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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Hepatol Res. Author manuscript; available in PMC 2010 July 22.
Published in final edited form as:
PMCID: PMC2908315
NIHMSID: NIHMS212844

The Matrix Metalloproteinase Inhibitor, CTS-1027, Attenuates Liver Injury and Fibrosis in the Bile Duct-Ligated Mouse

Abstract

Excessive matrix metalloproteinase (MMP) activity has been implicated in the pathogenesis of acute and chronic liver injury. CTS-1027 is a MMP inhibitor, which has previously been studied in humans as an anti-arthritic agent. Thus, our aim was to assess if CTS-1027 is hepato-protective and anti-fibrogenic during cholestatic liver injury. C57/BL6 mice were subjected to bile duct ligation (BDL) for 14 days. Either CTS-1027 or vehicle was administered by gavage. BDL mice treated with CTS-1027 demonstrated a 3-fold reduction in hepatocyte apoptosis as assessed by the TUNEL assay or immunohistochemistry for caspase 3/7-positive cells as compared to vehicle treated BDL animals (p<0.01). A 70% reduction in bile infarcts, a histologic indicator of liver injury, was also observed in CTS-1027 treated BDL animals. These differences could not be ascribed to differences in cholestasis as serum total bilirubin concentrations were nearly identical in the BDL groups of animals. Markers for stellate cell activation (α-smooth muscle actin) and hepatic fibrogenesis (collagen 1) were reduced in CTS-1027 versus vehicle-treated BDL animals (p<0.05). Overall animal survival following 14 days of BDL was also improved in the group receiving the active drug (p<0.05). In conclusion, in the BDL mouse, liver injury and hepatic fibrosis are attenuated by treatment with the MMP inhibitor CTS-1027. This drug warrants further evaluation as an anti-fibrogenic drug in hepatic injury.

Keywords: apoptosis, cholestasis, liver fibrosis, matrix metalloproteinase

Liver fibrosis and its end stage sequela of cirrhosis represent a major world-wide health problem. Hepatic fibrosis is the result of an exuberant wound healing response resulting in excessive collagen deposition in the liver. Hepatic stellate cells are the major source of collagen in the diseased liver.1 During liver injury, hepatic stellate cells undergo activation and transdifferentiation to myofibroblasts, which efficiently generate collagen I as part of their wound healing response.1 These activated cells, interestingly, also secrete matrix metalloproteinases which may degrade collagenous and non-collagenous substrates.2 Usually these potent proteases are held in check by the secretion of tissue inhibitors of metalloproteinases (TIMPS) 1 and 2.2 However, excessive and prolonged MMP secretion can alter the hepatic scaffolding. This alteration of hepatic architecture results in further liver injury which in turn elicits increased hepatic damage and fibrosis. It is this upregulated MMP activity which leads to a feed-forward damage response in liver injury. In particular, matrix metalloproteinase (MMP) -2, -3 and -9, are upregulated and thought to contribute to human liver disease.3-5

Inhibition of MMP activity is a potential strategy to minimize liver injury and reduce hepatic fibrogenesis. For example, inhibition of MMP activity by a MMP inhibitor, BB-94, blocks hepatocyte apoptosis and improves animal survival in a model of TNF-α induced acute liver injury.6 Genetic deletion of MMP13, collagenase-3, attenuates hepatic fibrogenesis in a cholestatic model of liver injury, the bile duct ligated (BDL) mouse.7 These observations suggest MMP inhibition may be hepato-protective in liver injury. CTS-1027, N-hydroxy-4-{[4-(4-chlorophenoxy) benzenesulfonyl] methyl}-2, 3, 5, 6-tetrahydropyran-4-carboxamide, is a reversible MMP inhibitor. It is an especially potent inhibitor of human MMP 2, 3, 8, 9, 12, 13 and 14 but not 1 or 7. The Ki for inhibiting these MMP is ≤9 nM. CTS-1027 appears to be selective for MMP and has little or no activity for other proteinases including caspases, the initiator and effector proteinases of apoptosis. This hydroxamate-based inhibitor has been studied in clinical trials as an anti-arthritic agent. The compound was well tolerated and side effects were generally mild, reversible and primarily limited to the musculoskeletal system. Thus, given its safety profile and selectivity in targeting MMP, CTS-1027 is an attractive agent to inhibit MMP as a potential hepato-protective agent. However, preclinical studies have not yet been reported addressing a potential hepato-protective effect, although CTS-1027 is currently in early phase 2 studies in HCV patients.

