Angiotensin-II type 1 receptor-mediated Janus kinase 2 activation induces liver fibrosis.

UNLABELLED: Activation of the renin angiotensin system resulting in stimulation of angiotensin-II (AngII) type I receptor (AT1R) is an important factor in the development of liver fibrosis. Here, we investigated the role of Janus kinase 2 (JAK2) as a newly described intracellular effector of AT1R in mediating liver fibrosis. Fibrotic liver samples from rodents and humans were compared to respective controls. Transcription, protein expression, activation, and localization of JAK2 and downstream effectors were analyzed by real-time polymerase chain reaction, western blotting, immunohistochemistry, and confocal microscopy. Experimental fibrosis was induced by bile duct ligation (BDL), CCl4 intoxication, thioacetamide intoxication or continuous AngII infusion. JAK2 was inhibited by AG490. In vitro experiments were performed with primary rodent hepatic stellate cells (HSCs), Kupffer cells (KCs), and hepatocytes as well as primary human and human-derived LX2 cells. JAK2 expression and activity were increased in experimental rodent and human liver fibrosis, specifically in myofibroblastic HSCs. AT1R stimulation in wild-type animals led to activation of HSCs and fibrosis in vivo through phosphorylation of JAK2 and subsequent RhoA/Rho-kinase activation. These effects were prevented in AT1R(-/-) mice. Pharmacological inhibition of JAK2 attenuated liver fibrosis in rodent fibrosis models. In vitro, JAK2 and downstream effectors showed increased expression and activation in activated HSCs, when compared to quiescent HSCs, KCs, and hepatocytes isolated from rodents. In primary human and LX2 cells, AG490 blocked AngII-induced profibrotic gene expression. Overexpression of JAK2 led to increased profibrotic gene expression in LX2 cells, which was blocked by AG490. CONCLUSION: Our study substantiates the important cell-intrinsic role of JAK2 in HSCs for development of liver fibrosis. Inhibition of JAK2 might therefore offer a promising therapy for liver fibrosis.

Activation of the MAS receptor by angiotensin-(1-7) in the renin-angiotensin system mediates mesenteric vasodilatation in cirrhosis.

BACKGROUND & AIMS: Splanchnic vascular hypocontractility with subsequent increased portal venous inflow leads to portal hypertension. Although the renin-angiotensin system contributes to fibrogenesis and increased hepatic resistance in patients with cirrhosis, little is known about its effects in the splanchnic vasculature, particularly those of the alternate system in which angiotensin (Ang) II is cleaved by the Ang-converting enzyme-2 (ACE2) to Ang-(1-7), which activates the G-protein-coupled Mas receptor (MasR). We investigated whether this system contributes to splanchnic vasodilatation and portal hypertension in cirrhosis. METHODS: We measured levels of renin-angiotensin system messenger RNA and proteins in splanchnic vessels from patients and rats with cirrhosis. Production of Ang-(1-7) and splanchnic vascular reactivity to Ang-(1-7) was measured in perfused mesenteric vascular beds from rats after bile-duct ligation. Ang-(1-7) and MasR were blocked in rats with cirrhosis to examine splanchnic vascular hemodynamics and portal pressure response. RESULTS: Levels of ACE2 and MasR were increased in splanchnic vessels from cirrhotic patients and rats compared with healthy controls. We also observed an ACE2-dependent increase in Ang-(1-7) production. Ang-(1-7) mediated splanchnic vascular hypocontractility in ex vivo splanchnic vessels from rats with cirrhosis (but not control rats) via MasR stimulation. Identical effects were observed in the splanchnic circulation in vivo. MasR blockade reduced portal pressure, indicating that activation of this receptor in splanchnic vasculature promotes portal inflow to contribute to development of portal hypertension. In addition, the splanchnic effects of MasR required nitric oxide. Interestingly, Ang-(1-7) also decreased hepatic resistance. CONCLUSIONS: In the splanchnic vessels of patients and rats with cirrhosis, increased levels of ACE2 appear to increase production of Ang-(1-7), which leads to activation of MasR and splanchnic vasodilatation in rats. This mechanism could cause vascular hypocontractility in patients with cirrhosis, and might be a therapeutic target for portal hypertension.

Atorvastatin inhibits proliferation and apoptosis, but induces senescence in hepatic myofibroblasts and thereby attenuates hepatic fibrosis in rats.

