Mast Cell Chymase Research Articles

TRPV4 Mediates Mast Cell Activation in Cathelicidin-Induced Rosacea Inflammation

TRPV4 Mediates Mast Cell Activation in Cathelicidin-Induced Rosacea Inflammation

The effect of dietary curcumin on hepatic chymase activity and serum fetuin-A levels in rats fed on a high-fat diet

The effects of curcumin on mast cell chymase activity in fatty liver and serum fetuin-A levels in rats fed a high-fat diet (HFD) were investigated. Male Sprague-Dawley rats received HFD (60% of total calories from fat) and 1 g curcumin/kg HFD for 16 weeks. Hepatic chymase activity was determined using spectrophotometric analysis while liver lipid levels were measured using colorimetric methods and serum fetuin-A, insulin, leptin, and adiponectin levels were detected using commercial enzyme-linked immunosorbent assay kits. Hepatic fat accumulation and fibrotic changes were ameliorated with curcumin treatment. Curcumin significantly reduced hepatic lipids, chymase activity, and serum fetuin-A levels. Decreased serum leptin and augmented adiponectin levels were also observed. These findings suggest that curcumin attenuated hepatic fat accumulation and regulated adipokine levels. The reduction of liver chymase activity and serum fetuin-A levels may also contribute to the beneficial effects of curcumin in fatty liver disease induced inflammatory status. Practical applications Curcumin (diferuloylmethane), which is extracted from the dried root of the rhizome Curcuma longa, is a popular dietary spice (turmeric) in Asia and used in curry. Turmeric is widely used as food component, flavoring agent, and colorant. This research revealed that dietary curcumin treatment reduces hepatic fat accumulation, ameliorates liver damage, and inflammation related to fat storage. Therefore, curcumin may be a potential protective agent in the prevention of fatty liver disease and the anti-inflammatory capacity of curcumin may reveal a beneficial application in medicine and also food technology.

Pso p27, a SERPINB3/B4-derived protein, is most likely a common autoantigen in chronic inflammatory diseases

Autoimmune diseases are characterized by chronic inflammatory reactions localized to an organ or organ-system. They are caused by loss of immunologic tolerance toward self-antigens, causing formation of autoantibodies that mistakenly attack their own body. Psoriasis is a chronic inflammatory autoimmune skin disease in which the underlying molecular mechanisms remain elusive. In this review, we present evidence accumulated through more than three decades that the serpin-derived protein Pso p27 is an autoantigen in psoriasis and probably also in other chronic inflammatory diseases.Pso p27 is derived from the serpin molecules SERPINB3 and SERPINB4 through non-canonical cleavage by mast cell chymase. In psoriasis, it is exclusively found in skin lesions and not in uninvolved skin. The serpins are cleaved into three fragments that remain associated as a Pso p27 complex with novel immunogenic properties and increased tendency to form large aggregates compared to native SERPINB3/B4. The amount of Pso p27 is directly correlated to disease activity, and through formation of complement activating immune-complexes, Pso p27 contribute to the inflammation in the skin lesions. SERPINB3/B4 are expressed in skin fibroblasts and keratinocytes, but normally absent in mast cells. Overexpression of the serpins may be induced by inflammation and hypoxia, resulting in mast cell uptake via yet unknown mechanisms. Here the generation and subsequent release of Pso p27 aggregates may promote an inflammatory loop that contributes to the chronicity of psoriasis and other autoimmune diseases.

