Chymase Activity Research Articles

Establishment of an assay method for human mast cell chymase

Chymase secreted by mast cells found in fibroblast-containing interstitial connective tissue has been implicated in collagen fiber formation and extracellular matrix production. We established a method for determination of human chymase activity, and applied this technique to measurements in serum and liver tissue. The mean chymase concentration in liver biopsy specimens from 26 patients with chronic hepatitis was 5.23±5.98 ng/mg (ranges 0.32–21.4). The serum chymase concentration was below the limit of detection, in both chronic hepatitis patients and healthy individuals. No significant relationship was seen between chymase activity in liver tissue and severity of liver fibrosis, but further investigation in larger numbers of patients is warranted.

Influence of vitamin E on mast cell mediator release.

We investigated the influence of vitamin E on mediator activity and release in a canine mastocytoma cell line (C2) as a model for canine atopic dermatitis. Cells were incubated without and with vitamin E (100 microm) for 24 h. The histamine and prostaglandin D2 (PGD2) release as well as the chymase and tryptase activity were measured. To stimulate the PGD2 and histamine release, cells were incubated with the wasp venom peptide mastoparan (50 microm) for 30 or 45 min. Nonstimulated as well as mastoparan-stimulated histamine and PGD2 release was reduced significantly in vitamin E-treated cells. The activity of chymase tended to decrease, but the tryptase activity of C2 cells was not influenced by vitamin E. These results indicate that vitamin E decreased the production and release of inflammatory mediators in C2 cells, suggesting that vitamin E might have a possible beneficial effect in inflammatory diseases.

Rodent alpha-chymases are elastase-like proteases.

Although the alpha-chymases of primates and dogs are known as chymotrypsin-like proteases, the enzymatic properties of rodent alpha-chymases (rat mast cell protease 5/rMCP-5 and mouse mast cell protease 5/mMCP-5) have not been fully understood. We report that recombinant rMCP-5 and mMCP-5 are elastase-like proteases, not chymotrypsin-like proteases. An enzyme assay using chromogenic peptidyl substrates showed that mast cell protease-5s (MCP-5s) have a clear preference for small aliphatic amino acids (e.g. alanine, isoleucine, valine) in the P1 site of substrates. We used site-directed mutagenesis and computer modeling approaches to define the determinant residue for the substrate specificity of mMCP-5, and found that the mutant possessing a Gly substitution of the Val at position 216 (V216G) lost elastase-like activity but acquired chymase activity, suggesting that the Val216 dominantly restricts the substrate specificity of mMCP-5. Structural models of mMCP-5 and the V216G mutant based on the crystal structures of serine proteases (rMCP-2, human cathepsin G, and human chymase) revealed the active site differences that can account for the marked differences in substrate specificity of the two enzymes between elastase and chymase. These findings suggest that rodent alpha-chymases have unique biological activity different from the chymases of other species.

Chymase Inhibitor, BCEAB, Suppressed Peritoneal Adhesion Formation in Hamster

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 (placebo-treated group, 2.80 ± 0.20; chymase inhibitor-treated group 1.60 ± 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 ± 2.69 mU/mg protein; chymase inhibitor-treated group 9.60 ± 0.89: P < 0.05). After scraping the utelus, the level of transforming growth factor-β 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.

Suppression of basic fibroblast growth factor-induced angiogenesis by a specific chymase inhibitor, BCEAB, through the chymase-angiotensin-dependent pathway in hamster sponge granulomas.

1. We investigated the profound involvement of mast cell chymase, an alternative angiotensin II-generating enzyme, in angiogenesis using a specific chymase inhibitor. We also studied the functional profiles of this novel inhibitor in basic fibroblast growth factor (bFGF)-induced angiogenesis. 2. In this study, angiogenesis was induced by daily injections of bFGF (0.3 micro g site(-1) day(-1)), angiotensin I (2 nmol site(-1) day(-1)) or angiotensin II (2 nmol site(-1) day(-1)) into sponges implanted to male hamsters subcutaneously for 7 days. Angiogenesis in the granulation tissue surrounding sponges was evaluated by measuring the haemoglobin (Hb) content and local blood flow as the parameters for angiogenesis. 3. A chymase inhibitor, BCEAB (4-[1-[[bis-(4-methyl-phenyl)-methyl]-carbamoyl]-3-(2-ethoxy-benzyl)-4-oxo-azetidine-2-yloxy]-benzoic acid), was simultaneously administered into the implanted sponges (2 or 5 nmol site(-1) day(-1), for 7 days) treated with bFGF and strongly suppressed the haemoglobin contents in sponge granulomas. In the studies using a laser doppler perfusion imager, BCEAB (5 nmol site(-1) day(-1)) also attenuated the bFGF-induced increase of local blood flow around the implanted sponge granuloma. 4. In bFGF-induced angiogenesis, chymase activity in sponge granulomas was substantially increased. It was also confirmed that the chymase activity increased by bFGF was significantly and dose-dependently inhibited by BCEAB (2, 5 nmol site(-1) day(-1)). 5. BCEAB inhibited the Hb contents and the expression of vascular endothelial growth factor (VEGF) mRNA induced by angiotensin I but not by angiotensin II. 6. These results suggest that the significance of chymase in bFGF-induced angiogenesis was confirmed, and a novel inhibitor, BCEAB, strongly suppresses the bFGF-induced angiogenesis through the chymase-angiotensin II-VEGF dependent pathway.

