exam ine whe ther AT-II and ARB (CA)affect the TGF-/31 exp ress ion in the act ivated HSCs, we per-form ed real - t ime PCR analysis . As shown in F ig. 5B, the m eanTGF-131 mRNA express ion increased 3.3-fold by the t re atmen twi th AT-II , and this ef fect was alm ost com pletely abol i shed byCA. These resul t s suggested that the AT-II and AT1-R interac-t ion plays an im por ta nt role in reg ulat ing TGF-]31 express ion

in act ivated HSCs. We also examin ed quiesc ent i solated HSCsand found that TGF-]31 mRNA was unal tered by AT-II (datano t shown) .D I S C U S S I O N

In this s tudy, we show ed that RAS inhibi to ry agents , ACE-I(PE) and ARB (CA), s igni ficant ly block ed he pat ic f ibrosis in-duced by p i g se rum. M oreover , t he se rum l evel s o f t hese i n -hibi tors af ter adminis t ra t ion of 8 mg/kg to ra ts were s imi lar tothose fou nd in clinical pra ctice. 13,14,27,2s The inhi bitor s we res tar ted 1 or 4 weeks af ter the pig serum t reatment , whichmodels the typical c l inical s i tuat ion bet ter than the adminis-t ra t ion of the inhibi tors a t onset of f ibrogenesis induct ion.F ur t he rmore , t hese i nh i b i to r s were e f fec t ive no t on l y i n t hep i g se rum- i nduced mode l bu t a l so i n f i b ros i s i nduced by achol ine-de f ic ient amino acid diet (data not shown) . In shor t ,the data ap pear qu i te re levant to c l inical pract ice .I t has been repor ted that another ACE-I , captopr i l , inhib-i ted the g rowth of f ibroblas ts in vitro. 29 I t a l so reduced col la-gen accumul a t i on i n t he p i g se rum- i nduced l i ve r f i b ros i smodel . 3 How ever , the d egree o f inhibi tory ef fect of captopr i lon hepa t i c hydro xypro l i ne accumul a t i on was a l mos t ha l f o fthat of PE and CA in the present s tudy. T he autho rs in thats t udy d i d no t exami ne t he poss ib l e i nvo l vemen t o f AT - II , and

FIG. 3. Immunohistochemical anal-ysis of c~-SMA. Immunoposi tivecells ofc~-SMA wer e significandy supp ressedin the liver of the PE-treated gro up (B)(G2) and CA-treated group (C) (G3)compared with the pig serum-treatedcontrol group (A) (G1). (Original mag-nification 40.) (D) Localization ofo~-SMA-positive cells in G1. Strong im-munopo sitiv e staining w as observed inthe fibrotic septa. (Original magnifica-tion 200.)


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i

= I

, ~ 20!rD

o Cont +PE +CA PBS

FI6.4. (A) Computer-assiste d semiquantitative analysis of a-SMA-posi-tire ceils in the liver, c>SMA-positive cells were significantly suppressed inthe PE-treated group (G2) and CA-treated grou p (G3) co mpared with the pigserum- treat ed control group (G1). (B) TGF-/31 protein expression levels inthe liver. The TGF-/31 protein concentration in the liver was significantlylower in the liver of the PE-treated group (G2) a nd CA-treated group (G3)compared with the pig serum-tr eated control group (G 1). The suppression ofTGF-/31 with treatme nt of PE and CA was of similar magnitude to i nhibitionof a-SMA-posit ive cells. The data rep resent means _+ SD. Each group con-sisted of 10 rats. *Statistically significant difference compared with the con-trol group (P < .01). Abbreviations: Cont, con trol rats (G1); PE, PE-treatedrats (8 mg/kg/d) (G2); CA, CA-treated rats (8 mg/kg/d) (G3).

t h e y s u g g e s t e d t h a t t h e a t t e n u a t i o n b y c a p t o p r i l w a s r e l a t e d t ot h e r e d u c t i o n o f m a s t c e l l s a n d e o s i n o p h i l a c c u m u l a t i o n i n t h eh e p a t i c t i s su e .A T - I I is n o w r e c o g n i z e d a s a m u h i f u n c t i o n a l p r o t e i n . 5 m I th a s b e e n r e p o r t e d t h a t A T - I I i n d u c e d i n a d o s e - d e p e n d e n tm a n n e r t h e v a s c u l a r e n d o t h e l i a l g r o w t h f a c t o r ( V E G F ) ,w h i c h i s o n e o f t h e m a j o r a n g i o g e n i c f a c t o r s . 32-34 T h e h e p a t i cV E G F l e v e l i n c h r o n i c l i v e r d i s e a s e s h a s b e e n s h o w n t o i n -c r e a s e w i t h d i s e a s e p r o g r e s s i o n . 35,36 R e c e n t l y , i t h a s b e e n r e -p o r t e d t h a t V E G F e x p r e s s i o n w a s s i g n if i c an t l y in c r e a s e d i nt h e e x p e r i m e n t a l l i v e r fi b r os i s m o d e l ) 7 V E G F s t i m u l a t e d H S Ca c t i v a t i o n a n d s i n u s o i d a l c a p i l l a r i z a t i o n ) 8,3 9 F u r t h e r m o r e , i th a s r e c e n t l y b e e n s h o w n t h a t a n t i - a n g io g e n i c c o m p o u n d sp r e v e n t t h e d e v e l o p m e n t o f e x p e r i m e n t a l l i v e r f i b r o s i s a n dH S C a c t iv a t io n . 4 W e p r e v i o u s l y r e p o r t e d t h a t P E e x e r t e d a na n t i - a n g i o g e n i c a c t i v i t y a n d t h a t i t s u p p r e s s e d V E G F e x p r e s -s i o n Y I t i s a l s o p o s s i b l e t h a t A T - I I i n h i b i t i o n b y P E a n d C A

angiotensin-ii type 1 receptor interaction is a major regulator for

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