Perlecan and Decorin in Carbon-Tetrachloride - NCBI

American Journal of Pathology, Vol. 148, No. 5, May 1996 Copyright C) American Society for Investigative Pathology

Expression of Extracellular Matrix Proteoglycans Perlecan and Decorin in Carbon-TetrachlorideInjured Rat Liver and in Isolated Liver Cells

Monika Gallai,* Ilona Kovalszky,* Thomas Knittel,t Katrin Neubauer,t Thomas Armbrust,t and Giuliano Ramadorit From the First Institute ofPathology and Experimental Cancer Research,* Semmelweis Medical University, Budapest, Hungary, and the Department of Gastroenterology and Endocrinology,t Georg August University, Gottingen, Germany

Proteoglycansare important components of the extracellular matrix. They are involved in liver regeneration as well as in liver fibrosis. The distribution and cellular source of proteoglycans under normal as well as pathological conditions is still under debate. Localization of decorin and perlecan was studied in normal, acutely damaged, and cirrhotic liver by histochemistry. Furthermore, their synthesis was analyzed in different liver cell populations isolated from normal rat liver. In normal liver, decorin positivity was observed in the perisinusoidal space and in the portal area. Perlecan was clearly detectable in the portal area (blood vessels and bile ducts); a moderate reaction was also seen along the sinusoids. Strong positivity for both proteoglycans was detectable in the necrotic areas of acutely damaged liver. Chronic liver damage was characterized by the deposition of decorin and perlecan in the fibrotic septa. Immunocytochemical reactions were positive for perlecan and decorin in cultured Ito and endothelial cells but not in hepatocytes and Kupffer cells. Northern hybridization confirmed the capacity of Ito cells and endothelial cells to express the two genes. Interestingly, although rat skin fibroblasts expressed strong messages for both proteoglycans, rat aortic smooth muscle cells did not synthesize decorin. (Am J Pathol 1996,

148:1463-1471)

Introduction In the last few years the great relevance of proteoglycans (PGs) in several fields of biology has been determined.1-3 PGs are known to function as cell surface receptors, as ligands for growth factors and cytokines, and as signals for cell growth, migration, and differentiation.4-7 Besides collagens, liver extracellular matrix contains several glycoproteins and PGs, but their exact nature was just recently determined.8 - PGs seem to be involved not only in the assembly of extracellular matrix but also in the replication of hepatocytes and expression of their tissue-specific function.12-15 Furthermore, it has been suggested that they could create an electrostatic barrier to the transfer of anionic molecules from the sinusoids to the hepatocytes within the subendothelial space.16 Although knowledge about distribution of different PGs in normal and diseased liver has been increased enormously during the last few years9 10 17-19 and the availability of isolation procedures for the different liver cell populations has allowed preliminary synthetic studies, the picture is far from complete. Perlecan is the most prominent multidomain heparan sulfate PG of the basement membrane.20-22 Immunohistochemical studies identified perlecan in all vascular basement membranes, in structural elements of connective tissue, and in sinusoidal regions of organs like the liver.10'23'24 Decorin is one of the collagen-fiber-associated PGs.25 It consists of a core protein and of a single chondroitin or dermatan sulfate chain.26'27 Decorin can bind and inhibit transforming growth factor (TGF)Supported by the Hungarian National Science Foundation (OTKA 017085), the Deutsche Forschung Gemeinschaft (SFB 402/C6), and the German-Hungarian Scientific and Technological Collaboration Project (89). Accepted for publication January 2, 1996. Address reprint requests to Dr. G. Ramadori, Department of Gastroenterology and Endocrinology, Georg August University, Robert Koch Strasse 40, 37075 G6ttingen, Germany.

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1.6,28 On the other hand, TGF-,B1 is able to regulate synthesis of decorin in many cell types.9'29'30 This underlines the role of decorin in a feedback system regulating the assembly of extracellular matrix. In this work the deposition of decorin and perlecan in normal and damaged liver has been studied. Furthermore, we tried to determine which cells of the liver have potential to produce these two PGs.

