Liver tissue samples
Male Wistar rats of 200-250 g body weight were obtained from Charles River Laboratories (Wilmington, MA, USA). Liver fibrosis was induced by administering 200 mg/kg thioacetamide for 2 wk as described before . If not noted otherwise, reagents were purchased from Merck (Darmstadt, Germany) or from Sigma (Deisenhofen, Germany) and were of the highest purity available. Tissue samples were either fixed by 4% (vol/vol) formalin and embedded in paraffin or prepared for cryostat sections. Animal protocols were approved by the regional animal study committee. Specimens of cirrhotic human livers were obtained from explanted livers from patients with alcoholic cirrhosis undergoing orthotopic liver transplantation. Informed consent was obtained prior to surgery. Immediately after explantation, tissue samples were snap-frozen and stored over liquid nitrogen.
In vitro activation of MMP-2 and MMP-9
The 62-kDa actMMP-2 (Invitek, Berlin, Germany) was released from 27.8 nM 72-kDa proMMP-2 in 1 mM APMA in MMP activity buffer consisting of 50 mM Tris·HCl, pH 7.5, 200 mM NaCl, 5 mM CaCl2, and 0.02% (vol/vol) Brij-35 for 1 h. The 86-kDa actMMP-9 (Invitek) was obtained from 217 nM 92-kDa proMMP-9 in 80-μl MMP activity buffer without Brij-35 incubated with 100 μg/ml chymotrypsin activity-blocked trypsin for 20 min. Tryptic digestion was terminated by 100 μg/ml aprotinin within 10 min. All steps were performed at 37°C. These completely activated MMPs as end products of the in vitro activation structurally and functionally correspond to those found in vivo[42, 43]. All MMPs were stored in aliquots at -80°C. The (pro)MMP activation state was routinely checked by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) substrate zymography.
Radiolabeling of pro- and actMMP-2 and pro- and actMMP-9
Human recombinant proMMPs and in vitro activated MMPs were obtained from Invitek  and were labeled with the [125I]-Bolton-Hunter reagent according to the manufacturer's instructions (PerkinElmer, Rodgau, Germany) before the buffer was exchanged for phosphate-buffered saline (PBS) with 0.05% (vol/vol) Tween 20 by gel filtration as described previously . Specific radioactivity for [125I]-MMPs was 3-9 × 104 cpm/ng. Precipitation with 10% (wt/vol) trichloroacetic acid and 200 μg of BSA recovered 96% to 100% of protein-bound radioactivity. Aliquots of labeled MMPs were frozen and stored at -80°C. The activity and integrity were checked by substrate gel zymography using SDS-PAGE and autoradiography with overnight exposure to Biomax MS film (Kodak, Stuttgart, Germany) (Figure 2A).
Preparation of collagens, CI derivatives and structural analogs
Native human CI, CIII and CVI were purified from skin tissue and placenta. The CI single chains α1(I) and α2(I) were obtained and modified as described previously . To prepare defined fragments, 2 mg of α1(I) were dissolved in 1 ml of 70% (vol/vol) formic acid at room temperature, the tubes were flushed with nitrogen for 10 min, and then 2 mg of CB were added. After incubation for 4 h at 37°C, free CB was neutralized and the samples were lyophilized. The peptides CB3, CB6, CB7 and CB8 were separated from the reaction mixture by gel filtration followed by ion-exchange chromatography. The resulting peptides were characterized by amino acid analysis and SDS-PAGE [45, 46]. The CB peptides had the following melting temperatures: CB3, 23.9°C; CB6, 26.7°C; CB7, 28.1°C; and CB8, 28.0°C .
The following collagen analogs and control peptides were synthesized as described previously : (GPO)10, H-Gly-Cys-Hyp-(Gly-Pro-Hyp)10-Gly-Cys-Hyp-Gly-NH2; (GPP)10, H-Gly-Cys-Pro-(Gly-Pro-Pro)10-Gly-Cys-Pro-Gly-NH2; and GAP, H-Gly-Ala-Cys-(Gly-Ala-Pro)5-Gly-Phe-Hyp-Gly-Glu-Arg-(Gly-Ala-Pro)5-NH2. Peptides (POG)10, (PPG)10 and (POG)5 were purchased from Peptide International (Louisville, KY, USA). Spontaneous triple-helix assembly was approved by polarimetry over a 10-cm path length at 1°C/min in 10 mM phosphate buffer, pH 7.4. At 5 mg/ml, midpoints of melting curves occurred at 82.3 ± 1.4°C for (GPO)10 and at 45.8 ± 0.8°C for (GPP)10. Peptide GAP was determined to be nonhelical even at 5°C. Graphs were calculated from the primary data using a custom fitting program written by D.A. Slatter (Department of Biochemistry, University of Cambridge, Cambridge, UK ) to model different possible transitions. All collagens, CI derivatives and peptides were stored in stock solutions of 2 mg/ml in 150 mM acetic acid at -20°C.