Chymase inhibition suppresses high-cholesterol diet-induced lipid accumulation in the hamster aorta.

The role of chymase (a mast cell-derived angiotensin II-forming serine proteinase) in aortic lipid deposition was investigated using an orally active, non-peptide chymase inhibitor, SUN-C8257. Male golden Syrian hamsters, 8 weeks old, were fed with a standard rodent meal supplemented with or without 0.5% cholesterol and 10% coconut oil for 12 weeks. The hamsters fed high cholesterol diet were further separated into two groups treated with or without SUN-C8257 for 12 weeks. The aortic lipid deposition was visualized by Oil red O staining and planimetrically measured. Immunohistochemical staining for angiotensin II (Ang II) of the aortic root region was performed. Aortic Ang II-forming activity was measured using Ang I as a substrate. Plasma total-, low-density lipoprotein (LDL)-, high-density lipoprotein (HDL)-cholesterol and triglyceride were quantified by enzymatic methods. Plasma Ang I and Ang II were measured by radioimmunoassay. After 12 weeks of high cholesterol diet, aortic chymase activity in the untreated group increased significantly and showed a positive correlation with plasma total- and LDL-cholesterol. This group of hamsters developed marked lipid deposits in the aortic intima. However, treatment with SUN-C8257 significantly suppressed aortic lipid deposition without changing body weight, blood pressure, plasma LDL-cholesterol and Ang II levels. The level of the adventitial Ang II-immunoreactivity was markedly inhibited in the group treated with SUN-C8257. Our results suggest that arterial chymase may participate in the acceleration of lipid deposition in arterial walls exposed to high plasma cholesterol and that inhibition of arterial chymase may retard the progression of atherosclerosis.

Increased local angiotensin II formation in aneurysmal aorta

We investigated the levels and locations of angiotensin II-forming enzymes, angiotensin converting enzyme (ACE) and chymase, in aneurysmal and normal aortas. Aneurysmal aortic specimens (n = 14) were obtained at the time of operative aneurysm repair from 14 patients ranging in age from 57 to 84 y. Normal aortic specimens (n = 16) were obtained from 16 patients (48 to 72 y) who underwent coronary artery bypass surgery. The ACE and chymase activities were determined using each specimen. Sections of each specimen were immunostained with antibodies for ACE and chymase. The ACE activities in the aneurysmal and normal aortas were 0.82 ± 0.10 and 0.14 ± 0.05 mU/mg protein, respectively, and this difference was significant. The chymase activities in the aneurysmal and normal aortas were 17.9 ± 2.40 and 1.02 ± 0.18 mU/mg protein, respectively, and this difference was also significant. In the aneurysmal aorta, ACE-positive cells were detected with macrophages in the intima and media and chymase-positive cells were detected with mast cells in the media and adventitia, whereas positive ACE and chymase cells in the normal aorta were located only in the endothelium and adventitia, respectively. Angiotensin II-forming enzymes, chymase and ACE, were significantly increased in the aneurysmal aorta, and increased angiotensin II may be associated with the development of aneurysmal formations.

Differential regulation of elevated renal angiotensin II in chronic renal ischemia.

