Fibrosis In Biliary Atresia Research Articles

The combination of conventional ultrasound and shear-wave elastography in evaluating the segmental heterogeneity of liver fibrosis in biliary atresia patients after Kasai portoenterostomy.

To retrospectively assess the value of the combination of conventional ultrasound and shear-wave elastography (SWE) in evaluating the segmental heterogeneity of liver fibrosis in biliary atresia (BA) patients after Kasai portoenterostomy. A total of 35 BA patients with liver segmental deformation were enrolled. The segmental deformation was assessed by conventional ultrasound followed with SWE examinations for evaluating the liver stiffness. Liver biopsy was performed in 11 patients in the region of SWE measurement and liver fibrosis was assessed using the Metavir classification. Aminotransferase to platelet ratio index (APRI) was calculated for comparison. The correlations between serum biochemical tests with SWE values were evaluated. Spearman's rank coefficient test was performed to evaluate the correlation between variables. The SWE values of the biopsy segments had significant positive correlations with liver fibrosis severity (r = 0.828, p = 0.001), which was better than APRI (r = 0.366, p = 0.242). The levels of bilirubin and transaminase showed significant correlations with the SWE values at hypertrophic segments in all patients (r from 0.336 to 0.576, all p < 0.05). Awareness of the segmental heterogeneity of liver fibrosis evaluated by conventional ultrasound and SWE may assist in selecting an appropriate biopsy location and predicting postoperative surveillance for patients with BA.

SOX9 in biliary atresia: New insight for fibrosis progression

BackgroundLiver fibrosis is a hallmark determinant of morbidity in biliary atresia (BA) even in successfully operated cases. Responsible factors for this rapid progression of fibrosis are not completely defined. Aberrant expression of the transcription factor SOX9 and hepatic progenitor cells (HPCs) proliferation have roles in fibrogenesis in cholestatic disorders. However, they were not investigated sufficiently in BA. We aimed to delineate the relation of SOX9 and HPCs to fibrosis and its progression in BA. MethodsForty-eight patients with BA who underwent an initial diagnostic liver biopsy (LB) and consequent intraoperative LB were recruited and compared to 28 cases with non-BA cholestasis that had an LB in their diagnostic workup. Liver fibrosis, tissue SOX9 and HPC expressions were studied in both BA and non-BA-cholestasis cases. Liver fibrosis, SOX9, and HPCs' dynamic changes in BA cases were assessed. Relation of fibrosis and its progression to SOX9 and HPCs in BA was assessed. ResultsSOX9 and HPCs in ductular reaction (DR) form were expressed in 100% of BA and their grades increased significantly in the second biopsy. The rapidly progressive fibrosis in BA, represented by fibrosis grade of the intraoperative LB, correlated significantly to SOX9-DR and HPC-DR at the diagnostic (r = 0.420, P = 0.003 and r = 0.405, P = 0.004, respectively) and the intraoperative (r = 0.460, P = 0.001 and r = 0.467, P = 0.001, respectively) biopsy. On the other hand, fibrosis, SOX9-DR, and HPC-DR were significantly lower in non-BA cases at a comparable age (P < 0.001, P = 0.006, and P = 0.014, respectively). ConclusionsFibrosis in BA is rapidly progressive within a short time and is significantly correlated to SOX9 and HPCs. Assessment of targeting SOX9 and HPCs on fibrosis progression is warranted.

Hepatic glypican-3 and alpha-smooth muscle actin overexpressions reflect severity of liver fibrosis and predict outcome after successful portoenterostomy in biliary atresia