Early Activation of Th2/Th22 Inflammatory and Pruritogenic Pathways in Acute Canine Atopic Dermatitis Skin Lesions

Determining inflammation and itch pathway activation in patients with atopic dermatitis (AD) is fraught with the inability to precisely assess the age of skin lesions, thus affecting the analysis of time-dependent mediators. To characterize inflammatory events occurring during early experimental acute AD lesions, biopsy samples were collected 6, 24, and 48 hours after epicutaneous application of Dermatophagoides farinae house dust mites to sensitized atopic dogs. The skin transcriptome was assessed using a dog-specific microarray and quantitative PCR. Acute canine AD skin lesions had a significant up-regulation of genes encoding T helper (Th) 2 (e.g., IL4, IL5, IL13, IL31, and IL33), Th9 (IL9), and Th22 (IL22) cytokines as well as Th2-promoting chemokines such as CCL5 and CCL17. Proinflammatory (e.g., IL6, LTB, and IL18) cytokines were also up-regulated. Other known pruritogenic pathways were also activated: there was significant up-regulation of genes encoding proteases cathepsin S (CTSS), mast cell chymase (CMA1), tryptase (TPS1) and mastin, neuromedin-B (NMB), nerve growth factor (NGF), and leukotriene-synthesis enzymes (ALOX5, ALOX5AP, and LTA4H). Experimental acute canine house dust mite-induced AD lesions exhibit an activation of innate and adaptive immune responses and pruritogenic pathways similar to those seen in humans with acute AD, thereby validating this model to test innovative therapeutics modalities for this disease.

Functional testing of topical skin formulations using an optimised ex vivo skin organ culture model.

A number of equivalent-skin models are available for investigation of the ex vivo effect of topical application of drugs and cosmaceuticals onto skin, however many have their drawbacks. With the March 2013 ban on animal models for cosmetic testing of products or ingredients for sale in the EU, their utility for testing toxicity and effect on skin becomes more relevant. The aim of this study was to demonstrate proof of principle that altered expression of key gene and protein markers could be quantified in an optimised whole tissue biopsy culture model. Topical formulations containing green tea catechins (GTC) were investigated in a skin biopsy culture model (n = 11). Punch biopsies were harvested at 3, 7 and 10 days, and analysed using qRT-PCR, histology and HPLC to determine gene and protein expression, and transdermal delivery of compounds of interest. Reduced gene expression of α-SMA, fibronectin, mast cell tryptase, mast cell chymase, TGF-β1, CTGF and PAI-1 was observed after 7 and 10 days compared with treated controls (p < 0.05). Histological analysis indicated a reduction in mast cell tryptase and chymase positive cell numbers in treated biopsies compared with untreated controls at day 7 and day 10 (p < 0.05). Determination of transdermal uptake indicated that GTCs were detected in the biopsies. This model could be adapted to study a range of different topical formulations in both normal and diseased skin, negating the requirement for animal models in this context, prior to study in a clinical trial environment.

Imatinib treatment attenuates growth and inflammation of angiotensin II induced abdominal aortic aneurysm

BackgroundAbdominal aortic aneurysm (AAA) is characterized by vascular remodeling with increased infiltration of inflammatory cells and apoptosis/modulation of vascular smooth muscle cells (SMCs). Imatinib is a selective inhibitor of several tyrosine kinases, including PDGF receptors, Abl, and c-kit. The objective of this study was to characterize the potential protective role of imatinib on AAA development and the molecular mechanisms involved. MethodsMale ApoE−/− mice were infused with angiotensin (Ang) II (1000 ng/kg/min) for 4 weeks to induce AAA or saline as controls. Daily treatment with 10 mg/kg imatinib, or tap water as control, was provided via gavage for 4 weeks. ResultsTreatment with imatinib was found to decrease the aortic diameter and vessel wall thickness, mediated by multiple effects. Imatinib treatment in AngII infused mice resulted in a reduced cellular infiltration of CD3ε positive T lymphocytes by 86% and reduced gene expression of mast cell chymase by 50% compared with AngII infused mice lacking imatinib. Gene expression analysis of SMC marker SM22α demonstrated an increase by 48% together with a more intact medial layer after treatment with imatinib as evaluated with SM22α immunostaining. ConclusionPresent findings highlight the importance of tyrosine kinase pathways in the development of AAA. Our results show, that imatinib treatment inhibits essential mast cell, T lymphocyte and SMC mediated processes in experimental AAA. Thus, our results support the idea that tyrosine kinase inhibitors may be useful in the treatment of pathological vascular inflammation and remodeling in conditions like AAA.

