Abstract Secreted protein, acidic and rich in cysteine(SPARC), which functions in tissue remodeling, hasbeen reported to be expressed by myofibroblasts in livercirrhosis and hepatocellular carcinoma. This study aimedto reveal its expression in chronic hepatitis. Immuno-light and electron microscopy demonstrated that SPARCwas expressed by nerve fibers and hepatic stellate cells(HSCs) in the liver parenchyma and myofibroblasts inthe fibrous septa. Reaction products were localized in the rough endoplasmic reticulum and nuclear envelope.Serial section analysis demonstrated that SPARC, platelet-derived growth factor receptor-beta, and alpha-smoothmuscle actin were co-expressed by HSCs. Quantitativeanalysis demonstrated that, while SPARC-positive HSCswere sparse in control livers, they significantly increasedin number in the livers with chronic hepatitis. Therewere, however, no significant differences in numberamong the grades of activity, the stages of fibrosis, oretiology (virus-infected or autoimmune, hepatitis B virusor hepatitis C virus). In liver cirrhosis, however, theysignificantly decreased in number. The present results indicate that SPARC is expressed by activated HSCs inchronic hepatitis, suggesting the involvement of SPARCin hepatic fibrogenesis after chronic injuries.

Keywords Human · SPARC/osteonectin · Chronic hepatitis · Activated hepatic stellate cells · Immunohistochemistry

Introduction

Chronic hepatic injuries usually lead to a prominent accumulation of extracellular matrix (ECM) materialscomposed of collagens, proteoglycans, and fibronectinsin the necrotic lesions. In the liver parenchyma, theseECM components are synthesized and released by acti-vated hepatic stellate cells (HSCs), which express ECM-binding proteins such as integrins [4], neural cell adhe-sion molecule (N-CAM) [20, 25, 26], and secreted pro-tein, acidic and rich in cysteine (SPARC) [10, 17] and induce fibrotic reconstruction of the liver lobule [1, 9, 11,30]. It is reported that SPARC, a Ca2+-binding 43-kDaglycoprotein also termed osteonectin and BM-40 [3, 6], isreleased from the platelets and mesenchymal cells as arethe osteoblasts and fibroblasts and functions in tissue re-modeling [36, 37, 38, 40, 43]. By binding collagens,SPARC enhances cell migration to lesions via its anti-adhesive property [34] and induces ECM remodelingthrough interactions with matrix metalloproteases [39]and a plasminogen-activating system [13]. Accordingly,this substance is likely to be deeply implicated in the de-velopment of fibrosis. SPARC expression has been re-ported in human livers with various fibrotic diseases suchas liver cirrhosis [2, 10], biliary cirrhosis of biliary atresia[22], and hepatocellular carcinoma [23]. As revealed byimmunohistochemistry and in situ hybridization, SPARCwas expressed by myofibroblasts in the fibrous septa ofliver cirrhosis [2, 22] and the capsule of hepatocellularcarcinoma [23], while it was generally weak [23] or scat-tered in the liver parenchyma [2]. Prior to the establish-ment of fibrotic tissue in liver cirrhosis, fibrogenesis pro-ceeds to various degrees with ECM deposition in the liverparenchyma during chronic hepatitis. There are, however,no reports on SPARC expression in the liver with chronichepatitis except a brief description on high SPARC ex-pression in a few patients [23]. In this study, in order toreveal SPARC expression in chronic hepatitis and its cor-relation with the grades of inflammatory activity and thestages of fibrosis, we immunohistochemically identifiedSPARC-positive cells in the liver parenchyma, and con-

K. Nakatani (✉) · K. Ikeda · K. KanedaDepartment of Anatomy, Osaka City University Graduate School of Medicine, 1-4-3 Asahimachi, Abeno-ku, Osaka 545-8585, Japane-mail: kazuki@med.osaka-cu.ac.jpTel.: +81-6-66453706, Fax: +81-6-66463603

S. Seki · N. Kawada · T. Kitada · T. Yamada · H. SakaguchiH. KadoyaDepartment of Hepatology, Osaka City University Graduate School of Medicine, 1-4-3 Asahimachi, Abeno-ku, Osaka, Japan

