Persistence of Liver Cirrhosis in Association with Proliferation of Nonparenchymal Cells and Altered Location of α -Smooth Muscle Actin-Positive Cells

Abstract

This study was carried out to achieve pathological understanding for the persistence of cirrhosis induced by thioacetamide (TAA). Forty-five, male, 21-day-old, F344 rats were randomly allocated to group 1 and received drinking water as a control, and groups 2 and 3 given 0.015% or 0.03% TAA, respectively for 12 weeks. Two-third of animals per group were sacrificed, and remainder were maintained for a further 4 weeks without TAA treatment. Liver cirrhosis was induced in all animals in group 3 at week 12, with obvious increase of collagen content, and this persisted after cessation of TAA. Proliferating cell nuclear antigen (PCNA) positive labeling indices of nonparenchymal cells were increased significantly after cessation in groups 2 and 3 (p < 0.01). RT-RCR analysis of α-smooth muscle actin (α-SMA) showed significant increase in group 3 compared to that of control at both time points (p < 0.05). Immunohistochemical staining of it demonstrated positive cells to mainly be located around regenerating hepatic nodules at week 12, however, they were focused into enlarged portal areas consisting of fibrous tissues and pseudo-bile ductular cells after the cessation. Taken together, we conclude persistence of liver cirrhosis could be associated with the proliferation of nonparenchymal cells and altered location of α-SMA positive cells.

Introduction

It is well known that cirrhosis results from excess deposition of extracellular matrix component, mainly type I collagen that is produced by hepatic stellate cells (HSCs) (Eng and Friedman, 2000), and during the process, there is an impaired capability for liver regeneration (Andiran et al., 2000). Most therapy for cirrhosis is focused on elimination of direct cause (Hammel et al., 2001; Kweon et al., 2001), and on inducing liver regeneration and increased extracellular matrix (ECM) degradation (Hernandez-Munoz et al., 2001).

It is generally considered that advanced cirrhosis in humans is irreversible after it was established (Bonis et al., 2001). Although some reports have pointed to therapeutic recovery (Dufour et al., 1997, 1998; Poynard et al., 2000), and some remodeling cases of fibrosis were reported in animal studies (Abdel-Aziz et al., 1990; Iredale et al., 1998; Iredale, 2001), it seems there are different recovery pattern depending on animal models used and amount of collagen deposition in the liver (Iredale et al., 1998; Muller et al., 1988).

This study was carried out to achieve further pathological understating for the persistence of cirrhosis induced by thioacetamide1 (TAA), which producing regenerative nodules (Dashti et al., 1989), and hepatic cirrhosis (Muller et al., 1988; Li et al., 2002). We compared the pathological lesions in liver treated with TAA for 12 weeks, and the lesions after its cessation, and investigated the proliferation of hepatic cells and nonparenchymal cells, separately, and expression of α-smooth muscle actin (α-SMA), a maker of activated HSCs between 2 time points.

As liver cirrhosis is one of the strong risk factors for hepatocellular carcinoma development in human (La Vecchia et al., 1998), we also examined the variation of glutathione S-transferase placental form (GST-P) positive foci, preneoplastic lesions, and 8-hydroxy-2′-deoxyguanosine (8-OHdG), a maker for DNA damage.

