Introduction

Fibrogenesis represents a uniform response of the liver to chronic insults of toxic and infectious agents as well as metabolic stress and is characterized by an excessive accumulation of extracellular matrix (ECM) components^1,2,3. The net accumulation of the ECM connective tissue results from enhanced synthesis, or diminished breakdown of the matrix, or both^4,5. Collagens, predominately types I and III, are the major fibrous proteins in ECM and their synthesis increases in the liver exposed to carbon tetrachloride (CCl₄)^6,7,8. The enzymes that are involved in the collagen synthesis include lysyl oxidase, lysyl hydroxylase, and prolyl hydroxylase⁹. The degradation of ECM is dependent on the activity of matrix metalloproteinases (MMPs)¹⁰. According to their substrate specificity, MMPs fall into five categories: collagenases (MMP-1, -8, -13), gelatinases (MMP-2, -9), stromelysins (MMP-3, -7, -10, -11), membrane-type MMPs (MMP-14, -15, -16, -17, -24, -25), and metalloelastase (MMP-12)¹⁰. Previous studies have demonstrated that the activities of these enzymes are altered during the processes of fibrogenesis and fibrinolysis^10,11,12. These alterations affect the outcome of the fibrogenesis and the reversibility of fibrosis^13,14. Prolyl hydroxylase, gelatinases, and stromelysins are increased in the fibrotic liver. When fibrosis is in the reversible phase the collagenase activities are detectable^13,14, and when fibrosis becomes irreversible the collagenase activities fall¹⁴.

Prolyl hydroxylase is an Fe²⁺-dependant enzyme¹⁵. Zinc is an effective competitive inhibitor, replacing Fe²⁺ at the active site of the enzyme¹⁶. In contrast, the catalytic activity of MMPs depends on zinc at the active center¹⁷. Therefore, the availability of zinc affects the activities of the zinc-dependent enzymes^16,17. There are several studies that have examined the beneficial effect of zinc supplementation on liver fibrosis^18,19. Zinc administration has been shown to inhibit prolyl hydroxylase activity and increase MMP activities in the liver of rats treated with chemicals that induce hepatic fibrosis¹⁹.

Metallothionein (MT) is an important zinc-binding protein and is involved in zinc metabolism and homeostasis^20,21,22. MT is inducible by numerous metals, cytokines, and other chemicals^22,23,24. It is believed that this property of MT is associated with protection from metal toxicity²⁵. The interaction of MT with a number of oxidants causes zinc release from the protein^21,26,27, and the released zinc would affect the activities of the enzymes involved in fibrogenesis and fibrinolysis in the liver. Thus, it is interesting to explore the role of MT in the process of hepatic fibrogenesis and fibrinolysis. The present study, by using an adenoviral delivery gene therapy approach and taking advantage of MT-knockout mice in which MT-I and MT-II are genetically disrupted, was undertaken to study the requirement of MT in the reversal of toxicant-induced liver fibrosis.

Results

Hepatotoxicity Induced by CCl₄

To evaluate the hepatotoxic effect of CCl₄ and its reversibility upon cessation of the exposure, we exposed both wild-type (WT) mice and MT-knockout (MT-KO) mice to CCl₄ at 1 ml/kg in corn oil by ip injection twice a week. After 4 weeks exposure, serum alanine aminotransferase (ALT) activity was increased significantly in WT mice compared to the oil-treated control mice (250 45 versus 7.8 5.6 U/ml). We observed typical necrotic changes in the tissue slices obtained from the left lobe of the liver through histopathological examination (Fig. 1). We observed the infiltration of inflammatory cells. Upon cessation of CCl₄ dosing for 4 weeks, these pathological changes in the liver were recovered. After the WT mice were treated with CCl₄ for 8 weeks, we observed the same hepatic pathological changes, but they were more extensive (data not shown). Treatment with CCl₄ for 4 weeks in the MT-KO mice caused the same liver injury as that observed in the WT mice treated with CCl₄ for 8 weeks (Fig. 1). Four weeks after the cessation of CCl₄ dosing, these pathological changes in the MT-KO mice (Fig. 1) and the WT mice treated with CCl₄ for 8 weeks (data not shown) were only partially recovered. These results demonstrate that either longer exposure to CCl₄ or the lack of MT in the liver made the CCl₄-induced liver pathological changes less reversible.

Figure 1.

