transforming-growth-factor-beta and Biliary-Atresia

Nephromegaly, assessed by calculating kidney volume using renal ultrasound, was studied in infants with biliary atresia, neonatal hepatitis, or fulminant hepatitis. We evaluated kidney volume in 29 patients with biliary atresia, 17 patients with neonatal hepatitis, and 10 patients with fulminant hepatitis, as well as 32 healthy infants. Levels of plasma hepatocyte growth factor (HGF) were measured in all infants. Levels of plasma transforming growth factor-beta1 (TGF-beta1) were also measured in diseased infants and 20 healthy infants. Significant nephromegaly was found in infants with biliary atresia compared with healthy infants (P < 0.001 by analysis of covariance). Marked nephromegaly was also noted in all infants with fulminant hepatitis and 35% of infants with neonatal hepatitis. No nephromegaly was found in infants at 2 months of age with biliary atresia or neonatal hepatitis despite mildly elevated plasma HGF levels. Regardless of the duration of HGF exposure and healthy renal growth by a certain age, a positive correlation existed between plasma HGF level and kidney volume (r = 0.529; P < 0.001), but an inverse correlation was found between plasma TGF-beta1 level and nephromegaly (r = -0.505; P < 0.001) in all diseased infants. There was a stronger positive correlation between plasma HGF-TGF-beta1 ratio and kidney volume (r = 0.666; P < 0.001) and degree of nephromegaly (r = 0.717; P < 0.001). These results confirm the presence of large kidneys not only in patients with biliary atresia but also in patients with fulminant hepatitis, which suggests the possible pathogenic role of HGF and manifests as elevated HGF-TGF-beta1 ratios in patients with such conditions. Nephromegaly in patients with severe or chronic liver dysfunction may provide a new in vivo model to study the mechanisms of renal growth.

Topics: Biliary Atresia; Hepatitis; Hepatitis B; Hepatocyte Growth Factor; Humans; Infant; Infant, Newborn; Kidney; Kidney Diseases; Transforming Growth Factor beta; Transforming Growth Factor beta1; Ultrasonography

Biliary atresia (BA) is a childhood liver disease characterized by fibrous obstruction and obstruction of the extrahepatic biliary system and is one of the most common and serious biliary disorders in infants. Significant inflammation and fibrosis of the liver and biliary tract are the most prominent features, regardless of the initial damage to the BA. Abnormalities in innate or adaptive immunity have been found in human patients and mouse models of BA. We previously reported that children with BA had abnormal lipid metabolism, including free serum carnitine.. To study gene and protein expression levels of the hepatic peroxisome proliferator-activated receptor-α (PPARα) signaling pathway and farnesoid X receptor (FXR) in BA and BA fibrosis, and assess their clinical values.. Low expression of PPARα and NR1H4 (FXR) in BA were validated in the Gene Expression Omnibus database. Functional differences were determined by gene set enrichment analysis based on of PPARα and NR1H4 expression. BA patients from GSE46960 were divided into two clusters by using consensus clustering according to PPARα, NR1H4, and SMAD3 expression levels, and immunoinfiltration analysis was performed. Finally, 58 cases treated in our hospital were used for experimental verification. (IHC: 10 Biliary atresia, 10 choledochal cysts; PCR: 10 Biliary atresia, 14 choledochal cysts; WB: 10 Biliary atresia, 4 choledochal cysts).. Bioinformatics analysis showed that the expression of PPARα, CYP7A1 and NR1H4 (FXR) in the biliary atresia group was significantly lower than in the control group. More BA-specific pathways, including TGFβ signaling pathway, P53 signaling pathway, PI3K-AKT-mTOR signaling pathway, etc., are enriched in BA patients with low PPARα and NR1H4 expression. In addition, low NR1H4 expression is abundant in inflammatory responses, IL6/STAT3 signaling pathways, early estrogen responses, IL2 STAT5 signaling pathways, and TGFβ signaling pathways. The TGFβ signaling pathway was significant in both groups. According to the expression of PPARα, NR1H4 and SMAD3, a key node in TGFβ pathway, BA patients were divided into two clusters using consensus clustering. In cluster 2, SMAD3 expression was high, and PPARα and NR1H4 expression were low. In contrast to cluster 1, immune cell infiltration was higher in cluster 2, which was confirmed by immunohistochemistry. The mRNA and protein levels of PPARα and NR1H4 in BA patients were lower than in the control group by immunohistochemistry, Western blot analysis and real-time PCR.. The downregulation of PPARα and NR1H4 (FXR) signaling pathway may be closely related to biliary atresia.

