title: K⁺-channel inhibition reduces portal perfusion pressure in fibrotic rats and fibrosis associated characteristics of hepatic stellate cells.
abstract: OBJECTIVE
  In liver fibrosis, activated hepatic stellate cells (HSC) secrete excess extracellular matrix, thus, represent key targets for antifibrotic treatment strategies. Intermediate-conductance Ca(2) (+) -activated K(+) -channels (KCa3.1) are expressed in non-excitable tissues affecting proliferation, migration and vascular resistance rendering KCa3.1 potential targets in liver fibrosis. So far, no information about KCa3.1 expression and their role in HSC exists. Aim was to quantify the KCa3.1 expression in HSC depending on HSC activation and investigation of antifibrotic properties of the specific KCa3.1 inhibitor TRAM-34 in vitro and in vivo.
  
  
  METHODS
  KCa3.1 expression and functionality were studied in TGF-β1-activated HSC by quantitative real time PCR, western-blot and patch-clamp analysis respectively. Effects of TRAM-34 on HSC proliferation, cell cycle and fibrosis-related gene expression were assessed by [(3) H]-thymidine incorporation, FACS-analysis and RT-PCR respectively. In vivo, vascular resistance and KCa3.1 gene and protein expression were determined in bile duct ligated rats by in situ liver perfusion, Taqman PCR and immunohistochemistry respectively.
  
  
  RESULTS
  Fibrotic tissues and TGF-β1-activated HSC exhibited higher KCa3.1-expressions than normal tissue and untreated cells. KCa3.1 inhibition with TRAM-34 reduced HSC proliferation by induction of cell cycle arrest and reduced TGF-β1-induced gene expression of collagen I, alpha-smooth muscle actin and TGF-β1 itself. Furthermore, TRAM-34 blocked TGF-β1-induced activation of TGF-β signalling in HSC. In vivo, TRAM-34 reduced the thromboxane agonist-induced portal perfusion pressure.
  
  
  CONCLUSIONS
  Inhibition of KCa3.1 with TRAM-34 downregulates fibrosis-associated gene expression in vitro, and reduces portal perfusion pressure in vivo. Thus, KCa3.1 may represent novel targets for the treatment of liver fibrosis.
authors.affiliation: Department of Gastroenterology, Infectiology and Rheumatology, Charité - University Medicine Berlin, Campus Benjamin Franklin, Berlin, Germany.
authors.forename: Christian
authors.initials: C
authors.lastname: Freise
authors.forename: Silke
authors.initials: S
authors.lastname: Heldwein
authors.forename: Ulrike
authors.initials: U
authors.lastname: Erben
authors.forename: Joachim
authors.initials: J
authors.lastname: Hoyer
authors.forename: Ralf
authors.initials: R
authors.lastname: Köhler
authors.forename: Korinna
authors.initials: K
authors.lastname: Jöhrens
authors.forename: Eleonora
authors.initials: E
authors.lastname: Patsenker
authors.forename: Martin
authors.initials: M
authors.lastname: Ruehl
authors.forename: Daniel
authors.initials: D
authors.lastname: Seehofer
authors.forename: Felix
authors.initials: F
authors.lastname: Stickel
authors.forename: Rajan
authors.initials: R
authors.lastname: Somasundaram
mesh_terms: D000199:Actins / Q000235:genetics / Q000378:metabolism; D000818:Animals; D002453:Cell Cycle / Q000187:drug effects; D002460:Cell Line; D049109:Cell Proliferation / Q000187:drug effects; D024042:Collagen Type I / Q000235:genetics / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D005786:Gene Expression Regulation; D055166:Hepatic Stellate Cells / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D051660:Intermediate-Conductance Calcium-Activated Potassium Channels / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D008099:Liver / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D008106:Liver Cirrhosis, Experimental / Q000188:drug therapy* / Q000235:genetics / Q000378:metabolism / Q000473:pathology / Q000503:physiopathology; D008297:Male; D017082:Portal Pressure / Q000187:drug effects*; D026902:Potassium Channel Blockers / Q000494:pharmacology*; D011720:Pyrazoles / Q000494:pharmacology*; D017207:Rats, Sprague-Dawley; D015398:Signal Transduction / Q000187:drug effects; D014162:Transfection; D053773:Transforming Growth Factor beta1 / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D014655:Vascular Resistance / Q000187:drug effects
chemical_list: D000199:Actins; D024042:Collagen Type I; D051660:Intermediate-Conductance Calcium-Activated Potassium Channels; D026902:Potassium Channel Blockers; D011720:Pyrazoles; D053773:Transforming Growth Factor beta1; C491575:Kcnn4 protein, rat; C411671:TRAM 34; C508962:Tgfb1 protein, rat; C498862:Acta2 protein, rat
keywords: HSC; KCa3.1; TGF-β1; TRAM-34; liver fibrosis