CCl₄-induced liver histopathological changes and their reversibility upon cessation of CCl₄ dosing in WT and MT-KO mice. Mice received CCl₄ in corn oil at 1.0 ml/kg through intraperitoneal injection twice a week for 4 weeks and were sacrificed on the second day or 4 weeks after the last dosing. (A, E) Corn oil only treated for 4 weeks and sacrificed on the second day after last dosing as control; (B, F) CCl₄ treated for 4 weeks and sacrificed on the second day after last dosing; (C, G) 4 weeks after cessation of corn oil injection; (D, H) 4 weeks after cessation of CCl₄ dosing (H inset is a 3 enlargement showing necrotic area). Arrows indicate infiltration of inflammatory cells and arrowheads necrotic hepatocytes.

Full figure and legend (224K)

CCl₄-Induced Fibrosis and Its Recovery

To examine the fibrogenic effect of CCl₄ in the liver, we detected collagen accumulation by picrosirius red staining and analyzed it semiquantitatively using a computer program. The results presented in Fig. 2 show that intrahepatic collagen accumulation was a common feature in WT mice treated with CCl₄ for 4 or 8 weeks and in MT-KO mice treated with CCl₄ for 4 weeks, although the fibrosis was less extensive in the WT mice treated for 4 weeks relative to other groups. Upon cessation of CCl₄ dosing for 4 weeks, we observed an almost complete reversal of the liver fibrosis in the WT mice treated with CCl₄ for 4 weeks. We also observed some reversal in the WT mice treated with CCl₄ for 8 weeks, but the level of the remaining fibrosis was the same as that observed in the WT mice at the end of 4 weeks treatment with CCl₄ (Fig. 2B). However, fibrosis in MT-KO mice treated with CCl₄ for 4 weeks was not reversed after cessation of the exposure (Fig. 2B). We confirmed these changes by measuring hepatic hydroxyproline concentrations (Fig. 2C). The results suggest that the reversibility of liver fibrosis is related to the length of CCl₄ exposure and the presence of MT in the liver; the longer the exposure, the less the reversibility, and the lack of MT in the liver makes the CCl₄-induced fibrosis persist after cessation of the exposure.

Figure 2.

CCl₄-induced liver fibrosis and its reversibility upon cessation of CCl₄ dosing in WT and MT-KO mice. (A) Hepatic collagens were stained by picrosirius red as shown in the microphotographs. WT mice received CCl₄ in corn oil at 1.0 ml/kg through intraperitoneal injection twice a week for 4 (4 wks) or 8 weeks (8 wks) and sacrificed on the second day or 4 weeks after the last dosing and the MT-KO mice were treated for 4 weeks only and sacrificed following the same schedules for WT mice. Images A, E, and I, corn oil only treated for 4 (A, I) or 8 weeks (E) and sacrificed on the second day after last dosing as control; B, F, and J, CCl₄ treated for 4 (B, J) or 8 weeks (F) and sacrificed on the second day after last dosing; C, G, and K, 4 weeks after cessation of corn oil injection; D, H, and L, 4 weeks after cessation of CCl₄ dosing. (B) Semiquantitative data obtained from a computer-assisted image analyzer for the picrosirius red-stained collagens. Each data point was obtained from five animals, and five slides from each animal (tissue block) and 10 random fields at 100 per slide were examined. (C) Data obtained from the analysis of hepatic hydroxyproline concentrations (n = 5). The mean values that do not share the same letter are significantly different (P < 0.05) from one another (for both (B) and (C)).

Full figure and legend (217K)

MT Concentrations in the Livers Treated with CCl₄ and after the Cessation

To examine further the association of the levels of MT in the liver and the reversibility of CCl₄-induced pathological changes, we determined MT concentrations in the liver under different conditions. The treatment with CCl₄ for 4 weeks significantly increased MT concentrations in the liver of WT mice; however, the hepatic MT concentrations were dramatically depressed after the cessation of CCl₄ dosing for 4 weeks (Fig. 3). The levels of MT in the liver of mice treated with CCl₄ for 8 weeks were significantly lower than those in the WT mice treated for 4 weeks (Fig. 3). The MT concentrations in the liver of MT-KO mice were about fivefold lower than those in the WT mice and the CCl₄ treatment or cessation did not cause any changes in these mice (Fig. 3). These differences in hepatic MT concentrations correlated with the pathological changes induced by CCl₄, namely, high levels of MT in the liver predicted less pathological change and higher reversibility.

Figure 3.

