Phosphodiesterase inhibition mediates matrix metalloproteinase activity and the level of collagen degradation fragments in a liver fibrosis ex vivo rat model
- Sanne Skovgård Veidal†1,
- Mette Juul Nielsen†1Email author,
- Diana Julie Leeming1 and
- Morten Asser Karsdal1
© Veidal et al.; licensee BioMed Central Ltd. 2012
Received: 23 May 2012
Accepted: 13 December 2012
Published: 18 December 2012
Accumulation of extracellular matrix (ECM) and increased matrix metalloproteinase (MMP) activity are hallmarks of liver fibrosis. The aim of the present study was to develop a model of liver fibrosis combining ex vivo tissue culture of livers from CCl4 treated animals with an ELISA detecting a fragment of type III collagen generated in vitro by MMP-9 (C3M), known to be associated with liver fibrosis and to investigate cAMP modulation of MMP activity and liver tissue turnover in this model.
In vivo: Rats were treated for 8 weeks with CCl4/Intralipid. Liver slices were cultured for 48 hours. Levels of C3M were determined in the supernatants of slices cultured without treatment, treated with GM6001 (positive control) or treated with IBMX (phosphodiesterase inhibitor). Enzymatic activity of MMP-2 and MMP-9 were studied by gelatin zymography. Ex vivo: The levels of serum C3M increased 77% in the CCl4-treated rats at week 8 (p < 0.01); Levels of C3M increased significantly by 100% in fibrotic liver slices compared to controls after 48 hrs (p < 0.01). By adding GM6001 or IBMX to the media, C3M was restored to control levels. Gelatin zymography demonstrated CCl4-treated animals had highly increased MMP-9, but not MMP-2 activity, compared to slices derived from control animals.
We have combined an ex vivo model of liver fibrosis with measurement of a biochemical marker of collagen degradation in the condition medium. This technology may be used to evaluate the molecular process leading to structural fibrotic changes, as collagen species are the predominant structural part of fibrosis. These data suggest that modulation of cAMP may play a role in regulation of collagen degradation associated with liver fibrosis.
KeywordsPrecision-cut liver slices Fibrosis Ex vivo cAMP
Liver fibrosis due to viral or alcohol-induced injury is one of the leading causes of death worldwide . Liver biopsy is the most commonly used method for fibrosis assessment, but it is invasive, associated with patient discomfort and, in rare cases, with serious complications [2–4]. Therefore, research has focused on the evaluation of non-invasive methods for the assessment of liver fibrosis , including a highly enforced effort in discovering and developing biochemical markers for liver fibrosis assessment. In alignment there is an urgent need for applied translational science, in which information from preclinical settings may be translated to clinical settings. One such approach may be to investigate the same biochemical analyte in liver explants as a model of liver fibrosis, in animal models of liver fibrosis, and then finally in clinical settings.
Fibrosis may be described as extensive scar formation, observed as increased deposition and abnormal distribution of extracellular matrix (ECM) components such as collagens and proteoglycans. ECM remodeling is a key process of tissue homoeostasis [6–8], and specific proteolytic activities are a prerequisite for a range of cellular functions and interactions during the process . The specific proteolytic activities are precisely coordinated under physiological situations, with a specified sequence of events resulting in controlled tissue turnover. In pathological situations, including inflammation, fibrosis and cancer, the normal damage/repair balance is displaced , leading to excessive remodeling. As a consequence of this tissue turnover, there is a release of several protein degradation fragments specific for the combination of the involved proteases, the affected organ and the disease. The fragmentation results in exposure of new peptide ends (so-called neo-epitopes) to which specific antibodies can be developed. These neo-epitopes may be used for the design of molecular biochemical markers .
In the healthy human liver the most abundant collagens are the fibril-forming type I and type III . Fibril-forming collagens are synthesized as precursor molecules with large propeptide extensions at both the N- and C-terminals of the molecule . The mature propeptides are cleaved from procollagen by N- or C-terminal proteinases, and mature collagen is integrated into the ECM [12, 13]. During fibrogenesis, type I and III collagen levels increase up to 8 times . Endopeptidases such as matrix metalloproteinases (MMPs) play a major part in the degradation of extracellular macromolecules such as collagens and during fibrogenesis the levels of MMPs increase. With respect to excessive proteolytic activity in the fibrous tissue, the gelatinases MMP-2 and MMP-9 have been investigated and documented to be highly regulated [15–17]. Thereby a fragment of type III collagen generated in vitro by MMP-2 and MMP-9 may be a biochemical marker for liver fibrosis. Thus by analysis of cleavage fragments generated by MMP-9 of type III collagen, and the development of a specific assay quantifying a validated fragment, novel tools with increased sensitivity and specificity for some types of fibrosis may be developed. We have previously identified fragments of other collagens, and developed assays for those [18–20], and very recently identified a unique type III collagen degradation product (C3M), in vitro generated by MMP-2 and MMP-9. We consequently developed a novel Enzyme-linked immunosorbent assay (ELISA) using monoclonal antibody to detect this specific fragment, which later on demonstrated to be highly associated with liver fibrosis [21–24].
