Transient expression of tenascin in experimentally induced cholestatic fibrosis in rat liver: an immunohistochemical study

Journal of Hepatology, 1993; 19:353-366

353

© 1993 ElsevierScientific Publishers Ireland Ltd. All rights reserved. 0168-8278/93/506.00 HEPAT 01443

Transient expression of tenascin in experimentally induced cholestatic fibrosis in rat liver: an immunohistochemical study

Hiroshi Miyazaki, Peter Van Eyken, Tania Roskams, Rita De Vos and Valeer J. Desmet Department of Pathology. Laboratory of Histo- and Cytochemistrv. University Hospital St. RafaEI. Tire Catholic University of Leuven. Minderbroedersstraat 12. B-3000 Leuven. Belgium

(Received 26 February 1992)

This study describes the sequential changes in tenascin expression in hepatic fibrosis induced by bile duct ligation (BDL) in the rat. Two days after BDL, tenascin was strongly expressed in the matrix surrounding interlobular bile ducts and also between proliferating ductules. From day 7 onwards, its distribution was restricted to the connective tissue-parenchymal interfaces where ductular proliferation was still active. A markedly increased number of desminand a-smooth muscle actin (o/-smA)-positive cells, considered myofibroblasts, was noted around interlobular bile ducts and between proliferating ductules during periductal fibrogenesis. Type IV collagen and laminin were strongly expressed on the basement membranes of proliferating ductules, and contributed to the development.of newly formed fibrous septa. The transient expression of tenascin around interiobular bile ducts in the early phase of BDL may be related to the onset of periductal fibrosis or to the mitogenic response of the biliary epithelium. The expression of tenascin between 'proliferating' ductules in contrast to its absence from 'mature' fibrous areas suggests a transient role in early matrix organization. Furthermore, o~-smA-positive ceils may modulate the synthesis of extracellular matrix components. Key words: Hepatic fibrosis; Tenascin; Myofibroblast; Matrix organization; Epithelial-mesenchymal interaction

In recent years, knowledge about the molecules of the extracellular matrix (ECM) has greatly increased (1-3). Numerous studies have revealed that the ECM is not only a structural support, but is also capable of influencing cell growth and differentiation. In the liver, the ECM modulates the morphology and function of epithelial cells (2-6) and mesenchymal cells (6). In altered physiological states, the ECM may change dramatically by replacement or restructuring of the normal ECM constituents. The altered ECM may differ in its biological activities from the normal one and be re-

sponsible in part for the disturbance of organ-specific functions associated with pathological fibrosis. The latter is a salient feature of the hepatic response to various injuries. Tenascin - - originally described as myotendinous antigen - - is an ECM glycoprotein with restricted tissue distribution both in embryonic and adult tissues (7-11). It has been suggested that tenascin may play a role in ECM organization (12-14), in epithelial-mesenchymal interactions during embryogenesis (7), in the migration of cells (11,15), and in cell attachment (16). Tenascin can

Correspondence to: Dr. Hiroshi Miyazaki, First Department of Internal Medicine. The Jikei UniversitySchool of Medicine. 3-25-8 Nishishinbashi.

Minato-ku, Tokyo 105, Japan. The results of this study were presented in part at the International Falk Symposium'Molecular and Cell Biologyof Liver Fibrogenesis'. 22-23 January 1992, Marburg, Germany. Abbreviations: BDL, bile duct ligation; ECM, extracellular matrix: FSCs. fat-storing cells: u-smA, u-smooth muscle actin; SMC, smooth muscle cells.

