Hepatocyte specific expression of an oncogenic variant of ß-catenin results in lethal metabolic dysfunction in mice.

BACKGROUND: Wnt/ß-catenin signaling plays a crucial role in embryogenesis, tissue homeostasis, metabolism and malignant transformation of different organs including the liver. Continuous ß-catenin signaling due to somatic mutations in exon 3 of the Ctnnb1 gene is associated with different liver diseases including cancer and cholestasis. RESULTS: Expression of a degradation resistant form of ß-catenin in hepatocytes resulted in 100% mortality within 31 days after birth. Ctnnb1CAhep mice were characterized by reduced body weight, significantly enlarged livers with hepatocellular fat accumulation around central veins and increased hepatic triglyceride content. Proteomics analysis using whole liver tissue revealed significant deregulation of proteins involved in fat, glucose and mitochondrial energy metabolism, which was also reflected in morphological anomalies of hepatocellular mitochondria. Key enzymes involved in transport and synthesis of fatty acids and cholesterol were significantly deregulated in livers of Ctnnb1CAhep mice. Furthermore, carbohydrate metabolism was substantially disturbed in mutant mice. CONCLUSION: Continuous ß-catenin signaling in hepatocytes results in premature death due to severe disturbances of liver associated metabolic pathways and mitochondrial dysfunction. METHODS: To investigate the influence of permanent ß-catenin signaling on liver biology we analyzed mice with hepatocyte specific expression of a dominant stable form of ß-catenin (Ctnnb1CAhep ) and their WT littermates by serum biochemistry, histology, electron microscopy, mRNA profiling and proteomic analysis of the liver.

Single-sex infection with female Schistosoma mansoni cercariae mitigates hepatic fibrosis after secondary infection.

BACKGROUND: Infection with Schistosoma spp. affects more than 258 million people worldwide. Current treatment strategies are mainly based on the anthelmintic Praziquantel, which is effective against adult worms but neither prevents re-infection nor cures severe liver damage. The best long-term strategy to control schistosomiasis may be to develop an immunization. Therefore, we designed a two-step Schistosoma mansoni infection model to study the immune-stimulating effect of a primary infection with either male or female cercariae, measured on the basis of TH1/TH2-response, granuloma size and hepatic fibrosis after a secondary bisexual S. mansoni challenge. METHODOLOGY/PRINCIPLE FINDINGS: As a first step, mice were infected with exclusively female, exclusively male, or a mixture of male and female S. mansoni cercariae. 11 weeks later they were secondarily infected with male and female S. mansoni cercariae. At week 19, infection burden, granuloma size, collagen deposition, serum cytokine profiles and the expression of inflammatory genes were analyzed. Mice initially infected with female S. mansoni cercariae displayed smaller hepatic granulomas, livers and spleens, less hepatic fibrosis and higher expression of Ctla4. In contrast, a prior infection with male or male and female S. mansoni did not mitigate disease progression after a bisexual challenge. CONCLUSIONS/SIGNIFICANCE: Our findings provide evidence that an immunization against S. mansoni is achievable by exploiting gender-specific differences between schistosomes.

Boosting the hypoxic response in myeloid cells accelerates resolution of fibrosis and regeneration of the liver in mice.

We have recently shown that targeting Vascular Endothelial Growth Factor (VEGF) specifically in scar-infiltrating myeloid cells prevented remodeling of the sinusoidal vasculature and abrogated the resolution of murine liver fibrosis, thereby unmasking an unanticipated link between angiogenesis and resolution of fibrosis. In a gain of function approach, we wanted to test the impact of VEGF overexpression in myeloid cells on fibrolysis. We observe that genetic inactivation of the von Hippel Lindau protein (VHL), a negative regulator of Hypoxia-inducible factors (HIF) in myeloid cells, leads to increased VEGF expression and most importantly, accelerated matrix degradation and reduced myofibroblast numbers after CCl4 challenge. This is associated with enhanced expression of MMP-2 and -14 as well as lower expression of TIMP-2 in liver endothelial cells. In addition, we report increased expression of MMP-13 in scar-associated macrophages as well as improved liver regeneration upon ablation of VHL in myeloid cells. Finally, therapeutic infusion of macrophages nulli-zygous for VHL or treated with the pharmacologic hydroxylase inhibitor and HIF-inducer Dimethyloxalylglycine (DMOG) accelerates resolution of fibrosis. Hence, boosting the HIF-VEGF signaling axis in macrophages represents a promising therapeutic avenue for the treatment of liver fibrosis.

