This work was approved by First Hospital of Jilin University and was carried out in accordance with the Declaration of Helsinki (2000) of the World Medical Association. Informed consent was obtained from all patients prior to sample collection.

Serum was collected from 146 patients with CLD-B who were either outpatients or inpatients at First Hospital (Jilin University, Changchun, China) from June 2008 to September 2011. Of the 146 patients, there were 107 cases with chronic hepatitis B (CHB) (69 men and 38 women; mean age: 43.9 years, range: 18-76 years), 39 cases with active liver cirrhosis (27 men and 12 women; mean age: 41.9 years, range: 26-68 years). As determined using the Child-Pugh score to assess the severity of liver cirrhosis, 25 cases were class A and 14 cases were class B.

Serum was collected from 30 healthy individuals (18 men and 12 women; mean age: 33.6 years, range: 19-56 years) for controls. Serum and liver samples were collected from 8 HBV carriers who had histological normal livers (6 men and 2 women; mean age: 28.6 years, range: 19-38 years), and were studied as normal controls for the assessment of liver fibrosis of CLD-B patients. Patients and HBV carriers meet the diagnostic criteria for chronic HBV infection[12].

Liver tissue samples from 39 CLD-B patients and 8 HBV carriers were obtained using a percutaneous needle. The length of each sample was more than 1.5 cm. There were 31 cases with CHB (23 men and 8 women; mean age: 29.8 years, range: 18-50 years) and 8 cases with HBV-induced active liver cirrhosis (6 men and 2 women; mean age: 36.6 years, range: 26-54 years). Importantly, initial studies showed that levels of serum CTGF, Collagen I, Collagen III or aminotransferases in CLD-B patients receiving a liver biopsy were not significantly different as compared to CLD-B patients without a biopsy.

Sera were stored at -70 °C for 1 to 6 mo before analysis. The level of CCN2 in sera from 146 patients with CLD-B, 8 HBV carriers and 30 healthy individuals were measured with a commercial enzyme-linked immunosorbent assay (ELISA) kit, according to manufacturer’s protocols (USCN Life Science and Technology Co, TX, United States). Briefly, microtiter wells were pre-coated for 2 h at 37 °C, with 100 μL of each standard or 1:20 dilutions of sera. The plates were then developed by sequential addition of biotinylated anti-CCN2 antibody, avidin-conjugated horseradish peroxidase and tetramethylbenzidine substrate solution, and the color reaction was measured at 450 nm.

Liver tissue samples from 31 CHB patients, 8 patients with HBV-induced liver cirrhosis and 8 HBV carriers were individually fixed, paraffin-embedded and subjected to H and E staining or Van Gieson’s method to determining histological inflammation and fibrosis which were scored using the Metavir system. Fibrosis was staged on a 4-point scale (F0: No fibrosis; F1: Minimal fibrosis; F2: Fibrosis with a few septa; F3: Numerous bridging fibrosis without cirrhosis; F4: Cirrhosis or advanced severe fibrosis). F1-F2 was defined as mild fibrosis and F3-F4 as significant fibrosis. Inflammation was graded on a four-point scale from A0, which indicated no inflammatory activity, up to A3, which indicated severe activity.

In situ hybridization (ISH) was performed using digoxigenin-labled sense or anti-sense probes for CCN2 or TGF-β1 (Boster Biotechnology Co. Ltd. Wuhan, China). In brief, liver tissue samples from CLD-B patients were formaldehyde-fixed and parafin-embeded. The tissue sections (5 μm) were deparaffinized, rehydrated with PBS, digested with pepsin (30 μg/mL) for 10 min at 37 °C, fixed in 4% paraformaldehyde in PBS and washed in 3×SSC. The samples were pre-hybridized at 40 °C for 2 h, and hybridization was performed overnight at 40 °C with sense or anti-sense probes. After hybridization, excess probes were removed by sequential washing in twice concentrated (2×) saline-solution citrate buffer (SSC), 0.5×SSC and then 0.2×SSC at 37 °C for 2 h. The tissue sections were incubated at 37 °C for 1 h with biotinylated mouse anti-digoxigenin, followed by addition of the streptavidin-biotin-peroxidase complex for 20 min. The slides were then developed with 3-amino-9-ethylcarbazole (Boster Biotechnology). Ten random images (original magnification ×400) of each slide underwent computer image analysis using Image-Pro Plus 6.0 software to assess the integrated optimal density (IOD) of CCN2-positive cells in liver tissues.

Formalin-fixed, paraffin-embedded sections (5 μm) were de-waxed and re-hydrated. Sections were incubated overnight at 4 °C with mouse anti-human α-SMA monoclonal antibody (Zhongshan Goldbridge Biotechnology, Beijing, China) or rabbit anti-human CCN2 polyclonal antibody (Santa Cruz, Heidelberg, Germany) or rabbit anti-human F4/80 polyclonal antibody (Spring Bioscience, United States). Sections were washed in PBS and incubated at room temperature for 10 min with biotinylated goat anti-mouse and rabbit IgG (Maixin Bio, Fuzhou, China). After washing with PBS, sections were incubated with streptavidin-peroxidase (Maixin Bio, Fuzhou, China) for 10 min and then developed with diaminobenzidine or 3-amino-9-ethylcarbazole.

The values reported represent the median [95% confidence interval (CI)] of the measurements. Statistical analysis of the data was performed using SPSS 13.0 for Windows (SPSS Inc, Chicago, IL, United States). The nonparametric Wilcoxon signed ranks test was used for pair-wise comparison of groups and Spearman’s rank correlation analysis was used to determine the relationship between two variables. Areas under the receiver operating characteristic (ROC) curves (AUC) were calculated for comparing the accuracy of the CCN2 in sera in different subgroups.

