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The effect of the chymase inhibitor TY-51469 on the development and progression of non-alcoholic steatohepatitis (NASH) was evaluated in rats fed a high-fat and high-cholesterol (HFC) diet. To evaluate the preventive effect of TY-51469 on the development of NASH, stroke-prone spontaneously hypertensive rat 5 (SHRSP5)/Dmcr rats were fed either a normal or HFC diet for 8 weeks, and concurrently administered either placebo or TY-51469 (1 mg/kg per day). To evaluate the effect of TY-51469 on the survival rate, TY-51469 was administered either concurrently with HFC diet (pretreated group) or 8 weeks after HFC diet at which point NASH had developed (posttreated group). Eight weeks after HFC diet, significant increases of steatosis, fibrosis and chymase-positive cells were observed in liver from the placebo-treated rats. Significant increases of myeloperoxidase, transforming growth factor-β, matrix metalloproteinase-9, and collagen I mRNA levels were also observed. However, all parameters were significantly attenuated in the TY-51469-treated group. A survival rate of the placebo-treated group fed the HFC diet was 0% at 14 weeks. In comparison, the rates of TY-51469-pretreated and TY-51469-posttreated groups were 100% and 50% at 14 weeks, respectively. Chymase inhibitor may be applicable to preventing the development and progression of NASH.

To study the role of periostin in adhesion formation, the effect of periostin antisense oligonucleotide (PAO) on adhesion formation was evaluated in mice. Under anesthesia, the serous membrane of the cecum was abraded, and the adhesion score and mRNA levels of periostin and its related factors were determined after surgery. Saline, 40 mg/kg of negative sense oligonucleotide (NSO), or 40 mg/kg of PAO were injected into the abdomen after surgery, and the adhesion score and mRNA levels were evaluated 14 days later. Filmy adhesion formation was observed 1 day after surgery, and the adhesion score increased gradually to 14 days. The mRNA levels of periostin, transforming growth factor (TGF)-β, and collagen I increased gradually from 3 days to 14 days. The adhesion score of PAO was significantly lower than of saline or NSO 14 days after surgery. The mRNA levels of periostin, TGF-β, and collagen I were also significantly attenuated by treatment with PAO compared with saline or NSO. Thus, these results demonstrated that the periostin mRNA level increased in the abraded cecum, and PAO prevented adhesion formation along with attenuation of the periostin mRNA level.

The effect of recombinant human soluble thrombomodulin (TM-α) on acute liver failure (ALF) is unclear, and we elucidated the effect of TM-α in lipopolysaccharide (LPS)/d-galactosamine (GalN)-induced ALF in mice. Placebo (saline) or TM-α (100 mg/kg) was administered 1 h after LPS/GalN administration. Survival rates were evaluated for 24 h after LPS/GalN administration. Plasma and liver samples were evaluated 1, 3, and 7 h after LPS/GalN administration. Survival rates were significantly higher in the TM-α-treated group than in the placebo group. A significant augmentation of plasma high-mobility group box 1 protein (HMGB1) was observed 7 h after LPS/GalN administration. In the TM-α-treated mice, plasma HMGB1 was significantly lower than in the placebo group. A significant augmentation of hepatic nuclear factor (NF)-κB p65 was observed in the placebo-treated group, whereas a significant reduction, relative to placebo, was observed in the TM-α-treated group. Hepatic expression of tumor necrosis factor (TNF)-α and myeloperoxidase were significantly increased in the placebo group, and were similarly significantly attenuated in the TM-α-treated group. TM-α treatment also produced a significant attenuation of liver neutrophil accumulation after LPS/GalN administration. Thus, TM-α may become a useful treatment strategy for reducing the symptoms of ALF via the attenuation of LPS/GalN-induced HMGB1 levels.

