Journal: Hepatology (Baltimore, Md.)

Article Title: Forkhead box A2 regulated biliary heterogeneity and senescence during cholestatic liver injury

doi: 10.1002/hep.28831

Figure Lengend Snippet: Panel A: Relative gene expression profile between small cholangiocytes (SMCCs) versus large cholangiocytes (LGCCs) is shown (n=3). The expression of a panel of diverse stemness-associated genes was evaluated by real-time PCR using Mice Stem Cell PCR Array from SABioscience. FoxA2 and Sox17 are the most up-regulated genes among the six stem cell signaling pathways in biliary committed progenitors. Panel B: Total RNA was extracted from SMCC, LGCC, H69-SM and H69-LG cells, FoxA2 and Sox17 expressions were assessed by quantitative real-time PCR. The relative mRNA expression was normalized to expression of LGCC or H69-LG as % of control. *p < 0.05 when compared with mRNA expression of LGCC or H69-LG cells. Panel C: Immunocytochemistry for FoxA2 and Sox17 was performed in TGF-β and Activin A (both at 10 ng/ml) treated mice liver progenitor cells in William's E medium for 7 days. A significant increase in the percentage of FoxA2 and Sox17 positive cells is observed in LPC cell lines after TGF-β + Activin A treatment. *p < 0.05, compared with expression in the TGF-β or Activin -only group. Panel D: TGF-β regulated differentiation process (MET) in human small and large cholangiocytes. Proteins were isolated from human small and large cholangiocytes treated with TGF-β (10 ng/ml) or diluents control for 7 days, Western blot confirmed reduced MET process after TGF-β treatment. Western blots of H69 cell lysates (TGF-β treated groups and controls) were performed and sequentially probed with antibodies against mesenchymal markers S100A4, Vimentin, CDH2; epithelial markers CDH1, and β-actin as a loading control as indicated. Representative immunoblots are shown on the Panel along with quantitative data that show the mean ± S.E. from four separate blots of independent experiments on Suppl Fig. 1A. Panel E: H69 human cholangiocytes were separated by counterflow elutriation into small and large populations. RNA was extracted from these separate populations and Cytokeratin-19 (CK-19) expression was analyzed with qPCR. No significant differences were observed between these two groups.

Article Snippet: Potential Molecular Mechanisms by which Biliary Cell Therapy Improves BDL-induced Biliary Damage and Liver Fibrosis To examine the molecular mechanisms by which biliary cell reversed BDL-induced hepatobiliary damages, we used the Mouse Fibrosis PCR array (PAMM-120ZA, Qiagen) and combined it with PCR of inflammation- and stem cell & development - related genes to evaluate mechanisms related to liver fibrosis, inflammation and stem cell signaling pathways. α-SMA, MMP-8 and MMP-9 were the most down-regulated genes among the fibrotic signaling pathways in cholestatic liver (BDL) coupled with SMCC therapy compared to those treated with LGCC therapy ( ).

Techniques: Gene Expression, Expressing, Real-time Polymerase Chain Reaction, Protein-Protein interactions, Control, Immunocytochemistry, Isolation, Western Blot

Journal: Hepatology (Baltimore, Md.)

Article Title: Forkhead box A2 regulated biliary heterogeneity and senescence during cholestatic liver injury