Material and Methods

Animals and Treatment

Forty-five, 18-day-old male F344 rats were obtained from Charles River Japan, Inc. (Atsugi, Japan), and housed in rooms maintained on a 12-hour light/dark cycle, at constant temperature and humidity. They were allowed free access to pellet chow diets (CE-2, Oriental Yeast Co., Tokyo, Japan) during the experiment. All procedures were approved by the Institutional Animal Care and Use Committee. Then, 21-day-old animals were randomly allocated to 3 groups: group 1 (n = 15) received tap water alone during the experiment; groups 2 (n = 15) and 3 (n = 15) were given 0.015% or 0.03% TAA, respectively, in drinking water. At week 12, TAA was withdrawn and 10 animals each in groups 1 and 2, and 6 in group 3 were sacrificed under ether anesthesia. The remaining rats were maintained for a further 4 weeks without TAA treatment, and then were sacrificed under ether anesthesia. Body weights, food consumption, and water intake of all animals were measured every week. At necropsy, blood was collected from the abdominal vein, and serum was separated by centrifugation for the estimation of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels, liver weights were measured, and liver tissues were fixed in 10% phosphate-buffered formalin. Tissues were processed routinely for embedding in paraffin, and staining of 4 μm sections with hematoxylin and eosin and Azan-Mallory staining for histopathological examination. Liver cirrhosis was the diagnosis on the basis of bridging and regenerating hepatic nodules. Samples of liver from all the animals were also snap-frozen in liquid nitrogen for subsequent RNA extraction and biochemical analysis.

Estimation of Collagen Content

Two sections of liver were cut at approximately 15 μm thick, deparaffinized, and hydrated. They were then kept in distilled water at 4°C and were incubated in aluminium foil-covered test tubes in the presence of a 0.04% solution of Fast green FCF (Sigma) in saturated picric acid for 15 minutes. They were then washed thoroughly with distilled water until the supernatant was clear. They were then incubated with in the presence of Fast green FCF 0.1% and Sirius red F3B (Aldrich) 0.04% in saturated picric acid for 30 minutes and further washed. One milliliter of 0.05N NaOH in 50% aqueous methanol was then added, and each tube was gently agitated. The color was read in a spectrophotometer (Ultraspec 3000, UV/Visible Spectrophotometer; Pharmacia Biotech, Tokyo, Japan) at 530 nm and 605 nm (corresponding to the maximal absorbance of Sirius red and Fast green, respectively). After quantitating the absorbance found for each dye, the collagen content was calculated according to the previously described method (Jimenez et al., 1985; Lopez-De Leon and Rojkind, 1985; Gascon-Barre et al., 1989).

Immunohistochemical Examination of Glutathione S-Transferase Placental Form (GST-P), Proliferating Cell Nuclear Antigen (PCNA), and α-Smooth Muscle Actin (α-SMA)

The avidin-biotin complex method was used to demonstrate GST-P, PCNA, and α-SMA in sections (4 μm) of liver tissue dewaxed with xylene and hydrated through a graded ethanol series. For GST-P staining, sections were treated sequentially with 0.3% hydrogen peroxide, normal goat serum, rabbit anti-GST-P antibody (MBL Co. Ltd, Nagoya, Japan) at 1:1000 dilution, biotin-labeled goat anti-rabbit IgG and avidin-biotin-peroxidase complex (ABC kit; Vector Laboratories, Burlingame, CA). For PCNA or α-SMA staining, sections in sodium citrate buffer (pH 6.0) were boiled in an autoclave for 25 minutes, and then were treated with 0.3% hydrogen peroxide, normal horse serum, anti-PCNA antibody (M 0744, Dako) at 1:500 dilution or α-SMA antibody (M0851, Dako) at 1:50 dilution, followed by ABC-peroxidase procedures (ABC kit, Vector Laboratories, Burlingame, CA). Immune complexes were visualized with 3,3′-diaminobenzidine tetrahydrochloride as a chromogen. As a negative control, normal serum was used instead of primary antibodies. The sections were counterstained with Mayer’s hematoxylin to facilitate examination under a light microscope.

Quantification of GST-P Positive Foci

GST-P positive foci (having more than 2 positive cells) were counted under a light microscope. Total area of GST-P positive foci and total areas of liver sections were measured using a color image processor (IPAP, Sumica Technos, Osaka, Japan) to allow calculation of the number of foci per cm² and the area (mm²) per cm² of liver section.

Quantification of PCNA Expression

Sections were analyzed by blinded observers for counts of PCNA positive cells. Quantification of PCNA positive hepatocytes and nonparenchymal cells was performed by scoring over 2000 cells from 8 random different fields from each animal at ×400 magnification. The results were expressed as PCNA positive labeling indices, relative to the total hepatocytes or total nonparenchymal cells, respectively.