Hepatic myofibroblasts (MFB) show increased proliferation, migration and collagen production, which are crucial for hepatic fibrogenesis. Atorvastatin treatment inhibits proliferation, apoptosis and cytokine production of MFB in bile duct-ligated (BDL) rats in vivo. Here, we have further investigated the underlying mechanisms. Primary rat hepatic stellate cells (HSC) were isolated and culture-activated to hepatic MFB. Following 3 days of incubation with atorvastatin (10(-4), 10(-5) and 10(-6) M), transcription levels of profibrotic cytokines (transforming growth factor-ß1, connective tissue growth factor and TIMP1) and procollagen Ia were analyzed by real time PCR. Proliferation was investigated by 5'-bromo-2'-deoxyuridine assays. α-Smooth muscle actin protein expression was examined by western blotting. Fluorescence-activated cell sorting analysis of Annexin V and propidium iodide were used to measure apoptosis. Furthermore, p21 western blotting and ß-galactosidase staining were investigated in MFB as senescence markers. Subsequently, hepatic expression of desmin and senescence markers were analyzed in the livers of rats receiving atorvastatin (15 mg/kg*d) for 1 week starting 3 and 5 weeks after BDL. Atorvastatin inhibited the activation of HSC to MFB and decreased cytokine and collagen production in MFB in vitro. In addition, proliferation, cytokine and collagen production of MFB were reduced by atorvastatin. Atorvastatin initiated apoptosis at 10(-4) M and attenuated it at 10(-5) M. Atorvastatin induced p21 protein expression and ß-galactosidase staining of MFB in vitro and in vivo. Atorvastatin elicits similiar effects on MFB as previously seen in vivo: it decreases MFB turnover and fibrogenesis. We suggest that a further mechanism explaining these effects is senescence of cells.

HSC-specific inhibition of Rho-kinase reduces portal pressure in cirrhotic rats without major systemic effects.

BACKGROUND & AIMS: Rho-kinase activation mediates cell contraction and increases intrahepatic resistance and consequently portal pressure in liver cirrhosis. Systemic Rho-kinase inhibition decreases portal pressure in cirrhosis, but also arterial pressure. Thus, liver-specific Rho-kinase inhibition is needed. The delivery of Rho-kinase inhibitor to activated hepatic stellate cells reduces fibrosis. It might also relax these contractile cells and therewith decrease intrahepatic resistance. We tested this hypothesis by performing acute experiments in cirrhotic rats. METHODS: Cirrhosis models were CCl(4)-intoxication and bile duct ligation. Three hours after injection of the Rho-kinase inhibitor (Y26732) coupled with a carrier (mannose-6-phosphate modified human serum albumin), which targets activated hepatic stellate cells, hemodynamics were analyzed by the colored microsphere technique and direct pressure measurements. The delivery site and effect of Rho-kinase inhibitor were investigated by immunohistochemical stainings, as well as Western blot. Experiments with Rho-kinase inhibitor coupled with unmodified human serum albumin served as untargeted control. RESULTS: In both models of cirrhosis, the carrier coupled Rho-kinase inhibitor lowered the portal pressure and decreased the hepatic-portal resistance. Immunohistochemical desmin-staining showed the carrier in hepatic stellate cells. The targeted therapy decreased the expression of the phosphorylated substrate of Rho-kinase (moesin) and abolished myosin light chains phosphorylation in fibrotic septae (collagen-staining). The targeted Rho-kinase inhibitor showed no major extrahepatic effects. By contrast, the untargeted Rho-kinase inhibitor elicited severe systemic hypotension. CONCLUSIONS: Activated hepatic stellate cells are crucially involved in portal hypertension in cirrhosis. Targeting of Rho-kinase in hepatic stellate cells not only decreased fibrosis, as previously shown, but also lowers portal pressure acutely without major systemic effects as demonstrated in this study.

The endocannabinoid N-arachidonoyl dopamine (NADA) selectively induces oxidative stress-mediated cell death in hepatic stellate cells but not in hepatocytes.