BackgroundGlypican-3 plays a vital role in regulating embryonic morphogenesis of the liver. This study aimed to investigate associations of hepatic expressions of glypican-3 and alpha-smooth muscle actin with clinical parameters in biliary atresia. MethodsLiver specimens were obtained from 20 biliary atresia infants and 7 non-biliary atresia controls. Relative mRNA expressions of glypican-3, alpha-smooth muscle actin, and signaling molecules of Wnt/β-catenin were measured using real-time polymerase chain reaction. Protein expressions of glypican-3 and alpha-smooth muscle actin were examined using immunohistochemistry. Masson’s trichrome staining was conducted to evaluate the stage of liver fibrosis. ResultsUp-regulation of glypican-3 mRNA expression was observed in biliary atresia livers, and its expression was positively associated with alpha-smooth muscle actin, β-catenin, c-Myc, and cyclin D-1. Immunostaining scores of glypican-3 and alpha-smooth muscle actin were significantly increased in biliary atresia livers. Biliary atresia patients with poor outcomes had significantly greater glypican-3 expression than those with good outcomes, consistent with hepatic alpha-smooth muscle actin expression analysis. Hepatic glypican-3 expression was associated with age, albumin, aspartate transaminase, and alkaline phosphatase in biliary atresia patients, while hepatic alpha-smooth muscle actin expression was correlated with alkaline phosphatase in the patients. Moreover, glypican-3 and alpha-smooth muscle actin expressions were positively associated with fibrosis stage in biliary atresia livers. There was a positive relationship between glypican-3 and alpha-smooth muscle actin expression in biliary atresia livers. Combined high expressions of glypican-3 and alpha-smooth muscle actin were associated with poor survival. ConclusionHepatic overexpressions of glypican-3 and alpha-smooth muscle actin were associated with hepatic dysfunction and the degree of liver fibrosis in biliary atresia.

胆道閉鎖症におけるductal plate malformationと肝線維化の関係

胆道閉鎖症におけるductal plate malformationと肝線維化の関係

Retraction Note: Interactions between Th1 cells and Tregs affect regulation of hepatic fibrosis in biliary atresia through the IFN-\u03b3/STAT1 pathway

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Correlation of Interleukin-33/ST2 Receptor and Liver Fibrosis Progression in Biliary Atresia Patients.

Background/Aims: Biliary Atresia (BA) is a devastating pediatric liver disease and characterized by aggressive liver fibrosis progression. The Interleukin-33 (IL-33)/ST2 receptor signaling axis has been demonstrated to be involved in several autoimmune and liver diseases. Since immune dysregulation is a contributor to BA pathogenesis, we aimed to investigate the role of IL-33/ST2 receptor in the progression of liver fibrosis in BA patients.Materials and Methods: The study included 36 BA patients (18 good- and 18 poor-prognosis BA patients); and 8 cholestasis infants as the control group. Patients' information and clinical data were retrospectively collected and compared. Liver fibrosis stage was determined by Masson's trichrome staining. Gene expression levels of IL-33, ST2 receptor, and TFG-β1 were detected by quantitative real-time PCR. MC count, IL-33, TGF-β1, and Interleukin-13 (IL-13) expressions were evaluated by immunohistochemistry. Serum IL-33 expression level was detected by enzyme-linked immunosorbent assay. Co-expression of MC and ST2 receptor was detected by immunofluorescence. In vitro mast cell was cultured with IL-33 stimulation, and ST2 receptor and TGF-β1 expressions were detected.Results: Compared with cholestasis control, BA patients had significantly higher GGT level and Masson score. Expression levels of IL-33, TGF-β1, and IL-13 were significantly increased in BA patients compared to control group, especially in poor-prognosis BA patients. Co-expression of ST2 receptor and MC was found in BA liver tissues. The MC count was markedly higher in BA patients especially in poor-prognosis subgroup. Serum IL-33 level was significantly elevated in poor-prognosis BA patients and related to a higher Masson score. In vitro mast cell culture exhibited significant upregulation of ST2 receptor and TGF-β1 mRNA expression after IL-33 stimulation.Conclusions: IL-33/ST2 receptor signaling axis is correlated with liver fibrosis progression in BA patients, and mast cells participates in this process. These indicate potential prognostic evaluation factors for BA patients and can help in the postoperative management to achieve better long-term prognosis in BA patients.

Long Noncoding RNA H19 Contributes to Cholangiocyte Proliferation and Cholestatic Liver Fibrosis in Biliary Atresia.