In this study we assessed the hepato-protective and anti-fibrogenic potential of CTS-1027 during cholestatic liver injury in a preclinical model, the BDL mouse. As compared to vehicle treated control animals, administration of CTS-1027 attenuates hepatocyte apoptosis, liver injury and hepatic fibrosis.

MATERIALS AND METHODS

Animal models

The care and use of the animals for the following experiments were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) at the Mayo Clinic. C57/BL6 wild-type (wt) mice (6 to 8 weeks of age, 20-25 g of body weight) were employed for these studies. Mice were maintained in a temperature-controlled (22°C), pathogen-free environment and fed a standard rodent chow diet and water ad libitum. For experimental procedures, mice were anesthetized with ketamine 60 mg/kg plus xylazine 10 mg/kg body weight by intraperitoneal injection. After a midline upper-abdominal incision, the peritoneal cavity was opened, the abdominal wall retracted, and the common hepatic bile duct was double-ligated below the bifurcation and transected between the ligatures as previously described by us in detail.8 Sham-operated mice, used as controls, also underwent similar laparotomy with exposure but without ligation of the common bile duct. The fascia and skin of the midline abdominal incision were closed with sterile surgical 5-0 sutures (Ethicon Inc., Somerville, New Jersey). Either CTS-1027 (Conatus Pharmaceuticals, San Diego, California) or the vector carboxymethylcellulose (CMC Sigma-Aldrich, St. Louis, Missouri) were administered by gavage in a dose of 10 mg/kg body weight once a day. Drugs were prepared freshly on the day of the study. After 14 days of BDL and gavage, mice were re-anesthetized, sacrificed and blood was obtained from the inferior vena cava for serum total bilirubin and ALT determinations and the liver was removed, cut into small pieces and either snap-frozen in liquid nitrogen for storage at −80°C or fixed in freshly prepared 4% paraformaldehyde in phosphate-buffered saline (PBS) for 48 hours at 4°C for additional studies (vide infra). Liver tissue was also subjected to RNA extraction using the Trizol reagent (Invitrogen, Carlsbad, California). Serum bilirubin and ALT determinations were performed as previously described.9

Histopathology

For histological review of haematoxylin and eosin (H&E)-stained liver sections by light microscopy (Nikon Eclipse Meta Morph V 5.0.7, West Lafayette, Indiana), the liver was diced into 5×5 mm sections, fixed in 4% paraformaldehyde for 48 hours and then embedded in paraffin (Curtin Matheson Scientific Inc., Houston, Texas). Tissue sections (4 μm) were prepared using a microtome (Reichert Scientific Instruments, Buffalo, New York) and placed on glass slides. H&E staining was performed according to standard techniques.

TUNEL assay and immunofluorescent identification of activated caspases 3/7

Apoptotic cells were quantitated by the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay which enzymatically labels free 3′-OH ends of damaged DNA with a fluorescently-labeled nucleotide as we have previously described in detail.10 TUNEL-labeled cells (i.e., fluorescent nuclei) were quantified by counting the number of positive cells per high-power field. A total of 10 high-power fields were analyzed for each animal with excitation and emission wavelengths of 490 and 520 nm, respectively, using an inverted laser scanning confocal microscope (LSM 510, Carl Zeiss Micro-Imaging Inc., Thornwood, New Jersey) equipped with a 40xNA 1.4 lens and LSM 510 imaging software. Data were expressed as the number of TUNEL-positive cells/10 high-power fields (hpf). Immunofluorescence analysis for activated caspases 3/7 was performed using a rabbit anti-active caspase 3/7 polyclonal antibody (BD Biosciences/Pharmingen, San Diego, California) recognizing a common neo-epitope shared by activated caspases 3 and 7 as we have previously described.10 Secondary goat-anti-rabbit antibody conjugated to Texas Red-X (Invitrogen) was used for visualization of staining. The liver specimens were viewed by confocal microscopy using excitation and emission wavelengths of 543 and 568 nm, respectively. The number of caspase 3/7-positive cells was quantified per 10 hpf as described above for the TUNEL assay. Negative control slides were incubated with non-immune immunoglobulin under same conditions.

