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PubMed2002

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.

Angioplasty, Balloon↗
PubMed2005

Effect of chymase inhibition on the arteriovenous fistula stenosis in dogs.

It was hypothesized that chymase may participate in hemodialysis vascular access dysfunction, as chymase has been known to be an effective enzyme in the conversion of angiotensin I (Ang I) to Ang II and in the latent TGF-beta1 to the active form. An arteriovenous (AV) fistula was created between the brachial artery and vein in dogs. In the AV anastomosis, when the walls of the venous and arterial sides were compared, the eccentric neointimal formation was most evident in the venous wall. Compared with the venous side downstream of the AV anastomosis, a severe neointimal hyperplasia was found in the venous side upstream of the AV anastomosis (intima/media, 153 +/- 25%). The chymase- and TGF-beta-positive mast cells were markedly accumulated in the proliferous neointima and media. In association with the reduction of chymase expression, a marked decrease in Ang II-, AT(1) receptor-, and TGF-beta-positive areas was achieved by NK3201 (a chymase inhibitor) treatment, and the neointima formation (intima/media: region A, 53 +/- 9%, P < 0.001; region B, 54 +/- 14%, P < 0.001) was also significantly suppressed in this group. Although lisinopril treatment also provided some beneficial effects with regard to the prevention of neointimal formation, the degree was less than that seen with chymase inhibition. These findings indicate that mast cell-derived chymase plays an essential role in the pathogenesis of the AV fistula access failure and that chymase inhibition may be a therapeutic target for the treatment of hemodialysis vascular access dysfunction in clinic settings.

Acetamides↗
PubMed2002

Mast cell alpha-chymase reduces IgE recognition of birch pollen profilin by cleaving antibody-binding epitopes.

In sensitized individuals birch pollen induces an allergic response characterized by IgE-dependent mast cell degranulation of mediators, such as alpha-chymase and other serine proteases. In birch and other plant pollens, a major allergen is profilin. In mammals, profilin homologues are found in an intracellular form bound to cytoskeletal or cytosolic proteins or in a secreted form that may initiate signal transduction. IgE specific to birch profilin also binds human profilin I. This cross-reactivity between airborne and endogenous proteins may help to sustain allergy symptoms. The current work demonstrates that cultured mast cells constitutively secrete profilin I, which is susceptible to degranulation-dependent proteolysis. Coincubation of chymase-rich BR mastocytoma cells with Ala-Ala-Pro-Phe-chloromethylketone (a chymase inhibitor) blocks profilin cleavage, which does not occur in degranulated HMC-1 mast cells, which are rich in tryptase, but chymase deficient. These data implicate chymase as the serine protease cleaving secreted mast cell profilin. Sequencing of chymase-cleaved profilins reveals hydrolysis at Tyr(6)-Val(7) and Trp(35)-Ala(36) in birch profilin and at Trp(32)-Ala(33) in human profilin, with all sites lying within IgE-reactive epitopes. IgE immunoblotting studies with sera from birch pollen-allergic individuals demonstrate that cleavage by chymase attenuates binding of birch profilin to IgE. Thus, destruction of IgE-binding epitopes by exocytosed chymase may limit further mast cell activation by this class of common plant allergens, thereby limiting the allergic responses in sensitized individuals.

Allergens↗
PubMed1997

[The expression of MLC2-chymase fusion gene in cultured neonatal myocardial cells].

A series of MLC2-chymase fusion genes consisting of nested 5' deletion of the chymase promoter region were constructed and transfected into primary cultured neonatal Wistar rat myocardial cells according to the modified calcium phosphate precipitation method. Total RNA Dot-blot hybridization and analysis of densitometric scanning demonstrated that MLC2 promoter is enough to direct human heart chymase gene transcription in cultured neonatal rat myocardial cells. The highest level of expression was found with the MLC2-chymase construct containing 6 bp of the chymase promoter region. More sequence of the chymase promoter region between MLC2 promoter and the ATG of chymase gene resulted in a subsequent decrease in the expression of human heart chymase gene.