Histological detection of connective tissue
In rat liver samples, connective tissue was visualized using Sirius red staining in thin sections of formalin-fixed, paraffin-embedded tissue samples . Cryostat sections of human liver samples were fixed with 1% (vol/vol) formalin for 10 min before being stained with Sirius red. Slides were assessed using standard light microscopy (Olympus, Hamburg, Germany).
In situ zymography
As described earlier, in situ zymography was performed with cryostat sections (6 μm) of rat cirrhotic liver [41, 49]. In brief, sections were dried, overlayed with 100 μg/ml DQ-gelatine (λex/em, 495/515 nm; Molecular Probes, Eugene, OR, USA) and 0.5% (wt/vol) low-melt agarose in MMP activity buffer. For negative controls, 10 mM ethylenediaminetetraacetic acid or 1 mM phenanthroline was included to the reaction mixture, after which no generation of bright green fluorescence was observed, implying inhibition of gelatinase activity . Samples were inserted into coverslips and incubated at 40°C for 1 h before being transferred to room temperature for an additional 2 to 16 h. Hoechst 33342 (Invitrogen, Carlsbad, California, USA) nuclear dye was used for counterstaining. Images were obtained by fluorescence microscopy using a Nikon E800 photodocumentation microscope (Nikon Imaging, Düsseldorf, Germany).
In situ binding of (pro)MMP-2 and (pro)MMP-9
Human cirrhotic liver cryostat sections (5 μm) were air-dried and fixed in ice-cold acetone for 10 min. Tissue sections were rehydrated with PBS and incubated with 25 ng/50 μl of the respective proMMP and actMMP for 30 min or were left untreated. After thorough washing with PBS, antibodies specific for human MMP-9 (clone MAB911; R&D Systems, Minneapolis, MN, USA) and human MMP-2 (clone 75-7F7; Oncogene, Cambridge, MA, USA) were applied, and primary antibody binding was detected using the alkaline phosphatase-antialkaline phosphatase detection system (Dako, Hamburg, Germany). An irrelevant primary mouse antibody served as control. Nuclei were counterstained with Hemalaun, and slides were examined by standard light microscopy.
Solid-phase binding studies
ProMMP-2 and proMMP-9 or actMMP-2 and actMMP-9 were bound to nitrocellulose and polystyrene-immobilized native collagens, CI chains, CB peptides or structural analogs. Serial dilutions of collagens or CI derivatives in 150 mM acetic acid were dotted at 3 × 3 μl to a nitrocellulose membrane with high protein-binding capacity (GE Healthcare, Munich, Germany). Air-dried membranes were blocked with PBS and 0.3% (vol/vol) Tween 20 overnight at 4°C, washed three times, and incubated with 1 ng/ml [125I]-pro- and actMMP-2 and pro- and actMMP-9 in PBS and 0.3% (vol/vol) Tween 20 for 2 h at room temperature. Membranes were washed again and air-dried, and bound MMP was monitored by autoradiography. In parallel, polystyrene microtiter plates (Dynex, Chantilly, VA, USA) were coated with collagen proteins and peptides. Here 2 μg/well or 200 ng/well proteins and peptides or BSA as control were immobilized in 100 μl of 50 mM ammonium bicarbonate buffer, pH 9.6, by overnight incubation at 4°C. Immobilization efficacies were 20% to 45% of total proteins . Wells were washed three times with PBS, and nonspecific binding sites were blocked with PBS and 0.05% (vol/vol) Tween 20 for 1 h at room temperature. All incubation steps were performed with 2 ng of [125I]-MMPs at 4°C for 2 h. Unbound reagents were removed by thorough washing with PBS and 0.05% (vol/vol) Tween 20, and residual radioactivity was determined using a gamma counter (Berthold, Bad Wildbach, Germany).