Several animal models for liver fibrosis have been developed, most of them in small rodents , each with individual strengths and weaknesses. These different rodent models are complementary as they represent different pathways to fibrosis, as also seen in human disease. Bile duct ligation (BDL) in rats has been used as a model of chronic liver injury due to its resemblance to hepatocyte damage, hepatic stellate cell (HSC) activation, and liver fibrosis observed in human cholestatic liver diseases [1, 25]. Carbon tetrachloride (CCl4) is a hepatotoxin that causes acute liver injury and, when given repetitively at a low dose, induces liver fibrosis. It is a highly reproducible and robust model which is used to resemble alcoholic and non-alcoholic steatohepatitis with the consequent fibrosis and cirrhosis in humans [1, 25].
Ex vivo models enables the study of complicated processes in a multicellular system in which cell-cell and cell-matrix interactions are maintained. The ECM holds various components, both inhibitors and promoters of cell function, which are absent in traditional plastic culture systems [26, 27]. In liver fibrosis where tissue turnover plays a fundamental role, the ECM has both structural and biochemical features, which are not easily accounted for by either hepatic stellate cell line (e.g. HSC-T6) or isolation of primary HSC . A potential ex vivo model for studying liver fibrosis (and HSC activation) are the precision-cut liver slices (PCLS). Several researches have investigated and used the liver explant model, first reported by Otto Warburg in 1923 . Since then, these experiments have for the major part been performed in healthy livers. Thus in the current study, we employed the CCl4 model, with disease affected livers, and cultured these under optimal condition compared to healthy control liver, assessed by the novel liver fibrosis marker C3M, to develop an ex vivo model system for investigation of the processes involved in liver fibrosis tissue turnover.
Induction of cyclic AMP (cAMP) by pan-specific phosphodiesterase (PDE) inhibition has been shown to modulate MMP activity in a cartilage ex vivo models . These studies clearly suggested that cAMP induction was essential for MMP activity and tissue turnover, both processes that are highly regulated in fibrotic diseases. We aimed at testing the hypothesis whether cAMP induction may in addition to cartilage turnover also be important for tissue turnover associated with liver fibrosis, and thus a more common regulator of MMP mediated tissue turnover.
In the present study, we aimed at using the novel molecular biochemical marker C3M to 1) develop an ex vivo model in which the same biochemical marker may be measured ex vivo, in animal settings and clinical setting to enable translational research and 2) to use this model to preliminary investigate modulation of MMP activity and liver tissue turnover by PDE inhibition.
Materials and methods
All reagents were standard high-quality chemicals from companies such as Merck and Sigma-Aldrich. Krebs-Heinseleit buffer used to preparation of PCLS was from Amplicon (Skovlunde, DK). The culture medium Williams medium E was from Lonza (Verviers, BG). IBMX was from Sigma (Poole, UK), while GM6001 was from AM Scientific (Pleasant Hill, CA). AlamarBlue assay was from AbD Serotec (Oxford, UK).
20 male Spraque-Dawley rats aged 4 months were housed at the animal facilities at Nordic Bioscience, Denmark. The experiments were approved by the Animal Ethics Committee of the Danish Ministry of Justice (2008/561-1450). The rats were housed in standard cages, with bedding and nest material at 18-22°C and fed with standard pellet diet and tap water ad libitum. The rats were kept under conditions of a 12 hr light/dark cycle. Liver fibrosis was induced by intraperitoneal injections (i.p.) of CCl4 twice a week for 8 weeks starting one week after acclimatization. The rats were divided in two groups, 14 rats were treated with 10% CCl4 in intralipid (0.5 ml/kg) and 6 vehicle rats were treated with intralipid (0.5 ml/kg).
Blood samples at baseline and termination were taken under inhalation anesthesia using isoflurane from the retro-orbital sinus of rats which had fasted for at least 14 hr. The blood were left for 30 min at room temperature to clot and centrifuged at 3000 rpm for 10 min. Liquid were transferred to new tubes and centrifuged again at 3000 rpm for 10 min. Serum were transferred into clean tubes and stored for −20°C until use.
After the rats were euthanized, the liver was carefully excised and weighted. A small part of one of the lobes was fixated in 4% formaldehyde for at least 24 hr and embedded in paraffin. Sections were cut 4–5 μm thick, mounted on glass slides and stained with 1% Sirius Red F3B (Sigma-Aldrich, St. Louis, MO) in saturated picric acid (Sigma-Aldrich). The liver sections were evaluated by histological changes in tissue architecture, presence of inflammation and proliferation of liver fibrosis.
