Characterization of glial fibrillary acidic protein (GFAP)-expressing hepatic stellate cells and myofibroblasts in thioacetamide (TAA)-induced rat liver injury

Experimental and Toxicologic Pathology 65 (2013) 1159–1171

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Characterization of glial fibrillary acidic protein (GFAP)-expressing hepatic stellate cells and myofibroblasts in thioacetamide (TAA)-induced rat liver injury Anusha Hemamali Tennakoon, Takeshi Izawa, Kavindra Kumara Wijesundera, Hossain M. Golbar, Miyuu Tanaka, Chisa Ichikawa, Mitsuru Kuwamura, Jyoji Yamate ∗ Laboratory of Veterinary Pathology, Division of Veterinary Sciences, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, 1-58, Rinku-ourai-kita, Izumisano City, Osaka 598-8531, Japan

a r t i c l e

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Article history: Received 18 February 2013 Accepted 28 May 2013 Keywords: Glial fibrillary acidic protein Hepatic stellate cells Myofibroblasts Thioacetamide Rat liver Stem cells

a b s t r a c t Hepatic stellate cells (HSCs), which can express glial fibrillary acidic protein (GFAP) in normal rat livers, play important roles in hepatic fibrogenesis through the conversion into myofibroblasts (MFs). Cellular properties and possible derivation of GFAP-expressing MFs were investigated in thioacetamide (TAA)induced rat liver injury and subsequent fibrosis. Seven-week-old male F344 rats were injected with TAA (300 mg/kg BW, once, intraperitoneally), and were examined on post single injection (PSI) days 1–10 by the single and double immunolabeling with MF and stem cell marker antibodies. After hepatocyte injury in the perivenular areas on PSI days 1 and 2, the fibrotic lesion consisting of MF developed at a peak on PSI day 3, and then recovered gradually by PSI day 10. MFs expressed GFAP, and also showed co-expressions such cytoskeletons (MF markers) as vimentin, desmin and ␣-SMA in varying degrees. Besides MFs co-expressing vimentin/desmin, desmin/␣-SMA or ␣-SMA/vimentin, some GFAP positive MFs co-expressed with nestin or A3 (both, stem cell markers), and there were also MFs co-expressing nestin/A3. However, there were no GFAP positive MFs co-expressing RECA-1 (endothelial marker) or Thy1 (immature mesenchymal cell marker). GFAP positive MFs showed the proliferating activity, but they did not undergo apoptosis. However, ␣-SMA positive MFs underwent apoptosis. These findings indicate that HSCs can proliferate and then convert into MFs with co-expressing various cytoskeletons for MF markers, and that the converted MFs may be derived partly from the stem cell lineage. Additionally, well-differentiated MFs expressing ␣-SMA may disappear by apoptosis for healing. These findings shed some light on the pathogenesis of chemically induced hepatic fibrosis. © 2013 Elsevier GmbH. All rights reserved.

1. Introduction Post-cell injury liver fibrogenesis, irrespective of the etiology, is a dynamic and highly integrated molecular and cellular process, leading to cirrhosis at the advanced stage. Activated hepatic stellate cells (HSCs) are believed to represent the principal fibroblastic cell type which plays important roles in hepatic fibrogenesis through the conversion into myofibroblasts (Forbes and Parola, 2011; Knittel et al., 1999). The myofibroblasts are the most activated cells which can produce extracellular matrices (ECMs) such as collagens (Desmouliere et al., 2003). Transformed HSC-derived myofibroblasts in rat hepatic fibrosis express such cytoskeletons as vimentin, desmin, and ␣-smooth muscle actin (␣-SMA) in varying degrees during the development; out of them, well-differentiated

∗ Corresponding author. E-mail address: [email protected] (J. Yamate). 0940-2993/$ – see front matter © 2013 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.etp.2013.05.008

myofibroblasts may express ␣-SMA (Desmouliere et al., 2003). Additionally, it is reported that HSC-derived myofibroblasts exhibit glial fibrillary acidic protein (GFAP), suggesting that myofibroblasts seen in hepatic fibrosis are heterogeneous in cell population, function and derivation (Forbes and Parola, 2011; Ide et al., 2004). GFAP is a type III intermediate filament (IF) protein; its expression is specific for astroglial cells in the brain (Eng and Ghirnikar, 1994). Although myofibroblasts, characterized by expressions of vimentin, desmin and ␣-SMA, have been reported in cutaneous fibrosis after wound, renal fibrosis and pulmonary fibrosis (Desmouliere et al., 2003; Juniantito et al., 2012b; Willis et al., 2006), GFAP-expressing myofibroblasts are not found in the fibrotic lesions in other organs and sites. As mentioned above, interestingly, GFAP expression is seen in HSC-derived myofibroblasts (Cassiman et al., 2002); likely, GFAP expression is limited in hepatic fibrosis. The reasons why hepatic myofibroblasts can exhibit a variety of cytoskeletal proteins remains to be investigated (Guyot et al., 2006).

