Smad3 depletion prevented TGF-β responses in Huh7 cells expressing or not the HCV core protein.

<p>(A) Smad3 expression determined by Western blot analysis in four independent clones selected after stable transfection with pRetroSuper-shRNA-Smad3 plasmid. Anti-p38 antibody was used as control loading. (B) Different clones were transfected with the CAGA-luc reporter plasmid and treated or not with TGF-β (5 ng/ml) for 18 h before determination of luciferase activity. Results were normalized with renilla luciferase and represent the mean of triplicates+/−SD. (C, D) Different clones were treated with TGF-β for 48 h before determination of cell viability (C) or caspase3 activity (D). Results represent the mean+/−SD of triplicates from a representative experiment. * p≤0.05, *** p≤0.0005, NS : not significant. (E) Different clones were treated with TGF-β for 48 h and αSMA polymerization was estimated by immunofluorescence analysis.</p

TGF-β responses in Huh7 cells expressing different levels of Smad3.

<p>(A) Huh7-shRNA-Smad3 cells (Clone 3) were transfected with increasing amounts of Myc-Smad3 expression vector together with pCMV renilla luciferase. Smad3 protein expression was evaluated 24 h later by Western blot analysis using an anti-Myc antibody and loading was normalized with renilla luciferase expression. (B) Huh7-shRNA-Smad3 cells (Clone 3) were cotransfected with the CAGA-luc reporter plasmid and increasing amounts of Myc-Smad3 vector together with pCMV renilla luciferase. 24 h later, they were treated or not with different doses of TGF-β for 18 h before determination of luciferase activity. Results were normalized with renilla luciferase and represent the mean+/−SD of triplicates from a representative experiment. (C, D) Huh7-shRNA-Smad3 cells (Clone 3) were transfected with increasing amounts of Myc-Smad3 vector together with pGFP plasmid and sorted by FACS 24 h later on the basis of GFP expression. Cells were then cultured for 24 h and treated with different doses of TGF-β for 48 h before determination of cell viability (C) or caspase3 activity (D). * p≤0.05, ** p≤0.005, *** p≤0.0005, NS : not significant. (E) Huh7-shRNA-Smad3 cells (Clone 3) were transfected with increasing amounts of Myc-Smad3 vector together with pGFP plasmid and sorted 24 h later on the basis of GFP expression. αSMA expression was estimated by immunofluorescence analysis after treatment with TGF-β (1 ng/ml) for 48 h.</p

The Reg3α (HIP/PAP) Lectin Suppresses Extracellular Oxidative Stress in a Murine Model of Acute Liver Failure

<div><p>Background and Aims</p><p>Acute liver failure (ALF) is a rapidly progressive heterogeneous illness with high mortality rate and no widely accessible cure. A promising drug candidate according to previous preclinical studies is the Reg3α (or HIP/PAP) lectin, which alleviates ALF through its free-radical scavenging activity. Here we study the therapeutic targets of Reg3α in order to gain information on the nature of the oxidative stress associated with ALF.</p><p>Methods</p><p>Primary hepatocytes stressed with the reactive oxygen species (ROS) inducers TNFα and H₂O₂ were incubated with a recombinant Reg3α protein. ALF was induced in C57BL/6J mice by an anti-CD95 antibody. Livers and primary hepatocytes were harvested for deoxycholate separation of cellular and extracellular fractions, immunostaining, immunoprecipitation and malondialdehyde assays. Fibrin deposition was studied by immunofluorescence in frozen liver explants from patients with ALF.</p><p>Results</p><p>Fibrin deposition occurs during experimental and clinical acute liver injuries. Reg3α bound the resulting transient fibrin network, accumulated in the inflammatory extracellular matrix (ECM), greatly reduced extracellular ROS levels, and improved cell viability. Hepatocyte treatment with ligands of death receptors, e.g. TNFα and Fas, resulted in a twofold increase of malondialdehyde (MDA) level in the deoxycholate-insoluble fractions. Reg3α treatment maintained MDA at a level similar to control cells and thereby increased hepatocyte survival by 35%. No antioxidant effect of Reg3α was noted in the deoxycholate-soluble fractions. Preventing fibrin network formation with heparin suppressed the prosurvival effect of Reg3α.</p><p>Conclusions</p><p>Reg3α is an ECM-targeted ROS scavenger that binds the fibrin scaffold resulting from hepatocyte death during ALF. ECM alteration is an important pathogenic factor of ALF and a relevant target for pharmacotherapy.</p></div

Abundant fibrin deposition in human ALFs.