Introduction:Chymase converts angiotensin I to angiotensin II and it can also convert precursors of TGF-ββ and MMP-9 to their active forms. Therefore, diseases related to angiotensin II TGF-ββ, and MMP-9 could potentially be treated with chymase inhibitors.Areas covered:This review discusses the appropriate targets and safety of chymase inhibitors. Six diseases with notable mortality or morbidity as targets of chymase inhibitors are focused on; abdominal aortic aneurysms (AAAs), nephropathy and retinopathy, cardiomyopathy, nonalcoholic steatohepatitis (NASH), organ fibrosis and intestinal diseases.Expert opinion:If chymase inhibition proves to be a useful strategy for the attenuation of angiotensin II, TGF-ββ and MMP-9 in vivo,the application of chymase inhibitors is likely to become widespread in various diseases in the clinical setting. Chymase inhibitors are anticipated not to interfere with the homeostasis of resting tissues, that is, those not affected by injury or inflammation.

The side effects of nonsteroidal anti-inflammatory drugs (NSAIDs) include gastrointestinal damage not only in the stomach but also in the small intestine. Chymase converts promatrix metalloproteinase-9 to matrix metalloproteinase (MMP)-9, which plays an important role in NSAID-induced gastric damage, but it has been unclear whether chymase-dependent MMP-9 activation is involved in the NSAID-induced small intestinal damage. To clarify the involvement of chymase-dependent MMP-9 activation on NSAID-induced small intestinal damage, the effect of a chymase inhibitor, 2-[4-(5-fluoro-3-methylbenzo[b]thiophen-2-yl)sulfonamido-3-methanesulfonylphenyl] thiazole-4-carboxylic acid (TY-51469), on indomethacin-induced small intestinal damage in rats was evaluated. Until 6 h after oral administration of indomethacin in rats, intestinal MMP-9 activity was unchanged compared with normal rats, but significant increases in MMP-9 activity were observed 12 and 24 h after indomethacin administration. Significant increases in the small intestinal damage score were also observed 12 and 24 h after indomethacin administration. In the extract from the small intestine 24 h after indomethacin administration, the MMP-9 activation was significantly attenuated by TY-51469. Intraperitoneal injection of TY-51469 (10 mg/kg) 3 h before indomethacin administration significantly attenuated the MMP-9 activity in the small intestine compared with placebo treatment. Myeloperoxidase activity, which indicates accumulation of neutrophils, was significantly increased in the small intestine in the placebo-treated rats, but its activity was significantly attenuated by TY-51469 treatment. The area of small intestinal damage was also significantly ameliorated by TY-51469 treatment. These findings suggest that chymase-dependent MMP-9 activation has a significant role in indomethacin-induced small intestinal damage in rats.

Chymase plays a crucial role in angiotensin II formation in various tissues. Angiotensin II induces gene expression of transforming growth factor (TGF)-βand matrix metalloproteinase (MMP)-9 precursors, and chymase can convert precursors of TGF-βand MMP-9 to their active forms. In cultured fibroblasts, significant increases in cell growth and TGF-βlevels were observed after chymase injection; these increases were inhibited by a chymase inhibitor, but not by an angiotensin II–receptor blocker. In apolipoprotein E–deficient mice, abdominal aortic aneurysm (AAA) development depends on an increase in MMP-9 activities induced by angiotensin II infusion, but the inhibition of MMP-9 activation by a chymase inhibitor resulted in attenuation of the angiotensin II–induced AAA development. The upregulation of MMP-9 and TGF-βlevels is involved in damage to various organs, but these gene expressions are not completely induced by angiotensin II alone. Therefore, chymase inhibition may be useful for attenuating MMP-9 and TGF-βlevels, in addition to reducing angiotensin II formation, and this function may provide powerful organ protection. In this review, we propose the possible use of chymase inhibitors as agents to prevent organ damage.

