Vatalanib was purchased from LC Laboratories (Woburn, MA, USA; cat. no. V-8303), and dissolved in 100 mg/ml distilled water. A total of 32 male BALB/c mice weighing 18–20 g were obtained from Beijing Vital River Laboratory Animal Technology (Beijing, China). The Animal Care and Use Committee of Harbin Medical University approved all protocols and procedures (Harbin, China). Animals were housed in an air-conditioned room at 23–25°C with a 12-h dark/light cycle for one week prior to the initiation of experiments. All animals received appropriate care during the study with unlimited access to food and water.

Liver fibrosis was induced in mice by intraperitoneal injection of CCl₄ (40% CCl₄ in olive oil, 0.2 ml/100 g body weight, twice weekly) for 6 weeks as previously reported (21). A total of four groups were studied: Group 1, normal control mice; group 2, CCl₄ alone; group 3, CCl₄ + vatalanib (50 mg/kg/d by gavage for 6 weeks, vatalanib was administered simultaneously with a CCl₄ injection for 6 weeks); and group 4, CCl₄ + vatalanib (50 mg/kg/d by gavage for 4 weeks, CCl₄ alone was administered to mice for 2 weeks, following which vatalanib was administered to mice simultaneously with CCl₄ injection for another 4 weeks). In the previous study, vatalanib (50 and 100 mg/kg/d) was well tolerated by the mice, and had no significant effects on body weight (22). Therefore, 50 mg/kg/d was used in the present study. Previous studies identified that CCl₄-induced hepatic fibrosis is present after 2 weeks (20). Group 1 received olive oil intraperitoneally at the same dose and conditions as the CCl₄-treated mice.

Mice were euthanized (via 2% pentobarbital i.p. at 0.3 ml/ 100 g body weight) and liver samples were immediately snap-frozen in liquid nitrogen and stored at −80°C until further use. An additional section was embedded in paraffin and sliced into 4–5 µm sections.

Sections were stained with hematoxylin and eosin and Sirius Red (Solarbio Life Sciences, Beijing, China) for histopathological analysis and liver fibrosis evaluation. Each sample was independently assessed and scored by two pathologists, blinded to the study protocol, according to a fibrosis score system (16). Liver fibrosis was divided into seven stages: 0, no fibrosis; 1, short fibrous tissue in central venule (C); 2, fibrous between central venule and central venule (C-C) septa appearance; 3, C-C fibrous septa incompletely developed; 4, C-C septa completely connected (pseudo-lobule); 5, C-P (portal tract) bridging fibrosis, nodular appearance ≤50%; and 6, nodular appearance >50%.

Hepatic hydroxyproline was measured using a hydroxyproline detection kit (Jiancheng Institute of Biotechnology, Nanjing, China) according to the manufacturer's protocol. The hydroxyproline content is expressed as mg/g wet liver.

Samples were processed for TEM as described previously (23). Fresh specimens were fixed in 3% glutaraldehyde (Beijing Brilliance Biochemical Company, Beijing, China), washed with PBS, and fixed in 1% osmic acid for 60 min. The samples were dehydrated through an alcohol series, embedded in EPON 812 epoxy resin (Hede Biotechnology Co., Ltd., Beijing, China), and then cut into 50 nm sections with an ultrathin microtome. Following staining with uranyl acetate and lead citrate for 30 min, the sections were examined using a transmission electron microscope (cat. no. H-7650; Hitachi, Ltd., Tokyo, Japan). A total of 10 hepatic sinusoids with a diameter of 2–3 µm were randomly selected from each group and subgroup, and the mean number of fenestrae per hepatic sinusoid was determined. Each sample was independently assessed and scored by two pathologists, both blinded to the study protocol.

Blood was collected from the inferior vena cava. Serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (TBIL) and albumin were assayed using an automatic biochemical analyzer (cat. no. 7600; Hitachi, Tokyo, Japan).

CD34 is a marker of newly-formed blood vessels. The vascular density in portal and peri-portal areas was assessed by determining the number of CD34-labeled vessel sections (24). Liver tissue samples were fixed in 10% formalin for 1 week at room temperature, sliced into 4–6 mm pieces, dehydrated in ethanol and embedded in paraffin wax. α-SMA and CD34 were detected in paraffin-embedded liver sections (4–5 µm thick) using the following specific primary antibodies: α-SMA (cat. no. A2547; Sigma-Aldrich; Merck Millipore, Darmstadt, Germany) and CD34 (cat no. ab81289; Abcam, Cambridge, MA, USA) and an avidin-biotin complex immunoperoxidase method (15). Following antigen retrieval by immersion in EDTA (1 mmol/l, pH 8.0) and boiling for 15 min, sections were pre-blocked with normal goat serum for 10 min, and incubated for 2 h at room temperature with specific antibodies (α-SMA, dilution 1:200; CD34, dilution, 1:200). PBS was used in place of the primary antibodies for the negative controls. Following rinsing, the biotinylated secondary antibody (goat anti-rabbit IgG, cat no. ZDR-5307, goat anti-mouse IgG, cat. no. ZDR-5306, OriGene Technologies, Inc., Beijing, China), avidin-biotin complex and horseradish peroxidase (Strept ABComplex; Dako; Agilent Technologies, Inc., Santa Clara, CA, USA) were applied. Finally, the sections were washed with PBS, developed with diaminobenzidine tetrahydrochloride substrate (Strept ABComplex; Dako; Agilent Technologies, Inc.) for 3 min, and counterstained with hematoxylin.