354 also interfere with the attachment of cells to fibronectin (17) and may exert immunomodulatory activities (18). We recently demonstrated that tenascin is a component of the ECM of normal human and rat livers and that changes in distribution occur in diseased human liver and carbon tetrachloride (CCI4)-induced hepatic fibrosis in the rat (19,20). Furthermore, evidence was provided that fat-storing cells (FSCs) are the most likely source of tenascin in normal and fibrotic rat livers (20,21). Hepatic fibrosis following bile duct obstruction is different from fibrosis induced by inflammation or treatment with CCI 4 (22), although the quantitative increase in collagen in terms of hydroxyproline content may be similar (23). With bile duct ligation (BDL), parenchymal cell necrosis is generally not extensive, especially in its early phase, and a fibrotic reaction in the centrilobular region is lacking (22). Hepatic fibrosis following BDL is portal in origin and closely related to the development of ductular proliferation (23,24). However, it has not been fully elucidated how the sequential changes in hepatic ECM formation occur, and which cells may be involved in ECM production in the absence of parenchymal cell necrosis and inflammation. In this study, we attempted to find out whether changes in the distribution of tenascin also occur in hepatic fibrosis induced by BDL. In addition, the distribution of u-smooth muscle actin (~-smA)-positive cells was studied in relationship to the presence of tenascin in the ECM. It has indeed become clear that ~-smA may serve as a marker for 'activated' FSCs (25-27).

Materials and Methods

Experimental animals Male Fischer rats weighing 130-150 g were fed standard laboratory chow with water ad libitum in a temperature-controlled room. Under intraperitoneal nembutal anesthesia, the common bile duct was exposed by blunt dissection, doubly ligated and sectioned between two ligatures as previously described (28). Normal and sham-operated rats were used as controls. Sham-operated rats underwent laparotomy, but the common bile duct was not ligated. At 6 h and 1, 2, 3, 7, 14, 21, 28 and 35 days after BDL or sham operation, 2 rats of each group were sacrificed under ether anesthesia.

Processing of liver tissue Biopsies were taken from the right liver lobe. Three blocks were fixed in Bouin's solution, B5-fixative and

1-1.MIYAZAKI et al. TABLE l Antibodies used in this study Antibodies

Source

Working dilution

Monoclonal antibodiesa Desmin a-Smooth muscle actin Cytokeratin-7 Cytokeratin-19

Boehringer Sigma Boehringer Amersham

1:50 1:200 I: I0 I : 10

Polyclonal antibodiesa Tenascin Collagen type IV Laminin

Dr. Chiquet-Ehrismann 1:50 Eurodiagnostics I:10 Eurodiagnostics 1:50

~AII antibodies were diluted in phosphate buffer saline (PBS, pH 7.2).

6% formalin, respectively, embedded in paraffin, and used for routine histological examination. Small blocks were snap-frozen in liquid nitrogen-cooled isopentane, and stored at -70°C until use for immunohistochemistry.

Immunohistochemical stainings Serially cut, 5-tzm thick cryostat sections were fixed in absolute acetone for 10 min, and stained with polyclonal and monoclonal antibodies listed in Table I. The polyclonal rabbit anti-chicken tenascin antibody (kindly provided by Dr. R. Chiquet-Ehrismann, Friedrich Miescher Institute, Basel, Switzerland) has been shown to cross-react with rat tenascin (7). The monoclonal antibodies specific for cytokeratin (CK)-7 and CK-19 were applied to decorate bile ductular cells (29,30). Monoclonal antibodies specific for desmin and c~-smA were used to identify FSCs (31) and myofibroblast-like cells (25-27), respectively, The polyclonal antibodies were used in an unlabelled antibody enzyme method (PAP). Incubation with the primary polyclonal antibody for 30 min at room temperature was followed by swine anti-rabbit immunoglobulins (DAKO, diluted 1:20), and finally by rabbit peroxidase-antiperoxidase complex (DAKO, diluted 1:300). Monoclonal antibodies were used in a 3-step indirect immunoperoxidase method. Sections were incubated with the primary monoclonal antibody for 30 min at room temperature. The secondary and tertiary antibodies consisted of peroxidaseconjugated rabbit anti-mouse and peroxidaseconjugated swine anti-r.abbit immunoglobulins, respectively (both obtained from DAKO, diluted 1:50 and 1:100, respectively). Incubations with the secondary and tertiary antibodies were carried out for 30 min at room temperature. The secondary antibodies were preabsorbed with rat liver powder (Sigma). The reaction

TRANSIENT EXPRESSION OF TENASCIN IN LIVER

product was developed by incubating sections in 3amino-9-ethylcarbazole/H202 solution for 10 minutes at room temperature. Sections were counterstained with Mayer's haemalum. Controls, consisting of the omission of the primary antibody, were always negative.