The endocannabinoid system is a crucial regulator of hepatic fibrogenesis. We have previously shown that the endocannabinoid anandamide (AEA) is a lipid mediator that blocks proliferation and induces death in hepatic stellate cells (HSCs), the main fibrogenic cell type in the liver, but not in hepatocytes. However, the effects of other endocannabinoids such as N-arachidonoyl dopamine (NADA) have not yet been investigated. The NADA-synthesizing enzyme tyrosine hydroxylase was mainly expressed in sympathetic neurons in portal tracts. Its expression pattern stayed unchanged in normal or fibrotic liver. NADA dose dependently induced cell death in culture-activated primary murine or human HSCs after 2-4 h, starting from 5 µM. Despite caspase 3 cleavage, NADA-mediated cell death showed typical features of necrosis, including ATP depletion. Although the cannabinoid receptors CB1, CB2, or transient receptor potential cation channel subfamily V, member 1 were expressed in HSCs, their pharmacological or genetic blockade failed to inhibit NADA-mediated death, indicating a cannabinoid-receptor-independent mechanism. Interestingly, membrane cholesterol depletion with methyl-ß-cyclodextrin inhibited AEA- but not NADA-induced death. NADA significantly induced reactive oxygen species formation in HSCs. The antioxidant glutathione (GSH) significantly decreased NADA-induced cell death. Similar to AEA, primary hepatocytes were highly resistant against NADA-induced death. Resistance to NADA in hepatocytes was due to high levels of GSH, since GSH depletion significantly increased NADA-induced death. Moreover, high expression of the AEA-degrading enzyme fatty acid amide hydrolase (FAAH) in hepatocytes also conferred resistance towards NADA-induced death, since pharmacological or genetic FAAH inhibition significantly augmented hepatocyte death. Thus the selective induction of cell death in HSCs proposes NADA as a novel antifibrogenic mediator.

The archaeal Lsm protein binds to small RNAs.

Proteins of the Lsm family, including eukaryotic Sm proteins and bacterial Hfq, are key players in RNA metabolism. Little is known about the archaeal homologues of these proteins. Therefore, we characterized the Lsm protein from the haloarchaeon Haloferax volcanii using in vitro and in vivo approaches. H. volcanii encodes a single Lsm protein, which belongs to the Lsm1 subfamily. The lsm gene is co-transcribed and overlaps with the gene for the ribosomal protein L37e. Northern blot analysis shows that the lsm gene is differentially transcribed. The Lsm protein forms homoheptameric complexes and has a copy number of 4000 molecules/cell. In vitro analyses using electrophoretic mobility shift assays and ultrasoft mass spectrometry (laser-induced liquid bead ion desorption) showed a complex formation of the recombinant Lsm protein with oligo(U)-RNA, tRNAs, and an small RNA. Co-immunoprecipitation with a FLAG-tagged Lsm protein produced in vivo confirmed that the protein binds to small RNAs. Furthermore, the co-immunoprecipitation revealed several protein interaction partners, suggesting its involvement in different cellular pathways. The deletion of the lsm gene is viable, resulting in a pleiotropic phenotype, indicating that the haloarchaeal Lsm is involved in many cellular processes, which is in congruence with the number of protein interaction partners.

Hepatic and HSC-specific sorafenib effects in rats with established secondary biliary cirrhosis.

Portal hypertension in cirrhosis depends on increased intrahepatic vascular resistance, which is explained by fibrosis and intrahepatic hyperresponsiveness to vasoconstrictors. Both are caused by activation and proliferation of hepatic stellate cells (HSCs). Portal hypertension of cirrhotic rats can be reduced by the multikinase inhibitor sorafenib, due to a reduction of intrahepatic vascular resistance. Therefore, the hepatic effects of sorafenib require further understanding. Here, we investigated hepatic and HSC-specific sorafenib effects in cirrhotic rats. Animal models of bile duct ligation-induced secondary biliary cirrhosis in rats were studied. The rats were treated with sorafenib (60 mg/kg/day) for 1 week, starting after established cirrhosis. Histological evaluation was carried out using hemalaun and eosin (HE) staining. Apoptosis was studied by PARP cleavage, colorimetric caspase-3 assay, and electrophoretic DNA detection. HSC activation was studied by hepatic Sirius red and immunohistochemical αSMA (α-smooth muscle actin) staining, and by in vitro experiments with culture-activated primary HSCs. Biochemical serum parameters suggested the occurrence of sorafenib-induced liver damage. HE staining revealed histological changes in livers of sham-operated and bile duct-ligated (BDL) rats in response to sorafenib, which were different in both groups. In BDL rats and isolated HSCs, the treatment with sorafenib reduced hepatic αSMA and procollagen-1α mRNA expression. As shown by immunohistochemical staining, perisinusoidal αSMA expression was reduced by sorafenib in BDL rats. This was associated with reduced perisinusoidal deposition of extracellular matrix, as revealed by Sirius red staining. Although no change in PARP cleavage and only a minor increase in hepatic caspase-3 activity were detected in BDL rats in response to sorafenib, livers of sorafenib-treated BDL rats contained small DNA fragments, which were not observed in untreated BDL rats. In conclusion, sorafenib treatment reduces the number of activated HSCs in cirrhotic livers. This leads to the decrease in intrahepatic vascular resistance, but also to liver damage in the dosage we used. Therefore, any translation to portal hypertensive patients who may profit from sorafenib should be done with particular care.