The present study was undertaken to clarify the role of intrarenal angiotensin (Ang) II and its generating pathways in clipped and nonclipped kidneys of 4-week unilateral renal artery stenosis in anesthetized dogs. After 4 weeks, renal plasma flow (RPF) decreased in clipped and nonclipped kidneys (baseline, 59+/-3; clipped, 16+/-1; nonclipped, 44+/-2 mL/min; P<0.01, n=22). Renal Ang I levels increased only in clipped, whereas intrarenal Ang II contents were elevated in both clipped (from 0.7+/-0.1 to 2.0+/-0.2 pg/mg tissue) and nonclipped kidneys (from 0.6+/-0.1 to 2.5+/-0.3 pg/mg tissue). Intrarenal ACE activity was increased in nonclipped kidneys but was unaltered in clipped kidneys. An angiotensin receptor antagonist (olmesartan medoxomil) given into the renal artery markedly restored RPF, and dilated both afferent and efferent arterioles (using intravital videomicroscopy). Furthermore, in clipped kidneys, the elevated Ang II was suppressed by a chymase inhibitor, chymostatin (from 2.1+/-0.6 to 0.8+/-0.1 pg/mg tissue; P<0.05), but not by cilazaprilat. In nonclipped kidneys, in contrast, cilazaprilat, but not chymostatin, potently inhibited the intrarenal Ang II generation (from 2.4+/-0.3 to 1.5+/-0.2 pg/mg tissue; P<0.05). Finally, [Pro11-D-Ala12]Ang I (an inactive precursor that yields Ang II by chymase but not by ACE; 1 to 50 nmol/kg) markedly elevated intrarenal Ang II in clipped, but not in nonclipped, kidneys. In conclusion, renal Ang II contents were elevated in both clipped and nonclipped kidneys, which contributed to the altered renal hemodynamics and microvascular tone. Furthermore, the mechanisms for intrarenal Ang II generation differ, and chymase activity is enhanced in clipped kidneys, whereas ACE-mediated Ang II generation is possibly responsible for elevated Ang II contents in nonclipped kidneys.

Differential ANG II generation in plasma and tissue of mice with decreased expression of the ACE gene.

We utilized mice with homozygous disruption of angiotensin-converting enzyme (ACE) (-/-), mice with heterozygous deletion of ACE (+/-), and wild-type mice (+/+) to test the hypothesis that genetic variation in ACE modulates tissue and plasma angiotensin (ANG) II concentrations. With the use of ANG I as substrate, kidney, heart, and lung ACE activity was reduced 80% in -/- mice compared with +/+ mice. However, ANG II concentrations and ANG II-to-ANG I ratios in the kidney, heart, and lung did not differ among genotypes. In contrast, plasma ANG II concentrations in -/- mice were <2 fmol/ml, whereas plasma ANG I concentrations were extremely high (765 fmol/ml). Chymase activity was increased 14-fold in the kidney (P < 0.05) and 1.5-fold in the heart (P < 0.05) of -/- versus +/+ mice but did not differ among genotypes in the lung. ANG II formation from enzymes other than ACE and chymase contributed <2% of total ANG II formation in all genotypes. These data suggest that ACE is essential to ANG II formation in the vascular space, whereas chymase may provide an important mechanism in maintaining steady-state ANG II levels in tissue.

Chymase inhibitors may prevent postoperative adhesion formation

Objective: To clarify the role of chymase produced from mast cells in adhesion formation, we measured chymase activity level and investigated the preventive effect of a chymase inhibitor, Suc-Val-Pro-Phe p(OPh) 2, on the postoperative adhesion formation. Design: Prospective randomized study using a surgical model for adhesion formation. Setting: Clean hamsters in an academic research environment. Animal(s): Sixty-seven female Syrian hamsters. Intervention(s): Hamsters were given a lesion, produced by uterus scraping, and the chymase inhibitor (10 μM) or placebo was injected into the abdomen. Chymase activities in uteri were measured 3 days after the operation, and the scores of adhesion formations were assessed at 2 weeks. Main Outcome Measure(s): Measurement of chymase activity and scoring of adhesion formation were performed. Result(s): A significant increase of chymase activity in the injured uterus reduced by treatment with the chymase inhibitor. The scores of adhesion formations in the chymase inhibitor-treated group were significantly decreased in comparison with those in the placebo-treated group. Conclusion(s): Chymase contained in mast cells plays an important role in adhesion formation, and a chymase inhibitor may be a useful drug for prevention of adhesion formation.