<p>Histological analysis using hematoxylin/eosin (H&E) staining (left), anti-fibrin immunofluorescence (right) of sections of liver explants from patients with end stage acute liver failure of different etiologies. Control: liver explants from patients with amyloid neuropathy. Scale bars: 50 μm.</p

Expression of HCV core proteins in primary mouse hepatocytes increase EMT induced by TGF-β.

<p>(A) Morphologic changes of mouse hepatocytes expressing or not HCV T core protein observed after 48 h of culture with or without TGF-β (2 ng/ml). (B) Hepatocytes isolated from transgenic mice expressing HCV core proteins were treated with TGF-β (2 ng/ml) or SB431542 (1 µM) for 48 h and expression of αSMA was examined by immunofluorescence using a αSMA antibody. Data are representative of three independent experiments. (C) Hepatocytes isolated from transgenic mice expressing HCV core proteins were treated with TGF-β or SB431542 for 48 h and expression of E-cadherin was determined by Western blotting. Anti-p38 western blotting was used as control loading. Data are representative of three independent experiments.</p

Expression of HCV core proteins in primary mouse hepatocytes reduce cell growth inhibition and apoptosis induced by TGF-β.

<p>(A,B,C) Mouse hepatocytes obtained from livers of transgenic mice expressing or not HCV core proteins isolated from tumor (T) or cirrhotic (NT) tissues were treated with TGF-β for 48 h before determination of cell proliferation, estimated by BrDU incorporation (A), cell viability (B) or caspase 3 activity (C). (D) Cells were treated with TRAIL (20 ng/ml) for 18 h before determination of caspase3 activity. Results represent the mean+/−SD of triplicates from a representative experiment. * p≤0.05, ** p≤0.005, *** p≤0.0005.</p

TGF-β increases EMT in primary human hepatocytes expressing HCV core proteins.

<p>(A) Expression of αSMA or Vimentin was estimated by immunofluorescence analysis after treatment with TGF-β (5 ng/ml) or SB431542 (1 µM). (B) Expression of Fibronectin or E-Cadherin was estimated by Western blot analysis in the same experimental conditions. Data are representative of three independent experiments.</p

Clinical characteristics of patients involved in the study.

<p>ALAT, <b>Alanine Aminotransferase;</b> ALF, Acute Liver Failure; AN, Amyloid neuropathy; ASAT, Aspartate Aminotransferase; FNA, Fine Needle Aspiration; MARS, Molecular Adsorbent Recirculating System; NAC, N-acetylcysteine; TP, Prothrombin level; Time, time in days from diagnostic to liver transplantation; UI, International Unit.</p><p>Clinical characteristics of patients involved in the study.</p

Smad activation is essential to induce TGF-β -mediated EMT.

<p>(A) Huh7 cells were co-transfected GFP together with TbR1act or TbR1l45M act plasmids in the presence or absence of HCV core vector. Immunofluorescence analysis was performed 48 h later with an anti αSMA antibody. (B) Expression of TbR1act or TbR1l45M act and HCV core protein were assessed by Western blotting using anti-HA, anti-Flag or anti-core antibodies respectively.</p

Immunochemical analyses of αSMA and γSMA expression in normal and HCC livers.

<p>(A) Serial sections of normal livers were immunochemically stained with anti-αSMA or anti-γSMA antibodies. BD: bile duct, A: artery, H: canal of Hering, PV: Portal Vein. x50. (B) Sections of normal livers were immunochemically stained with anti-αSMA or anti-γSMA antibodies. (<b>→</b>: canaliculi) x400. (C) (D) HCC nodules were immunochemically stained with different antibodies. Serial sections stained with γSMAand αSMA; x160 and x400 (C). γSMA and E-Cadherin (E-cad); γSMA and CK19 (D). x160.</p