Hepatocyte specific expression of an oncogenic variant of ß-catenin results in cholestatic liver disease.

BACKGROUND: The Wnt/ß-catenin signaling pathway plays a crucial role in embryonic development, tissue homeostasis, wound healing and malignant transformation in different organs including the liver. The consequences of continuous ß-catenin signaling in hepatocytes remain elusive. RESULTS: Livers of Ctnnb1CA hep mice were characterized by disturbed liver architecture, proliferating cholangiocytes and biliary type of fibrosis. Serum ALT and bile acid levels were significantly increased in Ctnnb1CA hep mice. The primary bile acid synthesis enzyme Cyp7a1 was increased whereas Cyp27 and Cyp8b1 were reduced in Ctnnb1CA hep mice. Expression of compensatory bile acid transporters including Abcb1, Abcb4, Abcc2 and Abcc4 were significantly increased in Ctnnb1CA hep mice while Ntcp was reduced. Accompanying changes of bile acid transporters favoring excretion of bile acids were observed in intestine and kidneys of Ctnnb1CA hep mice. Additionally, disturbed bile acid regulation through the FXR-FGF15-FGFR4 pathway was observed in mice with activated ß-catenin. MATERIALS AND METHODS: Mice with a loxP-flanked exon 3 of the Ctnnb1 gene were crossed to Albumin-Cre mice to obtain mice with hepatocyte-specific expression of a dominant stable form of ß-catenin (Ctnnb1CA hep mice). Ctnnb1CA hep mice were analyzed by histology, serum biochemistry and mRNA profiling. CONCLUSIONS: Expression of a dominant stable form of ß-catenin in hepatocytes results in severe cholestasis and biliary type fibrosis.

Sustained PI3K Activation exacerbates BLM-induced Lung Fibrosis via activation of pro-inflammatory and pro-fibrotic pathways.

Idiopathic pulmonary fibrosis (IPF) is a life-threatening disease with limited treatment options. Additionally, the lack of a complete understanding of underlying immunological mechanisms underscores the importance of discovering novel options for therapeutic intervention. Since the PI3K/PTEN pathway in myeloid cells influences their effector functions, we wanted to elucidate how sustained PI3K activity induced by cell-type specific genetic deficiency of its antagonist PTEN modulates IPF, in a murine model of bleomycin-induced pulmonary fibrosis (BIPF). We found that myeloid PTEN deficient mice (PTEN(MyKO)), after induction of BIPF, exhibit increased TGF-ß1 activation, mRNA expression of pro-collagens and lysyl oxidase as well as augmented collagen deposition compared to wild-type littermates, leading to enhanced morbidity and decreased survival. Analysis of alveolar lavage and lung cell composition revealed that PTEN(MyKO) mice exhibit reduced numbers of macrophages and T-cells in response to bleomycin, indicating an impaired recruitment function. Interestingly, we found dysregulated macrophage polarization as well as elevated expression and release of the pro-fibrotic cytokines IL-6 and TNF-α in PTEN(MyKO) mice during BIPF. This might point to an uncontrolled wound healing response in which the inflammatory as well as tissue repair mechanisms proceed in parallel, thereby preventing resolution and at the same time promoting extensive fibrosis.

