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(The FASEB Journal. 2006;20:444-454.)
© 2006 FASEB

Inhibition of hepatic fibrogenesis by matrix metalloproteinase-9 mutants in mice

Martin Roderfeld*,§, Ralf Weiskirchen{dagger}, Sandra Wagner*, Marie-Luise Berres*, Corinna Henkel*, Joachim Grötzinger{ddagger}, Axel M. Gressner{dagger}, Siegfried Matern* and Elke Roeb*,§

* Department of Internal Medicine III and
{dagger} Institute of Clinical Chemistry and Pathobiochemistry, RWTH University Hospital Aachen, Aachen, Germany;
{ddagger} Institute of Biochemistry, Christian-Albrechts-University Kiel, Kiel, Germany; and
§ Department of Internal Medicine II, Justus-Liebig University, Giessen, Germany

1Correspondence: Department of Internal Medicine II, Justus-Liebig University, Giessen Paul-Meimberg-Str. 5, D-35385 Giessen, Germany. E-mail: elke.roeb{at}innere.med.uni-giessen.de


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Tissue inhibitor of metalloproteinases-1 (TIMP-1) plays a crucial role in the pathogenesis of hepatic fibrosis and thus may represent an important therapeutic target in the design of anti-fibrotic strategies for chronic liver disease. We present an innovative therapy based on the assignment of inactivated enzymes acting as scavengers for TIMP-1. Hepatic fibrosis was induced in BALB/c mice by repetitive intraperitoneal CCl4 injection. The animals were treated with proteolytic inactive matrix metalloproteinase-9 mutants (E402Q, H401A, E402H/H411E) using adenovirus-mediated gene transfer. Application of these MMP-9 mutants inhibited fibrogenesis, which was indicated by decreasing portal and periportal accumulation of collagen. Total hydroxyproline of liver tissue, the morphometric stage of fibrosis as well as mRNA expression of marker proteins for hepatic fibrosis in livers of E402Q- and H401A-treated mice were significantly reduced. MMP-9 mutants suppressed transdifferentiation of hepatic stellate cells to the myofibroblast like phenotype in vitro and in vivo. Moreover, adenoviral application of the mutants MMP-9-H401A and -E402Q led to increased apoptosis of activated hepatic stellate cells, thought to be the main promoters of hepatic fibrosis. Application of MMP-9 mutants as TIMP-1 scavengers may provide a new therapeutic strategy for hepatic fibrosis.—Roderfeld, M., Weiskirchen, R., Wagner, S., Berres, M.-L., Henkel, C., Grötzinger, J., Gressner, A. M., Matern, S., Roeb, E. Inhibition of hepatic fibrogenesis by matrix metalloproteinase-9 mutants in mice.


Key Words: liver • fibrosis • MMP-9 • TIMP-1 • hepatic stellate cells


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
THE IMBALANCE BETWEEN MATRIX METALLOPROTEINASESand tissue inhibitors of matrix metalloproteinases (TIMPs) is considered to be a pivotal parameter of deposition and breakdown of the extracellular matrix (1) . TIMP-1, the most important endogenous inhibitor of most MMPs, plays a crucial role in the pathogenesis of liver fibrosis and may represent an important therapeutic target in the design of anti-fibrotic strategies for chronic liver disease (1 2 3 4) .

Previously we demonstrated that TIMP-1 is expressed in rat liver and regulated by inflammatory cytokines (5) . Furthermore, TIMP-1 is up-regulated by mouse oncostatin M, cardiotrophin-1, and transforming growth factor-ß (TGF-ß) (6 7 8 9) . Studies in cultured primary rat hepatocytes, hepatic stellate cells (HSC), and rat models of liver fibrosis revealed an association of progressive fibrosis and increased TIMP-1 expression (5 , 10 11 12 13) . In human liver disease, TIMP-1 expression is up-regulated 5-fold in cirrhotic compared with normal liver, and elevated TIMP-1 levels could be measured in plasma of such patients (14) . Its expression occurred predominantly in areas of inflammation, in mesenchymal cells, hepatocytes, and endothelial cells (12 , 15) . Chronic viral hepatitis B and C resulted in a significant increase in plasma TIMP-1 levels without elevation of TGF-ß1, a central mediator of fibrosis (16) . Aminotransferase activity, a surrogate parameter for the inflammatory activity of viral hepatitis, correlated significantly with TIMP-1 expression. Thus, TIMP-1 plays a pivotal role in liver fibrosis. TIMP-1 plasma levels may prove useful as an early noninvasive marker of fibrosis, eventually suitable for the clinical management of chronic hepatitis (17) . TIMP-1 overexpression does not result in liver fibrosis by itself, but strongly promotes liver fibrosis development (18) .