Beneficial effects of cardiac chymase inhibition during the acute phase of myocardial infarction

Recently, the presence of the chymase-dependent angiotensin (Ang) II-generating system in hamsters, dogs, monkeys, as well as human cardiovascular tissues has been identified. We have reported that the activation of cardiac chymase was more prominent than that of angiotensin converting enzyme (ACE) and that AT1 receptor antagonist treatment rather than ACE inhibitor treatment alone provided significant beneficial effects on cardiac function and survival after MI in hamsters. The aim of the present study was to determine whether this different effects between AT1 receptor antagonist and ACE inhibitor were due to the activation of cardiac chymase after MI in hamsters by using 4-[1-{[bis-(4-methyl-pheny)-methyl]-carbamoyl}-3-(2-ethoxy-benzyl)-4-oxo-azetidine-2-yloxy]-benzoic acid (BCEAB), a novel, orally active and specific chymase inhibitor. The ACE and chymase activities in the infarcted left ventricle were significantly increased 3 days after MI. BCEAB (100 mg/kg/day, p.o.) treatment starting 3 days before MI significantly suppressed the cardiac chymase activity, while it did not affect the plasma and cardiac ACE activities 3 days after MI. A significant improvement in hemodynamics (maximal negative and positive rates of pressure development; left ventricular systolic pressure) was observed for the treatment with BCEAB 3 days after MI. BCEAB (100 mg/kg/day, p.o.) treatment starting 3 days before MI significantly reduced the mortality rate during 14 days of observation following MI (vehicle, 61.1%, n = 18; BCEAB, 27.8%, n = 18; P < 0.05). These findings demonstrated for the first time that cardiac chymase participates directly in the pathophysiologic state after MI in hamsters.

Lys 40 but not Arg 143 influences selectivity of angiotensin conversion by human α-chymase

Human α-chymase is an efficient angiotensin (AT) converting enzyme, selectively hydrolyzing AT I at Phe 8 to generate bioactive AT II, which can promote cardiac hypertrophy, vascular stenosis, and hypertension. Some related enzymes, such as rat β-chymase 1, are much less selective, destroying AT by cleaving at Tyr 4. Comparisons of chymase structure and activity led to speculation that interaction between AT and the side chain of Lys 40 or Arg 143 accounts for the human enzyme’s marked preference for Phe 8 over Tyr 4. To test these hypotheses, we compared AT hydrolysis by wild-type chymase with that by mutants changing Lys 40 or Arg 143 to neutral residues. Lys 40 was exchanged for alanine, the residue found in canine α- and rat β-chymase 1, the latter being dramatically less selective for hydrolysis at Phe 8. Arg 143 was exchanged for glutamine found in rat β-chymase 1. The Lys 40Ala mutant is a dog-like enzyme retaining strong preference for Phe 8 but with Tyr 4 hydrolytic rates enhanced 16-fold compared to wild-type human enzyme. Thus, of 40 residues mismatched between dog and human enzymes, a single residue accounts for most of the difference in specificity between them. The Arg 143Gln mutant, contrary to prediction, remains highly Phe 8-selective. Therefore, Lys 40, but not Arg 143, contributes to human chymase’s remarkable preference for AT II generation over destruction.

Chymase and angiotensin converting enzyme activities in a hamster model of glaucoma filtering surgery

Purpose. Two pathways for the formation of angiotensin II (Ang II) in local tissues are known to exist, one involving angiotensin-converting enzyme (ACE), and the other in which chymase plays a role. It has been shown that chymase activity is present in monkey, dog, and hamster eyes. Because chymase activates various cytokines by increasing Ang II formation, thereby promoting the production of extracellular matrix, the role of Ang II in wound healing has attracted much interest. In this work, we created sclerocorneal wounds in hamster eyes and measured the levels of ACE and chymase activities in the eye during the wound healing process. Methods. Sclerocorneal wounds were made at 6 locations on the corneal limbus of one eye in each of 36 hamsters. Using the contralateral eyes as controls, we measured levels of chymase and ACE activities in the overall eye at 3, 7, and 21 days postoperatively. Histopathological evaluations of the sclerocorneal wounds in the treated eyes were also carried out for samples stained with toluidine blue. Results. Chymase activity in the treated eyes tended to be higher than that in the control at 21 days. ACE activity in the treated eyes was significantly higher than that in the control at 3, 7, and 21 days postoperatively. Histopathological examination revealed increased mast cells in the subconjunctival tissue and around the tunnel opening in the sclerocornea. Conclusions. These findings show that not only ACE but also chymase contributes to the formation of Ang II in the healing of sclerocorneal wounds in hamster eyes. This leads to the suggestion that ACE inhibitors or chymase inhibitors could potentially inhibit scarring in glaucoma filtering surgery.