The aim of this study was to identify the role of chymase in the conversion of exogenously administered Big endothelin-1 in the mouse in vivo. Real-time polymerase chain reaction analysis detected mRNA of mucosal mast cell chymases 4 and 5, endothelin-converting enzyme 1a, and neutral endopeptidase 24.11 in pulmonary, cardiac, and aorta homogenates derived from C57BL/6J mice, with the latter tissue expressing the highest levels of both chymase isoforms. Furthermore, hydrolysis of a fluorogenic peptide substrate, Suc-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin, was sensitive to the chymase inhibitors Suc-Val-Pro-Phe(P)(OPh)(2) (200 microM) and chymostatin [(S)-1-carboxy-2-phenylethyl]-carbamoyl-alpha-[2-iminohexahydro-4(S)-pyrimidyl]-(S)-Gly-X-Phe-al, where X can be the amino acid Leu, Val, or Ile) (100 microM) in supernatants extracted from the same tissue homogenates. In anesthetized mice, Big endothelin-1, endothelin-1 (1-31), and endothelin-1 triggered pressor responses (ED(50)s, 0.67, 0.89, and 0.16 nmol/kg) that were all reduced or potentiated by selective endothelin ET(A) or ET(B) receptor antagonists, respectively, BQ-123 (cyclo[D-Asp-Pro-D-Val-Leu-D-Trp]) or BQ-788 (N-[N-[N-[(2,6-dimethyl-1-piperidinyl)carbonyl]-4-methyl-l-leucyl]-1-(methoxycarbonyl)-D-tryptophyl]-d-norleucine sodium salt), each at 1 mg/kg. The pressor responses to big endothelin-1 were significantly reduced by the neutral endopeptidase inhibitor thiorphan (dl-3-mercapto-2-benzylpropanoylglycine) (1 mg/kg) or the endothelin-converting enzyme inhibitor CGS 35066 [alpha-[(S)-(phosphonomethyl)amino]-3-dibenzofuranopropanoic acid] (0.1 mg/kg). In contrast, the responses to endothelin-1 (1-31) were abolished by thiorphan but unaffected by CGS 35066. In addition, Suc-Val-Pro-Phe(P)(OPh)(2) (20-40 mg/kg) reduced, by more than 60%, the hemodynamic response to big endothelin-1 but not to endothelin-1 (1-31) and endothelin-1. Finally, intravenous administration of big endothelin-1 induced Suc-Val-Pro-Phe(P)-(OPh)(2)-sensitive increases in plasma-immunoreactive levels of endothelin-1 (1-31) and endothelin-1. The present study suggests that chymase plays a pivotal role in the conversion and cardiovascular properties of big endothelin-1 in vivo.

In the present study, the effects of long-term angiotensin (Ang) II antagonism on the development of cardiac and endothelial disorders were examined in Spontaneously Diabetic Torii (SDT) rats. Blood glucose concentration started to increase markedly in the untreated SDT rats from 20 weeks of age, while the blood glucose concentrations of candesartan cilexetil–treated SDT rats were significantly lower until 30 weeks of age. Cardiac function deteriorated in SDT rats and was accompanied by severe cardiac fibrosis, cardiac hypertrophy, and microstructural pathologic change in cardiomyocytes. Cardiac function was very well preserved in the age-matched Sprague Dawley (SD) rats, but cardiac fibrosis developed with aging. Candesartan cilexetil treatment improved cardiac structural remodeling and cardiac function in SDT rats. Surprisingly, the degree of cardiac fibrosis in candesartan cilexetil–treated SDT rats was less than that of SD rats. Immunohistological staining confirmed that in addition to collagen deposition, fibroblasts and myofibroblasts were the main cellular components in the cardiac fibrotic areas. The diabetic hearts showed positive staining for ACE, Ang II, and AT1receptors. SDT rats also showed decreased endothelial function, which was improved with candesartan cilexetil treatment. These findings indicate that Ang II is involved in the development of cardiac dysfunction by accelerating cardiac remodeling and cardiomyocyte damage in the presence of hyperglycemia. On the other hand, although the mechanisms responsible for the cardiac fibrosis that occurs under normal conditions may differ greatly from those responsible for cardiac fibrosis with hyperglycemia, Ang II seems to play an important role in both.