Virchows Arch (2002) 441:466–474DOI 10.1007/s00428-002-0631-z

O R I G I N A L A RT I C L E

Kazuki Nakatani · Shuichi Seki · Norifumi KawadaTakuya Kitada · Takao Yamada · Hiroki SakaguchiHirokazu Kadoya · Kazuo Ikeda · Kenji Kaneda

Expression of SPARC by activated hepatic stellate cells and its correlation with the stages of fibrogenesis in human chronic hepatitis

Received: 11 May 2001 / Accepted: 31 January 2002 / Published online: 26 April 2002© Springer-Verlag 2002

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ducted quantitative analyses. The results demonstratedthat SPARC was highly expressed by activated HSCs inhuman livers with chronic hepatitis and might be in-volved in the fibrogenesis in the liver parenchyma.

Patients and methods

Liver specimens

Human liver specimens were obtained by either surgical resectionor laparoscopy from three patients with Gilbert’s disease, 32 pa-

tients with chronic viral hepatitis [hepatitis C virus (HCV) 25,hepatitis B virus (HBV) 7], 14 patients with autoimmune chronichepatitis, eight patients with liver cirrhosis after viral hepatitis(HCV 7, HBV 1), three patients with liver cirrhosis after autoim-mune hepatitis, and one patient with liver metastasis. Informedconsent was obtained from each patient. The study was approvedby the local ethics committee and was carried out according to theprovisions of the Declaration of Helsinki.

Histological evaluation

Histological diagnosis was done in hematoxylin–eosin-stainedsections. The histology of the liver with Gilbert’s disease and anon-cancerous portion of liver metastasis were almost normal andtherefore used as control livers. Chronic hepatitis was scored forthe grades of inflammatory activity and stages of fibrosis accord-ing to Desmet and colleagues [5]. We also assessed the grade ofinflammatory activity using the histological activity index (HAI)according to Knodell and colleagues [21].

Immunohistochemistry

Fresh liver specimens were fixed in a periodate–lysine 2% parafor-maldehyde (PLP) solution for 6–24 h at 4°C. They were then im-mersed in 0.1 M phosphate buffer (pH 7.4) containing 8.5%, 15%,and 20% sucrose, successively, each for 1 day. For light microscopy,PLP-fixed materials were embedded in OCT compound (SakuraFinetek USA, Torrance, Calif.) and frozen in dry ice/99.5% ethanol.

Fig. 1 Immunohistochemistry for secreted protein, acidic and richin cysteine (SPARC) in control livers (a), livers with chronic hepati-tis (b, c) and livers with cirrhosis (d). a Positive staining was foundin the nerve fibers (arrowheads) in the portal tract. Inset is S-100-positive nerve fibers (arrowheads) in a serial section. Only a fewpositive hepatic stellate cells (HSCs) were located around the portaltract as indicated by an arrow. P portal vein, ×220. b In chronichepatitis, positive HSCs (arrows) increased in number and distribut-ed throughout the liver parenchyma. ×220. c Higher magnificationof a SPARC-positive HSC, which is star-shaped and contains vacu-oles representative of lipid droplets (small arrows). ×1900. d In liver cirrhosis, positive cells (arrowheads) are found in the fibroussepta and the liver parenchyma near the septa (arrows). PositiveHSCs decreased in number in the regenerative nodules. ×220