Quantification of 8-Hydroxy-2′-Deoxyguanosine (8-OHdG) Formation

DNA samples isolated from pieces of frozen liver weighing 500 mg were digested into deoxynycleosides by combined treatment nuclease P1 and alkaline phosphotase. Levels of 8-OHdG were determined by high-performance liquid chromatography according to the method of Nakae et al. (1997). Values were expressed as the number of 8-OHdG residues/10⁵ total deoxyguanosines.

RNA Preparation

Total RNA was isolated from frozen liver using ISOGEN (Nippon Gene Co. Ltd, Tokyo, Japan), isopropanol precipitated, dissolved in DEPC-treated distilled water and stored at −80°C until use. RNA concentrations were determined with a spectrophotometer (Ultraspec 3000, UV/Visible Spectrophotometer; Pharmacia Biotech, Tokyo, Japan). For cDNA synthesis, 3 μg of total RNA were heated to 70°C for 10 minutes and then placed immediately on ice for 10 minutes. To each sample, 4 μl of 5X first strand buffer, 2 μl of 0.1 M DTT, 4 μl of 2 mM each dNTP mix, and 1 μl of oligo(dT) primer, and 1 μl of Superscript II reverse transcriptase (Invitrogen, CA) were added. Reverse transcriptions were carried out at 42°C for 50 minutes followed by heating 70°C for 15 minutes and cDNA samples were stored at −20°C until assayed.

RT-PCR for α-SMA mRNA

cDNA were amplified using specific oligonucleotide primers for the rat α-SMA and β-actin genes using the following primer sequences: α-SMA 5′-ACTGGGACGACATGGAAAAg-3′(sense) and 5′-CATCTCCAGAGTCCAGCACA-3′(antisense); β-actin 5′-ACCACAGCTGAGAGGGAAATCG-3′(sense) and 5′-AGAGGTCTTTACGGATGTCAACG-3′(antisense).

The PCR program cycle was set to denature at 94°C for 1 minute, to anneal at 58°C for 1 minute, and to extend at 72°C for 5 minutes for total of 28 cycles for α-SMA mRNA. The β-actin mRNA was used as an internal standard and PCR products were amplified on a linear cycle. The α-SMA mRNA was determined by quantitative PCR and normalized against β-actin mRNA levels. The PCR products were electrophoresed on a 2% Nusieve agarose gel and stained with ethidium bromide. The gels were then scanned and analyzed using a FMBIO II Multi-View Image Analyzer Scanning Unit (Hitachi, Japan). Data are the mean ± SD from 5 samples per group and three independent experiments.

Statistical Analysis

Statistical analyses were performed with the Tukey-Kramer method using the JMP program (SAS Institute, Cary, NC). For all comparisons, probability values less than 5% (p < 0.05) were considered to be statistically significant.

Results

Body and Relative Liver Weights, and Serum Analysis

Comparing with group 1, marked growth retardation was noted during the TAA-treatment, with significant decrease in body weights and significant increase of relative liver weight in groups 2 and 3 at 12 weeks and at 4 weeks after cessation of TAA treatment (p < 0.01). In group 3, the serum AST level was significantly higher at both time points as compared with the control values (p < 0.01). In groups 2 and 3, there was a significant increase of ALT levels at 4 weeks after cessation of TAA compared with that at 12 weeks, and for group 3 as compared to the control group (p < 0.01) (Table 1).