doi: 10.1002/hep.28831

Figure Lengend Snippet: Panel A: The expression of DNMT3B and DNMT3A is down-regulated in small cholangiocytes. Relative gene expression profile between SMCCs vs. LGCCs is shown (n=3). The expression of a panel of diverse epigenetic-associated genes was evaluated by real-time PCR using Mouse Epigenetic Chromatin Modification Enzymes PCR Array from SABioscience. Gene expression relative to GAPDH was plotted as the Volcano Plots, depicting the relative expression levels (Log 2) for selected genes in SMCCs versus control LGCC panels (Left). The relative expression levels and p values for each gene in the related samples were also plotted against each other in the scatter plot (Right). DNMT3B and DNMT3A are the most down-regulated genes among the five epigenetic signaling pathways in cholangiocytes. Panel B: Real-Time PCR confirmed the reduced mRNA expression of DNMT3B and DNMT3A in cultured SMCCs, isolated SMCCs from BDL mice liver and small human cholangiocytes (H69-SM) compare to respective controls. The percentages shown represent the mean value (relative to control) normalized with GAPDH from four independent experiments. Panel C: Analysis of the promoter region using MethPrimer software revealed the presence of several CpG islands ~2000 basepairs upstream of the 5’-region of the FoxA2 and Sox17 sequence. Panel D: Small and large cholangiocytes were treated with 10 μM 5-aza-2'deoxy-cytidine (5-Aza-CdR) or diluent control for 72 hr. The expressions of FoxA2, Sox17 and Notch1 were assessed by Western blot analysis. 5-Aza-CdR increased FoxA2 and Sox17 expression in LGCCs but not SMCCs. Representative immunoblots are shown on the top right along with quantitative data that show the mean ± S.E. from four separate blots of independent experiments on bottom panel. * p < 0.05 relative to controls, # p < 0.05 relative to SMCCs. Panel E: DNMT3A was overexpressed in SMCCs. Top Panel, cells were harvested with trypsin, stained with trypan blue and cell size was measured compared to control as a percentage with the control set at 0. *p < 0.05 compared to control. Bottom Panel, RNA was extracted and markers of small and large cholangiocytes were measured with qPCR. *p < 0.05 compared to control. Panel F: DNMT3A was overexpressed in SMCCs, RNA was extracted and DE markers were measured with qPCR. *p < 0.05 compared to control.

Article Snippet: Potential Molecular Mechanisms by which Biliary Cell Therapy Improves BDL-induced Biliary Damage and Liver Fibrosis To examine the molecular mechanisms by which biliary cell reversed BDL-induced hepatobiliary damages, we used the Mouse Fibrosis PCR array (PAMM-120ZA, Qiagen) and combined it with PCR of inflammation- and stem cell & development - related genes to evaluate mechanisms related to liver fibrosis, inflammation and stem cell signaling pathways. α-SMA, MMP-8 and MMP-9 were the most down-regulated genes among the fibrotic signaling pathways in cholestatic liver (BDL) coupled with SMCC therapy compared to those treated with LGCC therapy ( ).

Techniques: Expressing, Gene Expression, Real-time Polymerase Chain Reaction, Modification, Control, Protein-Protein interactions, Cell Culture, Isolation, Software, Sequencing, Western Blot, Staining

Journal: Hepatology (Baltimore, Md.)

Article Title: Forkhead box A2 regulated biliary heterogeneity and senescence during cholestatic liver injury

doi: 10.1002/hep.28831

Figure Lengend Snippet: Panel A: Supernatants from liver tissue homogenates obtained from a surgical (Bile Duct Ligation) or transgenic (MDR2−/−) mouse model of cholestatic liver injury were harvested with respective controls (normal or wild type), and soluble TGF-β levels were measured by ELISA (n=4). There were significant increases in soluble TGF-β levels in BDL or MDR2−/− mice liver. Panel B&C: Verification of specific protein and mRNA expressions in BDL and MDR2−/− mice liver (n=4). Proteins and total RNAs were isolated from BDL/MDR2−/− mice liver tissue homogenates, the expressions of specific proteins and mRNAs were verified by Western blot (Panel B) and Taqman real-time PCR analysis (Panel C). In Panel B representative immunoblots are shown on top panel along with quantitative data that show the mean ± S.E. from four separate blots of independent experiments on bottom panel. Reduced expressions of FoxA2, BMP1, Sox17 and Notch1 after cholestatic liver injury were observed in both models. Panel D: TGF-β was overexpressed in SMCCs, RNA was extracted and levels of DE markers were measured with qPCR. *p < 0.05 compared to control. Panel E–F: Percentages of positive cells stained with Sirius red were counted in BDL and MDR2−/− mice liver with the respective controls (Panel E&F). Significantly increased intensity of liver fibrosis was confirmed in both models by enhanced Sirius red expression (n = 5). *p < 0.05 versus normal or WT controls.

