Chymase Activity Research Articles

Nafamostat mesilate attenuates colonic inflammation and mast cell infiltration in the experimental colitis

Serine proteases are important in the pathogenesis of intestinal inflammation. Recent studies have shown that nafamostat mesilate (NM) can inhibit the colonic mucosal inflammation induced by TNBS in rats. The aim of this study was to investigate the anti-inflammatory effects of NM on a DSS-induced colitis. Colitis was induced in female BALB/c mice by 5% dextran sulfate sodium (DSS) for 6 days. NM (2 or 20 mg/kg body weight) was orally administered once a day for 6 days during treatment of the mice with DSS. The inflammatory response of the colon was assessed 1 week after DSS treatment. NM at a high dose, but not at a low dose significantly decreased disease activity index (DAI) and myeloperoxidase (MPO) induced by DSS. Furthermore, NM (20 mg/kg) inhibited the production of tumor necrosis factor (TNF)-α, cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) in the colonic tissues treated with DSS. The increase in chymase activity by DSS treatment was also attenuated by the administration of NM (20 mg/kg). NM (20 mg/kg) significantly decreased the colonic mucosal injury and the infiltrated mast cell number induced by DSS. These results indicate that NM might inhibit the colonic inflammation through inhibition of both chymase activity and mast cell infiltration in colon tissues of DSS-induced colitis.

Mast Cell Fibroblastoid Differentiation Mediated by Airway Smooth Muscle in Asthma

Mast cell microlocalization to the airway smooth muscle (ASM) bundle is a key feature of asthma, but whether these mast cells have an altered phenotype is uncertain. In this paper, we report that in vivo, mast cells within the ASM bundle, in contrast to mast cells in the bronchial submucosa, commonly expressed fibroblast markers and the number of these cells was closely related to the degree of airway hyperresponsiveness. In vitro human lung mast cells and mast cell lines cultured with fibronectin or with primary human ASM cells acquired typical fibroblastic markers and morphology. This differentiation toward a fibroblastoid phenotype was mediated by ASM-derived extracellular matrix proteins, independent of cell adhesion molecule-1, and was attenuated by α5β1 blockade. Fibroblastoid mast cells demonstrated increased chymase expression and activation with exaggerated spontaneous histamine release. Together these data indicate that in asthma, ASM-derived extracellular matrix proteins mediate human mast cell transition to a fibroblastoid phenotype, suggesting that this may be pivotal in the development of airway dysfunction in asthma.

Mast cell chymase inhibition reduces atherosclerotic plaque progression and improves plaque stability in ApoE−/− mice

mast cells have been shown to accumulate in the adventitia of human atherosclerotic plaques and were recently demonstrated by us to contribute to plaque progression and instability. In this study, we investigated whether selective inhibition of mast cell chymases would affect the lesion development and stability. the protease inhibitor RO5066852 appeared to be a potent inhibitor of chymase activity in vitro and ex vivo. With this inhibitor, we provide three lines of evidence that chymase inhibition can prevent many pro-atherogenic activities. First, oral administration of RO5066852 reduced spontaneous atherosclerosis in the thoracic aorta of apoE(-/-) mice. Second, chymase inhibition prevented the accelerated plaque progression observed in apoE(-/-) mice that were exposed to repetitive episodes of systemic mast cell activation. Furthermore, RO5066852 enhanced lesional collagen content and reduced necrotic core size. Third, RO5066852 treatment almost completely normalized the increased frequency and size of intraplaque haemorrhages observed in apoE(-/-) mice after acute perivascular mast cell activation in advanced atherosclerosis. our data indicate that chymase inhibition can inhibit pro-atherogenic and plaque destabilizing effects which are associated with perivascular mast cell activation. Our study thus identifies pharmacological chymase inhibition as a potential therapeutic modality for atherosclerotic plaque stabilization.