Angiotensin II may be involved in pancreatic disorganization, but the involvement of chymase has been unclear. In the present study, we examined whether chymase is involved in pancreatic disorganization in hamsters with streptozotocin (STZ)-induced diabetes. Hamsters were injected with streptozotocin (60 mg/kg), and non-injected hamsters served as controls. To investigate the effect of a chymase inhibitor, TY-51469 (30 mg/kg per day), hamsters in the STZ group were administered TY-51469 or placebo from 2 weeks after STZ injection, for 1 week. A significant increase in blood glucose level was observed at 1 week after STZ injection. This was maintained at 2 weeks, and a further significant increase was observed at 3 weeks. Until 2 weeks after STZ injection, all angiotensin II–related enzyme activities were unchanged, but at 3 weeks pancreatic chymase and total angiotensin II–forming activities, but not angiotensin-converting enzyme activity, were significantly increased. TY-51469 significantly attenuated blood glucose level along with reductions of chymase and total angiotensin II–forming activities and malondialdehyde level. Furthermore, there were significantly more pancreatic islets in the TY-51469 group than in the placebo group. In conclusion, chymase inhibition might protect against pancreatic islet disorganization.

Matrix metalloproteinase (MMP)-9 plays an important role in the pathogenesis of colitis. Recent studies have demonstrated that chymase is involved in the conversion of promatrix metalloproteinase (proMMP)-9 to MMP-9. However, whether chymase contributes to the activation of proMMP-9 in colitis has remained unclear. In this study, we administered 5% dextran sodium sulfate (DSS) solution to mice for 7 days. At 7 days after starting administration, both chymase activity and MMP-9 activity were significantly increased. In extract from colitis in DSS-treated mice, MMP-9 activity was significantly increased after 8 h of incubation, but increased activity was almost completely suppressed in the presence of a chymase inhibitor, 2-(5-formylamino-6-oxo-2-phenyl-1,6-dihydropyrimidine-1-yl)-N-[{3,4-dioxo-1-phenyl-7-(2-pyridyloxy)}-2-heptyl] acetamide (NK3201). At 7 days after starting administration, intestinal length was significantly shorter in DSS-treated mice than in normal mice, but these changes were significantly prevented by NK3201 (10 mg/kg per day i.p.). Disease activity index and histological damage score were also significantly reduced by NK3201. The filtrated neutrophil number was significantly decreased by NK3201. Furthermore, NK3201 significantly attenuated not only chymase activity but also MMP-9 activity in DSS-treated mice. These findings suggest that chymase plays an important role in the development of colitis via MMP-9 activation.

Angiotensin II-forming chymase is expressed in the pulmonary arteries of the monocrotaline-induced pulmonary hypertensive rats, but its actual role is unclear. We studied chymase-dependent angiotensin II formation in the pulmonary arteries of the monocrotaline-induced pulmonary hypertensive rats and observed the effects of an angiotensin II receptor blocker on vascular remodeling. Four weeks after the administration of monocrotaline (60 mg/kg, s.q.), echocardiographic, hemodynamic, morphometric and biochemical analyses were performed. Age-matched rats were used as controls. To evaluate the effects of an angiotensin II receptor blocker, 2 wk after beginning of monocrotaline treatment, the rats were given candesartan (10 mg/kg per day) or placebo for 2 wk. In the monocrotaline-induced pulmonary hypertensive rats, the elevated systolic pulmonary arterial pressure and right ventricular hypertrophy were observed. Medial hypertrophy of lung arterioles was also observed. Chymase activity and angiotensin II concentration, but not angiotensin-converting enzyme activity, were significantly increased in the lung. In the angiotensin II receptor blocker-treated group, both systolic pulmonary arterial pressure and right ventricular hypertrophy were significantly reduced, and arteriolar hypertrophy was also prevented. Thus, angiotensin II-forming chymase may play a role in the proliferation of the medial layer in the lung arterioles of monocrotaline-induced pulmonary hypertensive rats.