Animals↗
PubMed2003

Modulation of enzymatic activity of human mast cell tryptase and chymase by protease inhibitors.

AIM: To investigate the actions of protease inhibitors on the enzymatic activities of tryptase and chymase in similar experimental systems. METHODS: Human lung tryptase and human skin chymase were purified by a similar procedure involving high salt extraction of tryptase, heparin agarose affinity chromatography, and S-200 Sephacryl gel filtration chromatography. Actions of protease inhibitors on tryptase and chymase activities were examined by enzyme assays. RESULTS: The specific activities of tryptase and chymase were 2.1 kU/g protein and 4.9 kU/g protein, respectively. Both preparations showed a single diffuse band on SDS-PAGE. Among non-native protease inhibitors, N-(1-hydroxy-2-naphthoyl)-L- arginyl-L-prolinamide hydrochloride (HNAP), leupeptin, antipain, benzamidine, and protamine inhibited more than 90 % enzymatic activity of tryptase, whereas soy bean trypsin inhibitor (SBTI), Z-Ile-Glu-Pro-Phe-CO2Me (ZIGPPM) and chymostatin inhibited more than 95 % enzymatic activity of chymase. Native protease inhibitors alpha 1-antitrypsin and secretory leukocyte protease inhibitor (SLPI) inhibited more than 90 % enzymatic activity of chymase, but lactoferrin appeared to enhance chymase enzymatic activity. All the 3 inhibitors had weak inhibitory actions on tryptase. CONCLUSION: The protease inhibitors tested had relatively good selectivity to either tryptase or chymase.

Chymases↗
PubMed2004

Inhibition of tryptase and chymase induced nucleated cell infiltration by proteinase inhibitors.

AIM: To investigate the ability of proteinase inhibitors to modulate nucleated cell infiltration into the peritoneum of mice induced by tryptase and chymase. METHODS: Human lung tryptase and skin chymase were purified by a similar procedure involving high salt extraction, heparin agarose affinity chromatography followed by S-200 Sephacryl gel filtration chromatography. The actions of proteinase inhibitors on tryptase and chymase induced nucleated cell accumulation were examined with a mouse peritoneum model. RESULTS: A selective chymase inhibitor Z-Ile-Glu-Pro-Phe-CO2Me (ZIGPPF) was able to inhibit approximately 90 % neutrophil, 73 % eosinophil, 87 % lymphocyte and 60 % macrophage accumulation induced by chymase at 16 h following injection. Soy bean trypsin inhibitor (SBTI), chymostatin, and alpha1-antitrypsin showed slightly less potency than ZIGPPF in inhibition of the actions of chymase. While all tryptase inhibitors tested were able to inhibit neutrophil, eosinophil, and macrophage accumulation provoked by tryptase at 16 h following injection, only leupeptin, APC366, and aprotinin were capable of inhibiting tryptase induced lymphocyte accumulation. The inhibitors of tryptase tested were also able to inhibit tryptase induced neutrophil and eosinophil accumulation at 6 h following injection. When being injected alone, all inhibitors of chymase and tryptase at the concentrations tested by themselves had no significant effect on the accumulation of nucleated cells in the peritoneum of mice at both 6 h and 16 h. CONCLUSION: Proteinase inhibitors significantly inhibited tryptase and chymase-induced nucleated cell accumulation in vivo, and therefore they are likely to be developed as a novel class of anti-inflammatory drugs.

Animals↗
PubMed1992

Protease composition of exocytosed human skin mast cell protease-proteoglycan complexes. Tryptase resides in a complex distinct from chymase and carboxypeptidase.