Topics: Animals; Bile Acids and Salts; Biliary Atresia; Child; Choledochal Cyst; Fibrosis; Humans; Infant; Liver; Mice; Phosphatidylinositol 3-Kinases; PPAR alpha; Receptors, Cytoplasmic and Nuclear; Transforming Growth Factor beta

In biliary atresia (BA), epithelial-mesenchymal hepatic progenitor cells (HPC) expressing the stem/progenitor cell marker PROMININ-1 (PROM1) undergo expansion and subsequent transdifferentiation into collagen-producing myofibroblasts within regions of evolving biliary fibrosis under the regulation of Transforming Growth Factor-β (TGFβ) signaling. We hypothesized that pro-inflammatory Toll-like Receptor-3 (TLR3) signal activation promotes the differentiation of PROM1+ HPC via TGFβ pathway activation in vitro.. PROM1+ Mat1a(-/-) HPC were treated with a double-stranded RNA analog, polyionosinic-polycytidylic acid (Poly I:C), ± small molecule inhibitors nafamostat, or SB431542.. Poly I:C induced myofibroblastic-like morphologic changes, degradation of IκB-α consistent with TLR3-NFκB activation, a 15-fold increase in the expression of Vimentin, a 9-fold increase in Collagen-1a, a 4.6-fold increase in Snail at 24h (p<0.05), and an 8.2-fold increase in Prom1 at 72h (p<0.0001) by qPCR. Immunofluorescence demonstrated nuclear phosphorylated SMAD3, TLR3, and COLLAGEN-1α staining following Poly I:C treatment. Degradation of IκBα was inhibited by nafamostat. Co-treatment with either nafamostat or SB431542 blocked the morphologic change and abrogated the increased expression of Cd133, Collagen, Vimentin, and Snail1.. TLR3 activation induces myofibroblastic differentiation of PROM1+ HPC in part via TGFβ pathway activation to promote BA-associated biliary fibrosis.

Topics: AC133 Antigen; Biliary Atresia; Biomarkers; Cell Proliferation; Cell Transdifferentiation; Cells, Cultured; Fibrosis; Hepatocytes; Humans; Mesenchymal Stem Cells; Myofibroblasts; Signal Transduction; Toll-Like Receptor 3; Transforming Growth Factor beta

Topics: Animals; Biliary Atresia; Down-Regulation; Humans; Liver; Liver Cirrhosis; Transforming Growth Factor beta; Up-Regulation

Biliary atresia (BA), the most common cause of end-stage liver disease and the leading indication for pediatric liver transplantation, is associated with intrahepatic ductular reactions within regions of rapidly expanding periportal biliary fibrosis. Whereas the extent of such biliary fibrosis is a negative predictor of long-term transplant-free survival, the cellular phenotypes involved in the fibrosis are not well established. Using a rhesus rotavirus-induced mouse model of BA, we demonstrate significant expansion of a cell population expressing the putative stem/progenitor cell marker, PROMININ-1 (PROM1), adjacent to ductular reactions within regions of periportal fibrosis. PROM1positive (pos) cells express Collagen-1α1. Subsets of PROM1pos cells coexpress progenitor cell marker CD49f, epithelial marker E-CADHERIN, biliary marker CYTOKERATIN-19, and mesenchymal markers VIMENTIN and alpha-SMOOTH MUSCLE ACTIN (αSMA). Expansion of the PROM1pos cell population is associated with activation of Fibroblast Growth Factor (FGF) and Transforming Growth Factor-beta (TGFβ) signaling. In vitro cotreatment of PROM1-expressing Mat1a-/- hepatic progenitor cells with recombinant human FGF10 and TGFβ1 promotes morphologic transformation toward a myofibroblastic cell phenotype with increased expression of myofibroblastic genes Collagen-1α1, Fibronectin, and α-Sma. Infants with BA demonstrate similar expansion of periportal PROM1pos cells with activated Mothers Against Decapentaplegic Homolog 3 (SMAD3) signaling in association with increased hepatic expression of FGF10, FGFR1, and FGFR2 as well as mesenchymal genes SLUG and SNAIL. Infants with perinatal subtype of BA have higher tissue levels of PROM1 expression than those with embryonic subtype.. Expansion of collagen-producing PROM1pos cells within regions of periportal fibrosis is associated with activated FGF and TGFβ pathways in both experimental and human BA. PROM1pos cells may therefore play an important role in the biliary fibrosis of BA.