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Frozen samples were cut into 5-µm-thick sections with a Cryostat(Leica, Nussloch, Germany) and air dried immediately. After beingwashed with 0.01 M phosphate-buffered saline (PBS) three timesfor 10 min, they were treated with 10% normal fetal bovine serumfor 1 h and incubated overnight with 10 µg/ml mouse anti-bovineosteonectin monoclonal antibody (IgG1, ON1–1 clone, Takara Bio-medicals, Shiga, Japan), which recognizes both bovine and humanSPARC [18], 20 µg/ml mouse anti-human platelet-derived growthfactor receptor-beta (PDGFRβ) monoclonal antibody (IgG1, Gen-zyme, Cambridge, Mass.), 1 µg/ml mouse anti-human alpha-smoothmuscle actin (αSMA) monoclonal antibody (IgG2a, Dako, Glos-trup, Denmark), or 0.0475 µg/ml rabbit anti-cow S-100 polyclonalantibody (Dako). As negative controls, specimens were incubatedwith mouse IgG1, IgG2a monoclonal antibody (Dako), or rabbitIgG polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz,Calif.), none of which recognize human proteins. They were rinsedwith PBS twice for 5 min. Endogenous peroxidase was thenblocked by incubating sections in methanol containing 0.3% hydro-gen peroxide for 30 min. They were rinsed with PBS once for5 min, with 0.075% BRIJ35 solution (Sigma diagnostics, St. Louis,Mo.) in PBS four times for 15 min [29], and then with PBS threetimes for 5 min. In following reactions, we used an LSAB-2 kit (forperoxidase method, Dako). They were then treated with biotinylateddonkey polyclonal antibody against mouse and rabbit Igs for 1 h.After being rinsed with BRIJ in PBS and then with PBS, the speci-mens were incubated with horseradish peroxidase-conjugated strep-tavidin for 1 h. After being rinsed with BRIJ in PBS and then withPBS, they were treated with 0.25 mg/ml 3,3’-diaminobenzidine tetrahydrochloride (DAB, Wako Pure Chemical Industries, Osaka,Japan) in the presence of 0.003% hydrogen peroxide in 0.05 M Tris buffer (pH 7.4) for 10 min. They were counterstained for nuclei with 5% methyl green (Muto Pure Chemicals, Tokyo, Japan).For electron microscopy, liver specimens fixed with a PLP solu-tion were cut into 50-µm-thick sections by a Microslicer (Dosaka,Kyoto, Japan). After being washed with PBS three times for 20 minat 4°C, they were treated with 10% normal fetal bovine serum for1 h at room temperature and incubated with anti-SPARC monoclo-nal antibody for two nights at 4°C. After rinsing with PBS threetimes for 20 min at 4°C, they were treated with biotinylated donkeypolyclonal antibody to mouse Igs overnight at 4°C. The sections

were rinsed with PBS three times for 20 min at 4°C and incubatedwith horseradish peroxidase-conjugated streptavidin for 1 h at roomtemperature. After rinsing three times for 20 min each at 4°C withPBS, sections were incubated with 0.5 mg/ml DAB in 0.05 M Trisbuffer for 30 min and then with the same medium in the presence of0.01% hydrogen peroxide for 1–2 min at room temperature. Theywere rinsed three times for 20 min each at 4°C with PBS, postfixedin 1% osmium tetroxide for 1 h at room temperature, and dehydrat-ed in ethanol series prior to embedding in Polybed (PolyscienceInc., Warrington, Penn.). Ultrathin sections were stained with satu-rated lead citrate and observed under a JEM-1200EX electron microscope (JEOL, Tokyo, Japan) at 100 kV.

Enumeration of SPARC-positive cells

The number of SPARC-positive cells and αSMA-positive cells in the liver parenchyma was counted under a light microscope. We counted the cells which displayed nuclei on the section. Foreach patient, several microscopic fields were randomly chosen. The average number of positive cells was calculated and expressed asthe number per unit square (1 mm2) of liver parenchyma. Data wereexpressed as the mean ±SD. Significant difference was obtained us-ing the unpaired Student’s t-test. The correlation between the num-ber of SPARC-positive cells and αSMA-positive cells was assessedusing Pearson’s correlation coefficient (r) with Fisher’s r to z.

Results

SPARC expression in control and diseased livers

In control livers, SPARC was expressed by fibrous struc-tures, which entered the liver parenchyma from the por-tal tract, and few sinusoidal cells in the periportal liverparenchyma (Fig. 1a). These fibrous structures wereidentified as nerve fibers from the positive immunostain-ing for S-100 protein in serial sections (Fig. 1a inset).Positive sinusoidal cells displayed the nuclei within themand had several vacuoles in the cytoplasm. In the liverswith chronic hepatitis, SPARC-positive sinusoidal cellsappeared not only in the periportal zone of the liver lob-ule but also in the intermediate and pericentral zones.They increased in number and were evenly distributed inthe liver parenchyma (Fig. 1b). In higher magnification,they extended well-developed cytoplasmic processes