Elevated Plasma Chymotrypsin-like Protease (Chymase) Activity in Women with Preeclampsia

Objective: Chymase, a chymotrypsin-like protease, is a non-ACE angiotensin II (Ang II) generating enzyme. We determined if maternal chymotrypsin-like protease/chymase activity was increased in women with preeclampsia (PE). Methods: Maternal plasma was extracted from venous blood of healthy nonpregnant women, women with normal and preeclamptic pregnancies. Chymotrypsin-like protease/chymase activity was measured by a colorimetric assay. Maternal vessel chymotrypsin-like protease/chymase expression was examined by immunohistochemistry. Results: Maternal plasma chymotrypsin-like protease/chymase activity was significantly higher in women with PE than in nonpregnant and normal pregnant women: 0.181 ± 0.011 vs. 0.097 ± 0.05 (p < 0.01) and 0.132 ± 0.013 (p < 0.05) μkat/mL. Chymotrypsin-like protease/chymase activity was markedly reduced 24 hours postpartum in women with PE, p < 0.05. Enhanced chymotrypsin-like protease/chymase expression was observed in vascular endothelium in women with PE compared with those in normal pregnancies. Conclusions: Elevated maternal chymotrypsin-like protease/chymase activity and enhanced protease immunostaining in the maternal vessel endothelium may constitute the exacerbated inflammatory state and account for the increased vascular Ang II sensitivity in PE.

Chymase inhibition attenuates tetrachloride‐induced liver fibrosis in hamsters

Chymase converts angiotensin I to angiotensin II, which may promote the development of liver fibrosis. In this study, whether a chymase inhibitor TY-51469 attenuated tetrachloride (CCl(4))-induced liver fibrosis was examined. Liver fibrosis was induced by the s.c. injection of 1 mL/kg of CCl(4) twice weekly for 8 weeks, and each hamster was given TY-51469 (1 mg/kg per day) or placebo. Untreated hamsters were used as a control group. Significant increases of serum alanine aminotransferase, total bilirubin and hyaluronic acid levels were observed in the placebo-treated group compared with the control group, but these levels were significantly attenuated in the TY-51469-treated group. Liver chymase activity was significantly higher in the placebo-treated group than in the control group, whereas the activity in the TY51469-treated group was not. Total angiotensin II-forming activity in the liver was also significantly higher in the placebo-treatedgroup than in the control group or the TY-51469-treated group. The ratio of the fibrotic area to the total area in the liver was significantly higher in the placebo-treated group than in the control group, but the ratio was significantly lower in the TY-51469-treated group than in the placebo-treated group. A significant decrease in the number of alpha-smooth muscle actin (SMA)-positive cells was seen in the TY-51469-treated group compared to the placebo-treated group. Significant correlations between the number of chymase-positive cells and the degree of fibrosis and between the numbers of chymase-positive cells and alpha-SMA-positive cells were observed. Thus, chymase inhibition may be a useful strategy for preventing liver fibrosis.

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

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. Hamsters were fed a methionine- and choline-deficient (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. 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 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 diet-fed hamsters. These findings demonstrate that the mast cell protease chymase may play a crucial role in the development of NASH in hamsters.

M1757 Impact of Luminal and Systemic Iron on the Development of Chronic Ileitis Targeting the Gut Microbial Composition and Stress Response in Intestinal Epithelial Cells

activity, but also MMP-9 activity in DSS-treated mice. Chymase activity was significantly lower in the NK3201-treated group than in the placebo-treated group. MMP-9 level was also significantly lower in the NK3201-treated group than in the placebo-treated group. In NSAID-induced small intestinal enteritis, MMP-9 level was significantly increased to 5.6 and 9.2 times of control intestinal. Chymase inhibitor significantly decreased to 13.9% the ulcer index compared with placebo. Conclusion: These findings suggest that chymase plays an important role in the development of gut inflammation via MMP-9 activation.

Chymase‐dependent generation of Angiotensin II from Angiotensin‐(1–12) in human atrial tissue

We showed that angiotensin‐(1–12) [Ang‐(1–12)] is a non‐renin dependent alternate precursor for the generation of cardiac Ang peptides in rat tissue. In this study, we investigated the metabolism of Ang‐(1–12) by plasma membranes (PM) isolated from human atrial appendage tissue from patients undergoing open‐heart surgery. PM were incubated with highly purified 125I‐Ang‐(1–12) at 37° C for 1 h with or without a cocktail of peptidase inhibitors for ACE, ACE2, neprilysin (NEP) and chymase (50 μM each). 125I‐peptide fractions were identified in the supernatant by HPLC coupled to an inline γ‐detector. In the absence of inhibitor, 125I‐Ang‐(1–12) was converted into Ang I (10±7%), Ang II (62±14%), Ang‐(1–7) (7±3%) and other smaller peptides (&lt;2%). In the presence of the all inhibitors, 90±4% of 125I‐Ang‐(1–12) remained intact. The relative contribution of selective inhibition of ACE, ACE2, NEP, and chymase enzyme showed that 125I‐Ang‐(1–12) was primarily converted into Ang II (77±24%) by chymase while its hydrolysis into Ang peptides by ACE, ACE2 and NEP was significantly lower. The activity of individual enzyme was then calculated based on the formation of Ang I, Ang II, Ang‐(1–7) peptides fractions. These results showed a very high chymase activity (54±22 fmol × min−1 × mg protein−1, N=5), followed by ACE2, and NEP (10.5±6 and 7.3±5.6 fmol × min−1 × mg protein−1, respectively) while ACE activity was negligible (2.8±2.1 fmol × min−1 × mg protein−1). In conclusion, we demonstrate for the first time in human cardiac tissue a dominant role of cardiac chymase in the formation of angiotensins peptides from Ang‐(1–12).