Topics: AC133 Antigen; Animals; Antigens, CD; beta Catenin; Biliary Atresia; Disease Models, Animal; Female; Fibroblast Growth Factors; Glycoproteins; Humans; Liver Cirrhosis; Male; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Peptides; Proto-Oncogene Proteins c-akt; Rotavirus Infections; Transforming Growth Factor beta

Our previous work demonstrated altered messenger RNA expression of integrin β-5 and -8, using an in silico analysis of publically available data from patients with biliary atresia (BA); however, we were unable to demonstrate statistically significant differences in protein expression because of sample size. In the present study, we repeated the analysis of liver fibrosis and protein expression of the integrins in a larger cohort of patients with BA and compared them with patients undergoing liver biopsy for other diagnoses, with the hypothesis that ≥ 1 of the integrins would be differentially expressed.. Liver specimens were obtained at 2 collaborating institutions. Samples from infants with BA (n = 23) were compared with samples from those who underwent liver biopsy for neonatal hepatitis (n = 9). All of the specimens were analyzed by 2 pathologists (C.R. and R.A.), who were blinded to the diagnoses. Standard Ishak scoring was performed to evaluate fibrosis and inflammation, and immunohistochemical (IHC) positivity was graded from 0 to 4. Comparisons between the IHC positivity and Ishak scoring for the BA and control groups were performed using the Student t test with P < 0.01 considered significant because of the multiple comparisons. Interobserver variability was assessed by intraclass correlation (ICC).. Pooled analysis from specimens from patients with BA showed significantly more fibrosis than controls based on Ishak scores (3.21 ± 1.82 vs 1.17 ± 1.00, P < 0.005). IHC evaluation showed increased integrin ανβ8 protein expression when compared with controls (2.67 ± 0.81 vs 1.72 ± 0.62, P < 0.005); however, there were no significant differences in integrin ανβ5 (1.93 ± 0.84 vs 1.50 ± 0.90, P = 0.23) or integrin ανβ6 (0.85 ± 1.20 vs 0.94 ± 0.85, P = 0.82) expression. These data were confirmed on individual analysis. Interobserver agreement was fair for integrin ανβ5 (ICC 0.52), good for integrin ανβ6 (ICC 0.72), and excellent for integrin ανβ8 (ICC 0.79) and fibrosis (ICC 0.89).. Our data show that integrin ανβ8, but not integrin ανβ5 or integrin ανβ6, protein expression is increased in liver specimens of patients with BA. These data support the mounting evidence that transforming growth factor-β (TGF-β) activation is responsible for the fibrosis found in BA. Anti-integrin ανβ8 or more global integrin blocking strategies may be therapeutic options in BA, but further work is clearly needed.

Topics: Biliary Atresia; Biopsy; Gene Expression; Humans; Immunohistochemistry; Infant, Newborn; Integrin beta Chains; Liver; Liver Cirrhosis; RNA, Messenger; Transforming Growth Factor beta

Our previous work demonstrated that the transforming-growth factor (TGF) β pathway plays a central role in the liver fibrosis associated with experimental biliary atresia (BA). To confirm these findings in humans, we performed an in silico analysis of publicly available microarray data from liver specimens from children with BA, with the hypothesis that the TGF-β pathway would be dysregulated.. We analyzed publicly available liver gene expression microarray data from 47 infants with BA. We re-analyzed the microarray image files and clinical data to compare gene expression differences between the fibrogenic and inflammatory cohorts identified in the initial study. Targets from the microarray analysis were confirmed using the animal model of BA.. Analysis of variance (ANOVA) detected 6903 transcripts (2822 distinct genes) differentially regulated between groups (p < 0.01; fold change >1.2). We used a targeted approach to identified a subgroup of 24 TGF-β-related transcripts. Expressions for procollagen transcripts were increased in the fibrogenic group (1.2-fold to 1.4-fold); expression of matrix metalloproteinase (MMP)-7 was similarly increased 2-fold, while MMP-9 and plasminogen activator inhibitor-1 were decreased 2-fold and 3-fold respectively. Integrins β5 (1.18-fold) and β8 (1.84-fold) also demonstrated increased expression in the fibrogenic group. Increased expression of β5 (3-fold) and β8 (5-fold) as well as Smad-3 (4-fold) and Smad interacting protein (SIP)-1 (3.5-fold) mRNA was confirmed in experimental BA. Phosphorylated Smad-3 protein in the experimental group was also nearly twice that of the control group, further implicating the TGF-β pathway.. Gene transcripts for known upstream and downstream TGF-β mediators are differentially expressed in liver specimens from children with BA and a fibrogenic gene signature. The same integrins that were dysregulated in the human specimens were also found to be up-regulated in our animal BA model, as were other intermediaries in the TGF-β pathway. Further investigation into whether these mediators may be attractive targets for future therapy in children with BA is warranted.

