Biliary atresia (BA) can cause neonatal cholestasis by undefined mechanisms, and is characterized by fibrosclerosing and inflammatory destruction of the extrahepatic and intrahepatic biliary system during the first few weeks of life (1,2). Neonatal cholestasis is a devastating disease that leads to cirrhosis, requiring liver transplantation as the only option for therapy in the majority of cases (3); however, despite surgical relief from the obstructive deposition of collagen, progressive hepatic fibrosis and portal hypertension usually occur in BA (4). The mechanisms responsible for increased collagen production and hepatic fibrosis in BA remain largely unknown. Hepatic stellate cells (HSCs) have been shown to be activated and responsible for the increased production of type I collagen leading to hepatic fibrosis in pathological conditions of the adult human liver, and in a number of experimental models of adult liver injury, including cholestasis. During liver injury, HSCs are transformed into myofibroblasts (activated HSCs), which produce increased levels of fibrillar collagen, and express an intracellular microfilament protein, α-smooth muscle actin (α-SMA), which has traditionally been used as a marker protein of the activated HSC phenotype (5–7); however, it is not clear to date whether the expression of α-SMA is associated with the hepatic fibrosis due to BA. Therefore, in the present study, we focused on the expression of α-SMA in infants with BA.
Patients and Controls
The study group consisted of infants with BA, and controls infants with choledochal cysts and cholestasis syndrome. All of the individuals were recruited prospectively from the Children's Hospital of Fudan University (from March 2011 to August 2011). In the BA group, there were 21 infants diagnosed by intraoperational cholangiography who underwent a classic Kasai procedure at ages 42 to 113 days (66 ± 20 days). All but 2 were younger than 70 days. In the control group, 5 who had cholestasis syndrome were diagnosed by the cholangiography and underwent laparotomy at age 59 to 88 days (72 ± 13 days). A total of 10 cases of choledochal cyst underwent a Roux-en-Y choledochojejunostomy at ages 59 to 88 days (72 ± 13 days). For BA, the remnant of the porta hepatis and a wedge liver biopsy were obtained. For the control group, liver biopsies were obtained. All of the studies were approved by the ethics committee of Fudan University, and voluntary written informed consent was obtained from the parents of all of the participants involved in the present study.
Histologic Grading of Fibrosis
Paraffin-embedded liver sections were stained with hematoxylin-eosin and with Gomori trichrome. A pathologist blinded to patient outcomes scored each set of slides for degree of fibrosis on 3 separate occasions. In the fibrosis analysis, we used a 3-grade staging system used previously in BA (8), defined as mild (grade I), if it ranged from portal fibrous expansion to bridging fibrosis involving <50% of portal tracts; moderate (grade II), if bridging fibrosis was present with >50% of portal tracts involved without nodular architecture; and severe (grade III), if there was bridging fibrosis with >50% of portal tracts involved, accompanied by nodular architecture. Because the intraobserver variation for the latter grading system was 0.8 compared with 1.8 for the 6-stage score, we used the 3-grade system in the present study (8).
All of the liver sections were incubated with a mouse monoclonal primary antibody directed against α-SMA (1:400, clone 1A4; Sigma Chemical Co, St Louis, MO). Bound antibodies were detected with biotinylated rabbit anti-mouse immunoglobulin G and a streptavidin-biotin complex/horseradish peroxidase kit (DAKO Glostrup, Denmark) using 3,3-diaminobenzidine tetrahydrochloride (Sigma Chemical Co) as the chromogenic substrate. Sections were counterstained with eosin (5).
A Leica DM microscope was used to acquire images of the sections. All of the pictures were taken at one time in to keep the conditions identical for all. A total of 5 random fields of view where the antigens were positive were chosen for each section (×200 magnification for CD68, monocyte chemoattractant protein-1, intercellular adhesion molecule [ICAM], and ×100 magnification for SMA). The proportion of the antibody-positive area and integrated optical density was measured by using Image Pro Plus software. The former stands for the total amount of expressed antibody and the latter for intensity.
Analysis of Prognosis
A total of 19 cases (2 cases were lost to follow-up) were reviewed. The rates of decline of serum-conjugated bilirubin (CB) were calculated: (1-postoperative CB at 3 months/preoperative CB) × 100% (8).
Data are expressed as the mean ± standard deviation. The Student t test was used to determine statistically significant differences between groups, and P < 0.05 was considered significant. The correlations were analyzed by Spearman coefficient analysis. All of the analyses were performed using SPSS version 13.0 (SPSS Inc, Chicago, IL) software.
In the fibrous cone of the porta hepatis, α-SMA staining localized in fibrous collagen surrounding the bile ducts and biliary epithelial cells of BA. In liver tissue, α-SMA staining was localized in biliary epithelial cells and fibroblasts of BA, and was relatively weaker in the control liver tissue. Staining was positive in dilated bile ducts in some cases of choledochal cyst (Fig. 1).