Materials and Methods Reagents Normal and methionine-free Dulbecco's modified Eagle's medium (DMEM) and fetal calf serum (FCS) were purchased from Flow (Bonn, Germany); L-glutamine, penicillin, and streptomycin were from Seromed (Berlin, Germany); collagenase of clostridium histolyticum was from Boehringer (Mannheim, Germany); pronase E was from Merck (Darmstadt, Germany); Nycodenz was from Nyegaard & Co. (Oslo, Norway); EDTA, ethidium bromide, 2-mercaptoethanol, collagenase type (125 to 250 U/mg), and sodium dodecyl sulfate (SDS) were from Bio-Rad Laboratories (Munich, Germany); guanidinium isothiocyanate was from Fluka (Bern, Switzerland); CsCI2, agarose, and the random priming kit were from Boehringer; and [32P]dCTP was from Amersham Buchler (Frankfurt, Germany). Acetylated low density protein labeled with dioctadecyltetramethyl indocarbocyanine perchlorate was purchased from Paesel and Lorei (Frankfurt, Germany). Endothelial cell (EC) medium kit was purchased from Promo Cell

(Heidelberg, Germany).

Antibodies and cDNA Probes The antiserum against human decorin was purchased from Telios (La Jolla, CA). To prepare an antiserum against mouse perlecan, the large heparan sulfate PG of EHS tumor was isolated and purified. The purity of the isolated PG was determined by polyacrylamide gel electrophoresis and column chromatography according to standard methods.31 Polyclonal antibody was raised in white New Zealand rabbits by subcutaneous injection of 0.5 mg of purified PG with Freund adjuvant. Four and six weeks later, the treatment was repeated. The titer of antiserum was followed by immunoprecipitation. After isolation, the reactivity of the antiserum was studied on Western and dot-blot hybridizations. It recognized the large (>400 kd) PG of EHS tumor as well as the isolated human and rat perlecan.32 The antibody did not cross-react with laminin, fibronectin, or

type IV collagen. The monoclonal antibody directed against the ED-2 -epitope was a gift from C. Dijkstra.33 The monoclonal antibody against desmin and the peroxidase-labeled immunoglobulin (1g)G fractions directed against murine or rabbit Igs were obtained from Dako. The monoclonal antibodies directed against a-smooth muscle actin and albumin were obtained from Sigma (Munich, Germany). A partial cDNA clone for human perlecan34 PG40 cDNA clone,26 a kind gift from E. Rouslahti, and a ,B-actin cDNA clone35 as standard are used for hybridization.

Induction of Acute and Chronic Liver Damage Acute liver damage was induced in 8-week-old female Wistar rats (body weight approximately 200 g) by oral administration of a carbon tetrachloride (CCI4)/corn oil solution (50% v/v) as previously described36 but modified by reduction of the CCI4 dose to 75 ,ul/100 g body weight. Animals were sacrificed 48 hours after a single high dose of poisoning. Control animals were treated with corn oil only. The liver was perfused with saline solution (0.9% NaCI) and snap frozen in liquid nitrogen. For induction of liver cirrhosis, animals were pretreated with phenobarbital-containing drinking water (0.03%) for 2 weeks and then exposed to oral application of CCI4 once a week up to 10 weeks, according to the method of Proctor and Chatamra,37 starting with 0.04 ml of CCI4. An additional 0.04 ml of CCI4 was added weekly. In our experiments, chronic liver injury was studied 48 hours after the actual administration of CCI4.

Isolation of Liver Cells Rat hepatocytes were isolated from normal liver according to Seglen,38 as described elsewhere,39 and were cultured on collagen-coated tissue culture plates in DMEM, supplemented with 10% FCS, 0.05% insulin, and 10-7 mol/L dexamethasone. Four hours after plating, medium was supplemented with 0.2% bovine serum albumin, 0.05% insulin, and 10-7 mol/L dexamethasone to replace the plating medium. Ito cells and Kupffer cells were isolated according to the method of De Leeuw et al,40 as described previously.41 Rat sinusoidal ECs were obtained according to Knook et al.42 Ito cells were cultured in DMEM with 10% FCS. Kupffer cells were cultured in M199 supplemented with 15% FCS, and ECs were cultured in EC medium supplemented

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with epidermal growth factor, basic fibroblast growth factor, 0.15% insulin, and 2% FCS. Medium was replaced 1 day (Kupffer cells and ECs) or 2 days (Ito cells) after plating and every other day thereafter. Cells were kept in culture at 370C in a 5% C02 atmosphere and 100% humidity. Purity of the cell isolations was determined by phase contrast microscopy, by indirect immunofluorescence staining, vitamin A autofluorescence, and desmin staining of the Ito cells, by acetylated low density lipoprotein incorporation of ECs, and by ED-2 staining of Kupffer cells.

