- Email: [email protected]
Chymase Inhibitor, BCEAB, Suppressed Peritoneal Adhesion Formation in Hamster
Journal of Surgical Research 107, 219 –222 (2002) doi:10.1006/jsre.2002.6515
Chymase Inhibitor, BCEAB, Suppressed Peritoneal Adhesion Formation in Hamster Yukiko Okamoto, M.D., Shinji Takai, Ph.D., and Mizuo Miyazaki, M.D. 1 Department of Pharmacology, Osaka Medical College, Takatsuki City, Osaka 569-8686, Japan Submitted for publication January 9, 2002; published online August 29, 2002
Background. Mast cells are closely related to adhesion formation, while it has been unclear which factor in mast cells plays an important role in the development of adhesion formation. To clarify the role of chymase produced from mast cells in adhesion formation, we investigated the preventive effect of a specific chymase inhibitor, BCEAB, on adhesion formation in a hamster experimental model. Materials and methods. Hamsters were administered orally once daily with 100 mg/kg of BCEAB or placebo from the operated day to 1 week after the operation. The uterus was grasped and denuded by a swab. Results. One week after the operation, the scores for adhesion formation in the chymase inhibitor-treated group were significantly decreased in comparison with those in the placebo-treated group (placebotreated group, 2.80 ⴞ 0.20; chymase inhibitor-treated group 1.60 ⴞ 0.31: P < 0.01). The chymase activity in the injured uterus was also significantly suppressed in the chymase inhibitor-treated group (placebo-treated group, 17.3 ⴞ 2.69 mU/mg protein; chymase inhibitortreated group 9.60 ⴞ 0.89: P < 0.05). After scraping the utelus, the level of transforming growth factor- in the peritoneal fluid was significantly increased in the placebo-treated group, while it was suppressed to 70% by the treatment with BCEAB. Conclusions. The specific chymase inhibitor BCEAB may be a useful drug for prevention of adhesion formation. © 2002 Elsevier Science (USA) Key Words: adhesion; chymase; mast cells; chymase inhibitor; hamster.
gest that mast cells may be involved in peritoneal adhesion formation [1, 2]. For example, the number of mast cells is increased around wounds in the late stages of the healing process [1, 2]. A mast cell stabilizer, which inhibits the activation and accumulation of mast cells, is effective in attenuating adhesion formation in rat models [3]. Recently, we also reported that adhesion formation in mast cell-deficient mice was significantly less severe than that in normal control mice [4]. These reports suggest that mast cells are closely related to adhesion formation. However, mast cells release a large number of inflammatory mediators such as histamine, serotonin, chemotactic factors, cytokines, and serine proteases during the repair phase of adhesion formation [5, 6], and it has been unclear which factor plays an important role in the development of adhesion formation. Chymase is a chymotrypsin-like serine protease contained in the secretory granules of mast cells. Previously, we reported that the number of mast cells containing chymase significantly increased in the healing sites after cecal scraping in mice [4]. However, the pathophysiological role of chymase in the development of adhesion formation remains unclear. In this study, to elucidate the relation between chymase and adhesion formation, we investigated the effect of a specific chymase inhibitor, BCEAB [7], on adhesion formation. MATERIALS AND METHODS
Drugs and Animals INTRODUCTION
Postoperative intraperitoneal adhesions are a wellknown complication of surgery. Previous reports sugTo whom correspondence should be addressed. Fax: ⫹81-726-846518. E-mail: [email protected]. 1
A specific chymase inhibitor, 4-[1-[bis-(4-methyl-pheny)-methyl]-3(2-ethoxy-benzyl)-4-oxo-azetidine-2-yloxy]-benzoic acid (BCEAB), was a gift from Shionogi Co. (Osaka). Mature female Syrian hamsters (n ⫽ 44, SLC, Shizuoka, Japan), 6 weeks of age, weighing 85 to 90 g were maintained in an environmentally controlled room with a 12-h light, 12-h dark cycle. The experimental procedure for the animals was in accordance with the Guide for the Care and Use of
219
0022-4804/02 $35.00 © 2002 Elsevier Science (USA) All rights reserved.