Changes in MT concentrations in the livers treated with CCl₄ and after the cessation of CCl₄ dosing in WT and MT-KO mice. The treatment and animal sacrificing schedules were the same as presented in Fig. 2. (A, E, I) Corn oil only treated for 4 (A, I) or 8 weeks (E) and sacrificed on the second day after last dosing as control; (B, F, J) CCl₄ treated for 4 (B, J) or 8 weeks (F) and sacrificed on the second day after last dosing; (C, G, K) 4 weeks after cessation of corn oil injection; (D, H, L) 4 weeks after cessation of CCl₄ dosing. Each data point was obtained from five animals and expressed as the mean SD, and the mean values that do not share the same letter are significantly different (P < 0.05) from one another.

Full figure and legend (36K)

MT Gene Therapy for the Irreversible Liver Fibrosis

The above results suggest that MT would be involved in the liver fibrinolysis. To test this, we applied a gene therapy approach using an adenovirus (Ad5-MT) to deliver a human MT-II gene (approved gene symbol MT2A) into the liver through intravenous injection. The results shown in Fig. 4 demonstrate that the adenoviral delivery system highly efficiently increased MT expression in the liver 3 days after the injection in both WT and MT-KO mice with irreversible liver fibrosis. The results in Fig. 5 show that 3 days after the administration of Ad5-MT, a significant fibrinolysis was observed in both WT and MT-KO mice. Further examination revealed that MT gene therapy caused hepatic cell regeneration as determined by PCNA staining (Fig. 5).

Figure 4.

Expression of MT in the fibrotic liver 3 days after Ad5-MT gene delivery through intravenous injection in WT and MT-KO mice. Top: Immunohistochemical detection of MT expression and distribution in the hepatic cells. (A, C) CCl₄ treated for 8 (A) or 4 weeks (C) followed by Ad5-lacZ gene transfer and sacrificed on the fourth day after the gene transfer. (B, D) CCl₄ treated for 8 (B) or 4 weeks (D) followed by Ad5-MT gene transfer and sacrificed on the fourth day after the gene transfer. Bottom: Quantitative analysis by a cadmium–hemoglobin affinity assay of the MT concentrations in the livers under the same treatment protocol above (n = 5). The mean values that do not share the same letter are significantly different (P < 0.05) from one another.

Full figure and legend (89K)

Figure 5.

MT gene transfer-induced reduction in liver fibrosis and hepatocyte regeneration in WT and MT-KO mice. Mice received CCl₄ in corn oil at 1.0 ml/kg through intraperitoneal injection twice a week for 8 (WT) or 4 weeks (MT-KO) and (A, E, I, M) were sacrificed on the second day or (B, F, J, N) on the seventh day after the last dosing, or (C, G, K, O) were subjected to Ad5-lacZ gene transfer on the fourth day and sacrificed on the seventh day after the last dosing, or (D, H, L, P) were subjected to Ad5-MT gene transfer on the fourth day and sacrificed on the seventh day after the last dosing. (A to H) Sirius red staining; (I to P) PCNA staining. Bottom: Quantitative analysis of hepatic hydroxyproline concentrations from the same treatments as indicated for A to H. Each data point was obtained from five animals and is expressed as mean SD, and the mean values that do not share the same letter are significantly different (P < 0.05) from one another.

Full figure and legend (190K)

Activation of Enzymes Involved in Fibrinolysis by MT Gene Therapy

The results above indicate that MT would enhance the fibrinolytic pathway leading to the observed acceleration of fibrinolysis and the recovery of the irreversible liver fibrosis. Since collagenases (MMP-1, -8, and -13) are responsible mainly for collagenolysis, we measured the total enzymatic activities of collagenases in the liver of the WT mice treated with CCl₄ for 4 weeks, at which time point the CCl₄-induced hepatic fibrosis was reversible. In comparison, we also measured the enzyme activities in the liver of the MT-KO mice treated with CCl₄ for 4 weeks. As shown in Fig. 6, the activities of the hepatic collagenases (predominately MMP-13 in mouse) were almost the same at the end of the treatment with CCl₄ for 4 weeks in WT and MT-KO mice. These enzyme activities were elevated in the WT mice, measured on the third day after the cessation of the CCl₄ dosing, but decreased in the MT-KO mice at the same time. On the fourth day after the cessation of CCl₄ dosing, the MT-KO mice were injected with Ad5-MT. Three days after the Ad5-MT administration, these enzyme activities in the liver of MT-KO mice were significantly elevated in comparison with that of the untreated controls. Adenoviral vector (Ad5-lacZ)-injected control mice did not show significant increase in the enzyme activities (Fig. 6). These results demonstrate that high levels of MT in the liver are associated with higher activities of collagenases (predominately MMP-13 in the mouse liver).