Results

Liver histology and CK-7 and CK-19 immunostaining The histological changes of the liver following BDL in the rat have been well-described (32,33). The reported speed of progression to secondary biliary cirrhosis varies widely (34). In the livers of control (normal and sham-operated) rats, no morphological abnormalities were seen. In bile duct-ligated rats, there were no obvious histological changes 6 h after BDL, compared with controls. At day 1, the cellularity in medium and large portal tracts was slightly increased, but this finding was not uniform. Portal connective tissue was slightly edematous. Biliary epithelial cells were tall and irregular, and the nuclei were larger" and more crowded. At day 2, ductular proliferation with mild infiltration of polymorphonuclear cells was observed in portal tracts, and was prominent at the margin of portal tracts at day 3. The epithelial cells of proliferating ductules showed irregularity and mitoses. In the liver parenchyma, small foci of necrosis appeared at day 2 and enlarged afterwards. Some hepatocytes showed mitotic figures, but this finding was less common after day 2. Sinusoidal cellularity was slightly increased. From day 7 onwards, the proliferating ductules extended into the liver parenchyma, accompanied by fibrous expansion of the portal tracts. The cellularity in the sinusoids was markedly increased. On day 14, periportal and concentric periductal fibrosis with rather dense collagenous fibers was noted, while collagenous fibers appeared loose around actively proliferating ductules. Bile infarcts characterized by necrosis of hepatocytes and accumulation of fibrin were observed at day 21. At day 28, the proliferating ductules had almost replaced the liver parenchyma, and occasionally connected neighbouring portal tracts. The portal-portal connections from proliferating ductules accompanied by newly formed fibrous septa were best seen on Sirius Red stained slides. As a result, the normal Iobular architecture was disorganized. Bilirubinostasis was not observed. CK-7 and CK-19, which decorate strongly bile ductular cells (29,30), were consistently expressed by both normal and proliferating biliary structures. In bile duct-ligated rats, 'proliferating' ductules were

355

strongly immunoreactive for CK-7 and CK-19 (Fig. 1). In addition, a few small ductular cells located at a various distances from portal tracts and at least one hepatocyte neighbouring a portal tract was positive for CK-7 and CK-19 at all experimental stages.

Tenascin immunostaining In control rats, staining for tenascin was discontinuous along the sinusoidal walls. Occasionally, perisinusoidal lining cells with fat droplets in their cytoplasm revealed positive staining. Reactivity on the walls of centrilobular veins ranged from negative to weakly positive. Portal connective tissue was negative (Fig. 2a). This staining pattern for tenascin in normal rat liver has already been reported (20). In bile duct-ligated rats, an accumulation of tenascinimmunoreactivity was observed in the matrix surrounding interlobular bile ducts at day 1 after BDL. At day 2 and 3, tenascin was more intensely expressed in the matrix both surrounding interlobular bile ducts and between proliferating ductules at the margin of portal tracts. The interfaces between portal tract and liver parenchyma were also positive for tenascin (Fig. 2b). In the liver parenchyma, some accumulation of tenascin was occasionally present in areas of spotty necrosis and increased cellularity in sinusoids. Reactivity on sinusoidal walls was increased. At day 7, the positivity of the matrix between proliferating ductules had obviously decreased, compared with the positivity at day 2 and 3. Tenascin was still intensely expressed at the interfaces between portal tract and liver parenchyma (Fig. 2c). Portal connective tissue was weakly positive. Reactivity on sinusoidal walls and perisinusoidal lining cells was more easily detectable. Occasionally, enhanced tenascin staining on sinusoidal walls was observed and decorated the extended cytoplasmic processes of tenascin-positive sinusoidal cells. Some of these cells had fat droplets in their cytoplasm. From day 14 onwards, the distribution of tenascin was more restricted to and most intense at the connective tissue-parenchymal interfaces where ductular proliferation was still active. Increased positivity on all sinusoidal walls remained. Desmin immunostaining The distribution of desmin positivity in control livers was similar to that previously described (31,35) (Fig. 3a). At 6 h and at 1 day after BDL, perisinusoidal desminpositive cells increased slightly in the periportal regions. An increased number of desmin-positive cells were noted both in portal tracts and between proliferating