Role of chymase in cigarette smoke-induced pulmonary artery remodeling and pulmonary hypertension in hamsters

BackgroundCigarette smoking is an important risk factor for pulmonary arterial hypertension (PAH) in chronic obstructive pulmonary disease (COPD). Chymase has been shown to function in the enzymatic production of angiotensin II (AngII) and the activation of transforming growth factor (TGF)-β1 in the cardiovascular system. The aim of this study was to determine the potential role of chymase in cigarette smoke-induced pulmonary artery remodeling and PAH.MethodsHamsters were exposed to cigarette smoke; after 4 months, lung morphology and tissue biochemical changes were examined using immunohistochemistry, Western blotting, radioimmunoassay and reverse-transcription polymerase chain reaction.ResultsOur results show that chronic cigarette smoke exposure significantly induced elevation of right ventricular systolic pressures (RVSP) and medial hypertrophy of pulmonary arterioles in hamsters, concurrent with an increase of chymase activity and synthesis in the lung. Elevated Ang II levels and enhanced TGF-β1/Smad signaling activation were also observed in smoke-exposed lungs. Chymase inhibition with chymostatin reduced the cigarette smoke-induced increase in chymase activity and Ang II concentration in the lung, and attenuated the RVSP elevation and the remodeling of pulmonary arterioles. Chymostatin did not affect angiotensin converting enzyme (ACE) activity in hamster lungs.ConclusionsThese results suggest that chronic cigarette smoke exposure can increase chymase activity and expression in hamster lungs. The capability of activated chymase to induce Ang II formation and TGF-β1 signaling may be part of the mechanism for smoking-induced pulmonary vascular remodeling. Thus, our study implies that blockade of chymase might provide benefits to PAH smokers.

The one-two punch: knocking out angiotensin II in the heart

Ang II plays an important role in the pathophysiology of cardiovascular disease. Angiotensin-converting enzyme (ACE) inhibitors lower Ang II levels by inhibiting conversion of Ang I to Ang II, but Ang II levels have been shown to return to normal with chronic ACE inhibitor treatment. In this issue of the JCI, Wei et al. show that ACE inhibition induces an increase in chymase activity in cardiac interstitial fluid, providing an alternate pathway for Ang II generation.

Mast cell chymase limits the cardiac efficacy of Ang I–converting enzyme inhibitor therapy in rodents

Ang I-converting enzyme (ACE) inhibitors are widely believed to suppress the deleterious cardiac effects of Ang II by inhibiting locally generated Ang II. However, the recent demonstration that chymase, an Ang II-forming enzyme stored in mast cell granules, is present in the heart has added uncertainty to this view. As discussed here, using microdialysis probes tethered to the heart of conscious mice, we have shown that chronic ACE inhibitor treatment did not suppress Ang II levels in the LV interstitial fluid (ISF) despite marked inhibition of ACE. However, chronic ACE inhibition caused a marked bradykinin/B2 receptor-mediated increase in LV ISF chymase activity that was not observed in mast cell-deficient KitW/KitW-v mice. In chronic ACE inhibitor-treated mast cell-sufficient littermates, chymase inhibition decreased LV ISF Ang II levels substantially, indicating the importance of mast cell chymase in regulating cardiac Ang II levels. Chymase-dependent processing of other regulatory peptides also promotes inflammation and tissue remodeling. We found that combined chymase and ACE inhibition, relative to ACE inhibition alone, improved LV function, decreased adverse cardiac remodeling, and improved survival after myocardial infarction in hamsters. These results suggest that chymase inhibitors could be a useful addition to ACE inhibitor therapy in the treatment of heart failure.