Recently, researchers have paid much attention to the reversal of hepatic fibrosis (3 , 19) . Previous experiments demonstrated that TIMP-1 attenuates spontaneous resolution of liver fibrosis by the combination of a net reduction of the MMP activity and suppression of apoptosis in HSC (20) . Murphy et al. demonstrated that TIMP-1 has anti-apoptotic effects in HSC, strongly depending on the level of MMP inhibition (21) . They also demonstrated that persistent TIMP-1 expression in a model of carbon tetrachloride- (CCl4) -induced cirrhosis is associated with persistence of HSC and decreased recovery of liver fibrosis. Recovery from comparatively advanced cirrhosis is possible and results in remodeling from micronodular cirrhosis to macronodular cirrhosis. Nevertheless, resolution of fibrosis is limited by a failure of HSC apoptosis (19) . Increased TIMP-1 production in HSC and their central role in the pathogenesis of liver fibrosis may represent a promising therapeutic target in the design of anti-fibrotic strategies (2) . Therefore, a specific neutralization of TIMP-1 would be beneficial. Many stimulators of TIMP-1 expression have been characterized, but little is known about blockade of TIMP-1 expression. Since the cloning of TIMP-1 in 1986, a reduction of TIMP-1 expression could only be demonstrated by dexamethasone (5) and concanavalin A (22) . Histological findings from patients with liver fibrosis and from animal models of fibrosis indicate that recovery from liver fibrosis with diminution of excess ECM proteins is possible (3 , 19 , 20) . During recovery, an increased collagenase activity could be detected as a key mechanism in spontaneous resolution of fibrosis in rat liver homogenates (3) . As fibrotic liver already exhibits an increased proteolytic activity, which is inhibited by high TIMP-1 activity, a possible therapeutic approach to interfere with fibrogenesis may be the specific neutralization of TIMP-1. We have already shown that a catalytically inactive MMP-9 (MMP-9-H401A) that still binds TIMP-1 can be used as a specific antagonist of TIMP-1 activity in vitro (23) . The primary objective of this study was to investigate the anti-fibrotic potential of WT-MMP-9 and MMP-9 mutants. We show that several adenovirally (Ad) transferred, proteolytically inactive MMP-9 mutants inhibit hepatic fibrogenesis in vivo. This is the first time that an inactivated enzyme acting as a TIMP-1 scavenger is used as a novel therapeutic agent against fibrosis.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Construction of recombinant adenoviruses
The vector p{Delta}E1sp1A-CMV-EGFP expressing the enhanced green fluorescent protein (EGFP) has been described (24) . For generation of the different adenoviruses expressing MMP-9 derivatives, the respective ~2.9 kbp fragments of pRc/CMV-mGelB, pRc/CMV-mGelB(E402Q), pRc/CMV-mGelB(H401A), or pRc/CMV-mGelB(E402H/H411E) were integrated into the vector p{Delta}E1sp1A-CMV-EGFP, depleted for EGFP. The integrity of all cloning boundaries was verified by sequencing. The integration of cloned sequences into the Ad5-backbone vector pJM17 (25) was performed by in vitro homologous recombination as described elsewhere (24) .

Cell culture and adenovirus transfection
Human hepatoma cells (HepG2) were cultured as described before (23) and transfected using a virus concentration of 2 x 108 pfu/mL in DMEM-F12 containing 5% (v/v) heat inactivated fetal calf serum, 100 mg/L streptomycin, and 60 mg/L penicillin for 16 h. Cells were cultured for 24 h with renewed medium, and thereafter grown under serum-free conditions for 48 h before harvest.

Protein analysis
Silver staining of polyacrylamide gels and Western blot (ECL Kit, Amersham, Freiburg, Germany) were performed as described before (26) . Immunodetection of proteins was achieved using goat anti-mouse MMP-9 IgG antibodies with a final dilution of 4.3 µg/mL (gift from G. Murphy, University of East Anglia, School of Biological Sciences, UK). For visualization of immunodetected proteins a horseradish peroxidase coupled donkey anti-sheep/goat antibody with a final dilution of 1 µg/mL (Biodesign Int., Saco, ME, USA) was used.

Purification of MMP-9 mutants
The pooled HepG2 conditioned media were sterile-filtered, and dialyzed against "native buffer" (NB) containing 100 mM Tris/HCl pH 7.5; 50 mM NaCl; 1 mM CaCl2; 0.02% (w/v) PEG6000. The MMP-9 variants in the prepurified samples were isolated in a one step affinity chromatography using a gelatin-Sepharose column (27) .

Activity and inhibition analysis
Proteolytic inactivity of the recombinant MMP-9 mutants was shown by gelatin zymography (28) and a MMP activity assay (29) . For gelatin zymography 1 mg/mL gelatin was copolymerized in a 7.5% PAGE gel and 10 ng of each purified mutant was loaded. The MMP activity assay was performed on a 96-well plate using a Tecan Spectra Fluor fluorescence spectrometer (Tecan, Crailsheim, Austria) with the synthetic fluorogenic MMP-substrate MCA-Pro-Leu-Gly-Leu-Dpa-Ala-Arg (1 µM final concentration) and a MMP-9 concentration of 0.5 nM (in a final volume of 150 µL). The TIMP-1 antagonistic effect of the MMP-9 mutants was measured by addition of mutants (360 nM) to MMP-9 (0.5 nM) inhibited by TIMP-1 (19 nM, R&D Biosystems, Abingdon, UK).

Animal treatment
Six-wk-old male BALB/c mice (17–27 g, Taconic M&B Ry, Denmark) were injected twice a week intraperitoneally with CCl4 (0.8 mL/kg as a 1:1 mixture with mineral oil and mineral oil solely for control) for 4 wk to induce liver fibrosis. After sacrifice, liver tissue samples of the right median lobe were snap-frozen in liquid nitrogen or fixed in 10% buffered formalin. Animal studies were conducted according to the national animal welfare regulations and approved by the local Animal Care and Use Committee of Aachen University.

Application of adenoviruses
Analysis of time-dependent expression of EGFP in mouse liver after a single injection of Ad5-CMV-EGFP (1.5x109 pfu) via tail vein displayed a maximum protein expression after 3–4 days. Therefore, mice were infected twice a week with recombinant adenovirus (1.5x109 pfu, 50 µL), followed by i.p. injection of either CCl4 or pure mineral oil the day after.