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Thioacetamide (TAA)-induced rat hepatic lesions are used as a useful animal model to know the pathogenesis of post-hepatocyte injury fibrosis (Ide et al., 2002, 2003). Using this animal model, to shed some light behind the chemically induced hepatic fibrogenesis, we analyzed GFAP expression patterns by means of single and double immunolabelings with antibodies for cytoskeletal marker antibodies (desmin, vimentin, and ␣-SMA). Furthermore, we pursued the possible derivation of GFAP-expressing myofibroblasts using double immunofluorescence with GFAP antibody and antibodies for endothelial marker (rat endothelial cell antigen-1 [RECA-1]), neurogenic stem cell marker (nestin) or stromal stem cell markers (Thy-1 and A3). This study shows that hepatic myofibroblasts may be derived partly from HSCs in the stem cell lineage. 2. Materials and methods 2.1. Experimental animals Seven-week-old 28 male F344 rats (110–120 g body weight; Charles River Japan, Hino, Shiga, Japan) were housed in an animal room at 21 ± 3 ◦ C and with a 12 h light–dark cycle, and fed a standard diet and tap water ad libitum. Twenty-four rats in TAA group were injected intraperitoneally (IP) with TAA (Wako Pure Chemical, Osaka, Japan) dissolved in physiological saline at a dose of 300 mg/kg body weight. Four rats were examined on each of postsingle injection (PSI) days 1, 2, 3, 5, 7 and 10. The remaining four rats received an equivalent volume of physiological saline in the same manner and served as controls. One hour before euthanasia, all the rats received IP injection of 5-bromo-2 -deoxyuridine (BrdU: Sigma–Aldrich Co., St. Louis, MO, USA) at the dose of 50 mg/kg body weight. These experiments complied with our institutional guidelines for animal care and were approved by the local ethic committee. 2.2. Histopathology All rats were euthanized under isoflurane anesthesia. Liver samples were immediately fixed in 10% neutral buffered formalin, PLP-AMeX (Suzuki et al., 2000) or in Zamboni’s solution (0.21% picric acid and 2% paraformaldehyde in 130 mM phosphate buffer, pH 7.4). The fixed tissues were embedded in paraffin and sectioned at 3–5 ␮m in thickness. Formalin-fixed, deparaffinized sections were stained with hematoxylin and eosin (HE) for histopathological observations and with the Azan–Mallory stain for collagen deposition. 2.3. Single immunohistochemical labeling Livers fixed in Zamboni’s solution or PLP-AMeX was analyzed immunohistochemically by the indirect immunoperoxidase method (Histofine Simple Stain Kit: Nichirei Corp., Tokyo, Japan). Primary antibodies, pre-treatment and dilution for each antibody are listed in Table 1. After pre-treatment, sections were treated with 3% H2 O2 in phosphate buffered saline (PBS) for 30 min to quench endogenous peroxidase and then with 5% skimmed milk in PBS for 30 min at room temperature (RT) to inhibit non-specific reactions, the sections were incubated with the primary antibody overnight at 4 ◦ C. The sections were then incubated for 30 min with peroxidase-conjugated secondary antibody, which consisted of goat anti-mouse IgG Fab fragment antibody (Histofine simplestain MAX PO® ; Nichirei, Tokyo, Japan) or goat anti-rabbit IgG Fab fragment (Histofine simplestain MAX PO® ; Nichirei, Tokyo, Japan). Positive reactions were visualized with 3,3 -diaminobenzidine (DAB; Vector Laboratories Inc., Burlingame, CA, USA) and the sections were lightly counterstained with hematoxylin. As negative

controls, tissue sections were treated with mouse or rabbit nonimmunized serum instead of the primary antibody. 2.4. Double immunohistochemical labeling To investigate co-localization between cell-specific antigens, the double immunofluorescence labeling was carried out using cryostat tissue sections (10 ␮m thick) from the controls and TAA group on PSI days 2 and 3. The combinations in the dual immunofluorescence labeling were GFAP/vimentin, GFAP/desmin, GFAP/␣-SMA, vimentin/desmin, vimentin/␣-SMA, desmin/␣-SMA, GFAP/nestin, GFAP/A3, GFAP/RECA-1 and GFAP/BrdU. The sections except for GFAP/BrdU were fixed in methanol/acetone (1:1) for 10 min at −4 ◦ C and washed in PBS. For GFAP/BrdU staining, sections were fixed in 4% paraformaldehyde for 10 min. Blocking was performed with 10% normal goat serum for 1 h. Subsequently, appropriate primary antibodies were applied (Table 1). After overnight incubation at 4 ◦ C, the sections were washed and exposed for 45 min to appropriate flourochrome-conjugated secondary antibodies. Goat anti-rabbit-Alexa 488 (Invitrogen, Carlsbad, CA, USA) was used for rabbit antibodies. Mouse monoclonal antibodies were visualized with goat anti-mouse IgG-Cy3 (Jackson Immunoresearch, West Grove, PA, USA) or goat anti-mouse IgG2a-Alexa 568 (Invitrogen, Carlsbad, CA, USA). Additionally, isotype specific antibody, goat anti-mouse IgG2a-Alexa 488 (Invitrogen, Carlsbad, CA, USA) was used for the ␣-SMA antibody. Vimentin and RECA-1 directly labeled with HyLyte flour 555 labeling Kit-NH2 (Dojindo Laboratories, Kumamoto, Japan) were used for desmin/vimentin and GFAP/RECA-1 combinations. Slides were mounted with mounting medium including 4 ,6-diamino-2-phenylindole (DAPI; VECTASHIELD® ; Vector Laboratories, Burlingame, CA, USA). Fluorescence signal and co-localization were examined by a laser scanning confocal imaging microscope (Nikon C1Si; Nikon, Tokyo, Japan) and processed with EZ-C1 3.20 Free Viewer (Nikon). Negative control immunostainings were performed, either by omission of the primary antibody or by treatment with non-immune rabbit or mouse IgG. To identify the co-expressions of antigens recognized by nestin and A3, the modified double immunohistochemistry was performed according to the method described by Hasui et al. (2003). Firstly, the sections were immune-labeled with nestin antibody and visualized red by the Fuchsin substrate–chromogen system (Dako, Carpinteria, CA, USA). Secondly, sections used for nestin were reacted with A3. The positive reactions at the second labeling were visualized brown with DAB as mentioned above. 2.5. Terminal deoxyribonucleotide transferase (TdT)-mediated deoxyuridine triphosphate nick end labeling (TUNEL) A standard in situ TUNEL (Appop Tag® Peroxidase In situ Apoptosis Detection Kit, Millipore, Bedford, USA) method was used for detection of DNA fragmentation in apoptotic cells according to manufacturer’s instructions. The formalin-fixed and deparaffinized tissue sections were retrieved with proteinase K (100 ␮g/ml) for 20 min, and then, with 3% H2 O2 for 20 min to inactivate endogenous peroxidase. Afterwards, sections were incubated with TdT enzyme and digoxigenin DNA labeling mixture in the TdT reaction buffer for 60 min at 37 ◦ C, and then, treated with horseradish peroxidase-conjugated anti-digoxigenin for 60 min. The sections were visualized with DAB (Vector Laboratories). Negative control sections were incubated with distilled water instead of TdT enzyme. 2.6. TUNEL and GFAP or ˛-SMA double labeling Double labeling was carried out with the TUNEL method for apoptosis and GFAP or ␣-SMA using cryostat tissue sections (10 ␮m