Figure 6.

(Top) Dynamic changes in collagenase activities in the livers following CCl₄ treatment and cessation of CCl₄ dosing in WT and MT-KO mice and (bottom) the effects of MT gene transfer in the MT-KO mice. Top: Both WT and MT-KO mice received CCl₄ in corn oil at 1.0 ml/kg through intraperitoneal injection twice a week for 4 weeks and were sacrificed on the second day (A), on the third day (B), or on the fifth day (C) after cessation of CCl₄ dosing. The collagenase activities in the WT and MT-KO control mice without CCl₄ treatment were 5.7 0.4 units/mg protein. The activity in mice on the second day after cessation of CCl₄ dosing was 8.6 0.6 units/mg protein, which is expressed as 100% of control, and the rest are expressed as relative increase or decrease compared to the control (% of control). Bottom: MT-KO mice were treated with CCl₄ for 4 weeks, followed by Ad5-MT gene transfer on the fourth day and sacrifice on the seventh day after the cessation of CCl₄ dosing (A) or followed by Ad5-lacZ gene transfer on the fourth day and sacrifice on the seventh day (B), and compared with CCl₄-treated MT-KO mice that received no further treatment and were sacrificed on the seventh day after the cessation of CCl₄ dosing (C). The enzymatic changes are expressed as relative (%) to C (100%). Each data point was obtained from five mice and expressed as the mean SD, and the mean values that do not share the same letter are significantly different (P < 0.05).

Full figure and legend (41K)

Discussion

The results obtained from this study showed that the reversibility of CCl₄-induced liver fibrosis was related to hepatic MT concentrations. MT was inducible in the liver by CCl₄; the reversal of CCl₄-induced liver fibrosis was accompanied by the spending of MT. High levels of MT at the time of cessation of CCl₄ dosing predicted a reversible and low levels were associated with a less reversible fibrosis. MT gene therapy made the irreversible liver fibrosis become reversible and the activation of collagenases (predominately MMP-13 in the mouse liver) would be highly responsible for the recovery of the irreversible fibrosis. In addition, the enhanced hepatocyte regeneration would also make a significant contribution to the MT gene therapy-induced recovery.

The CCl₄-induced liver fibrogenesis involves marked infiltration of inflammatory cells, mimicking the changes in chronic viral hepatitis-associated fibrosis in humans²⁸. Thus it has been widely used in experimental approaches to the pathogenesis of liver fibrosis in rodent models^29,30,31. The difference in the collagen deposition in the liver between WT mice treated with CCl₄ for 4 weeks and those treated for 8 weeks suggested that the length of the chemical exposure would make a major contribution to this difference. However, it is important to define what are the possible factors determining the exposure time-dependent fibrogenesis. MT, a major protein involved in the regulation of zinc homeostasis, is stress inducible and plays an important role in protection against chemical-induced pathogenesis in the liver^18,32,33. The results obtained from this study strongly suggest that MT is also a critical factor in hepatic resistance to CCl₄-induced fibrosis. First, MT concentrations in the liver were induced to a high level by the end of CCl₄ treatment for 4 weeks in the WT mice and significantly decreased to a barely detectable level 4 weeks after the cessation of the CCl₄ dosing, suggesting the spending of MT during the recovery. Second, MT concentrations in the liver of the WT mice treated with CCl₄ for 8 weeks were much lower than those in the WT mice treated with CCl₄ for 4 weeks and further decreased to the same barely detectable level after cessation of CCl₄ dosing for 4 weeks. Third, the extent of liver fibrosis induced by CCl₄ treatment for 4 weeks in the MT-KO mice was the same as that in the WT mice treated with CCl₄ for 8 weeks. These observations thus indicate that the level of MT in the liver is highly related to the progression of CCl₄-induced fibrosis, although other factors might be also involved in the more severe fibrogenesis effect of a longer exposure to CCl₄.