Topics: Animals; Biliary Atresia; Biomarkers; Down-Regulation; Enzyme-Linked Immunosorbent Assay; Humans; Immunohistochemistry; Infant; Liver; Liver Cirrhosis; Mice, Inbred BALB C; Oligonucleotide Array Sequence Analysis; Reverse Transcriptase Polymerase Chain Reaction; Transforming Growth Factor beta; Up-Regulation

Topics: Biliary Atresia; Biomarkers; Cytokines; Humans; Inflammation; Postoperative Complications; Transforming Growth Factor beta

Biliary atresia (BA) may be characterized as an occlusive cholangiopathy affecting both intra- and extra-hepatic parts of the biliary tree, together with a pronounced inflammatory response consisting of hepatic infiltration of (predominantly) CD4+ lymphocytes and macrophages. Soluble cellular adhesion molecules are also known to be raised at the time of portoenterostomy, presumably reflecting intrahepatic disease. We investigated this measurable inflammatory component longitudinally by studying a panel of cellular adhesion molecules (soluble intercellular adhesion molecule-1 [sICAM-1], soluble vascular cell adhesion molecule-1 [sVCAM-1]) and soluble proinflammatory mediators (T helper 1 [interleukin [IL]-2 and interferongamma] and T helper 2 [IL-4 and IL-10]) cytokines and macrophage markers (tumor necrosis factor [TNF] alpha and IL-18) in 21 consecutive infants with BA post-Kasai portoenterostomy (KP). The levels of all adhesion molecules and cytokines (except IL-10) increased progressively by 6 months post-portoenterostomy. The response was non-polarized but with 100-fold increases in IL-2, TNFalpha and IL-18 particularly but only modest elevations in IL-10. When proinflammatory profiles were related to outcome, we found poor discrimination if assessed as clearance of jaundice but markedly higher values for IL-2, interferongamma, IL-4, IL-10, TNFalpha and sICAM-1 for those who would be transplanted by 1 year. Using ROC curve analysis for sICAM-1 levels at 1 month post-KP, a cutoff level of 1,779 ng/ml was determined to predict the need for transplantation at 1 year with 92% specificity and 87% sensitivity.. The early circulating inflammatory process in BA is persistent, progressive and involves a non-polarized T cell, macrophage and cell adhesion molecule response only partially ameliorated by KP.

Topics: Biliary Atresia; Biomarkers; Cholestasis; Cytokines; Female; Humans; Infant; Inflammation; Postoperative Complications; Reference Values; Spleen; Syndrome; Transforming Growth Factor beta

Connective tissue growth factor (CTGF) is a newly described protein that stimulates transforming growth factor-beta1 (TGF-beta1). We evaluated the expression of CTGF mRNA in operative biopsy specimens from biliary atresia (BA) patients and normal controls to assess the role of CTGF in BA. Liver biopsy specimens were taken from BA patients at the time of portoenterostomy (n=22) and compared with specimens taken from normal controls (n=6). In situ hybridization was used to stain CTGF mRNA in all specimens. The distribution of collagen type IV (C-IV) was also assessed in the same specimens as an indicator of the severity of fibrosis present at the time of biopsy. Results were analyzed to determine whether there was any correlation between CTGF and C-IV and outcome. Of the 22 postoperative BA patients, eventual outcome was good in 17 (group I), and five (group II) subsequently required liver transplantation. Control specimens (n=6) had no apparent CTGF mRNA expression, and median C-IV positive immunoreactivity was 1.23+/-0.25%. CTGF mRNA was weakly expressed in hepatic stellate cells (HSC) and hepatocytes in specimens from group I, and median C-IV-positive immunoreactivity was 3.18+/-0.86%. However, in specimens from group II, there was increased CTGF mRNA in HSC and hepatocytes. Median C-IV-positive immunoreactivity was 6.31+/-0.96%. There was a significant correlation between CTGF mRNA intensity and the amount of C-IV, which implies that CTGF expression reflects prognosis. This study provides the first evidence that CTGF is strongly expressed in BA, in particular in HSC and hepatocytes, suggesting that they may be a source of CTGF. The strong correlation with C-IV indicates that CTGF plays a major role in the pathogenesis of progressive fibrosis in BA.