Angiotensin II-forming chymase is expressed in the pulmonary arteries of the monocrotaline-induced pulmonary hypertensive rats, but its actual role is unclear. We studied chymase-dependent angiotensin II formation in the pulmonary arteries of the monocrotaline-induced pulmonary hypertensive rats and observed the effects of an angiotensin II receptor blocker on vascular remodeling. Four weeks after the administration of monocrotaline (60 mg/kg, s.q.), echocardiographic, hemodynamic, morphometric and biochemical analyses were performed. Age-matched rats were used as controls. To evaluate the effects of an angiotensin II receptor blocker, 2 wk after beginning of monocrotaline treatment, the rats were given candesartan (10 mg/kg per day) or placebo for 2 wk. In the monocrotaline-induced pulmonary hypertensive rats, the elevated systolic pulmonary arterial pressure and right ventricular hypertrophy were observed. Medial hypertrophy of lung arterioles was also observed. Chymase activity and angiotensin II concentration, but not angiotensin-converting enzyme activity, were significantly increased in the lung. In the angiotensin II receptor blocker-treated group, both systolic pulmonary arterial pressure and right ventricular hypertrophy were significantly reduced, and arteriolar hypertrophy was also prevented. Thus, angiotensin II-forming chymase may play a role in the proliferation of the medial layer in the lung arterioles of monocrotaline-induced pulmonary hypertensive rats.

Chymase forms angiotensin II (Ang II) which plays a crucial role in vascular diseases. In dog grafted veins, chymase activity and Ang II concentration along with vascular proliferation were significantly increased, while they were completely suppressed by a chymase inhibitor. After balloon injury in dog arteries, vascular chymase activity was significantly increased, and a chymase inhibitor and an Ang II receptor blocker were effective in preventing the vascular proliferation, but an angiotensin-converting enzyme inhibitor was ineffective. In clinical studies, an Ang II receptor blocker was successful in preventing restenosis after percutaneous coronary intervention, but an Ang II-converting enzyme inhibitor was not. In human and animal atherosclerosis, chymase activity was significantly increased, and a chymase inhibitor prevented experimental atherosclerosis. These observations suggest chymase may promote vascular proliferation and atherosclerosis. Chymase also activates matrix metalloproteinase-9 which promotes aortic aneurysm and angiogenesis. In human aortic aneurysms, chymase activity was significantly higher than in the normal aortas. In a hamster aneurysm model, chymase activity was also higher in the aneurysmal aortas than in the normal aortas, whereas a chymase inhibitor significantly prevented the aortic aneurysm. Chymase also acts as a pro-angiogenic factor, because the injections of the chymase gene or of purified chymase into implanted sponges strongly facilitate angiogenesis in hamsters. The mechanism of chymase- induced angiogenesis may depend not only on Ang II formation but also on activation of matrix metalloproteinase- 9. In this review, we propose chymase inhibitors as a novel therapeutic strategy for anti-vascular remodeling.

To clarify the regressive effect of an angiotensin II type 1 receptor blocker (ARB) on already formed fatty streaks, we investigated the effect of the administration of an ARB, olmesartan, on formed fatty streaks in monkeys fed a high-cholesterol diet.

Chymase generates angiotensin II which plays a crucial role in vascular remodeling. In clinical studies, an angiotensin II receptor blocker was successful in preventing restenosis after percutaneous coronary intervention, but an angiotensin II-converting enzyme inhibitor was not. In dog, chymase activity was significantly increased in vessels injured by a balloon catheter, and a chymase inhibitor and an angiotensin II receptor blocker were effective in preventing the vascular proliferation, but an angiotensin-converting enzyme inhibitor was ineffective. Chymase also activates matrix metalloproteinase-9, and their function may play an important role in development of atherosclerosis and aneurysmal aorta. In human atherosclerosis and aneurysmal aorta, chymase activity was significantly increased. In the experimental models, chymase activity and mRNA level were also significantly increased in atherosclerotic lesions and aneurysmal aorta, but chymase inhibitors prevented the vascular remodeling. On the other hand, chymase activates latent transforming growth factor--binding protein to transforming growth factor-, and its function may be related in promoting tissue fibrosis. Chymase promotes activations of angiotensin II, matrix metalloproteinase-9 and transforming growth factor-, and chymase inhibitors may promise to prevent vascular remodeling and tissue fibrosis.