Estrogen receptors in mast cells from arterial walls

We examined the presence of estrogen receptors (ER) in vascular mast cells and a possible genomic effect of estrogens on the expression of mast cell (MC) mediators such as chymase, TNF alpha, NOS and IL-10, which are known to affect the course of atherosclerosis. Immunocytochemical detection of mast cell tryptase and the co-localization of ERs in MCs from abdominal aortic vessels from 10 fertile woman, 10 postmenopausal women and 15 men was performed. The genomic expression of IL-10, TNF alpha, and NOS was analyzed by RT-PCR and chymase activity by spectrophotometry after 24 h incubation with 17-beta estradiol (0.2-0.5 ng/mL) in rat purified peritoneal MCs. A similar number of MCs were found in both intima and adventitia layers from men, and fertile and postmenopausal women, while ERs were detected only in the arterial walls from fertile women. The mRNA expressions of IL-10 and TNF alpha, as well as chymase activity, were not affected. A moderate increment of NO and both NOS, and a reduction in TNF alpha cytotoxicity was observed after incubating peritoneal MCs with 17-beta estradiol at a concentration of 0.5 ng/mL. Taken together, these results indicate that vascular MCs express ERs. The data demonstrate that estrogens can directly modify vascular MC activity. This is a novel mechanism of synergistic cooperation for the protective role of estrogens in the genesis of atherosclerosis.

The Role of Chymase in Vascular Proliferation.

In vascular tissues, angiotensin II is potentially cleaved from angiotensin I by chymase and angiotensin-converting enzyme (ACE). Chymase is stored in mast cells and has no enzymatic activity in the normal state. Chymase is activated immediately upon its release into the extracellular matrix in vascular tissues after mast cells have been activated by stimuli such as vessel injury by grafting or a balloon catheter. In dog grafted veins, chymase activity is increased, and the vascular proliferation is suppressed by either a chymase inhibitor or an angiotensin II receptor blocker. After balloon injury in dog vessels, chymase activity is locally increased in the injured artery, and a chymase inhibitor is effective in preventing the vascular proliferation, but an ACE inhibitor is ineffective. Chymase plays an important role in the development of vascular proliferation via the induction of local angiotensin II formation in injured vessels. (c) 2002 Prous Science. All rights reserved.

Development and Application of Chymase Inhibitors: Effect of Chymase Inhibitor on Vascular Proliferation

In vascular tissues, angiotensin II is potentially cleaved from angiotensin I by chymase and angiotensin-converting enzyme (ACE). In the normal state, vascular ACE regulates local angiotensin II formation and plays a crucial role in the regulation of blood pressure, whereas chymase is stored in mast cells and has no enzymatic activity. Chymase is activated immediately upon its release into the extracellular matrix in vascular tissues after mast cells have been activated by stimuli such as vessel injury by grafting or a balloon catheter. In dog grafted veins, chymase activity is increased, and the vascular proliferation is suppressed by either a chymase inhibitor or an angiotensin II receptor blocker. After balloon injury in dog vessels, chymase activity is significantly increased in the injured artery, and a chymase inhibitor is effective in preventing the vascular proliferation, but an ACE inhibitor is ineffective. Chymase plays an important role in the development of vascular proliferation via the induction of local angiotensin II formation in injured vessels.

Beneficial Effects of Angiotensin-Converting Enzyme Inhibition in Adriamycin-Induced Cardiomyopathy in Hamsters

This study was performed to determine whether angiotensin (Ang) II-forming enzymes, angiotensin converting enzyme (ACE) and chymase might contribute to the development of adriamycin-induced cardiomyopathy in hamsters. Hamsters were administered adriamycin (2.0 mg/kg per day, i.p.) three times weekly for 2 weeks. In the ACE inhibitor-treated group, the hamsters received lisinopril (20 mg/kg per day, p.o.) for 2 weeks after the last injection of adriamycin. The 4-week mortality rates of the vehicle- and ACE inhibitor-treated hamsters were 44% and 12%, respectively. In comparison to the age-matched hamsters used as the control hamsters, a significant decrease in cardiac function and a significant increase in the ratio of the heart weight to the body weight were observed in the vehicle hamsters. Cardiac ACE activity, but not the chymase activity, in the vehicle hamsters was significantly increased in comparison to that in the control hamsters. In the ACE inhibitor-treated group, the increased ACE activity was reduced significantly, and the cardiac hypertrophy and dysfunction were improved significantly. In adriamycin-induced cardiomyopathic hamsters, cardiac ACE activity was increased and ACE inhibition significantly improved cardiac function and survival rate, indicating that cardiac ACE, but not the chymase, plays the pivotal role in the development of the adriamycin-induced cardiomyopathy.