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Table 1 Primary antibodies used in single and double immunohistochemical labeling. Antibody

Clone

Dilution

Pre treatment

Source

Specificity

␣-SMA Vimentin Desmin GFAPa RECA-1 Nestin MFH Thy-1 BrdU

1A4 V9 D33

1:500 (1:1000) 1:200 Pre-diluted 1:300 (1:500) 1:100 1:200 1:500 1:500 1:200

No Microwave in citrate buffer, 20 min Microwave in citrate buffer, 20 min 0.1% Trypsin, 15 min at 37 ◦ C

Dako, Carpinteria, USA Dako, Carpinteria, USA Dako, Carpinteria, USA Dako, Carpinteria, USA AbD Serotec, Oxford UK Millipore, Temecula, CA, USA TransGenic Inc., Kobe, Japan Cedarlane, Burlington, USA Dako, Carpinteria, USA

Myofibroblasts Mesenchymal cells Smooth muscle cells and myofibroblasts Astrocytes and HSC Endothelial cells Neuroepithelial stem cells Mesenchymal stem cells Mesenchymal stem cells Proliferating cells

HIS52 Rat-401 A3 OX-7

Microwave in citrate buffer, 20 min Microwave in citrate buffer, 20 min Microwave in citrate buffer, 20 min 0.2 N HCl, 10 min at 37 ◦ C

␣-SMA: alpha smooth muscle actin; RECA-1: rat endothelial cell antigen-1; MFH: malignant fibrous histiocytoma; BrdU: 5-bromo-2 -deoxyuridine; HSC: hepatic stellate cells. Parentheses indicate the dilution used for double immunofluorescence. a Rabbit polyclonal, others are mouse monoclonal.

thick) from frozen tissue blocks in the TAA group on PSI day 2. Briefly, the cryostat sections were fixed in 1% paraformaldehyde for 10 min, and after PBS wash, post-fixed in 2:1 ethanol:acetic acid solution at −20 ◦ C for 5 min. Sections were incubated with TdT enzyme and digoxigenin DNA labeling mixture in the TdT reaction buffer for 60 min at 37 ◦ C, and then, primary antibody to GFAP or ␣-SMA was applied for 60 min. After that, the sections were washed, and goat anti-rabbit IgG Alexa 568 (Invitrogen, Carlsbad, CA, USA) was used for GFAP, and goat anti-mouse IgG Cy3 (Jackson Immunoresearch, West Grove, PA, USA) was applied for ␣-SMA for 40 min. Then, the sections were treated with antidigoxigenin antibody conjugated to fluorescein for 30 min. Slides were mounted with DAPI (Vector Laboratories). Fluorescence signal and co-localization were examined by a laser scanning confocal imaging microscope (Nikon C1Si) and processed with EZ-C1 3.20 Free Viewer (Nikon). Negative control sections were incubated with distilled water instead of TdT enzyme. 2.7. Quantitative real time polymerase chain reaction (RT-PCR) Total RNA was extracted from liver samples in the controls and TAA group using a SV Total RNA isolation system (Promega, Madison, WI, USA), and concentration of RNA was adjusted to 0.5 ␮g/␮l. Briefly, first strand cDNA was synthesized by using SuperScriptTM first strand synthesis system (Invitrogen). Quantitative PCR was performed with detection of SYBR green real-time PCR master mix (Toyobo, Osaka, Japan) by a LineGene system detector (BioFlux, Tokyo, Japan). The oligonucleotide sequences used for PCR are listed in Table 2. The following conditions were used for the amplification: for TGF-␤1, 40 cycles of 15 s of denaturation at 95 ◦ C, 15 s of annealing at 64 ◦ C, and 20 s of extension at 72 ◦ C: for the other primers, 40 cycles of 15 s of denaturation at 95 ◦ C, 15 s of annealing at 60 ◦ C and 20 s of extension at 72 ◦ C. Data were calculated using the comparative Ct method. Relative quantitation of mRNA was normalized by that of 18s rRNA as the internal control. The PCR products were electrophoresed in 2% agarose gel, and DNA was stained with ethidium bromide in the gel.

2.8. Statistical evaluation Cells showing a distinct immunopositive-reaction for ␣-SMA, desmin, vimentin and GFAP in the affected perivenular area were semi-quantitatively analyzed with scoring criteria (number and intensity) as follows: − = negative, ± = slightly positive, 1+ = positive, 2+ = moderately positive, 3+ = highly positive; the scoring was evaluated in comparison with that of normal rat liver samples. Liver sections from four rats were examined at each examination point. The percentage of double labeled myofibroblasts (desmin+ /GFAP+ , vimentin+ /GFAP+ , ␣-SMA+ /GFAP+ , ␣-SMA+ /vimentin+ , vimentin+ /desmin+ , ␣-SMA+ /desmin+ , nestin+ /GFAP+ , A3+ /GFAP+ , A3+ /nestin+ , BrdU+ /GFAP+ and TUNEL+ /GFAP+ ) was calculated per 0.2 mm2 of tissue in the affected areas. Data obtained in the RT-PCR methods were expressed as means ± standard deviation (SD), and were statistically evaluated by Dunnett’s comparison test. Value of P < 0.05 was considered significant. 3. Results 3.1. Histopathology and mRNA expressions of inflammatory factors No histopathological lesions were seen in control livers (Fig. 1A). Hepatic lesions induced by TAA were characteristically seen in the perivenular areas. Hepatocyte degeneration/necrosis was seen as early as PSI day 1 and showed the maximum on PSI day 2 (Fig. 1B). On PSI day 2, fibrotic lesions with inflammatory cell reactions began to develop in the damaged areas and peaked on PSI day 3; the collagen deposition was demonstrated by the Azan–Mallory stain (Fig. 1C). On PSI day 5, the perivenular lesions began to decrease in size, indicating healing process. On PSI days 7 and 10, the affected areas were almost replaced with regenerating hepatocytes, but a small amount of collagen fibers were seen around the central vein and along sinusoid without inflammatory cells (Fig. 1D).