Histology, immunohistochemistry, and hydroxyproline assay
For detection of collagen fibers, paraffin sections of 5 µm thickness were stained in 0.1% Sirius red F3B (SR) in saturated picric acid (Chroma, Münster, Germany) (30) . Morphometric analysis of hepatic fibrosis was performed using semiquantitative fibrosis scores in similarity to the score system suggested by Batts and Ludwig (31) and Ishak et al. (32) for hepatic fibrosis in humans. Healthy liver was classified as 0. Fibrous expansion of the portal areas was scored as 1. Stage 2 denotes septal fibrosis with marked fibrous septa, and stage 3 was characterized by portal-portal septa (bridging fibrosis but intact architecture). The advanced fibrotic stage 4 (i.e., cirrhosis, characterized by bridging fibrosis with nodules) was not observed in BALB/c mice after 4 wk of CCl4 treatment. Three different blinded persons determined the fibrotic scores, which were used in mean for the analysis. Anti-mouse {alpha}-smooth muscle actin ({alpha}-SMA) antibodies (PROGEN, Heidelberg, Germany) and polyclonal anti collagen type I IgG (M-19-sc-8788, Santa Cruz, CA, USA) were used for immunohistochemistry in combination with the Vectastain Elite ABC kit (Vector Laboratories, Burlingame, CA, USA). Entire collagen was determined by hydroxyproline quantification (33) .

Determination of mRNA using light cycler quantitative PCR
Real-time quantitative RT-PCR was executed using the LightCycler Fast Start DNA Master SYBR Green I kit (Roche, Mannheim, Germany). The amounts of mRNA for actin, MMP-2, MMP-13, TGF-ß, type I collagen, and TIMP-1 were quantified. Only data providing significant results were discussed. All primers were obtained from MWG (Ebersberg, Germany).

HSC isolation and culturing
HSC were isolated from male Sprague-Dawley rats by pronase-collagenase method followed by a density centrifugation on a Nycodenz gradient (34 , 35) . Purified cells were seeded and cultured following established protocols (36) .

Transdifferentiation and Annexin-V-FLUOS assay
To determine the influence of the different MMP-9 mutants on the grade of transdifferentiation of HSC, cells were transfected (36) and harvested 4 days later. {alpha}-SMA expression was quantified by densitometric Western blot analysis of lysates. The beginning of early apoptosis was monitored by the Annexin-V-FLUOS Assay (Roche) using assay conditions as published before (36) .

Molecular modeling
The structure of MMP-3 in complex with TIMP-1 (37) (pdb code: 1UEA) was used as template to build a 3-dimensional model of the MMP-9/TIMP-1 complex. Alignment was performed using the Clustal W algorithm (http://www.ebi.ac.uk/clustalw). Modeling was elaborated using the software package WHATIF (38). Model structures were energy-minimized using the steepest descent algorithm implemented in the GROMOS force field (van Gunsteren, W. F., BIOMOS biomol. software b.v., Laboratory of Physical Chemistry, University of Groningen, The Netherlands). The structural representations were generated with the RIBBON 2.0 program developed by M. Carson (39) .

Statistics
The number of experiments and animals is indicated by n and used for calculation of mean values and statistical variation. Results are mean ± standard error of the mean (SEM). ANOVA or an unpaired Student’s t test was used for statistical comparisons. Differences at P< 0.05 were considered to be significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Adenoviral expression of MMP-9 mutants in HepG2 cells
In recent years vectors based on adenovirus-2 (Ad2) or adenovirus-5 (Ad5) have emerged as the most efficient vehicles of transferring genes into liver cells. In particular, the introduction of genes into HSC and their transdifferentiated phenotype, i.e., the myofibroblasts (MFB) is difficult to achieve, in part due to the quiescent and fragile phenotype of HSC or the extracellular matrix in which the MFB are embedded. To investigate the effect of MMP-9 and TIMP-1 in liver fibrogenesis, we constructed several Ad5-derivatives expressing proteolytically inactive MMP-9 mutants and MMP-9 (Fig. 1 a). All experiments were performed with murine MMP-9 and mutants thereof. Adenoviral vectors were checked for functionality in culture using the human hepatoma cell line HepG2 as host cell. After adenoviral infection, protein expression was verified by SDS-PAGE and Western blot analysis of cell culture media (lanes 1–4 in Fig. 1b ). We found that the expression amounts of MMP-9 mutants H401A, E402Q, and E402H/H411E as well as wild-type (WT) enzyme in conditioned medium varied. WT-MMP-9 protein was quantified by ELISA 48 h after cell seeding in a range of 5–10 nM, while mutants were found in concentrations of 1–5 nM. Adenoviral expression of MMP-9 variants had no effect on cell viability in comparison to mock-transfected cells overexpressing EGFP (data not shown).


Figure 1
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  		Figure 1. Cloning, expression, purification, and in vitro characterization of MMP-9 mutants. a) Schematic representation of the cloning strategy. The expression cassette of pRc/CMV-mMMP-9 was transferred into the shuttle vector p{Delta}E1sp1A-CMV-EGFP depleted for CMV and EGFP. BGHpA, bovine growth hormone polyadenylation signal, CMV, cytomegalovirus immediate early gene 1 promoter/enhancer; SV40pA, simian virus 40 polyadenylation signal. b) In lanes 1–4 conditioned cell media bearing the respective mutants were loaded. Lanes 5–8 display the purified and enriched samples of the MMP-9 mutants. Secretion medium of mock-infected cells served as negative control (lane 9) and 20 ng recombinant murine MMP-9 as positive control (lane 10). c) Proteolytic inactivity of the MMP-9 mutants in lanes 2–4 was shown in comparison to WT-MMP-9 in lane 1 by gelatin zymography. d) White bar displays the normalized activity of MMP-9. The addition of TIMP-1 caused an inhibition of MMP-9 activity, indicated by the black bar, which was abrogated by addition of the MMP-9 mutants (halftone bars).

