shown to contribute to an acceleration of liver fibrosis by mediating initial neutrophil infiltration into the cholestatic liver [12]. MMP-9-deficient mice exhibited moderate protection against early fibrosis [13]. Thus, the question arises, do MMP-9 and 13 account for all the hepatoprotective effects of general MMP inhibitors, or do other MMPs also play important roles? MMP-19 appears to be widely expressed at the mRNA level [14,15], however, the expression of the protein seems to be restricted to several cell types and tissues [16–19]. MMP-19 is able to cleave components of ECM such as laminin 5c2 chain, nidogen-1, tenascin C, collagen IV, and aggrecan among others [20–23]. The role of MMP-19 appears to be prominent in cell types or in compartments where these substrates as well as MMP- 19 are simultaneously available, as was documented in the study showing that MMP-19-deficiency causes an accumulation of tenascin-C in bronchial walls of mice suffering from asthma [24]. MMP-19 was also shown to exhibit an anti-tumor effect as it suppresses tumor angiogenesis and invasion [25,26]. Of note, it has recently been reported that MMP19 may play a protective role during the development of lung fibrosis [27]. However, although MMP-19 is highly expressed in liver [28,29], its contribution to the pathologic processes in this organ is completely unknown. In this study, we used MMP-19-deficient mice (MMP19KOs) to establish the role of this MMP in the development and resolution of liver fibrosis in mice using CCl4 intoxication. We show that MMP-19-deficiency leads to a reduction in the extent of hepatocellular injury and, thus also to a faster recovery from fibrosis. Materials and Methods Animal Model of CCl4-induced Liver Fibrosis Generation of MMP19KOs was previously described [30]. Experiments were performed with male MMP19KO homozygotes on C57BL/6NCrl background (backcross 21) and control (wild- type, WT) C57BL/6NCrl male mice derived from the backcross breeding. The high number of backcrosses to C57BL/6N background and stable genetic background is important in mouse experimental studies as documented in other studies [31]. MMP19WT littermate controls were used in some experiments with similar results as C57BL/6NCrl mice. To induce fibrogen- esis, mice were injected intraperitoneally with 1 ml/g CCl4 (diluted 1:3 with olive oil) or with the vehicle (olive oil control) twice a week for 4 or 6 weeks. Liver and blood samples were collected 48 hours after the last injection under anesthesia. For more details on sample collection see Materials and Methods S1 section. Ethic Statement Animal care and experiments were approved by the Animal Care Committee of the Institute of Molecular Genetics in compliance with national and institutional guidelines. Histological Analysis Specimens were fixed in 4% buffered paraformaldehyde, embedded in paraffin, sectioned, and stained with hematoxylin and eosin (H&E), Sirius Red [32], or processed for immunohis- tochemistry to detect Ki67 antigen (details in Materials and Methods S1 section). Analysis of Serum Parameters Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were assessed from serum using kits from Roche Diagnostics (Prague, Czech Republic). Serum levels of MCP1, IL-6, and KC MMP-19 Deficiency Is Hepatoprotective In Vivo were assayed by ELISA using antibodies from R&D Systems (Minneapolis, MN). Quantitative Reverse-Transcriptase Polymerase Chain Reaction (qRT-PCR) RNA was isolated from liver samples stored in RNAlaterH (Ambion, Austin, TX) using the RNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. RNA from samples from the 6-week experiment was isolated from frozen tissue using TRIreagent (Sigma, St.Louis, MO). See supplementary data for further details. Expression of genes of interest was normalized to GAPDH. Hydroxyproline analysis Liver samples were analyzed on the JASCO HPLC (Essex, UK) system equipped with a SC-5ODS column (200 mm64 mm; Waters) at 0.8 mL/min and 30uC using 470 nm excitation and 540 nm emission with 0.05 M phosphate buffer (pH 5.0) contain- ing 8% acetonitrile as a mobile phase as described [33]. The peak area of hydroxyproline was normalized to the total amino acid content in lysates and expressed as mM per mg of amino acid (mM/ AA). Immunoblotting and gelatin zymography Frozen liver tissues in RIPA buffer or TRI ReagentH (Sigma) were homogenized using TissueLyzer II (Qiagen). Equal amounts of protein were resolved on SDS polyacrylamide gels and transferred to nitrocellulose membranes; proteins were detected using antibodies as detailed in the Supporting Information section. For zymography, lysates were prepared by homogenization of frozen tissue in PBS containing 1% Triton X-100, 0.5% deoxycholate, and 0.1% SDS and the zymography was performed as described previously [34]. Primary hepatocyte isolation Hepatocytes were isolated from WTs and MMP19KOs as described [35]. Hepatocytes were plated on collagen I (0.3 mg/ mL; BD, Franklin Lakes, NJ) and cultured in DMEM (Sigma) containing 10% fetal bovine serum (FBS, Invitrogen Life Technologies, Carlsbad, CA), 1% penicillin-streptomycin (PAA, Pasching, Austria) and 0.08 U/mL insulin (Eli Lilly, Indianapolis, IN). After 24 h, cells were serum-starved and then stimulated with recombinant human TGF-ß1 (2.5 ng/mL; R&D Systems). Cells were harvested after 0.5, 1, 6, 24, 48, and 72 h of stimulation for RNA and protein isolation by TRIreagent and processed for qRT- PCR (see Materials and Methods S1 section) and immunoblotting. Results MMP19KOs exhibit ameliorated injury after chronic CCl4- administration MMP19KOs exhibit no apparent changes in liver histology [30] and their ALT and AST serum levels are comparable to WTs (Figure S1). To assess the role of MMP-19 in liver fibrosis, we compared liver damage in MMP19KO and WT male mice after 4- or 6-week exposure to CCl4. A single dose of CCl4 did not result in any significant difference in ALT or AST levels between WT and MMP19KO mice (not shown). However, after 4 weeks of intoxication, while both WT and MMP19KO mice developed serious hepatocellular damage as judged from high serum ALT and AST levels compared to olive oil-treated controls, both markers were significantly higher in WT than in MMP19KO mice (Figure 1A). 6-week CCl4-treatment resulted in further increases in PLOS ONE | www.plosone.org 2 October 2012 | Volume 7 | Issue 10 | e46271