Chymase inhibitor prevents the nonalcoholic steatohepatitis in ...

Hepatology Research 2010; 40: 514–523

doi: 10.1111/j.1872-034X.2010.00627.x

Original Article

Chymase inhibitor prevents the nonalcoholic steatohepatitis in hamsters fed a methionine- and choline-deficient diet hepr_627

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Keitaro Tashiro,1,2 Shinji Takai,1 Denan Jin,1 Hiromi Yamamoto,1,3 Koji Komeda,2 Michihiro Hayashi,2 Kazuhiko Tanaka,3 Nobuhiko Tanigawa2 and Mizuo Miyazaki1 1

Departments of Pharmacology, 2Department of General and Gastroenterological Surgery, Osaka Medical College, Daigaku-machi, Takatsuki, Japan, and 3Department of Clinical Pharmacy and Clinical Pharmacokinetics, Osaka University of Pharmaceutical Sciences, Nasahara, Takatsuki, Japan

Aim: Mast cells may be involved in the pathogenesis of nonalcoholic steatohepatitis (NASH). The mast cell protease chymase contributes to the formation of angiotensin II and matrix metalloproteinase (MMP)-9, both of which are intimately involved in liver fibrosis. Therefore, we hypothesized that chymase plays an important role in the development of NASH. Methods: Hamsters were fed a methionine- and cholinedeficient (MCD) diet for 8 weeks. These animals were divided into two groups and received either TY-51469 (1 mg/kg per day) or placebo. A third group was fed a normal diet as a control. Results: Total plasma bilirubin, triglycerides, and hyaluronic acid levels were significantly higher in the MCD diet-fed hamsters than in the normal diet-fed hamsters, but the levels were significantly lower in chymase inhibitor-treated MCD diet-fed hamsters than in placebo-treated MCD diet-fed hamsters. Using histological analysis, marked steatosis and fibrosis

INTRODUCTION

N

ONALCOHOLIC FATTY LIVER disease (NAFLD) has been recognized as the most common form of chronic liver disease in the United States and other developed countries.1,2 A wide spectrum of liver pathology, ranging from simple steatosis to steatohepatitis and cirrhosis, has been observed. Nonalcoholic steatohepatitis (NASH), the progressive form of NAFLD, is a

Correspondence: Dr Shinji Takai, Department of Pharmacology, Osaka Medical College, 2-7 Daigaku-machi, Takatsuki 569-8686, Japan. Email: [email protected] Received 7 August 2009; revision 28 October 2009; accepted 8 November 2009.

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were observed in MCD diet-fed hamsters, but these changes were significantly attenuated by treatment with the chymase inhibitor. Increases in mast cells and chymase-positive cells were observed in the liver after the MCD diet, but the increases disappeared in the chymase inhibitor-treated group. The significant increase observed in chymase activity in liver tissue extract from the MCD diet-fed group was also reduced by treatment with the chymase inhibitor. Chymase inhibition significantly reduced not only angiotensin II expression but also matrix metallopeptidase 9 activity in MCD dietfed hamsters.

Conclusion: These findings demonstrate that the mast cell protease chymase may play a crucial role in the development of NASH in hamsters. Key words: angiotensin II, chymase, fibrosis, nonalcoholic steatohepatitis, steatosis

distinct clinical entity characterized by varying degrees of progressive steatosis, lobular inflammation, and fibrosis of the liver.3,4 Patients with NASH are considered to be at high risk for developing advanced fibrosis, cirrhosis, and hepatocellular carcinoma.5 Little is known about the mechanisms underlying the transition from steatosis to steatohepatitis. A conceptual model referred to as the “two-hit hypothesis” has been proposed.6,7 The first “hit” is the accumulation of fatty acids and triglycerides in hepatocytes. The second “hit”, possibly of environmental or genetic origin, may lead to the development of an inflammatory process that ultimately triggers a fibrogenic response resulting in collagen deposition in the liver.8 Several therapies, including diet adjustment, anti-oxidants, and