Carboxyl-Terminal Cleavage of Apolipoprotein A-I by Human Mast Cell Chymase Impairs Its Anti-Inflammatory Properties.

Apolipoprotein A-I (apoA-I) has been shown to possess several atheroprotective functions, including inhibition of inflammation. Protease-secreting activated mast cells reside in human atherosclerotic lesions. Here we investigated the effects of the neutral proteases released by activated mast cells on the anti-inflammatory properties of apoA-I. Activation of human mast cells triggered the release of granule-associated proteases chymase, tryptase, cathepsin G, carboxypeptidase A, and granzyme B. Among them, chymase cleaved apoA-I with the greatest efficiency and generated C-terminally truncated apoA-I, which failed to bind with high affinity to human coronary artery endothelial cells. In tumor necrosis factor-α-activated human coronary artery endothelial cells, the chymase-cleaved apoA-I was unable to suppress nuclear factor-κB-dependent upregulation of vascular cell adhesion molecule-1 (VCAM-1) and to block THP-1 cells from adhering to and transmigrating across the human coronary artery endothelial cells. Chymase-cleaved apoA-I also had an impaired ability to downregulate the expression of tumor necrosis factor-α, interleukin-1β, interleukin-6, and interleukin-8 in lipopolysaccharide-activated GM-CSF (granulocyte-macrophage colony-stimulating factor)- and M-CSF (macrophage colony-stimulating factor)-differentiated human macrophage foam cells and to inhibit reactive oxygen species formation in PMA (phorbol 12-myristate 13-acetate)-activated human neutrophils. Importantly, chymase-cleaved apoA-I showed reduced ability to inhibit lipopolysaccharide-induced inflammation in vivo in mice. Treatment with chymase blocked the ability of the apoA-I mimetic peptide L-4F, but not of the protease-resistant D-4F, to inhibit proinflammatory gene expression in activated human coronary artery endothelial cells and macrophage foam cells and to prevent reactive oxygen species formation in activated neutrophils. The findings identify C-terminal cleavage of apoA-I by human mast cell chymase as a novel mechanism leading to loss of its anti-inflammatory functions. When targeting inflamed protease-rich atherosclerotic lesions with apoA-I, infusions of protease-resistant apoA-I might be the appropriate approach.

Mast Cells: Key Players in the Shadow in Oral Inflammation and in Squamous Cell Carcinoma of the Oral Cavity

Although mast cells (MCs) have been discovered over 130 years ago, their function was almost exclusively linked to allergic affections. At the time being, it is well known that MCs possess a great variety of roles, in both physiologic and pathologic conditions. In the oral tissues, MCs release different proinflammatory cytokines, tumor necrosis factor alpha (TNF-α), that promote leukocyte infiltration in various inflammatory states of the oral cavity. These cells play a key role in the inflammatory process and, as a consequence, their number changes in different pathologic conditions of the oral cavity, like gingivitis, periodontitis, and so on. MCs also represent a rich source of proteases, especially of mast cell tryptase and chymase, which directly degrade the extracellular matrix through their proteolytic activity and thus indirectly stimulate angiogenesis and facilitate invasion and metastasis. It may be stated that mast cells could have an impact on primary tumor development, progression, and metastases in oral squamous cell carcinoma. By understanding the role of mast cells in the pathogenesis of different inflammatory and tumor diseases of the oral cavity, these cells may become therapeutic targets that could possibly improve the prognosis and survival of these patients.

Ultramicronized palmitoylethanolamide reduces viscerovisceral hyperalgesia in a rat model of endometriosis plus ureteral calculosis: role of mast cells.

