Hepatic and serum levels of miR-122 after chronic HCV-induced fibrosis.

BACKGROUND & 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.

Telmisartan plus propranolol improves liver fibrosis and bile duct proliferation in the PSC-like Abcb4-/- mouse model.

BACKGROUND: 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. AIMS: We investigated the antifibrotic potential of telmisartan, an angiotensin II type 1 receptor antagonist, and the ß-adrenoceptor blocker propranolol in the PSC-like Abcb4 knockout mouse model. METHODS: Sixty-five Abcb4 (-/-) mice were treated with telmisartan for 3 or 5 months (T) and with telmisartan plus propranolol for 3, 5, or 8 months (TP), or for 2 or 5 months starting with a delay of 3 months (TP delayed). Liver hydroxyproline content, inflammation, fibrosis, and bile duct proliferation were assessed; fibrosis-related molecules were analyzed by real-time polymerase chain reaction and Western blotting. RESULTS: Compared to controls, telmisartan monotherapy had no significant influence on hydroxyproline; however, telmisartan plus propranolol reduced hydroxyproline (TP 3 months, p = 0.008), fibrosis score (TP 3 months and TP 8 months, p = 0.043 and p = 0.008, respectively; TP delayed 8 months, p < 0.0005), bile duct proliferation (TP 8 months and TP delayed 8 months, p = 0.006 and p < 0.0005, respectively), and procollagen α1(I), endothelin-1, TIMP-1 and MMP3 mRNA as well as α-SMA, CK-19, and TIMP-1 protein. CONCLUSIONS: Telmisartan plus propranolol reduces liver fibrosis and bile duct proliferation in the PSC-like Abcb4 (-/-) mouse model, even when started at late stages of fibrosis, and may thus represent a novel therapeutic option for cholestatic liver diseases such as PSC.

Hepatocyte growth factor (HGF) inhibits collagen I and IV synthesis in hepatic stellate cells by miRNA-29 induction.

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-ß (TGF-ß) 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-ß 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-ß, HGF, collagen-I and -IV mRNA, in addition to miR-29a and miR-29b were determined after HGF and TGF-ß 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-ß stimulation of HSC, in vitro. Finally, we show that during myofibroblastic transition and TGF-ß exposure the HGF-receptor, Met, is upregulated in HSC. Thus, whereas TGF-ß 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-ß take part in the anti- or profibrogenic response of HSC, respectively.

ß-Adrenoceptor blockade in sclerosing cholangitis of Mdr2 knockout mice: antifibrotic effects in a model of nonsinusoidal 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 ß-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 ß-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-ß, 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 ß-blockade was most obvious. The transcript levels of procollagen 1A1, TNF-α, TGF-ß, 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 ß-adrenoceptors is a promising option to support future therapeutic strategies in the treatment of human PSC.

Combined paracrine and endocrine AAV9 mediated expression of hepatocyte growth factor for the treatment of renal fibrosis.

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.

Profiling of anti-fibrotic signaling by hepatocyte growth factor in renal fibroblasts.

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 TGFbeta. 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.