Total Collagenase Activity Research Articles

Insight into the spoilage heterogeneity of meat-borne bacteria isolates with high-producing collagenase

Chilled chicken is inevitably contaminated by microorganisms during slaughtering and processing, resulting in spoilage. Cutting parts of chilled chicken, especially wings, feet, and other skin-on products, are abundant in collagen, which may be the primary target for degradation by spoilage microorganisms. In this work, a total of 17 isolates of spoilage bacteria that could secrete both collagenase and lipase were determined by raw-chicken juice agar (RJA) method, and the results showed that 7 strains of Serratia, Aeromonas, and Pseudomonas could significantly decompose the collagen ingredients. The gelatin zymography showed that Serratia liquefaciens (F5) and Pseudomonas saponiphila (G7) had apparent degradation bands around 50 kDa, and Aeromonas veronii (G8) and Aeromonas salmonicida (H8) had a band around 65 and 95 kDa, respectively. The lipase and collagenase activities were detected isolate-by-isolate, with F5 showing the highest collagenase activity. For spoilage ability on meat in situ, F5 performed strongest in spoilage ability, indicated by the total viable counts, total volatile basic nitrogen content, sensory scores, lipase, and collagenase activity. This study provides a theoretical basis for spoilage heterogeneity of strains with high-producing collagenase in meat.

Influence of TEGDMA monomer on MMP-2, MMP-8, and MMP-9 production and collagenase activity in pulp cells

ObjectivesResin-based composites may leach monomers such as triethylene-glycol dimethacrylate (TEGDMA), which could contribute to intrapulpal inflammation. The aim of this investigation was to examine whether various concentrations of TEGDMA are able to influence dentally relevant Matrix metalloproteinase (MMP)-2, MMP-8, and MMP-9 production, total collagenase/gelatinase activity in pulp cells, and suggest possible signaling mechanisms.Materials and methodsPulp cells were cultured, followed by a 1-day exposure to sublethal TEGDMA concentrations (0.1, 0.2, and 0.75 mM). Total MMP activity was measured by an EnzCheck total collagenase/gelatinase assay, while the production of specific MMPs and the relative changes of phosphorylated, i.e., activated signaling protein levels of extracellular signal-regulated kinase (ERK)1/2, p38, c-Jun N-terminal kinase (JNK) were identified by western blot. Immunocytochemistry image data was also plotted and analyzed to see whether TEGDMA could possibly alter MMP production.ResultsAn increase in activated MMP-2, MMP-8, and MMP-9 production as well as total collagenase activity was seen after a 24-h exposure to the abovementioned TEGDMA concentrations. Increase was most substantial at 0.1 (P = 0.002) and 0.2 mM (P = 0.0381). Concurrent p-ERK, p-p38, and p-JNK elevations were also detected.ConclusionsResults suggest that monomers such as TEGDMA, leached from resin-based restorative materials, activate and induce the production of dentally relevant MMPs in pulp cells. Activation of ERK1/2, p38, or JNK and MMP increase may play a role in and/or can be part of a broader stress response.Clinical relevanceInduction of MMP production and activity may further be components in the mechanisms of intrapulpal monomer toxicity.