The effects of ultramicronized palmitoylethanolamide were evaluated on pain behaviours and markers of mast cell (MC) activity in a rat model of endometriosis plus ureteral calculosis (ENDO+STONE)-induced viscerovisceral hyperalgesia (VVH). Female Sprague-Dawley rats that underwent surgical induction of endometriosis were randomly assigned to receive active (ultramicronized palmitoylethanolamide 10 mg·kg(-1)·d(-1), orally) or placebo treatment for 25 days. At day 21, they underwent ureteral stone formation and were video-recorded till day 25 to evaluate ureteral and uterine pain behaviours. At autopsy (day 25), ureteral condition and number and diameter of endometrial cysts were evaluated. The following were then measured: number and percentage of degranulating MCs, number of vessels, chymase, nerve growth factor (NGF), vascular endothelial growth factor (VEGF), and Flk-1 (VEGF receptor) in cysts, and NGF in dorsal root ganglia (DRG). Ultramicronized palmitoylethanolamide-treated vs placebo-treated rats showed significantly lower number, duration and complexity of ureteral crises, shorter duration of uterine pain, and smaller cyst diameter (0.0001 < P < 0.004); a significantly higher percentage of expelled stones (P < 0.0001); significantly lower MC number (P < 0.01), vessel number (P < 0.01), chymase (P < 0.05), NGF (P < 0.05), VEGF (P < 0.01), and Flk-1 (P < 0.01) expression in cysts and NGF expression in DRG (P < 0.01). In all animals, the global duration of ureteral crises correlated linearly and directly with cyst diameter, MC number and chymase in cysts, and NGF in cysts and DRG (0.02 < P < 0.0002). Ultramicronized palmitoylethanolamide significantly reduces VVH from ENDO+STONE, probably by modulating MC expression/activity in cysts, thus reducing central sensitization due to noxious signals from endometriotic lesions. The results suggest potential utility of the compound for VVH in clinics.

Mast cell chymase in experimentally induced psoriasis.

Mast cell chymase can have a pro-inflammatory or an immunosuppressive function in psoriasis, but the outcome may depend on the level of chymase activity. Therefore, mast cells showing chymase activity (Chyact ) and immunoreactivity (Chyprot ) were studied during the Köbner reaction (0 days, 2 h, 1 day, 3 days and 7 days) of psoriasis induced by the tape-stripping technique. Also, the effect of recombinant human chymase (rh-chymase) or human LAD2 mast cells (LAD2) on the (3) H-thymidine uptake of psoriatic peripheral blood mononuclear cells (PBMC) or total T cells was studied. The Chyact /Chyprot ratio tended to be higher in all time-point biopsies in the Köbner-negative (n = 10) than -positive (n = 8) group (P = 0.073), although chymase activity decreased significantly at 2 h to 1 day only in the Köbner-negative group. rh-chymase (0.05-0.5 μg/mL) stimulated to a varying extent PBMC in eight out of nine cultures, but in all cultures 5 μg/mL rh-chymase turned the stimulation towards inhibition. The effect of rh-chymase on T cells varied from stimulation to inhibition, but in 11 of 15 cultures rh-chymase, at least at 5 μg/mL, produced a change to inhibition. In co-cultures, LAD2 inhibited PBMC in the absence of soybean trypsin inhibitor (SBTI). In the presence of SBTI, LAD2 stimulated PBMC in the majority of seven cultures. In summary, the psoriatic immunopathogenesis may be promoted at low, but controlled at high, activity status of chymase.