Inhibition of intestinal bile acid absorption improves cholestatic liver and bile duct injury in a mouse model of sclerosing cholangitis.

BACKGROUND AND AIMS: Approximately 95% of bile acids (BAs) excreted into bile are reabsorbed in the gut and circulate back to the liver for further biliary secretion. Therefore, pharmacological inhibition of the ileal apical sodium-dependent BA transporter (ASBT/SLC10A2) may protect against BA-mediated cholestatic liver and bile duct injury. METHODS: Eight week old Mdr2(-/-) (Abcb4(-/-)) mice (model of cholestatic liver injury and sclerosing cholangitis) received either a diet supplemented with A4250 (0.01% w/w) - a highly potent and selective ASBT inhibitor - or a chow diet. Liver injury was assessed biochemically and histologically after 4weeks of A4250 treatment. Expression profiles of genes involved in BA homeostasis, inflammation and fibrosis were assessed via RT-PCR from liver and ileum homogenates. Intestinal inflammation was assessed by RNA expression profiling and immunohistochemistry. Bile flow and composition, as well as biliary and fecal BA profiles were analyzed after 1week of ASBT inhibitor feeding. RESULTS: A4250 improved sclerosing cholangitis in Mdr2(-/-) mice and significantly reduced serum alanine aminotransferase, alkaline phosphatase and BAs levels, hepatic expression of pro-inflammatory (Tnf-α, Vcam1, Mcp-1) and pro-fibrogenic (Col1a1, Col1a2) genes and bile duct proliferation (mRNA and immunohistochemistry for cytokeratin 19 (CK19)). Furthermore, A4250 significantly reduced bile flow and biliary BA output, which correlated with reduced Bsep transcription, while Ntcp and Cyp7a1 were induced. Importantly A4250 significantly reduced biliary BA secretion but preserved HCO3(-) and biliary phospholipid secretion resulting in an increased HCO3(-)/BA and PL/BA ratio. In addition, A4250 profoundly increased fecal BA excretion without causing diarrhea and altered BA pool composition, resulting in diminished concentrations of primary BAs tauro-ß-muricholic acid and taurocholic acid. CONCLUSIONS: Pharmacological ASBT inhibition attenuates cholestatic liver and bile duct injury by reducing biliary BA concentrations in mice.

Epiplakin attenuates experimental mouse liver injury by chaperoning keratin reorganization.

BACKGROUND & AIMS: Epiplakin is a member of the plakin protein family and exclusively expressed in epithelial tissues where it binds to keratins. Epiplakin-deficient (Eppk1(-/-)) mice displayed no obvious spontaneous phenotype, but their keratinocytes showed a faster keratin network breakdown in response to stress. The role of epiplakin in the stressed liver remained to be elucidated. METHODS: Wild-type (WT) and Eppk1(-/-) mice were subjected to common bile duct ligation (CBDL) or fed with a 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC)-containing diet. The importance of epiplakin during keratin reorganization was assessed in primary hepatocytes. RESULTS: Our experiments revealed that epiplakin is expressed in hepatocytes and cholangiocytes, and binds to keratin 8 (K8) and K18 via multiple domains. In several liver stress models epiplakin and K8 genes displayed identical expression patterns and transgenic K8 overexpression resulted in elevated hepatic epiplakin levels. After CBDL and DDC treatment, Eppk1(-/-) mice developed a more pronounced liver injury and their livers contained larger amounts of hepatocellular keratin granules, indicating impaired disease-induced keratin network reorganization. In line with these findings, primary Eppk1(-/-) hepatocytes showed increased formation of keratin aggregates after treatment with the phosphatase inhibitor okadaic acid, a phenotype which was rescued by the chemical chaperone trimethylamine N-oxide (TMAO). Finally, transfection experiments revealed that Eppk1(-/-) primary hepatocytes were less able to tolerate forced K8 overexpression and that TMAO treatment rescued this phenotype. CONCLUSION: Our data indicate that epiplakin plays a protective role during experimental liver injuries by chaperoning disease-induced keratin reorganization.