Effect of long-term treatment with urocortin on the activity of somatic angiotensin-converting enzyme in spontaneously hypertensive rats

Our previous acute study on urocortin (Ucn) demonstrated that Ucn altered serum and tissue angiotensin-converting enzyme (ACE) activity in rats. Therefore, the present investigation was designed to explore the effect of long-term treatment with Ucn on somatic ACE (sACE) and other components of the renin-angiotensin system (RAS). After 8 weeks of intravenous administration of Ucn in spontaneously hypertensive rats (SHR), serum and tissue sACE, angiotensin II (Ang II), nitric oxide (NO), Ang-(1-7), and tissue chymase activities were evaluated. RT-PCR analysis was performed to determine the quantity of tissue sACE mRNA. Serum sACE activity was reduced by Ucn, although tissue sACE activity and tissue sACE mRNA were elevated. Chymase activity was observed to be enhanced by Ucn, whereas the ACE inhibitor enalapril failed to influence chymase. Serum and tissue Ang II activity was reduced, but NO and Ang-(1-7) production was increased in a concentration-dependent manner after Ucn treatment. Meanwhile, a significant decrease of the systolic blood pressure (SBP) was observed after the long-term Ucn administration, and there was a significant positive correlation (r2 = 0.6993) between serum ACE activity and SBP. Pretreatment with the corticotropin-releasing factor (CRF) blocker astressin and the extracellular signal-regulated kinase 1/2 (ERK1/2) pathway blocker PD98059 abolished these effects of Ucn. Our findings further support the hypothesis that the changes of sACE activity and the production of other RAS components may play roles in the vasodilatory property of Ucn via the activation of the ERK1/2 pathway.

Production of proteolytic enzymes in mast cells, fibroblasts, vascular smooth muscle and endothelial cells cultivated under normoxic or hypoxic conditions

Matrix metalloproteinases (MMPs) is a family of proteolytic enzymes involved in remodeling of extracellular matrix. Although proteolytic enzymes are produced by many cell types, mast cells seem to be more important than other types in remodeling of pulmonary arteries during hypoxia. Therefore, we tested in vitro production of MMPs and serine proteases in four cell types (mast cells, fibroblasts, vascular smooth muscle cells and endothelial cells) cultivated for 48 h under normoxic or hypoxic (3% O2) conditions. MMP-13 was visualized by immunohistochemistry, MMP-2 and MMP-9 were detected by zymography in cell lysates. Enzymatic activities (MMPs, tryptase and chymase) were estimated in the cultivation media. Hypoxia had a minimal effect on total MMP activity in the cultivation media of all types of cells, but immunofluorescence revealed higher intensity of MMP-13 in the cells exposed to hypoxia except of fibroblasts. Tryptase activity was three times higher and chymase activity twice higher in mast cells cultivated in hypoxia than in those cultured in normoxia. Among all cell types studied here, mast cells are the most abundant source of proteolytic enzymes under normoxic and hypoxic conditions. Moreover, in these cells hypoxia increases the production of both specific serine proteases tryptase and chymase, which can act as MMPs activators.

Homocysteine-induced oxidative stress upregulates chymase in mouse mastocytoma cells

Reactive oxygen species (ROS) such as hydrogen peroxide (H(2)O(2)), O(*-)(2) and OH(*) participate in the pathogenesis of ischemia/reperfusion injury, inflammation and atherosclerosis. Our previous studies have suggested that increased angiotensin II (Ang II)-forming chymase may be involved in the development of atherosclerosis. However, the regulatory mechanism of chymase expression has not yet been clarified. In this study, we tested whether oxidative stress upregulates mouse mast cell proteinase chymase, mouse mast cell proteinase (MMCP)-5 or MMCP-4. We also examined the expression and activity of these proteins after treatment. Cultured mouse mastocytoma cells (MMC) displaying chymase-dependent Ang II-forming activity were treated with H(2)O(2) and several aminothiols with or without anti-oxidants. The levels of MMCP-5 and MMCP-4 expression were determined by quantitative RT-PCR; the level of chymase-dependent Ang II-forming activity was measured by high performance liquid chromatography using Ang I as a substrate. Treatment of MMC with homocysteine (0.1-3 mmol l(-1)) significantly increased MMCP-5 and MMCP-4 expression, as well as Ang II-forming activity. These effects were significantly inhibited by the addition of catalase and further suppressed by the combination of catalase and superoxide dismutase. Incubation with hydrogen peroxide alone caused a significant increase in Ang II-forming activity, which was completely suppressed by co-treatment with catalase. Furthermore, MMCP-5 and MMCP-4 expression levels were drastically suppressed and chymase induction by homocysteine was diminished under the GATA-inhibited condition. Homocysteine increased mast cell chymase expression and activity through the mechanism of oxidative stress. Our results suggest that there is a biochemical link between oxidative stress and the local Ang II-forming system.