Topics: Bile Ducts, Intrahepatic; Biliary Atresia; Biomarkers; Biopsy; Child; Collagen Type IV; Connective Tissue Growth Factor; Disease Progression; Female; Follow-Up Studies; Gene Expression; Humans; Immediate-Early Proteins; Immunohistochemistry; In Situ Hybridization; Intercellular Signaling Peptides and Proteins; Liver Cirrhosis; Male; Prognosis; RNA, Messenger; Transforming Growth Factor beta; Transforming Growth Factor beta1

We previously showed a positive correlation between nephromegaly and plasma hepatocyte growth factor (HGF)/transforming growth factor beta1 (TGF-beta1) ratio in children with biliary atresia. The purpose of this study is to examine the possible reversibility of nephromegaly in patients with biliary atresia.. We evaluated kidney volume in 13 patients with biliary atresia before and after liver transplantation, 6 patients with hepatoblastoma, and 26 healthy children. Plasma HGF and TGF-beta1 levels were determined for all children.. We noted significant nephromegaly in children with biliary atresia before liver transplantation compared with healthy children and children after liver transplantation (P < 0.001 and P = 0.006 for intercepts, P = 0.064 and P = 0.753 for slopes by analysis of covariance, respectively). The highest plasma HGF levels and HGF/TGF-beta1 ratios and the lowest TGF-beta1 concentrations were found in children with biliary atresia before liver transplantation (P < 0.001). No statistically significant nephromegaly was observed in children with biliary atresia after liver transplantation or those with hepatoblastoma despite the presence of a mildly increased plasma HGF level and HGF/TGF-beta1 ratio. Plasma HGF/TGF-beta1 ratio correlated positively with degree of nephromegaly in all patients (r = 0.717; P < 0.001).. Our data suggest that liver transplantation reverses the nephromegaly present in children with biliary atresia and that plasma HGF/TGF-beta1 ratio may be associated with the development of nephromegaly in patients with biliary atresia.

Topics: Biliary Atresia; Biomarkers; Child; Child, Preschool; Female; Hepatoblastoma; Hepatocyte Growth Factor; Humans; Infant; Kidney; Liver Neoplasms; Liver Transplantation; Male; Organ Size; Postoperative Period; Transforming Growth Factor beta; Transforming Growth Factor beta1; Ultrasonography

Transforming growth factor-beta (TGF-beta) 1 and 2 and their receptors TbetaR-I, TbetaR-II, and TbetaR-III are powerful profibrogenic mediators in the body. Their expression has not been completely elucidated in the progress of liver fibrosis associated with biliary atresia (BA).. The authors compared the cytokine expression in the liver of 3 patients with BA at Kasai's procedure (KP) and in 3 patients at liver transplantation (LT). Two liver samples from children with no liver disorders served as normal controls (CO). Real-time quantitative reverse transcriptase polymerase chain reaction (QRT-PCR) was used to confirm the findings of relative mRNA expression of TGF-beta1 and 2 and their receptors. An immunohistochemistry and an enzyme-linked immunoassay (ELISA) were used to localize the liver cells that express TGF-beta2 and to quantitate the protein expression among groups.. Compared with controls, both TGF-beta1 and TGF-beta2 mRNA expression increased in the liver during the progress of liver fibrosis in patients with KP and LT on the array. Only TGF-beta2 showed a significant increase in expression in LT compared with KP and CO (P =.001 for TGF-beta2 and P = 0.054 for TGF-beta1). Both TbetaR-I and TbetaR-II showed no significant change among groups; TbetaR-III decreased significantly in LT compared with CO (P =.011). TGF-beta2 immunostaining was mainly localized in the bile duct epithelium and was remarkably higher in LT in which the proliferating bile ductules and the hepatocytes contributed to the increase in immunostaining and possibly to significantly higher plasma TGF-beta2 protein levels in LT than in KP.. This study identified TGF-beta2 as the most actively transcribed TGF-beta gene during the progress of liver fibrosis in BA and found a reciprocal relationship of upregulation of TGF-beta2 with downregulation of TbetaR-III in LT.