Liver sections (4–5 μm) were de-paraffinised, hydrated and further peroxidase activity was blocked with the addition of 0.4% hydrogen peroxide. Sections were the incubated with a polyclonal antibody against α-SMA (1:400, Abcam, Cambridge, UK). Sections were then rinsed and the antibody binding was depicted using the Super Sensitive Polymer-HRP IHC Detection System combined with AEC substrate, according to the supplier’s instructions (Biogenex, Taby, Sweden). Sections were counterstained with Mayer’s haematoxylin. Digital photographs were taken using an Olympus Bx60 microscope with x40 magnification and an Olympus 5050-zoom digital camera (Olympus, Tokyo, Japan).
Ex vivo experiments
For ex vivo liver tissue culture experiments, fibrosis was induced as described. Rat livers were excised from adult male Spraque-Dawley rats, weighted, stored in cold PBS and liver fibrosis was evaluated by gross appearance. PCLS were prepared from the livers in ice-cold Krebs-Heinseleit buffer containing 25 mM glucose, 10 mM HEPES and 25 mM NaHCO3, and sliced on the TSE Krumdieck tissue slicer MD 4000 as previously described . The PCLS were cultured for 2 hr at 37°C under carbogen atmosphere in sterile 48 well plates containing 300 μl Williams Medium E containing 25 mM glucose, 10 mg/ml gentamycin and 10% fetal bovine serum (FBS). After 2 hr the serum media was removed and the slices were washed in serum-free media. New serum-free media was added containing the respective treatments and the PCLS were cultured for 48 hr. Treatments used in the experiments were 100 μM IBMX, a non-specific PDE inhibitor, and 10 μM GM6001, a MMP inhibitor. After the culturing period supernatants were collected and stored at −20°C until use.
Cell viability measured by AlamarBlue
AlamarBlue is a sterile non-toxic, aqueous oxidation-reduction indicator that yields colorimetric changes and a fluorescent signal in response to metabolic activity. For the quantification of cell viability, we used the AlamarBlue assay. The active compound, resazurin, is an oxidation-reduction indicator that changes from the oxidized non-fluorescent form (indigo) to the reduced fluorescent form (purple) according to the viability and proliferating activity of cells . In short, 300 μl 10% AlamarBlue was added to each well of the microtiter plate containing PCLS and incubated for 2 hr at 37°C while shaking. The results were measured by ELISA reader at 540–590 nm.
Activity and expression of MMP-2 and MMP-9 were investigated using gelatinase zymography. Supernatants from the ex vivo experiment were diluted 3:4 in 4x sample buffer, heated at 37°C for 15 min and applied for gel-electrophoresis on 10% SDS-polyacrylamide gel using the substrate 3 mg/ml gelatin. After electrophoresis, gels were washed three times before four days incubation at 37°C with gentle agitation in 1% Triton-X, 100 mM Tris Base, 13 mM CaCl2, 0.2 mM ZnCl2, 6 mM NaN3, and pH 7.5. Gels were stained for 15 min with a solution containing 0.25% Coomassie R-250 with 45% methanol and 3% acetic acid. The gels were destained in a solution of 20% methanol, 17% ethanol, 7% acetic acid, and 0.6% diethyl-ether for 45 min until clear bands were obtained. Gels were finally soaked in 2% glycerol, dried in plastic bags and scanned for documentation. The intensity of the bands was quantified by densitometry analysis shown as average pixel number.
Results are shown as mean ± standard error of mean (SEM). Five vehicle rat livers and 9 CCl4 treated rat livers were used in the ex vivo experiment with 10 replicas for each treatment. Differences between mean values were compared by nonparametric Mann-Whitney’s t-test for two-tailed observations. All statistical analyses were performed in GraphPad Prism software v.5 (GraphPad Software, San Diego, CA). P values less than 0.05 were considered significant.
Evaluation of in vivo experiment
Ex vivo tissue culture
AlamarBlue was investigated as a measure for metabolic activity (Figure 3B). A 60% increase was observed in fibrotic liver slices compared to vehicle without intervention (p = 0.035). Adding IBMX or GM6001 to the fibrotic liver slices had no effect on metabolic activity.
Liver injury is a multifactor process, involving many cell types, mediators and cell-cell and cell-matrix interactions. The main advantages of the PCLS model are that the cells are the maintained in the 3-dimensional organ structure, with the same relative cell number and orientation towards other cells , which makes the model a valuable tool for studying tissue turnover, due to the high in vivo likeness . The PCLS model has been applied recently to the study of HSC activation , and in addition it has also been shown that endothelial cells  and Kupffer cells  remain viable in these cultures. Especially for studying ECM remodeling in the liver during fibrosis, the PCLS model provide several advantages that seem essential .
This study provides the first measurement of a liver fibrosis marker in ex vivo cultures that in other settings have been shown to correlate to the extent of liver fibrosis in vivo. Furthermore, we used a PDE inhibitor, that in other settings have been shown to modulate MMP activity , to demonstrate that this ex vivo liver fibrosis model in combination with the biochemical marker may provide further insights into ECM remodeling in the liver during pathology. Hopefully this will enable other researchers to investigate and validate key processes in liver fibrosis and enable more developments of treatments for liver diseases.