Surface plasmon resonance analysis
Sensor chip preparations and SPR measurements were performed using a BiacoreX device and the Bia-evaluation software (version 3.2; Biacore, Uppsala, Sweden). The pepsin-resistant triple-helical part of human fibrillar CI (100 μg/ml) in 10 mM acetate coupling buffer, pH 4.8, was immobilized to a dextran matrix-sensor chip at a flow rate of 5 μl/min, resulting in 5,500 resonance units from CI covalently linked via its primary amino groups. The control flow cell was prepared using the coupling buffer without CI. Surfaces were activated and blocked as described previously . Immediately after thawing, pro- and actMMP-2 and pro- and actMMP-9 were diluted to 100 to 250 nM in MMP activity buffer or in PBS and 0.05% (vol/vol) Tween 20. For SPR measurements, flow rates were 10 μl/min at 25°C, and equilibrium was typically reached after 30 to 60 s. The effects of (GPO)10, GAP, (POG)5, (GPP)10 and P33-42 on MMP-2 and MMP-9 binding to CI and their enzymatic activity were determined by adding the binding competitors to the (pro- and act)MMP-2 and MMP-9 solution in 10- to 150-fold molar excesses. Effects independent of MMP-2 and MMP-9 activity were monitored in the presence of Ro 28-2653 (1 μM) during SPR analysis. The gelatinase inhibitor Ro 28-2653 was a generous gift from H.-W. Krell (Roche, Grenzach-Wyhlen, Germany). Sensor surfaces were regenerated with 10 mM glycine, pH 2.3, for 1 min between runs, and sensor chips were used up to 25 times. Kinetic parameters were analyzed using the 1:1 binding model with drifting baseline and subtraction of the control flow cell binding from sensorgrams obtained with immobilized CI. Binding constants (K
) were calculated from the association (k
) and dissociation rates (k
) obtained from individual binding curves at different concentrations. Individual drifts of the resonance signal were fitted locally, and χ2 values of 0.2% to 1.0% of the maximum resonance value were considered good fits.
Fluorogenic MMP activity assay
Enzymatic activities of MMPs were studied spectrofluorimetrically by cleavage of fluorogenic substrates in MMP activity buffer within 2 to 5 h. For gelatinases 800 nM MCA-Pro-Leu-Gly-Leu-Dnp-Dap-Ala-Arg-NH2 (λex/em 328/393 nm; Bachem, Bubendorf, Switzerland) or 10 μg/ml DQ-gelatine were used, according to the method described by Knight et al. for collagenases 800 nM MCA-Pro-Cha-Gly-Nva-His-Ala-Dpa-NH2 (λex/em 280/360 nm; Anaspec, San José, CA, USA). In some experiments, proMMP-2 was fully activated in the presence of 1 mM APMA prior to the kinetic measurements. A quantity of 50 ng pro- or actMMP-2 alone or mixed with 10- to 150-fold molar excesses of (GPO)10, GAP, P33-42 or mixtures of (GPO)10 and P33-42, were added to CI-coated, BSA-coated (1 μg/well both) or uncoated wells containing 150 μl of the respective substrate solution. The peptide P33-42 was purchased from the Institute of Biochemistry (Humboldt-University, Berlin, Germany). The influence of CI on MMP-2 enzymatic activity against the quenched fluorescent substrate could be excluded . Background subtraction (measurement without MMPs) was applied to all curves. All experiments were performed with a fluorescence microplate reader (Molecular Devices, Sunnyvale, CA, USA) and black 96-well microtiter plates with a clear bottom (Greiner bio-one, Frickenhausen, Germany).
Samples containing MMP-2 were diluted with zymogram sample buffer (Bio-Rad, Munich, Germany) and separated on homogeneous 10% SDS-PAGE gels containing 1 mg/ml (wt/vol) gelatine (Bio-Rad), washed with excess MMP activity buffer containing 2.5% (vol/vol) Triton X-100 to remove SDS, and incubated with MMP activity buffer for 24 h. Gels were stained with Coomassie Blue R-250. Gels showing proteolytic bands corresponding to proMMP-2 (72 kDa) or actMMP-2 (62 kDa) were scanned (Plustek, Norderstedt, Germany) and analyzed from inverted grayscale images.
Release of in situ bound proMMP-2
ProMMP-2 was labeled using the FluoroLink Cy2 Labeling Kit according to the manufacturer's instructions (Amersham Biosciences, Freiburg, Germany). Unbound fluorescent dye was removed by ultrafiltration (Nanosep, Lund, Sweden), and labeling success was monitored using a fluorescence microplate reader (λex/em, 489/506 nm). Serial cirrhotic human liver sections were covered with 1.2 μg/ml Cy2-proMMP-2 in 50 mM Tris·HCl, pH 7.4, containing 1 mM CaCl2 or with buffer alone, and were incubated in a dark humidified chamber for 24 h at 4°C. To study effects of (GPO)10, a 10-fold molar excess in relation to proMMP-2 was added to slides prior to or after Cy2-proMMP-2 binding. Slides were washed with PBS, air-dried, and rinsed with deionized water. Bound Cy2-proMMP-2 was detected by fluorescence microscopy (Olympus, Hamburg, Germany).
One-way analysis of variance and Tukey's tests were performed using SigmaStat for Windows version 2.03 (Sigmaplot, Erkrath, Germany), and P < 0.05 was considered significantly different.