Isolation and Culture of Rat Aortic Smooth Muscle Cells and Skin Fibroblasts Isolation of arterial smooth muscle cells (SMCs) from pieces of aorta was performed according to Oakes et al.43 Skin fibroblasts were obtained after digestion of pieces of epidermis and dermis in solution containing collagenase (0.5% w/v) for 30 minutes at 37°C. After isolation, cells were washed with DMEM and plated. SMCs were cultured in DMEM containing 15% FCS, and skin fibroblasts were cultured in DMEM containing 7.5% FCS. Culture medium was changed every 24 hours. Cells were kept in culture at 37°C in a 5% CO2 atmosphere and 100% humidity, as described previously.44

Immunocytochemistry and Immunohistochemistry Freshly isolated hepatocytes, Ito cells, ECs, and Kupffer cells were centrifuged onto slides in a cytospin centrifuge Shandon (London, UK) at 300 rpm (hepatocytes and Ito cells) and 1500 rpm (Kupffer cells and ECs). For immunocytochemistry, cells were cultured in Labtec dishes. Cells were fixed in methanol (5 minutes) and acetone (10 seconds). Frozen sections (5 ,um) were prepared by cryostat. Samples were air dried, fixed in methanol (10 minutes) and acetone (10 minutes) at -20°C, washed with phosphate-buffered saline (PBS), and preincubated with FCS for 20 minutes at 37°C. After three washes in PBS, cells were incubated with antigen-specific antibodies (perlecan 1:100 and decorin 1:200) for 1 hour at 370C and then again rinsed three times in PBS and incubated with peroxidase-labeled IgGs directed against murine or rabbit Igs.

Northern Blot Analysis of Total RNA Total RNA was isolated from freshly isolated and cultured hepatocytes, Ito cells, Kupffer cells, and

ECs by the guanidinium isothiocyanate and CsCI2 ultracentrifugation method of Chirgwin, as described previously.46 Total RNA was separated by agarose gel electrophoresis, transferred onto nylon membrane, and finally hybridized with specific braclets [32P]dCTP-labeled cDNA probes. Probes were labeled by random priming. Hybridization and posthybridization washes were carried out under low stringency conditions. Hybridization was performed overnight at 42°C in 2 x standard saline citrate (SSC) 0.1% SDS for 30 minutes each. Filters were exposed to Kodak X-omat film at -70°C. Filters were successively hybridized with cDNAs specific for decorin, perlecan, and ,B-actin. Data presented in this manuscript are representative of four parallel studies.

Results Immunolocalization of Perlecan and Decorin in Normal and Damaged Rat Liver In normal rat liver, perlecan was found mainly in the basement membranes of liver vasculature and bile ducts. Very faint immunostaining could be observed along the sinusoids. In CCI4-induced acute injury, the sinusoidal positivity increased especially in the centrolobular injured areas. In cirrhotic livers, a pronounced perisinusoidal deposition and a positivity of fibrotic septa occurred (Figure 1, a-c). Decorin in normal liver outlined the sinusoids and could be identified also as a component of the periportal connective tissue. In acute damage, increased positivity for decorin was observed in the necrotic areas and reactivity was observed along the sinusoidal space of the nondamaged parenchyma. In cirrhotic livers, the perisinusoidal reaction became fiber-like and positivity around individual hepatocytes developed. The strong decorin staining of the fibrotic septa was the most characteristic change (Figure 1, d-f).

Immunocytochemistry of Isolated Liver Cells To determine which cells could be the source of decorin and perlecan in the liver, immunocytochemistry was carried out on freshly isolated and cultured cells that were kept for 1 or 3 days in culture. Previously, the purity of the isolated liver cells was determined by established markers (a-smooth muscle actin and desmin for Ito cells, albumin for hepatocytes, ED-2 for Kupffer cells, acetylected low density lipoprotein incorporation for ECs; data not shown). For

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Figure 1. Immunobistochemical localization ofperlecan (a to c) and decorin (d to f) in normal (a and d), acutely damaged (b and e), and cirrhotic (c and f) rat liver. Polyclonal rabbit antiserum against perlecatn ( 1: 100) and decorin ( 1:200) was used as primary antibody. The reaction was detected by peroxidase-labeled anti-rabbit IgG. In normal liver, perlecan localizes in the liver vasculatulre and in the perisinusoidal space (arrowhead); decorin is strongly detectable in the same locations. In acute liver damage, the necrotic areas are strongly positivefor botb PGs. Cirrbotic livers are cbaracterized by clear perlecan positivity in the perisinusoidal space (smaller arrowheads) and in fibrotic septa (larger arrowhead). Decorin deposition showed a similar pattern. Oniginal magnification, X 250 (a and C) and x 100 (b, d, e, and f).