Sputum mast cell subtypes relate to eosinophilia and corticosteroid response in asthma

Mast cells are a resident inflammatory cell of the airways, involved in both the innate and adaptive immune response. The relationship between mast cells and inflammatory phenotypes and treatment response of asthma is not clear.Clinical characteristics of subjects with stable asthma (n=55), inflammatory cell counts and gene expression microarrays in induced sputum were analysed. Sputum mast cell subtypes were determined by molecular phenotyping based on expression of mast cell biomarkers (tryptase (TPSAB1), chymase (CMA1) and carboxypeptidase A3 (CPA3)). Effects of mast cell subtypes on steroid response were observed in a prospective cohort study (n=50).MCT(n=18) and MCT/CPA3(mRNA expression of TPSAB1 and CPA3; n=29) subtypes were identified, as well as a group without mast cell gene expression (n=8). The MCT/CPA3 subtype had elevated exhaled nitric oxide fraction, sputum eosinophils, bronchial sensitivity and reactivity, and poorer asthma control. This was accompanied by upregulation of 13 genes. Multivariable logistic regression identified CPA3(OR 1.21, p=0.004) rather than TPSAB1(OR 0.92, p=0.502) as a determinant of eosinophilic asthma. The MCT/CPA3 subtype had a better clinical response and reduced signature gene expression with corticosteroid treatment.Sputum mast cell subtypes of asthma can be defined by a molecular phenotyping approach. The MCT/CPA3 subtype demonstrated increased bronchial sensitivity and reactivity, and signature gene expression, which was associated with airway eosinophilia and greater corticosteroid responsiveness.

Low numbers of tryptase+ and chymase+ mast cells associated with reduced survival and advanced tumor stage in melanoma.

The role of mast cells in cutaneous melanoma remains unclear. Tryptase and chymase are serine proteinases and major proteins in mast cell secretory granules. Therefore, this study aimed to investigate the presence of tryptase and chymase mast cells in benign and malignant cutaneous melanocytic lesions and in lymph node metastases of melanomas. The presence of positively stained mast cells was correlated with clinicopathological characteristics in invasive melanomas. Paraffin-embedded sections of 28 benign (13 intradermal, 10 compound, and five junctional nevi) and 26 dysplastic nevi, 15 in-situ melanomas, 36 superficially (pT1, Breslow's thickness<1 mm), and 49 deeply (pT4, Breslow's thickness>4 mm) invasive melanomas and 30 lymph node metastases were immunohistochemically stained for mast cell tryptase and chymase, and immunopositive cells were counted using the hotspot counting method. The mean count of tryptase and chymase mast cells was lower in invasive melanomas compared with in-situ melanomas and dysplastic and benign nevi. In deeply invasive melanomas, the difference was statistically significant compared with dysplastic nevi (P=0.003 for tryptase and P=0.009 for chymase) and in-situ melanomas (0.043 for tryptase). Low numbers of tryptase mast cells were associated with poor overall survival (P=0.031) in deeply invasive melanomas and with a more advanced stage (T1b, P=0.008) in superficially invasive melanomas. Low numbers of chymase mast cells were associated with microsatellites (P=0.017) in deeply invasive melanomas. The results suggest that these serine proteinases of mast cells may be protective in the pathogenesis of melanoma.

The autocrine role of tryptase in pressure overload-induced mast cell activation, chymase release and cardiac fibrosis

BackgroundCardiac mast cell (MC) proteases, chymase and tryptase, increase proliferation and collagen synthesis in cultured cardiac fibroblasts. However, the question as to why preventing individually the actions of either protease prevents fibrosis when both are released upon MC activation remains unanswered. Since tryptase has the ability to activate MCs in noncardiac tissues via the protease-activated receptor-2 (PAR-2), there is the possibility that its, in vivo, fibrotic role is due to its ability to induce MC degranulation thereby amplifying the release of chymase. MethodsThis study sought to delineate the interactions between tryptase and chymase in myocardial remodeling secondary to transverse aortic constriction (TAC) for 5weeks in male Sprague–Dawley rats untreated or treated with either the tryptase inhibitor, nafamostat mesilate or MC membrane stabilizing drug, nedocromil (n=6/group). In addition, ventricular slices from 6 rat hearts were incubated with tryptase, tryptase plus nafamostat mesilate or the chymase inhibitor chymostatin for 24h. Results and conclusionThe results indicate the presence of PAR-2 on MCs and that tryptase inhibition and nedocromil prevented TAC-induced fibrosis and increases in MC density, activation, and chymase release. Tryptase also significantly increased chymase concentration in ventricular slice culture media, which was prevented by the tryptase inhibitor. Hydroxyproline concentration in culture media was significantly increased with tryptase incubation as compared to the control group and the tryptase group incubated with nafamostat mesilate or chymostatin. We conclude that tryptase contributes to TAC-induced cardiac fibrosis primarily via activation of MCs and the amplified release of chymase.