Purification and characterization of MMP-9 mutants
Before MMP-9 mutants were used to antagonize TIMP-1 in mice, they were characterized in vitro. Recombinant MMP-9 mutants were enriched and purified from secretion medium by affinity chromatography using gelatin-Sepharose. We extracted 50–200 µg (of ~80–90% purity) of MMP-9 mutants from 200 mL medium. Cell culture media samples (lanes 1–4) were compared with purified material (lanes 5–8) in silver-stained PAGE gel and MMP-9 Western blot (Fig. 1b ).

Two main characteristics of the MMP-9 mutants developed as TIMP-1 antagonists had to be proven in vitro: 1) proteolytical inactivity, and 2) the ability to complex TIMP-1 with high affinity. The complete loss of proteolytic activity of all MMP-9 mutants tested was shown in comparison to WT-MMP-9 using two different activity assays. First we used a gelatin zymography assay loaded with equal amounts (10 ng) of WT-MMP-9 (lane 1), H401A mutant (lane 2), E402Q mutant (lane 3), and E402H/H411E mutant (lane 4) (Fig. 1c ). Only WT-MMP-9 was able to degrade gelatin and produced the characteristic degradation band of 110 kDa for latent murine MMP-9. A synthetic fluorogenic MMP substrate composed of a hexapeptide with a specific MMP cleavage site carrying a highly fluorescent 7-methoxycoumarin group N-terminally that is efficiently quenched by energy transfer to the 2, 4-dinitrophenyl group at the C-terminal site was used for the second assay (29) . Proteolytic cleavage of the peptide abrogates the quenching effect. The increase in fluorescence was measured indicating MMP activity. All three mutants exhibited no proteolytic activity against the synthetic substrate. The activity of WT-MMP-9 was determined as kcat/KM value to 1.26 x 104 M–1s–1 (data not shown).

Antagonization of TIMP-1 was measured using similar conditions. In the respective assay, MMP-9 activity was set as 100% (Fig. 1d ). Addition of murine TIMP-1 led to significant inhibition of ~32 ± 7.7%, which was abrogated significantly by addition of the MMP-9 mutants (Fig. 1d ). The mutant E402H/H411E showed the highest potential for TIMP-1 antagonization in this quantitative in vitro assay. 91 ± 1.3% of the primary MMP-9 activity could be restored by surplus addition of MMP-9-E402H/H411E. Addition of MMP-9-E402Q lead to 88 ± 2.0% of MMP-9 activity and when MMP-9-H401A mutant was added 80 ± 4.4% of the original MMP-9 activity was regained. Thus, we were able to prove the TIMP-1 antagonizing effect for all three inactive MMP-9 mutants in vitro.

Intrahepatic expression of MMP-9 mutants
Intrahepatic expression efficiency of MMP-9 mutants after adenoviral infection was analyzed by quantitative RT-PCR. Three mice per MMP-9 variant were treated with a single infection of 2 x 109 virions via tail vein injection. Livers were harvested 3 days after infection. mRNA of all mutants was significantly up-regulated (factor 5-200 to control group, from 1350 copies/µg mRNA in the control group to 5750 copies/µg mRNA for WT-MMP-9 and 119000, 289000, and 8750 copies/µg mRNA for the H401A-, E402Q- and E402H/H411E-MMP-9 mutants, respectively; data not shown).

Histological alterations of fibrotic liver due to TIMP-1 antagonization
To exclude viral effects all results were referred to a CCl4 control group infected with Ad5-CMV-EGFP. Hepatic collagen content was assessed by Sirius red (SR) staining. In control mice exposed to mineral oil only, deposition of collagen was found surrounding central veins and enclosing portal triads representing the collagen distribution of healthy liver (Fig. 2 a, SR). Microvesicular fat debris, probably caused by mineral oil injections, was found to be the only alteration in comparison to untreated mouse liver.


Figure 2
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  		Figure 2. Histological alterations caused by CCl4 administration and the effect of MMP-9 mutants. Sirius red (SR), hematoxylin eosin (HE), and {alpha}-SMA immunoreactivity-stained histological liver sections of control mice treated with mineral oil and the control vector (a) in comparison to fibrotic liver sections of mice treated with CCl4 and the control vector (b). Fibrogenesis and the expression of {alpha}-SMA were abolished more or less by the expression of WT-MMP-9 or the MMP-9 mutants (c–f). A representative liver section of each respective mouse group is shown in SR, HE staining, and {alpha}-SMA immunoreactivity.

Mice being treated with CCl4 and control virus Ad5-CMV-EGFP over 4 wk displayed a periportal fibrosis (F3) characterized by portal-portal septa surrounding the lobules (Fig. 2b , SR). Liver sections of mice receiving the control virus plus CCl4 did not differ in morphology or grade of fibrosis in comparison to mice receiving CCl4 alone (data not shown). In all mice subjected to CCl4 injection, inflammation and small areas of necrotic hepatocytes in the collagenized zones were observed (Fig. 2b-f , SR). Adenoviral expression of wild-type MMP-9, however, resulted in a reduction of the fibrotic stage (F2). Collagen bridges appeared to be limbered and more diffuse in comparison to the control groups (Fig. 2c , SR). The necrotic areas within the septa showed a higher grade of inflammation with an increased infiltration of mainly macrophages.

Application of proteolytically inactive MMP-9 mutants reduced the stage of fibrosis to a different extent as indicated by the decreasing portal and periportal accumulation of collagen (Fig. 2d-f , SR). These areas displayed less inflammation than observed for mice treated with WT-MMP-9 and CCl4. Liver sections of control mice that received mineral oil and the adenoviral expression vectors of the MMP-9 mutants but no CCl4 were histologically unaffected (not shown).