Biliary atresia (BA) is a neonatal liver disease featuring cholestasis and severe liver fibrosis (LF). Despite advances in the development of surgical treatment, lacking an early diagnostic marker and intervention of LF invariably leads to death from end-stage liver disease in the early years of life. We previously reported that knockout of sphingosine 1-phosphate receptor 2 (S1PR2) protected mice from bile duct ligation (BDL)-induced cholangiocyte proliferation and LF. Our recent studies further showed that both hepatic and serum exosomal long noncoding RNA H19 (lncRNAH19) levels are correlated with cholestatic injury in multidrug resistance 2 knockout (Mdr2-/- ) mice. However, the role of lncRNAH19 in BA progression remains unclear. Here, we show that both hepatic and serum exosomal H19 levels are positively correlated with severity of fibrotic liver injuries in BA patients. H19 deficiency protects mice from BDL-induced cholangiocyte proliferation and LF by inhibiting bile-acid-induced expression and activation of S1PR2 and sphingosine kinase 2 (SphK2). Furthermore, H19acts as a molecular sponge for members of the microRNA let-7 family, which results in up-regulation of high-mobility group AT-hook 2 (HMGA2), a known target of let-7 and enhancement of biliary proliferation. Conclusion: These results indicate that H19 plays a critical role in cholangiocyte proliferation and cholestatic liver injury in BA by regulating the S1PR2/SphK2 and let-7/HMGA2 axis. Serum exosomal H19 may represent a noninvasive diagnostic biomarker and potential therapeutic target for BA.

Divergent expression of liver transforming growth factor superfamily cytokines after successful portoenterostomy in biliary atresia

Pathogenesis of progressive liver fibrosis in biliary atresia after successful portoenterostomy remains unclear. We related hepatic expression of transforming growth factor beta (TGF-β) superfamily cytokines to histologic liver injury after successful portoenterostomy. Enrolled in our study were 28 patients with biliary atresia who had liver biopsies obtained during and after successful portoenterostomy, which normalized serum bilirubin (<20 µmol/l). Biopsies were evaluated for cholestasis, inflammation, ductal reaction, and fibrosis and were stained immunohistochemically for transforming growth factor beta 1, transforming growth factor beta 2, connective tissue growth factor, and decorin. Respective gene expression (TGFB1, TGFB2, TGFB3, CTGF, DCN) was analyzed at follow-up using quantitative reverse transcription polymerase chain reaction (qRT-PCR). Results were compared with fibrotic and healthy control livers. After median follow-up of 3.0 years, histologic cholestasis resolved, whereas fibrosis had progressed only in isolated biliary atresia. Liver protein expression of transforming growth factor beta 1 and connective tissue growth factor (P < .001 for both), but not that of transforming growth factor beta 2 or decorin, decreased after successful portoenterostomy, although expression of all four cytokines remained elevated. In accordance with postportoenterostomy changes in protein expression, follow-up ribonucleic acid expression of TGFB2 and DCN, but not that of TGFB1 and CTGF, was upregulated when compared with the controls. Both protein and gene expression of transforming growth factor beta 1 and protein expression of transforming growth factor beta 2, connective tissue growth factor and decorin correlated with METAVIR fibrosis stage. Syndromic patients (n = 12) showed milder fibrosis and lower transforming growth factor beta 1 expression than patients with isolated biliary atresia. These findings support a central role of transforming growth factor beta superfamily in mediating continuing liver fibrogenesis after successful portoenterostomy. Transforming growth factor beta pathway cytokines responded divergently to clearance of jaundice, which was reflected by differential progression of fibrosis between syndromic and isolated patients.

Staging of liver fibrosis in biliary atresia : Comparison of Chevallier and Ishak score as well as automated evaluation

Biliary atresia (BA) is arare disease of the newborn, resulting in liver cirrhosis due to obliterative cholangiopathy. Liver biopsies are commonly performed in order to confirm the diagnosis and in order to stage fibrosis. The present study intended to analyze two established scores for evaluating liver fibrosis focusing on the interobserver variability as well as the prognostic reliability towards the time of liver transplantation. Liver biopsies of BA patients between 2012 and 2015 were evaluated retrospectively by two pathologists at the Hannover Medical School (MHH) and the RWTH Aachen University Hospital. Fibrosis was measured using Ishak and Chevallier scores. Furthermore, acomputerized automatically algorithm-based analyzation (ABAA) was performed. Results were evaluated towards the time point of liver transplantation and hepatoportoenterostomy (HPE). Overall, 34liver biopsies were analyzed. The Ishak score showed aremarkable interobserver variability (ΚW = 0.68) while the Chevallier score was proven to have apoor interobserver variability (Fleiss' Κappa = -0.01). However, both scores were correlated positively, as was the ABAA (p < 0.001). Regarding prognostic reliability, ROC analyses of the Ishak score revealed the best validity towards an early liver transplantation within 12months (AUC 0.813, p = 0.011). In addition, an increased Ishak score ≥4 reduced the survival time with the native liver (hazard ratio6.6 [95% CI1.9-23.3]). The Ishak score was revealed to have the best interobserver variability as well as prognostic validity towards an early liver transplantation in BA patients. Due to its easy applicability, the Ishak score was proven superior in comparison to the Chevallier score and ABAA. Therefore, use of the Ishak score is recommended in daily clinical routine for analyzing liver biopsies in BA patients.