356

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Fig. 1. Biliary epithelial cells decorated by CK-19. F o l l o w i n g BDL, CK-19-positive d u c t u a l r structures extended from the portal tracts into the liver parenchyma. (a, control; b, at day 35) (Indirect i m m u n o p e r o x i d a s e , counterstained with Mayer's haemalum, magnification x95.

357

TRANSIENT EXPRESSION OF TENASCIN IN LIVER

ductules at days 2 and 3, compared with the control liver. Desmin-positive cells also increased in the lobules, especially in acinar zone 1. From day 7 onwards, numerous desmin-positive cells were seen in the portal tracts and between proliferating ductules extending into the liver parenchyma. This finding correlated closely with the ductular proliferation accompanied by periductal and periportal fibrosis (Fig. 3b). Unlike the matrix surrounding actively proliferating ductules, with the development of periductal fibrosis the concentric layers of fibrosis around interiobular bile ducts contained less desmin-positive cells. In the liver parenchyma, desminpositive cells accumulated in areas of spotty necrosis. Tenascin was also observed in serially cut sections, as well as in the periportal regions where ductules were actively proliferating. Using serial sections, the staining pattern for tenascin along the sinusoids seemed to be the similar pattern observed for desmin.

ct-smA staining In control rats, vessel walls in the portal tracts were stained for c~-smA. Positivity of the walls of hepatic vein branches was discontinuous. A~ small number of the mesenchymal cells in the large portal tracts was positive for t~-smA. No positivity was seen in the parenchyma (Fig. 4a). In bile duct-ligated rats, a small number of perisinusoidal c~-smA-positive cells was present in periportal regions at 6 h after BDL. Perisinusoidal asmA-positive cells were clearly less numerous than perisinusoidal desmin-positive cells. At day 1, ct-smApositive cells were present around interlobular bile ducts. The same area was weakly positive for tenascin. At day 2 and day 3, t~-smA-positive cells markedly increased in the portal tracts and accumulated in the matrix surrounding interlobular bile ducts where tenascin was intensely expressed. ~-smA was weakly expressed between the proliferating ductules (Fig. 4b). From day 7 onwards, the expression of ct-smA was more intense between proliferating ductules than at day 2 and day 3. a-smA was still expressed on the cells accumulating around interlobular bile ducts and ductules with 'immature' concentric periductal fibrosis more intensely than desmin. As periductai fibrosis progressed, the expression of et-smA gradually decreased. At day 21, slight positivity on sinusoidal walls was observed throughout the acini. Afterwards positivity became more pronounced near the edge of active ductular proliferation (Fig. 4c). c~-smA was expressed in some areas of parenchymal cell necrosis where positive staining for tenascin and desmin was also found. Immunoreactivity for c~-smA was less intense than for desmin in these areas. The dis-

tribution of t~-smA-positive cells in relationship to the presence of tenascin in the ECM is summarized in Table 2.