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I Figure 2. Immunocytochemical reaction forperlecan (a and c) and decorin (b and d) on sinusoidal ECs (a and b) and on Ito cells (c and d) after 3 days in culture. The cells were reacted with polyclonal rabbit and antiserum against perlecan (1:100) and decoin (1:200). After reacting with anti-rabbit secondary antibody, the reaction was detected by peroxidase reaction. Cytoplasm of both cell types is positive for decorin and perlecan. No reacton was found in controls in which the primary antibody was replaced by rabbit serum (not shown). Original magnification, x 250.

control, the first antibody was replaced by rabbit or mouse serum. Under the conditions described, neither freshly isolated nor cultured hepatocytes nor Kupffer cells reacted with the antiserum against perlecan or decorin (data not shown). In contrast, the immunostaining for both PGs were positive in the cytoplasm of Ito cells and ECs. The modest reaction for decorin and perlecan in the freshly isolated Ito cells and ECs gradually increased during the time in culture resulting in strong positivity at the third day (Figure 2,

a-d).

Northern Hybridization Total RNAs, isolated from hepatocytes, Kupffer cells, Ito cells, ECs, aortic SMCs, and rat skin fibroblast cells, were hybridized with decorin and perlecan cDNA probes. Hybridization with decorin revealed transcripts of 1.6 and 1.9 kb at similar

abundance. Among the cells, studied, Ito cells, ECs, and fibroblasts expressed these transcripts. Their amount was at least three times more abundant in fibroblast RNA than in the other cells. Neither freshly isolated nor cultured hepatocytes nor Kupffer cells expressed decorin mRNA. Surprisingly, aortic SMCs were also negative for decorin. Hybridization with the perlecan probe resulted in a 12-kb transcript in fibroblasts, Ito cells, ECs, and SMCs but not in hepatocytes and Kupffer cells (Figure 3). In accordance with the immunocytochemistry, the steady-state level of decorin and perlecan mRNA increased in Ito cells during the first 3 days in culture. Interestingly, whereas the amount of perlecan-specific transcripts increased during the culture up to 7 days (Figure 4), decorin gene expression decreased after the 3rd day after plating. An increase of perlecan gene expression in ECs during the time in culture was also detectable (Figure 4).

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Discussion In this work three questions have been studied: 1) the distribution pattern of perlecan and decorin in normal and damaged rat liver, 2) the potential cellular source of these proteoglycans; and 3) the possible differences in perlecan and decorin gene expression of activated Ito cells, skin fibroblasts, and aortic SMCs. By an immunohistochemical method, both PGs were found in normal liver. Our antibody localized the molecule mainly into the basement membrane of liver vasculature and bile ducts of normal liver. A weak but clear reactivity was also detectable along the sinusoids. The same localization of perlecan in normal liver was described by other authors.10"18 The weak perisinusoidal perlecan reaction was in contrast to the findings on human liver10'21 but was in agreement with the results obtained by Rescan et al18 studying rat liver, which suggests an interspecies difference in the expression of this basement membrane PG. A similar tendency has been described for collagens, demonIto cell

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1 2 3 4 Figure 4. Deconln and perlecan genle exprevssion in fresbly isolated a nd c ulftu red Ito cells a nd FCs fo r up to 7 days. For Ito cells Ila ne 1 is fresbly isolated ancl lanes 2, 3, and 4, are 3, 5, and 7 days ini cufture, respectively. For ECs, lanes 1, 2, and 3 are 3, 5, and 7 days in culture, respectively. Interestingly, deconin gene expression increases u4p to day 3 of cuilture and decreases thereafter. At the same time, the amounlt of perlecan-specific transcripts increases up to day 7.