Article Snippet: Potential Molecular Mechanisms by which Biliary Cell Therapy Improves BDL-induced Biliary Damage and Liver Fibrosis To examine the molecular mechanisms by which biliary cell reversed BDL-induced hepatobiliary damages, we used the Mouse Fibrosis PCR array (PAMM-120ZA, Qiagen) and combined it with PCR of inflammation- and stem cell & development - related genes to evaluate mechanisms related to liver fibrosis, inflammation and stem cell signaling pathways. α-SMA, MMP-8 and MMP-9 were the most down-regulated genes among the fibrotic signaling pathways in cholestatic liver (BDL) coupled with SMCC therapy compared to those treated with LGCC therapy ( ).

Techniques: Ligation, Transgenic Assay, Enzyme-linked Immunosorbent Assay, Isolation, Western Blot, Real-time Polymerase Chain Reaction, Control, Staining, Expressing

Journal: Hepatology (Baltimore, Md.)

Article Title: Forkhead box A2 regulated biliary heterogeneity and senescence during cholestatic liver injury

doi: 10.1002/hep.28831

Figure Lengend Snippet: Panel A–E: Altered FoxA2, Sox17, Notch1 and BMP-1 expression along with enhanced DNA methyltransferases in human PSC and PBC liver tissues. Total RNA was isolated from human liver from normal controls or patients with PSC/PBC. Real-time PCR analysis was performed, and the ratio of specific mRNAs to GAPDH mRNA expression in human liver samples was determined. The PCR products were also verified by 1.8% agarose gel electrophoresis. FoxA2 was significantly reduced in 5 among 6 PSC/PBC livers relative to normal controls (Panel A). Meanwhile, the expression of Sox17 was only silenced in PBC liver tissues but increased in PSCs (Panel B&C). DNA methylation enzymes DNMT1 and DNMT3B were also increased in both PSC and PBC livers, suggested the potential hypermethylation mechanisms of FoxA2 gene during PSC/PBC development. Additionally, both Notch-1 and BMP-1 were significantly reduced in PBC and PSC livers compare to controls (Panel D&E). Data represent mean ± SE from liver samples from three PSC or three PBC patients relative to three normal controls with three separate experiments. *p < 0.05 relative to normal controls.

Article Snippet: Potential Molecular Mechanisms by which Biliary Cell Therapy Improves BDL-induced Biliary Damage and Liver Fibrosis To examine the molecular mechanisms by which biliary cell reversed BDL-induced hepatobiliary damages, we used the Mouse Fibrosis PCR array (PAMM-120ZA, Qiagen) and combined it with PCR of inflammation- and stem cell & development - related genes to evaluate mechanisms related to liver fibrosis, inflammation and stem cell signaling pathways. α-SMA, MMP-8 and MMP-9 were the most down-regulated genes among the fibrotic signaling pathways in cholestatic liver (BDL) coupled with SMCC therapy compared to those treated with LGCC therapy ( ).

Techniques: Expressing, Isolation, Real-time Polymerase Chain Reaction, Agarose Gel Electrophoresis, DNA Methylation Assay

Journal: Hepatology (Baltimore, Md.)