Collagen type IV and laminin immunostainings In control rats, basement membranes of blood vessels and bile ducts were immunoreactive for collagen type IV and laminin. Sinusoidal reactivity for collagen type IV was detectable, but ranged from negative to weakly positive for laminin (Fig. 5a). In bile duct-ligated rats, collagen type IV was strongly expressed on the basement membranes of proliferating ductules and interlobular bile ducts throughout the experiment (Fig. 5b). Collagen type IV-positive layers corresponded well to the development of ductular proliferation and newly formed fibrous septa. Sinusoidal reactivity for collagen type IV became more pronounced with the increasing duration of biliary obstruction. Portal connective tissue was weakly positive for collagen type IV. The proliferating ductules with collagen type IV-positive layer replaced the liver parenchyma in the later phase. The staining pattern for laminin was similar to collagen type IV except for immunoreactivity on the sinusoidal walls, where laminin was expressed less than collagen type IV. When the serial sections stained for CK-7 and CK-19 were compared, at least one liver parenchymal cell without lumen was found at a certain distance from the portal tracts. These cells were positive for CK-7 and CK-19 and surrounded by a thin layer which was positive for both collagen type IV and iaminin. Desminpositive cells accumulated in these areas and the matrix nearby was positive for tenascin, c~-smA expression was almost never seen at day 2 and day 3, and was detectable from day 7 onwards.

Discussion

In this immunohistochemical study, dramatic changes in the expression of tenascin were demonstrated following BDL. At day 2 after BDL, tenascin was intensely expressed in the matrix surrounding the interlobular bile ducts and also between the proliferating ductules at the margin of the portal tracts. From day 7 onwards, the distribution of tenascin was limited to the connective tissueparenchymal interfaces at the 'front' of the ductular proliferation. Several studies have shown that tenascin has a much more restricted spatial and temporal pattern during the development of various organs than other

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Fig. 2. The expression of tenascin in rat liver following BDL. In control liver, the portal connective ttssue was negative for tenascin. (a, control. ×237.5) At day 2, tenascin expression was intense around interlobular bile ducts and between proliferating ductules (arrows). (b, at day 2, x237.5) At day 7, the immunoreactivity for tenasein was restricted to the interfaces between porliferating ductules and liver parenchyma (arrows). (c, at day 7, x380) (Indirect immunoperoxidase, counterstained with Mayer's haemalum.)

ECM glycoproteins such as fibronectin or laminin (36). Since tenascin accumulates predominantly in the mesenchyme around actively growing epithelium, it has been suggested that this glycoprotein is important in epithelial-mesenchymal interactions during morphogenesis (7). In contrast to embryonic tissues, tenascin expression often decreases in normal adult tissues and its spatial distribution changes (7,8,37), while in pathological conditions in adult tissues a prominent reexpression of tenascin has been noted in the mesenchyme (7,19,36,38-40). In vitro experiments suggest that proliferating epithelium induces tenascin production by mesenchymal cells (36). The results of this study on tenascin expression following BDL support the hypothesis that actively growing, migrating and differentiating epithelium may locally produce active factors to stimulate tenascin synthesis in the nearby mesenchyme (41). Although tenascin remained present at the interface between proliferating ductules and liver parenchyma, it was no longer present in the 'mature' fibrous areas. This suggests that tenascin may play a transient role in early matrix organization (19). Previous studies on tenascin expression in CCl4-induced hepatic fibrosis (20) and pathological human livers (19) also support this hypothesis. Finally, in healing skin wounds, tenascin is

no longer detectable in the fibrous scar (36). The transient expression of tenascin around the interlobular bile ducts, located in the center of the portal tracts, is intriguing and might be related to the onset of periductal fibrosis or to the mitogenic response of the biliary epithelium to BDL. Using in situ hybridization, Nakatsukasa and colleagues (42,43) have recently demonstrated abundant transcripts of transforming growth factor (TGF)-~ in cells in fibrous septa following CCl4-administration in rat. Ramadori et al. (44) reported that TGF-~I transcript distribution resembles tenascin distribution as detected by immunohistochemistry in the CCI4-treated rat liver. Pearson et al. (45) and Chiquet-Ehrismann et ai. (46) have shown an increase in tenascin secretion by chick embryo fibroblasts after TGF-/3 treatment. It is therefore tempting to speculate whether TGF-/3 is also one of the local factors stimulating the synthesis of tenascin in the nearby mesenchyme following BDL. Recently, 'fibroblastic' cells have been found to express a repertoire of muscle differentiation features in both physiological conditions and pathological settings characterized by tissue remodeling and fibrosis (47), and to exhibit morphological (48) and biological (49) pr °perties which intermediate between fibroblasts and