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JOURNAL OF SURGICAL RESEARCH: VOL. 107, NO. 2, OCTOBER 2002
Laboratory Animals (Animal Research Laboratory, Osaka Medical College).
Surgical Technique Hamsters were anesthetized with intraperitoneal sodium pentobarbital (50 mg/kg). An abdominal midline incision was made, and the right uterus was grasped and denuded of serosa over half the length of the uterine body until punctate hemorrhage occurred, using a swab [4]. Then, the abdomen was closed in two layers with silk. Hamsters were administered orally once daily with 100 mg/kg of BCEAB or placebo from the operated day to 1 week after the operation. One week after the surgery, the animals (placebo group, n ⫽ 10; chymase inhibitor group, n ⫽ 10) were anesthetized with sodium pentobarbital (50 mg/kg, ip), and then adhesions were assessed. After the scoring, the uterus was removed for the measurement of chymase activity.
Scoring of Adhesions After anesthetization, the hamster was incised at the abdominal midline and the intraperitoneal adhesion formations were scored. The adhesions were graded blindly according to a modified classification of Hulka et al. [8]: Score 0, no adhesions; Score 1, mild adhesions; Score 2, localized moderate adhesions; Score 3, moderate and wide adhesions; Score 4, severe adhesions, impossible to separate.
Tissue Preparation A tissue extract for measurement of chymase activity was prepared as described previously [9]. The uterus was minced and homogenized in 10 vol (w/v) of 20 mM Na-phosphate buffer (pH 7.4). The homogenate was centrifuged at 10,000 rpm for 30 min. The supernatant was discarded. These steps were repeated twice. The pellets were resuspended and homogenized in 5 vol (w/v) of 10 mM Na-phosphate buffer (pH 7.4) containing 2 M KCI and 0.1% Nonidet P-40. The homogenate was stored overnight at 4°C and then centrifuged at 10,000 rpm for 30 min. The supernatant was used as the tissue extract for the measurement of chymase activity.
Measurement of Chymase Activity Chymase activity was measured as described previously by Takai et al. [7]. Aliquots (20 L) of tissue extract were incubated for 30 min at 37°C with 4 mM angiotensin (Ang) I in 150 mM borax– borate buffer, pH 8.5 (final incubation volume of 0.1 mL), containing 5 mM ethylenediaminetetraacetic acid, 8 mM dipyridyl, and 0.77 mM diisopropyl phosphorofluoridate. Reactions were terminated by addition of 0.15 mL of 15% trichloroacetic acid, and then centrifugation was conducted at 10,000 rpm for 10 min. For fluorometric quantification of His–Leu as an Ang II metabolite, 10% o-phthaldialdehyde (dissolved in neat methanol) was added to the supernatant under alkaline conditions, and then 6 N HCl was added to stabilize the fluorescence for the measurement at 340 nm excitation and 455 nm emission. A blank was carried out with the addition of 0.5 mM chymostatin (dimethylformamide solution). Protein concentration was measured by bicinchoninic acid protein assay reagent (Pierce Chemical, Rockford, IL) using bovine serum albumin as a standard.
Measurement of TGF- Concentration After scraping the uterus, 1 mL of saline was injected into the abdominal cavity, and the peritoneal fluid was collected immediately (0 min) and 60 min after the injection. The level of transforming growth factor (TGF)- in the collected peritoneal fluid was measured with an ELISA kit (R&D Systems, Minneapolis, MN).
FIG. 1. Typical photographs of adhesion lesions in the placeboand the chymase inhibitor (BCEAB)-treated hamsters 1 week after the operation. The arrows indicate the uterus.