In this study, we evaluated whether a specific chymase inhibitor, TY-51184 (2-[4-(5-fluoro-3-methylbenzo[b]thiophen-2-yl)sulfonamido-3-methanesulfonylphenyl]oxazole-4-carboxylicacid), prevents the vascular proliferation in canine grafted veins. In the placebo- and chymase inhibitor-treated groups, the external jugular vein was infiltrated with saline and 10 μM TY-51184, respectively, and then it was grafted to the ipsilateral carotid artery. The non-surgical dogs were used as the control group. By 28 days after grafting, the chymase and ACE activities were significantly increased in the injured arteries. TY-51184 significantly reduced the chymase activity in the grafted veins, while it did not affect the ACE activity. The intimal areas in the placebo- and TY-51184-treated groups were 3.32 ± 0.16 and 1.96 ± 0.52 mm2, respectively, and this difference was significant. The ratios of intimal area to medial area in the placebo- and TY-51184-treated groups were 66.8 ± 3.5% and 34.9 ± 9.2%, respectively, and this difference was also significant. There was a significant relationship between vascular proliferation and chymase activity, but not ACE activity. In this study, we demonstrated that a single treatment with a specific chymase inhibitor, TY-51184, could prevent the vascular proliferation in canine grafted veins.

J Thorac Cardiovasc Surg 2002;124:621-5

We investigated the inhibitory mechanisms of daphnodorins for human chymase using three-dimensional molecular modeling. In daphnodorin A–human chymase complex, daphnodorin A was fixed to the active site via hydrogen bonds with Ala177, Phe29, and Gly199 in human chymase, and it formed hydrogen bonds with Ser182 and Gly180, and this complex was formed stably. In daphnodorin B–human chymase complex, daphnodorin B formed hydrogen bonds with Lys28 and Phe29 in human chymase, but it could not form hydrogen bonds with Gly199, Ala177, and Lys179. The phenyl group of daphnodorin B shifted from the P1 hole in human chymase in comparison with that of daphnodorin A. For the inhibition of human chymase by daphnodorins, we indicated that it was significant whether daphnodorins formed hydrogen bonds with Ala177 located in the P1 hole, Ser182 located in the active site, Gly180 located in the anion hole, and with Gly199, Phe29, and Lys28 in human chymase.

Objectives:The great saphenous vein graft is known to be less patent than the internal thoracic artery graft. Recently, we reported that chymase-dependent angiotensin II formation plays an important role in the development of intimal hyperplasia in dog grafted veins. In this study we investigated the levels of angiotensin II–forming enzymes, angiotensin-converting enzyme, and chymase in human saphenous veins and internal thoracic arteries. Methods:The saphenous vein and internal thoracic artery specimens were obtained from coronary artery bypass grafts of patients during surgical procedures (saphenous vein, n = 16; internal thoracic artery, n = 16). Activities of angiotensin-converting enzyme and chymase were determined by using the extract from the saphenous vein or internal thoracic artery. Sections of the saphenous vein or internal thoracic artery were stained with van Gieson's elastin stain and were immunostained with anti-human chymase antibody. Results:The activities of angiotensin-converting enzyme in the saphenous vein and internal thoracic artery were 0.34 ± 0.12 and 0.32 ± 0.17 mU/mg protein, respectively, and the difference was not significant. The chymase activity in the saphenous vein was significantly higher than that in the internal thoracic artery (saphenous vein, 10.1 ± 0.81 mU/mg protein; internal thoracic artery, 6.21 ± 1.86 mU/mg protein). Chymase-positive cells in the saphenous vein were located in both the media and adventitia, and those in the internal thoracic artery were located only in the adventitia. The number of chymase-positive cells in the saphenous vein was about 2.6 times that in the internal thoracic artery. Conclusion:The chymase activity, but not the angiotensin-converting enzyme activity, was significantly higher in the saphenous vein, suggesting that the high levels of chymase activity may be related to the poorer performance of the saphenous vein for use as a bypass conduit. (J Thorac Cardiovasc Surg 2001;121:729-34)

We investigated the effect of a chymase inhibitor Suc‐Val‐Pro‐PheP(OPh)2on the proliferation of the grafted vein in dog. By 28 days after the operation, the mean intimal area of the grafted vein in the placebo group was 3.24±0.32 mm2. The intimal area of the grafted vein in the chymase inhibitor‐treated group was reduced to 63.9%. In the placebo group, the activities of chymase and angiotensin‐converting enzyme in grafted vein were significantly increased 15‐ and 2‐fold, respectively. In the chymase inhibitor‐treated group, chymase activity in the grafted veins was decreased significantly. These findings suggest that inhibition of chymase appears useful for preventing vascular proliferation.