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Fig. 3. The distribution of desmin in the time course of BDL. In control liver, desmin-positive cells were irregularly distributed in portal tracts. Vessel walls were positive for desmin. (a. control rat liver, x237.5) Numerous desmin-positive cells were seen in portal tracts, in periportal regions, and between proliferating ductules extending into the liver parenchyma, correlating closely to the ductular proliferation after BDL accompanying periductal and periportal fibrosis. (b, at day 7, x237.5) (Indirect immunoperoxide, counterstained with Mayer's haemalum.)

T R A N S I E N T EXPRESSION OF TENASCIN IN LIVER

smooth muscle cells (SMC) (47). 'Fibroblastic' cells with SMC differentiation appear transiently in the granulation tissue of normally healing wounds (50), and more permanently in chronic pathological settings such as liver fibrosis, thus suggesting that differences in fibroblastic phenotypes might be related to differences in biological behaviour (47). It has been suggested that FSCs belong to the same cell lineage as fibroblasts and myofibroblast precursor cells, due to functional and morphological similarities (51,52). There are several studies on the permutations of vimentin, desmin and usmA expression on the 'fibroblastic' cells of the liver (25-27,35,48,53). 'Fibroblastic' cells in the various organs, including the liver (54), are thought to express vimentin (47). Yokoi and colleagues (31,53) have demonstrated that desmin, which is an intermediate filament protein predominantly expressed in myogenic cells, can be found in the cytoplasm of FSCs - - even in so-called empty FSCs or activated FSCs - - and suggested that these cells belong to the myogenic cell lineage and develop into myofibroblasts (53). Burt et al. (35) found desmin-positive cells around the hepatic vein branches and distributed irregularly within the portal tracts in the normal rat liver. The perivenular cells may represent myofibroblasts which have been shown to have a predominantly perivenular distribution in the human and baboon liver (55,56), and which have been isolated from the rat liver (57). The nature of the stellate desmin-positive cells in the portal tracts, however, is still uncertain. In CCl4-induced liver injury, the desminpositive cells are probably equivalent to the so-called transitional cells between classical FSCs and fibroblasts, since these cells exhibit many of the ultrastructural features of FSCs, including the presence of microfilaments (56,58). Mak et al. (58) have drawn attention to ultrastructural similarities between myofibroblasts and FSCs and suggested that the two cell types are related. Recently, c~-smA has been shown to be a specific marker of smooth muscle differentiation (59,60), and a good marker for the detection of myofibroblast-like cells (26). Expression of ~-smA was found in cultured FSCs from the rat liver (54). The presence of o~-smA-positive cells has also been reported in fibrotic rat livers (25,26). a-smApositive cells always co-express desmin in vivo (25) and in vitro (54). Since Iobular desmin-positive cells are identified as FSCs in normal (31) and fibrotic rat livers (25,35,53), some of FSCs may undergo a phenotypic change and express a-smA in hepatic fibrosis (25). In hepatic fibrosis, myofibroblasts have been found in fibrotic bands (61), and perivenular areas (56). The distribution of these cells is similar to that of a-smApositive FSCs (25). FSCs can probably differentiate into