Article Title: Forkhead box A2 regulated biliary heterogeneity and senescence during cholestatic liver injury

doi: 10.1002/hep.28831

Figure Lengend Snippet: Panel A: Localization of transplanted small cholangiocytes inside the mice liver: An additional group of mice was used for detecting transplanted small cholangiocytes location. Locally transplanted small cholangiocytes were labeled using PKH26 red fluorescent to detect small cholangiocytes which migrated to the liver in normal mice. A piece of mouse liver (~1 cm) was removed and fixed. The fluorescent signals from labeled SMCCs were predominantly detected in the mice liver 14 days after small cholangiocytes injection. Panel B: ELISA assay for TGF-β was carried out in the bile from small or large cholangiocytes transplanted mice after BDL. The TGF-β secretion in bile was significantly reduced after small cholangiocytes transplantation when compared to the large cholangiocytes and ECM control group after BDL. Panel C: Enhanced expressions of FoxA2 were detected in SMCC-BDL mice liver relative to ECM control mice liver by real-time PCR assay. Panel D&E: Reduced staining of Sirius Red was seen in SMCC-BDL mice liver when compared to the controls. Area of collagen present in liver sections stained with Sirius red were quantified in BDL mice liver with small or large cholangiocyte cell therapy relative to ECM control (Panel E). Significantly decreased intensity of liver fibrosis was detected in SMCC-BDL mice liver relative to ECM controls by enhanced Sirius red expression (n = 5). Original magnifications ×200. *p < 0.05 versus respective controls. Panel F: LGCC markers (AE2, Secretin (Sec), Secretin Receptor (SR)) and CK-19 were measured in cell therapy animals via qPCR (n=5) *p<0.05 vs ECM.

Article Snippet: Potential Molecular Mechanisms by which Biliary Cell Therapy Improves BDL-induced Biliary Damage and Liver Fibrosis To examine the molecular mechanisms by which biliary cell reversed BDL-induced hepatobiliary damages, we used the Mouse Fibrosis PCR array (PAMM-120ZA, Qiagen) and combined it with PCR of inflammation- and stem cell & development - related genes to evaluate mechanisms related to liver fibrosis, inflammation and stem cell signaling pathways. α-SMA, MMP-8 and MMP-9 were the most down-regulated genes among the fibrotic signaling pathways in cholestatic liver (BDL) coupled with SMCC therapy compared to those treated with LGCC therapy ( ).

Techniques: Labeling, Injection, Enzyme-linked Immunosorbent Assay, Transplantation Assay, Control, Real-time Polymerase Chain Reaction, Staining, Expressing

Journal: Hepatology (Baltimore, Md.)

Article Title: Forkhead box A2 regulated biliary heterogeneity and senescence during cholestatic liver injury

doi: 10.1002/hep.28831

Figure Lengend Snippet: Panel A: Relative gene expression profile between small cholangiocytes versus large cholangiocytes treated BDL liver is shown. The expression of a panel of diverse fibrosis-associated genes was evaluated by real-time PCR using Mice Fibrosis PCR Array from SABioscience. Gene expression relative to GAPDH was plotted as the Volcano Plots, depicting the relative expression levels (Log 10) for selected genes in BDL + Small versus BDL + Large control panels. α-SMA, MMP-2 and MMP-9 are the most down-regulated genes among the fibrotic signaling pathways in small cholangiocytes cell therapy after BDL injury. Panel B-C;E-F(left): Altered cellular senescence after small cholangiocytes cell therapy. Panel B presents Ingenuity Pathway analysis (IPA) of differentially regulated gene network after small cholangiocytes cell therapy. IPA was performed to understand the cellular context of the differentially expressed genes related to the recovery effects. Several genes implicated in cellular senescence are regulated by FoxA2, including p16 (CDKN2A), PAI-1 (SERPINE1), CCL2 and EGR1. Liver sections are stained with SA-β-Gal (Panel C) as described to reveal cellular senescence in BDL mice +small relative to +ECM and +Large controls. Small cholangiocytes cell therapy significantly reduced cellular senescence after BDL as quantified by SA-β-Gal staining (Panel C) and flurometric detections (Panel E, left), whereas the enhanced cellular senescence in isolated hepatic stellate cells by LCM was observed (Panel E. right). The alterations of cellular senescence markers p16, PAI-1, CCL2 and EGR1 after small cholangiocytes therapy in BDL mice liver were also verified (Panel F, left). *p < 0.05 versus normal controls. #p < 0.05 versus BDL+ECM controls. Panel D: p16 overexpression was performed in SMCCs, RNA was extracted and FoxA2 levels were measured with qPCR. *p < 0.05 versus control. Panel F (Right): Cholangiocyte supernatants derived from BDL mice treated with ECM, SMCC or LGCC were used to treat cultured stellate cells and inflammatory markers (CCL2 and IL-8) and a senescence marker (p16) were measured with qPCR. *p < 0.05 versus basal. #p < 0.05 versus BDL+ECM ECM.