Statistical Analysis Adhesion scores were evaluated in a nonparametric test, and statistically analyzed by the Mann–Whitney U test. Chymase activity and TGF- concentration were evaluated by Student’s t and Fisher’s tests, respectively. Values are given as means ⫾ standard error (SE). Differences were considered statistically significant at P ⬍ 0.05.
RESULTS
Animal Model No difference in the body weight was found between placebo-treated and the chymase inhibitor-treated hamsters in this study of the 1-week model. Scores of Adhesion Typical photographs of the adhesion lesions are shown in Fig. 1. The scores of adhesion formation in hamsters treated with placebo and in those treated with the chymase inhibitor were 2.80 ⫾ 0.20 and 1.60 ⫾ 0.31, respectively, and this difference is significant (Fig. 2). Chymase Activity The uterus chymase activities in the hamsters treated with the chymase inhibitor and placebo were 17.3 ⫾ 2.69 mU/mg protein and 9.60 ⫾ 0.89 mU/mg protein, respectively, and this difference was significant (Fig. 3). TGF- Concentration The level of TGF- in the peritoneal fluid was significantly increased 60 min after scraping the utelus, compared with the level at 0 min in the placebo-treated group (Fig. 4). In the chymase inhibitor-treated group, the significant increase in TGF- was suppressed up to about 70%, but this difference was not significant (Fig. 4).
OKAMOTO, TAKAI, AND MIYAZAKI: BCEAB SUPPRESSES ADHESION
FIG. 2. Scores for adhesion formation in the placebo- and the chymase inhibitor (BCEAB)-treated hamsters 1 week after the operation. Values are means ⫾ SE. *P ⬍ 0.05 vs placebo-treated group.
DISCUSSION
In this study, we demonstrated that the chymase activity in the uterus 1 week after the operation was significantly increased in comparison with that in the normal uterus. The chymase activity of the injured uterus in the chymase inhibitor-treated hamsters was significantly decreased in comparison with that in placebo-treated hamsters, and adhesion formation was also decreased. These findings suggest that chymase contained in mast cells plays an important role in the development of adhesion formation. It is well known that the number of mast cells increases where inflammation occurs after surgery. We also reported that an accumulation of chymase-positive mast cells were observed in adhesion lesions, suggesting that the accumulation of chymase-positive mast cells may play an important role in the development of adhesion formation [4]. Chymase activates stem cell factor, a typical cytokine that has the ability to induce
FIG. 3. Chymase activities in the extract from the uterus in the placebo- and the chymase inhibitor (BCEAB)-treated hamsters 1 week after the operation. Values are means ⫾ SE. *P ⬍ 0.05 vs placebo-treated group.
221
FIG. 4. TGF- concentrations in the peritoneal fluid of the placebo-treated hamsters immediately after scraping the uterus and of the placebo- and the chymase inhibitor (BCEAB)-treated hamsters 60 min after the scraping. **P ⬍ 0.01 vs 0 min in placebo-treated group.