The levels of the C3M fragment was 100% elevated in the ex vivo cultures of fibrotic liver slices. In the present study we used the general MMP inhibitor GM6001 as a positive control . Following exposure of the liver explants to GM6001 the level of the C3M was restored to normal levels, suggesting that this marker is both specific for MMP activity and pathology relevant. Furthermore, in the in vivo experiment a 77% increase of serum C3M fragment in CCl4 rats at termination compared to baseline was observed. The two experiments combined illustrate the accuracy for this marker both in vivo and ex vivo. Additionally, our data are in alignment with previous investigation of C3M as a marker for liver fibrosis, which demonstrated C3M was elevated in serum from a BDL rat model [23, 38] and a CCl4 inhalation model  respectively.
cAMP modulation has in other settings been shown to modulate MMP activity [30, 40, 41]. In particular in ex vivo models, cAMP induction by either the PDE inhibitor IBMX or induction of adenylate cyclase by forskolin, resulted in inhibition of MMP expression and activity . A similar pattern was seen in the current studies, suggesting that 1) cAMP is a general regulator of MMP activity and 2) that this model and biomarker may be used for important hypothesis testing and aid in the understanding of the processes of ECM remodeling in the liver during pathology. One drawback of the study is lack of intracellular cAMP measurement, thus we can only hypothesize that inhibition of PDE modulate cAMP levels as previously reported . Further research is needed to understand the effect of the PDE inhibition in hepatocellular models. The current findings suggest a possibly path of investigation in terms of using the described tools allowing for a deeper molecular understanding of fibrogenesis and fibrolysis.
Interesting, inhibition of MMP activity seemed to increase the MMP-2 and MMP-9 expressions, suggesting a regulatory feedback loop which augments MMP production in the absence of MMP activity. Such a feedback loop have previously been reported for protease inhibition, and suggest further caution when targeting protease inhibition on various diseases [27, 42]. Whether MMP-2 and MMP-9 play different roles in regulating fibrosis has not yet been elucidated; however it is generally accepted that both are involved in fibrogenesis and fibrosis resolution [43–45]. Gelatin zymography performed with acute liver injury studies showed an over-expression of both MMP-2 and MMP-9 in BDL rats compared to sham , while others have shown that only MMP-2 was up-regulated in CCl4 rats . In the current experiment, MMP-9 was highly expressed compared to MMP-2, in fibrotic livers. When interpreting the levels of MMP-2 and MMP-9, it is important to take into account the levels of Tissue Inhibitor of Matrix Metalloproteinase (TIMP) for a more complete picture of the proteolytic capacity . Further studies are needed to investigate this balance in different models of liver fibrosis, to understand the molecular action of each of the individual players that might be highly stage dependent [48, 49]. There was no clear inhibition of MMP activity in the presence of IBMX, albeit a significant lower release of the C3M fragment was demonstrated. The measurement of MMP-2 and MMP-9 is done by zymography in the conditioned medium. There may be a range of differences in the matrix of the fibrotic liver, and in that local activation milieu, compared to that of the “simple” conditioned medium. Thus, the cascade of activation proteases and generating the C3M fragment is dependent on MMP activity, and when only one single protease is present on MMP-9 activity. However, while in a more complex environment multiple protease are present, and both activation cascades and local positioning is complex and may be involved, while general MMP activity still is essential for generating the C3M fragment. This suggests that other MMPs are responsible for generation of this fragment than MMP-2 and MMP-9, and that other MMPs may have been attenuated. Thorough characterization of C3M is ongoing in our group. Investigation of in vitro cleaved type III collagen with ADAM-TS’s, MMP-1, MMP-3, MMP-7, MMP-12, and MMP-13 showed no reactivity using the C3M antibody (data not shown). However, further biological characterization is needed in order to understand the generation of the C3M fragment in vivo and ex vivo.
In the present study AlamarBlue was used for quantitative measurements of cell viability and proliferation. This dye has previously been undertaken in other settings by other researchers . Interestingly we found a 60% higher metabolic activity in fibrotic liver slices compared to vehicle, suggesting that the liver toxicity by CCl4 induced high activity within the liver tissue. This clearly demonstrated the grave pathological induction in the tissue by CCl4 that may be part of the processes leading to liver fibrosis. Furthermore, the cell viability remained the same as for fibrotic rat liver slices after addition of IBMX and GM6001 suggesting that the decreased levels of the C3M fragment released into the supernatants is not caused by compound toxicity but rather altered MMP activity. This phenomenon can be explained by the increased proliferation and ECM deposition by activated HSCs in liver fibrosis. Furthermore, during fibrogenesis hepatocytes undergo apoptosis thus releasing intracellular substances. Mitochondrial, cytosolic, and microsomal enzymes have been shown to reduce Alamar Blue , which might explain the increase in Alamar Blue activity in fibrotic liver slices compared to vehicle liver slices. Further studies are needed to understand this high metabolic activity in details.