24-nor-ursodeoxycholic acid ameliorates inflammatory response and liver fibrosis in a murine model of hepatic schistosomiasis.

BACKGROUND & AIMS: Intrahepatic granuloma formation and fibrosis characterize the pathological features of Schistosoma mansoni infection. Based on previously observed substantial anti-fibrotic effects of 24-nor-ursodeoxycholic acid (norUDCA) in Abcb4/Mdr2(-/-) mice with cholestatic liver injury and biliary fibrosis, we hypothesized that norUDCA improves inflammation-driven liver fibrosis in S. mansoni infection. METHODS: Adult NMRI mice were infected with 50 S. mansoni cercariae and after 12 weeks received either norUDCA- or ursodeoxycholic acid (UDCA)-enriched diet (0.5% wt/wt) for 4 weeks. Bile acid effects on liver histology, serum biochemistry, key regulatory cytokines, hepatic hydroxyproline content as well as granuloma formation were compared to naive mice and infected controls. In addition, effects of norUDCA on primary T-cell activation/proliferation and maturation of the antigen-presenting-cells (dendritic cells, macrophages) were determined in vitro. RESULTS: UDCA as well as norUDCA attenuated the inflammatory response in livers of S. mansoni infected mice, but exclusively norUDCA changed cellular composition and reduced size of hepatic granulomas as well as TH2-mediated hepatic fibrosis in vivo. Moreover, norUDCA affected surface expression level of major histocompatibility complex (MHC) class II of macrophages and dendritic cells as well as activation/proliferation of T-lymphocytes in vitro, whereas UDCA had no effect. CONCLUSIONS: This study demonstrates pronounced anti-inflammatory and anti-fibrotic effects of norUDCA compared to UDCA in S. mansoni induced liver injury, and indicates that norUDCA directly represses antigen presentation of antigen presenting cells and subsequent T-cell activation in vitro. Therefore, norUDCA represents a promising drug for the treatment of this important cause of liver fibrosis.

Resolution of liver fibrosis requires myeloid cell-driven sinusoidal angiogenesis.

UNLABELLED: Angiogenesis is a key feature of liver fibrosis. Although sinusoidal remodeling is believed to contribute to fibrogenesis, the impact of sinusoidal angiogenesis on the resolution of liver fibrosis remains undefined. Myeloid cells, particularly macrophages, constantly infiltrate the fibrotic liver and can profoundly contribute to remodeling of liver sinusoids. We observe that the development of fibrosis is associated with decreased hepatic vascular endothelial growth factor (VEGF) expression as well as sinusoidal rarefication of the fibrotic scar. In contrast, the resolution of fibrosis is characterized by a rise in hepatic VEGF levels and revascularization of the fibrotic tissue. Genetic ablation of VEGF in myeloid cells or pharmacological inhibition of VEGF receptor 2 signaling prevents this angiogenic response and the resolution of liver fibrosis. We observe increased expression of matrix metalloproteases as well as decreased expression of tissue inhibitor of metalloproteases confined to sinusoidal endothelial cells in response to myeloid cell VEGF. Remarkably, reintroduction of myeloid cell-derived VEGF upon recovery restores collagenolytic acitivity and the resolution of fibrosis. CONCLUSION: We identify myeloid cell-derived VEGF as a critical regulator of extracellular matrix degradation by liver endothelial cells, thereby unmasking an unanticipated link between angiogenesis and the resolution of fibrosis.

Evaluation of genome-wide loci of iron metabolism in hereditary hemochromatosis identifies PCSK7 as a host risk factor of liver cirrhosis.