the accumulation of mast cells. He and Walls [10] reported that chymase induces the accumulation of inflammatory cells such as neutrophils and eosinophils, both of which are known to be related to tissue remodeling, in addition to mast cells. Therefore, in this study, the inhibition of chymase by BCEAB may suppress the accumulation of mast cells and other inflammatory cells, resulting in inhibition of adhesion formation. The development and progression of adhesion formation are also known to cause tissue degradation and the growth of extracellular matrix. Chymase processes and activates promatrix metalloproteinase to matrix metalloproteinase (MMP), which is a strong tissuedegradative protease [11]. However, generally, MMP is balanced by the tissue inhibitor of metalloproteinase (TIMP), and tissue degradation is dependent on an imbalance between MMP and TIMP. Chymase cleaves TIMP to inactive fragments, and it also cleaves complexes of MMP and TIMP, which have no MMP activity, to form active MMP [12]. MMP activation by chymase may contribute to the tissue degradation of inflammatory lesions. On the other hand, chymase directly processes matrix-bound latent TGF- to its active forms, and it also cleaves type I procollagen to induce collagen-fibril formation [13, 14]. TGF- stimulates gene expression of collagen I, collagen III, and fibronection, all of which are related to the growth of the extracellular matrix [15]. In the present study, for the first time we demonstrated a significant increase in the level of TGF- after scraping the uterus and the inhibition of this increase by the administration of the chymase inhibitor. In a previous article, in the rat adhesion model, adhesion was significantly suppressed by adding neutralizing antibodies against TGF- into the abdominal cavity [16]. Therefore, the suppression
222
JOURNAL OF SURGICAL RESEARCH: VOL. 107, NO. 2, OCTOBER 2002
of TGF- production by chymase inhibitor may be important in preventing adhesions due to chymase. In conclusion, chymase plays an important role in the development of adhesion formation, and a specific chymase inhibitor, BCEAB, may be a useful drug for suppression of peritoneal adhesions. ACKNOWLEDGMENTS We thank Shionogi Co. (Osaka) for kindly supplying BCEAB. This study was supported in part by Grant-in-Aid 12770048 for Encouragement of Young Scientists from the Ministry of Education, Science, Sports and Culture of Japan and by a grant from the Hoan-sha Foundation.
7.
8.
9.
10.
11.
REFERENCES 1.
2.
3.
4.
5.
6.
Persinger, M. A., Lepage, P., Simard, J. P., and Parker, G. Mast cell numbers in incisional wounds in rat skin as a function of distance, time and treatment. Br. J. Dermatol. 108: 179, 1983. Liebman, S. M., Langer, J. C., Marshall, J. S., and Collins, S. M. Role of mast cells in peritoneal adhesion formation. Am. J. Surg. 165: 127, 1993. Adachi, S., Maruyama, T., Kondo, T., Todoroki, T., and Fukao, K. The prevention of postoperative intraperitoneal adhesions by tranilast: N-(3⬘,4⬘-dimethoxycinnamoyl) anthranilic acid. Surg. Today 29: 51, 1999. Yao, Y. L., Ishihara, T., Takai, S., Miyazaki, M., and Mita, S. Association between the expression of mast cell chymase and intraperitoneal adhesion formation in mice. J. Surg. Res. 92: 40, 2000. Ramos, B. F., Zhang, Y., Jakschik, B. A., and Qureshi, R. Mast cells are critical for the production of leukotrienes responsible for neutrophils recruitment in immune complex-induced peritonitis in mice. J. Immunol. 147: 1636, 1991. Das, A. M., Flower, R. J., and Perretti, M. Eotaxin-induced eosinophil migration in the peritoneal cavity of ovalbumin-
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15.
16.