The C3M assay was designed with that purpose, as the epitope is a conserved sequence through many species, and has been identified in: human, monkey, rat, mouse, and sheep. We used the C3M assay in order to investigate the type III collagen degradation in both ex vivo and in vivo model of liver fibrosis. We found a highly significant, and more than 100% fold up regulation of the neo-epitope in fibrotic liver tissue compared to the vehicle tissue in the ex vivo liver tissue cultures.
In the current experiments we used fibrotic livers, suggesting that the model may reflect parts of the processes of ECM remodeling in liver pathology. In alignment, as fibrotic livers represent a highly altered metabolic profile with activated cellular phenotypes [31, 36, 52], such systems may allow for improved translational research, compared to that of healthy livers, allowing a more predictive outcome. Further research is needed to understand whether this model may both be used for investigation of the processes involved in liver fibrogenesis as well as those processes involved in liver fibrosis resolution.
In conclusion, we have developed a novel ex vivo model system with pathological affected livers in a combination with a biochemical marker that is the result of extensive matrix remodeling associated with fibrosis. We found a highly significant, and more than 100% fold up regulation of the neo-epitope in fibrotic liver tissue compared to the vehicle tissue in the ex vivo liver tissue cultures. Following exposure of the liver explants to GM6001 or IBMX, C3M levels was restored to control levels. Gelatin zymography demonstrated CCl4-treated animals had highly increased MMP-9, but not MMP-2 activity, compared to slices derived from control animals. These data suggest that modulation of cAMP through PDE inhibition may play a role in the regulation of collagen turnover during liver fibrosis. Further studies are needed to proof this hypothesis, such as induction of adenylate cyclase by forskolin. As the biochemical marker C3M can be assessed in both ex vivo and preclinical, this may allow for better translational research assisting drug discovery and development in the liver fibrosis field.
A Disintegrin and metalloproteinase with trombospondin motifs
Bile duct ligation
Enzyme-linked immunosorbent assay
Fetal bovine serum
Food and drug administration
Hepatic stellate cell
Precision-cut liver slices
Standard error of mean
Tissue inhibitor of matrix metalloproteinase.
We thank the animal technicians Christina Hansen and Mia Jørgensen for performing the in vivo experiment and Henrik Simonsen for aiding the histology section. We also thank Per Engstrup for his keen interest and indispensable help with installing equipment.
Lastly, we acknowledge the funding from the Danish Science Foundation (Den Danske Forskningsfond).
- Friedman SL: Liver fibrosis – from bench to bedside. J Hepatol. 2003, 38 (Suppl 1): S38-S53.PubMedView ArticleGoogle Scholar
- Gressner OA, Weiskirchen R, Gressner AM: Biomarkers of liver fibrosis: clinical translation of molecular pathogenesis or based on liver-dependent malfunction tests. Clin Chim Acta. 2007, 381: 107-113. 10.1016/j.cca.2007.02.038.PubMedView ArticleGoogle Scholar
- Bedossa P, Dargere D, Paradis V: Sampling variability of liver fibrosis in chronic hepatitis C. Hepatology. 2003, 38: 1449-1457.PubMedView ArticleGoogle Scholar
- Maharaj B, Maharaj RJ, Leary WP, Cooppan RM, Naran AD, Pirie D: Sampling variability and its influence on the diagnostic yield of percutaneous needle biopsy of the liver. Lancet. 1986, 1: 523-525.PubMedView ArticleGoogle Scholar
- Veidal SS, Bay-Jensen AC, Tougas G, Karsdal MA, Vainer B: Serum markers of liver fibrosis: combining the BIPED classification and the neo-epitope approach in the development of new biomarkers. Dis Markers. 2010, 28: 15-28.PubMedPubMed CentralView ArticleGoogle Scholar
- Kumar VAAKFN: Tissue renewal and repair: regeneration, healing, and fibrosis. Pathologic basis of disease. 2005, Philadelphia, Pennsylvania, USA: Elsevier Saunders, 87-118. Ref Type: GenericGoogle Scholar