Pathophysiological Role of Skin Mast Cells in Wound Healing after Scald Injury: Study with Mast Cell-Deficient W/WV Mice

Background: The major role of mast cells in wound healing process has not been identified. In this study, we used mast cell-deficient W/W^V mice and their congenic control (+/+) mice to examine the role of mast cells in scald wound healing. Methods: The size of the scald wound, thickness of the dermis, collagen deposition, vascularization, number of mast cells and chymase activity were measured before and at 3, 7, 14 and 21 days after inducing scald injury. Results: Although the process of wound closure and re-epithelialization was not markedly different between W/W^V mice and +/+ mice, the degree of fibrous proliferation at the wound edge and wound vascularization in the proliferative phase was significantly lower in W/W^V mice than in +/+ mice, and no vascular regression in the late remodeling phase was observed in W/W^V mice. Mast cells producing chymase, FGF2, TGF-β1 and VEGF were highly accumulated at the edge of scald wound in +/+ mice during the proliferative and remodeling phases at days 14 and 21. Chymase activity in the injured tissues of +/+ mice decreased in the acute phase, but recovered to no-injury level at days 14 and 21. The number of mast cells and chymase activity were very low in the injured tissues of W/W^V mice throughout the experiment. Conclusions: Wound healing after skin scald injury was partially impaired in mast cell-deficient mice. Mast cells may contribute to the wound healing process, especially in the proliferative and remodeling phases after scald injury.

Cleaved secretory leucocyte protease inhibitor as a biomarker of chymase activity in allergic airway disease

Secretory leucocyte protease inhibitor (SLPI), which is present in many physiological fluids including saliva, sputum and nasal discharge, is the most effective inhibitor of chymase. Previously, we demonstrated that chymase is able to cleave SLPI and that the cleaved portion, cSLPI, is a biomarker of chymase activity. We investigated the potential of cSLPI as a biomarker of chymase activity in subjects with allergic rhinitis (AR) and asthmatic airway disease. Baseline sputum samples were collected from atopic asthmatics and healthy controls (HC). Nasal lavages (NAL) were performed in subjects with AR both at baseline and following a nasal challenge with allergen or placebo. Levels of cSLPI and chymase were determined by Western analysis, and tryptase and alpha-2 macroglobulin were measured by immunoassay. As compared with HC, asthmatics showed a significant increase in baseline cSLPI/total SLPI ratios and an increase in chymase levels. There was a high correlation of cSLPI/SLPI ratios to chymase levels in normal individuals and untreated asthmatics. In the NAL of patients with AR, as compared with placebo, allergen challenge increased inflammatory biomarkers, including cSLPI/SLPI ratios, chymase levels, tryptase levels and alpha2-macroglobulin levels. Correlations were observed between cSLPI/SLPI ratios and chymase levels and cSLPI/SLPI ratios and alpha2-macroglobulin levels; no correlation was seen between cSLPI/SLPI ratios and tryptase levels. Our data indicate that cSLPI reflects chymase activity in AR and asthma. Hence, cSLPI may serve as a biomarker for disease activity and for monitoring the efficacy of novel anti-inflammatory treatments in chymase-mediated diseases.

Chymase Plays an Important Role in Left Ventricular Remodeling Induced by Intermittent Hypoxia in Mice

Intermittent hypoxia caused by sleep apnea is associated with cardiovascular disease. Chymase has been reported to play an important role in the development of cardiovascular disease, but it is unclear whether chymase is involved in the pathogenesis of left ventricular remodeling induced by intermittent hypoxia. The aim of this study was to evaluate the effect of a novel chymase inhibitor (NK3201) on hypoxia-induced left ventricular remodeling in mice. Male C57BL/6J mice (9 weeks old) were exposed to intermittent hypoxia or normoxia and were treated with NK3201 (10 mg/kg per day) or the vehicle for 10 days. Left ventricular systolic pressure showed no significant differences among all of the experimental groups. Exposure to intermittent hypoxia increased left ventricular chymase activity and angiotensin II expression, which were both suppressed by treatment with NK3201. Intermittent hypoxia also increased the mean cardiomyocyte diameter, perivascular fibrosis, expression of inflammatory cytokines, oxidative stress, and NADPH-dependent superoxide production in the left ventricular myocardium. These changes were all suppressed by NK3201 treatment. Therefore, chymase might play an important role in intermittent hypoxia-induced left ventricular remodeling, which is independent of the systemic blood pressure.