sensitized mice: Mechanism of action. J. Immunol. 159: 1466, 1997. Takai, S., Jin, D., Sakaguchi, M., Kirimura, K., and Miyazaki, M. Oral dministration of a novel chymase inhibitor, BCEAB, suppresses heart chymase activity. Jpn. J. Pharmacol. 86: 124, 2001. Hulka, J. F., Omran, K., and Berger, G. S. Classification of adnexal adhesions: A proposal and evaluation of its prognostic value. Fertil. Steril. 30: 661, 1978. Takai, S., Yuda, A., Jin, D., Nishimoto, M., Sakaguchi, M., Sasaki, S., and Miyazaki, M. Inhibition of chymase reduces vascular proliferation in dog grafted veins. FEBS Lett. 467: 141, 2000. He, S., and Walls, A. F. Human mast cell chymase induces the accumulation of neutrophils, eosinophils and other inflammatory cells in vivo. Br. J. Pharmacol. 125: 1491, 1998. Saarinen, J., Kalkkinen, N., Welgus, H. G., and Kovanen, P. T. Activation of human interstitial procollagenase through direct cleavage of Leu83–Thr84 bond by mast cell chymase. J. Biol. Chem. 269: 18134, 1994. Frank, B. T., Rossall, J. C., Caughey, G. H., and Fang, K. C. Mast cell tissue inhibitor of metalloproteinase-1 is cleaved and inactivated extracellularly by alpha-chymase. J. Immunol. 166: 2783, 2001. Kofford, M. W., Schwartz, L. B., Schechter, N. M., Yager, D. R., Diegelmann, R. F., and Graham, M. F. Cleavage of type I procollagen by human mast cell chymase initiates collagen fibril formation and generates a unique carboxyl-terminal propeptide. J. Biol. Chem. 272: 7127, 1997. Taipale, J., Lohi, J., Saarinen, J., Kovanen, P. T., and KeskiOja, J. Human mast cell chymase and leukocyte elastase release latent transforming growth factor-beta 1 from the extracellular matrix of cultured human epithelial and endothelial cells. J. Biol. Chem. 270: 4689, 1995. Kim, S., and Iwao, H. Molecular and cellular mechanisms of angiotensin II-mediated cardiovascular and renal diseases. Pharmacol. Rev. 52: 11, 2000. Lucas, P. A., Warejcka, D. J., Young, H. E., and Lee, B. Y. of abdominal adhesions is inhibited by antibodies to transforming growth factor-beta1. J. Surg. Res. 65: 135, 1999.
Chymase Inhibitor, BCEAB, Suppressed Peritoneal Adhesion Formation in Hamster Yukiko Okamoto, M.D., Shinji Takai, Ph.D., and Mizuo Miyazaki, M.D. 1 Department of Pharmacology, Osaka Medical College, Takatsuki City, Osaka 569-8686, Japan Submitted for publication January 9, 2002; published online August 29, 2002
Background. Mast cells are closely related to adhesion formation, while it has been unclear which factor in mast cells plays an important role in the development of adhesion formation. To clarify the role of chymase produced from mast cells in adhesion formation, we investigated the preventive effect of a specific chymase inhibitor, BCEAB, on adhesion formation in a hamster experimental model. Materials and methods. Hamsters were administered orally once daily with 100 mg/kg of BCEAB or placebo from the operated day to 1 week after the operation. The uterus was grasped and denuded by a swab. Results. One week after the operation, the scores for adhesion formation in the chymase inhibitor-treated group were significantly decreased in comparison with those in the placebo-treated group (placebotreated group, 2.80 ⴞ 0.20; chymase inhibitor-treated group 1.60 ⴞ 0.31: P < 0.01). The chymase activity in the injured uterus was also significantly suppressed in the chymase inhibitor-treated group (placebo-treated group, 17.3 ⴞ 2.69 mU/mg protein; chymase inhibitortreated group 9.60 ⴞ 0.89: P < 0.05). After scraping the utelus, the level of transforming growth factor- in the peritoneal fluid was significantly increased in the placebo-treated group, while it was suppressed to 70% by the treatment with BCEAB. Conclusions. The specific chymase inhibitor BCEAB may be a useful drug for prevention of adhesion formation. © 2002 Elsevier Science (USA) Key Words: adhesion; chymase; mast cells; chymase inhibitor; hamster.