Table 2 Oligonucleotide sequences used for quantitative RT-PCR. Gene

Oligonucleotide sequence (forward/reverse)

18s rRNA GFAP TGF-␤1 TNF-␣ PDGF-␤ MMP-2 TIMP-2

5 -GTAACCCGTTGAACCCCATT-3 ; 5 -CCATCCAATCGGTAGTAGCG-3 5 -GCTGGAGGGCGAAGAAAAC-3 ; 5 -GCATCTCCACCGTCTTTACCA-3 5 -CTTCAGCTCCACAGAGAGAAGAACTGC-3 ; 5 CACGATCATGTTGGACAACTGCTCC-3 5 -TGCCTCAGCCTCTTCTCATTC-3 ; 5 -GCTCCTCTGCTTGGTGGTTT-3 5 -TCTGGCCTGCAAGTGTGAG-3 ; 5 -CCCGAGTTTGAGGTGTCTTG-3 5 -GGGTCTATTCTGCCAGCACTTT-3 ; 5 -GGGGTCCATTTTCTTCTTTACTT-3 5 -CGTTTTGCAATGCAGACGTA-3 ; 5 -GGCCGTGTAGATAAATTCGATG-3

GFAP: glial fibrillary acidic protein; TGF-␤1: transforming growth factor-beta 1; TNF-␣: tumor necrosis factor-alpha; PDGF-␤: platelet derived growth factor BB; MMP-2: matrix metaloproteinases-2; TIMP-2: tissue inhibitor of matrix metaloproteinases-2.

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Fig. 1. Histopathology of control liver (A) and thioacetamide (TAA)-treated liver lesion (B–D) in rats. Control liver shows normal hepatic architecture (A). In TAA-treated livers on post-single injection (PSI) day 2, the perivenular lesion consists of hepatocyte coagulation necrosis and inflammatory cell reaction (B, asterisks). On PSI day 3, collagen deposition is seen in the perivenular injured lesion, indicative of development of fibrosis (C, asterisks). On PSI day 7, collagen deposition is resolved in the perivenular fibrotic lesion (D), indicating healing process. A and B, HE stain; C and D, the Azan–Mallory stain. CV, central vein; bar = 100 ␮m.

To know the molecular events of inflammatory factors in the TAA-induced rat hepatic lesions, we investigated mRNA expressions of the following factors: transforming growth factor-␤1 (TGF-␤1), tumor necrosis factor-␣ (TNF-␣), platelet derived growth factor-BB (PDGF-␤), tissue inhibitor of matrix metaloproteinases-2 (TIMP-2) and matrix metaloproteinases-2 (MMP-2). TGF-␤1 mRNA was significantly increased consistently on PSI days 1–10, showing a peak on PSI day 3 (Fig. 2A). mRNAs of TNF-␣ (Fig. 2B) and PDGF-␤ (Fig. 2C) was significantly increased on PSI days 1 and 2, with the maximum on PSI day 1; there were no significant changes on PSI days 3–10. mRNAs of TIMP-2 (Fig. 2D) and MMP-2 (Fig. 2E) showed a significant increase on PSI days 2 and 3, respectively. 3.2. Single immunohistochemistry for vimentin, desmin and ˛-SMA In control livers, cells positive to vimentin (Fig. 3A) and desmin (Fig. 3B) were seen along the sinusoids, depicting HSCs (Forbes and Parola, 2011); these cells were oval or spindloid in shape. Cells reacting to ␣-SMA were seen around the central veins (Fig. 3C); these cells were spindloid in shape and also reacted to vimentin (Fig. 3A), indicating pre-existing mesenchymal cells around the central vein. There were no cells reacting to ␣-SMA along the sinusoids. These findings indicate that HSCs can express vimentin and desmin, but not ␣-SMA. In the TAA group, the numbers of cells reacting to vimentin (Fig. 3D), desmin (Fig. 3E) and ␣-SMA (Fig. 3F) were increased on PSI days 1–5 in the affected perivenular area, showing the maximum on PSI day 3 (Table 3) when the fibrotic lesion was the greatest. On PSI days 7 and 10 during healing process, the numbers were gradually decreased. The configuration of cells reacting to vimentin, desmin and ␣-SMA in the fibrotic lesions was round, oval or spindloid (Fig. 3D, E, F, respective insets).