Genome-wide association studies (GWAS) have revealed genetic determinants of iron metabolism, but correlation of these with clinical phenotypes is pending. Homozygosity for HFE C282Y is the predominant genetic risk factor for hereditary hemochromatosis (HH) and may cause liver cirrhosis. However, this genotype has a low penetrance. Thus, detection of yet unknown genetic markers that identify patients at risk of developing severe liver disease is necessary for better prevention. Genetic loci associated with iron metabolism (TF, TMPRSS6, PCSK7, TFR2 and Chr2p14) in recent GWAS and liver fibrosis (PNPLA3) in recent meta-analysis were analyzed for association with either liver cirrhosis or advanced fibrosis in 148 German HFE C282Y homozygotes. Replication of associations was sought in additional 499 Austrian/Swiss and 112 HFE C282Y homozygotes from Sweden. Only variant rs236918 in the PCSK7 gene (proprotein convertase subtilisin/kexin type 7) was associated with cirrhosis or advanced fibrosis (P = 1.02 × 10(-5)) in the German cohort with genotypic odds ratios of 3.56 (95% CI 1.29-9.77) for CG heterozygotes and 5.38 (95% CI 2.39-12.10) for C allele carriers. Association between rs236918 and cirrhosis was confirmed in Austrian/Swiss HFE C282Y homozygotes (P = 0.014; ORallelic = 1.82 (95% CI 1.12-2.95) but not in Swedish patients. Post hoc combined analyses of German/Swiss/Austrian patients with available liver histology (N = 244, P = 0.00014, ORallelic = 2.84) and of males only (N = 431, P = 2.17 × 10(-5), ORallelic = 2.54) were consistent with the premier finding. Association between rs236918 and cirrhosis was not confirmed in alcoholic cirrhotics, suggesting specificity of this genetic risk factor for HH. PCSK7 variant rs236918 is a risk factor for cirrhosis in HH patients homozygous for the HFE C282Y mutation.

Adenoma-linked barrier defects and microbial products drive IL-23/IL-17-mediated tumour growth.

Approximately 2% of colorectal cancer is linked to pre-existing inflammation known as colitis-associated cancer, but most develops in patients without underlying inflammatory bowel disease. Colorectal cancer often follows a genetic pathway whereby loss of the adenomatous polyposis coli (APC) tumour suppressor and activation of ß-catenin are followed by mutations in K-Ras, PIK3CA and TP53, as the tumour emerges and progresses. Curiously, however, 'inflammatory signature' genes characteristic of colitis-associated cancer are also upregulated in colorectal cancer. Further, like most solid tumours, colorectal cancer exhibits immune/inflammatory infiltrates, referred to as 'tumour-elicited inflammation'. Although infiltrating CD4(+) T(H)1 cells and CD8(+) cytotoxic T cells constitute a positive prognostic sign in colorectal cancer, myeloid cells and T-helper interleukin (IL)-17-producing (T(H)17) cells promote tumorigenesis, and a 'T(H)17 expression signature' in stage I/II colorectal cancer is associated with a drastic decrease in disease-free survival. Despite its pathogenic importance, the mechanisms responsible for the appearance of tumour-elicited inflammation are poorly understood. Many epithelial cancers develop proximally to microbial communities, which are physically separated from immune cells by an epithelial barrier. We investigated mechanisms responsible for tumour-elicited inflammation in a mouse model of colorectal tumorigenesis, which, like human colorectal cancer, exhibits upregulation of IL-23 and IL-17. Here we show that IL-23 signalling promotes tumour growth and progression, and development of a tumoural IL-17 response. IL-23 is mainly produced by tumour-associated myeloid cells that are likely to be activated by microbial products, which penetrate the tumours but not adjacent tissue. Both early and late colorectal neoplasms exhibit defective expression of several barrier proteins. We propose that barrier deterioration induced by colorectal-cancer-initiating genetic lesions results in adenoma invasion by microbial products that trigger tumour-elicited inflammation, which in turn drives tumour growth.

Interleukin-17 signaling in inflammatory, Kupffer cells, and hepatic stellate cells exacerbates liver fibrosis in mice.