Liver fibrosis in biliary atresia.

Biliary atresia (BA) is the most common cause of obstructive jaundice in infants. Although the Kasai procedure has greatly improved the prognosis, most patients still need liver transplantation (LT) for long-term survival. The pathogenesis of BA has not been fully clarified, and liver fibrosis in BA is far beyond biliary obstructive cirrhosis. Literature reviews were underwent through PubMed. Persistent inflammation, immune response, biliary epithelial-mesenchymal transition, matrix deposition, decompensated angiogenesis, and unique biliary structure development all contribute to the fibrosis process. Observed evidences in such fields have been collected and form the backbone of this review. Interactions of the multiple pathways accelerate this process. Understanding the mechanisms of the liver fibrosis in BA may pave the way to improved survival after the Kasai procedure.

Bile duct ligature in young rats: A revisited animal model for biliary atresia

Biliary atresia leads to cirrhosis in the vast majority of patients and constitutes the first cause of paediatric liver transplantation. Animal models allow us to understand the molecular basis and natural history of diseases. The aim of this study is to describe a surgically created animal model of biliary atresia with emphasis in long-term liver function. Forty-two 3-week-old Sprague- Dawley rats were randomly divided into two groups: bile duct ligature (BDL) and control. The animals were sacrificed on the 2nd, 4th, and 6th postoperative weeks. Blood samples were collected for liver function analysis. The spleen to body weight ratio was determined. Histopathological examination of liver tissue was performed by hematoxylin-eosin and Sirius red staining. Collagen quantification was determined by using colorimetric digital image analysis and was expressed as a percentage of total liver tissue area. Quantitative real-time polymerase chain reaction was performed to analyse gene expression levels of transforming growth factor-β1 (Tgfb1) and apeline (Apln) genes. Statistical analysis was performed where P<0.05 was considered significant. Animals from BDL group developed increasing cholestasis with clinical and laboratory features. Splenomegaly was detected at 4th and 6th week (P<0.05). Histological evaluation of the liver showed ductular reaction, portal fibrosis and bile plugs. Collagen area to total liver tissue area had a median of 2.5% in the control group and 6.5 %, 14.3 % and 37.7 % in BDL rats at 2nd, 4th and 6th weeks, respectively (P<0.001). Tgfb1 mRNA expression level was significantly higher at 6th week (P<0.001) in BDL group when compared to control. Apln mRNA expression level was significantly higher at 4th and 6th week (P<0.001) and showed a positive linear correlation (r = 0.975, P<0.05) in BDL group when compared to control. Bile duct ligature in young rats is an animal model that recreates clinical, laboratory, histological and molecular findings of biliary atresia. Bile duct ligature constitutes a good animal model to investigate therapeutic approaches for modifying the progression of liver fibrosis in biliary atresia.

Downregulation of microRNA-145 may contribute to liver fibrosis in biliary atresia by targeting ADD3.

Background and objectivesBiliary atresia (BA) is a pediatric liver disease characterized by fibro-obliteration and obstruction of the extrahepatic biliary system, that invariably leads to cirrhosis and even death, if left untreated for extended time. However, its pathology and etiology still remained unknown. In this study, we tested the expression of adducin 3 (ADD3), the gene identified as a susceptibility gene in BA by GWAS, and uncovered its upstream regulatory microRNA in the pathogenesis of BA.MethodsIn this study, 14 infants with BA and 14 infants with choledochal cyst (CC) were enrolled as experimental group and control group, respectively. ADD3 and microRNA-145 (miR-145) expression profiles in liver tissues of BA and CC were determined using qPCR. Luciferase reporter assay was performed to verify the direct interaction between miR-145-5p and ADD3 3’ Untranslated Regions (3’UTR). The Lentiviral vectors containing miR-145, miR-145-3p inhibitor, miR-145-5p inhibitor, empty vector were transfected into human hepatic stellate cell line (LX-2) to determine the functional effect of miR-145 on ADD3 expression at both mRNA and protein level.ResultsMiR-145 was shown to be down-regulated in liver tissues of infants with BA compared to CC (p = 0.0267). ADD3, verified as a target of miR-145-5p, was shown to be overexpressed in infants with BA at the mRNA level (p = 0.0118). Transfection of lentiviruses containing miR-145 into LX-2 cells decreased the expression of ADD3 at both mRNA and protein level compared to negative control group, and suppressed the expression of p-Akt at protein level.ConclusionsOur study has shown that overexpressed ADD3 and downregulated miR-145 were detected in BA liver tissues. MiR-145-5p was confirmed to target ADD3 by luciferase reporter assay. The downregulation of miR-145 may contribute to liver fibrosis in BA by upregulating the expression of ADD3.