© 2010 The Japan Society of Hepatology


approaches that improve insulin resistance, have been tried, but no commonly accepted therapeutic protocol has been established.9–11 Mast cell numbers increase in human chronic liver diseases associated with fibrosis.12–14 Recently, N-(3′,4′dimethoxycinnamoyl)-anthranilic acid (tranilast) was reported to inhibit the activation of mast cells and prevent the development of hepatic fibrosis in a rat NASH model.15 However, histamine, growth factors, cytokines, and proteases are released from mast cells after activation, and which factor plays an important role in hepatic fibrosis is unclear. Chymase is a chymotrypsin-like serine protease located in the secretory granules of mast cells. Chymase converts angiotensin I to angiotensin II, and it activates the precursor of matrix metalloprotease (MMP)-9 to the active form. In a rat model, chymase inhibition reduces the progression to heart failure after autoimmune myocarditis via a reduction in the area of myocardial fibrosis and fibrogenesis.16 In mice, chymase may also play a role in heart remodeling by increasing angiotensin II formation and activating MMP-9, both of which are involved in tissue fibrogenesis.17 In addition, we and other groups have demonstrated that the accumulation of chymasepositive cells is observed in fibrotic regions in the liver of chronic cirrhosis patients.18–20 In this study, we hypothesized that a chymase inhibitor would attenuate the progression of NASH. To investigate this hypothesis, we investigated the effect of a chymase inhibitor by using a dietary animal model of NASH in hamsters fed a methionine- and cholinedeficient (MCD) diet.

Chymase inhibitor prevents the NASH in hamsters

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minipump (model 2ML4; Durect, Cupertino CA) to 20-week-old hamsters for 8 weeks. Normal-fed hamsters were used as a control group (n = 7). All procedures involving animals were conducted in accordance with the guidelines of Osaka Medical College.

Biochemical markers Total bilirubin level and triglycerides were automatically measured (7180 model; Hitachi, Ibaragi,). Hyaluronic acid level was measured with a latex agglutination immunoassay (JCA-BM8000; JEOL, Tokyo).

Chymase activity

METHODS

Each liver specimen was homogenized in 20 mmol/L Na phosphate buffer, pH 7.4. The homogenate was centrifuged at 14 000 rpm for 30 min. The supernatant was discarded, and the pellet was resuspended and homogenized in 5 volumes (w/v) of 10 mmol/L Na phosphate buffer, pH 7.4 containing 2 mol/L KCl and 0.1% Nonidet P-40. The homogenate was stored overnight at 4°C, and was then centrifuged at 14 000 rpm for 30 min. The supernatant was used to measure chymase activity.21 Chymase activity was measured by incubating tissue extracts for 30 min at 37°C with 4 mmol/L angiotensin I in 150 mmol/L borax-borate buffer, pH 8.5 containing 8 mmol/L dipyridyl, 0.77 mmol/L diisopropylfluorophosphate, and 5 mmol/L ethylene diamine tetraacetic acid, as described previously.21 One unit of chymase activity was defined as the amount of enzyme that formed 1 mmol of angiotensin II/min. Protein concentration of the extract was assayed using the bicinchoninic acid Protein Assay Reagent (Pierce, Rockford IL), with bovine serum albumin as the standard.

Drugs

Malondialdehide (MDA) level

2

-[4-(5-fluoro-3-methylbenzo[b]thiophen-2-yl) sulfonamido-3-methanesulfonylphenyl]thiazole-4carboxylic acid (TY-51469) was synthesized as a specific chymase inhibitor (Toaeiyo, Tokyo).16

Animal model Twenty-week-old male F1B hamsters (n = 21) were obtained from Jackson Laboratories (Bar Harbor ME) and were housed in a temperature-, humidity-, and light-controlled room. MCD diet-fed hamsters were fed ad libitum MCD diet (Oriental Yeast, Tokyo). Then, MCD diet-fed hamsters were divided into two groups, and TY-51469 (1 mg/kg/day, n = 7) or saline (n = 7) was administered subcutaneously using an Alzet osmotic

The level of MDA, which is a product of lipid peroxidation in liver extracts, was measured by incubating tissue extracts for 1 h at 100°C with 20 mmol/L thiobarbituric acid in 300 mmol/L phosphoric acid. The reaction was terminated by cooling on ice, and the mixture was then centrifuged at 14 000 rpm for 5 min at 4°C. The absorbance of the supernatant was recorded at 532 nm.