gest that mast cells may be involved in peritoneal adhesion formation [1, 2]. For example, the number of mast cells is increased around wounds in the late stages of the healing process [1, 2]. A mast cell stabilizer, which inhibits the activation and accumulation of mast cells, is effective in attenuating adhesion formation in rat models [3]. Recently, we also reported that adhesion formation in mast cell-deficient mice was significantly less severe than that in normal control mice [4]. These reports suggest that mast cells are closely related to adhesion formation. However, mast cells release a large number of inflammatory mediators such as histamine, serotonin, chemotactic factors, cytokines, and serine proteases during the repair phase of adhesion formation [5, 6], and it has been unclear which factor plays an important role in the development of adhesion formation. Chymase is a chymotrypsin-like serine protease contained in the secretory granules of mast cells. Previously, we reported that the number of mast cells containing chymase significantly increased in the healing sites after cecal scraping in mice [4]. However, the pathophysiological role of chymase in the development of adhesion formation remains unclear. In this study, to elucidate the relation between chymase and adhesion formation, we investigated the effect of a specific chymase inhibitor, BCEAB [7], on adhesion formation. MATERIALS AND METHODS
Drugs and Animals INTRODUCTION
Postoperative intraperitoneal adhesions are a wellknown complication of surgery. Previous reports sugTo whom correspondence should be addressed. Fax: ⫹81-726-846518. E-mail: [email protected]. 1
A specific chymase inhibitor, 4-[1-[bis-(4-methyl-pheny)-methyl]-3(2-ethoxy-benzyl)-4-oxo-azetidine-2-yloxy]-benzoic acid (BCEAB), was a gift from Shionogi Co. (Osaka). Mature female Syrian hamsters (n ⫽ 44, SLC, Shizuoka, Japan), 6 weeks of age, weighing 85 to 90 g were maintained in an environmentally controlled room with a 12-h light, 12-h dark cycle. The experimental procedure for the animals was in accordance with the Guide for the Care and Use of
219
0022-4804/02 $35.00 © 2002 Elsevier Science (USA) All rights reserved.
220
JOURNAL OF SURGICAL RESEARCH: VOL. 107, NO. 2, OCTOBER 2002
Laboratory Animals (Animal Research Laboratory, Osaka Medical College).
Surgical Technique Hamsters were anesthetized with intraperitoneal sodium pentobarbital (50 mg/kg). An abdominal midline incision was made, and the right uterus was grasped and denuded of serosa over half the length of the uterine body until punctate hemorrhage occurred, using a swab [4]. Then, the abdomen was closed in two layers with silk. Hamsters were administered orally once daily with 100 mg/kg of BCEAB or placebo from the operated day to 1 week after the operation. One week after the surgery, the animals (placebo group, n ⫽ 10; chymase inhibitor group, n ⫽ 10) were anesthetized with sodium pentobarbital (50 mg/kg, ip), and then adhesions were assessed. After the scoring, the uterus was removed for the measurement of chymase activity.
Scoring of Adhesions After anesthetization, the hamster was incised at the abdominal midline and the intraperitoneal adhesion formations were scored. The adhesions were graded blindly according to a modified classification of Hulka et al. [8]: Score 0, no adhesions; Score 1, mild adhesions; Score 2, localized moderate adhesions; Score 3, moderate and wide adhesions; Score 4, severe adhesions, impossible to separate.
Tissue Preparation A tissue extract for measurement of chymase activity was prepared as described previously [9]. The uterus was minced and homogenized in 10 vol (w/v) of 20 mM Na-phosphate buffer (pH 7.4). The homogenate was centrifuged at 10,000 rpm for 30 min. The supernatant was discarded. These steps were repeated twice. The pellets were resuspended and homogenized in 5 vol (w/v) of 10 mM Na-phosphate buffer (pH 7.4) containing 2 M KCI and 0.1% Nonidet P-40. The homogenate was stored overnight at 4°C and then centrifuged at 10,000 rpm for 30 min. The supernatant was used as the tissue extract for the measurement of chymase activity.