Topics: Adolescent; Biliary Atresia; Child, Preschool; Female; Humans; Infant; Liver; Liver Cirrhosis; Liver Transplantation; Male; Oligonucleotide Array Sequence Analysis; Receptors, Transforming Growth Factor beta; Reference Values; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Transcription, Genetic; Transforming Growth Factor beta; Transforming Growth Factor beta1; Transforming Growth Factor beta2

Topics: Biliary Atresia; Biomarkers; Disease Progression; Humans; Liver Cirrhosis; Postoperative Complications; Transforming Growth Factor beta; Transforming Growth Factor beta1

To demonstrate nephromegaly in children with biliary atresia and children with compensatory renal hypertrophy and to examine their plasma hepatocyte growth factor (HGF), transforming growth factor beta1 (TGF-beta1), and the difference of total kidney volume, 11 children with biliary atresia (age range 5 months to 10 years), 11 with compensatory renal hypertrophy, and 11 age-matched healthy controls were investigated. Kidney volume was measured by renal ultrasonography and plasma HGF and TGF-beta1 levels were studied. To clarify the significance of nephromegaly in biliary atresia, creatinine clearance was also measured in 9 children with biliary atresia and 9 healthy children. The unilateral kidney in biliary atresia and the solitary kidney in compensatory renal hypertrophy had significantly higher kidney volumes compared with those of healthy children (P<0.001 by analysis of covariance). However, a significant increase in total kidney volume was noted only in children with biliary atresia (P<0.001 by analysis of covariance). Although this was actually associated with increased creatinine clearance (117.3+/-22.0 ml/min per 1.73 m(2) vs. 98.3+/-13.6 ml/min per 1.73 m(2) in controls, P<0.05), corrected creatinine clearance was not correlated with total kidney volume (r=0.199, P=0.61) in biliary atresia. Plasma HGF levels and HGF/TGF-beta1 ratios were elevated in children with biliary atresia (2,648+/-1,215 pg/ml and 233.8+/-139.1 pg/ng vs. 493+/-131 pg/ml and 35.9+/-15.7 pg/ng in compensatory renal hypertrophy and 468+/-194 pg/ml and 24.0+/-19.6 pg/ng in controls, P<0.001) and had a positive correlation with total kidney volume by multiple regression analysis (P=0.006 and P=0.002, respectively). These results show that nephromegaly in biliary atresia is associated with increased total kidney volume and a higher glomerular filtration rate, and is positively correlated with plasma HGF and plasma HGF/TGF-beta1 ratio, implying a role of HGF in this situation. However, nephromegaly in compensatory renal hypertrophy may have different mechanisms in terms of normal total kidney volume, transient elevation of plasma HGF followed by normal plasma HGF, and normal plasma HGF/TGF-beta1 ratio. These data also suggest a common mechanism (HGF) for initial renal hypertrophy (as in compensatory renal growth), with dysregulation of control of this process later in the course (as in biliary atresia). The detailed mechanisms for nephromegaly in these two conditions should be fu

Topics: Adaptation, Physiological; Biliary Atresia; Child; Child, Preschool; Creatinine; Hepatocyte Growth Factor; Humans; Hyperplasia; Hypertrophy; Infant; Kidney; Nephrectomy; Transforming Growth Factor beta; Transforming Growth Factor beta1