We characterized the release and the protease composition of high m.w. complexes released from dispersed human skin mast cells, under conditions that did not disrupt the binding of proteases to proteoglycan. The net percent release ratio of tryptase to histamine, after anti-IgE and calcium ionophore A23187 stimulation was higher than those for chymase or carboxypeptidase. This was explained by the greater cell association of carboxypeptidase and chymase, compared with tryptase, after mast cell degranulation and/or differential cosedimentation of the proteases with mast cells, because treatment of activated mast cells with 1 M NaCl increased the release ratios of chymase and carboxypeptidase more than that of tryptase. Tryptase, after release, was stable in 0.12 M NaCl and had a molecular mass of approximately 200 to 250 kDa, suggesting that it was bound to proteoglycan. We demonstrated that complexes containing chymase and carboxypeptidase were separable from tryptase-containing complexes by gel filtration and by affinity chromatography. First, on fast protein liquid chromatography, released tryptase filtered at a molecular mass of approximately 200 to 250 kDa, compared with chymase and carboxypeptidase at 400 to 560 kDa. Second, by using affinity chromatography with immobilized antitryptase mAb in 0.15 M NaCl, carboxypeptidase and chymase activities were recovered primarily in the effluent and washes of an antitryptase antibody affinity column and cofiltered at 400 to 560 kDa. Tryptase was recovered only in the eluate. Finally, by using potato tuber carboxypeptidase inhibitor-Sepharose affinity chromatography, tryptase activity was found primarily in the effluent and washes, filtered at a molecular mass of 200 kDa on fast protein liquid chromatography, and was stable in 0.12 M NaCl buffer at 37 degrees C. Carboxypeptidase and chymase activities were found primarily in the eluate. These findings suggest that tryptase and carboxypeptidase/chymase reside in distinct macromolecular complexes. Separate complexes containing these proteases may help explain previous ultrastructural observations in which the distributions of chymase and tryptase within a single granule did not always coincide.

Carboxypeptidases↗
PubMed1994

Activation of human interstitial procollagenase through direct cleavage of the Leu83-Thr84 bond by mast cell chymase.

In inflamed tissue sites characterized by on-going matrix degradation, the matrix metalloproteinases are secreted as latent precursors which are capable of proteolysis only after extracellular activation. Such areas often contain locally increased numbers of mast cells capable of releasing complexes between heparin proteoglycans and fully active endopeptidases with either tryptic (tryptase) or both tryptic and chymotryptic (chymase) activity. We have examined the ability of purified human skin chymase to activate human interstitial procollagenase (matrix metalloproteinase-1) in the absence and presence of heparin, the physiologic associate of chymase. Our studies indicate that chymase activates procollagenase in a time- and concentration-dependent manner. Heparin was found to increase markedly the rate at which chymase activates procollagenase both by accelerating the cleavage of procollagenase and also by preventing its further degradation. Moreover, we found that chymase activates procollagenase directly by cleaving the Leu83-Thr84 bond, without formation of any intermediate species. This is a novel mechanism for procollagenase activation, which contrasts sharply with the activation mechanisms of other activators studied so far. The ability of chymase to activate procollagenase suggests that chymase plays an active role in matrix degradation at tissue sites where mast cells coexist with extracellular procollagenase.

Amino Acid Sequence↗
PubMed1993

Reaction of human chymase with reactive site variants of alpha 1-antichymotrypsin. Modulation of inhibitor versus substrate properties.