RETRACTED ARTICLE:Interactions between Th1 cells and Tregsaffect regulation of hepatic fibrosis in biliary atresia through the IFN-\u03b3/STAT1 pathway

Regulatory T cells (Tregs) and CD4+ Thelper (Th) cells have important roles in bile duct injury of biliary atresia (BA).However, their impacts on liver fibrosis are undefined. Between 2013 and 2016, 146patients with various stages of BA were enrolled in this study. Peripheral blood,liver biopsy and lymph node samples were collected. Flow cytometry, magnetic cellsorting and immunostaining were used to characterize lymphocytes from BA patients.Deficiency of Tregs was observed along with increased Th1, Th2 and Th17 frequenciesin the peripheral blood and livers of BA patients. The levels of peripheral andintrahepatic Th1 cells positively correlated with the stage of liver fibrosis.Furthermore, Th1 cells were located in close proximity to activated hepatic stellatecells (HSCs) and areas of fibrosis in BA livers. In culture, Th1 cells acceleratedthe proliferation and secretion of profibrogenic markers of HSCs through theIFN-γ/STAT1 pathway. Of note, Tregs blocked theTh1-stimulated effects on HSCs by inhibiting Th1-induced activation of STAT1.Consistent with the results of in vitro study,intrahepatic IFN-γ/STAT1 levels increased inrelation to the severity of liver fibrosis in BA patients, and the altered balancebetween MMP2 and TIMP1 expressions in livers may contribute to increased depositionof extracellular matrix and fibrosis. Finally, to identify the effects of Th1 cellson Tregs, we demonstrated that Th1 cells upregulated the proportion of aTreg cellsby secreting IFN-γ cytokine. Thus, aberrant Th1immune responses in BA promote the proliferation and secretion of HSCs through theIFN-γ/STAT1 pathway. The regulation of HSCs bythe interactions between Tregs and Th1 cells might be part of the mechanismunderlying progressive liver fibrosis and may be a suitable target fortherapy.

Forkhead box A3 attenuated the progression of fibrosis in a rat model of biliary atresia

Biliary atresia is a rare, devastating disease of infants where a fibroinflammatory process destroys the bile ducts, leading to fibrosis and biliary cirrhosis, and death if untreated. The cause and pathogenesis remain largely unknown. We tried to investigate factors involved in biliary atresia, especially forkhead box A3 (Foxa3), which might exert a role in the treatment of liver disease. We used RNA sequencing to sequence the whole transcriptomes of livers from six biliary atresia and six choledochal cysts patients. Then, we employed a rat disease model by bile duct ligation (BDL) and adenovirus transduction to address the function of Foxa3 in biliary atresia. We found that tight junction, adherence junction, cell cycle, apoptosis, chemokine singling, VEGF and MAPK signaling pathways were enriched in biliary atresia livers. We showed that Foxa3 expression was notably decreased in liver samples from biliary atresia patients. More importantly, we found that its lower expression predicted a poorer overall survival of biliary atresia patients. Rats that received BDL surgery and Foxa3 expression adenovirus resulted in a significant decrease in the deposition of collagen, and expression of profibrotic cytokines (transforming growth factor-β and connective tissue growth factor) and fibrosis markers (α-smooth muscle actin, collagen I and collagen III), as compared with rats that received BDL surgery and control adenovirus. Our data suggested a protection role for Foxa3 during the progression of liver fibrosis in biliary atresia, and thereby supported increasing Foxa3 as a targeted treatment strategy.