Role of cannabinoid receptors in alcoholic hepatic injury: steatosis and fibrogenesis are increased in CB2 receptor-deficient mice and decreased in CB1 receptor knockouts.

BACKGROUND: Alcohol is a common cause of hepatic liver injury with steatosis and fibrosis. Cannabinoid receptors (CB) modulate steatosis, inflammation and fibrogenesis. To investigate the differences between CB(1) and CB(2) in the hepatic response to chronic alcohol intake, we examined CB knockout mice (CB(1)(-/-), CB(2)(-/-)). METHODS: Eight- to 10-week-old CB(1)(-/-), CB(2)(-/-) and wild-type mice received 16% ethanol for 35 weeks. Animals receiving water served as controls. We analysed triglyceride and hydroxyproline contents in liver homogenates. mRNA levels of CBs, pro-inflammatory cytokines [tumour necrosis factor (TNF)-α, monocyte chemotactic protein (MCP)-1, interleukin (IL)-1ß] and profibrotic factors [α-smooth muscle actin (α-SMA), procollagen-Ia, platelet-derived growth factor ß receptor (PDGFß-R)] were analysed by reverse transcription-polymerase chain reaction (RT-PCR). Histology (hemalaun and eosin, oil-red O, CD3, CD45R, CD45, F4/80, Sirius red) characterized hepatic steatosis, inflammation and fibrosis. Activation of lipogenic pathways, activation and proliferation of hepatic stellate cell (HSC) were assessed by western blot [fatty acid synthase (FAS), sterol regulatory element binding protein 1c (SREBP-1c), α-SMA, proliferating cell nuclear antigen (PCNA), cathepsin D]. RESULTS: Hepatic mRNA levels of the respective CBs were increased in wild-type animals and in CB(1)(-/-) mice after ethanol intake. Ethanol intake in CB(2)(-/-) mice induced much higher steatosis (SREBP-1c mediated) and inflammation (B-cell predominant infiltrates) compared with wild-type animals and CB(1)(-/-) mice. HSC activation and collagen production were increased in all groups after forced ethanol intake, being most pronounced in CB(2)(-/-) mice and least pronounced in CB(1)(-/-) mice. DISCUSSION: The fact that CB(2) receptor knockout mice exhibited the most pronounced liver damage after ethanol challenge indicates a protective role of CB(2) receptor expression in chronic ethanol intake. By contrast, in CB(1) knockouts, the effect of ethanol was attenuated, suggesting aggravation of fibrogenesis and SREBP-1c-mediated steatosis via CB(1) receptor expression after ethanol intake.

Atorvastatin attenuates hepatic fibrosis in rats after bile duct ligation via decreased turnover of hepatic stellate cells.

BACKGROUND & AIMS: Activation of hepatic stellate cells (HSC) and transdifferentiation to myofibroblasts following liver injury is the main culprit for hepatic fibrosis. Myofibroblasts show increased proliferation, migration, contraction, and production of extracellular matrix (ECM). In vitro, HMG-CoA reductase inhibitors (statins) inhibit proliferation and induce apoptosis of myofibroblastic HSC. To investigate the antifibrotic effects of atorvastatin in vivo we used bile duct ligated rats (BDL). METHODS: BDL rats were treated with atorvastatin (15 mg/kg/d) immediately after ligation (prophylactically) or in on-going fibrosis (therapeutically). Fibrosis was assessed by hydroxyproline content and Sirius-red staining. The activation of HSC was investigated by analysis of alphaSMA expression. mRNA levels of cytokines and procollagen were analyzed by RT-PCR, and MMP-2 activity by zymography. Proliferation was assessed by expression of cathepsins (B and D), proliferating cell nuclear antigen (PCNA), and Ki67-staining. Apoptosis was characterized by caspase-3 activity, cleavage of PARP-1, and TUNEL assay. Hepatic inflammation was investigated by serum parameters and liver histology. RESULTS: Prophylactic and early therapy with atorvastatin significantly attenuated fibrosis and HSC activation. Later therapy lacked significant effects on fibrosis but reduced profibrotic cytokine expression and led to a more quiescent state of HSC with less proliferation and apoptosis, while hepatic inflammation did not change. CONCLUSIONS: This study shows that very early atorvastatin treatment inhibits HSC activation and fibrosis in the BDL model in vivo, while late treatment reduces HSC turnover and activity. Our findings underline that long-term studies in humans are warranted.

