Biglycan fragmentation in pathologies associated with extracellular matrix remodeling by matrix metalloproteinases (original) (raw)
Related papers
PLoS ONE, 2011
Background and Aims: During fibrogenesis, in which excessive remodeling of the extracellular matrix occurs, both the quantity of type VI collagen and levels of matrix metalloproteinases, including MMP-2 and MMP-9, increase significantly. Proteolytic degradation of type VI collagen into small fragments, so-called neo-epitopes, may be specific biochemical marker of liver fibrosis. The aim of this study was to develop an ELISA detecting a fragment of type VI collagen generated by MMP-2 and MMP-9, and evaluate this assay in two preclinical models of liver fibrosis. Methods: Mass spectrometric analysis of cleaved type VI collagen revealed a large number of protease-generated neoepitopes. A fragment unique to type VI collagen generated by MMP-2 and MMP-9 was selected for ELISA development. The CO6-MMP assay was evaluated in two rat models of liver fibrosis: bile duct ligation (BDL) and carbon tetrachloride (CCl4)treated rats. Results: Intra-and inter-assay variation was 4.1% and 10.1% respectively. CO6-MMP levels were significantly elevated in CCl 4-treated rats compared to vehicle-treated rats at weeks 12 (mean 30.9 ng/mL vs. 12.8 ng/mL, p = 0.002); week 16 (mean 34.0 ng/mL vs. 13.7 ng/mL, p = 0.0018); and week 20 (mean 35.3 ng/mL vs. 13.3 ng/mL, p = 0.0033) with a tight correlation between hepatic collagen content and serum levels of CO6-MMP (R 2 = 0.58, p,0.0001) in CCl 4-treated rats. In BDL rats, serum levels of CO6-MMP were significantly elevated compared to the levels in sham-operated animals both at 2 weeks (mean 29.5 ng/mL vs. 14.2 ng/mL, p = 0.0001) and 4 weeks (mean 33.0 ng/mLvs. 11.8 ng/mL, p = 0.0003). Conclusions: This novel ELISA is the first assay enabling assessment of MMP degraded type VI collagen, allowing quantification of type VI collagen degradation, which would be relevant for different pathologies. The marker was highly associated with liver fibrosis in two liver fibrosis animal models, suggesting type VI turnover to be a central player in fibrogenesis.
Liver International, 2010
Background: During fibrogenesis in the liver, in which excessive remodelling of the extracellular matrix (ECM) occurs, both the quantity of type III collagen (CO3) and levels of matrix metalloproteinases (MMPs), including MMP-9, increase significantly. MMPs play major roles in ECM remodelling, via their activity in the proteolytic degradation of extracellular macromolecules such as collagens, resulting in the generation of specific cleavage fragments. These neo-epitopes may be used as markers of fibrosis. Aims: The current study investigated whether a novel enzyme-linked immunosorbent assay (ELISA) assay specifically measuring an MMP-9-cleaved sequence of type III collagen located at position 610 (CO3-610C) may be used as a marker of liver fibrosis. Material and methods: Bile duct ligation (BDL) was performed in 20 rats, with sham operations performed on another 20 rats. Serum levels of the neoepitope CO3-610C (MMP-mediated type III collagen degradation) were determined with an ELISA at 14 and 28 days post-surgery. Liver fibrosis was evaluated by quantitative digital image analysis of Sirius red-stained formalinfixed and paraffin-embedded sections. Western blot and densitometry were performed to confirm the CO3-610C ELISA data. Results: CO3-610C levels in serum increased significantly in BDL rats compared with those undergoing sham operations (% increase: 14 days = 153%, P o 0.0001; 28 days = 134%, P = 0.0014). This increase was confirmed by Western blot and densitometry of the identified bands. The CO3-610C levels correlated to liver fibrosis (R 2 = 0.23 and P = 0.01), as evaluated by quantitative digital histology. Discussion and conclusion: The data suggest that MMP-9-mediated CO3 turnover is a central event in the pathogenesis of fibrosis, and that the neo-epitope generated may be a novel biochemical marker.
Arthritis research & therapy, 2006
A major and early feature of cartilage degeneration is proteoglycan breakdown. Matrix metalloprotease (MMP)-13 plays an important role in cartilage degradation in osteoarthritis (OA). This MMP, in addition to initiating collagen fibre cleavage, acts on several proteoglycans. One of the proteoglycan families, termed small leucine-rich proteoglycans (SLRPs), was found to be involved in collagen fibril formation/interaction, with some members playing a role in the OA process. We investigated the ability of MMP-13 to cleave members of two classes of SLRPs: biglycan and decorin; and fibromodulin and lumican. SLRPs were isolated from human normal and OA cartilage using guanidinium chloride (4 mol/l) extraction. Digestion products were examined using Western blotting. The identities of the MMP-13 degradation products of biglycan and decorin (using specific substrates) were determined following electrophoresis and microsequencing. We found that the SLRPs studied were cleaved to differing ex...