Quantitative Real Time-Polymerase Chain Reaction (qrt-PCR)

Total RNA was isolated from liver tissue using the Trizol reagent (Invitrogen). For each RNA sample, a 10 μg aliquot was reverse-transcribed into complementary DNA (cDNA) using random primers and Maloney murine leukaemia virus (MMLV) reverse transcriptase (Invitrogen) as previously described in detail.11 After the reverse transcription reaction, the cDNA template was amplified by real time PCR with Plantinum Taq DNA polymerase (Invitrogen) using standard protocols. All amplified PCR products were confirmed by electrophoresis in 1% low-melting temperature agarose gel, stained with ethidium bromide and photographed using UV-illumination. The expected base pair PCR products were identified and the bands cut from the gel. Next, PCR products were eluted into Tris-HCl using a DNA elution kit (Qiagen, Valencia, California). The concentration of DNA in the extracted PCR product was measured spectrophotometrically at 260 nm (Beckman DU 4400, Palo Alto, California) and converted to copies/μl. Extracted PCR products were prepared as standards. Quantitative real time PCR was performed using the Light Cycler (Roche Diagnostics Corp., Mannheim, Germany) and SYBR green as the fluorophore (Invitrogen) as previously described by us in detail.12, 13 PCR primers (all obtained from the Mayo DNA Synthesis Core Facility, Rochester, Minnesota) were as follows: α-SMA forward 5′-ACT ACT GCC GAG CGT GAG AT-3′, reverse 5′-AAG GTA GAC AGC GAA GCC AG-3′ (yielding a 452-bp product) and collagen 1α (I) forward 5′-GAA ACC CGA GGT ATG CTT GA-3′, reverse 5′-GAC CAG GAG GAC CAG GAA GT-3′ (yielding a 276-bp product).

Immunohistochemistry for α-smooth muscle actin (α-SMA) and determination of liver fibrosis by Sirius red staining

The sections were stained for α-SMA using a mouse monoclonal antibody (Neo Markers, Fremont, California). The sections were incubated with the primary antibody overnight at 4° C. Negative control slides were incubated with non-immune immunoglobulin under same conditions. Secondary reagents were obtained from the Dako Cytomation En Vision + System-HRP ready-to-use kit (Dako Cytomation Inc., Carpinteria, California) and 3,3′-diaminobenzidine (DAB) chromogen solution was used for visualization by light microscopy. Finally, the tissue was counterstained with haematoxylin for 3 minutes. Liver fibrosis was quantified using Sirius red FSB (also called Direct Red 80) as described.14 Direct red 80 and Fast green FCF (counterstain) were obtained from Sigma-Aldrich Diagnostics. Liver sections were stained and red-stained collagen fibers were quantified by digital image analysis as previously described by us in detail.12

Statistical analysis

All data are expressed as the mean ± standard error unless otherwise indicated. Differences between groups were compared using analysis of variance (ANOVA) for repeated measurements and post-hoc Bonferroni test to correct for multiple comparisons. A p-value less than 0.05 was considered to be statistically significant. All statistical analyses were performed using In-Stat Software (Graph Pad, San Diego, California).

RESULTS AND DISCUSSION

Hepatocyte apoptosis is significantly reduced in the BDL mouse with CTS-1027 treatment

Hepatocyte apoptosis is a prominent feature of cholestatic liver injury and occurs in the bile duct ligated liver.8 If an agent is to exert hepato-protective properties, it should attenuate hepatocyte apoptosis. To examine the effects of the MMP inhibitor CTS-1027 on hepatic apoptosis, mice were subjected to BDL for 14 days. Liver specimens from BDL animals treated with vehicle demonstrated numerous clusters of apoptotic cells characterized by condensation of chromatin at the nuclear membrane and fragmentation of the cell into subcellular bodies in a background of altered hepatic micro-architecture (Fig. 1A). The MMP inhibitor significantly modified the extent of cell death from numerous clusters of apoptotic hepatocytes to reduced numbers of isolated apoptotic hepatocytes. Quantitation of these TUNEL-positive cells demonstrated a 3-fold decrease in BDL mice receiving CTS-1027 as compared to mice treated with vehicle (Fig. 1A). The activation of executioner caspases, especially caspases 3 and 7, is a biochemical hallmark of apoptosis.15 Therefore, to further confirm hepatocyte apoptosis in animals following BDL, we next performed immunohistochemistry for activated caspases 3/7. Immunoreactive product was readily identified in liver tissues from mice following BDL, but not in sham-operated controls (Fig. 1B). Consistent with the TUNEL assay, BDL animals receiving CTS-1027 demonstrated a 5-fold decrease in the number of caspase 3/7-positive hepatocytes versus mice treated with the vehicle (Fig. 1B). Taken together, these data demonstrate that treatment with the MMP inhibitor CTS-1027 reduces hepatocyte apoptosis in the BDL mouse.