Article Snippet: Potential Molecular Mechanisms by which Biliary Cell Therapy Improves BDL-induced Biliary Damage and Liver Fibrosis To examine the molecular mechanisms by which biliary cell reversed BDL-induced hepatobiliary damages, we used the Mouse Fibrosis PCR array (PAMM-120ZA, Qiagen) and combined it with PCR of inflammation- and stem cell & development - related genes to evaluate mechanisms related to liver fibrosis, inflammation and stem cell signaling pathways. α-SMA, MMP-8 and MMP-9 were the most down-regulated genes among the fibrotic signaling pathways in cholestatic liver (BDL) coupled with SMCC therapy compared to those treated with LGCC therapy ( ).

Techniques: Gene Expression, Expressing, Real-time Polymerase Chain Reaction, Control, Protein-Protein interactions, Staining, Isolation, Over Expression, Derivative Assay, Cell Culture, Marker

Journal: Hepatology (Baltimore, Md.)

Article Title: Forkhead box A2 regulated biliary heterogeneity and senescence during cholestatic liver injury

doi: 10.1002/hep.28831

Figure Lengend Snippet: Schematic representation of laser capture microdissection (LCM) procedures was displayed in Panel A. Frozen liver sections from control, BDL, BDL-SMCC and BDL-LGCC mice were sectioned with a cryostat and affixed the membrane side of nuclease and human nucleic acid free 2.0mm PEN membrane slides. A LCM system LMD7000 (Leica, Buffalo Grove, IL) was then used to capture desmin positive hepatic stellate cells and collect them in a thin walled PCR tube. The collected cells were then used to isolate RNA with the Arcturus Pico Pure RNA Isolation Kit, and real-time PCR analysis for cellular senescence markers p16, PAI-1, EGR1 and CCL2 were carried out. Enhanced expressions of p16 and EGR1 were discovered in isolated hepatic stellate cells from BDL-SMCC mice liver when compared to control BDL groups (Panel B), suggested SMCC cell therapy inhibited liver fibrosis through the induction of cellular senescence in hepatic stellate cells. *p < 0.05 versus normal or WT controls. #p < 0.05 versus Control-BDL mice liver. Panel C: Formalin fixed-paraffin embedded liver sections from PSC patients were sectioned at 6 μm and affixed to the membrane side of PEN membrane slides. Sections were stained for desmin and a LCM system LMD7000 was used to capture desmin positive cells. The collected cells were used to extract RNA as described above and the senescence marker PAI-1 and the inflammation marker IL-8 were quantified with qPCR. *p < 0.05 versus normal.

Article Snippet: Potential Molecular Mechanisms by which Biliary Cell Therapy Improves BDL-induced Biliary Damage and Liver Fibrosis To examine the molecular mechanisms by which biliary cell reversed BDL-induced hepatobiliary damages, we used the Mouse Fibrosis PCR array (PAMM-120ZA, Qiagen) and combined it with PCR of inflammation- and stem cell & development - related genes to evaluate mechanisms related to liver fibrosis, inflammation and stem cell signaling pathways. α-SMA, MMP-8 and MMP-9 were the most down-regulated genes among the fibrotic signaling pathways in cholestatic liver (BDL) coupled with SMCC therapy compared to those treated with LGCC therapy ( ).

Techniques: Laser Capture Microdissection, Control, Membrane, Isolation, Real-time Polymerase Chain Reaction, Formalin-fixed Paraffin-Embedded, Staining, Marker