Male C57BL/6 mice (Harlan Laboratories, Eystrup, Germany) aged 10–12 weeks were kept under controlled environmental conditions with a 12-h light–dark cycle. Mice were fed on a standard laboratory diet with food and water ad libitum. FHL2−/− knockout mice were bread into C57Bl/6 background for more than ten generations. All animal experiments were approved by the Landesamt für Natur, Umwelt und Verbraucherschutz NRW (LANUV), Recklinghausen, Germany under reference number 8.87-51.04.20.09.
Induction of liver fibrosis
Six to eight weeks old wt mice (n = 9) or FHL2-deficient mice (n = 12) weighing approximately 18–20 g received intraperitoneal injections twice weekly of 0.7 ml/kg body weight of CCl4 in equal volume of mineral oil for up to 6 weeks (i.e. a total of 12 i.p. injections), whereas mineral oil alone was used as controls in wt animals (n = 4) or FHL2-deficient mice (n = 4).
After 6 weeks of treatment, the animals were sacrificed by cervical dislocation. The livers were removed and samples snap-frozen in liquid nitrogen and stored at −80°C or fixed in formaldehyde (4%). Blood was drawn from the right ventricle, centrifuged and the serum was stored at −80°C until further analysis.
Measurement of serum parameters
Blood biochemical parameters (bilirubin, ALT, AST and total protein) were measured using the Modular Pre-Analytics (MPA) system (Roche Diagnostics, Mannheim, Germany).
Histology and immunohistochemistry
After 24 hours fixation in 4% buffered formalin, the liver specimens were embedded into paraffin. Histological quantitative examination of liver fibrosis was performed on sections after standard Sirius Red staining. For the morphometric analysis of Sirius Red-stained specimens, at least 10 mm2 of liver tissue was analysed by means of computational analysis (Histoquant, 3DHistech, Budapest, Hungary). Large bile ducts and vessels were excluded. The principle of computational analysis has been described elsewhere [10, 11]. For the analysis of activated HSC in wt and FHL2−/− livers, paraffin liver sections were co-stained with antibodies for collagen III (anti-collagen III, Biozol/SouthernBiotech, Eching, Germany) and α-SMA (anti-α-SMA, clone E184, Biomol/Epitomocs, Hamburg, Germany).
Hepatic hydroxyproline content
Hepatic hydroxyproline content was determined in analogue segments (50 mg) of snap-frozen livers using standard methods. The results are calculated as mg/g of wet liver tissue.
RNA isolation, reverse transcription with 0.5 μg total RNA, and detection by RT-PCR were performed as previously described . Most of the primers and probes for RT-PCR were obtained as a ready-to-use mix (α-SMA, Fibronectin, TGF-β, Procollagen Iα, from Applied Biosystems, Foster City, USA). Murine GFAP was amplified using primers mGFAPf (5′-CCT TCT GAC ACG GAT TTG GT-3′) and mGFAPr (5′-ACA GAC TTT CTC CAA CCT CCA G-3′) and murine fibulin-2 was amplified using primers mFbln2f (5′-CCA TCA AAC ACT CGT CTT GGT-3′) and mFbln2r (5′-TGT TGT TGG GGA CAC AGC TA-3′), respectively. GAPDH served as endogenous control (primers and probes ready-to-use mix by Applied Biosystems). RT-PCR (ABI 7300 sequence detector) and PCR reaction (2x TaqMan-PCR-mastermix, Applied Biosystems) were performed as previously described . For each of the genes, a validation experiment was performed. Efficiencies of RT-PCR for the target gene and the endogenous control were approximately equal. -ΔCT expresses the difference between number of cycles (CT) of the target genes and the endogenous control. Results were expressed as 2-ΔΔCt, and express the x-fold increase of gene expression compared to control group.
Primary HSC from wt and FHL2 deficient mice were isolated by pronase-collagenase perfusion following by FACS-based purification protocols . 10 × 104 cells each were seeded onto glass coverslips and cultivated in Dulbecco’s modified Eagle medium (Lonza BioWhittaker, Verviers, Belgium) supplemented with 10% FCS (PAA Laboratories, Pasching, Austria), 4 mM L-Glutamine (Lonza), 100 U/ml penicillin and streptomycin (Lonza). For generation of fully transdifferentiated MFB, the cells were passaged once and cultured for an additional 2 or 7 day period. At indicated time points, the cells were fixed in 4% paraformaldehyde for 10 min and stained with a rhodamin phalloidin.
FHL2 immunohistochemistry in human liver fibrosis
We investigated the expression of FHL2 immunohistochemically in three samples of human liver fibrosis. Normal liver served as control. The staining procedure was carried out as described earlier . In brief, immunohistochemistry was performed on 4-μm-thick paraffin-embedded sections by use of the peroxidase-conjugated avidin–biotin method. Primary antibodies included FHL2 (kind gift of R. Schüle, University Hospital Freiburg, Freiburg, Germany) and α-SMA (clone 1A4, DAKO, Hamburg, Germany, 1:4000).
Data are presented as mean±standard error of the mean (SEM). Student’s t-test was used for comparison where appropriate, p-values < 0.05 were considered statistically significant.