Time-dependent expression of chymase and angiotensin converting enzyme in the hamster heart under pressure overload.

The role of a dual angiotensin (Ang) II-forming pathway from the local renin angiotensin system (RAS) of the cardiac tissue was determined in a hamster model of cardiac hypertrophy. Time-dependent expressions of chymase and angiotensin converting enzyme (ACE) genes and their enzymes activities, and Ang II levels were measured in the hamster heart at 3 days, and at 4 and 8 weeks after pressure overload. Cardiac hypertrophy was induced by an operation to constrict the abdominal aorta. Compared to the sham-operated group, the cardiomyocyte diameters of hamster hearts at 3 days after overload underwent no obvious changes, while those at 4 and 8 weeks after overload increased markedly (p<0.01), and both transcriptional expressions of chymase and ACE genes gradually increased in the hamster hearts at 3 days, and at 4 and 8 weeks after overload, but the transcriptional expressions of angiotensin II type 1 receptor (AT1R) gene gradually decreased. Chymase and ACE activities (U/mg) (0.441+/-0.040 vs. 0.175+/-0.014, 0.446+/-0.036 vs. 0.160+/-0.016 and 0.522+/-0.014 vs. 0.148+/-0.038) (p<0.01) and (0.142+/-0.023 vs. 0.056+/-0.038, 0.317+/-0.017 vs. 0.079+/-0.016 and 0.466+/-0.010 vs. 0.098+/-0.003) (p<0.01), respectively and Ang II levels (pg/g) (98.7+/-4.5 vs. 71.2+/-4.9, 134.4+/-7.8 vs. 71.9+/-12.8 and 151.6+/-10.1 vs. 80.7+/-3.0) gradually increased in the hamster hearts, vs. sham treatment, respectively, at 3 days, and at 4 and 8 weeks after overload. However, the increases in chymase and ACE activities were much higher than those in their respective mRNA levels, and the levels of chymase activities were also higher than those of ACE activities during the development of cardiac hypertrophy. The results suggested that the increase in Ang II levels via the dual pathway of Ang II formation by chymase and ACE plays an important role in the cardiac hypertrophy of hamsters caused by the overloaded state. Importantly, in the non-hypertrophied hamster heart in the early stage after overload (at 3 days), chymase could be activated by mechanical stress in advance of an increase in its mRNA, and the Ang II level increased significantly.

Mast Cells as a Target of Rheumatoid Arthritis Treatment

Rheumatoid arthritis (RA) is a chronic inflammatory disease and its exact cause and pathophysiological process remain unclear. Fibroblast-like synoviocytes, macrophages and T lymphocytes are considered to be the major contributors in the pathophysiological process of RA; however, an increasing number of papers have drawn attention to the potential role of mast cells (MCs) in the process. In an animal model of RA, we reported an increase in MC numbers in the arthritic region, which agreed with the observation in human RA. In addition, a good correlation between the number of MCs and the development of disease was observed. However, there has been little experimental or clinical evidence of the beneficial effects of the modification of MC activity on the pathogenesis of RA and this is the weak point of the hypothesis. We therefore studied the effects of a MC-stabilizing compound, cromoglicate lisetil (CL), which is an orally deliverable prodrug of cromolyn sodium, on the RA disease model. The MC-stabilizer had efficacy in a mouse model. The beneficial effects of CL in this animal model further suggested the contribution of MCs in the pathophysiological process of RA. Concerning the contributive mechanism of MC on the pathogenesis of RA, our results using a disease model suggested that activation of MC chymase and matrix metalloproteinases might be involved. MC is now considered to be one of the targets of RA treatment.

Oral Administration of a Novel Chymase Inhibitor, NK3201, Prevents Peritoneal Adhesion Formation in Hamsters

We investigated the preventive effect of an orally active chymase inhibitor, NK3201 (2-(5-formylamino-6-oxo-2-phenyl-1,6-dihydropyrimidine-1-yl)-N-[[3,4-dioxo-1-phenyl-7-(2-pyridyloxy)]-2heptyl]acetamide), on the adhesion formation in a hamster experimental model. Hamsters were administered orally once daily with 30 mg/kg of NK3201 or placebo from 3 days before uterus scraping to 7 days after it. A significant increase of chymase activity in the injured uterus was reduced by treatment with NK3201. The score of adhesion formations in the chymase inhibitor-treated group was significantly decreased in comparison with that in the placebo-treated group (P < 0.01). Oral administration of NK3201 may be a useful drug for prevention of peritoneal adhesion formation.