Fig. 2 Immunoelectron microscopy for secreted protein, acidicand rich in cysteine (SPARC) in the liver parenchyma. a, b Hepat-ic stellate cells (HSCs, SC) with lipid droplets (asterisks) in thespace of Disse between the hepatocytes (H) and sinusoidal endo-thelial cells (E). Immune reactions are found in dilated rough en-doplasmic reticulum (arrows) and nuclear membrane (arrow-heads). Hepatocytes, endothelial cells, and Kupffer cells (KC) arenegative for SPARC. a ×5000, b ×14,000

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along the sinusoidal wall and contained several vacuolesindicative of lipid droplets in the cytoplasm (Fig. 1c). Inthe livers with cirrhosis, SPARC was preferentially ex-pressed by myofibroblasts within the thick fibrous septa.In the regenerative lobules, however, SPARC-positiveHSCs were only detected near the fibrous septa(Fig. 1d). In the negative control specimens treated withmouse IgG1 antibody, mouse IgG2a antibody, or rabbitIgG antibody, there were no detectable immuno-positivestructures (data not shown).

Fig. 3 Immunohistochemistry for secreted protein, acidic and richin cysteine (SPARC, a, d), platelet-derived growth factor receptor-beta (PDGFRβ, b, e) and alpha-smooth muscle actin (αSMA, c) inserial sections from two portions (a–c, d, e) of the livers with chron-ic hepatitis. a–c SPARC-positive cells, PDGFRß-positive cells, andαSMA-positive cells showed similar distribution in the liver paren-chyma, while the latter two are more abundant than the former inthe fibrous septa as indicated by arrows. ×220. d, e SPARC andPDGFRβ are co-expressed by HSCs as indicated by arrowheads.×950

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Fig. 4 Quantitative analysis of secreted protein, acidic and rich in cys-teine (SPARC)-positive cells per unit square (1 mm2) of liver paren-chyma. a Comparison with control livers (n=4) including Gilbert’s dis-ease and non-cancerous portions of liver metastasis, livers with virus-infected (VI) chronic hepatitis (n=32), those with autoimmune (AI)chronic hepatitis (n=14), those with cirrhosis after virus-infected hepa-titis (n=8), and those with cirrhosis after autoimmune hepatitis (n=3).Significant differences were given between control and virus-infectedand autoimmune hepatitis, virus-infected hepatitis and virus-infectedcirrhosis, and autoimmune hepatitis and autoimmune cirrhosis. Therewas a significant difference between controls and autoimmune cirrho-sis, while no significant difference was detected between controls andvirus-infected cirrhosis. There were no significant differences betweenvirus-infected hepatitis and autoimmune hepatitis and between virus-infected cirrhosis and autoimmune cirrhosis. CH chronic hepatitis, LC liver cirrhosis, * P<0.01, ** P<0.05. NS, no significant difference.b Type of viruses in virus-infected chronic hepatitis. No significant dif-ference was given between hepatitis B virus (HBV, n=7) and hepatitisC virus (HCV, n=25). c Grades of inflammatory activity in chronichepatitis. There were no significant differences among minimal (n=7),mild (n=17), moderate (n=20), and severe (n=2) activities. d Stages offibrosis in chronic hepatitis. There were no significant differencesamong no fibrosis (n=3), mild (n=21), moderate (n=14), and severe(n=8) fibrosis. e Histological activity index (HAI) in chronic hepatitis.There are no significant differences among HAI

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Subcellular localization of SPARC-positive reactions

Immunoelectron microscopy revealed that SPARC-posi-tive sinusoidal cells were HSCs as indicated by charac-teristic lipid droplets (Fig. 2a). Immunoprecipitates werelocalized in dilated rough endoplasmic reticulum and thenuclear envelope (Fig. 2a, b). There were, however, noreaction products in ECM around HSCs. However,Kupffer cells, sinusoidal endothelial cells, and hepa-tocytes were negative for SPARC (Fig. 2a).