Corrigendum

CorrigendumCorrigendumPublished Online:15 Oct 2014https://doi.org/10.1152/ajpcell.zh0-7613-corr.2014Original articleMoreSectionsPDF (663 KB)Download PDF ToolsExport citationAdd to favoritesGet permissionsTrack citations ShareShare onFacebookTwitterLinkedInEmailWeChat Volume 298, March 1, 2010Volume 67, March 1, 2010Awad AE, Kandalam V, Chakrabarti S, Wang X, Penninger JM, Davidge ST, Oudit GY, Kassiri Z. Tumor necrosis factor induces matrix metalloproteinases in cardiomyocytes and cardiofibroblasts differentially via superoxide production in a PI3Kγ-dependent manner. Am J Physiol Cell Physiol 298: C679–C692, 2010. First published December 9, 2009; doi: 10.1152/ajpcell.00351.2009. In results, Figs. 3, 5, and 8 were incorrect. The zymogram gel loading controls presented in Figs. 3A and 5C have been replaced with Coomassie blue-stained gel loading controls from the same experiments to correct errors identified in the images originally presented. Figure 8A has been revised to declare that the lanes presented are not contiguous. Legends to the figures have been updated to address these changes. The corrections do not alter the conclusions of this study. The corrected figures are shown below. Fig. 3.TNF stimulates expression of matrix metalloproteinase (MMP)9, but not MMP2, in a superoxide-dependent fashion. A: gelatin zymography on cell homogenates and conditioned media from WT cardiomyocyte and cardiofibroblast control, rTNF-, or rTNF + apocynin-treated groups after 1 h (i) or 24 h (ii). Averaged band intensities for MMP9 are presented as bar graphs for control (white), rTNF (black), and rTNF + apocynin (horizontal hatch). Coomassie blue-stained parallel gels, using the same protein samples and dilution as in the zymograms, were used as the loading controls. B: MMP9 mRNA levels measured by Taqman RT-PCR in cardiomyocytes and cardiofibroblasts treated as in A. *P < 0.05 compared with all other groups. #P < 0.05 compared with rTNF-treated group. Ctrl, control; rTNF, recombinant TNF; APO, apocynin; MW, molecular weight. RE, relative expression.Download figureDownload PowerPointFig. 5.TNF-mediated superoxide production and MMP activation is ablated in phosphatidylinositol 3-kinase (PI3K)γ−/− neonatal mouse cardiomyocytes and cardiofibroblasts. A: lucigenin chemiluminescence assay in WT and PI3Kγ−/− cultures before and after 1 h or 24 h of rTNF treatment. Values were normalized to the control within each group. B: representative images of DHE-stained PI3Kγ−/− cultures and the corresponding counterstained nuclei. Bar graphs show averaged superoxide levels for each group (n = 12 fields/group). C: gelatin zymography in PI3Kγ−/− cardiomyocytes (i) and cardiofibroblasts (ii) after 24 h of rTNF treatment. Coomassie blue-stained parallel gels, using the same protein samples and dilution as in the zymograms, were used as the loading controls. D: mRNA expression of MMP9 in WT and PI3Kγ−/− cardiomyocytes and cardiofibroblasts. Values were normalized to the control group within each genotype. E: total collagenase activity in PI3Kγ−/− compared with WT cultures. *P < 0.05 compared with the control group within each genotype. #P < 0.05 compared with WT-rTNF group. MFI, mean fluorescent intensity. RE, relative expression.Download figureDownload PowerPointFig. 8.PI3K activity is suppressed in TNF−/− mice following pressure overload. A: representative Western blots showing PI3Kγ (i) and protein loading controls α-tubulin (ii) and Coomassie blue staining (iii) in WT and TNF−/− mice after sham operation or 10 wk post-TAC. All samples were run on the same gel but lanes were removed for final presentation as indicated by the space inserted at the splice sites. B: averaged PI3Kγ protein levels normalized to corresponding α-tubulin in WT and TNF−/− mice post-TAC (n = 4). C: PI3K activity in WT and TNF−/− mice following TAC (n = 6). *P < 0.05 compared with corresponding sham. #P < 0.05 compared with WT-TAC. AU, arbitrary units.Download figureDownload PowerPointThis article has no references to display. Download PDF Previous Back to Top FiguresReferencesRelatedInformationRelated ArticlesTumor necrosis factor induces matrix metalloproteinases in cardiomyocytes and cardiofibroblasts differentially via superoxide production in a PI3Kγ-dependent manner 01 Mar 2010American Journal of Physiology-Cell Physiology More from this issue > Volume 307Issue 8October 2014Pages C760-C762 Copyright & PermissionsCopyright © 2014 the American Physiological Societyhttps://doi.org/10.1152/ajpcell.zh0-7613-corr.2014History Published online 15 October 2014 Published in print 15 October 2014 Metrics

IKKα/CHUK Regulates Extracellular Matrix Remodeling Independent of Its Kinase Activity to Facilitate Articular Chondrocyte Differentiation