Gelatin zymography Equal volumes of tissue extract (25 mg of protein) were resolved by electrophoresis on 10% sodium dodecyl sulphate (SDS)-polyacrylamide gels containing 1 mg/mL gelatin.22 Gels were then renatured in 50 mmol/L Tris-HCl, pH 7.5 containing 100 mmol/L


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NaCl and 2.5% Triton X-100 for 90 min to remove SDS, and then incubated in 50 mmol/L Tris-HCl, pH 7.5 containing 10 mmol/L CaCl2 for 32 h. Gels were stained with Coomassie Brilliant Blue, and gelatinolytic activity was quantified using NIH-Image 1.61 software (National Institutes of Health, Bethesda MD).

Histological analysis Liver tissue specimens were fixed with Carnoy’s fixative in 10% methanol overnight. Fixed liver tissues were embedded in paraffin, and then cut at a thickness of 5 mm. The sections were mounted on silanized slides (Dako Japan, Kyoto) and deparaffinized with xylene and ethanol. The severity of hepatic histological changes was assessed using hematoxylin and eosin (HE) and Syrius red staining, and was blindly measured by two observers. The degree of steatosis was quantitated as the percentage of lipid droplet area of the total hepatic area.15 Another specimen was stained with Syrius red, and the red area was defined as the fibrotic area,23 which was measured with computerized morphometry using the Fuji-BSA 2000 image analyzing system (Fuji, Tokyo). Mast cells were stained with 0.5% toluidine blue (Chroma-Gesellschaft, Stuttgart, Germany) at pH 4.8.24 The procedures for immunohistochemical analysis of hamster chymase and angiotensin II have been previously described.24 To detect chymase-positive cells, sections were incubated for 1 h at room temperature with anti-hamster chymase antibody (raised in rabbit by immunizing animals with SPYVPWINIVIKASS, which is a C-terminal peptide comprising amino acid residues 212–226 of hamster chymase), followed by reaction with appropriate reagents from a streptavidin-biotin peroxidase kit (Dako LSABkit; Dako, Carpinteria CA) and 3-amino-9-ethylcarbazole, which was used for color development. The sections were lightly counterstained with hematoxylin. To detect angiotensin II-positive cells, rabbit polyclonal antibody against angiotensin II (IgG, Nashville TN) was used. The sections were incubated for 1 h at room temperature with anti-angiotensin II antibody, then reacted with appropriate reagents from a streptavidin-biotin peroxidase kit (Dako) and 3-amino-9-ethylcarbazole, which was used for color development. The sections were lightly counterstained with hematoxylin. The numbers of chymase-positive and angiotensin II-positive cells and mast cells at the sites where they had accumulated in the liver specimens were determined using light microscopy. The average number of



chymase-positive cells and angiotensin II-positive cells counted in three selected fields was determined.

Statistical analysis Data are expressed as the mean 1 standard error of the mean (SEM). Significant differences among the mean values of multiple groups were evaluated using one-way analysis of variance (ANOVA) followed by Fisher’s test. Values of P < 0.05 were considered statistically significant.

RESULTS Effects of chymase inhibitor on macroscopic fatty liver and liver weight : body weight ratio

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HE CHYMASE INHIBITOR did not affect physical appearance, stool composition, coat maintenance, or food or water intake, which indicated that the chymase inhibitor was well tolerated. Macroscopically, the color of the livers was pale in MCD diet-fed hamsters compared with that in normal hamsters, which suggested that fatty liver was induced by the MCD diet. In hamsters treated with the chymase inhibitor, the color of the livers was similar to normal hamster liver (Fig. 1). This observation indicated that the chymase inhibitor may prevent development of a fatty liver in hamsters fed the MCD diet. The body weight of animals in the three groups were not significantly different (Fig. 1). Although there was no significant difference, the liver weight in MCD dietfed hamsters treated with chymase inhibitor tended to be lower than that in MCD diet-fed hamsters treated with placebo (P = 0.07, Fig. 1).

Effect of chymase inhibitor on biochemical parameters Total plasma bilirubin, triglycerides, and hyaluronic acid levels are shown in Fig. 2. Hamsters fed the MCD diet had higher plasma levels of all three parameters compared to levels of hamsters fed a normal diet. The chymase inhibitor significantly decreased to the normal level all three parameters.