Serial section analysis on SPARC, PDGFRβ, and αSMA expression

To investigate the relationship of SPARC expressionwith HSC activation, we performed immunohistochemi-cal staining of the liver for SPARC, PDGFRβ, andαSMA in serial sections. In control livers, PDGFRβ-pos-itive HSCs were sparsely distributed in the liver paren-chyma at a frequency similar to that of SPARC-positiveHSCs, while αSMA-positive HSCs were more frequent,preferentially distributing in the periportal and interme-diate zones of the liver lobule (data not shown). In thelivers with chronic hepatitis, both SPARC-positive HSCs(Fig. 3a) and PDGFRβ-positive HSCs (Fig. 3b) in-creased in number, distributing throughout the liver lob-ule. In higher magnification of serial sections, it wasclearly shown that SPARC (Fig. 3d) and PDGFRβ(Fig. 3b) were co-expressed in HSCs. Alpha-SMA-posi-tive HSCs (Fig. 3c) also increased in number, showing

Fig. 5 Quantitative analysis on the correlation between the num-ber of secreted protein, acidic and rich in cysteine (SPARC)-posi-tive cells and that of alpha-smooth muscle actin (αSMA)-positivecells. a In the stages of no and mild fibrosis, there was no signifi-cant correlation. b In the stages of moderate and severe fibrosis, asignificant correlation was detected

the intralobular distribution similar to that of SPARC-and PDGFRβ-positive HSCs. In the fibrous septa, how-ever, there were more abundant PDGFRβ-positive cellsand αSMA-positive cells than SPARC-positive cells.

Quantitative analysis

Quantitative analysis demonstrated that the number ofSPARC-positive HSCs per square unit of liver paren-chyma significantly increased in the livers with chronichepatitis (both virus-infected and autoimmune) com-pared with control livers (Fig. 4a). There was, however,a significant difference between control livers and livercirrhosis after autoimmune hepatitis. Between virus-in-fected and autoimmune hepatic diseases (Fig. 4a) or be-tween HBV and HCV (Fig. 4b), no significant differ-ences in the number of positive HSCs were found inchronic hepatitis. In liver cirrhosis, there was also no significant difference between virus-infected and auto-immune hepatic diseases. It was noted that, as the dis-ease advanced from chronic hepatitis to liver cirrhosis,the number of positive HSCs significantly decreased independent of etiology, either virus-infected or auto-immune. Values were also compared among the gradesof inflammation activity (Fig. 4c) and stages of fibrosis(Fig. 4d) in chronic hepatitis. Statistic analysis was con-ducted by joining virus-infected and autoimmune diseas-es and by joining HBV and HCV because of no signifi-cant differences between them. The results demonstratedthat no significant differences were found among thegrades (Fig. 4c) and the stages (Fig. 4d). Furthermore,SPARC expression was not related to HAI in chronichepatitis (Fig. 4e). Moreover, no significant differencesin HAI were detected among the livers with HBV infec-tion (9.43±3.05, n=7), those with HCV infection(8.56±3.00, n=25), and those with autoimmune hepatitis(7.43±5.23, n=14). By serial section analysis, significantcorrelation was found between the number of SPARC-

positive HSCs and that of αSMA-positive HSCs in latestages (moderate and severe) of fibrosis (Fig. 5b), whilenot in early stages (no and mild, Fig. 5a).

Discussion

A previous in situ hybridization study of SPARC expres-sion in human cirrhotic livers demonstrated that signalswere the strongest in myofibroblasts within the fibroussepta, but were weaker or scattered within the liver pa-renchyma [2]. As shown using immunostaining, howev-er, SPARC was only weakly expressed by the sinusoidalcells of non-cirrhotic livers in contrast to intense stain inthe stromal cells of hepatocellular carcinoma [23]. Thepresent immunohistochemical study was designed to re-veal SPARC expression in pre-cirrhotic stages of chronichepatitis. The results demonstrated that SPARC washighly expressed by sinusoidal cells within the liver pa-renchyma as well as myofibroblast-like cells within thefibrous septa. Immunoelectron microscopy revealed thatSPARC-positive sinusoidal cells in the parenchyma wereHSCs. Reaction products were localized in the endoplas-mic reticulum and nuclear envelope, representing thesynthetic process of this protein. They were, however,not detected in ECM components around HSCs, which isin agreement with a previous study [2]. The reason forthis is not known, but the concentration of SPARC atthese sites might be lower or it is more easily washedaway during preparation of samples.