3.3. Double immunofluorescence between vimentin, desmin and ˛-SMA In control livers, HSCs present along the sinusoids showed double positive reactions to vimentin and desmin (Fig. 4A). Double positive cells reacting to vimentin and ␣-SMA (Fig. 4B) or desmin and ␣-SMA were limited around the central veins (Fig. 4C). These findings corresponded to those in the single immunohistochemistry; that is, HSCs reacted exclusively to vimentin and desmin, but they did not express ␣-SMA. In the TAA group, on PSI day 3 when the fibrotic lesion was the greatest, approximately 60% of desmin-positive cells showed a positive reaction to vimentin (Fig. 5A). Interestingly, the majority of cells reacting to vimentin (Fig. 5B) or desmin (Fig. 5C) reacted simultaneously to ␣-SMA. 3.4. GFAP expression and double immunofluorescence with GFAP and vimentin, desmin or ˛-SMA In control liver, some GFAP-positive cells were observed only along the sinusoids (Fig. 6A). In the TAA group, the number of GFAPpositive cells was increased in the affected perivenular lesions on PSI days 2 (Fig. 6B), 3 (Fig. 6C), 5 and 7, with the greatest expression level on PSI days 3 and 5 (Table 3). On PSI day 10, the distribution of GFAP-positive cells was almost similar to that of controls (Fig. 6D). In agreement with GFAP immunoexpression pattern, mRNAs of GFAP was significantly increased on PSI days 2–7 (Fig. 6E). To identify the GFAP-positive cells, double immunofluorescence was carried out. In control livers, 70–100% of GFAP-positive cells reacted to vimentin (Fig. 7A) or desmin (Fig. 7B); these findings indicated that HSCs reacting to vimentin or desmin also react to GFAP (Forbes and Parola, 2011). Interestingly, ␣-SMA-positive cells present around the central vein did not show a positive reaction

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Fig. 2. mRNA expressions for TGF-␤1 (A), TNF-␣ (B), PDGF-␤ (C), TIMP-2 (D), MMP-2 (E) in livers of controls and TAA-treated livers on PSI days 1–10. Expression level was normalized to that of 18s rRNA level. Bar represents the mean ± SD; *Significantly different from controls at P < 0.05.

Table 3 The appearance of cells reacting to vimentin, desmin, ␣-SMA, GFAP, nestin, A3, BrdU (proliferating cells) and TUNEL (apoptosis) in thioacetamide (TAA)-induced rat liver fibrosis.

Vimentin Desmin ␣-SMA GFAP Nestin A3 BrdU TUNEL

Control

Day 1

Day 2

Day 3

Day 5

Day 7

Day 10

1+ 1+ ± 1+ − − − ±

2+ 2+ 1+ 1+ 2+ − 1+ 1+

2+ 3+ 2+ 2+ 3+ 2+ 2+ 2+

3+ 3+ 3+ 3+ 3+ 2+ 2+ 2+

3+ 2+ 2+ 3+ 1+ − − ±

2+ 1+ 1+ 2+ 1+ − − ±

1+ 1+ 1+ 1+ ± − − ±

−: negative; ±: slightly positive; 1+: positive; 2+: moderately positive; 3+: highly positive. ␣-SMA: alpha-smooth muscle actin; GFAP: glial fibrillary acidic protein; BrdU: 5-bromo-2 -deoxyuridine; TUNEL: terminal deoxyribonucleotide transferase (TdT)-mediated deoxyuridine triphosphate nick end labeling.

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Fig. 3. Immunoreactivity for vimentin (A, D), desmin (B, E) and ␣-smooth muscle actin (␣-SMA) (C, F) in control livers (A–C) and TAA-induced perivenular lesions on PSI day 3 (D–F). In controls, cells labeled for vimentin (A) and ␣-SMA (C) are present around the central vein; some cells (arrows) reacting to vimentin (A) and desmin (B) are seen along the sinusoids, depicting hepatic stellate cells (HSCs). However, HSCs do not react to ␣-SMA (C). In TAA-induced liver lesions on PSI day 3, cells (arrows) positive to vimentin (D), desmin (E) and ␣-SMA (F) are seen in the perivenular fibrotic lesions; these cells are round and fusiform in shape (D–F, insets), indicating development of myofibroblasts. Single immunohistochemistry with DAB, counterstained with hematoxylin. CV, central vein; A–C, bar = 50 ␮m; D–F, bar = 100 ␮m, inset bar = 100 ␮m.

to GFAP (Fig. 7C). In the affected perivenular lesions on PSI day 3, there were cells reacting simultaneously to vimentin/GFAP (approximately 50%; Fig. 8A), desmin/GFAP (approximately 70%; Fig. 8B), and ␣-SMA/GFAP (approximately 40%; Fig. 8C). 3.5. Relationship between GFAP reactivity and endothelial marker (RECA-1) or stem cell markers (nestin, Thy-1 and A3), as well as proliferating activity (BrdU) or apoptosis (TUNEL) The fibrogenesis includes newly formed blood vessels. RECA1-positive endothelial cells did not show any positive reaction to GFAP in controls or in fibrotic lesions on PSI day 3 (Fig. 10A). Although there were no positive cells reacting to nestin in control livers, nestin-positive cells were seen in the injured perivenular areas, with the maximum expression on PSI day 3 (Table 3); the positive cells were round or spindloid in shape (Fig. 9A). On

PSI day 3, approximately 30% of GFAP-positive cells reacted to nestin in the fibrotic area (Fig. 10B). A few Thy-1 positive cells were seen in the Glisson’s sheath both in the control and TAA groups; however, Thy-1-positive cells were not seen in the affected perivenular lesions. Although there were no HSCs reacting to A3 in control livers, A3-positive cells were seen sporadically in the affected perivenular lesions on PSI day 2 and 3 (Fig. 9B); approximately 10% of GFAP-positive cells also reacted to A3 (Fig. 10C). Additionally, approximately 20% of nestin-positive cells showed a positive reaction to A3 (Fig. 10D). To investigate the proliferating activity or apoptosis, BrdU immunohistochemistry and TUNEL method were carried out (Table 3). In control livers, there were no BrdU-positive HSCs. A small number of BrdU-positive myofibroblasts were seen on PSI days 1–3 (Fig. 9C), showing more increased number on days 2 and day 3. Double immunofluorescence revealed that approximately

Fig. 4. Double immunofluorescence for desmin/vimentin (A), ␣-SMA/vimentin (B) and ␣-SMA/desmin (C) in control livers. There are HSCs reacting simultaneously to desmin/vimentin (A, arrows), indicating that HSCs can express desmin and vimentin. Cells (arrows) reacting simultaneously to ␣-SMA/vimentin (B) and ␣-SMA/desmin (C) may be smooth muscles present around the central veins; however, there are no HSCs positive to both ␣-SMA/vimentin (B) and ␣-SMA/desmin (C) along the sinusoids. DAPI for nuclear stain. CV, central vein; bar = 40 ␮m.