- Schuppan D: Structure of the extracellular matrix in normal and fibrotic liver: collagens and glycoproteins. Semin Liver Dis. 1990, 10: 1-10. 10.1055/s-2008-1040452.PubMedView ArticleGoogle Scholar
- Schuppan D, Ruehl M, Somasundaram R, Hahn EG: Matrix as a modulator of hepatic fibrogenesis. Semin Liver Dis. 2001, 21: 351-372. 10.1055/s-2001-17556.PubMedView ArticleGoogle Scholar
- Lochter A, Bissell MJ: An odyssey from breast to bone: multi-step control of mammary metastases and osteolysis by matrix metalloproteinases. APMIS. 1999, 107: 128-136. 10.1111/j.1699-0463.1999.tb01535.x.PubMedPubMed CentralView ArticleGoogle Scholar
- Karsdal MA, Madsen SH, Christiansen C, Henriksen K, Fosang AJ, Sondergaard BC: Cartilage degradation is fully reversible in the presence of aggrecanase but not matrix metalloproteinase activity. Arthritis Res Ther. 2008, 10: R63-10.1186/ar2434.PubMedPubMed CentralView ArticleGoogle Scholar
- Karsdal MA, Henriksen K, Leeming DJ, Mitchell P, Duffin K, Barascuk N: Biochemical markers and the FDA Critical Path: how biomarkers may contribute to the understanding of pathophysiology and provide unique and necessary tools for drug development. Biomarkers. 2009, 14: 181-202. 10.1080/13547500902777608.PubMedView ArticleGoogle Scholar
- Gelse K, Poschl E, Aigner T: Collagens–structure, function, and biosynthesis. Adv Drug Deliv Rev. 2003, 55: 1531-1546. 10.1016/j.addr.2003.08.002.PubMedView ArticleGoogle Scholar
- Garnero P, Ferreras M, Karsdal MA, Nicamhlaoibh R, Risteli J, Borel O: The type I collagen fragments ICTP and CTX reveal distinct enzymatic pathways of bone collagen degradation. J Bone Miner Res. 2003, 18: 859-867. 10.1359/jbmr.2003.18.5.859.PubMedView ArticleGoogle Scholar
- Gressner AM, Weiskirchen R: Modern pathogenetic concepts of liver fibrosis suggest stellate cells and TGF-beta as major players and therapeutic targets. J Cell Mol Med. 2006, 10: 76-99. 10.1111/j.1582-4934.2006.tb00292.x.PubMedPubMed CentralView ArticleGoogle Scholar
- Hemmann S, Graf J, Roderfeld M, Roeb E: Expression of MMPs and TIMPs in liver fibrosis - a systematic review with special emphasis on anti-fibrotic strategies. J Hepatol. 2007, 46: 955-975. 10.1016/j.jhep.2007.02.003.PubMedView ArticleGoogle Scholar
- Kirimlioglu H, Kirimlioglu V, Yilmaz S: Expression of matrix metalloproteinases 2 and 9 in donor liver, cirrhotic liver, and acute rejection after human liver transplantation. Transplant Proc. 2008, 40: 3574-3577. 10.1016/j.transproceed.2008.09.033.PubMedView ArticleGoogle Scholar
- Gieling RG, Wallace K, Han YP: Interleukin-1 participates in the progression from liver injury to fibrosis. Am J Physiol Gastrointest Liver Physiol. 2009, 296: G1324-G1331. 10.1152/ajpgi.90564.2008.PubMedPubMed CentralView ArticleGoogle Scholar
- Olsen AK, Sondergaard BC, Byrjalsen I, Tanko LB, Christiansen C, Muller A: Anabolic and catabolic function of chondrocyte ex vivo is reflected by the metabolic processing of type II collagen. Osteoarthritis Cartilage. 2007, 15: 335-342. 10.1016/j.joca.2006.08.015.PubMedView ArticleGoogle Scholar
- Bay-Jensen AC, Liu Q, Byrjalsen I, Li Y, Wang J, Pedersen C: Enzyme-linked immunosorbent assay (ELISAs) for metalloproteinase derived type II collagen neoepitope, CIIM–increased serum CIIM in subjects with severe radiographic osteoarthritis. Clin Biochem. 2011, 44: 423-429. 10.1016/j.clinbiochem.2011.01.001.PubMedView ArticleGoogle Scholar
- Leeming DJ, Larsen DV, Zhang C, Hi Y, Veidal SS, Nielsen RH: Enzyme-linked immunosorbent serum assays (ELISAs) for rat and human N-terminal pro-peptide of collagen type I (PINP)–assessment of corresponding epitopes. Clin Biochem. 2010, 43: 1249-1256. 10.1016/j.clinbiochem.2010.07.025.PubMedView ArticleGoogle Scholar
- Vassiliadis E, Larsen DV, Clausen RE, Veidal SS, Barascuk N, Larsen L: Measurement of CO3-610, a Potential Liver Biomarker Derived from Matrix Metalloproteinase-9 Degradation of Collagen Type III, in a Rat Model of Reversible Carbon-Tetrachloride-Induced Fibrosis. Biomark Insights. 2011, 6: 49-58.PubMedPubMed CentralView ArticleGoogle Scholar