Effect of chymase inhibitor on liver steatosis Histological analysis of HE-stained liver sections from MCD diet-fed hamsters showed development of hepatic lipid droplets (Fig. 3). In contrast, the chymase inhibitor dramatically decreased the development of hepatic lipid



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with the chymase inhibitor than in MCD diet-fed hamsters treated with placebo (Fig. 3).

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Sirius red staining represents hepatic collagen deposits and was performed to examine the degree of hepatic fibrosis. The Sirius red-stained area was clearly larger in MCD diet-fed hamsters than in the normal diet-fed hamsters, and the size of the area was reduced by the chymase inhibitor (Fig. 4). Quantitative analysis of the ratio of fibrotic area to total liver area showed that the ratio was significantly increased (over twofold) in the MCD diet-fed group compared with the normal

(a) 0.16 Total bilirubin (mg/dL)

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Figure 1 (a) Typical whole livers from the control, methionine- and choline-deficient (MCD) diet-fed + placebotreated and MCD diet-fed + 2-[4-(5-fluoro-3-methylbenzo [b]thiophen-2-yl)sulfonamido-3-methanesulfonylphenyl] thiazole-4-carboxylic acid (TY-51469)-treated hamsters (black bars represent 1 cm). (b) Body weights in the control, MCD diet-fed + placebo-treated and MCD diet-fed + TY-51469treated groups. (c) Liver weights in the control, MCD dietfed + placebo-treated and MCD diet-fed + TY-51469-treated groups. Values represent mean 1 standard error of the mean.

droplets (Fig. 3). Quantitative analysis showed that the ratio of the lipid droplet area to the total liver area in MCD diet-fed hamsters treated with the chymase inhibitor was reduced by about 98.5% when compared with that of MCD diet-fed hamsters treated with placebo (Fig. 3). Further, under high magnification, we observed some accumulation of neutrophils in MCD diet-fed hamsters treated with placebo, but this accumulation was not observed in other groups of hamsters (Fig. 3). The MDA level in the liver was significantly higher in MCD diet-fed hamsters treated with placebo than in normal diet-fed hamsters. However, the MDA level was significantly lower in MCD diet-fed hamsters treated

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Figure 2 Plasma levels in total bilirubin (a), tryglycerides (b) and hyaluronic acid (c) in the control, methionine- and choline-deficient (MCD) diet-fed + placebo-treated and MCD diet-fed + 2-[4-(5-fluoro-3-methylbenzo[b]thiophen2-yl)sulfonamido-3-methanesulfonylphenyl]thiazole-4carboxylic acid (TY-51469)-treated groups. Values represent mean 1 standard error of the mean. *P < 0.05 and **P < 0.01 vs. MCD diet-fed + placebo-treated group.


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Chymase activity in liver extract from hamsters fed the MCD diet was significantly elevated compared with the activity in extract from those fed a normal diet, and the activity was reduced in MCD diet-fed hamsters treated with the chymase inhibitor (Fig. 6).

Effect of chymase inhibitor on angiotensin II-positive cells The number of angiotensin II-positive cells was significantly higher in hamsters fed the MCD diet compared to

2 0

nificant reduction in mast cell number compared with placebo treatment of MCD diet-fed hamsters (Fig. 5). We performed immunohistochemical analysis of chymase-positive cells. The number of chymase-positive cells tended to be increased in MCD diet-fed hamsters compared with normal diet-fed hamsters. However, the number of chymase-positive cells was significantly reduced in MCD diet-fed hamsters treated with the chymase inhibitor (Fig. 5).

Control

Placebo TY-51469 MCD

Figure 3 (a) Typical photographs of the hematoxylin and eosin-stained liver sections from the control, MCD dietfed + placebo-treated and methionine- and choline-deficient (MCD) diet-fed + 2-[4-(5-fluoro-3-methylbenzo[b]thiophen2-yl)sulfonamido-3-methanesulfonylphenyl]thiazole-4carboxylic acid (TY-51469)-treated hamsters (magnification ¥40). The expanded photograph in the MCD dietfed + placebo-treated hamster shows the accumulation of neutrophils (A: the lower right of the middle photograph). The original magnification was 100¥ (A: the lower right of the middle photograph). Ratios of the lipid droplet area to the total liver area in the control, MCD diet-fed + placebo-treated and MCD diet-fed + TY-51469-treated groups (b). Values represent mean 1 standard error of the mean. **P < 0.01 vs. MCD diet-fed + placebo-treated group.