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Fig. 5. Double immunofluorescence for desmin/vimentin (A), ␣-SMA/vimentin (B), and ␣-SMA/desmin (C) in TAA-induced fibrotic lesions on PSI day 3. Many desmin-positive cells show a positive reaction to vimentin (A, arrows, inset). The majority of ␣-SMA-positive cells react to vimentin (B, arrows, inset) and desmin (C, arrows, inset) in the perivenular fibrotic lesions. Inset show a higher magnification of positive cells. Right side image (A–C) is merged. DAPI for nuclear stain. CV, central vein; bar = 40 ␮m; inset, bar = 40 ␮m.

10% of GFAP-positive cells gave a positive reaction to BrdU (Fig. 11A). Cells undergoing apoptosis was seen exclusively on PSI days 2 and 3 (Fig. 9D); however, there were no GFAP-positive cells undergoing apoptosis at any examination point (Fig. 11B). Interestingly, a few cells reacting to ␣-SMA (Fig. 11C), vimentin or desmin showed apoptosis in the TAA group. 4. Discussion 4.1. TAA-induced hepatic lesions and inflammatory factors TAA-induced liver injury is widely used as an animal model. The initial lesion in the present TAA group developed in the perivenular areas as early as on PSI day 1; the lesions on PSI days 1 and 2 were characterized by coagulation necrosis/degeneration of hepatocytes, accompanied by inflammatory cells. Then, on PSI days 2 and 3, collagen deposition began to be seen in the injured areas, indicative of commencement of fibrosis as the healing process, and the fibrosis was the greatest on PSI day 3. On PSI day 5 onwards, the fibrotic lesions were gradually reduced until control level by PSI day 10. These histopathological findings corresponded to those reported in previous studies (Fujisawa et al., 2011; Ide et al., 2004).

It is reported that myofibroblasts take part in the hepatic fibrosis (Forbes and Parola, 2011); the myofibroblasts are considered to be mesenchymal cells having an intermediate nature between smooth muscles and fibroblasts (Desmouliere et al., 2003). The development of myofibroblasts may be induced by inflammatory factors produced in the injured areas (Reeves and Friedman, 2002). As reported previously as fibrogenic factors (Czochra et al., 2006; Reeves and Friedman, 2002), mRNAs of PDGF-␤ and TNF␣ were significantly increased on PSI days 1 and 2. Additionally, TGF-␤1 mRNA showed a consistent increase on PSI days 1–10. The fibrotic lesion in the affected perivenular area was maximum on PSI day 3, corresponding to the peaked expression of TGF-␤1 mRNA. TGF-␤1 is well known to be the major fibrotic factor (Reeves and Friedman, 2002). Consistent increase of TGF-␤1 mRNA expression has been reported in cirrhotic lesions (Ide et al., 2002). Taken these findings together, it was considered that TNF-␣ and PDGF␤ might influence fibrogenesis at early stages, and TGF-␤1 could persistently act on advancement of fibrosis. MMP-2 and TIMP-2 are involved in the formation of fibrotic lesions through regulating myofibroblastic cell development. MMPs have functions to degrade ECMs, whereas TIMPs are inhibitors of the MMPs (Knittel et al., 2000). Simultaneous increase

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Fig. 6. Immunoreactivity for glial fibrillary acidic protein (GFAP) in control liver (A) and in TAA-induced perivenular lesions (B–D), as well as mRNA expressions for GFAP (E). In control liver, GFAP-positive cells are seen along the sinusoids, indicating that HSCs can express GFAP (A, arrows). On PSI day 2, GFAP-positive cells appear in the injured perivenular area (B, arrows). There are more increased GFAP-positive cells (arrows) in the perivenular fibrotic lesion on PSI day 3, and these cells are fusiform or stellate in shape (C, inset). On PSI day 7, GFAP-positive cells are gradually decreased, and they are seen mainly along the sinusoids, like distribution in controls (D, arrows). Immunohistochemistry with DAB, counterstained with hematoxylin. CV, central vein; bar = 100 ␮m; inset, bar = 100 ␮m. mRNA expression in control livers and TAA-treated livers on PSI days 1–10 (E); expression level was normalized to that of 18s rRNA level; bar represents the mean ± SD; *Significantly different from controls at P < 0.05.

Fig. 7. Double immunofluorescence for GFAP/vimentin (A), GFAP/desmin (B) and GFAP/␣-SMA (C) in control livers. HSCs react simultaneously to GFAP/vimentin (A, arrows) and GFAP/desmin (B, arrows). However, there are no HSCs positive to both ␣-SMA/GFAP (C). DAPI for nuclear stain. CV, central vein; bar = 40 ␮m.

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Fig. 8. Double immunofluorescence for GFAP/vimentin (A), GFAP/desmin (B) and GFAP/␣-SMA (c) in the TAA-induced liver lesions on PSI day 3. GFAP-positive cells show positive reactions to vimentin (A, arrows), and to desmin (B, arrows). Furthermore, ␣-SMA-positive cells react to GFAP (C, arrows). Right side image (A–C) is merged. DAPI for nuclear stain. CV, central vein; bar = 40 ␮m.

