Cell viability was measured by trypan blue exclusion and exceeded 90%. The purity of HSC was higher than 99%, as assessed by fluorescence of retinoid-containing vacuoles under ultraviolet excitation.9 This study was performed following the regulations
of the local Animal Care Ethical Committee. Cirrhosis was induced by weekly intragastric administration of CCl4 for 8 weeks (Supporting Fig. 1A)10 or by intraperitoneal administration of 200 mg/kg of thioacetamide (TAA) 3 times per week for 7 weeks. SV40 vectors encoding IGF-I (SVIGF-I) and luciferase (SVLuc) have been produced as described7 and a single dose of 1 × 1011 viral particles DNA Damage inhibitor was administered through the hepatic artery 1 week after the last dose see more of hepatotoxicant. For the CCl4 model of liver cirrhosis four experimental groups of animals were analyzed in two independent experiments: healthy rats (n = 11), cirrhotic rats injected with saline (Ci)
(n = 14), and cirrhotic rats treated with either of SVLuc (Ci+Luc) (n = 8) or SV-IGF-I (Ci+IGF-I) (n = 16). For the TAA model animals were divided into the same groups (6 healthy rats, 5 Ci, 5 Ci+Luc, 5 Ci+IGF-I). Animals were sacrificed 8 weeks after virus injection. Blood samples were collected at different timepoints and analyzed as indicated (Supporting Fig. 1). Liver samples were processed for histology and purification of RNA and proteins for further analysis. Liver collagen content was assessed and quantified as described (Supporting Fig. 1).7 Immunohistochemical staining for α-smooth muscle actin (αSMA) was done with antibody 1A4 (M0851, Dako) diluted 1:100, and for IGF-1Rβ with antibody sc-713 (Santa Cruz Biotechnology) diluted 1:50. Total liver IGF-I (OCTEIA Rat/mouse IGF-I, Vitro) was measured in serum and liver extracts by ELISA. Total MMP activity was measured using a fluorogenic peptide substrate (R&D Systems). TIMP-1 was evaluated with antibody from R&D Systems diluted 1:500 and the western blot was quantified with Image Quant ECL selleck inhibitor (GE). Total RNA was extracted as described.7 RNA was also extracted from laser dissected
liver sections with Absolutely RNA nanoprep (Stratagene). Quantitative reverse-transcription polymerase chain reactions (qRT-PCRs) were done as described (Supporting Table 1).7 Data are expressed as means ± standard deviation. Statistical significance was estimated with Student’s t test. A P-value < 0.05 was considered significant (*). All statistical analyses were carried out with SPSS v. 11.0. To evaluate IGF-I effect in rat cirrhotic livers, cirrhosis was induced by intragastric administration of CCl4 for 8 weeks (Supporting Fig. 1A). Transaminases increased at the end of CCl4 treatment and remained higher than healthy controls more than half a year after completion of cirrhosis induction (Supporting Fig. 1B).