A further demonstration of the role of MT in the liver fibrogenesis is the reversibility of the liver fibrosis 4 weeks after cessation of CCl₄ exposure in the WT mice treated with CCl₄ for 4 weeks, but not in the mice treated for 8 weeks. This difference was associated with a higher hepatic MT level in the mice treated with CCl₄ for 4 weeks and a lower level in the mice treated with CCl₄ for 8 weeks at the end of the exposure or at the beginning of the recovery. The possible cause-and-effect relation between low MT concentrations and irreversibility of liver fibrosis was indicated by the fact that liver fibrosis was not reversible in the MT-KO mice treated with CCl₄ for only 4 weeks. An important, and the most convincing, piece of evidence that MT promotes liver fibrolysis was obtained from the MT gene therapy result. In particular, MT gene therapy converted the irreversible liver fibrosis to reversible in both WT and MT-KO mice, indicating the significance of MT in the fibrinolysis.

It is important to understand the mechanism by which MT gene therapy made the irreversible liver fibrosis reversible. Interstitial collagenases, including MMP-1, -8 and -13, are important metalloproteases involved in the liver fibrinolysis. In mouse and rat, a homologue of MMP-1 has not been identified, and MMP-13 is considered to be the major interstitial collagenase in these species^34,35. The role of these collagenases in the recovery of established liver fibrosis induced by CCl₄ in rat livers has been studied using gene therapy approaches. For instance, human MMP-8 delivered by an adenovirus vector (AdMMP-8) has been shown to reverse liver fibrosis effectively³⁶. The same AdMMP-8 system was also shown to be effective in reducing liver fibrosis in bile duct-ligated cirrhosis rats³⁷. A similar approach, but using MMP-1 delivered by an AdMMP-1 construct, was also shown to be effective in reversing liver fibrosis induced by CCl₄ in rats³⁸. In addition to these interstitial collagenases, other gene therapies using different regulators in collagen synthesis and degradation have been tested and demonstrated to be effective in inhibition or reversal of liver fibrosis. These factors include urokinase-type plasminogen activator³⁹, telomerase³¹, and hepatic growth factor⁴⁰.

Collagenases are zinc-dependent for their catalytic activities¹⁶. MT is a major protein involved in the intracellular zinc homeostasis, and under stress conditions, MT releases zinc to make zinc available for many other cellular processes triggered by the stress responses^21,41. In this context, we determined the enzyme activities of collagenases (predominately MMP-13 in the mouse liver) in the WT and MT-KO mice treated with CCl₄ for 4 weeks. It appeared that reversible liver fibrosis in the WT mice was associated with higher levels of the enzyme activities after the cessation of the CCl₄ dosing and the irreversibility with reduced enzyme activities. The association of MT with the enzyme activities was revealed by the fact that MT gene therapy in the MT-KO mice led to a significant elevation in the activities of the hepatic collagenases.

Regardless of these interesting and important findings, there are some limitations in the present study. First, the mechanistic insights into the MT gene therapy-induced recovery of irreversible liver fibrosis need to be further approached. Although the important fibrinolytic enzyme activities were determined, other factors that are possibly involved in the liver fibrinolysis and activated by the MT gene therapy need to be explored. Second, a detailed study examining zinc transport from one protein to another and its regulation by MT is a major task for future undertakings. However, the present study indeed paved a new avenue for the study of the role of MT in hepatic responses to chemical toxicity.

In conclusion, the present study demonstrates that MT is a critical factor determining the reversibility of the CCl₄-induced liver fibrosis in the mouse model. Importantly, MT gene therapy made the irreversible liver fibrosis recoverable. The action of MT in the liver fibrinolysis was highly related to the activation of the fibrinolytic enzyme activities. However, further mechanistic insights into the beneficial effect of MT gene therapy in the chemical-induced liver fibrosis need to be obtained from future studies. Importantly, the results obtained from this study strongly suggest the therapeutic potential of MT for patients with certain liver fibrosis.

Materials and methods

Animal experimental procedure

Homozygous metallothionein-knockout mice, produced on the 129/Sv genetic background⁴², were obtained from The Jackson Laboratory (Bar Harbor, ME, USA) along with the wild-type mice and housed in cages with a 12-h light/dark cycle and provided with rodent chow and tap water ad libitum. All animals received humane care in compliance with the institution's guidelines. The animal procedures were approved by the Institutional Animal Care and Use Committee, which is certified by the American Association for Accreditation of Laboratory Animal Care. The experimental mice at 10 weeks of age started receiving an intraperitoneal injection of 1 ml/kg CCl₄ (Sigma Chemical, St. Louis, MO, USA) in corn oil (1:4 ratio) or corn oil alone as control twice a week for 4 or 8 weeks for the WT mice, or for 4 weeks only for the MT-KO mice. Mice were sacrificed on the day following the last dose, 4 weeks after the last dose, or at the time specified in the text.