Frontiers in Physiology, 2013
Background: Progression of liver fibrosis is characterized by synthesis and degradation of extracellular matrix (ECM). Matrix-metalloproteinases (MMP) cleave collagen fibers at a specific site and thereby generate soluble fragments of ECM (neo-epitopes). The levels of these neo-epitopes might reflect the stage of liver fibrosis and may allow monitoring of anti-fibrotic therapies. Here we analyzed these neo-epitopes as read-out for a liver directed therapy with statins. Methods: Bile duct ligation (BDL) was performed on wild type rats, which received atorvastatin (15 mg/kg * d) for 1 week starting at 1, 2, 3, 4 and 5 weeks after BDL (T1-T5), while controls remained untreated. Hepatic fibrosis was analyzed by immunohistochemistry and hepatic hydroxyproline content. TGFβ levels were measured by RT-PCR. Proteolytic activity of MMP-2 was examined by zymography. Levels of degradation MMP driven type I, III, IV and VI collagen degradation (C1M, C3M, C4M, and C6M) and type III and IV collagen formation (PRO-C3 and P4NP7S) markers were assessed by specific ELISAs in serum probes. Results: Serum markers of ECM neo-epitopes reflected significantly the deposition of ECM in the liver and were able to distinguish between early (T1-T3) and severe fibrosis (T4-T5). Statin treatment resulted in reduction of neo-epitope markers, especially when therapy was started in the stage of severe fibrosis (T4-T5). Furthermore, these markers correlated with hepatic expression of profibrotic cytokines TGFβ1 and TGFβ2. Formation markers of type III and IV collagen (PRO-C3 and P4NP7S) and degradation markers C4M and C6M correlated significantly with hepatic MMP-2 activity in rats with severe fibrosis. Conclusion: Determination of ECM remodeling turnover markers in serum allowed a distinction between mild and severe fibrosis. With respect to statin therapy, the markers may serve as read-out for efficacy of anti-fibrotic treatment.
Biomarker Insights, 2011
Background and aim: The current study utilized a carbon tetrachloride (CCl 4 )-induced liver fibrosis model to measure levels of the MMP9-mediated collagen type III degradation fragment CO3-610 (site of cleavage: KNGETGPQGP), during disease progression and regression, and to investigate a potential prognostic role of the biomarker. Materials and methods: 72 female Sprague-Dawley rats aged 6 months old were injected with CCl 4 twice a week over different periods of time to induce varying degrees of liver fibrosis. After 4, 6 and 8 weeks of treatment, administration of CCl 4 was stopped. The 6-and 8-week treatment groups were left to regress for a further 6 or 12 weeks at which point they were sacrificed and livers removed and sectioned. Liver fibrosis was quantified using Visiopharm software to analyse Sirius red-stained sections. Serum levels of CO3-610 were measured in all animals using an ELISA assay as described by Barascuk et al. 1 Results: Quantitative histology revealed total collagen deposition in the liver increased as fibrosis progressed. In animals treated with CCl 4 for 4 weeks, collagen comprised on average 4.94% of the total tissue in liver sections, while after 6 weeks the mean was 8.25%, and after 8 weeks, 9.11%. During the regression phase, the total collagen deposition gradually decreased to a mean of 6.9% and 5.09% for animals regressing 6 and 12 weeks respectively after 6 weeks treatment, and 6.27% for animals regressed 12 weeks after 8 weeks treatment. CO3-610 values increased progressively in rats treated for 4 weeks (by a mean of 55.0 ng/ml), 6 weeks (mean 61.1 ng/ml) and 8 weeks (mean 70.2 ng/ml). During the regression phase, CO3-610 values rapidly decreased by a mean of 28.9 ng/ml at 6 weeks and 21.6 ng/ml at 12 weeks in animals previously treated for 6 weeks, and by a mean of 19.52 ng/ml in animals treated for 8 weeks and regressed for 12 weeks. CO3-610 levels were statistically significantly correlated with total collagen during disease progression (r = 0.5701, P , 0.0001). No statistically significant correlation was observed during regression (r = 0.2081, P = 0.1138). Conclusion: Levels of the MMP-9 generated fragment of collagen type III, CO3-610, correlated with the degree of liver fibrosis in rats during the progression phase, but were not correlated with total collagen levels during regression. CO3-610 seems to be produced only under the CCL 4 stimulus, and signifies CO3-610 as a potential marker of progression rather than regression. The corresponding steep elevations in levels of CO3-610 total collagen and collagen type III during liver fibrosis progression underline a potential prognostic capacity of the biomarker.