Fn14 hepatic progenitor cells are associated with liver fibrosis in biliary atresia.

The liver in biliary atresia (BA) is characterized by progressing fibrosis which is promoted by unclear reasons. We aimed to understand the factors influencing liver fibrosis. This study hypothesized that HPCs (hepatic progenitor cells) are activated and associated with liver fibrosis in biliary atresia. Liver samples from biliary atresia patients are as BA group, and the normal liver derived from hepatoblastoma infants during operation are control group. The extent of fibrosis in liver samples was blindly evaluated by two experienced pathologists depending on Ishak system. The BA liver samples were divided into mild liver fibrosis group (grade I-IV, BAa) and severe liver fibrosis group (grade V-VI, BAb) to detect Fn14 protein expression. In mRNA level, Fn14 expression was 21.23 ± 8.3 vs. 1.00 ± 0.17, p = 0.023 < 0.05 and CD133 expression was 6.02 ± 2.16 vs. 1.14 ± 0.75, p = 0.008 < 0.01 between BA group and control group. Fn14 cells co-expressed the progenitor marker CD133 in liver, and activated in BA. Fn14 andα-SMA were co-location in fibrous area in liver. Compared to the control group, Fn14, CD133, and α-SMA protein expression were 2.10 ± 0.53 vs. 0.97 ± 0.2, p = 0.001, 2.23 ± 0.57 vs. 1.00 ± 0.03, p = 0.000, 4.96 ± 2.4 vs. 1.00 ± 0.22, p = 0.001. The Fn14 protein expression was 2.60 ± 0.35 vs. 1.86 ± 0.42, p = 0.012, between BAb and BAa group. Fn14 cells, which co-express the progenitor marker CD133 in liver, are HPCs and activated in BA. Fn14 + HPCs are associated with liver fibrosis in BA.

Hepatic Prominin-1 expression is associated with biliary fibrosis

Intrahepatic biliary fibrosis, as seen with cholestatic liver injuries such as biliary atresia, is mechanistically distinct from fibrosis caused by hepatocyte toxicity. We previously demonstrated the expansion of cells expressing the stem/progenitor cell marker Prominin-1, within regions of developing fibrosis in biliary atresia. Thus, we hypothesized that Prominin-1 expression is biliary fibrosis-specific. Gene expression of Prominin-1 was analyzed in adult mice undergoing either cholestatic bile duct ligation or hepatotoxic carbon tetrachloride administration by quantitative polymerase chair reaction. Lineage tracing of Prominin-1-expressing cells and Collagen-1α-expressing cells was performed after bile duct ligation in Prominin-1cre-ert2-lacz;Gfplsl and Collagen-1αGfp transgenic mice, respectively. Prominin-1 expression increased significantly after bile duct ligation compared with sham (6.6±0.9-fold change at 2weeks, P<.05) but not with carbon tetrachloride (-0.7±0.5-fold change, not significant). Upregulation of Prominin-1 was observed histologically throughout the liver as early as 5days after bile duct ligation in Prominin-1cre-ert2-lacz mice by LacZ staining in nonhepatocyte cells. Lineage tracing of Prominin-1-expressing cells labeled prior to bile duct ligation in Prominin-1cre-ert2-lacz;Gfplsl mice, demonstrated increasing colocalization of GREEN FLUORESCENT PROTEIN with biliary marker CYTOKERATIN-19 within ductular reactions up to 5weeks after bile duct ligation consistent with biliary transdifferentiation. In contrast, rare colocalization of GREEN FLUORESCENT PROTEIN with mesenchymal marker α-SMOOTH MUSCLE ACTIN in Prominin-1cre-ert2-lacz;Gfplsl mice and some colocalization of GREEN FLUORESCENT PROTEIN with PROMININ-1 in Collagen-1αGfp mice, indicate minimal contribution of Prominin-1 progenitor cells to the pool of collagen-producing myofibroblasts. During biliary fibrosis Prominin-1-expressing progenitor cells transdifferentiate into cells within ductular reactions. This transdifferentiation may promote fibrosis.

Association between Promoter Hypomethylation and Overexpression of Autotaxin with Outcome Parameters in Biliary Atresia.