- Barascuk N, Vassiliadis E, Larsen L, Wang J, Zheng Q, Xing R: Development and validation of an enzyme-linked immunosorbent assay for the quantification of a specific MMP-9 mediated degradation fragment of type III collagen-A novel biomarker of atherosclerotic plaque remodeling. Clin Biochem. 2011, 44: 900-906. 10.1016/j.clinbiochem.2011.04.004.PubMedView ArticleGoogle Scholar
- Barascuk N, Veidal SS, Larsen L, Larsen DV, Larsen MR, Wang J: A novel assay for extracellular matrix remodeling associated with liver fibrosis: An enzyme-linked immunosorbent assay (ELISA) for a MMP-9 proteolytically revealed neo-epitope of type III collagen. Clin Biochem. 2010, 43: 899-904. 10.1016/j.clinbiochem.2010.03.012.PubMedView ArticleGoogle Scholar
- Vassiliadis E, Veidal SS, Barascuk N, Mullick JB, Clausen RE, Larsen L: Measurement of matrix metalloproteinase 9-mediated collagen type III degradation fragment as a marker of skin fibrosis. BMC Dermatol. 2011, 11: 6-10.1186/1471-5945-11-6.PubMedPubMed CentralView ArticleGoogle Scholar
- Weiler-Normann C, Herkel J, Lohse AW: Mouse models of liver fibrosis. Z Gastroenterol. 2007, 45: 43-50. 10.1055/s-2006-927387.PubMedView ArticleGoogle Scholar
- Sondergaard BC, Wulf H, Henriksen K, Schaller S, Oestergaard S, Qvist P: Calcitonin directly attenuates collagen type II degradation by inhibition of matrix metalloproteinase expression and activity in articular chondrocytes. Osteoarthritis Cartilage. 2006, 14: 759-768. 10.1016/j.joca.2006.01.014.PubMedView ArticleGoogle Scholar
- Sondergaard BC, Henriksen K, Wulf H, Oestergaard S, Schurigt U, Brauer R: Relative contribution of matrix metalloprotease and cysteine protease activities to cytokine-stimulated articular cartilage degradation. Osteoarthritis Cartilage. 2006, 14: 738-748. 10.1016/j.joca.2006.01.016.PubMedView ArticleGoogle Scholar
- Friedman SL: Hepatic stellate cells: protean, multifunctional, and enigmatic cells of the liver. Physiol Rev. 2008, 88: 125-172. 10.1152/physrev.00013.2007.PubMedPubMed CentralView ArticleGoogle Scholar
- Ekins S: Past, present, and future applications of precision-cut liver slices for in vitro xenobiotic metabolism. Drug Metab Rev. 1996, 28: 591-623. 10.3109/03602539608994019.PubMedView ArticleGoogle Scholar
- Karsdal MA, Sumer EU, Wulf H, Madsen SH, Christiansen C, Fosang AJ: Induction of increased cAMP levels in articular chondrocytes blocks matrix metalloproteinase-mediated cartilage degradation, but not aggrecanase-mediated cartilage degradation. Arthritis Rheum. 2007, 56: 1549-1558. 10.1002/art.22599.PubMedView ArticleGoogle Scholar
- Van de Bovenkamp M, Groothuis GM, Draaisma AL, Merema MT, Bezuijen JI, van Gils MJ: Precision-cut liver slices as a new model to study toxicity-induced hepatic stellate cell activation in a physiologic milieu. Toxicol Sci. 2005, 85: 632-638. 10.1093/toxsci/kfi127.PubMedView ArticleGoogle Scholar
- Al-Nasiry S, Geusens N, Hanssens M, Luyten C, Pijnenborg R: The use of Alamar Blue assay for quantitative analysis of viability, migration and invasion of choriocarcinoma cells. Hum Reprod. 2007, 22: 1304-1309. 10.1093/humrep/dem011.PubMedView ArticleGoogle Scholar
- Van de Bovenkamp M, Groothuis GM, Meijer DK, Olinga P: Liver fibrosis in vitro: cell culture models and precision-cut liver slices. Toxicol In Vitro. 2007, 21: 545-557. 10.1016/j.tiv.2006.12.009.PubMedView ArticleGoogle Scholar
- Vickers AE, Fisher RL: Precision-cut organ slices to investigate target organ injury. Expert Opin Drug Metab Toxicol. 2005, 1: 687-699. 10.1517/17425255.1.4.687.PubMedView ArticleGoogle Scholar
- Olinga P, Groen K, Hof IH, De KR, Koster HJ, Leeman WR: Comparison of five incubation systems for rat liver slices using functional and viability parameters. J Pharmacol Toxicol Methods. 1997, 38: 59-69. 10.1016/S1056-8719(97)00060-9.PubMedView ArticleGoogle Scholar
- Olinga P, Merema MT, de Jager MH, Derks F, Melgert BN, Moshage H: Rat liver slices as a tool to study LPS-induced inflammatory response in the liver. J Hepatol. 2001, 35: 187-194. 10.1016/S0168-8278(01)00103-9.PubMedView ArticleGoogle Scholar
