Background and Aim: Myofibroblastic hepatic stellate cells (HSC) are the central cell types of liver fibrosis due to their excessive matrix production. By deep sequencing of the Ago2 interacting transcriptome we have demonstrated that neuronal miR-9, miR-125b, miR -128 are involved in regulation of chemokine signaling during myofibroblastic HSC activation (Noetel et al., 2013). In the present study, we aimed to analyse further putative targets of the Ago2 neuronal miRNA complex, namely Notch1, KLF4, and Lin28.

Primary sclerosing cholangitis (PSC) is a chronic cholestatic liver disease leading to cirrhosis and cholangiocellular carcinoma. Inhibitors of the renin–angiotensin system or the sympathetic nervous system delay liver fibrogenesis in animal models.

Background 82 Aims: The progression of liver fibrosis in patients with chronic hepatitis C (CHC) is important to decide on the treatment of the virus. As liver biopsy and liver stiffness measurement for staging of fibrosis present limitations, circulating levels of miR-122 have been suggested as a novel biomarker to predict the extent of liver injury. We evaluated the potential of miR-122 as an indicator of fibrosis progression in CHC infection and performed, for the first time, a comprehensive analysis of hepatic and circulating miR-122 levels in patients with CHC.Methods: Patients with well-documented CHC infection were selected from the database of HepNet, the German-Competence-Network on Viral Hepatitis. All patients underwent blood sampling and liver biopsy with grading of inflammation and staging of fibrosis. RNA was extracted from 84 liver biopsies and 164 serum samples of CHC patients. miR-122 levels in liver and serum samples were quantified by real-time PCR normalized to RNU6 or spiked-in RNA, respectively.Results: Hepatic levels of miR-122 decreased significantly with the severity of fibrosis (p = 0.001). In addition, circulating miR-122 levels correlated negatively with increasing stages of fibrosis, although the inverse correlation was moderate due to a two-phase miR-122 pattern during fibrosis progression. Thus, circulating miR-122 levels decreased in patients with severe fibrosis (F3, F4), while at early stages with distinct fibrotic structures (F2) and high inflammatory activity, miR-122 serum levels were elevated.Conclusions: We conclude that during progression of fibrosis less miR-122 is released into the blood stream due to the loss of liver cells and the decrease of hepatic miR-122 levels. Although the release of circulating miR-122 possibly mirrors acute liver injury, in chronic liver disease and fibrosis, the loss of liver cells and the decreased hepatocellular miR-122 expression render miR-122 an inappropriate marker, when exclusively used for interpretation of fibrosis progression. (C) 2012 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

Background and Aim: Activated hepatic stellate cells (HSC), which transdifferentiate into myofibroblastic cells, are the main producers of extracellular components upon chronic liver disease. Myofibroblastic transition is associated with an altered profile of profibrogenic growth factors, but also of neuronal signal transducers. MicroRNAs are small non-coding RNAs that regulate gene expression by translational inhibition. Therefore, we studied the role of microRNAs known to be involved in neuronal differentiation during HSC transdifferentiation. Methods: RNA was extracted by the Trizol method from primary rat HSC cultured for up to 10 days. miRNA expression profiling was performed by microarray analyses of miRNA extracted from HSC during myofibroblastic differentiation. Further polyadenylation combined reverse transcription was carried out and neuronal miR-9, miR-125b and miR-128 were quantified by Real-Time PCR. Putative targets were searched using various algorithms and a novel immunoprecipitation assay of the Argonaute 2 protein. Potential miRNA binding sites of the 3'-UTR were verified by luciferase reporter analyses. Results: During myofibroblastic HSC differentiation an upregulation for miR-9, miR-125b and miR-128 was observed. In silico target prediction identified putative binding sites of the neuronal miRNAs in transcripts encoding for several pluripotency factors such as LIN28. To evaluate the targets functional reporter assays were performed by cloning the wild type in comparison to corresponding mutated sequences with two point mutations in the binding consensus. We identified for all three neuronal miRNAs the potential target LIN28 which triggers pluripotency in embryonic stem cells. Conclusion: In conclusion, the neuronal microRNAs miR-9, miR-125b and miR-128 are highly upregulated during HSC transdifferentiation and are suggested to play an important role in myofibroblastic transition and expression of neuronal features by targeting LIN28 mRNA in HSC.

BACKGROUND:In chronic liver disease, hepatic stellate cells (HSC) transdifferentiate into myofibroblasts, promoting extracellular matrix (ECM) synthesis and deposition. Stimulation of HSC by transforming growth factor-β (TGF-β) is a crucial event in liver fibrogenesis due to its impact on myofibroblastic transition and ECM induction. In contrast, hepatocyte growth factor (HGF), exerts antifibrotic activities. Recently, miR-29 has been reported to be involved in ECM synthesis. We therefore studied the influence of HGF and TGF-β on the miR-29 collagen axis in HSC.METHODOLOGY:HSC, isolated from rats, were characterized for HGF and Met receptor expression by Real-Time PCR and Western blotting during culture induced myofibroblastic transition. Then, the levels of TGF-β, HGF, collagen-I and -IV mRNA, in addition to miR-29a and miR-29b were determined after HGF and TGF-β stimulation of HSC or after experimental fibrosis induced by bile-duct obstruction in rats. The interaction of miR-29 with 3'-untranslated mRNA regions (UTR) was analyzed by reporter assays. The repressive effect of miR-29 on collagen synthesis was studied in HSC treated with miR-29-mimicks by Real-Time PCR and immunoblotting.PRINCIPAL FINDINGS:The 3'-UTR of the collagen-1 and -4 subtypes were identified to bind miR-29. Hence, miR-29a/b overexpression in HSC resulted in a marked reduction of collagen-I and -IV synthesis. Conversely, a decrease in miR-29 levels is observed during collagen accumulation upon experimental fibrosis, in vivo, and after TGF-β stimulation of HSC, in vitro. Finally, we show that during myofibroblastic transition and TGF-β exposure the HGF-receptor, Met, is upregulated in HSC. Thus, whereas TGF-β stimulation leads to a reduction in miR-29 expression and de-repression of collagen synthesis, stimulation with HGF was definitely associated with highly elevated miR-29 levels and markedly repressed collagen-I and -IV synthesis.CONCLUSIONS:Upregulation of miRNA-29 by HGF and downregulation by TGF-β take part in the anti- or profibrogenic response of HSC, respectively.