α2-Macroglobulin Capture Allows Detection of Mast Cell Chymase in Serum and Creates a Reservoir of Angiotensin II-Generating Activity

Human chymase is a highly efficient angiotensin II-generating serine peptidase expressed by mast cells. When secreted from degranulating cells, it can interact with a variety of circulating antipeptidases, but is mostly captured by alpha(2)-macroglobulin, which sequesters peptidases in a cage-like structure that precludes interactions with large protein substrates and inhibitors, like serpins. The present work shows that alpha(2)-macroglobulin-bound chymase remains accessible to small substrates, including angiotensin I, with activity in serum that is stable with prolonged incubation. We used alpha(2)-macroglobulin capture to develop a sensitive, microtiter plate-based assay for serum chymase, assisted by a novel substrate synthesized based on results of combinatorial screening of peptide substrates. The substrate has low background hydrolysis in serum and is chymase-selective, with minimal cleavage by the chymotryptic peptidases cathepsin G and chymotrypsin. The assay detects activity in chymase-spiked serum with a threshold of approximately 1 pM (30 pg/ml), and reveals native chymase activity in serum of most subjects with systemic mastocytosis. alpha(2)-Macroglobulin-bound chymase generates angiotensin II in chymase-spiked serum, and it appears in native serum as chymostatin-inhibited activity, which can exceed activity of captopril-sensitive angiotensin-converting enzyme. These findings suggest that chymase bound to alpha(2)-macroglobulin is active, that the complex is an angiotensin-converting enzyme inhibitor-resistant reservoir of angiotensin II-generating activity, and that alpha(2)-macroglobulin capture may be exploited in assessing systemic release of secreted peptidases.

Chymase activity is closely related with plaque vulnerability in a hamster model of atherosclerosis

Objective To test the hypothesis that stimulation of chymase secretion may contribute to plaque vulnerability and inhibition of chymase activity may enhance plaque stability. Methods and results Sixty eight-week-old male Syrian golden hamsters were randomly divided into normal control group, high-cholesterol (HC) treated group, HC + ovalbumin treated group and HC + tranilast treated group. The normal control group received a normal diet while the other three intervention groups received a high-cholesterol diet for 15 weeks. Hamsters in the HC + ovalbumin treated group underwent transcatheter pharmacological triggering at the end of week 15 after antigen sensitization and those in the HC + tranilast treated group were given tranilast intragastrically for 3 weeks before euthanasia. Serological, ultrasonographic, pathologic, immunohistochemical, and gene expression studies were performed in all animals. The total number of mast cells, proportion of degranulated mast cells and the number of extracellular granules in plaques, the apoptosis rate of vascular smooth cells, the local activities of chymase, the concentration of Ang II and the expression levels of inflammatory markers as well as plaque vulnerability index all increased significantly in HC + ovalbumin treated group, but remarkably decreased in HC + tranilast treated group, in comparison with the HC treated group. These results suggest that stimulation of chymase secretion contributes to plaque vulnerability while inhibition of chymase activity enhances plaque stability. We conclude that chymase activity provides a promising therapeutic target in the stabilization of atherosclerotic plaques.

Structure of granzyme C reveals an unusual mechanism of protease autoinhibition

Proteases act in important homeostatic pathways and are tightly regulated. Here, we report an unusual structural mechanism of regulation observed by the 2.5-A X-ray crystal structure of the serine protease, granzyme C. Although the active-site triad residues adopt canonical conformations, the oxyanion hole is improperly formed, and access to the primary specificity (S1) pocket is blocked through a reversible rearrangement involving Phe-191. Specifically, a register shift in the 190-strand preceding the active-site serine leads to Phe-191 filling the S1 pocket. Mutation of a unique Glu-Glu motif at positions 192-193 unlocks the enzyme, which displays chymase activity, and proteomic analysis confirms that activity of the wild-type protease can be released through interactions with an appropriate substrate. The 2.5-A structure of the unlocked enzyme reveals unprecedented flexibility in the 190-strand preceding the active-site serine that results in Phe-191 vacating the S1 pocket. Overall, these observations describe a broadly applicable mechanism of protease regulation that cannot be predicted by template-based modeling or bioinformatic approaches alone.