BACKGROUND & AIMS: Interleukin (IL)-17 signaling has been implicated in lung and skin fibrosis. We examined the role of IL-17 signaling in the pathogenesis of liver fibrosis in mice. METHODS: Using cholestatic and hepatotoxic models of liver injury, we compared the development of liver fibrosis in wild-type mice with that of IL-17RA(-/-) mice and of bone marrow chimeric mice devoid of IL-17 signaling in immune and Kupffer cells (IL-17RA(-/-) to wild-type and IL-17A(-/-) to wild-type mice) or liver resident cells (wild-type to IL-17RA(-/-) mice). RESULTS: In response to liver injury, levels of Il-17A and its receptor increased. IL-17A increased appeared to promote fibrosis by activating inflammatory and liver resident cells. IL-17 signaling facilitated production of IL-6, IL-1, and tumor necrosis factor-α by inflammatory cells and increased the expression of transforming growth factor-1, a fibrogenic cytokine. IL-17 directly induced production of collagen type I in hepatic stellate cells by activating the signal transducer and activator of transcription 3 (Stat3) signaling pathway. Mice devoid of Stat3 signaling in hepatic stellate cells (GFAPStat3(-/-) mice) were less susceptible to fibrosis. Furthermore, deletion of IL-23 from immune cells attenuated liver fibrosis, whereas deletion of IL-22 exacerbated fibrosis. Administration of IL-22 and IL-17E (IL-25, a negative regulator of IL-23) protected mice from bile duct ligation-induced liver fibrosis. CONCLUSIONS: IL-17 induces liver fibrosis through multiple mechanisms in mice. Reagents that block these pathways might be developed as therapeutics for patients with cirrhosis.

Xenobiotic-induced liver injury and fibrosis.

INTRODUCTION: Many different drugs and xenobiotics (chemical compounds foreign to an organism) can injure the bile duct epithelium and cause inflammatory bile duct diseases (cholangiopathies) ranging from transient cholestasis to vanishing bile duct syndrome, sclerosing cholangitis with development of biliary fibrosis and cirrhosis. Animal models of xenobiotic-induced liver injury have provided major mechanistic insights into the molecular mechanisms of xenobiotic-induced cholangiopathies and biliary fibrosis including primary biliary cirrhosis and primary sclerosing cholangitis. AREAS COVERED: In this review, the authors discuss the basic principles of xenobiotic-induced liver and bile duct injury and biliary fibrosis with emphasis on animal models. A PubMed search was performed using the search terms "xenobiotic," "liver injury," "cholestasis," and "biliary fibrosis." Reference lists of retrieved articles were also searched for relevant literature. EXPERT OPINION: Xenobiotic-induced cholangiopathies are underestimated and frequently overlooked medical conditions due to their often transient nature. However, biliary disease may progress to vanishing bile duct syndrome, biliary fibrosis, and cirrhosis. Moreover, xenobiotics may prime the liver for subsequent liver disease by other agents and may also contribute to the development of hepatobiliary cancer though interaction with resident stem cells.

Animal models of biliary tract injury.

PURPOSE OF REVIEW: Cholestatic liver diseases with bile duct injury and biliary fibrosis account for a significant percentage of patients with end-stage liver disease and undergoing liver transplantation. A number of different animal models have been established and have added substantially to our understanding of the molecular mechanisms underlying this group of chronic liver diseases. In the present review, we discuss recent findings and new insight derived from different animal models of biliary tract injury and fibrosis. RECENT FINDINGS: Cholangiocytes do not undergo epithelial to mesenchymal transition and do not contribute to the pool of biliary fibroblasts involved in extracellular matrix deposition. Rather cholangiocytes can acquire a reactive phenotype activating fibrogenesis through secretion of proinflammatory and profibrogenic mediators. Bile acid homeostasis is controlled by a gut-liver axis playing a crucial role in the adaptive response to bile duct injury and cholestasis. The nuclear factor-kappa B and hedgehog signaling pathways play a critical role in cholestatic liver injury and the emergence of liver cancer. Nuclear receptors are key mediators of adaptive response mechanisms in cholestasis and potential therapeutical targets. SUMMARY: Recent progress and mechanistic insights from mouse models have added to our understanding of the molecular mechanisms underlying cholestatic liver and biliary tract injury and pointed to new therapeutic options.