BackgroundThe non-canonical NF-κB activating kinase IKKα, encoded by CHUK (conserved-helix-loop-helix-ubiquitous-kinase), has been reported to modulate pro- or anti- inflammatory responses, cellular survival and cellular differentiation. Here, we have investigated the mechanism of action of IKKα as a novel effector of human and murine chondrocyte extracellular matrix (ECM) homeostasis and differentiation towards hypertrophy.Methodology/Principal FindingsIKKα expression was ablated in primary human osteoarthritic (OA) chondrocytes and in immature murine articular chondrocytes (iMACs) derived from IKKαf/f:CreERT2 mice by retroviral-mediated stable shRNA transduction and Cre recombinase-dependent Lox P site recombination, respectively. MMP-10 was identified as a major target of IKKα in chondrocytes by mRNA profiling, quantitative RT-PCR analysis, immunohistochemistry and immunoblotting. ECM integrity, as assessed by type II collagen (COL2) deposition and the lack of MMP-dependent COL2 degradation products, was enhanced by IKKα ablation in mice. MMP-13 and total collagenase activities were significantly reduced, while TIMP-3 (tissue inhibitor of metalloproteinase-3) protein levels were enhanced in IKKα-deficient chondrocytes. IKKα deficiency suppressed chondrocyte differentiation, as shown by the quantitative inhibition of.Alizarin red staining and the reduced expression of multiple chondrocyte differentiation effectors, including Runx2, Col10a1 and Vegfa,. Importantly, the differentiation of IKKα-deficient chondrocytes was rescued by a kinase-dead IKKα protein mutant.Conclusions/SignificanceIKKα acts independent of its kinase activity to help drive chondrocyte differentiation towards a hypertrophic-like state. IKKα positively modulates ECM remodeling via multiple downstream targets (including MMP-10 and TIMP-3 at the mRNA and post-transcriptional levels, respectively) to maintain maximal MMP-13 activity, which is required for ECM remodeling leading to chondrocyte differentiation. Chondrocytes are the unique cell component in articular cartilage, which are quiescent and maintain ECM integrity during tissue homeostasis. In OA, chondrocytes reacquire the capacity to proliferate and differentiate and their activation results in pronounced cartilage degeneration. Τηυσ, our findings are also of potential relevance for defining the onset and/or progression of OA disease.

N-acetyl-Ser-Asp-Lys-Pro inhibits interleukin-1β-mediated matrix metalloproteinase activation in cardiac fibroblasts

Myocardial matrix turnover involves a dynamic balance between collagen synthesis and degradation, which is regulated by matrix metalloproteinases (MMPs). N-acetyl-Ser-Asp-Lys-Pro (Ac-SDKP) is a small peptide that inhibits cardiac inflammation and fibrosis. However, its role in MMP regulation is not known. Thus, we hypothesized that Ac-SDKP promotes MMP activation in cardiac fibroblasts and decreases collagen deposition via this mechanism. To that end, we tested the effects of Ac-SDKP on interleukin-1β (IL-1β; 5 ng/ml)-stimulated adult rat cardiac fibroblasts. We measured total collagenase activity, MMP-2, MMP-9, and MMP-13 expressions, and activity along with their inhibitors, tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2. In order to examine the effects of Ac-SDKP on the signaling pathway that controls MMP transcription, we also measured nuclear factor-κB (NFκB) and p42/44 mitogen-activated protein kinase (MAPK) activation. Ac-SDKP did not alter collagenase or gelatinase activity in cardiac fibroblasts under basal conditions, but blunted the IL-1β-induced increase in total collagenase activity. Similarly, Ac-SDKP normalized the IL-1β-mediated increase in MMP-2 and MMP-9 activities and MMP-13 expression. Inhibition of MMPs by Ac-SDKP was associated with increased TIMP-1 and TIMP-2 expressions. Collagen production was not affected by Ac-SDKP, IL-1β, or a combination of both agents. Ac-SDKP blocked IL-1β-induced p42/44 phosphorylation and NFκB activation in cardiac fibroblasts. We concluded that the Ac-SDKP-inhibited collagenase expression and activation was associated with increased expression of TIMP-1 and TIMP-2. These pharmacological effects of Ac-SDKP may be linked to the inhibition of MAPK and NFκB pathway.

Vaginal degeneration following implantation of synthetic mesh with increased stiffness