Transforming growth factor-beta 1 (TGF beta-1) is an important mediator of liver-cell proliferation and replication that is implicated in hepatic fibrosis (HF). Hepatic stellate cells (HSC) are activated by TGF beta-1 and are the main precursor cells involved in fibrogenesis. The correlation between serum TGF beta-1, activated HSC in liver-biopsy specimens, and liver biochemistry was investigated to determine the value of TGF beta-1 as an indicator of clinical status in postoperative biliary atresia (BA) patients. Thirty-two postoperative BA patients (mean age 11.2 +/- 2.8 years) and 13 normal controls (mean age 10.3 +/- 3.7 years) were studied. Based on average liver function test (LFT) results over a 3-month period immediately prior to this study, the BA patients were divided into group I (anicteric, normal LFT; n = 10); group II (anicteric, elevated liver transaminases; n = 12), and group III (jaundiced end-stage liver fibrosis awaiting liver transplantation; n = 10). Serum TGF beta-1 was determined using ELISA. Liver-biopsy specimens were examined with antibody against TGF beta-1 and alpha-smooth muscle actin (SMA) antibody for detection of activated HSC. Serum TGF beta-1 was significantly higher in groups I (11.4 +/- 3.7 ng/ml; P < 0.01) and II (23.3 +/- 11.3 ng/ml; P < 0.001) than in group III (3.0 +/- 1.5 ng/ml) and controls (4.5 +/- 2.5 ng/ml) despite normal LFT in group I. The 3 subjects with the highest serum TGF beta-1 in group II had bile lakes. Biopsies from groups I and II were strongly positive for TGF beta-1 in hepatocytes and Kupffer cells and for activated HSC detected by SMA compared with group III and controls. Because serum TGF beta-1 and activated HSC are only present during active fibrosis, we conclude that there is progressive fibrogenesis even in seemingly normal postoperative BA patients. In particular, bile lakes should be regarded as a key sign of progressive HF, the presence of which should be regarded with extreme caution. We suggest that serum TGF beta-1 could be used as an accurate indicator of progressive fibrogenesis in postoperative BA patients.

Topics: Adolescent; Biliary Atresia; Biopsy; Child; Female; Follow-Up Studies; Humans; Liver; Liver Cirrhosis; Liver Function Tests; Male; Postoperative Complications; Transforming Growth Factor beta

Progressive fibrosis, despite successful surgical treatment, is one of the serious complications of biliary atresia. To understand the mechanism of this fibrosis, the in situ expression of fibrogenic growth factors (TGF-beta and PDGF) and their corresponding receptors was studied by immunohistochemistry using frozen sections. The results were compared between the early (n=12) and late (n=6) stages. The early stage was characterized by abundant expression of all ligands and receptors, together with type I procollagen (PC-I). The major cellular sources were activated fibroblasts/myofibroblasts distributed mostly in the portal tracts. Macrophages also expressed all the ligands and the receptors, but to a lesser degree. Bile duct cells strongly expressed TGF-beta RI and RII and PDGF AA and BB, but focally expressed TGF-beta. All of these decreased in the late stage of biliary atresia. These results suggest that TGF-beta and PDGF play important roles in the fibrogenesis of biliary atresia, especially in its early stage, acting either by autocrine or paracrine mechanisms involving activated fibroblasts/myofibroblasts, bile duct cells, and macrophages.

Topics: Adult; Aged; Biliary Atresia; Disease Progression; Female; Fluorescent Antibody Technique; Humans; Immunoenzyme Techniques; Liver; Male; Middle Aged; Platelet-Derived Growth Factor; Procollagen; Receptors, Platelet-Derived Growth Factor; Receptors, Transforming Growth Factor beta; Transforming Growth Factor beta

Biliary atresia and paucity of intrahepatic bile ducts are the main causes of neonatal cholestasis leading to hepatic fibrosis. Fibrotic evolution is slow in paucity of bile ducts as compared to the rapid progression to biliary cirrhosis in biliary atresia when cholestasis persists despite hepatoportoenterostomy. Our aim was to compare the expression of collagens type I and IV, alpha-smooth muscle actin, osteonectin and transforming growth factor beta1 in biliary atresia and paucity of bile ducts.. Liver biopsies were obtained in 12 children with biliary atresia and in five with paucity of bile ducts. Collagens type I and IV, alpha-smooth muscle actin were detected with immunostaining. Collagens type I and IV, osteonectin and transforming growth factor beta1 mRNAs were detected by in situ hybridization.. Expression of mRNA and proteins was roughly parallel. In ductular proliferation areas of biliary atresia: (1) the expression of collagens type I and IV and osteonectin was increased, and was localized to periductular myofibroblasts; (2) transforming growth factor beta1 was expressed around biliary ductules, probably in inflammatory cells, and also in biliary cells. Osteonectin expression was also increased in the lobules. In paucity of bile ducts, there was no overexpression of collagens type I and IV and transforming growth factor beta1, except in the only child with marked fibrosis. However, osteonectin expression was enhanced at the periphery of the lobules, even when fibrosis was mild or absent.. These findings suggest that in biliary atresia ductular proliferation areas are the site of a marked production of extracellular matrix proteins in periductular myofibroblasts, probably secondary to transforming growth factor beta1 production by inflammatory cells and by biliary cells. The weak expression of transforming growth factor beta1 could explain the slow progression of fibrosis in paucity of bile ducts.