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group, and that the chymase inhibitor significantly reduced (Fig. 4).

Effects of chymase inhibitor on accumulation of mast cells and chymase-positive cells The mast cell number was significantly higher in MCD diet-fed hamsters than in normal diet-fed hamsters. However, chymase inhibitor treatment induced a sig-


Figure 4 (a) Typical photographs of the Sirius red-stained liver sections from the control, methionine- and choline-deficient (MCD) diet-fed + placebo-treated and MCD diet-fed + 2[4-(5-fluoro-3-methylbenzo[b]thiophen-2-yl)sulfonamido3-methanesulfonylphenyl]thiazole-4-carboxylic acid (TY51469)-treated hamsters (magnification ¥40). (b) Ratios of fibrotic area to total liver area in the control, MCD dietfed + placebo-treated and MCD diet-fed + TY-51469-treated groups. Values represent mean 1 SEM. *P < 0.05 and **P < 0.01 vs. MCD diet-fed + placebo-treated group.


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Figure 5 Typical photographs of the liver sections stained with (a) toluidine blue and (b) immunostained with anti-chymase antibodies from the methionine- and choline-deficient (MCD) diet-fed + placebo-treated hamsters. Arrows indicate (a) toluidine blue-stained cells and (b) chymase-positive cells (magnification ¥40). Numbers of (c) mast cells and (d) chymase-positive cells in the liver sections from the control (Cont), MCD diet-fed + placebo-treated (Pl) and MCD diet-fed + 2-[4-(5-fluoro-3-methylbenzo[b]thiophen-2-yl) sulfonamido-3-methanesulfonylphenyl]thiazole-4-carboxylic acid (TY-51469)-treated (TY) hamsters. Values represent mean 1 standard error of the mean. *P < 0.05 vs. MCD dietfed + placebo-treated group.

that of hamsters fed a normal diet, and the cell number was attenuated in MCD diet-fed hamsters treated with the chymase inhibitor (Fig. 7).

Effect of chymase inhibitor on matrix metallopeptidase 9 (MMP-9) activity To determine the effect of the chymase inhibitor on MMP-9 activity in liver, we evaluated the activity using in situ gelatin zymography. Augmentation of MMP-9 activity was found in livers from hamsters fed the MCD diet compared with the normal diet group, and this activity was significantly decreased by the chymase inhibitor (Fig. 8).

DISCUSSION N THIS STUDY, we examined the role of chymase in NASH pathogenesis. Chymase, which is contained in the granules of mast cells, has been reported to be involved in the development of tissue fibrosis, including that seen in liver cirrhosis.20 In this study, we used the

Figure 6 Chymase activities in the liver extracts from the control, methionine- and choline-deficient (MCD) diet-fed + placebo-treated and MCD diet-fed + 2-[4(5-fluoro-3-methylbenzo[b]thiophen-2-yl)sulfonamido-3methanesulfonylphenyl]thiazole-4-carboxylic acid (TY51469)-treated hamsters. Values represent mean 1 standard error of the mean. **P < 0.01 vs. MCD diet-fed + placebotreated group.

specific chymase inhibitor TY-51469 at a dose of 1 mg/kg per day, as previously reported.16 TY-51469 inhibits human chymase with an IC50 of 7 nmol/L. Chymase is a chymotrypsin-like serine protease, but TY-51469 does not inhibit other chymotrypsin-like serine proteases, such as bovine chymotrypsin or human cathepsin G, even at a concentration as high as

(a)

(b) Angiotensin II-positive cells(cells/mm2)

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Figure 7 (a) A typical photograph of the liver section stained with immunostained with anti-angiotensin II antibodies from the methionine- and choline-deficient (MCD) dietfed + placebo-treated hamster (arrows indicate angiotensin II-positive cells; magnification ¥40). Numbers of angiotensin II-positive cells in the liver sections from the control (Cont), MCD diet-fed + placebo-treated (Pl) and MCD diet-fed + 2[4-(5-fluoro-3-methylbenzo[b]thiophen-2-yl)sulfonamido-3methanesulfonylphenyl]thiazole-4-carboxylic acid (TY51469)-treated (TY) hamsters (b). Values represent mean 1 standard error of the mean. *P < 0.05 vs. MCD dietfed + placebo-treated group.