Epidermal Expression of Filaggrin/Profilaggrin Is Decreased in Atopic Dermatitis: Reverse Association With Mast Cell Tryptase and IL-6 but Not With Clinical Severity.

A decrease in filaggrin expression contributes to the pathogenesis of atopic dermatitis (AD) and can be modified by inflammatory factors. The aim of this study was to determine the correlation of (pro)filaggrin (filaggrin and profilaggrin) expression with clinical severity in AD and with mast cell (MC) tryptase, chymase, and IL-6. Punch biopsies were collected from 17 patients with moderate-to-severe AD and from 10 psoriatic patients. Atopic dermatitis severity was measured using different clinical parameters. (Pro)filaggrin, MC tryptase, chymase, and IL-6 were stained using immunohistochemical, enzymehistochemical, and sequential double-staining methods. (Pro)filaggrin expression was lower in the lesional than in the nonlesional granular layer in AD and was correlated negatively with itch severity but not with other severity parameters. (Pro)filaggrin expression was also decreased in the psoriatic lesions. In AD, (pro)filaggrin expression correlated negatively with the number of tryptase MCs in the nonlesional granular layer and with IL-6 MCs in both the nonlesional and lesional granular layers. (Pro)filaggrin expression is decreased in AD and is reversely associated with MC tryptase and IL-6. However, it does not associate with disease severity, and it was also decreased in psoriasis.

Mast cell tryptase and chymase in the progress of cutaneous vasculitis.

In animal models of vasculitis, mast cells are essential in the pathogenesis, but their involvement in human skin vasculitis is obscure. Because tryptase and chymase are potent serine proteinases in the secretory granules of mast cells, the purpose was to examine the number of mast cells expressing tryptase and chymase during the progress of cutaneous vasculitis. These numbers were correlated with the appearance of immunoreactants (C3c, fibrin, IgM, IgA and IgG) in vessel walls. For this, skin biopsies were taken from the healthy-looking skin, initial petechial lesion (IP), and palpable purpura (PP) of the leg of patients with leukocytoclastic vasculitis (n=10). The frozen biopsies were analysed using enzyme- and immunohistochemistry and direct immunofluorescence (IF) staining. The results show that there are no marked changes in the numbers of mast cells expressing chymase or tryptase proteins. Instead, chymase enzyme activity decreased, but the score of α1-antichymotrypsin staining increased, during the progress of vasculitis. The IF positivity of fibrin correlated positively with chymase activity (p=0.01) and the ratio of chymase activity to tryptase protein in IP (p=0.03), as well as with mast cells showing tryptase (p=0.03) and chymase (p=0.01) proteins in PP. The IF positivity of C3c correlated with the ratio of chymase activity to tryptase protein in IP (p=0.01). In conclusion, chymase is partially inactivated in vasculitis possibly due to α1-antichymotrypsin. Several positive correlations between chymase and fibrin and/or C3c in IP or PP suggest that this enzyme is involved in the deposition of immunoreactants in the vessel wall.