JAK-STAT signaling in hepatic fibrosis.

Chronic liver injury, liver fibrosis and formation of hepatocellular carcinoma are intimately linked and represent a major medical challenge since treatment options are limited. Therefore, it is important to identify cellular and molecular pathways that promote liver damage or provide hepatoprotection for development of therapeutic approaches. Recently, the transcription factors STAT3 and STAT5 have been implicated in liver fibrosis induced by cholestatic liver damage. In this review, we summarize our current knowledge about STAT proteins in liver fibrosis and focus on common activities that underlie the hepatoprotective mechanisms regulated by IL-6/gp130/STAT3 and GH/STAT5/IGF-1 signaling pathways.

Fibrosis in autoimmune and cholestatic liver disease.

Autoimmune and cholestatic liver disease account for a significant part of end-stage liver disease and are leading indications for liver transplantation. Especially cholestatic liver diseases (primary biliary cirrhosis and primary sclerosing cholangitis) appear to be different from other chronic liver diseases with regards to pathogenesis. Portal fibroblasts located in the connective tissue surrounding bile ducts appear to be different from hepatic stellate cells with regards to expression of marker proteins and response the profibrogenic and mitogenic stimuli. In addition there is increasing evidence for a cross talk between activated cholangiocytes and portal myofibroblasts. Several animal models have improved our understanding of the mechanisms underlying these chronic liver diseases. In the present review, we discuss the current concepts and ideas with regards to myofibroblastic cell populations, mechanisms of fibrosis, summarize characteristic histological findings and currently employed animal models of autoimmune and cholestatic liver disease.

The nicotinamide adenine dinucleotide phosphate oxidase (NOX) homologues NOX1 and NOX2/gp91(phox) mediate hepatic fibrosis in mice.

UNLABELLED: Nicotinamide adenine dinucleotide phosphate oxidase (NOX) is a multicomponent enzyme that mediates electron transfer from nicotinamide adenine dinucleotide phosphate to molecular oxygen, which leads to the production of superoxide. NOX2/gp91(phox) is a catalytic subunit of NOX expressed in phagocytic cells. Several homologues of NOX2, including NOX1, have been identified in nonphagocytic cells. We investigated the contributory role of NOX1 and NOX2 in hepatic fibrosis. Hepatic fibrosis was induced in wild-type (WT) mice, NOX1 knockout (NOX1KO) mice, and NOX2 knockout (NOX2KO) mice by way of either carbon tetrachloride (CCl(4) ) injection or bile duct ligation (BDL). The functional contribution of NOX1 and NOX2 in endogenous liver cells, including hepatic stellate cells (HSCs), and bone marrow (BM)-derived cells, including Kupffer cells (KCs), to hepatic reactive oxygen species (ROS) generation and hepatic fibrosis was assessed in vitro and in vivo using NOX1 or NOX2 BM chimeric mice. Hepatic NOX1 and NOX2 messenger RNA expression was increased in the two experimental mouse models of hepatic fibrosis. Whereas NOX1 was expressed in HSCs but not in KCs, NOX2 was expressed in both HSCs and KCs. Hepatic fibrosis and ROS generation were attenuated in both NOX1KO and NOX2KO mice after CCl(4) or BDL. Liver fibrosis in chimeric mice indicated that NOX1 mediates the profibrogenic effects in endogenous liver cells, whereas NOX2 mediates the profibrogenic effects in both endogenous liver cells and BM-derived cells. Multiple NOX1 and NOX2 components were up-regulated in activated HSCs. Both NOX1- and NOX2-deficient HSCs had decreased ROS generation and failed to up-regulate collagen α1(I) and transforming growth factor ß in response to angiotensin II. CONCLUSION: Both NOX1 and NOX2 have an important role in hepatic fibrosis in endogenous liver cells, including HSCs, whereas NOX2 has a lesser role in BM-derived cells.