Measurement of Chymase Activity Chymase activity was measured as described previously by Takai et al. [7]. Aliquots (20 L) of tissue extract were incubated for 30 min at 37°C with 4 mM angiotensin (Ang) I in 150 mM borax– borate buffer, pH 8.5 (final incubation volume of 0.1 mL), containing 5 mM ethylenediaminetetraacetic acid, 8 mM dipyridyl, and 0.77 mM diisopropyl phosphorofluoridate. Reactions were terminated by addition of 0.15 mL of 15% trichloroacetic acid, and then centrifugation was conducted at 10,000 rpm for 10 min. For fluorometric quantification of His–Leu as an Ang II metabolite, 10% o-phthaldialdehyde (dissolved in neat methanol) was added to the supernatant under alkaline conditions, and then 6 N HCl was added to stabilize the fluorescence for the measurement at 340 nm excitation and 455 nm emission. A blank was carried out with the addition of 0.5 mM chymostatin (dimethylformamide solution). Protein concentration was measured by bicinchoninic acid protein assay reagent (Pierce Chemical, Rockford, IL) using bovine serum albumin as a standard.
Measurement of TGF- Concentration After scraping the uterus, 1 mL of saline was injected into the abdominal cavity, and the peritoneal fluid was collected immediately (0 min) and 60 min after the injection. The level of transforming growth factor (TGF)- in the collected peritoneal fluid was measured with an ELISA kit (R&D Systems, Minneapolis, MN).
FIG. 1. Typical photographs of adhesion lesions in the placeboand the chymase inhibitor (BCEAB)-treated hamsters 1 week after the operation. The arrows indicate the uterus.
Statistical Analysis Adhesion scores were evaluated in a nonparametric test, and statistically analyzed by the Mann–Whitney U test. Chymase activity and TGF- concentration were evaluated by Student’s t and Fisher’s tests, respectively. Values are given as means ⫾ standard error (SE). Differences were considered statistically significant at P ⬍ 0.05.
RESULTS
Animal Model No difference in the body weight was found between placebo-treated and the chymase inhibitor-treated hamsters in this study of the 1-week model. Scores of Adhesion Typical photographs of the adhesion lesions are shown in Fig. 1. The scores of adhesion formation in hamsters treated with placebo and in those treated with the chymase inhibitor were 2.80 ⫾ 0.20 and 1.60 ⫾ 0.31, respectively, and this difference is significant (Fig. 2). Chymase Activity The uterus chymase activities in the hamsters treated with the chymase inhibitor and placebo were 17.3 ⫾ 2.69 mU/mg protein and 9.60 ⫾ 0.89 mU/mg protein, respectively, and this difference was significant (Fig. 3). TGF- Concentration The level of TGF- in the peritoneal fluid was significantly increased 60 min after scraping the utelus, compared with the level at 0 min in the placebo-treated group (Fig. 4). In the chymase inhibitor-treated group, the significant increase in TGF- was suppressed up to about 70%, but this difference was not significant (Fig. 4).
OKAMOTO, TAKAI, AND MIYAZAKI: BCEAB SUPPRESSES ADHESION
FIG. 2. Scores for adhesion formation in the placebo- and the chymase inhibitor (BCEAB)-treated hamsters 1 week after the operation. Values are means ⫾ SE. *P ⬍ 0.05 vs placebo-treated group.
DISCUSSION
In this study, we demonstrated that the chymase activity in the uterus 1 week after the operation was significantly increased in comparison with that in the normal uterus. The chymase activity of the injured uterus in the chymase inhibitor-treated hamsters was significantly decreased in comparison with that in placebo-treated hamsters, and adhesion formation was also decreased. These findings suggest that chymase contained in mast cells plays an important role in the development of adhesion formation. It is well known that the number of mast cells increases where inflammation occurs after surgery. We also reported that an accumulation of chymase-positive mast cells were observed in adhesion lesions, suggesting that the accumulation of chymase-positive mast cells may play an important role in the development of adhesion formation [4]. Chymase activates stem cell factor, a typical cytokine that has the ability to induce
FIG. 3. Chymase activities in the extract from the uterus in the placebo- and the chymase inhibitor (BCEAB)-treated hamsters 1 week after the operation. Values are means ⫾ SE. *P ⬍ 0.05 vs placebo-treated group.