- Guyot C, Combe C, Balabaud C, Bioulac-Sage P, Desmouliere A: Fibrogenic cell fate during fibrotic tissue remodelling observed in rat and human cultured liver slices. J Hepatol. 2007, 46: 142-150. 10.1016/j.jhep.2006.08.013.PubMedView ArticleGoogle Scholar
- Veidal SS, Vassiliadis E, Barascuk N, Zhang C, Segovia-Silvestre T, Klickstein L: Matrix metalloproteinase-9-mediated type III collagen degradation as a novel serological biochemical marker for liver fibrogenesis. Liver Int. 2010, 30: 1293-1304. 10.1111/j.1478-3231.2010.02309.x.PubMedView ArticleGoogle Scholar
- Segovia-Silvestre T, Reichenbach V, Fernandez-Varo G, Vassiliadis E, Barascuk N, Morales-Ruiz M: Circulating CO3-610, a degradation product of collagen III, closely reflects liver collagen and portal pressure in rats with fibrosis. Fibrogenesis Tissue Repair. 2011, 4: 19-10.1186/1755-1536-4-19.PubMedPubMed CentralView ArticleGoogle Scholar
- Fantidis P: The role of intracellular 3'5'-cyclic adenosine monophosphate (cAMP) in atherosclerosis. Curr Vasc Pharmacol. 2010, 8: 464-472. 10.2174/157016110791330843.PubMedView ArticleGoogle Scholar
- Schippers M, Schmidt M, Spijkers K, Post E, Beljaars L, Poelstra K: 40th Keystone Symposia on Molecular and Cellular Biology. Prostaglandin E2-activated Epac-1 expression reduces collagen deposition in CCl4-induced liver fibrosis in mice. 2012, Ref Type: AbstractGoogle Scholar
- Schurigt U, Hummel KM, Petrow PK, Gajda M, Stockigt R, Middel P: Cathepsin K deficiency partially inhibits, but does not prevent, bone destruction in human tumor necrosis factor-transgenic mice. Arthritis Rheum. 2008, 58: 422-434. 10.1002/art.23224.PubMedView ArticleGoogle Scholar
- Iredale JP, Benyon RC, Pickering J, McCullen M, Northrop M, Pawley S: Mechanisms of spontaneous resolution of rat liver fibrosis. Hepatic stellate cell apoptosis and reduced hepatic expression of metalloproteinase inhibitors. J Clin Invest. 1998, 102: 538-549. 10.1172/JCI1018.PubMedPubMed CentralView ArticleGoogle Scholar
- Woessner JF: Matrix metalloproteinases and their inhibitors in connective tissue remodeling. FASEB J. 1991, 5: 2145-2154.PubMedGoogle Scholar
- Han YP: Matrix metalloproteinases, the pros and cons, in liver fibrosis. J Gastroenterol Hepatol. 2006, 21 (Suppl 3): S88-S91.PubMedPubMed CentralView ArticleGoogle Scholar
- Kossakowska AE, Edwards DR, Lee SS, Urbanski LS, Stabbler AL, Zhang CL: Altered balance between matrix metalloproteinases and their inhibitors in experimental biliary fibrosis. Am J Pathol. 1998, 153: 1895-1902. 10.1016/S0002-9440(10)65703-3.PubMedPubMed CentralView ArticleGoogle Scholar
- Takahara T, Furui K, Funaki J, Nakayama Y, Itoh H, Miyabayashi C: Increased expression of matrix metalloproteinase-II in experimental liver fibrosis in rats. Hepatology. 1995, 21: 787-795. 10.1002/hep.1840210328.PubMedView ArticleGoogle Scholar
- Lee UE, Friedman SL: Mechanisms of hepatic fibrogenesis. Best Pract Res Clin Gastroenterol. 2011, 25: 195-206. 10.1016/j.bpg.2011.02.005.PubMedPubMed CentralView ArticleGoogle Scholar
- Jiao J, Sastre D, Fiel MI, Lee UE, Ghiassi-Nejad Z, Ginhoux F: Dendritic cell regulation of carbon tetrachloride-induced murine liver fibrosis regression. Hepatology. 2012, 55: 244-255. 10.1002/hep.24621.PubMedPubMed CentralView ArticleGoogle Scholar
- Karsdal MA, Larsen L, Engsig MT, Lou H, Ferreras M, Lochter A: Matrix metalloproteinase-dependent activation of latent transforming growth factor-beta controls the conversion of osteoblasts into osteocytes by blocking osteoblast apoptosis. J Biol Chem. 2002, 277: 44061-44067. 10.1074/jbc.M207205200.PubMedView ArticleGoogle Scholar
- Gonzalez RJ, Tarloff JB: Evaluation of hepatic subcellular fractions for Alamar blue and MTT reductase activity. Toxicol In Vitro. 2001, 15: 257-259. 10.1016/S0887-2333(01)00014-5.PubMedView ArticleGoogle Scholar
- Van de Bovenkamp M, Groothuis GM, Meijer DK, Slooff MJ, Olinga P: Human liver slices as an in vitro model to study toxicity-induced hepatic stellate cell activation in a multicellular milieu. Chem Biol Interact. 2006, 162: 62-69. 10.1016/j.cbi.2006.05.006.PubMedView ArticleGoogle Scholar
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