To compare the impact of the prototype prolapse mesh Gynemesh PS with that of two new-generation lower stiffness meshes, UltraPro and SmartMesh, on vaginal morphology and structural composition. A mechanistic study employing a nonhuman primate model. Magee-Womens Research Institute at the University of Pittsburgh. Parous rhesus macaques, with similar age, weight, parity and Pelvic Organ Prolapse-Questionnaire scores. Following Institutional Animal Care Use Committee approval, 50 rhesus macaques were implanted with Gynemesh PS (n = 12), UltraPro with its blue line perpendicular to the longitudinal axis of vagina (n = 10), UltraPro with its blue line parallel to the longitudinal axis of vagina (n = 8) or SmartMesh (n = 8) via sacrocolpopexy following hysterectomy. Sham-operated animals (n = 12) served as controls. The mesh-vagina complex was removed after 12 weeks and analysed for histomorphology, in situ cell apoptosis, total collagen, elastin, glycosaminoglycan content and total collagenase activity. Appropriate statistics and correlation analyses were performed accordingly. Relative to sham and the two lower stiffness meshes, Gynemesh PS had the greatest negative impact on vaginal histomorphology and composition. Compared with sham, implantation with Gynemesh PS caused substantial thinning of the smooth muscle layer (1557 ± 499 μm versus 866 ± 210 μm, P = 0.02), increased apoptosis particularly in the area of the mesh fibres (P = 0.01), decreased collagen and elastin content (20%, P = 0.03 and 43%, P = 0.02, respectively) and increased total collagenase activity (135%, P = 0.01). Glycosaminoglycan, a marker of tissue injury, was highest with Gynemesh PS compared with sham and other meshes (P = 0.01). Mesh implantation with the stiffer mesh Gynemesh PS induced a maladaptive remodelling response consistent with vaginal degeneration.

ANGIOTENSIN II-MEDIATED MYOCARDIAL EXPRESSION OF MMP2, MMP9 AND MT1-MMP WERE ENHANCED IN ACE2-NULL MICE

ObjectivesINTRODUCTION: Angiotensin (Ang) II is known to activate matrix metalloproteinases (MMPs), leading to degradation of extracellular matrix (ECM) proteins and myocardial remodelling. Angiotensin-converting enzyme-2 (ACE2) is a carboxypeptidase that metabolises...

Lack of Tissue Inhibitor of Metalloproteinases 2 Leads to Exacerbated Left Ventricular Dysfunction and Adverse Extracellular Matrix Remodeling in Response to Biomechanical Stress

Remodeling of the extracellular matrix (ECM) is a key aspect of myocardial response to biomechanical stress and heart failure. Tissue inhibitors of metalloproteinases (TIMPs) regulate the ECM turnover through negative regulation of matrix metalloproteinases (MMPs), which degrade the ECM structural proteins. Tissue inhibitor of metalloproteinases 2 is unique among TIMPs in activating pro-MMP2 in addition to inhibiting a number of MMPs. Given this dual role of TIMP2, we investigated whether TIMP2 serves a critical role in heart disease. Pressure overload by transverse aortic constriction (TAC) in 8-week-old male mice resulted in greater left ventricular hypertrophy, fibrosis, dilation, and dysfunction in TIMP2-deficient (TIMP2(-/-)) compared with wild-type mice at 2 weeks and 5 weeks post-TAC. Despite lack of MMP2 activation, total collagenase activity and specific membrane type MMP activity were greater in TIMP2(-/-)-TAC hearts. Loss of TIMP2 resulted in a marked reduction of integrin β1D levels and compromised focal adhesion kinase phosphorylation, resulting in impaired adhesion of cardiomyocytes to ECM proteins, laminin, and fibronectin. Nonuniform ECM remodeling in TIMP2(-/-)-TAC hearts revealed degraded network structure as well as excess fibrillar deposition. Greater fibrosis in TIMP2(-/-)-TAC compared with wild-type TAC hearts was due to higher levels of SPARC (secreted protein acidic and rich in cysteine) and posttranslational stabilization of collagen fibers rather than increased collagen synthesis. Inhibition of MMPs including membrane type MMP significantly reduced left ventricular dilation and dysfunction, hypertrophy, and fibrosis in TIMP2(-/-)-TAC mice. Lack of TIMP2 leads to exacerbated cardiac dysfunction and remodeling after pressure overload because of excess activity of membrane type MMP and loss of integrin β1D, leading to nonuniform ECM remodeling and impaired myocyte-ECM interaction.

Repetitive mechanical stretch increases extracellular collagenase activity in vaginal fibroblasts.