of both TIMP-2 and MMP-2 were reported in CCl4 -induced rat liver lesions (Knittel et al., 2000), indicating importance of appropriate balance between these factors for the fibrotic lesion. The present study also confirmed the increased expression of these factors at early stages on PSI days 2 and 3. 4.2. Expression of vimentin, desmin and ˛-SMA in hepatic fibrosis Vimentin is a type III intermediate filament protein expressed mainly by mesenchymal cells (Geerts et al., 2001), and desmin is a subunit of the intermediate filament in cardiac, skeletal and smooth muscles (Paulin and Li, 2004). Corresponding to findings reported previously (Desmouliere et al., 2003), it was confirmed that HSCs in control livers expressed vimentin and desmin; on the contrary, ␣-SMA expression was not seen in control HSCs. ␣-SMA-positive cells around the central vein may be smooth muscles, because the central vein has contractibility and ␣-SMA is the most abundant protein in smooth muscles (Hu et al., 2003). It has been reported that myofibroblasts appearing in hepatic fibrogenesis can express such cytoskeletons as vimentin, desmin and ␣-SMA in varying degrees (Guyot et al., 2006; Knittel et al., 1999). In single immunohistochemistry, vimentin-, desmin- and ␣SMA-positive cells were diffusely observed in the present affected perivenular areas; the greatest expression corresponded to the peaked fibrosis on PSI day 3. Detailed double immunofluorescence analyses for these intermediate cytoskeletons have not been carried out in hepatic lesions. In the present hepatic fibrosis, the

majority (almost 100%) of vimentin- and desmin-positive cells showed a positive reaction to ␣-SMA. This indicated that HSCs could come to express ␣-SMA as the fibrosis progressed. Double positive cells to vimentin and desmin were approximately 60%. Based on the expression pattern for intermediate cytoskeletons, myofibroblasts in fibrotic lesions are usually divided into following types: VA type (co-expressing vimentin and ␣-SMA), VD type (co-expressing vimentin and desmin), and VAD type (co-expressing vimentin, ␣SMA, and desmin) (Chaurasia et al., 2009). Thus, all types of the myofibroblasts might be present in the present hepatic fibrosis. In a rat model of bleomycin-induced scleroderma, desmin expression was not seen in developing myofibroblasts, and the most predominant type was the VA (Juniantito et al., 2012a). There may be differences in the expression patterns of intermediate cytoskeletons of myofibroblasts between organs or sites. 4.3. Co-expression of GFAP with other cytoskeletons such as vimentin, desmin and ˛-SMA in hepatic fibrosis Besides astrocytes in the brain, GFAP was found to be expressed in HSCs (Cassiman et al., 2002). In double immunofluorescence, HSCs reacting to vimentin and desmin also showed a positive reaction to GFAP in control livers, confirming that rat HSCs can originally express vimentin, desmin and GFAP as immunohistochemical markers (Guyot et al., 2006). In the present fibrotic lesions, the number of GFAP-positive cells was markedly increased, in agreement with the kinetics of vimentin- and desmin-positive

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Fig. 9. Immunoreactivity for nestin (A) and A3 (B), proliferating marker (BrdU) (C), as well as TUNEL method for apoptosis (D) in TAA-induced hepatic lesions on PSI day 3. In the injured perivenular lesions, nestin-positive cells are seen, showing round and spindloid in morphology (A, arrows). A small number of A3-positive cells are seen in the injured perivenular area (B, arrows). There are cells reactive for BrdU in the injured perivenular area (C, arrows), indicating proliferating activity of myofibroblasts. A few cells undergo apoptosis in the injured perivenular area (D, arrows). Immunohistochemistry with DAB (A–C) and TUNEL method (D), counterstained with hematoxylin (A–D). CV, central vein; bar = 100 ␮m.

cells; the greatest expression for GFAP, vimentin and desmin was on PSI days 3 and 5. Additionally, the GFAP-immuno-expression pattern agreed with that of its mRNA. Double immunofluorescence showed that 40–70% of GFAP-positive cells in the fibrotic lesions gave a positive reaction to vimentin, desmin or ␣-SMA. In hepatic fibrosis, HSCs can transform into myofibroblasts, and activated HSCs may express higher level of GFAP (Maubach et al., 2006). The addition of TGF-␤1 and PDGF-␤ to rat primary HSC increased GFAP mRNA expression (Maubach et al., 2006; Zhang and Zhuo, 2006). TNF-␣ may be related to the induction of increased GFAP expression in injured brain (Zhang et al., 2000). The increases in TNF-␣ and PDGF-␤ mRNAs seen on PSI days 1 and 2 might have influence on increased number of GFAP-expressing HSCs in the fibrotic lesions. In addition, GFAP mRNA, which showed a significant increase on PSI days 2–7, might be related to the consistent increase of TGF␤1 mRNA on PSI days 1–10. GFAP-expressing myofibroblasts have not been found in pulmonary and renal fibrosis, as well as cutaneous fibrosis in rats (Desmouliere et al., 2003; Hinz et al., 2007; Juniantito et al., 2012b). HSCs are believed to be ontogenically differentiated from neural/neuroectodermal tissues in the ectodermal layer; GFAP expression in HSCs may be partly because of its embryological origin (Morrison et al., 1999). The molecular mechanisms of GFAP expression in hepatic myofibroblasts should be investigated further.

labeled with BrdU, indicating the proliferating activity of GFAPexpressing HSCs. It was clearly shown that quiescence HSCs could participate into hepatic fibrogenesis through the conversion into myofibroblasts with proliferating activity. In the resolution of hepatic fibrosis, activated myofibroblasts should disappear. In skin wound healing, myofibroblasts disappeared via apoptosis (Moulin et al., 2004). Because apoptotic cells should be cleared dramatically by macrophages, the apoptosis number detectable by the TUNEL method was usually small (Krysko et al., 2006). In the present hepatic fibrosis, myofibroblasts undergoing apoptosis were observed by the TUNEL assay mainly on PSI days 2 and 3 (Iredale et al., 1998). In double immunofluorescence, interestingly, GFAP-positive myofibroblasts did not react to the TUNEL method, whereas apoptosis was seen only in myofibroblasts reacting to ␣-SMA, vimentin and desmin. Out of these cytoskeletons, ␣-SMA may be expressed in well-developed myofibroblasts as the final stage (Desmouliere et al., 2003). Besides apoptosis, loss of activated myofibroblasts in hepatic fibrosis may be due to reversion into the quiescent phenotype (Friedman, 2000; Kisseleva et al., 2012). GFAP-expressing myofibroblasts could reverse to a more quiescent phenotype without undergoing apoptosis in healing process in the present hepatic lesion.