TIMP-1 antagonization reduces extracellular matrix deposition in vivo
A gradual characterization of fibrogenesis was performed quantitatively by morphometric analysis of SR-stained liver sections (Fig. 3 a) and quantification of the hydroxyproline level in the liver (Fig. 3b ). Quantitative morphometric data proved the anti-fibrotic effectiveness of the MMP-9 mutants with a significant reduction of fibrosis by 42 ± 15% (P=0.005) for E402Q and 29 ± 9% (P=0.038) for the mutant H401A (Fig. 3a ). We found a trend of reduction for the E402H/H411E mutant (30±21%, P=0.37) and for WT-MMP-9 (11±13%, P=0.24). Fibrotic scores for control groups of uninjured mice treated with oil were 0. Control mice injured with CCl4 or CCl4 plus control virus (Ad5-CMV-EGFP) scored at an average fibrosis level of 2.5 each (Fig. 3a ).


Figure 3
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  		Figure 3. Reduction of ECM-deposition after treatment with MMP-9 mutants in CCl4 induced fibrosis. The grade of fibrosis was determined by semiquantitative fibrosis scores in similarity to the score system suggested by Batts and Ludwig (31) and Ishak et al. (32) for hepatic fibrosis in humans (a). CCl4 injured control mice are indicated by black bars. A lower staging was determined for mice additionally treated with WT-MMP-9 or one of its mutants (halftone bars). b) Quantification of hydroxyproline (Hyp) indicated different grades of fibrogenesis between controls (white and black bars) and the MMP-9 mutant-treated mice (halftone bars). Statistical significance is referred to the CCl4/Ad-CMV-EGFP-treated group.

Quantification of the collagen-specific amino acid hydroxyproline (Fig. 3b ) supported the tendency of fibrotic stage assessed morphometrically. The hydroxyproline level in livers of E402Q-treated mice was significantly reduced from 430 µg/g to 317 µg/g (26±5.8%, P=0003) and in H401A-treated mice to 373 µg/g (15±7.2%, P=0.02). E402H/H411E-treated mice, however, did not show a decreased hydroxyproline level in liver tissue. CCl4 injured mice treated with WT-MMP-9 also showed a significantly reduced hydroxyproline level to 310 µg/g (28±14%, P=0001) in comparison to the control group (430 µg/g). These results provide evidence for an effective anti-fibrotic impact of MMP-9 mutants E402Q and H401A.

MMP-9 mutants reduce type I collagen mRNA expression in vivo
To elucidate the anti-fibrotic effect of the MMP-9 mutants, we examined the influence of TIMP-1 antagonization on type I collagen mRNA expression in mouse livers by quantitative RT-PCR (Fig. 4 a). Type I collagen mRNA expression was found not to be altered in WT-MMP-9-treated mice in comparison to CCl4-treated controls. Treatment of CCl4-injured mice with MMP-9 mutants, however, resulted in a reduction of type I collagen mRNA. In livers of E402Q- and H401A-treated mice a significant reduction of type I collagen mRNA of 58 ± 12% (P=0.035) and 52 ± 21% (P=0.049), respectively, was observed. Contrarily, the mutant E402H/H411E did not cause any significant effect at all.


Figure 4
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  		Figure 4. Reduction of fibrosis relevant genes. The influence of CCl4 or additional treatment with the MMP-9 mutants on mRNA expression of type I collagen (a), TIMP-1 (b), and MMP-2 (c) was determined by quantitative RT-PCR. All data were normalized to the actin mRNA content. Statistical significance is referred to the CCl4/Ad-CMV-EGFP-infected control group.

H401A- and E402Q mutants reduce TIMP-1 mRNA expression in vivo
MMP-9 mutants were primarily created to reduce the TIMP-1 protein level. We observed a significant reduction of TIMP-1 mRNA expression for the E402Q mutant by 65 ± 5.5% (P=0.024) and by 58 ± 7.3% for the H401A mutant (P=0.036). WT-MMP-9 and the E402H/H411E mutant did not change TIMP-1 mRNA level significantly (Fig. 4b ).

MMP-9 mutants reduce MMP-2 mRNA expression in vivo
After CCl4 treatment, MMP-2 mRNA expression decreased by 56 ± 16% in the H401A group (P=0.023), by 50 ± 15% in E402Q-treated mice (P=0.033), and in the E402H/H411E group by 90 ± 5% (P=0.005) (Fig. 4c ). E402H/H411E mutant has not shown any effect on the development of fibrosis although it acted as TIMP-1 antagonist in vitro.

MMP-9 mutants reduce type I collagen protein expression in vivo
To determine hepatic fibrosis and the specific collagen type I protein content, immunohistochemistry using polyclonal anti-collagen type I was carried out (Fig. 5 ).


Figure 5
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  		Figure 5. Expression of col{alpha}I in mouse liver (100x). a) Expression of col{alpha}I in healthy mouse liver. In healthy control mice treated with oil, col{alpha}I expression could be detected in sinusoids only, especially along with HSC or Kupffer cells (arrows). b) Expression of col{alpha}I in injured mouse liver. Control mice treated with CCl4 showed enhanced col{alpha}I expression in sinusoids especially along with HSC or Kupffer cells (black arrows). Elevated expression levels could be detected in periportal and septal parts of the lobuli especially along with HSC or Kupffer cells (red arrows) but sometimes also with hepatocytes (red arrows). c–f) Expression of col{alpha}I in mouse liver treated with CCl4 plus Ad5-CMV-MMP-9 (or MMP-9 mutants H401A, E402Q, E402H/H411E, respectively). Expression of col{alpha}I could be detected in sinusoidal-type cells (black arrows) and to a much less degree (compared with CCl4 control mice) in periportal and septal areas (red arrows, collagenized parts of the lobulus).