OBJECTIVES: The objectives were 1) to determine whether human vaginal fibroblasts are mechanosensitive and 2) to study the impact of mechanical stretch on these cells in the presence and absence of hormones. METHODS: Fibroblasts obtained from biopsies of full thickness vagina of 3 women were cyclically biaxially stretched at a magnitude of 8 and 16% for 72 hours with or without 17-β-estradiol plus progesterone. Culture media was collected and total collagenase activity was measured in duplicate using a fluorogenic substrate degradation assay. Data were analyzed at the 0.05 level of significance using Student t-test. RESULTS: Cells remained 90% viable throughout the experiments. Relative to the controls, hormonal treatment alone decreased collagenase activity (P=0.008). In the presence of mechanical stretch and in the absence of hormones, collagenase activity was increased (8% elongation, P=0.04; 16% elongation, P=0.001, respectively). The increase in collagenase activity was linearly correlated with magnitude (P<0.001). In the presence of hormones, the increase in enzyme activity by mechanical stretch was suppressed to baseline control levels (P=0.46). There was no difference in suppression by hormones by magnitude (P=0.48). CONCLUSIONS: Vaginal connective tissue fibroblasts are mechanosensitive with increased collagenase activity in the presence of stretch. This degradative behavior is inhibited in the presence of hormones. The data provide a mechanism by which events that induce vaginal stretch may lead to progression of pelvic organ prolapse, particularly, in the absence of hormones. Further studies are needed to determine whether these events lead to tissue with inferior mechanical properties.

Association of Gingival Crevicular Fluid Biomarkers During Periodontal Maintenance With Subsequent Progressive Periodontitis

The analysis of biomarkers in gingival crevicular fluid (GCF) may be helpful in forecasting patient vulnerability to future attachment loss. The purpose of this study is to correlate GCF biomarkers of inflammation and bone resorption with subsequent periodontal attachment and bone loss in a longitudinal trial of a matrix metalloproteinase (MMP) inhibitor. GCF was collected from two periodontal pockets (mean +/- SD: 5.1 +/- 1.0 mm) at baseline and annually in postmenopausal females with moderate to advanced periodontitis undergoing periodontal maintenance every 3 to 4 months during a 2-year double-masked, placebo-controlled, randomized clinical trial of subantimicrobial dose doxycycline (SDD; 20 mg two times a day). Subjects were randomized to SDD (n = 64) or a placebo (n = 64). GCF was analyzed for the inflammation markers interleukin (IL)-1beta (using enzyme-linked immunosorbent assay), total collagenase activity (using hydrolysis of a synthetic octapeptide), and MMP-8 (using a Western blot) and the bone-resorption marker carboxyterminal telopeptide cross-link fragment of type I collagen (ICTP) (using a radioimmunoassay). Generalized estimating equations were used to associate these biomarkers, categorized into tertiles, with subsequent clinical attachment (using an automated disk probe) or interproximal bone loss (using radiography). Odds ratio (OR) values compared highest to lowest tertile groups. Increases in GCF IL-1beta and MMP-8 during the first year of periodontal maintenance were associated with increased odds of subsequent (year 2) periodontal attachment loss (OR = 1.67; P = 0.01 and OR = 1.50; P = 0.02, respectively) driven by the placebo group. Elevated baseline ICTP was also associated with increased odds of 1- and 2-year loss of alveolar bone density (OR = 1.98; P = 0.0001) in the placebo group, not the SDD group, and a loss of bone height (OR = 1.38; P = 0.06), again driven by the placebo group. These data support the hypothesis that elevated GCF biomarkers of inflammation and bone resorption from a small number of moderate/deep sites have the potential to identify patients who are vulnerable to progressive periodontitis, and SDD may modify that risk.

Tumor necrosis factor induces matrix metalloproteinases in cardiomyocytes and cardiofibroblasts differentially via superoxide production in a PI3Kγ-dependent manner