Topics: Actins; Bile Duct Diseases; Bile Ducts, Intrahepatic; Biliary Atresia; Collagen; Female; Humans; Immunohistochemistry; In Situ Hybridization; Infant; Infant, Newborn; Infant, Newborn, Diseases; Male; Muscle, Smooth, Vascular; Osteonectin; RNA, Messenger; Transforming Growth Factor beta

Extrahepatic biliary atresia is a severe neonatal liver disease resulting from a sclerosing cholangiopathy of unknown etiology. Although biliary obstruction may be surgically corrected by a "Kasai" hepatoportoenterostomy, most patients still develop progressive hepatic fibrosis, although the source of increased collagen deposition is unclear. This study examined the role of hepatic stellate cells (HSCs) and assessed the source of transforming growth factor-beta (TGF-beta) production in hepatic fibrogenesis in patients with biliary atresia. Liver biopsies from 18 biliary atresia patients (including 5 pre- and post-Kasai) were subjected to immunohistochemistry for alpha-smooth muscle actin and in situ hybridization for either procollagen alpha1 (I) mRNA or TGF-beta1 mRNA. Sections were also subjected to immunohistochemistry for active TGF-beta1 protein. The role of Kupffer cells in TGF-beta1 production was assessed by immunohistochemistry for CD68. Procollagen alpha1 (I) mRNA was colocalized to alpha-smooth muscle actin-positive HSCs within the region of increased collagen protein deposition in fibrotic septa and surrounding hyperplastic bile ducts. The number of activated HSCs was decreased in only one post-Kasai biopsy. TGF-beta1 mRNA expression was demonstrated in bile duct epithelial cells and activated HSCs and in hepatocytes in close proximity to fibrotic septa. Active TGF-beta1 protein was demonstrated in bile duct epithelial cells and activated HSCs. This study provides evidence that activated HSCs are responsible for increased collagen production in patients with biliary atresia and therefore play a definitive role in the fibrogenic process. We have also shown that bile duct epithelial cells, HSCs, and hepatocytes are all involved in the production of the profibrogenic cytokine, TGF-beta1.

Topics: Actins; Biliary Atresia; Child; Child, Preschool; Collagen; Female; Humans; Immunoenzyme Techniques; In Situ Hybridization; Infant; Kupffer Cells; Liver; Liver Cirrhosis; Male; Procollagen; RNA, Messenger; Transforming Growth Factor beta

Biliary atresia is an important cause of neonatal obstructive jaundice in which there is inflammation, sclerosis and eventual obliteration of the bile duct system. Its onset may be antenatal, affecting the normal development of the biliary system. The intrahepatic biliary system is derived from the ductal plate, a sheath of cuboidal epithelium that appears at the hepatocyte-mesenchymal junction around the portal vein branches at 6 weeks gestation. This epithelial structure is moulded into a network of tubular bile ducts by the proliferating mesenchyme. Certain portions of the ductal plate are selected to become definitive bile ducts, while redundant biliary epithelium is deleted. The molecular dynamics controlling the intra-uterine development of the biliary system in humans are not yet clearly understood. Transforming growth factor-beta 1 is a cytokine that stimulates mesenchymal proliferation and inhibits epithelial growth, and has been shown to be important in organogenesis. In the present study, the pattern of TGF beta 1 peptide immunolocalization was investigated with the aid of computerized image analysis, in normal human bile duct development and in biliary atresia. TGF beta 1 peptide was detected within hepatocytes and ductal plate epithelium from 7 weeks gestation; increased TGF beta 1 immunoreactivity was present within the epithelium of developing bile ducts at 13 weeks gestation, and apical polarization of the cytokine was observed from 16 weeks gestation. In biliary atresia, the TGF beta 1 immunoreactivity pattern within the bile duct structures at the porta hepatis and within intrahepatic portal tracts resembled that of the primitive ductal plate, and there was no significant apical polarization. This may indicate a developmental arrest in the normal ductal plate remodelling process in biliary atresia, and suggests an underlying epithelial-mesenchymal interactive disorder.

Topics: Adolescent; Adult; Bile Ducts, Intrahepatic; Biliary Atresia; Female; Fetus; Humans; Image Processing, Computer-Assisted; Immunohistochemistry; Infant; Infant, Newborn; Transforming Growth Factor beta