ObjectiveBiliary atresia (BA) is a progressive fibroinflammatory liver disease. Autotaxin (ATX) has a profibrotic effect resulting from lysophosphatidic acid activity. The purpose of this study was to examine ATX expression and ATX promoter methylation in peripheral blood leukocytes and liver tissues from BA patients and controls and investigate their associations with outcome parameters in BA patients.MethodsA total of 130 subjects (65 BA patients and 65 age-matched controls) were enrolled. DNA was extracted from circulating leukocytes and liver tissues of BA patients and from and age-matched controls. ATX promoter methylation status was determined by bisulfite pyrosequencing. ATX expression was analyzed using quantitative real-time polymerase chain reaction and enzyme-linked immunosorbent assay.ResultsDecreased methylation of specific CpGs were observed at the ATX promoter in BA patients. Subsequent analysis revealed that BA patients with advanced stage had lower methylation levels of ATX promoter than those with early stage. ATX promoter methylation levels were found to be associated with hepatic dysfunction in BA. In addition, ATX expression was significantly elevated and correlated with a decrease in ATX promoter methylation in BA patients compared to the controls. Furthermore, promoter hypomethylation and overexpression of ATX were inversely associated with jaundice status, hepatic dysfunction, and liver stiffness in BA patients.ConclusionAccordingly, it has been hypothesized that ATX promoter methylation and ATX expression in peripheral blood may serve as possible biomarkers reflecting the progression of liver fibrosis in postoperative BA. These findings suggest that the promoter hypomethylation and overexpression of ATX might play a contributory role in the pathogenesis of liver fibrosis in BA.

PROMININ-1 Expression Is Associated with Biliary Fibrosis in Biliary Atresia

PROMININ-1 Expression Is Associated with Biliary Fibrosis in Biliary Atresia

Histamine is correlated with liver fibrosis in biliary atresia

Background and aimsBiliary atresia (BA) is a severe neonatal cholestasis disease that is caused by obstruction of extra bile ducts. Liver fibrosis progresses dramatically in BA, and the underlying molecular mechanism is largely unknown. MethodsAmino acids and biogenic amines were quantified by targeted metabolomic methods in livers of 52 infants with BA and 16 infants with neonatal hepatitis syndrome (NHS). Normal adjacent nontumor liver tissues from 5 hepatoblastoma infants were used as controls. Orthogonal partial least-squares discriminant analysis was used to identify the differences between BA, NHS, and control tissues. Histamine metabolism enzymes and receptors were analyzed by immunohistochemistry and Western blot. ResultsThe orthogonal partial least-squares discriminant analysis clearly separated BA from NHS and the controls using amino acid and biogenic amine profiles. Histamine was significantly increased in the livers of BA infants and was positively correlated with the severity of fibrosis. This finding was supported by the elevated l-histidine decarboxylase and reduced monoamine oxidase type B expressions in the BA infants with severe fibrosis. Furthermore, histamine receptor H1 was observed in the cholangiocytes of BA livers. ConclusionsHistamine was positively correlated with fibrosis and may be a potential target to prevent liver fibrosis in BA.

MiR‐222 Overexpression May Contribute to Liver Fibrosis in Biliary Atresia by Targeting PPP2R2A

Biliary atresia (BA) is a devastating liver disease in infants. Progressive hepatic fibrosis is often observed in postoperative patients with BA even after a successful Kasai portoenterostomy procedure. MicroRNA-222 (miRNA) has been linked to the activation of stellate cells and the progression of liver fibrosis. In this study, the miR-222 expression profile in BA and infants with anicteric choledochal cyst (CC) was determined. The functional effect of miR-222 inhibition on the growth of the human hepatic stellate cell line LX-2 was also evaluated. The downstream signaling pathways and target of miR-222 were determined by coupling gene expression profiling and pathway analysis and by in silico prediction, respectively. In addition, we demonstrated miR-222 overexpression in patients with BA compared with choledochal cyst controls. Inhibition of miR-222 in the LX-2 cell line significantly decreased cell proliferation. We also identified protein phosphatase 2A subunit B as a target of miR-222. The downstream signaling pathway, Akt, was also influenced by miR-222. A consistent reduction of Akt phosphorylation and Ki67 in the LX-2 line was shown following miR-222 suppression. Our results show that miR-222 overexpression is common in BA and contributes to LX-2 cell proliferation by targeting protein phosphatase 2A subunit B and Akt signaling.