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(a) MMP-9 Control

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MMP-9 activity (% of control)

600

*

500 400 300 200 100 0 Control

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Figure 8 (a) Typical zymographic images of the liver extracts from the control, methionine- and choline-deficient (MCD) diet-fed + placebo-treated and MCD diet-fed + 2-[4(5-fluoro-3-methylbenzo[b]thiophen-2-yl)sulfonamido-3methanesulfonylphenyl]thiazole-4-carboxylic acid (TY51469)-treated hamsters. (b) Matrix metallopeptidase 9 (MMP-9) activities in the liver extracts from the control, MCD diet-fed + placebo-treated and MCD diet-fed + TY-51469treated hamsters. Values represent mean 1 standard error of the mean. *P < 0.05 and **P < 0.01 vs. MCD dietfed + placebo-treated group.

10 mmol/L. Thus, TY-51469 has a high specificity for chymase. Using a hamster NASH model, we observed macroscopically that the color of the livers was pale, indicating development of fatty liver after 8 weeks of the MCD diet. The levels of the biochemical markers total bilirubin, hyaluronic acid, and triglycerides, all of which are known to increase in NASH, were significantly increased in MCD diet-fed hamsters. Histologically, marked steatosis and fibrosis were also observed in MCD diet-fed hamsters. However, all the biochemical markers and the histological changes were significantly attenuated in MCD diet-fed hamsters treated with the chymase inhibitor. These findings suggest a usefulness of chymase inhibition for prevention of the development of NASH. Mast cells synthesize and secrete various biologically active substances, including histamine, serotonin, arachidonic acid derivates, proteases such as cathepsin G, tryptase, and chymase, growth factors, and cytokines, suggesting the involvement of mast cells in various biological processes. In a previous report, mast cells were found to be involved in fibrogenesis in chronic liver disease, and to play an important role in liver fibrosis of


primary biliary cirrhosis and alcoholic liver disease.12 The mast cell stabilizer tranilast significantly ameliorated a dietary rat model of NASH.15 However, the underlying mechanism of how tranilast affects the molecules released from mast cells is unknown. Tranilast significantly attenuated the accumulation of mast cells, resulting in reduced chymase activity in a dog vascular proliferative model.25 In our recent paper, the number of chymase-positive cells was significantly higher in the liver of patients with chronic cirrhosis than in noncirrhosis patients, and we observed significant correlations between the number of chymase-positive cells and the degree of fibrosis.19 In this study, not only the mast cell number but also the chymase-positive cell number in the liver were significantly increased after the MCD diet. Further, chymase activity was also significantly augmented in liver extract after the MCD diet. However, the chymase inhibitor induced reductions in the number of chymase-positive cells and in chymase activity in liver, which may result in the attenuation of the severity of NASH. Chymase converts angiotensin I to angiotensin II, and angiotensin II may be involved in the pathogenesis of NASH. Angiotensin II induces the proliferation of hepatic stellate cells and induces transforming growth factor (TGF)-b gene expression in fibroblasts.26,27 Further, the angiotensin II receptor blocker (ARB) losartan decreases blood markers of hepatic fibrosis, TGF-b levels, and hepatic fibrosis in patients with NASH.28 In a rat NASH model, the ARBs olmesartan and L158809 significantly reduced the severity of NASH and reduced TGF-b expression.29,30 In this study, angiotensin II-positive cells was significantly increased in the liver of NASH model animals. On the other hand, chymase inhibition significantly reduced the increase in angiotensin II-positive cells. Further, although chymase is an angiotensin II-forming enzyme, it also contributes to the release of TGF-b from its precursor, human fibroblast latent TGF-b-binding protein.31 In human dermal fibroblasts, chymase significantly increases fibroblast proliferation, and the increased proliferation can be completely suppressed by a chymase inhibitor.31 In media supernatants of cultured fibroblasts, the TGF-b concentration was significantly increased after chymase injection, and this increase in TGF-b concentration was inhibited by a chymase inhibitor.31 Anti-TGF-b neutralizing antibodies completely suppress cell proliferation induced by human chymase, indicating that chymase induces cell proliferation via TGF-b activation.31 To our knowledge, there is no information regarding the sequence of TGF-b in hamster, and anti-hamster TGF-b