In normal livers, only mild CCN2 mRNA staining was detected in portal tracts and there was no staining in the central vein or lobule (data not shown). However, CCN2 mRNA and protein were localized to fibrotic septa in CLD-B patients with hepatic fibrosis (Figure 1A and B) or cirrhosis (data not shown), and the pattern of CCN2 staining was well correlated with the distribution of collagen fibers (Figure 1D). By comparison to normal livers, CHB patients demonstrated strong α-SMA-positive staining in presumptive activated HSC (Figure 2A and B) and these cells also stained strongly for CCN2 mRNA (Figure 2C and D).

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Figure 1 Production of connective tissue growth factor or transforming growth factor β-1 in fibrous septa of hepatitis B virus-infected livers. Connective tissue growth factor mRNA (A) or protein (B) were detected by in situ hybridization (ISH) or immunohistochemistry respectively. transforming growth factor β-1 mRNA (C) was detected by ISH while collagen bundles (D) were stained red using Van Gieson’s method. Original magnification, × 200 in A, B, C and D. Examples of positively stained cells or structures in each panel are arrowed.

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Figure 2 Cellular localization of key fibrotic markers in hepatitis B virus -induced liver fibrosis. α-SMA-positive hepatic stellate cells (HSCs) were not detectable in hepatitis B virus carriers who had normal liver histology (A) but were present in chronic hepatitis B (CHB) liver (B). In CHB liver samples, there was no staining when the in situ hybridization probes were omitted (C) but their inclusion demonstrated the presence of either connective tissue growth factor mRNA in activated HSCs (D) or transforming growth factor β-1 mRNA in activated HSCs (E) or Kupffer cells (F). F4/80 antigen-positive Kupffer cells (G). Original magnification, × 400 in A-G. Examples of postively stained cells in each panel are arrowed.

Since CCN2 acts downstream of TGF-β1 to drive fibrosis[13], we examined the expression and distribution of TGF-β1 mRNA in the liver of CLD-B patients. TGF-β1 mRNA was detected in the fibrotic septa of patients with hepatic fibrosis (Figure 1C) or cirrhosis (data not shown) and was localized to activated HSCs in the lobule (Figure 2E) and Kupffer cells (Figure 2F and G) within inflammatory area in the livers of CHB patients.

Since CCN2 is synthesized with a signal peptide and can exist extracellularly in soluble or matrix-associated forms, we examined serum from CLD-B patients for the presence of CCN2 protein by ELISA. As shown in Table 1, serum CCN2 concentrations were, respectively, 4.0- or 4.9-fold higher in patients with CHB or active liver cirrhosis as compared to healthy individuals (P < 0.01). There was no difference in the serum CCN2 levels between the HBV carriers and healthy individuals.

Having shown that hepatic and serum CCN2 concentrations were higher in CLD-B patients than in healthy individuals, we next investigated if there was a correlation between CCN2 and fibrosis stage. As shown in Table 2, serum concentrations and hepatic content of CCN2 increased in proportion to the severity of fibrosis; Spearman’s rank correlation analysis showed that correlation coefficients were 0.85 and 0.89 (both P < 0.01), respectively. However, the levels of CCN2 in sera were not correlated with the degree of inflammation in CHB patients.

We further analyzed the diagnostic performance of serum CCN2 for assessing liver fibrosis using the ROC curves. Calculation of the areas under ROC curves (AUC) showed that serum CCN2 could be used to distinguish either normal liver controls from patients with F1 stage liver fibrosis (AUC = 0.94) or mild fibrosis (F1/F2) from significant fibrosis (F3/F4) (AUC = 0. 85) (Figure 3).

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Figure 3 Receiver operating characteristic curve of connective tissue growth factor. Receiver operating characteristic (ROC) curves of connective tissue growth factor distinguishing normal liver controls from patients with F1 stage liver fibrosis (A) or discriminating between mild and significant fibrosis (B) with areas under the ROC curves of 0.94 or 0.85, respectively.

Millions of individuals around the world are infected with hepatitis B virus (HBV), resulting in chronic liver disease. In many cases, affected individuals suffer from hepatic fibrosis, a highly debilitating pathology in which the normal cellular architecture and function in the liver are severely compromised through the deposition of collagen and other insoluble extracellular matrix molecules. This process is driven by connective tissue growth factor (CCN2) which is known to be produced at high levels in fibrotic livers and which acts to drive fibrogenic pathways in hepatic stellate cells (HSCs), a principal fibrotic cell type in the liver.

Currently, parameters used to assess liver fibrosis are inaccurate. There is optimism that measurement of CCN2 levels in either the livers or serum of affected patients will have useful diagnostic or prognostic value.

To date, there have been a limited number of studies regarding the value of serum CCN2 for assessment of hepatic fibrosis. In this study, the authors employed more systemic detection techniques to evaluate the relationship among serum CCN2 levels, hepatic CCN2 content and liver fibrosis severity in patients with chronic liver diseases. Furthermore, the authors described the expression characteristics of CCN2 in liver tissues and its role and mechanism in HBV-induced hepatic fibrosis.

These studies suggest that serum CCN2 concentrations are a reliable diagnostic indicator of HBV-induced liver fibrosis and that CCN2 can be used a part of the platform for evaluation of the severity of liver fibrosis.

CCN2: a pro-fibrogenic molecule that is over-expressed in many fibrotic diseases and which stimulates collagen synthesis in HSC.

This is an interesting and important issue in the utility of CCN2 for assessing hepatic fibrosis. Correlations of the serum levels of CCN2 in HBV infected patients with hepatic fibrosis have been well documented in literature.