strating that the liver of rodents contained considerably less of these matrix proteins than that of humans.47 Necrotic areas in acute injury strongly reacted with perlecan antiserum. In chronic liver injury, deposition of perlecan became abundant in fibrotic septa. This deposition presumably reflects the events of sinusoidal capillarization and the development of classical basement membrane underlining the sinusoidal ECs. Despite the biochemical measurement indicating that normal liver contains low amounts of dermatan or chondroitin sulfate, decorin positivity was found in considerable intensity in the perisinusoidal region and moderate reaction was observed in the sparse connective tissue of portal triads. Similarly to perlecan, strong decorin positivity was found in the necrotic areas of acutely damaged liver. In liver cirrhosis, decorin characteristically was localized into the fibrotic septa, and the perisinusoidal reaction was comparable to that found in normal liver. In human liver cirrhosis, a four- to five-fold increase of glycosaminoglycans with the selective enhancement of dermatan and chondroitin sulfate was demonstrated.48 On the basis of our results, these glycosaminoglycan chains may represent the sugar moieties of decorin. The detection of decorin and perlecan in the perisinusoidal area suggests that cells of this region could be involved in PG production in normal liver. The synthetic capacity of these cells increased when they became involved in the generation of fibrotic septa in the chronically damaged liver. Immunocytochemistry as well as Northern hybridization confirmed that both Ito cells and sinusoidal ECs from normal liver are able to synthesize increasing amounts of perlecan during time in culture. The results of previous studies dealing with localization and/or synthesis of PGs in liver and/or in liver cells are contradictory. In fact, whereas Rescan et al18 hypothesize the role of Ito cells in the production of perlecan in human liver, as detected by immunohistochemistry. The expression of decorin in sinusoidal ECs was unexpected. On the other hand, it has been published that ECs, under certain conditions, are able to express this PG.49 This finding, however, further stresses the capability of sinusoidal ECs to produce a previously unsynthesized protein, as described for von Willebrand factor, thus indicating that sinusoidal ECs of fibrotic septa could acquire the characteristics of the blood vessel ECs.50 None of the PGs studied could be detected in hepatocytes or in Kupffer cells either by immunohistochemistry or by Northern blot analysis. Our results did not support

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the observation of others18 who described perlecan in cultured hepatocytes, which, however, at the same time did not appear to be able to express the albumin gene. During the process of activation, isolated Ito cells have been shown to produce increasing amounts of extracellular matrix proteins whereas the amount of GFAP-specific51 and of HGF-specific52 transcripts decreased. In this work, we observed for the first time a different behavior. In fact, decorin gene expression first increased and then decreased after the 3rd day of culture. We do not have any explanation for this interesting finding at the moment, but experiments to clarify it are underway in the laboratory. The significance of Ito cells in the development of liver fibrosis is more and more emphasized.5354 Our results are in agreement with this theory. On the other hand, it is noteworthy to mention that damaged liver also contains fibroblasts and SMCs. Fibroblasts may be present in considerable number in periportal fibrotic areas and in fibrotic septa of cirrhotic liver, and they are found to synthesize matrix proteins.55 Similarly to activated Ito cells, fibroblasts express both perlecan and decorin. Furthermore, the message for the latter is stronger in fibroblasts. SMCs contain perlecan mRNA. Consequently, the role of fibroblasts and SMCs in the increased deposition of decorin and perlecan in damaged liver has to be considered. There is no explanation for the lack of decorin mRNA in rat aortic SMCs so far, as these cells of human or monkey origin produce decorin.56,57 On the other hand, this characteristic of rat SMCs might be useful to differentiate this cell type from activated rat Ito cells and fibroblasts. Determination of alternatively spliced mRNA of tenascin was also offered for the better identification of cells involved in liver fibrogenesis.53 An additional question to be addressed concerns the biological consequence of increased PG expression. These PGs are not just structural elements of extracellular matrix, but both decorin and perlecan bind, store, activate, or inactivate growth factors and cytokines, which play important roles in fibrogenesis. 6,59-63 Increased decorin deposition might inactivate TGF-1l, with subsequent inhibition of the fibrotic process, as was found in glomerulonephritis.61 However, the potential of decorin to activate TGF-,81 has also been published.62 Increased expression of perlecan may interfere with the action of basic fibroblast growth factor, which is a potent stimulator of Ito cells and is found to be synthesized in high quantity after CC14 administration.63

All of these questions are still open, and additional studies are needed to understand the significance of decorin and perlecan in the development of liver fibrosis.

Acknowledgment We thank Dr. E. Rasb and Dr. G. Pogany for the preparation of polyclonal antiserum against perlecan.

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