Computer-assisted semi-quantitative analysis was used to evaluate the areas of positive α-SMA and CD34 staining using Image-ProPlus software (version 4.5; Media Cybernetics, Inc., Rockville, MD, USA). The data for the α-SMA and CD34 staining are expressed as the mean percentage of the positively stained area relative to the total tissue section area (17).

Total RNA was extracted from liver tissues and HSC-T6 cells using a TRIzol^® Reagent kit (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) according to the manufacturer's protocol. The quality of total RNA was verified by ultraviolet absorbance spectrophotometry at 260 and 280 nm. cDNA was reversed transcribed using the High-Capacity cDNA Reverse Transcription kit (Applied Biosystems; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. The thermocycling parameters were as follows: 25°C for 10 min, 37°C for 150 min, 85°C for 5 sec, and 4°C for 5 min for one cycle, before chilling on ice. cDNA was stored at −20°C until further required. Collagen I, α-SMA, TGF-β1, VEGFR1 and VEGFR2 mRNA levels were quantified using the primers presented in Tables I and II. GAPDH, a housekeeping gene, was used as an internal control primer for target genes. All primers were obtained from Invitrogen; Thermo Fisher Scientific, Inc. The expression of mRNA was measured by SYBR Green Real-Time PCR using an ABI 7500 instrument (Applied Biosystems; Thermo Fisher Scientific, Inc.). PCR was performed in 20 µl buffer that contained 2 µg cDNA, 1 µl each primer and 10 µl SYBR Green PCR Master Mix (Applied Biosystems; Thermo Fisher Scientific, Inc.). Comparative quantitation threshold (Cq) calculations were all relative to the control group. The expression of mRNA relative to the control was derived using the equation 2^−∆∆Cq (25).

The results are expressed as the mean ± standard deviation. Statistical analysis was performed by analysis of one way analysis of variance and unpaired Student's t-test as appropriate. Non-parametric data were analyzed by the Mann-Whitney U-test. P<0.05 was considered to indicate a statistically significant difference. Statistical analyses were performed using SPSS (version 17.0; SPSS Inc., Chicago, IL, USA). Data are presented as the mean ± standard deviation.

There were no collagen fibers along the central vein in control group 1 (Fig. 1A). In group 2, collagenous fibers extended from the central vein and the portal area to the hepatic lobules with pseudo-lobule formation (Fig. 1B). With administration of vatalanib (50 mg/kg/d, for 4 or 6 weeks) in groups 3 (Fig. 1C) and 4 (Fig. 1D), collagenous fibers were decreased compared with group 2. The fibrosis scores following vatalanib administration in groups 3 and 4 were significantly reduced when compared with those in group 2 (P<0.05; Table II). In addition, vatalanib reduced hydroxyproline concentrations in liver tissue, reflecting decreased total hepatic collagen content (P<0.01; Table III).

Serum ALT and AST levels were significantly decreased following vatalanib treatment (P<0.01), whereas no significant alterations in serum TBIL and albumin levels were observed (P>0.05; Table III).

Levels of α-SMA, a typical marker of activated HSCs, were assessed by immunohistochemistry to evaluate the effect of vatalanib on HSC activation during hepatic fibrosis. There was no positive staining in control group 1 (Fig. 2A). CCl₄ alone led to considerable increases in the amount of α-SMA positive cells, which were distributed throughout the fibrotic septa (Fig. 2B). Computer-assisted semi-quantitative analysis revealed that vatalanib-treated groups (groups 3 and 4) presented significantly decreased α-SMA-positive areas compared with group 2 (P<0.05; Fig. 2C-E). These results demonstrated that vatalanib decreased the number of activated HSCs.

There was little positive CD34 staining in the control group 1 (Fig. 2F). Following six weeks of CCl₄ induction, this led to significant increases in the number of CD34 positive cells, while groups 3 and 4 presented significantly decreased CD34 positive areas compared with group 2 (P<0.05; Fig. 2G-J).

There were significantly increased fenestrae per SEC in group 1 (Fig. 3A) compared with group 2 (Fig. 3B; P<0.01; Table IV). In group 2, the microvilli of hepatocytes in the perisinusoidal space and the intra-lobular bile ducts (cholangioles) disappeared (Fig. 3C), and the junctions between hepatocytes were destroyed (Fig. 3D). In group 3, the number of fenestrae was significantly increased compared with group 2 (P<0.01; Fig. 3E; Table IV), and cholangioles appeared morphologically healthy (Fig. 3F). Group 4 additionally had an increased number of fenestrae compared with group 2 (Fig. 3G; Table IV), and presented with cell junctions similar to normal control mice (Fig. 3H). There were no significant differences in the numbers of fenestrae between groups 3 and 4 (P>0.05). The extent of hepatocyte necrosis and inflammation was less so in groups 3 and 4 compared with group 2.

mRNA expression levels of collagen I, α-SMA, TGF-β1, VEGFR1 and VEGFR2 were increased in group 2 compared with group 1 (P<0.01; Fig. 4). mRNA levels were reduced in groups 3 and 4 compared with group 2 (P<0.01).