Histopathological Examination of Liver

During the TAA treatment, 4 animals died in group 3, but none in groups 1 and 2. In group 1, all animals had normal-appearing livers. In group 2, there were 7 cases of fibrosis and 3 of cirrhosis at week 12, and 3 of fibrosis and 2 of cirrhosis at 4 weeks after cessation. In group 3, liver cirrhosis was induced in all animals at week 12, and was also evident at 4 weeks after cessation. Cirrhosis cases demonstrated marked hepatic nodules, separated by fibrous septa (Figure 1A, 1B) with a dense collagenous matrix, and many myofibroblast-like cells and fibroblasts. The aggregates appeared to rise in portal areas and to enlarge by expansion. Dysplastic bile-ductule-like structures and metaplastic intestinal-like glands were evident, surrounded by spindle shape cells and collagenous materials, with cell debris and/or blue-colored materials in their lumens at both time points.

Collagen Content

Collagen content of liver in TAA-treated animals was significantly increased in groups 2 and 3 at week 12 and 4 weeks after cessation as compared to control levels (p < 0.05) (Figure 2). In group 3, there was significant increase of collagen content at 4 weeks after cessation compared with that at week 12 (p < 0.05), but in group 2, there was no difference between 2 time points.

Quantitative Data for GST-P Positive Foci

Significant increase of number of GST-P positive foci was noted in groups 2 and 3 at week 12 (p < 0.01), and also 4 weeks after cessation compared with the control values (p < 0.05, p < 0.01, respectively, for groups 2 and 3) (Table 2). Similar finding were obtained for area. In groups 2 and 3, there was significant decrease of number but not area of GST-P positive foci at 4 weeks after cessation compared with that at week 12 (p < 0.01).

Quantitative Data for 8-OHdG

HPLC analysis of 8-OHdG formation showed significant increase in groups 2 and 3 at week 12 compared with that of control (p < 0.01), and to a lesser extent at 4 weeks after cessation (p < 0.05) (Table 2).

PCNA Expression

Some of both of hepatocytes and nonparenchymal cells were PCNA positive at week 12, with significant increase evident in group 3 (p < 0.01). However, at 4 weeks after cessation, the majority of staining was evident in nonparenchymal cells (Figure 3A, 3B).

PCNA positive labeling index of hepatocytes in group 3 was increased significantly at week 12 compared with that of control (p < 0.01), but showed significantly lower values at 4 weeks after cessation compared with that at week 12 (p < 0.01) (Figure 4). In contrast, PCNA positive labeling indices of nonparenchymal cells in groups 2 and 3 did not differ from that of control at week 12, but showed significant higher values at 4 weeks after cessation compared with that of control (p < 0.01). Total PCNA positive labeling index was significant increase in group 3 at week 12, and further in groups 2 and 3 at 4 weeks after cessation (p < 0.01).

α-SMA Expression

Immunohistochemical staining showed α-SMA positive cells to be located around regenerating hepatic nodules at week 12, and in enlarged portal areas consisting of fibrous tissues and dysplastic bile-ductule-like structures, along with metaplastic intestinal-like glands, at 4 weeks after cessation (Figure 3C, 3D).

RT-PCR analysis showed a significant increase of α-SMA mRNA expression in group 3 at week 12 compared with that of control (p < 0.01), and there was also a higher level 4 weeks after cessation (p < 0.05) (Figure 5).

Discussion

The present study confirmed TAA-induced liver cirrhosis to persist to some extent for 4 weeks and provided evidence that the persistence of liver cirrhosis could be associated with the proliferation of nonparenchymal cells and altered location of α-SMA positive cells.