Inhibition of human chymase by alpha 1-antichymotrypsin produces 3.5 mol of degraded inhibitor for every mol of chymase inhibited, resulting in a stoichiometry of inhibition (SI) of 4.5. In the present study, the substrate versus inhibitor properties of this reaction were examined further using wild type and mutant recombinant antichymotrypsins (rACT). Titration of chymase hydrolytic activity with rACT-L358 (wild type) and reactive site (P1) variants of ACT, L358W, L358M, and L358F revealed that the SI was sensitive to P1 residue replacements. SI values increased in the order of Trp < Met < Leu < Phe where SI values were 1.5, 2, 4, and 7, respectively. Chymase inhibitor complex and cleaved inhibitor were demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis for all variants; the relative intensities of each band were consistent with SI values established by titration. NH2-terminal sequence analyses of the products formed in the reaction of chymase with rACT-L358F indicated that the P1-P1' bond was the primary site of cleavage resulting in the hydrolysis and inactivation of this variant. The apparent second-order rate constant for chymase inhibition (k'/[I]) by rACT also was affected by P1 substitution. k'/[I] values increased in an order opposite that obtained for SI values (Phe < Leu < Met < Trp). The reactive loop mutant (rACT-P3P3') produced by replacing the reactive site region of ACT (Thr356-Val361) with that of alpha 1-proteinase inhibitor (Ile356-Pro361) revealed a different reaction pattern. Although its SI was near 1, the value for k'/[I] was the lowest among variants. rACT-L358R, another P1 variant, did not inhibit chymase. These results are evaluated with respect to the substrate preferences of human chymase and with respect to partitioning schemes proposed to explain SI values greater than 1.

Amino Acid Sequence↗
PubMed1998

Interleukin-4 promotes the development of tryptase and chymase double-positive human mast cells accompanied by cell maturation.

Human cultured mast cells (HCMCs) grown from cord blood mononuclear cells in the presence of stem cell factor (SCF) and interleukin-6 (IL-6) expressed tryptase but no or low chymase in their cytoplasm. The addition of IL-4 to these cells strikingly increased chymase expression. Consequently, the activity of chymase was significantly higher in IL-4-treated mast cells than that in IL-4-nontreated mast cells, whereas the activity of tryptase and histamine content were comparable in both cells. Electron microscopic immunocytochemistry also showed that secretary granules containing chymase increased in IL-4-treated mast cells. Interestingly, the IL-4-induced increase of chymase expression in HCMCs was accompanied by morphological maturation of the cells. Cytoplasmic projections were few in IL-4-nontreated HCMCs, and a small number of secretary granules were observed, most of which were empty or partially filled with discrete scrolls with rough particles showing immaturity. In contrast, IL-4-treated HCMCs had extremely abundant cytoplasmic projections and had many secretary granules filled with electron-dense crystal materials. Taken together, immature HCMCs grown only with SCF and IL-6 expressed tryptase with no or a low amount of chymase, and addition of IL-4 promoted cell maturation together with the expression of both tryptase and a high amount of chymase. Our findings will raise a possibility of a linear pathway of human mast cell development from tryptase single positive mast cells into tryptase and chymase double positive mast cells as the cells mature and will suggest that this maturation process is promoted by IL-4.

Cell Differentiation↗
PubMed2002

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.

Journal Article↗
PubMed1984

Activation of rat serosal mast cells by chymase, an endogenous secretory granule protease.

Chymase, the major neutral protease of the rat serosal mast cell (RMC) secretory granule, causes RMC to release their secretory granules and to oxidatively metabolize endogenous arachidonic acid to prostaglandin D2 (PGD2). The granule markers, endogenous beta-hexosaminidase and exogenously added [3H]serotonin, were released from 2.5 X 10(5) RMC in 50 microliters in parallel and in dose-response fashion, reaching a maximum net percent release of approximately 50% with 0.5 to 1.0 units chymase (15 U/mg)/ml. With incremental concentrations of chymase, the release of granule markers occurred with a shorter lag period and in a greater maximal net percent, whereas the release of PGD2 was dose-related without a reduction in latency to detectable generation. Inhibition of the esterase activity of chymase with lima bean trypsin inhibitor decreased the subsequent mast cell response, indicating that the active site of chymase was required to initiate granule secretion and PGD2 generation. The monophasic indomethacin-resistant rise in cellular cAMP at 15 to 45 sec coincident with the onset of chymase-induced mediator release and PGD2 secretion is similar to that observed with IgE receptor-initiated coupled activation-secretion. The ability of heparin to block the activation function of chymase without inhibition of esterase activity reveals a possible physiologic regulatory mechanism for limiting the potential action of secreted chymase.