Role of beta3-adrenoceptors for intrahepatic resistance and portal hypertension in liver cirrhosis.

UNLABELLED: Increased intrahepatic resistance and splanchnic blood flow cause portal hypertension in liver cirrhosis. Nonselective beta-adrenoceptor (beta-AR) antagonists have beneficial effects on hyperdynamic circulation and are in clinical use. In this context, the role of the beta(3)-AR is undefined. Here we investigated their expression and role in portal hypertension in patients and rats with liver cirrhosis. We analyzed cirrhotic human and rat tissues (liver, splanchnic vessels) and primary rat cells. Protein expression of beta(3)-AR was determined by western blot and messenger RNA (mRNA) levels by reverse-transcription polymerase chain reaction (RT-PCR). Activities of Rho-kinase and the nitric oxide (NO) effector protein kinase G (PKG) were assessed by way of substrate phosphorylation (moesin, vasodilator-stimulated phosphoprotein [VASP]). Cyclic 3',5' adenosine monophosphate (cAMP) accumulation was determined by an enzyme-immunoassay kit. The effects of selective beta(3)-AR agonists (CGP12177A, BRL37344) and antagonist (SR59230A) were investigated by collagen matrix contraction of hepatic stellate cells (HSCs), in situ liver perfusions, and in vivo hemodynamic parameters in bile duct ligation and carbon tetrachloride intoxication in cirrhotic rats. In cirrhosis of humans and rats, beta(3)-AR expression is markedly increased in hepatic and in splanchnic tissues. Stimulation of beta(3)-AR leads to relaxation of HSCs by way of cAMP accumulation, and by inhibition of Rho-kinase activity; any role of NO and its effector PKG was not observed. beta(3)-AR agonists decrease intrahepatic resistance and portal pressure in cirrhotic rats. CONCLUSION: There is a marked hepatic and mesenteric up-regulation of beta(3)-ARs in human cirrhosis and in two different animal models of cirrhosis. The beta(3)-AR-agonists should be further evaluated for therapy of portal hypertension.

Small RNAs of the halophilic archaeon Haloferax volcanii.

In recent years, sRNAs (small non-coding RNAs) have been found to be abundant in eukaryotes and bacteria and have been recognized as a novel class of gene expression regulators. In contrast, much less is known about sRNAs in archaea, except for snoRNAs (small nucleolar RNAs) that are involved in the modification of bases in stable RNAs. Therefore bioinformatic and experimental RNomics approaches were undertaken to search for the presence of sRNAs in the model archaeon Haloferax volcanii, resulting in more than 150 putative sRNA genes being identified. Northern blot analyses were used to study (differential) expression of sRNA genes. Several chromosomal deletion mutants of sRNA genes were generated and compared with the wild-type. It turned out that two sRNAs are essential for growth at low salt concentrations and high temperatures respectively, and one is involved in the regulation of carbon metabolism. Taken together, it could be shown that sRNAs are as abundant in H. volcanii as they are in well-studied bacterial species and that they fulfil important biological roles under specific conditions.

TGR(mREN2)27 rats develop non-alcoholic fatty liver disease-associated portal hypertension responsive to modulations of Janus-kinase 2 and Mas receptor.

Prevalence of non-alcoholic fatty liver disease (NAFLD) is increasing. Resulting fibrosis and portal hypertension, as a possible secondary event, may necessitate treatment. Overexpression of mouse renin in the transgenic rat model, TGR(mREN2)27, leads to spontaneous development of NAFLD. Therefore, we used TGR(mREN2)27 rats as a model of NAFLD where we hypothesized increased susceptibility and investigated fibrosis and portal hypertension and associated pathways. 12-week old TGR(mREN2)27 rats received either cholestatic (BDL) or toxic injury (CCl4 inhalation). Portal and systemic hemodynamic assessments were performed using microsphere technique with and without injection of the Janus-Kinase 2 (JAK2) inhibitor AG490 or the non-peptidic Ang(1-7) agonist, AVE0991. The extent of liver fibrosis was assessed in TGR(mREN2)27 and wild-type rats using standard techniques. Protein and mRNA levels of profibrotic, renin-angiotensin system components were assessed in liver and primary hepatic stellate cells (HSC) and hepatocytes. TGR(mREN2)27 rats developed spontaneous, but mild fibrosis and portal hypertension due to the activation of the JAK2/Arhgef1/ROCK pathway. AG490 decreased migration of HSC and portal pressure in isolated liver perfusions and in vivo. Fibrosis or portal hypertension after cholestatic (BDL) or toxic injury (CCl4) was not aggravated in TGR(mREN2)27 rats, probably due to decreased mouse renin expression in hepatocytes. Interestingly, portal hypertension was even blunted in TGR(mREN2)27 rats (with or without additional injury) by AVE0991. TGR(mREN2)27 rats are a suitable model of spontaneous liver fibrosis and portal hypertension but not with increased susceptibility to liver damage. After additional injury, the animals can be used to evaluate novel therapeutic strategies targeting Mas.