Our data showed an increase of PCNA positive labeling index of nonparenchymal cells after cessation of TAA. Most proliferative nonparenchymal cells are myofibroblast-like cells (α-SMA positive cells) and fibroblasts. At 12 weeks of TAA treatment, α-SMA positive cells were seen surrounding hepatic nodules, but at 4 weeks thereafter they were together with nonparenchymal cells, especially, around dysplastic pseudo-bile ductular cells or metaplastic intestinal-like glands structures. In general, α-SMA-positive cells accumulated within and around postnecrotic area (Ballardini et al., 1983). Usually, α-SMA positive cells detected in injured liver are often classified as activated hepatic stellate cells (HSCs). In normal liver, HSCs are perisinusoidal fat-and retinoid acid-storing cells, but upon this activation, which can be caused by multiple signals resulting from liver cell damage, they may undergo proliferation and differentiation, including a phenotypic shift to myofibroblast-like cells that are positive for α-SMA, and produces extracellular matrix (ECM) proteins, such as collagen type I (Geerts, 2001). Activation of HSCs is associated with secretion of several growth factors, cytokines, chemokines, products of oxidative stress, and change of composition and organization of ECM (Pinzani et al., 1998), and can be inhibited by anti-inflammatory therapy (Hellerbrand et al., 1998). Activated HSCs are believed to represent the principal fibroblastic cell type involved in liver fibrogenesis. It has been reported that they have the potential to migrate, mediated by elements such as Kupffer cells, and migrating stellate cells exhibited elevated proliferation activity (Ikeda et al., 1999). Several factors may influence the migration of HSCs (Friedman and Arthur, 1989; Pinzani et al., 1989), and it is possible that some α-SMA positive cells move into the portal area after cessation of TAA treatment. However, it should be considered that other cell types of the fibroblastic lineage, such as portal fibroblasts or vascular myofibroblasts, might also have fibrogenic potential (Ballardini et al., 1994; Tang et al., 1994; Tuchweber et al., 1996), and functionally different fibroblastic population can be derived from liver cell cultures (Knittel et al., 1999a). Therefore, we cannot exclude the possibility that α-SMA positive cells were generated in this way. Whatever the cellular origin, our data suggest α-SMA positive cells may aggregate in enlarged portal area in association with proliferation of nonparenchymal elements.

From the findings of increased ALT at 4 weeks after cessation in group 3, there may be mechanical destruction of hepatic cells, which may be associated with the observed increase of collagen levels. This would be expected to involve continuous deposition of ECM, with insufficient breakdown of collagen and lack of access to digest collagen fibrils within thick, cross-linked collagen bundles (Vater et al., 1979; Rojkind, 1999). Activated HSCs express type I collagen, and also collagen receptors, such as discoidin domain tyrosine kinase receptor (DDR), which may be upregulated with type I collagen, forming a positive feedback in activated HSCs (Olaso et al., 2001). Both paracrine or autocrine mechanisms may result in activation of downstream signal transduction, and proliferation of HSCs (Vogel et al., 1997; Ikeda et al., 2002). Another possibility is that insufficient breakdown of collagen may be caused by decrease of collagenase activity or by the action of specific inhibitory molecules (TIMP) (Knittel et al., 1999b; Arthur, 2000). Indeed, a tendency for decrease in collagenase activity has been reported after cessation of TAA treatment (Muller et al., 1988).

Our data revealed a decrease of PCNA positive labeling index in hepatocytes at 4 weeks after withdrawal of TAA. It seems this may not be mediated by TGF-β1, a well-known fibrogenic factor and anti-proliferative factor, since RT-PCR analysis of TGF-β1 showed its expression to be down-regulated (data not shown). Many growth factors and cytokines, such as HGF, TNF-α, and IL-6, modulate hepatocyte proliferation by providing both stimulatory and inhibitory signals (Fausto et al., 1995; Michalopoulos and DeFrances, 1997). MAPKs also participate in response to grwoth factors and cytokines (Talarmin et al., 1999).

Many GST-P positive foci, considered to be preneoplastic (Sato et al., 1992; Ito et al., 2003), were induced in the present study and it is well established that long-term feeding of TAA causes hepatic neoplasm in rats (Dasgupta et al., 1981; Becker, 1983). From our finding of proliferation of non parenchymal and persistence of large GST-P positive foci, as well as a high level of 8-OHdG formation, a marker of DNA damage, after withdrawal of TAA, treatment of liver cirrhosis by hepatoproliferative agents would not appear to be indicated, even though some cytokines such as HGF showed beneficial effect in experimental fibrosis model (Xue et al., 2003). Clearly, further investigation of this area is warranted.