These analysis revealed that hepatic collagen type I expression corresponded to the hydroxyproline content. It was found to be pronounced in CCl4-treated mice (Fig. 5B ) and was substantially reduced in mice treated with TIMP-1 antagonists and CCl4 (Fig. 5D-F ).

mTIMP-1 antagonization effects stellate cell activation in vivo
To explain the protective effect of MMP-9 mutants on liver fibrogenesis, we investigated the influence of these mutants on HSC in vivo. We analyzed the expression of {alpha}-SMA histologically (Fig. 2a-f , {alpha}-SMA). Liver sections of control mice treated with oil only showed a slight expression of {alpha}-SMA at the border of blood vessels (Fig. 2a , {alpha}-SMA). CCl4 and CCl4 plus Ad-CMV-EGFP-treated mice displayed a strong {alpha}-SMA expression in periportal regions where collagen accumulation was also shown by SR staining. Treatment of CCl4-mice with WT-MMP-9 and the MMP-9 mutants H401A and E402Q resulted in a reduced {alpha}-SMA expression (Fig. 2c-f , {alpha}-SMA) and periportal septae were stained to a lesser extent. Livers of mice treated with E402H/H411E hardly discerned from CCl4 injured control mice reflecting the slight effect of this mutant on fibrolysis. This suggests that the MMP-9 mutants H401A and E402Q, acting as TIMP-1 antagonists, inhibit fibrogenesis by affecting HSC transdifferentiation.

TIMP-1 antagonization effects stellate cell activation and apoptosis in cell culture
Due to the effects determined in vivo, we investigated the effect of MMP-9 mutants on activation and apoptosis of isolated HSC in cell culture (Fig. 6 ). Culturing of quiescent HSC on uncoated plastic surfaces causes activation similar to that seen in liver fibrogenesis (16 , 40) . We measured the activation of HSC untreated or treated with the MMP-9 mutants by densitometrical quantification of {alpha}-SMA in Western blot.


Figure 6
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  		Figure 6. Influence of MMP-9 mutants on HSC apoptosis and transdifferentiation. a) The amounts of {alpha}-SMA in HSC lysates of control groups (black bars) and MMP-9 mutant-treated groups (halftone bars) were measured densitometrically by Western blot analysis. Statistical significance is referred to the Ad-CMV-EGFP-treated group only. b) Annexin assay. Representative fluorescence microscopy images of HSC of a low apoptosis control group (left: expression-deficient Ad5-EGFP control), of HSC of a high apoptosis control group (middle: Ad5-p53 control) and E402Q-treated HSC (right). c) HSC-treated as described under panel a, the amount of apoptosis was quantified by counting Annexin-FITC-labeled cells at day 5 (randomly chosen fields of view). Statistical significance is referred to the mock-infected control. d) TIMP-1 antagonistic effect on HSC apoptosis in cell culture. The Annexin apoptosis assay revealed that apoptosis could be induced in plated HSC by treatment with TIMP-1 antagonists (c). Additional transfection of HSC with a vector coding for TIMP-1 reduced the number of apoptotic cells significantly (white bars; randomly chosen fields of view).

Transdifferentiation of HSC into the myofibroblastic phenotype was inhibited significantly by WT-MMP-9 and MMP-9 mutants (Fig. 6a ). All three mutants led to a significant decrease in the activation process (H401A: 46±1.6%, P=0.00002; E402Q: 49±3.5%, P=0.000004; E402H/H411E: 48±3.9%, P=0.00004, Fig. 6a ). The expression of {alpha}–SMA was reduced by 28 ± 3.4% for WT-MMP-9 (P=0.0003). We quantified apoptosis by counting Annexin V-stained cells in randomly chosen display sections (Fig. 6b ). In comparison to mock-transfected cells, which were infected with a nonexpressing Ad5-CMV-EGFP vector, all MMP-9 mutants increased apoptosis significantly (Fig. 6c ). Comparable to the positive control (Ad5-CMV-p53), showing a 3.4-fold increase in apoptosis, the H401A mutant induced a 3-fold increase in apoptosis (P=0.00001). Expression of WT-MMP-9 elevated the apoptotic level by a factor of 2.9 (P=0.0002) while the E402Q mutant achieved a factor of 2.7 (P=0.00001). The E402H/H411E mutant obtained a significant enhancement of apoptosis with a factor of 2.1 (P=0.00001) in comparison to mock-transfected cells.

Subsequently, an experiment was carried out to determine the specificity of TIMP-1 antagonism. Therefore, the effect of TIMP-1 antagonist expression has been compared with the co-expression of both TIMP-1 and TIMP-1 antagonist on HSC (Fig. 6d ). A significant reduction of apoptosis was observed when TIMP-1 was co-expressed in HSC. For WT-MMP-9, H401A, and E402Q apoptosis decreased significantly by 1.7- (P=0.009), 1.8- (P=0.017), and 2.3-fold (P=0.0002), respectively. Although not statistically significant, a slight decrease of HSC apoptosis was observed for the E402H/H411E mutant (1.4-fold decrease, P=0.12). TIMP-1 decreased the induction of apoptosis in HSC caused by infection with MMP-9 or MMP-9 mutants (Fig. 6d ). The effects of MMP-9 mutants/WT may well be antagonized by TIMP-1 overexpression. Nonspecific effects of MMPs on HSC apoptosis were not observed.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Here we demonstrated that proteolytic inactive MMP-9 mutants, acting as TIMP-1 antagonists in vitro, inhibit CCl4-induced liver fibrosis in mice. Thus, we present a new and innovative therapy based on inactivated enzymes acting as scavengers for a strong promoter of liver fibrosis. The anti-fibrotic properties of MMP-9 mutants are based on their inhibitory effect on the transdifferentiation of HSC to the myofibroblast-like phenotype. Moreover, adenoviral application of some mutants (MMP-9-H401A and -E402Q) lead to an increased apoptosis of activated HSC in vitro and in vivo.