Mast cells produce factor XIIIA and mast cell chymase reduces factor XIIIa activity during sepsis through proteolytic degradation (HYP5P.318)

Abstract Previously, we reported that mast cell (MC) chymase, and its mouse homologue, mast cell protease 4 (MCPT4), protect from septic morbidity and mortality by limiting inflammation via TNF degradation. Here, we identified another potential MCPT4 substrate; factor XIIIa (FXIIIa; the essential fibrin-crosslinking/clot stabilizing FXIII catalytic subunit). We found that extracellular FXIIIA (the pro-form of FXIIIa) protein levels were significantly increased in IgE-stimulated MCPT4-/- peritoneal MC (PMC) supernatants compared with WT PMCs (FXIIIA mRNA levels were unchanged). These data suggest that MCs both secrete FXIIIA and are capable of degrading it via MCPT4. Based on these results, and on published data demonstrating that FXIIIa activity correlated with sepsis severity, we hypothesize that chymase/MCPT4 mediated cleavage of FXIIIa participates in the previously-noted protective effect of chymase during sepsis. First, we verified that chymase is capable of degrading FXIIIA and reducing FXIIIa activity in-vitro and in-vivo. Additionally, we identified 6 chymase-driven primary cleavage sites in human FXIIIA. Finally, we observed that following experimentally-induced sepsis, plasma and peritoneal FXIIIA levels as well as plasma FXIIIa activity were significantly diminished in WT compared with MCPT4-/- mice. In conclusion, these data demonstrate that MCs produce FXIIIA and that MCPT4 reduces FXIIIa activity during experimental sepsis.

Expression and activity levels of chymase in mast cells of burn wound tissues increase during the healing process in a hamster model

The present study aimed to investigate the changes in the expression levels and activity of mast cell chymase in the process of burn wound healing in a hamster model of deep second-degree burn. The hamster model was established by exposing a ~3 cm diameter area of bare skin to hot water (75°C) for 0, 6, 8, 10 or 12 sec. Tissue specimens were collected 24 h after burning and histological analysis revealed that hot water contact for 12 sec was required to produce a deep second-degree burn. Quantitative polymerase chain reaction and a radioimmunoassay were used to the determine changes in chymase mRNA expression levels and activity. The mRNA expression levels and activity of chymase were increased in the burn wound tissues when compared with the normal skin. However, no statistically significant differences were observed in mast cell chymase activity amongst the various post-burn stages. Chymase mRNA expression levels peaked at day 1 post-burn, subsequently decreasing at days 3 and 7 post-burn and finally increasing again at day 14 post-burn. In summary, a hamster model of deep second-degree burn can be created by bringing the skin into contact with water at 75°C for 12 sec. Furthermore, the mRNA expression levels and activity of chymase in the burn wound tissues increased when compared with those in normal skin tissues.

Effects of Mast Cell Chymase on Cardiac Fibroblast Function

Mast cells are bone marrow‐derived effector cells that have been described historically for their role in mediating allergic reactions. More recently, they have received considerable attention due to their involvement in innate defense, tissue remodeling and other processes. Several studies have illustrated roles for mast cells in cardiovascular diseases including ventricular remodeling associated with myocardial infarction, hypertension and volume overload. Mast cells produce a variety of secretory components including cytokines, proteases, growth factors and fatty acid metabolites. The compliment of components synthesized by a given mast cell appears to be dictated by the tissue microenvironment giving these cells diverse roles in tissue homeostasis and disease. Mast cell proteases including tryptase and chymase are some of the most abundant proteins produced by mast cells. Several studies have illustrated that mast cell chymase plays a significant role in the formation of angiotensin II in the heart and thus this protease may indirectly promote a fibrotic response via angiotensin II‐induced TGF‐beta production. However, the direct effect of this protease on cardiac fibroblast function has not been evaluated. The present studies were performed to elucidate the effects of chymase on isolated cardiac fibroblasts. Fibroblasts were treated for 24 hours with varying doses of chymase (0 to 1000 ng/ml) and the effects on proliferation, collagen scaffold remodeling, myofibroblast formation and expression of extracellular matrix components assayed. Treatment of cardiac fibroblasts with chymase significantly reduced the conversion of quiescent fibroblasts to a myofibroblast phenotype. Chymase also impaired contraction of three‐dimensional collagen gels and the expression of specific extracellular matrix components by fibroblasts. These studies illustrate that treatment of isolated cardiac fibroblasts with mast cell chymase elicits an anti‐fibrotic effect, which may contribute to the transition to heart failure in vivo.