antibodies do not exist. Therefore, we could not measure TGF-b levels in this study. In a dog model of vascular proliferation, a chymase inhibitor significantly reduced not only angiotensin II-positive cell number but also TGF-b-positive cell number.32 Therefore, the mechanism of attenuation of NASH development by the chymase inhibitor may be due to a reduction in angiotensin II formation. In this study, we observed a dramatic attenuation of hepatic steatosis following treatment with the chymase inhibitor. Previous reports have demonstrated that angiotensin II plays an important role in the development of steatosis.29,33 Angiotensin II has been shown to induce hepatic oxidative stress, and in fact, blockade of the angiotensin II receptor results in attenuation of not only oxidative stress but also hepatic steatosis in a rat NASH model.29 Further, in angiotensin II type 1 receptor-deficient mice, significant attenuation of both oxidative stress and hepatic steatosis was observed.33 In this study, we observed a significant reduction in the MDA level, which is an indicator of lipid peroxidation. Therefore, up-regulation of chymase-dependent angiotensin II formation may induce hepatic steatosis, but in contrast, chymase inhibition may inhibit angiotensin II formation in liver, resulting in attenuation of hepatic steatosis. The chymase inhibitor significantly reduced Sirius red-stained hepatic collagen deposits in liver tissue in this study. Liver fibrosis is characterized by increased deposition of extracellular matrix (ECM) components due to ECM overproduction. MMP-9 is considered to play an important role in fibrogenesis and tissue remodeling, and a significant increase in MMP-9 was found in patients with NASH.34 Chymase has been shown to activate promatrix metalloproteinase-9 to MMP-9, both in vitro and in vivo.35–37 In aneurism models, both chymase and MMP-9 activities are significantly increased in aneurismal aortas, but a chymase inhibitor significantly attenuates not only the chymase activity but also the MMP-9 activity.22,38 In our study, MMP-9 activity was significantly increased in the liver of hamsters fed the MCD diet, and MMP-9 activity was dramatically reduced by the chymase inhibitor. Thus, the mechanism by which the chymase inhibitor prevents the development of liver fibrosis may depend on the inhibition of MMP-9 activation in the liver with NASH. A significant decrease in the number of liver mast cells was observed in animals treated with TY-51469 as compared with those in the MCD diet-fed, placebo-treated group in this study. Chymase may play an important role in the recruitment of mast cells. Stem cell factor


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(SCF) is a membrane-bound molecule that is proteolytically cleaved by chymase at a site close to the membrane, producing a soluble bioactive protein.39 Chymase-activated SCF may stimulate mast cell development and proliferation. We have previously shown that chymase inhibitors result in the reduction of mast cell number in experimental models,40,41 similar to what was observed in this study. Therefore, the increase in chymase activity may be dependent on the accumulation of mast cells in the liver after the MCD diet. In contrast, the inhibitory effect of TY-51469 may also contribute to the attenuation of mast cell accumulation. Chymase inhibition may also contribute to the attenuation of inflammatory cell accumulation. In this study, we observed accumulation of neutrophils only in MCD diet-fed hamsters treated with placebo, and no accumulation was observed in control and MCD dietfed hamsters treated with chymase inhibitor. He and Walls42 demonstrated the accumulation of neutrophils in the skin where purified human chymase had been injected. Terakawa et al.43 also reported that chymase induced the accumulation of neutrophils after the injection of purified mouse chymase into mouse skin. These reports suggest that chymase may provide a potent stimulus for recruitment of neutrophils. On the other hand, a chymase inhibitor, NK3201, significantly reduced the accumulation of neutrophils in colitis in mice treated with dextran sodium sulfate.44 Therefore, the attenuation of inflammatory cell accumulation may be part of the mechanism of prevention of steatosis and fibrosis following chymase inhibition. In conclusion, mast cells have been shown to be intimately involved in liver fibrosis. We demonstrate that the mast cell protease chymase may play an important role in the development of NASH in hamsters fed an MCD diet via increases in angiotensin II formation and MMP-9 activity. Our findings suggest that use of a chymase inhibitor may be a novel strategy to prevent NASH development in humans.

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