Hepatic fibrosis represents a transitional stage toward cirrhosis and is characterized by an accumulation of ECM-forming scars as a response to chronic liver disease. Main reasons for hepatic fibrogenesis are chronic hepatitis B and C, chronic toxic liver injury including alcoholism, nonalcoholic steatohepatitis, and chronic cholestasis. Some anti-fibrotic agents like silymarin (41) , ursodeoxycholic acid (42) , or IFN-{alpha} (43) were tested in clinical trials but as yet a successful therapeutic treatment has not prevailed. A central aspect of progression in liver fibrosis is represented by the accelerated transdifferentiation of perisinusoidal HSC to the activated MFB phenotype. In activated HSC, apoptosis is mainly suppressed by TIMP-1, mediated via effects on MMP inhibition (20 , 21) . Furthermore, the reversibility of micronodular cirrhosis is characterized by a decrease of TIMP-1 mRNA expression and an increase of HSC apoptosis (19) .

Recently different approaches to inhibit increased hepatic TIMP-1 levels were presented: Liu et al. investigated the effect of antisense TIMP-1 mRNA on activated HSC resulting in a decrease in collagen expression (44) . Different {alpha}vß3-integrin antagonists preventing TIMP-1 mRNA expression were tested in HSC (45) . Janoschek et al. used the TIMP-1 promoter in combination with the ganciclovir/thymidine kinase system for specific induction of programmed cell death in fibrogenic HSC (46) . In general, our data agree with some recent papers (47 , 48) . These results suffer from the fact that neither approach on HSC biology was investigated or elucidated (e.g., apoptosis). However, we could show that the antifibrotic effect of MMP-9 mutants was clearly caused by apoptosis of HSC.

The gene therapeutical application of MMP-1 and MMP-8 to induce fibrolysis (49 , 50) was discussed recently in the context of matrix degradation as a potential therapy for liver fibrosis (51) . However, MMP activity is a crucial target in the therapy of cancer growth and metastasis (52) , particularly of hepatocellular carcinoma invasion (53) . In contrast, we examined the TIMP-1 antagonistic effect of a proteolytically inactive MMP apart from the fibrolytic properties of the wild-type enzyme. Fibrotic liver exhibits increased proteolytic activity, which is inhibited by high TIMP-1 activity. Our therapeutic approach aimed toward the inactivation of TIMP-1. We chose MMP-9 for construction of TIMP-1 antagonists due to its high affinity (Ki values 50 pM) to TIMP-1 (54) . Our aim was to mutate MMP-9 in order to delete the aggressive proteolytic activity without disturbing the tight binding to TIMP-1. All mutations were realized in the catalytic center for specific creation of inactive mutants (Fig. 7 ).


Figure 7
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  		Figure 7. Molecular model of the MMP-9/TIMP-1 complex showing the active center of MMP-9. The MMP-9 mutant H401A was created by an exchange of histidine 401 against alanine resulting in a loss of Zn2+ coordination. Glutaminic acid 402 has an essential function for the catalytic mechanism and was substituted by glutamine to build the second proteolytic inactive MMP-9 mutant. In the third mutant a slightly disturbed architecture of the catalytic center was expected by the permutation of glutaminic acid 402 against histidine, and histidine 411 against glutaminic acid.

The single or double amino acid substitutions (H401A, E402Q, E402H/H411E) inside the catalytic cleft should not have altered the 3-dimensional structure, and thus should not have caused any significant antigenic reaction in mice. We expected a modified substrate or inhibitor binding due to changes within the binding site: the catalytic zinc ion may have been lost in the mutant H401A, and the previously charged carboxylate residue may have been replaced by the neutral amide group in the mutant E402Q. In contrast, we expected a lower influence of the E402H/H411E mutation. Modeling studies suggested that E402H/H411E should still be able to bind Zn2+, but the permutation of histidine and glutamic acid side chain may have resulted in a slight displacement of the catalytic center. This should lead to an inactive enzyme without changes in electrostatic properties. We chose adenoviral vectors and the CMV promoter for liver targeted overexpression, recently shown to be effective (55) .

Immunogenicity was assumed to be a potential problem that presumably occurs after repeated infection with adenoviruses (56) . However, a possible immunogenicity did not interfere with the anti-fibrotic effect of the MMP-9 mutants. We did not observe a significant influence of immunogenicity: all experiments were performed with a control virus (Ad5-CMV-EGFP). This control virus did not alter the extent of fibrosis as evidenced by liver histology (Fig. 3) . Histology of CCl4 and CCl4/EGFP liver sections showed no significant differences concerning fibrosis (data not shown). By immunohistochemistry we discovered that HSC and hepatocytes were infected by the adenoviruses (data not shown) and conclude that both cells are target cells for MMP-9 mutants. To prove a direct interaction of TIMP-1 and MMP-9 mutants expressed in mouse liver in vivo co-immunoprecipitation from liver lysates was carried out and demonstrated enhanced amounts of MMP-9 mutants co-immunoprecipitated with and bound to TIMP-1 in animals treated with the MMP-9 mutants H401A and E402Q (data not shown).