Animals↗
PubMed1997

Quantitative analysis of tryptase and chymase containing mast cells in benign and malignant breast lesions.

The aim of our work was to study the number and distribution of tryptase- and chymase-containing mast cells in benign and malignant breast lesions. Tryptase positivity reflects the total number of mast cells, whereas chymase is not present in all mast cells. Active forms of tryptase and chymase were demonstrated enzymo-histochemically in 30 benign and 98 malignant fresh frozen breast lesions, which were graded and analysed morphometrically and statistically. The exclusive presence of tryptase and chymase in mast cells was confirmed in 5 cases by a sequential double-staining method. In benign lesions, the number of mast cells exhibiting tryptase activity was similar to that of chymase-active mast cells. Malignant tumours, however, had 2 to 3 times more tryptase-containing than chymase-containing mast cells, while the number of mast cells with tryptase activity was significantly higher (p < 0.02) than in benign lesions. In malignant lesions, tryptase-containing mast cells were concentrated at the tumour edge, i.e., the "invasion zone," whereas chymase-containing mast cells were not increased in this area.

Adult↗
PubMed2004

Mast cell chymase is a possible mediator of neurogenic bladder fibrosis.

AIMS: Urinary bladders of patients with myelomeningocele, owing to spina bifida, are often functionally impaired, fibrotic organs. Common to this condition are repeated occurrences of bladder infection and inflammation. Since mast cells have been associated with a fibrogenic response in inflammatory conditions, we investigated the role of mast cell granule product, chymase, as a mediator of myleodysplastic bladder fibrosis. METHODS: Human control and myelodysplastic bladder tissues were stained with Unna's stain and chymase antibody to determine mast cell number and localization. Cell specific localization of collagen mRNAs was determined by in situ hybridization (ISH). In vitro, normal human bladder fibroblasts were treated with recombinant chymase, heparin and inhibitors, and collagen subtype concentration was determined by enzyme linked immunosorbent assay (ELISA). RESULTS: Myelodysplastic bladders were characterized by increased mast cells in the detrusor muscle layer compared to control bladders, as well as mast cell degranulation and increased connective tissue deposition. Both types I and III collagen mRNA localized to fibroblasts surrounding detrusor muscle fascicles, whereas only collagen III mRNA localized to cells within connective tissue infiltrated muscle bundles in myelomeningocele bladder tissue. Chymase treatment of bladder fibroblasts, in vitro, was dose-dependent and resulted in significant increases in both types I and III collagen. Heparin did not alter collagen protein expression, whereas heparin-chymase combination modulated type III collagen expression. Serine protease inhibitor, phenylmethylsulfonlyfluoride, did not inhibit collagen synthesis, whereas denatured chymase resulted in decreased collagenous protein levels. CONCLUSIONS: Bladder fibrosis may be mediated by mast cell chymase stimulation of collagen synthesis.

Adolescent↗
PubMed2002

Mast cell chymase regulates dermal mast cell number in mice.

Chymase inhibitor reduced the increase in the number of dermal mast cells in 2,4-dinitrofluorobenzene-induced dermatitis in a dose-dependent manner. Intradermal injection of human chymase to mouse ear significantly increased histamine content, the marker for mast cell number in the skin. These results suggest that chymase released by mast cells may participate in local mast cell accumulation in a positive feedback fashion. Immunohistochemical analysis revealed that the intradermal injection of chymase reduces expression of stem cell factor (SCF) on surface of the skin keratinocytes. In addition, incubation of human keratinocytes with chymase in vitro resulted in release of SCF into the culture medium. Since soluble SCF is thought to regulate mast cell number, the chymase-induced mast cell accumulation may occur via the ability of chymase to process membrane-bound SCF on the epidermal keratinocytes.

Animals↗
PubMed1998

Skin mast cell promotion of matrix remodeling in burn wound healing in mice: relevance of chymase.