Tumor necrosis factor (TNF) is an inflammatory cytokine that is upregulated in a number of cardiomyopathies. Adverse cardiac remodeling and dilation result from degradation of the extracellular matrix by matrix metalloproteinases (MMPs). We investigated whether TNF can directly trigger expression and activation of MMPs in cardiac cells. We compared MMP expression profile and activities between primary cultures of mouse neonatal cardiomyocytes and cardiofibroblasts and in cellular and extracellular compartments. In response to recombinant TNF (rTNF, 20 ng/ml), cardiomyocytes exhibited faster and more pronounced superoxide production compared with cardiofibroblasts, concomitant with increased expression of several MMPs. MMP9 levels increased more rapidly and about twofold more in cardiomyocytes than in cardiofibroblasts. TNF did not induce MMP2 expression. Expression of collagenases (MMP8, MMP12, MMP13, and MMP14) increased significantly, while total collagenase activity increased to a greater degree in conditioned medium of cardiomyocytes than in cardiofibroblasts. rTNF-mediated MMP expression and activation were dependent on superoxide production and were blocked by apocynin, an NADPH oxidase inhibitor. We identified phosphatidylinositol 3-kinase (PI3K)gamma as a key factor in TNF-mediated events since TNF-induced superoxide production, MMP expression, and activity were significantly suppressed in cardiomyocytes and cardiofibroblasts deficient in PI3Kgamma. We further demonstrated that the TNF-superoxide-MMP axis of events is in fact activated in heart disease in vivo. Wild-type and TNF(-/-) mice subjected to cardiac pressure overload revealed that TNF deficiency resulted in reduced superoxide levels, collagenase activities, PI3K activity, and fibrosis leading to attenuated cardiac dilation and dysfunction. Our study demonstrates that TNF triggers expression and activation of MMPs faster and stronger in cardiomyocytes than in cardiofibroblasts in a superoxide-dependent manner and via activation of PI3Kgamma, thereby contributing to adverse myocardial remodeling in disease.

Sulfasalazine blocks the release of proteoglycan and collagen from cytokine stimulated cartilage and down-regulates metalloproteinases

To investigate the effect of SSZ on the release of GAG and collagen fragments from bovine nasal cartilage and MMP and ADAMTS (a disintegrin and metalloproteinase domain with thrombospondin motifs) proteinases from human articular chondrocytes (HACs) stimulated with IL-1alpha and oncostatin M (OSM). SSZ was added to bovine nasal explant cultures stimulated to resorb with IL-1alpha and OSM, and the release of GAG and collagen has been determined. Collagenolytic activity was measured using the radio-labelled collagen bioassay. HACs were treated with IL-1alpha and OSM with and without SSZ, and MMP-1 and -13 and ADAMTS-4 and -5 were measured for protein and gene expression by ELISA and RT-PCR, respectively. SSZ blocked GAG and collagen fragment release from bovine cartilage, and reduced active and total collagenase activity in a dose-dependent manner. SSZ transcriptionally blocked MMP-1, -13 and ADAMTS-4, and reduced the protein levels of MMP-1 and -13 in a dose-dependent manner following stimulation of HACs with IL-1alpha and OSM. This study shows for the first time that SSZ blocks release of proteoglycan and collagen fragments from resorbing cartilage and lowers the levels of proteoglycan and collagen-degrading enzymes. These results indicate that in addition to acting as an anti-inflammatory agent, SSZ may have a therapeutic role in protecting cartilage from damage in OA.

Post‐treatment of Ganoderma lucidum reduced liver fibrosis induced by thioacetamide in mice

The present study was aimed at assessing the effects of Ganoderma lucidum extract (GLE) on an established liver fibrosis model with reference to the previously reported hepatoprotective effect of GLE against CCl(4)-induced fibrosis in rats. Repeated administration of thioacetamide (TAA) for 12 weeks to mice induced liver fibrosis. Treatment with GLE after the induction of liver fibrosis decreased the hepatic hydroxyproline content and improved liver histology. RT-qPCR analysis showed that GLE treatment reduced the mRNA expression of collagen (alpha1)(I), smooth muscle alpha-actin, tissue inhibitor of metalloproteinase 1 and metalloproteinase-13. In addition, the TAA-induced decrease in total collagenase activity was reversed by GLE treatment. In conclusion, oral administration of GLE reversed TAA-induced liver fibrosis, the mechanism of which might be related to the enhancement of collagenase activity.

Epidermal collagenase activity and its induction by 20-hydroxyecdysone in the fiddler crab Uca pugilator

Abstract The epidermal collagenase activity and its induction by 20- hydroxyecdysone in Uca pugilator were investigated. Zymographic electrophoresis showed four bands of collagenase activity, 16, 19, 22 and 29 kDa in molecular weight, with the former two accounting for 60% and 36%, respectively, of the total collagenase activity. The collagenase activity varies during the molting cycle. Among the molt stages tested, Premolt Stage D0 exhibited the highest epidermal collagenase activity for both the 16 and 19 kDa isoenzymes and, as the molt stage proceeded, the enzymatic activity of these two isoenzymes decreased, with the lowest activity for both found in Premolt Stage D3-4. Injection of 20-hydroxyecdysone significantly induced the activity of the 16 kDa collagenase in the epidermis of Uca pugilator, suggesting that the activity of this isoenzyme is under molting hormone control. Although 20-hydroxyecdysone injection did not result in a statistically significant increase in the activity of the 19 kDa isoenzyme, a tendency of the induction was nonetheless demonstrated. This is the first report on epidermal collagenase activity and its induction by the molting hormone in a crustacean.