It is reported that SPARC is secreted by various kindsof ECM-producing cells [34] and modulates the cell-to-matrix interactions after binding collagens [12, 43]. Be-cause activated HSCs are capable of producing an excessamount of ECM during liver fibrosis [30], SPARC ex-pression by activated HSCs in chronic hepatitis must berelated to ECM production by them [30]. Serial sectionanalysis demonstrated that SPARC and PDGFRβ wereco-localized in HSCs, exhibiting similar intralobular distribution between them both in control livers and thelivers with chronic hepatitis. Because PDGFRβ is amarker for HSC activation [8, 41], this finding indicatesthat SPARC is expressed by activated HSCs. BothPDGF-AB, a heterodimer of PDGF A- and B-chain, and-BB, a homodimer of PDGF B-chain, bind PDGFRβ onHSCs and induce migration, proliferation, and activationin them [14, 15].

It is reported that SPARC binds a B-chain of PDGFand thereby interferes with the binding of PDGF-AB and-BB to PDGFRβ [33]. As the consequence, SPARC in-hibits the augmentation effect of PDGF on activatedHSCs, raising the possibility that SPARC might be anendogenous inhibitor for PDGF. This interpretation issupported by the previous observation that SPARC in-hibited PDGF-induced proliferation of cultured mesan-gial cells that expressed both SPARC and PDGFRβ [31].It is also reported that SPARC induces expression ofplasminogen activator inhibitor (PAI)-1 [2]. ActivatedHSCs possess a plasminogen-activating system com-

posed of PAI-1, an urokinase plasminogen activator (u-PA), and a u-PA receptor, which contributes to the remodeling of ECM components in liver fibrosis throughthe proteolytic activity of plasminogen [24], suggestingthat SPARC-induced PAI-1 expression in activated HSCsmay inhibit the degradation of ECM deposition. Thus,SPARC possibly constitutes both positive and negativefeedback systems in HSCs during the progression of liver fibrogenesis.

To quantitatively analyze SPARC expression in theliver parenchyma, we counted the number of positivecells in immunostained sections. Although immunostain-ing does not necessarily correlate with the quantity ofprotein expression, it is well established that the increasein the number of positive cells must parallel enhancedproduction. In this study, SPARC-positive cells in theliver parenchyma significantly increased in number inchronic hepatitis independent of etiology, i.e., virus-infected or autoimmune. There were, however, no signif-icant differences among the grades of inflammatory ac-tivity and stages of fibrosis. It was also noted that posi-tive cells in the liver parenchyma significantly decreasedin number in liver cirrhosis compared with chronic hepa-titis, indicating that SPARC seems to be expressed by activated HSCs during active fibrogenesis before thecompletion of fibrotic reconstruction in cirrhotic livers.

We previously observed that another marker for HSCactivation, prion protein, similarly showed no differencesin expression among the stages of fibrosis in chronichepatitis and a significant decline in liver cirrhosis [19].However, a well known activation marker αSMA is al-ready shown to be expressed by several HSCs in normalhuman livers and is enhanced by HSC activation duringchronic hepatitis [7, 35, 42]. This molecule gives con-tractility to activated HSCs, which contribute to the con-traction of fibrous tissue. A significant correlation wasfound between the number of SPARC-positive cells andthat of αSMA-positive cells in late stages (moderate andsevere) of fibrosis, while not in early stages (no andmild). Such differences in the manner of expression be-tween SPARC and αSMA suggest that their expressionmay represent different phases of activation in HSCs.This fact may represent that in late stages activatedHSCs acquire both the capacities of collagen productionand cell contraction.

It was also observed that SPARC was expressed bynerve fibers in the liver consistent with the previousfinding of SPARC expression by human astrocytes [32].It has been recently proposed that HSCs are related tothe neural crest cells in origin [28] based on the fact thatHSCs and neural cells have several common markerslike N-CAM [20, 25, 26], prion protein [16, 19], glial fi-brillary acidic protein [27], and nestin [28]. The presentfinding of SPARC expression by neural cells and activat-ed HSCs is consistent with this view. In conclusion, thisstudy has demonstrated that SPARC expression dramati-cally increases in activated HSCs independent of stagesof fibrosis during chronic inflammation of human liver,while it declines in liver cirrhosis, indicating that

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SPARC expression by activated HSCs represents activefibrogenesis in the liver parenchyma before establish-ment of fibrotic reconstruction in liver cirrhosis.

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