221
FIG. 4. TGF- concentrations in the peritoneal fluid of the placebo-treated hamsters immediately after scraping the uterus and of the placebo- and the chymase inhibitor (BCEAB)-treated hamsters 60 min after the scraping. **P ⬍ 0.01 vs 0 min in placebo-treated group.
the accumulation of mast cells. He and Walls [10] reported that chymase induces the accumulation of inflammatory cells such as neutrophils and eosinophils, both of which are known to be related to tissue remodeling, in addition to mast cells. Therefore, in this study, the inhibition of chymase by BCEAB may suppress the accumulation of mast cells and other inflammatory cells, resulting in inhibition of adhesion formation. The development and progression of adhesion formation are also known to cause tissue degradation and the growth of extracellular matrix. Chymase processes and activates promatrix metalloproteinase to matrix metalloproteinase (MMP), which is a strong tissuedegradative protease [11]. However, generally, MMP is balanced by the tissue inhibitor of metalloproteinase (TIMP), and tissue degradation is dependent on an imbalance between MMP and TIMP. Chymase cleaves TIMP to inactive fragments, and it also cleaves complexes of MMP and TIMP, which have no MMP activity, to form active MMP [12]. MMP activation by chymase may contribute to the tissue degradation of inflammatory lesions. On the other hand, chymase directly processes matrix-bound latent TGF- to its active forms, and it also cleaves type I procollagen to induce collagen-fibril formation [13, 14]. TGF- stimulates gene expression of collagen I, collagen III, and fibronection, all of which are related to the growth of the extracellular matrix [15]. In the present study, for the first time we demonstrated a significant increase in the level of TGF- after scraping the uterus and the inhibition of this increase by the administration of the chymase inhibitor. In a previous article, in the rat adhesion model, adhesion was significantly suppressed by adding neutralizing antibodies against TGF- into the abdominal cavity [16]. Therefore, the suppression
222
JOURNAL OF SURGICAL RESEARCH: VOL. 107, NO. 2, OCTOBER 2002
of TGF- production by chymase inhibitor may be important in preventing adhesions due to chymase. In conclusion, chymase plays an important role in the development of adhesion formation, and a specific chymase inhibitor, BCEAB, may be a useful drug for suppression of peritoneal adhesions. ACKNOWLEDGMENTS We thank Shionogi Co. (Osaka) for kindly supplying BCEAB. This study was supported in part by Grant-in-Aid 12770048 for Encouragement of Young Scientists from the Ministry of Education, Science, Sports and Culture of Japan and by a grant from the Hoan-sha Foundation.
7.
8.
9.
10.
11.
REFERENCES 1.
2.
3.
4.
5.
6.
Persinger, M. A., Lepage, P., Simard, J. P., and Parker, G. Mast cell numbers in incisional wounds in rat skin as a function of distance, time and treatment. Br. J. Dermatol. 108: 179, 1983. Liebman, S. M., Langer, J. C., Marshall, J. S., and Collins, S. M. Role of mast cells in peritoneal adhesion formation. Am. J. Surg. 165: 127, 1993. Adachi, S., Maruyama, T., Kondo, T., Todoroki, T., and Fukao, K. The prevention of postoperative intraperitoneal adhesions by tranilast: N-(3⬘,4⬘-dimethoxycinnamoyl) anthranilic acid. Surg. Today 29: 51, 1999. Yao, Y. L., Ishihara, T., Takai, S., Miyazaki, M., and Mita, S. Association between the expression of mast cell chymase and intraperitoneal adhesion formation in mice. J. Surg. Res. 92: 40, 2000. Ramos, B. F., Zhang, Y., Jakschik, B. A., and Qureshi, R. Mast cells are critical for the production of leukotrienes responsible for neutrophils recruitment in immune complex-induced peritonitis in mice. J. Immunol. 147: 1636, 1991. Das, A. M., Flower, R. J., and Perretti, M. Eotaxin-induced eosinophil migration in the peritoneal cavity of ovalbumin-
12.
13.
14.
15.
16.
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