Adenoviral delivery of an antisense RNA complementary to the 3' coding sequence of transforming growth factor-beta1 inhibits fibrogenic activities of hepatic stellate cells.
Liver fibrosis occurs as a consequence of the transdifferentiationof hepatic stellate cells into myofibroblasts and is associated with an increased expression and activation of transforming growth factor (TGF)-beta1. This pluripotent, profibrogenic cytokine stimulates matrix synthesis and decreases matrix degradation, resulting in fibrosis. Thus, blockade of synthesis or sequestering of mature TGF-beta1 is a primary target for the development of antifibrotic approaches. The purpose of this study was to investigate whether the administration of adenoviruses constitutively expressing an antisense mRNA complementary to the 3' coding sequence of TGF-beta1 is able to suppress the synthesis of TGF-beta1 in culture-activated hepatic stellate cells. We demonstrate that the adenoviral vehicle directs high-level expression of the transgene and proved that the transduced antisense is biologically active by immunoprecipitation, Western blot, quantitative TGF-beta1 ELISA, and cell proliferation assays. Additionally, the biological function of the transgene was confirmed by analysis of differential activity of TGF-beta1-responsive genes using cell ELISA, Northern blotting, and by microarray technology, respectively. Furthermore, we examined the effects of that transgene on the expression of TGF-beta2, TGF-beta3, collagen type alpha1(I), latent transforming growth factor binding protein 1, types I and II TGF-beta receptors, and alpha-smooth muscle actin. Our results indicate that the administration of antisense mRNA offers a feasible approach to block autocrine TGF-beta1 signaling in hepatic stellate cells and may be useful and applicable in future to the treatment of fibrosis in chronic liver diseases.
Institute of Clinical Chemistry and Pathobiochemistry, RWTH- University Hospital, D-52074 Aachen, Germany.
Monica
M
Arias
Birgit
B
Lahme
Eddy
E
Van de Leur
Axel M
AM
Gressner
Ralf
R
Weiskirchen
D030201:Activin Receptors, Type I / Q000378:metabolism; D000256:Adenoviridae / Q000235:genetics*; D000818:Animals; D015152:Blotting, Northern; D015139:Blotting, Southern; D002352:Carrier Proteins / Q000378:metabolism; D002454:Cell Differentiation / Q000187:drug effects; D002478:Cells, Cultured; D024042:Collagen Type I / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D047908:Intracellular Signaling Peptides and Proteins; D053779:Latent TGF-beta Binding Proteins; D008099:Liver / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D008103:Liver Cirrhosis / Q000517:prevention & control; D008297:Male; D016366:Open Reading Frames; D017346:Protein Serine-Threonine Kinases; D016372:RNA, Antisense / Q000494:pharmacology*; D018075:RNA, Complementary; D051381:Rats; D017207:Rats, Sprague-Dawley; D000077293:Receptor, Transforming Growth Factor-beta Type I; D000077294:Receptor, Transforming Growth Factor-beta Type II; D018125:Receptors, Transforming Growth Factor beta / Q000378:metabolism; D016212:Transforming Growth Factor beta / Q000235:genetics* / Q000378:metabolism; D053773:Transforming Growth Factor beta1; D053781:Transforming Growth Factor beta2; D053782:Transforming Growth Factor beta3
D002352:Carrier Proteins; D024042:Collagen Type I; D047908:Intracellular Signaling Peptides and Proteins; D053779:Latent TGF-beta Binding Proteins; D016372:RNA, Antisense; D018075:RNA, Complementary; D018125:Receptors, Transforming Growth Factor beta; C508962:Tgfb1 protein, rat; C509046:Tgfb3 protein, rat; D016212:Transforming Growth Factor beta; D053773:Transforming Growth Factor beta1; D053781:Transforming Growth Factor beta2; D053782:Transforming Growth Factor beta3; D017346:Protein Serine-Threonine Kinases; D030201:Activin Receptors, Type I; D000077293:Receptor, Transforming Growth Factor-beta Type I; D000077294:Receptor, Transforming Growth Factor-beta Type II; C000625843:Tgfbr1 protein, rat