Inflammation, granulation, and collagen accumulation, which are observed in the wound healing process, occasionally lead to hypertrophic scarring. Several in vitro reports have suggested that skin mast cells (MCs) and their major protease, chymase, participate in the healing process as well as in fibrotic skin diseases. The present study examined the potential involvement of MCs and MC chymase in the healing of burns in mouse dorsal skin. The size of the burn wounds, density of the capillaries, collagen accumulation, MC number, and chymase activity were measured before and 1, 3, 7, and 14 days after burning. The healing process corresponded strongly with MC density and chymase activity in both acute and subacute phases. The maximum decrease in MC number and chymase activity occurred on day 3 when tissue loss due to necrosis was maximal. From day 7 to 14, the burn wounds retracted rapidly accompanied by increases in capillaries and collagen fibers, in correspondence with fast increments in MC numbers and chymase activity at the wound edges. The present results combined with previous in vitro results strongly support the contention that skin MC chymase plays a role in the normal wound healing process, and presumably in dermal fibrotic disorders.

Animals↗
PubMed2006

Mast cell chymase induces expression of chemokines for neutrophils in eosinophilic EoL-1 cells and mouse peritonitis eosinophils.

Human chymase induced release of interleukin-8 (IL-8) in human EoL-1 cells that had been differentiated into eosinophil-like cells with butyric acid. The chymase-induced IL-8 production was specific in that other cytokines/chemokines examined were not induced. Human chymase also increased mRNA for IL-8 in the differentiated EoL-1 cells, showing involvement of mRNA synthesis. The chymase-induced IL-8 release was inhibited by pertussis toxin as well as U0126 (an inhibitor for extracellular signal-regulated kinase pathway) and SB203580 (p38 inhibitor), suggesting that the chymase-induced IL-8 production is mediated by G protein-coupled receptor and mitogen-activated protein kinases. Mouse mast cell protease-4 (mMCP-4), a mouse chymase, induced macrophage-inflammatory protein-2 (MIP-2), a mouse homologue for IL-8, in mouse eosinophils in vitro. Intradermal injection of mMCP-4 not only induced skin edema but increased MIP-2 content and neutrophil number at the injection site. Taken together, our findings demonstrate that mast cell chymase may contribute to the interaction between eosinophils and neutrophils by inducing IL-8/MIP-2 in eosinophils at allergic inflamed sites.

Animals↗
PubMed2006

Mast cell chymase induces smooth muscle cell apoptosis by disrupting NF-kappaB-mediated survival signaling.

Chymase released from activated mast cells induces apoptosis of vascular smooth muscle cells (SMCs) in vitro by degrading the pericellular matrix component fibronectin, so causing disruption of focal adhesion complexes and Akt dephosphorylation, which are necessary for cell adhesion and survival. However, the molecular mechanisms of chymase-mediated apoptosis downstream of Akt have remained elusive. Here, we show by means of RT-PCR, Western blotting, EMSA, immunocytochemistry and confocal microscopy, that chymase induces SMC apoptosis by disrupting NF-kappaB-mediated survival signaling. Following chymase treatment, the translocation of active NF-kappaB/p65 to the nucleus was partly abolished and the amount of nuclear p65 was reduced. Pretreatment of SMCs with chymase also inhibited LPS- and IL-1beta-induced nuclear translocation of p65. The chymase-induced degradation of p65 was mediated by active caspases. Loss of NF-kappaB-mediated transactivation resulted in downregulation of bcl-2 mRNA and protein expression, leading to mitochondrial swelling and release of cytochrome c. The apoptotic process involved activation of both caspase 9 and caspase 8. The results reveal that, by disrupting the NF-kappaB-mediated survival-signaling pathway, activated chymase-secreting mast cells can mediate apoptosis of cultured arterial SMCs. Since activated mast cells colocalize with apoptotic SMCs in vulnerable areas of human atherosclerotic plaques, they may participate in the weakening and rupture of atherosclerotic plaques.

Active Transport, Cell Nucleus↗