4.5. Possible derivation of myofibroblasts based on immunolabeling with Thy-1, nestin, A3 and RECA-1 4.4. Relationship between GFAP-expressing myofibroblasts and proliferating activity or apoptosis Hepatic myofibroblasts may be derived from HSCs, and they increase the number in the fibrotic lesions by proliferating activity. In the present fibrotic lesions, around 10% of GFAP-positive cells

Thy-1 is a 25–37 kDa glycophosphatidylinositol (GPI) anchored cell surface glycoprotein and known to be expressed in mesenchymal stem cells in the bone marrow and pericytes (Bae et al., 2008; Yuasa et al., 2012). Thy-1 expression has been reported to correlate with myofibroblastic differentiation from the stromal stem cells

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Fig. 10. Double immunofluorescence for GFAP/rat endothelial cell antigen (RECA-1) (A), GFAP/nestin (B), and GFAP/A3 (for mesenchymal stem cell marker) (C) in TAA-induced liver lesions on PSI day 3, as well as double immunolabeling for nestin/A3 (D) in TAA-induced liver lesions on day 2. GFAP-positive cells do not react to RECA-1 (A). There are cells reacting to GFAP/nestin (B, arrows) and GFAP/A3 (C, arrows). Additionally, there are cells reacting to both nestin (red) and A3 (brown) (D, arrows; inset shows a representative double positive cell to nestin/A3). Right side image (A–C) is merged. DAPI for nuclear stain (A–C). CV, central vein; A–C, bar = 40 ␮m; D, bar = 100 ␮m; inset, bar = 100 ␮m. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

(Dezso et al., 2007; Rege and Hagood, 2006). In fact, myofibroblasts expressing Thy-1 were predominant in cutaneous fibrosis induced by bleomycin (Juniantito et al., 2012a), and Thy-1 expression was regarded as myofibroblasts at the early stages in cisplatin-induced rat renal fibrosis (Yuasa et al., 2012). In hepatic fibrosis, Thy-1positive cells in the Glisson’s sheath may migrate into injured areas as the precursor of myofibroblasts in hepatic fibrosis (Dezso et al., 2007). In the present study, Thy-1-positive cells were not seen in

the injured perivenular areas and there were no cells reacting to Thy-1, or both Thy-1 and GFAP. On the other hand, 30% and 10% of GFAP-expressing myofibroblasts reacted also to nestin and A3, respectively, in the present fibrotic lesion. Nestin is a class VI intermediate filament originally identified as a marker for neural stem cells (Namiki et al., 2012). Nestin is known to be expressed in activated HSCs in rats (Niki et al., 1999). Although nestin expression was not seen in control

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Fig. 11. Double labeling for GFAP/BrdU (A), TUNEL/GFAP (B) and TUNEL/␣-SMA (C) in TAA-induced lesions on PSI days 2 and 3. Some GFAP-positive cells show a positive reaction to BrdU in the perivenular fibrotic area (A, arrow; inset show a representative cells reacting to both GFAP/BrdU). GFAP-positive cells do not undergo apoptosis by the TUNEL method in the perivenular fibrotic lesion (B), whereas some ␣-SMA-positive cells show apoptosis (C, arrow). Right side image (A–C) is merged. DAPI for nuclear stain. CV, central vein; bar = 40 ␮m; inset, bar = 40 ␮m.

HSCs, there were cells reacting to both GFAP and nestin in the fibrotic lesion. A3-expressing cells may be regarded as stromal stem cells originating from the bone marrow, although the molecular properties of A3-recognizing antigens have not yet been decided (Yamate et al., 2007). Recently, it is considered that hepatic myofibroblasts may originate partly from bone marrow-derived stem cells (Henderson and Forbes, 2008). There were myofibroblasts reacting to both GFAP and A3. Furthermore, it is interesting to note that some nestin-positive cells in the fibrotic lesions showed a positive reaction to A3. Taken together, hepatic myofibroblasts may be recruited partly from the lineage of neural/neuroectodermal and bone marrow stem cells. Recently, the endothelial–mesenchymal transition (EnMT) has been proposed as a novel mechanism for generating myofibroblasts in fibrotic lesions (Kizu et al., 2009). There were no cells reacting simultaneously to GFAP and RECA-1 in the present fibrotic lesions, denying the possibility of EnMT of myofibroblasts in the present fibrotic lesions. In conclusion, the present study shows that GFAP-expressing HSCs can convert into myofibroblastic cells in chemically induced fibrotic lesions of rat livers. During the development, the GFAPpositive myofibroblasts proliferate and express intermediate cytoskeletons such as vimentin, desmin and ␣-SMA in varying

degrees. These characteristics of myofibroblasts would become useful tool to know the hepatotoxicity. As a phenomenon in degradation of fibrotic lesions, ␣-SMA-positive myofibroblasts undergo apoptosis, although GFAP-positive myofibroblasts did not show apoptosis. The appearance of myofibroblasts expressing both nestin and GFAP supports the notion that myofibroblasts may ontogenetically differentiate from stem cells in the neural/neuroectodermal tissues. Furthermore, the presence of myofibroblasts reacting simultaneously to GFAP and A3 suggested that hepatic myofibroblasts might be recruited partly from bone marrow stem cells. Inflammatory factors such as TGF-␤1, TNF-␣, PDGF-␤, MMP-2 and TIMP-2, of which mRNAs were significantly increased in agreement with hepatic injury and subsequent fibrosis, might have related to the phenotypical alteration of myofibroblasts. The factors influencing change of intermediate cytoskeletons and the molecular mechanisms of developing hepatic myofibroblasts should be investigated in relation with bone marrow and neural stem cells. Acknowledgements This study was supported in part by Grant-in-Aids for Scientific Research (B) (No. 22380173), and for Challenging Exploratory

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