Figure 1
Hepatocyte apoptosis is reduced in 14 day BDL treated with CTS-1027. A. The number of TUNEL-positive cells was quantitated and expressed as apoptotic cells/10 high-power fields (hpf). Data are from 10 independent animals per group and are expressed as ...

Features of cholestatic liver injury are significantly reduced in BDL mice treated with CTS-1027

To further examine the effects of CTS-1027 on liver injury during BDL, histopathological examination of liver specimens was performed. After 14 days of BDL, mice receiving vehicle displayed severe cholestatic hepatitis with evidence of widespread bile infarctions (Fig. 2A) - a pathognomic feature of large bile duct obstruction in this animal.16 This histopathologic feature of obstructive cholestasis is recognized as confluent regions of hepatocellular degeneration and was reduced in liver specimens from animals receiving CTS-1027. Remarkably, these animals displayed almost intact liver morphology with excellent architectural preservation (Fig. 2A). This difference in cholestatic liver injury could not be ascribed to alterations in cholestasis as total bilirubin levels were almost identical in treated and untreated groups of animals (Fig. 2B). Interestingly, after 14 days of BDL, serum ALT values were not different between the two groups (599±68 vs. 527±89 U/L, N=8 per group, p>0.05). This observation is consistent with the fact that serum ALT values are a surrogate marker for liver injury and do not necessarily correlate with tissue injury as has been observed in human hepatitis C infection.17 Taken together, these observations suggest MMP inhibition by CTS-1027 is hepato-protective during murine obstructive cholestasis.

Figure 2
Cholestatic liver injury is attenuated in animals receiving CTS-1027 during BDL. A. Representative photomicrographs of conventional H & E-stained liver sections (magnification 20 x) are demonstrated. Liver specimens of BDL mice treated with the ...

Markers of hepatic fibrogenesis are attenuated in animals following BDL receiving CTS-1027

If the reduction of liver injury in BDL mice treated with CTS-1027 is significant, it should also translate into reduced hepatic fibrogenesis - a sequela of liver damage. Because stellate cells are the principal hepatic cell type responsible for collagen deposition in the liver,18 we next quantified α-smooth muscle actin transcripts, markers for stellate cell activation, by quantitative real time PCR. After 14 days of BDL, mRNA for α-smooth muscle actin was reduced 60% in CTS-1027 treated animals as compared to those receiving vehicle (Fig. 3A). Immunoreactivity for α-SMA, which was increased in the sinusoid lining cells of BDL mice, was reduced in BDL mice treated with CTS-1027 (Fig. 3B). To ascertain if stellate cell activation was also associated with enhanced hepatic fibrogenesis, mRNA for hepatic collagen 1α (I) was quantified. Indeed, collagen 1α (I) mRNA expression was decreased 60% in BDL animals following daily administration of CTS-1027 as compared to mice receiving the vehicle (Fig. 3C). Hepatic collagen protein deposition was further identified in liver specimens by Sirius red staining (Fig. 3D) and subjected to computer-assisted quantitative morphometry for quantification.12 Collagen staining by Sirius red was decreased approximately 70% treated versus untreated mice in BDL mice (Fig. 3D). Collectively, these data suggest that in BDL animals, stellate cell activation and hepatic fibrogenesis are attenuated by administration of the MMP inhibitor CTS-1027.

Figure 3Figure 3
Hepatic fibrogenesis is reduced in BDL animals upon treatment with CTS-1027. A. α-SMA and collagen 1α (I) mRNA expression, markers for stellate cell activation and hepatic fibrogenesis, were quantified by quantitative real time-PCR after ...

Overall animal survival following 14 days of BDL is improved in mice receiving the active drug CTS-1027

Given that liver injury and also hepatic fibrogenesis are significantly reduced in BDL mice treated with CTS-1027, we reasoned that animal survival should also be enhanced in this experimental group. Therefore, in our final study, we examined overall animal survival after BDL following treatment with CTS-1027 versus the CMC vehicle. By day 14 following BDL, 50% of the animals treated with CTS-1027 were alive as compared to only 20% of the mice receiving CMC (Fig. 4). Taken together, this study indicates that during obstructive cholestasis, treatment with the MMP inhibitor CTS-1027 exerts a survival advantage.