361 myofibroblasts and a transition which is reflected by the expression of ct-smA (26,27). The appearance of ct-smA in liver mesenchymal cells seems closely related to the active process of hepatic fibrogenesis in both the rat and man, and can be detected earlier than overt hepatic fibrosis (26,27). Ramadori et al. (54) suggest that since activated FSCs are vimentin-, desminand c~-smA-positive, these cells are smooth muscle-related myo-fibroblasts with a special function. In this study, desmin- and c~-smA-positive cells found in the portal tracts, especially around interlobular bile ducts, were markedly increased at day 2 after BDL. Based on the permutations of cytoskeletal filament proteins described above, u-smA-positive cells in the portal tracts may represent myofibroblasts participating in ECM formation. At day 2 following BDL numerous ~smA-positive cells accumulated around the interlobular bile ducts where tenascin was strongly expressed, and from day 7 onwards c~-smA-positive cells were more pronounced between the proliferating ductules whereas the distribution of tenascin was restricted to the connective tissue-parenchymal interfaces in areas of active ductular proliferation. This suggests that these 'fibroblastic' stromal cells may modulate the synthesis of ECM components, actively synthesizing tenascin.in the early phase of this experimental process, and synthesizing other ECM components (for example, basement membrane constituents) in the later phase. Lobular c~-smA-positive cells, considered activated FSCs-myofibroblasts, may also participate in fibrogenesis in the later phase of this experimental model since perisinusoidal c~-smA-positive cells reappeared close to the proliferating ductules at this point. We have recently shown that FSCs may be the cellular source of tenascin in CCl4-treated rat liver (20,21). Similar observations have been reported by Ramadori et al. (44). In this study, t~-smA and tenascin were coexpressed around interlobular bile ducts at day 2 following BDL whereas a-smA was not expressed in the lobules. It is tempting to speculate that ot-smA-positive cells, probably myofibroblasts, are involved in tenascin synthesis in the portal tracts, at least in the early phase of this experimental model. Which liver cell is the origin of the numerous t~-smA-positive cells which accumulate around interlobular bile ducts? Our results cannot exclude the possibility that these cells are derived from FSCs in the periportal regions. A 'fibroblastic' cell origin in the portal tracts is also possible. Few scattered c~smA-positive cells were present within the portal tracts of control rat livers, in contrast to the findings by Tanaka et al. and Nouchi et al. (25,26). While FSCs are generally considered the main source of hepatic ECM,

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Fig. 4. a-smA expression in the rat liver after BDL. In control rat liver, a small number ofct-smA-positwe cells was scattered in large portal tracts (arrows). Vessel walls were stained for a-smA. (a, control, x380) At day 2, an accumulation of u-smA-positive cells in the areas around interlobular bile ducts was observed, a-smA was weakly expressed between the ductules. (b, at day 2, x237.5) At day 21, ct-smA-positive cells were seen around interlobular bile ducts and between the proliferating ductules with newly formed fibrous septa. Perisinusoidal ~-smA-positive cells were present in the Iobules near the edge of active ductular proliferation. (c, at day 21, x237.5) (Indirect immunoperoxidase, counterstained with Mayer's haemalum.) n o t o n l y F S C s b u t a l s o ' f i b r o b l a s t i c ' cells o f t h e p o r t a l

M a c k i e et al. (36) h y p o t h e s i z e t h a t t h e b a l a n c e b e t w e e n

t r a c t s m a y p a r t i c i p a t e in f i b r o g e n e s i s , e s p e c i a l l y in in-

tenascin and fibronectin may modulate the mobility of

f l a m m a t o r y liver d i s e a s e s a s s o c i a t e d w i t h p e r i p o r t a l fi-

m y o f i b r o b l a s t s a n d a l l o w t h e m to m i g r a t e i n t o t h e s k i n

brosis.

w o u n d a r e a . In this e x p e r i m e n t a l m o d e l , cells p o s i t i v e

Further

elucidation

of the

exact

nature

of

d e s m i n - a n d a - s m A - p o s i t i v e cells in t h e p o r t a l t r a c t s is

for d e s m i n a n d c~-smA a c c u m u l a t e d

therefore necessary.

p a r e n c h y m a l n e c r o s i s . F u r t h e r m o r e , in t h e e a r l y p h a s e ,

in t h e a r e a s o f

T e n a s c i n h a s b e e n s h o w n to i n t e r f e r e w i t h cell a t t a c h -

c~-smA-positive cells in b o t h p e r i p o r t a l r e g i o n s a n d p o r -

m e n t to f i b r o n e c t i n in v i t r o (17). B a s e d o n t h i s e v i d e n c e ,

tal t r a c t s a p p e a r e d to p r o l i f e r a t e a n d m i g r a t e i n t o t h e

TABLE 2 The sequential changes in the expression of tenascin and the distribution of a-smA-positive cells, following BDL 6h

I day

2 day

3 day

7 day

Tenascin ~ Around ILBD* Between ductules At the interface**

-

+ -

++ ++ ++

++

.