Biomarker Insights, 2011
Aim Arterial extracellular matrix (ECM) remodeling by matrix metalloproteinases (MMPs) is one of the major hallmarks of atherosclerosis. Mimecan, also known as osteoglycin has been implicated in the integrity of the ECM. This study assessed the validity of an enzyme-linked immunosorbent assay (ELISA) developed to measure a specific MMP12-derived fragment of mimecan, MMCN-151, in apolipoprotein-E knockout (ApoE-KO) mice. Methods and Results A mouse monoclonal antibody raised against MMCN-151 was used to develop a competitive ELISA. The assay was validated using samples from 20 ApoE-KO and 20 wild type [C57 BL/6] male mice fed a normal or high-fat diet (HFD) for up to 20 weeks. The technical reliability of the assay was established with intra-assay variability <2% and inter-assay variability < 10%. The lowest limit of quantification of MMCN-151 was 0.5 ng/ml. ApoE-KO mice fed a HFD for 20 weeks had four-fold increased circulating levels of MMCN-151 compared to baseline, whereas ...
Digestive Diseases and Sciences, 2011
Background and Aims-Matrix metalloproteinase-2 (MMP-2), a type IV collagenase secreted by activated hepatic stellate cells (HSCs), is upregulated in chronic liver disease and is considered a profibrotic mediator due to its proliferative effect on cultured HSCs and ability to degrade normal liver matrix. Although associative studies and cell culture findings suggest that MMP-2 promotes hepatic fibrogenesis, no in vivo model has definitively established a pathologic role for MMP-2 in the development and progression of liver fibrosis. We therefore examined the impact of MMP-2 deficiency on liver fibrosis development during chronic CCl 4 liver injury and explored the effect of MMP-2 deficiency and overexpression on collagen I expression.
The American Journal of Pathology, 1998
A rat model of common bile duct ligation (BDL)-induced hepatic fibrosis was used to assess the expression and activities of collagen-degrading proteinases and their inhibitors during the progression of fibrosis. Expression of four members of the matrix metalloproteinase (MMP) family (MMP-2/gelatinase A, MMP-3, MMP-9/gelatinase B, and MMP-13) and three tissue inhibitors of metalloproteinases-1, -2, and -3 (TIMP-1, TIMP-2, and TIMP-3) were evaluated by Northern blot analysis of RNA from liver tissue isolated at 0, 2, 5, 10, 20, and 30 days after either a BDL or sham operation. In addition, we analyzed free gelatinase and TIMP activities by zymography and reverse zymography, respectively. We found that the proteolytic activities of MMP-2 and MMP-9 increased by 2 days after ligation, reached maximal levels at day 10, and remained high through the study period, whereas the gelatinolytic activities in plasma were unchanged. The increase in gelatinase activities was accompanied by an increase in the TIMP mRNA transcripts. TIMP-1 transcripts appeared at day 2, increased until day 10, and remained elevated throughout the study period. TIMP-2 and TIMP-3 transcripts become detectable on day 10 and remained stable afterwards. No corresponding increase in TIMP protein activity was detected by reverse zymography. This appears to result from the formation of TIMP/MMP complexes. These findings indicate a likely surplus in the BDL model of fibrosis of free gelatinases as compared with the TIMPs. Thus, excessive TIMP production is not a sufficient explanation for the observed extracellular matrix accumulation, but complex changes in the local MMP/TIMP balance may underlie the pathomechanisms of fibrosis.
PLOS One, 2010
Background: Liver fibrosis is characterized by excessive synthesis of extracellular matrix proteins, which prevails over their enzymatic degradation, primarily by matrix metalloproteinases (MMPs). The effect of pharmacological MMP inhibition on fibrogenesis, however, is largely unexplored. Inflammation is considered a prerequisite and important co-contributor to fibrosis and is, in part, mediated by tumor necrosis factor (TNF)-a-converting enzyme (TACE). We hypothesized that treatment with a broad-spectrum MMP and TACE-inhibitor (Marimastat) would ameliorate injury and inflammation, leading to decreased fibrogenesis during repeated hepatotoxin-induced liver injury.