After four weeks of BDO both hepatic inflammation and collagen accumulation was evident in histological sections. Hydroxyproline determination indicates significant enhancement of matrix deposition in BDO treated livers. Thus, BDO induced severe liver fibrosis, when compared to livers of sham-operated animals. Consistent with the antifibrogenic and inhibitory function of miR-29 in collagen I and IV synthesis and the increase of col1A2 and col4A2, we observed a significant reduction in hepatic miR-29a and miR-29b levels in this experimental BDO model. This loss of the miR- 29a and miR-29b in fibrotic BDO treated livers is attended by reduced levels of HGF upon fibrosis. Stimulation of primary HSC and myofibroblastic HSC-T6 cells with TGF-b decreased significantly the expression of miR-29a and miR-29b in vitro. In contrast, the incubation of primary and transdifferentiated HSC with the recombinant hHGF elicited a marked upregulation of the miR-29a/b levels.

To address the question if the antagonistic influence of TGF-b and HGF can be also observed in other species or fibroblastic cells of other organs, we stimulated primary skin fibroblasts with both TGF-b and HGF. In agreement with the data on rat HSC, miR- 29 is repressed by TGF-b, but upregulated by HGF. Therefore, miR-29 expression appears to be reciprocally regulated by profibrogenic and antifibrogenic growth factors in HSC, suggesting that miR-29 occupies a central role in responding to the antagonistic actions of HGF and TGF-b in regulating collagen synthesis in activated and transdifferentiated HSC. After demonstrating that repression of the collagen-inhibiting miR-29 is an important downstream TGF-b effect, we studied if miR-29 overexpression can overcome the profibrogenic features mediated by TGF-b such as col1A1 induction. For this purpose, HSC-T6 cells were transfected with miR-29a mimics in comparison to LDN-193189 ALK inhibitor scrambled or miR-29-silencing miRNA.

Transfection of HSC with miR-29a mimics lead to a 10-fold overexpression of miR-29a. In HSC that express low levels of miR-29a due to transfection with scrambled miRNA or with a miR-29a silencer, TGF-b treatment highly induced col1A1 synthesis. However, col1A1 induction by TGF-b was only marginal in HSC, that highly overexpress miR-29a after transfection with miR-29a mimics. Thus, miR-29a is able to lower significantly the profibrogenic features of TGF-b in collagen induction. In the present study we investigated the effects of the opposing action of TGF-b and HGF on miR-29 regulated collagen synthesis by HSC during liver fibrosis. After transdifferentiation into myofibroblasts, HSC constitute the main matrix producing cell type of the fibrotic liver. Our findings demonstrate that myofibroblastic transdifferentiation is accompanied by the loss of HGF synthesis on the one hand, and a tremendous increase of Met receptor expression on the other hand.