Amount and Intensity of α-SMA Expression
The proportion and intensity of α-SMA in BA liver tissue were 6.4% ± 0.8%, and 17.8 ± 1.8, respectively, and 3.4% ± 0.7%, 12.0 ± 2.1 in controls, respectively. The amount and intensity of α-SMA in BA was higher than in controls (P < 0.05). The proportion (area%) of α-SMA in BA liver tissue positively correlated with the intensity (integrated optical density/area) (r = 0.549, P = 0.022).
Correlation Between CD68 and α-SMA BA in Liver Tissue
The proportion of CD68 in liver tissue of BA was 8.4% ± 1.1%. The correlation between the α-SMA and CD-68 expression was not significantly different (r = 0.444, P = 0.057).
Correlation Between Amount of α-SMA and Liver Fibrosis in BA Liver Tissue
According to the 3-grade scoring system in BA, 2 cases were defined as mild (grade I), 12 cases were moderate (grade II), and 7 cases severe (grade III). The positive areas of α-SMA in the 3-grade cases were 1.79% ± 2.02%, 5.49% ± 0.86%, and 8.01% ± 1.17%, respectively. An analysis of variance showed that the more severe the BA grade, the more positive the areas of α-SMA (Fig. 2).
Correlation Between the Expression of α-SMA and Prognosis
A total of 19 cases were reviewed after 3 months of operation (2 cases were lost to follow-up). The range of decline of serum CB at 3 months was 28.9% to 97.4%. The proportions of α-SMA in the livers of patients with BA negatively correlated with the reduction of direct bilirubin, 3 months postoperatively (r = −0.653, P = 0.029) (Fig. 3).
BA is a most common neonatal cholestatic disease, and is characterized by progressive destruction of bile ducts (9). Although the precise pathogenesis has not been determined, the most likely explanation at present is an abnormality in T-cell–mediated immunity against antigen-presenting biliary epithelial cells (10). Antigen-presenting cells (APCs) are likely to be central to the entire process of immunity. Professional APCs include macrophages, dendritic cells, and B lymphocytes. Nonprofessional APCs include endothelial and epithelial cells (of the bile duct). In BA, it is the macrophages (Kupffer cells in the liver), not B lymphocytes, which are prevalent in the porta hepatis (11,12). APCs produce an ICAM linking APCs to T cells. Recent studies have confirmed the expression of ICAM in epithelial cells in BA (13,14). Therefore, Kupffer cells and epithelial cells are the main APCs as far as BA is concerned. CD68 is a 110-kDa transmembrane glycoprotein, which is highly expressed in human monocytes including tissue macrophages. We assessed the amount and intensity of CD68 expression to show how many APCs (Kupffer cells) are activated. In the present experiments, CD68 was found to be expressed diversely in the liver of BA, and also expressed in the livers of the control group; however, the staining was weaker in the controls, which is consistent with the findings of Mack et al (10). Neither the amount nor the intensity of CD68 expression was significantly higher in the livers of BA compared with those in the control group.
Liver fibrosis, apoptosis of epithelial cells, and intracellular cholestasis are the main pathological features of BA. Liver fibrosis is stimulated by cytokines, and its presence is considered to be a prognostic factor in BA. Transforming growth factor-β1 (TGF-β1) is a potent fibrogenic cytokine well known to regulate a wide range of effects including activation of stellate cells, induction of synthesis of extracellular matrix proteins, and inhibition of production of matrix metalloproteinases (15–19). With regard to advanced liver fibrosis in BA, the present results are consistent with those of a previous study by Farrington et al (20) in which TGF-β1 levels were found to be significantly higher in the livers of patients with advanced stages of BA than healthy controls. Furthermore, it was demonstrated that the expression of TGF-β1 was significantly higher in centrolobular compared with portal regions. In contrast, α-SMA levels were found to be significantly elevated in portal regions in the liver parenchyma. Because TGF-β1 activates HSCs in which α-SMA is a marker of differentiation to myofibroblasts (21,22), it is not surprising that elevated α-SMA levels correlated with elevated TGF-β1 levels; however, in the present study, the observation of particular importance was the finding that α-SMA was present at elevated levels in epithelial cells and in periductular collagen fibers of the fibrous cone of the porta hepatis. Furthermore, the levels of α-SMA in BA were positively correlated with liver fibrosis scores, but not with CD-68 expression, suggesting that macrophage activation did not appear to be involved in myofibroblast activation in this anatomical location that is critically involved in BA.
Prevention and treatment of liver fibrosis in BA has been a challenge. The Kasai procedure makes it possible to decrease cholestasis and remove the remnant of the porta hepatis; however, the Kasai procedure is not always carried out in time or is successful. In addition, in most cases, the fibrogenesis continues even if serum bilirubin is normal (4). A potential advantage in the use of α-SMA levels over conventional histological analysis is that because α-SMA levels appear to be associated with early fibrosis, elevated levels may reflect progression of fibrosis before it is detectable histologically.
In summary, we have shown that α-SMA expression may be an early marker for hepatic fibrosis, and may, therefore, be important in determining the prognosis of the native liver after the Kasai procedure in children with BA.
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