GPR30 decreases cardiac chymase/angiotensin II by inhibiting local mast cell number

Chronic activation of the novel estrogen receptor GPR30 by its agonist G1 mitigates the adverse effects of estrogen (E2) loss on cardiac structure and function. Using the ovariectomized (OVX) mRen2.Lewis rat, an E2-sensitive model of diastolic dysfunction, we found that E2 status is inversely correlated with local cardiac angiotensin II (Ang II) levels, likely via Ang I/chymase-mediated production. Since chymase is released from cardiac mast cells during stress (e.g., volume/pressure overload, inflammation), we hypothesized that GPR30-related cardioprotection after E2 loss might occur through its opposing actions on cardiac mast cell proliferation and chymase production. Using real-time quantitative PCR, immunohistochemistry, and immunoblot analysis, we found mast cell number, chymase expression, and cardiac Ang II levels were significantly increased in the hearts of OVX-compared to ovary-intact mRen2.Lewis rats and the GPR30 agonist G1 (50 mg/kg/day, s.c.) administered for 2 weeks limited the adverse effects of estrogen loss. In vitro studies revealed that GPR30 receptors are expressed in the RBL-2H3 mast cell line and G1 inhibits serum-induced cell proliferation in a dose-dependent manner, as determined by cell counting, BrdU incorporation assay, and Ki-67 staining. Using specific antagonists to estrogen receptors, blockage of GPR30, but not ERα or ERβ, attenuated the inhibitory effects of estrogen on BrdU incorporation in RBL-2H3 cells. Further study of the mechanism underlying the effect on cell proliferation showed that G1 inhibits cyclin-dependent kinase 1 (CDK1) mRNA and protein expression in RBL-2H3 cells in a dose-dependent manner.

Chymase inhibitor-sensitive synthesis of endothelin-1 (1–31) by recombinant mouse mast cell protease 4 and human chymase

Important structural differences imply that human and mouse mast cell chymases may differ with respect to their enzymatic properties. We compared in this study the catalytic efficiencies of recombinant human chymase (rCMA1) and its functional murine homologue recombinant mouse mast cell protease-4 (rmMCP-4) toward a fluorogenic chymase substrate (Suc-Ala-Ala-Pro-Phe-7-amino-4-methylcoumarin (AMC) and by their ability to convert Big-endothelin (ET)-1 into ET-1 (1–31) using a LC/MS/MS system. Activities toward a fluorogenic substrate (Suc-Leu-Leu-Val-Tyr-AMC) and Big ET-1 were also measured in extracts from mouse peritoneal mast cells, LUVA human mast cell-like cells and human aortas. The specificity of these activities was assessed with the chymase inhibitor TY-51469 (2-[4-(5-fluoro-3-methylbenzo[b]thiophen-2-yl)sulfonamido-3-methanesulfonyl-phenyl]thiazole-4-carboxylic acid).For similar affinities, rmMCP-4 showed a higher activity toward the fluorogenic substrate and a higher ability to process Big ET-1 as compared to recombinant CMA1 (chymase activity (kcat/KM in μM−1s−1): 2.29×10−4vs. 6.41×10−6; ET-1 (1–31) production: 2.19×10−3vs. 6.57×10−5), and both of these activities of mouse and human chymase were sensitive to TY-51469. Furthermore, extracts from mouse peritoneal mast cells, LUVA cells and human aorta homogenates contained processing activities toward the fluorogenic chymase substrate as well as Big ET-1, all of which were sensitive to TY-51469. Finally, the pressor responses to Big ET-1 but not to ET-1 were significantly reduced in conscious and free moving mMCP-4 KO mice when compared to wild type congeners.Our results suggest that both mouse and human chymases have potent ET-1 (1–31)-producing abilities, with the murine isoform being more efficient.