Detrimental effect of excessive collagenase class II on human islet isolation outcome

Commercially available purified collagenases, derived from Clostridium histolyticum, contain two different classes of collagenase: class I collagenase (CI) and class II collagenase (CII) at a predetermined ratio. In this study, using purified CI and CII in separate vials, we had a unique opportunity to investigate the effect of the proportion between two collagenase classes on clinical human islet isolation. Pancreas organs derived from deceased donors were prospectively assigned to one of three different enzyme protocols: group A--CII:CI = 1:1 vial; group B--1:2; group C--1.5:1. As a result, their total collagenase activities were 2116, 2230, and 3117 Wunsch units/pancreas in groups A, B, and C, respectively but thermolysin dosage was adjusted to 624-988 Units/g pancreas. The pancreas was not efficiently digested in group C in spite of a relatively longer digestion time and significantly higher Wunsch activity, resulting in the poorest islet isolation outcome among the three groups. Additional retrospective analysis revealed that this suboptimal outcome in group C was not because of the absolute excessive amount of collagenase activity but as a result of the relatively high proportion of CII (i.e., unbalanced CII/CI ratio). Our study suggests that an excessive CII is ineffective in releasing islets from human pancreas, and rather a balanced CII/CI ratio is of paramount importance.

MMP-9 protein level does not reflect overall MMP activity in the airways of patients with COPD

Animal and human studies have implicated an imbalance of matrix metalloproteinases (MMPs) and tissue inhibitors of matrix metalloproteinases (TIMPs) in the pathogenesis of chronic obstructive pulmonary disease (COPD). MMP-9 protein is increased in COPD and we hypothesized that total MMP activity would be raised although this has not previously been measured. Using fluorescence and biotin labelled MMP assays, RT-PCR, western blotting and enzyme-linked immunosorbent assay we examined total MMP activity, specific gelatinase, elastase, collagenase activity, TIMP-1 and TIMP-2 in induced sputum from smokers with COPD and smokers without COPD. Induced sputum was obtained from 15 smokers with COPD and 14 smokers without COPD. MMP-9 levels were higher in those with COPD compared with controls (p<0.05). Total MMP activity, specific gelatinase, collagenase and elastase activities were not higher in COPD patients. In addition, reduced MMP activity was correlated with increasing airflow obstruction in COPD (p=0.016). MMP-9 protein but not MMP activity was higher in sputum of COPD patients compared with controls. These results suggest that MMP-9 levels may not reflect the overall MMP activity in the airways of patients with COPD suggesting a complex relationship between MMP-9 protein and activity. Further studies of MMPs in COPD should comprise activity measures in addition to protein levels.

Halofuginone Induces Matrix Metalloproteinases in Rat Hepatic Stellate Cells via Activation of p38 and NFκB

The semisynthetic plant alkaloid halofuginone (HAL) was reported to prevent and partly reverse experimental liver fibrosis. However, its mechanisms of action are poorly understood. We therefore aimed to determine the antifibrotic potential of HAL and to characterize involved signal transduction pathways in hepatic stellate cells (HSCs). Results were compared with its in vivo effects in a rat model of reversal of established liver fibrosis induced by thioacetamide. In vitro HAL inhibited HSC proliferation and migration dose dependently at submicromolar concentrations. HAL (200 nm) up-regulated matrix metalloproteinase (MMP)-3 and MMP-13 expression between 10- and 50-fold, resulting in a 2- to 3-fold increase of interstitial collagenase activity. Procollagen alpha1(I) and MMP-2 transcript levels were suppressed 2- to 3-fold, whereas expression of other profibrogenic mRNAs remained unaffected. p38 mitogen-activated protein kinase (p38 MAPK) and nuclear factor kappaB(NFkappaB) pathways were activated by HAL, and specific inhibitors of p38 MAPK and NFkappaB dose dependently inhibited MMP-13 induction. Treatment with HAL did not affect HSC viability, and observed effects were reversible after its removal. In vivo HAL up-regulated MMP-3 and -13 mRNA expression 1.5- and 2-fold, respectively, in cirrhotic rats, whereas tissue inhibitor of metalloproteinase-1 was suppressed by 50%. In conclusion, submicromolar concentrations of HAL inhibit HSC proliferation and migration and up-regulate their expression of fibrolytic MMP-3 and -13 via activation of p38 MAPK and NFkappaB. The remarkable induction of MMP-3 and -13 makes HAL a promising agent for antifibrotic combination therapies.