++

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++

t~-smA-positive cells b Around ILBD Between ductules In periportal region

+

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_

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All items were graded as: - , +, ++. ~-, negative; +, weakly positive; ++, strongly positive. b'l'he number of cells with cytoplasmic staining for a-smA. *ILBD, interlobular bile duct. **Between connective tissue and liver parenchyma.

++

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Fig. 5. The behavior of the basement membrane component, collagen type IV, during BDL. In control rat liver, basement membranes of blood vessels and bile ducts were immunoreactive for collagen type IV. Sinusoidal reactivity for collagen type IV was detectable. (a, control, x237.5) Collagen type IV was strongly expressed on basement membranes of the proliferating ductules (arrows) as well as that of interlobular bile ducts. Collagen type IV-positive layers correspond well to the development of ductular proliferation and the newly formed fibrous septa (b, at day 2. x 237.5) (Indirect immunoperoxide, counterstained with Mayer's haemalum.)

TRANSIENT EXPRESSION OF TENASCIN IN LIVER matrix

surrounding

interlobular

bile

ducts.

365 Thus,

9

tenascin m a y also be i m p o r t a n t in m e d i a t i n g the m o v e m e n t o f m y o f i b r o b l a s t s in the liver (19). Slott et al. (62) h a v e p r o v i d e d c o n c l u s i v e e v i d e n c e that

l0

the i n c r e a s e d n u m b e r o f d u c t u l e s f o l l o w i n g B D L is d u e to the m u l t i p l i c a t i o n o f e x t a n t ducts. In fact, the increased t o r t u o s i t y a n d g r o w t h o f e x t a n t d u c t s after B D L led to the a p p e a r a n c e o f c r o s s - s e c t i o n e d d u c t u l e s w h i c h are i m m u n o r e a c t i v e for C K - 7 a n d C K - 1 9 at the m a r g i n o f the p o r t a l tracts. O u r C K - i m m u n o h i s t o c h e m i c a l

II 12

data

c o n f i r m S l o t t et al. (62) a n d can fit this hypothesis. T h e

13

presence o f a few h e p a t o c y t e s e x p r e s s i n g bile d u c t type

14

C K has been r e p o r t e d by G a l l a n d Bathal (63). W e also only f o u n d a few h e p a t o c y t e s i m m u n o r e a c t i v e for C K - 7 and

CK-19.

This

suggests

that

ductular

15

metaplasia

might play a m i n o r role in the d u c t u l a r increase in this experimental model.

16

In c o n c l u s i o n , this s t u d y s h o w e d s t r i k i n g s e q u e n t i a l

17

changes in the e x p r e s s i o n o f t e n a s c i n a n d in the d i s t r i b u tion o f u - s m A - p o s i t i v e cells in h e p a t i c fibrosis i n d u c e d by B D L .

18 19

Acknowledgements 20 T h e a u t h o r s are greatly i n d e b t e d to Dr. R. C h i q u e t E h r i s m a n n , Basel, S w i t z e r l a n d , w h o g e n e r o u s l y p r o v i d ed the a n t i - t e n a s c i n a n t i s e r u m . T h e technical assistance of P a u l a A e r t s e n , M o n i k P a t t o u , S u z a n n e T a e l e m a n s ,

21

and Christel V a n den B r o e c k , a n d the p h o t o g r a p h i c a l assistance of acknowledged.

Michel

Rooseleers

are

gratefully

References I 2 3 4

5 6 7

8

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366 31 32 33 34 35

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39

40

41

42

43

44 45

46

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