Adenovirus preparation

Recombinant adenoviruses containing the transgene for either -galactosidase (Ad5-lacZ) or human MT-II (Ad5-MT) under the control of the CMV promoter were received as a kind gift from Dr. Bruce Pitt of the University of Pittsburgh. Adenoviruses were propagated in HER 293 cells and purified by banding twice on CsCl gradients. Viral titers were determined by optical densitometry (particles per milliliter) and by plaque assay.

Adenoviral gene transfer

Ad5-lacZ and Ad5-MT (2 10⁸ pfu per mice) were diluted in 200 l saline before use. On the fourth day after the last dosing with CCl₄ for 4 (MT-KO) or 8 weeks (WT), mice were injected with either Ad5-lacZ or Ad5-MT through the femoral vein under anesthetization with avertin (0.4 mg/g). Mice were sacrificed on the fourth day after the gene delivery.

Measurement of serum ALT

Serum ALT level was measured spectrophotometrically by a standard enzymatic method using a commercial kit (Sigma Chemical).

Histopathological examination and imaging analysis

Mice were sacrificed under anesthetization with avertin (0.4 mg/g). Left lobes of livers were removed immediately and fixed in 10% buffered formalin, dehydrated in graded ethyl alcohol, and embedded in paraffin. Sections 5 m thick were stained with hematoxylin/eosin (H/E) or picrosirius red. The H/E-stained sections were analyzed under light microscope for histopathological assessment. For the picrosirius red staining, the sections were stained with 0.1% Sirius red F3BA and 0.25% Fast green FCF⁴³. The picrosirius red-stained sections were assessed for the proportion of fibrosis in the liver tissues using a computer-assisted image analyzer (SigmaScanPro5.0; SPSS, Inc., Chicago, IL, USA). There were five slides obtained from each sample, and 10 random fields at 100 magnification per slide were examined and the ratio of collagen fiber to the whole area of the liver was calculated and expressed as a percentage of the liver tissue.

Immunohistochemical staining

Paraffin sections 5 m thick were deparaffinized in xylene and dehydrated in alcohol. After treatment with 3% (vol/vol) hydrogen peroxide in methanol to eliminate nonspecific reactions, the samples were incubated overnight at 4°C with a 1:100 dilution of a mouse anti-human MT antibody (Zymed Laboratories, Inc.) and a 1:100 dilution of a rabbit anti-human PCNA antibody (Santa Cruz Biotechnology, Inc.). After incubation with the avidin–biotin complex, the antibody labeling was visualized with diaminobenzidine and photographed using a light microscope.

Hepatic hydroxyproline concentration

Livers were removed and immediately stored in liquid nitrogen. The tissue sample (50 mg) was hydrolyzed in 4 ml 6 N HCl at 110°C for 24 h. After being filtered through a 0.45-m filter, 2 ml of samples was extracted and analyzed by the method of Kivirikko et al.⁴⁴.

Measurement of MT concentrations in the liver

Mice were sacrificed at the indicated times and MT was measured by a cadmium–hemoglobin affinity assay described previously⁴⁵. Briefly, liver tissues were homogenized in 4 vol of 10 mmol/L Tris–HCl buffer, pH 7.4, at 4°C. After centrifugation of the homogenate at 10,000g for 15 min, 200 l of supernatant was transferred to microtubes for MT analysis.

Determination of collagenase activity

The total enzyme activity of collagenases (MMP-1, -8, and -13) was determined in liver homogenates using an EnzCheck Gelatinase/Collagenase Assay kit (Molecular Probes, Eugene, OR, USA) according to the manufacturer's instructions. A fluorescently labeled collagen type I was used as substrate and the purified collagenase from Clostridium histolyticum as control. A general inhibitor of metalloproteinases (1,10-phenanthroline) was used to define specific activity of the collagenases through subtraction from the total proteolytic activity in the liver samples.

Statistical analysis

Data were expressed as means SD. Statistical differences between groups were determined by a paired Student t test. Otherwise, data were analyzed using one-way ANOVA, followed by Duncan's multiple range tests. P < 0.05 was selected to reflect significance.

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Acknowledgements

The authors thank Dr. Bruce Pitt at the University of Pittsburgh for kindly providing the Ad5-MT construct and Dr. Zhanxiang Zhou for technical advice. Y.J.K. is a Distinguished University Scholar of the University of Louisville.