K⁺-channel inhibition reduces portal perfusion pressure in fibrotic rats and fibrosis associated characteristics of hepatic stellate cells.

Abstract

BACKGROUND & AIMS:

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.

CONCLUSION:

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: Christian Freise, Silke Heldwein, Ulrike Erben, Joachim Hoyer, Ralf Köhler, Korinna Jöhrens, Eleonora Patsenker, Martin Ruehl, Daniel Seehofer, Felix Stickel, Rajan Somasundaram
• Journal: Liver international : official journal of the International Association for the Study of the Liver (Liver Int) Vol. 35 Issue 4 Pg. 1244-52 (Apr 2015) ISSN: 1478-3231 [Electronic] United States
• PMID: 25212242 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
• Copyright: © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Chemical References

• Actins
• Collagen Type I
• Intermediate-Conductance Calcium-Activated Potassium Channels
• Kcnn4 protein, rat
• Potassium Channel Blockers
• Pyrazoles
• TRAM 34
• Tgfb1 protein, rat
• Transforming Growth Factor beta1
• smooth muscle actin, rat

Topics

• Actins (genetics, metabolism)
• Animals
• Cell Cycle (drug effects)
• Cell Line
• Cell Proliferation (drug effects)
• Collagen Type I (genetics, metabolism)
• Dose-Response Relationship, Drug
• Gene Expression Regulation
• Hepatic Stellate Cells (drug effects, metabolism, pathology)
• Intermediate-Conductance Calcium-Activated Potassium Channels (antagonists & inhibitors, genetics, metabolism)
• Liver (drug effects, metabolism, pathology)
• Liver Cirrhosis, Experimental (drug therapy, genetics, metabolism, pathology, physiopathology)
• Male
• Portal Pressure (drug effects)
• Potassium Channel Blockers (pharmacology)
• Pyrazoles (pharmacology)
• Rats, Sprague-Dawley
• Signal Transduction (drug effects)
• Transfection
• Transforming Growth Factor beta1 (genetics, metabolism, pharmacology)
• Vascular Resistance (drug effects)