Statins activate the canonical hedgehog-signaling and aggravate non-cirrhotic portal hypertension, but inhibit the non-canonical hedgehog signaling and cirrhotic portal hypertension.

Liver cirrhosis but also portal vein obstruction cause portal hypertension (PHT) and angiogenesis. This study investigated the differences of angiogenesis in cirrhotic and non-cirrhotic PHT with special emphasis on the canonical (Shh/Gli) and non-canonical (Shh/RhoA) hedgehog pathway. Cirrhotic (bile duct ligation/BDL; CCl4 intoxication) and non-cirrhotic (partial portal vein ligation/PPVL) rats received either atorvastatin (15 mg/kg; 7d) or control chow before sacrifice. Invasive hemodynamic measurement and Matrigel implantation assessed angiogenesis in vivo. Angiogenesis in vitro was analysed using migration and tube formation assay. In liver and vessel samples from animals and humans, transcript expression was analyzed using RT-PCR and protein expression using Western blot. Atorvastatin decreased portal pressure, shunt flow and angiogenesis in cirrhosis, whereas atorvastatin increased these parameters in PPVL rats. Non-canonical Hh was upregulated in experimental and human liver cirrhosis and was blunted by atorvastatin. Moreover, atorvastatin blocked the non-canonical Hh-pathway RhoA dependently in activated hepatic steallate cells (HSCs). Interestingly, hepatic and extrahepatic Hh-pathway was enhanced in PPVL rats, which resulted in increased angiogenesis. In summary, statins caused contrary effects in cirrhotic and non-cirrhotic portal hypertension. Atorvastatin inhibited the non-canonical Hh-pathway and angiogenesis in cirrhosis. In portal vein obstruction, statins enhanced the canonical Hh-pathway and aggravated PHT and angiogenesis.

Serum markers of the extracellular matrix remodeling reflect antifibrotic therapy in bile-duct ligated rats.

BACKGROUND: Progression of liver fibrosis is characterized by synthesis and degradation of extracellular matrix (ECM). Matrix-metalloproteinases (MMP) cleave collagen fibers at a specific site and thereby generate soluble fragments of ECM (neo-epitopes). The levels of these neo-epitopes might reflect the stage of liver fibrosis and may allow monitoring of anti-fibrotic therapies. Here we analyzed these neo-epitopes as read-out for a liver directed therapy with statins. METHODS: Bile duct ligation (BDL) was performed on wild type rats, which received atorvastatin (15 mg/kg(*)d) for 1 week starting at 1, 2, 3, 4 and 5 weeks after BDL (T1-T5), while controls remained untreated. Hepatic fibrosis was analyzed by immunohistochemistry and hepatic hydroxyproline content. TGFß levels were measured by RT-PCR. Proteolytic activity of MMP-2 was examined by zymography. Levels of degradation MMP driven type I, III, IV and VI collagen degradation (C1M, C3M, C4M, and C6M) and type III and IV collagen formation (PRO-C3 and P4NP7S) markers were assessed by specific ELISAs in serum probes. RESULTS: Serum markers of ECM neo-epitopes reflected significantly the deposition of ECM in the liver and were able to distinguish between early (T1-T3) and severe fibrosis (T4-T5). Statin treatment resulted in reduction of neo-epitope markers, especially when therapy was started in the stage of severe fibrosis (T4-T5). Furthermore, these markers correlated with hepatic expression of profibrotic cytokines TGFß1 and TGFß2. Formation markers of type III and IV collagen (PRO-C3 and P4NP7S) and degradation markers C4M and C6M correlated significantly with hepatic MMP-2 activity in rats with severe fibrosis. CONCLUSION: Determination of ECM remodeling turnover markers in serum allowed a distinction between mild and severe fibrosis. With respect to statin therapy, the markers may serve as read-out for efficacy of anti-fibrotic treatment.