The TIMP-1 antagonizing potential of the MMP-9 mutants was quantified in vitro and a higher inhibitory effect for the mutants E402Q and E402H/H411E in comparison to H401A was found. The antagonizing effect of MMP-9-H401A on TIMP-1 activity in cell culture has been published (23) . In the present study we demonstrated that the isolated effect of scavenging TIMP-1 without increase of collagenolytic activity may be used successfully to treatment liver fibrosis. The significant reduction of liver fibrosis was shown by the loss of collagen deposition (Figs. 3 , 5) . By staging of hepatic fibrosis (31 , 32) as well as by quantification of the hepatic hydroxyproline content, the MMP-9 mutant E402Q revealed the greatest reduction of fibrosis. Although exhibiting a strong TIMP-1-antagonistic property in vitro (Fig. 1d , Fig. 6 ), application of the mutant E402H/H411E did not improve the fibrotic score nor reduce the hepatic hydroxyproline content (Fig. 3) . Changes in the catalytic cleft in MMP-9-E402H/H411E may have led to higher substrate affinity. Subsequent irreversible binding of components of the ECM could then have introduced clearing of these complexes and thus explain the surprisingly limited anti-fibrotic effect in mice.

WT-MMP-9 still exhibiting proteolytic activity did not alter stage of fibrosis histologically. However, a higher grade of inflammation with an increased infiltration of macrophages was observed after the adenoviral application of WT-MMP-9. The inflammation itself is considered a profibrogenic stimulus and may thereby enhance fibrosis. Our study has indeed shown that WT-MMP-9 was less effective in limiting fibrosis in vivo. This may well be due to the direct involvement of MMP-9 in the activation of TGF-ß (57) , a cytokine known to increase fibrogenesis and inflammation. The higher degree of inflammation observed in liver tissue after adenoviral application of WT-MMP-9 may have attenuated the anti-fibrotic effect of WT-MMP-9 in comparison to MMP-9-H401A and MMP-9-E402Q in the present study. Owing to its proinflammatory property and the involvement in tumor cell invasion, we deem WT-MMP-9 an unsuitable candidate for an anti-fibrotic approach.

In accordance with the SR staining results, we found a marked expression of {alpha}-SMA in overlapping distribution pattern with collagen deposition in the periportal areas of CCl4-treated animals. Pools of {alpha}-SMA directly indicate areas of HSC transdifferentiation to the activated phenotype that is mainly involved in ECM production (40) . According to the decreased hepatic collagen content, treatment with MMP-9 mutants, particularly with E402Q, resulted in a marked decline of {alpha}-SMA. In animals treated with E402Q and H401A, a reduced expression of fibrosis-associated genes like collagen type I, TIMP-1, and MMP-2 was found (Fig. 4) . Our experiments on transdifferentiation and apoptosis of isolated HSC in cell culture indicated the direct influence of MMP-9 mutants on HSC (Fig. 6) . These data suggest that autocrine inhibition of apoptosis by TIMP-1 in activated HSC was significantly antagonized by MMP-9 and MMP-9 mutants. In parallel, immunohistochemistry assays for detection of cleaved PARP as a marker for apoptosis demonstrated increased HSC death by TIMP-1 antagonists in mouse liver in vivo (data not shown).

In summary, our results suggest the following scenario of liver damage and protection by the MMP-9 mutants: CCl4 applied intraperitoneally is transported to the liver and is metabolized by hepatocytes (58) . Subsequent cell damage or cell death results in the production of cytokines like TNF-{alpha}, TGF-ß, IL-6, and IL-10, triggering the transdifferentiation process of HSC in paracrine and autocrine loops (59) . Finally, activated HSC accelerate production of extracellular matrix (ECM), TIMPs, cytokines, and several MMPs promoting fibrogenesis of the liver (60) . TIMP-1 was found to suppress apoptosis in activated HSC (20 , 21) . However, if the level of free TIMP-1 is reduced by complex formation with the MMP-9 mutants E402Q or H401A, apoptosis of activated HSC is induced. This decrease of activated HSC may then interrupt the fibrogenic process and stimulation of HSC transdifferentiation. At this stage the inherited increased proteolytic activity in fibrotic liver becomes obvious and fibrolysis will be accelerated.

In vitro and in inbreed mice models we have shown that inactive MMP-9 mutants inhibit hepatic fibrosis by TIMP-1 interception. On the basis of the present study, we expect that the application of MMP-9 mutants as TIMP-1 scavengers will open a new avenue for therapeutic treatment of hepatic fibrosis.


   ACKNOWLEDGMENTS
 
The authors thank O. Hiller (Institute for Biochemistry I, University Bielefeld) and J. Graf (Department of Anaesthesiology and Intensive Care Medicine, University Hospital Marburg) for critical reading of the manuscript, and A. Eisenträger (Institute of Hygiene and Environmental Medicine, RWTH University Hospital Aachen) for measurements at the Tecan Spectra Fluor Fluorescence Photometer. We thank J. Lorenzen (Molecular and Ultrastructural Pathology, RWTH University Hospital Aachen) for making available the microscopy imaging system. The authors gratefully acknowledge the technical assistance of Bettina Jansen, Judith Dahmen, Birgit Lahme, Karin Maschke-Neuss, and Annette Tschuschner. This work was supported by grants from the Deutsche Forschungsgemeinschaft (SFB 542, TP C3 and RO 957/6-1), the Federal Ministry of Education and Research of Germany (Network of Competence in Medicine, Kompetenznetz Hepatitis), and Aachen University (START project Identification of Molecular Markers and Gene Therapy of Fibrosis and Wound Healing).

Received for publication August 9, 2005. Accepted for publication November 15, 2005.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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