Fibroblast-specific protein 1 identifies an inflammatory subpopulation of macrophages in the liver.

Cirrhosis is the end result of chronic liver disease. Hepatic stellate cells (HSC) are believed to be the major source of collagen-producing myofibroblasts in cirrhotic livers. Portal fibroblasts, bone marrow-derived cells, and epithelial to mesenchymal transition (EMT) might also contribute to the myofibroblast population in damaged livers. Fibroblast-specific protein 1 (FSP1, also called S100A4) is considered a marker of fibroblasts in different organs undergoing tissue remodeling and is used to identify fibroblasts derived from EMT in several organs including the liver. The aim of this study was to characterize FSP1-positive cells in human and experimental liver disease. FSP1-positive cells were increased in human and mouse experimental liver injury including liver cancer. However, FSP1 was not expressed by HSC or type I collagen-producing fibroblasts. Likewise, FSP1-positive cells did not express classical myofibroblast markers, including αSMA and desmin, and were not myofibroblast precursors in injured livers as evaluated by genetic lineage tracing experiments. Surprisingly, FSP1-positive cells expressed F4/80 and other markers of the myeloid-monocytic lineage as evaluated by double immunofluorescence staining, cell fate tracking, flow cytometry, and transcriptional profiling. Similar results were obtained for bone marrow-derived and peritoneal macrophages. FSP1-positive cells were characterized by increased expression of COX2, osteopontin, inflammatory cytokines, and chemokines but reduced expression of MMP3 and TIMP3 compared with Kupffer cells/macrophages. These findings suggest that FSP1 is a marker of a specific subset of inflammatory macrophages in liver injury, fibrosis, and cancer.

Role and cellular source of nicotinamide adenine dinucleotide phosphate oxidase in hepatic fibrosis.

UNLABELLED: Reactive oxygen species (ROS) generated by nicotinamide adenine dinucleotide phosphate oxidase (NOX) is required for liver fibrosis. This study investigates the role of NOX in ROS production and the differential contribution of NOX from bone marrow (BM)-derived and non-BM-derived liver cells. Hepatic fibrosis was induced by bile duct ligation (BDL) for 21 days or by methionine-choline-deficient (MCD) diet for 10 weeks in wild-type (WT) mice and mice deficient in p47phox (p47phox knockout [KO]), a component of NOX. The p47phox KO chimeric mice were generated by the combination of liposomal clodronate injection, irradiation, and BM transplantation of p47phox KO BM into WT recipients and vice versa. Upon BDL, chimeric mice with p47phox KO BM-derived cells, including Kupffer cells, and WT endogenous liver cells showed a ∼25% reduction of fibrosis, whereas chimeric mice with WT BM-derived cells and p47phox KO endogenous liver cells, including hepatic stellate cells, showed a ∼60% reduction of fibrosis. In addition, p47phox KO compared to WT mice treated with an MCD diet showed no significant changes in steatosis and hepatocellular injury, but a ∼50% reduction in fibrosis. Cultured WT and p47phox KO hepatocytes treated with free fatty acids had a similar increase in lipid accumulation. Free fatty acids promoted a 1.5-fold increase in ROS production both in p47phox KO and in WT hepatocytes. CONCLUSION: NOX in both BM-derived and non-BM-derived cells contributes to liver fibrosis. NOX does not play a role in experimental steatosis and the generation of ROS in hepatocytes, but exerts a key role in fibrosis.