Figure 4
Overall animal survival following 14 days of BDL is enhanced in mice upon treatment with the MMP inhibitor CTS-1027. Initially, on day 7 after bile duct ligation 90 % of animals treated with CTS-1027 were still alive whereas 100 % of the mice receiving ...

In this study we demonstrated that inhibition of matrix metalloproteinases pharmacologically is beneficial in cholestatic liver injury. Cholestatic liver injury is likely mediated by a cascade of events. First, the acute retention of toxic bile acids promote hepatocyte injury, in part by death receptor-mediated processes.19 This primary hepatocyte injury elicits a secondary phase of injury characterized by infiltration of inflammatory cells, activation of Kupffer cells and transformation of quiescent stellate cells to activated myofibroblasts. A component of the secondary phase of injury is due to matrix metalloproteinases which induce remodelling of the extracellular matrix. This structural alteration of the liver further promotes liver injury and enhances hepatocyte apoptosis. Therefore, it is very likely in our current study that CTS-1027 inhibits the secondary injurious process in the liver. Interruption of this process attenuates further apoptosis and hepatic fibrosis. These data are consistent with the ability of matrix metalloproteinase inhibition to decrease inflammation in tumor necrosis factor-α/galactosamine treated mice.6 Our study extends these observations by demonstrating an antifibrogenic effect of a MMP inhibitor in a subacute process of liver injury. There are no data to suggest that these agents directly enter hepatocytes or alter the primaryphase of bile acid-mediated liver injury. Consistent with the elevated ALT values observed in both untreated and CTS-1027 treated BDL animals. Interestingly, in addition to their extracellular localization and function, MMPs have been demonstrated to be present and functional in the cell nucleus,20, 21 including in the liver.22 Further, they may have a role in apoptosis via their intracellular activity.20-22 Because CTS-1027 is cell-permeable (unpublished observation), we cannot exclude a role for intracellular MMP inhibition in the protection of hepatocyes from apoptosis.

Inhibition of MMP has been amply demonstrated to attenuate acute and chronic liver injury.6, 7 The current study extends these observations by employing the pharmacologic compound CTS-1027 in a preclinical model of obstructive cholestasis. This model was selected because BDL mice consistently undergo hepatocyte apoptosis and hepatic fibrosis over a subacute time frame, permitting assessment of liver injury and remodeling. In this model, CTS-1027 was able to attenuate both hepatic injury and liver fibrosis. This model displays features similar to human liver injury where hepatocyte damage promotes inflammation, stellate cell activation and hepatic fibrogenesis. However, what is less clear, is what the effect of CTS-1027 would be in a model where hepatic fibrosis is already established. One possibility is that CTS-1027 may limit further injury, as in the current model. Alternatively, because MMP-2, -8 and -13 also degrade collagen,23 broad-spectrum inhibition of MMP’s could impair the resolution phase of hepatic fibrosis. This concept could not be explored in the BDL mouse model given the limited survival observed in these animals. Nonetheless, preventing further fibrogenesis and hepatic injury would have a salutary effect in human liver disease, even if the previously deposited collagen present in the matrix could not be degraded. Additional studies using models of chronic fibrosis and cirrhosis will be necessary to evaluate the effects of CTS-1027 in established fibrotic disease models. Based on these data, CTS-1027 warrants further study as a hepatoprotective, antifibrogenic pharmacologic agent in human liver disease. Indeed, a limited clinical trial with CTS-1027 in hepatitis C patients is currently ongoing.

ACKNOWLEDGMENTS

The authors thank Erin Nystuen-Bungum for her excellent secretarial assistance and James Tarara from the Division of Biochemistry and Molecular Biology (Mayo Clinic, Rochester) for quantitation of the Sirius red images.

Grants: This work was supported by a fellowship grant from the Association for Scientific Research and Science at the Department of Gastroenterology and Hepatology, University Clinic Essen, Duisburg-Essen University, 45122 - Germany to A. K. and by grant DK 41876 from the National Institute of Health to G. J. G., Conatus Pharmaceuticals, as well as the Mayo Foundation Rochester, Minnesota, USA.

ABBREVIATIONS

α-SMA
α-smooth muscle actin
BDL
bile duct ligation/-ligated
CMC
carboxymethylcellulose
ECM
extracellular matrix
HSC
hepatic stellate cell
MMP
matrix metalloproteinase
TIMP
tissue inhibitor of metalloproteinase
TUNEL
terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling

Footnotes

DECLARATION OF COMPETING INTERESTS. This study was, in part, supported by Conatus Pharmaceuticals.

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