We investigated the effect of a chymase inhibitor Suc-Val-Pro-PheP(OPh)2on the proliferation of the grafted vein in dog. By 28 days after the operation, the mean intimal area of the grafted vein in the placebo group was 3.24±0.32 mm2. The intimal area of the grafted vein in the chymase inhibitor-treated group was reduced to 63.9%. In the placebo group, the activities of chymase and angiotensin-converting enzyme in grafted vein were significantly increased 15- and 2-fold, respectively. In the chymase inhibitor-treated group, chymase activity in the grafted veins was decreased significantly. These findings suggest that inhibition of chymase appears useful for preventing vascular proliferation.

Chronic pressure overload induces cardiac tissue remodeling. Chymase is known to regulate matrix metabolism and angiotensin II formation. In the present study, we investigated the pathophysiological functions of chymase in the pressure‐overloaded hamster heart induced by a two‐kidney, one‐clip (2K1C) hypertension procedure. Fibrosis and apoptosis were observed in the pressure‐overloaded hearts of 2K1C hamsters 32 weeks after clipping, but these histological changes were not detected at 16 weeks. Heart chymase‐like activity of 2K1C hamsters at 32 weeks increased 5.2‐fold compared with that at 16 weeks, while angiotensin‐converting enzyme was not activated. Chymase might be involved in cardiac tissue remodeling during the chronic stage of hypertension.

Recent reports suggested that human heart chymase contributed little to angiotensin (Ang) II formation in the presence of natural protease inhibitors such as α-antitrypsin. We studied whether chymase could contribute to Ang II formation in the presence of natural protease inhibitors in the homogenate, the extract, and slices of human vascular tissue, and whether these inhibitors affect Ang I-induced vasocontractile responses due to chymase. In the homogenate, lisinopril, chymostatin, and α-antitrypsin inhibited the formation of Ang II by 14, 92, and 74%, respectively. In the extract, the inhibition of Ang II formation by lisinopril, chymostatin, and α-antitrypsin was 18, 94, and 93%, respectively. In the slices, lisinopril and chymostatin inhibited Ang II formation by 5 and 90%, respectively. However, unlike the homogenate and the extract experiments, only 8% of the Ang II formation was suppressed by α-antitrypsin. In isolated human gastroepiploic artery, 30% of Ang I-induced vasoconstriction was blocked by lisinopril, and the rest was completely eliminated by a combination of lisinopril and chymostatin. On the other hand, α-antitrypsin was ineffective in blocking Ang I-induced vasoconstriction in the presence of lisinopril, which demonstrates that Ang II formation is dependent on chymase. These findings suggest that chymase in human vascular tissue plays a functional role in Ang II formation in the presence of natural protease inhibitors such as α-antitrypsin.

A chymase-dependent angiotensin II-forming pathway is present in human vascular tissues; however, the role, if it plays any, of chymase in the pathogenesis of hypertension is not known. When investigating the role of chymase, it is important to recognize marked differences in vascular angiotensin II-forming systems among species. We found recently that hamsters, like humans, possess the dual angiotensin II-forming system.

Purified monkey chymase cleaved the Tyr31–Gly32bond of big-endothelin-1 and big-endothelin-2 to yield endothelin-1-(1–31) and endothelin-2-(1–31), respectively. In the isolated monkey trachea, endothelin-1-(1–31) and endothelin-2-(1–31), as well as big-endothelin-1 and big-endothelin-2, induced contractile responses. Chymostatin, which inhibits chymase, suppressed the contractile response induced by big-endothelin-2 to 16.6% but not the responses induced by big-endothelin-1, endothelin-1-(1–31) and endothelin-2-(1–31). These results suggest that the contractile response of big-endothelin-2 is predominantly dependent on the conversion of big-endothelin-2 to endothelin-2-(1–31) by chymase.