Islet isolation for allotransplantation: variables associated with successful islet yield and graft function

Efficient islet isolation is an important prerequisite for successful clinical islet transplantation. Although progressively improved, islet yield and quality are, however, unpredictable and variable and require standardisation. Since 1989 we have processed 437 pancreases using the automated method. The donor characteristics, pancreas procurement, and digestion and purification procedures including a wide enzyme characterisation of these pancreases were analysed and correlated with islet yield and transplant outcome. By univariate analysis, islet yield was significantly associated with donor age (r=0.16; p=0.0009), BMI (r=0.19; p=0.0004), good pancreas condition (p=0.0031) and weight (r=0.15; p=0.0056), total collagenase activity (r=0.22; p=0.0001), adjusted collagenase activity/mg (r=0.18; p=0.0002), collagenase activity/solution volume (r=0.18; p=0.0002) and neutral protease activity/solution volume (r=0.14; p=0.0029). A statistically significant contribution to the variability of islet yield in a multivariate analysis performed on donor variables was found for donor BMI (p=0.0008). In a multivariate analysis performed on pancreas variables a contribution was found for pancreas weight (p=0.0064), and for a multivariate analysis performed on digestion variables we found a contribution for digestion time (p=0.0048) and total collagenase activity (p=0.0001). Twenty-four patients with type 1 diabetes received single islet preparations from single donors. In these patients, multivariate analyses showed that the reduction in insulin requirement was significantly associated with morphological aspects of islets (p=0.0010) and that 1-month C-peptide values were associated with islet purity (p=0.0071). These data provide baseline donor, digestion and purification selection criteria for islet isolation using the automated method and indicate that the morphological aspect may be a clinically relevant measure of islets on which the decision for transplant can be based.

Altered bleomycin-induced lung fibrosis in osteopontin-deficient mice

Osteopontin is a multifunctional matricellular protein abundantly expressed during inflammation and repair. Osteopontin deficiency is associated with abnormal wound repair characterized by aberrant collagen fibrillogenesis in the heart and skin. Recent gene microarray studies found that osteopontin is abundantly expressed in both human and mouse lung fibrosis. Macrophages and T cells are known to be major sources of osteopontin. During lung fibrosis, however, osteopontin expression continues to increase when inflammation has receded, suggesting alternative sources of ostepontin during this response. In this study, we demonstrate immunoreactivity for osteopontin in lung epithelial and inflammatory cells in human usual interstitial pneumonitis and murine bleomycin-induced lung fibrosis. After treatment with bleomycin, osteopontin-null mice develop lung fibrosis characterized by dilated distal air spaces and reduced type I collagen expression compared with wild-type controls. There is also a significant decrease in levels of active transforming growth factor-beta(1) and matrix metalloproteinase-2 in osteopontin null mice. Type III collagen expression and total collagenase activity are similar in both groups. These results demonstrate that osteopontin expression is associated with important fibrogenic signals in the lung and that the epithelium may be an important source of osteopontin during lung fibrosis.

The role of matrix metalloproteinases in rheumatoid tendon disease.

In rheumatoid arthritis (RA) invasive tenosynovitis is associated with an increase in tendon rupture, although little is known about the mechanisms involved. We obtained specimens of noninvasive encapsulating tenosynovium, invasive tenosynovium, and wrist joint synovium from 28 rheumatoid patients. In vitro production of the matrix metalloproteinase (MMP) enzymes, MMP-8 and -9, and total collagenase activity were measured. Invasive tenosynovium produced highest levels of the collagenase MMP-8 and displayed significantly greater ability to degrade collagen type I than encapsulating tenosynovium. Levels of the gelatinase enzyme MMP-9 were similar in all groups. These results show that invasive tenosynovium is more destructive than encapsulating tenosynovium at a molecular level, providing an explanation for the increased tendon rupture associated with invasive tenosynovitis in RA.