In chronic renal disease, tubulointerstitial fibrosis is a leading cause of renal failure. Here, we made use of one of the most promising gene therapy vector platforms, the adeno-associated viral (AAV) vector system, and the COL4A3-deficient mice, a genetic mouse model of renal tubulointerstitial fibrosis, to develop a novel bidirectional treatment strategy to prevent renal fibrosis. By comparing different AAV serotypes in reporter studies, we identified AAV9 as the most suitable delivery vector to simultaneously target liver parenchyma for endocrine and renal tubular epithelium for paracrine therapeutic expression of the antifibrogenic cytokine human hepatocyte growth factor (hHGF). We used transcriptional targeting to drive hHGF expression from the newly developed CMV-enhancer-Ksp-cadherin-promoter (CMV-Ksp) in renal and hepatic tissue following tail vein injection of rAAV9-CMV-Ksp-hHGF into COL4A3-deficient mice. The therapeutic efficiency of our approach was demonstrated by a remarkable attenuation of tubulointerstitial fibrosis and repression of fibrotic markers such as collagen1alpha1 (Col1A1), platelet-derived growth factor receptor-beta (PDGFR-beta), and alpha-smooth muscle actin (SMA). Taken together, our results show the great potential of rAAV9 as an intravenously applicable vector for the combined paracrine and endocrine expression of antifibrogenic factors in the treatment of renal failure caused by tubulointerstitial fibrosis.

Primary sclerosing cholangitis (PSC) is a cholestatic liver disease with high propensity to develop into cholangiocarcinoma. The hepatobiliary disorder of PSC is due to progressive fibrosis surrounding the intra- and extrahepatic bile ducts. Until now, no effective medical therapy exists. To study the progression of sclerosing cholangitis after inhibition of the sympathetic nervous system by blockade of the β -adrenoceptors, we used the Mdr2 −/− mouse model, which develops periportal fibrosis similar to human PSC. Liver tissues of Mdr2 −/− mice untreated or treated with the β -adrenoceptor antagonist propranolol were analyzed for inflammation and fibrosis progression at different time points by histological scoring and immunostaining for α -smooth muscle actin ( α -SMA), CD45 and S100A4. Transaminases and hydroxyproline contents were determined. Expression of angiotensinogen, endothelin-1, TGF- β , TNF- α , CTGF and procollagen 1A1 was studied by real-time PCR on laser-microdissected areas of acinar zones I and II–III. After 3 months, periportal fibrosis had developed in Mdr2 −/− mice, but immunostaining revealed no sinusoidal and only minor periportal contribution of myofibroblasts with prominent fibroblasts. Propranolol treatment of Mdr2 −/− mice improved liver architecture. Additionally, inflammation and fibrosis were significantly reduced. After 3 months of treatment, the antifibrotic effect of the β -blockade was most obvious. The transcript levels of procollagen 1A1, TNF- α , TGF- β , CTGF and endothelin-1 were markedly repressed in the portal areas of treated mice. Taken together, these data show that propranolol efficiently delays progression of sclerosing cholangitis. Therefore, the blockade of β -adrenoceptors is a promising option to support future therapeutic strategies in the treatment of human PSC.

Hepatocyte growth factor (HGF) is a multifunctional growth factor affecting cell proliferation and differentiation. Due to its mitogenic potential, HGF plays an important role in tubular repair and regeneration after acute renal injury. However, recent reports have shown that HGF also acts as an anti-inflammatory and anti-fibrotic factor, affecting various cell types such as renal fibroblasts and triggering tubulointerstitial fibrosis of the kidney.The present study provides evidence that HGF stimulation of renal fibroblasts results in the activation of both the Erk1/2 and the Akt pathways. As previously shown, Erk1/2 phosphorylation results in Smad-linker phosphorylation, thereby antagonizing cellular signals induced by TGF beta. By siRNA mediated silencing of the ErK1/2-Smad linkage, however, we now demonstrate that Akt signaling acts as an auxiliary pathway responsible for the anti-fibrotic effects of HGF. In order to define the anti-fibrotic function of HGF we performed comprehensive expression profiling of HGF-stimulated renal fibroblasts by microarray hybridization. Functional cluster analyses and quantitative PCR assays indicate that the HGF-stimulated pathways transfer the anti-fibrotic effects in renal interstitial fibroblasts by reducing expression of extracellular matrix proteins, various chemokines, and members of the CCN family. (C) 2009 Elsevier Inc. All rights reserved.