Dietary and genetic obesity promote liver inflammation and tumorigenesis by enhancing IL-6 and TNF expression.

Epidemiological studies indicate that overweight and obesity are associated with increased cancer risk. To study how obesity augments cancer risk and development, we focused on hepatocellular carcinoma (HCC), the common form of liver cancer whose occurrence and progression are the most strongly affected by obesity among all cancers. We now demonstrate that either dietary or genetic obesity is a potent bona fide liver tumor promoter in mice. Obesity-promoted HCC development was dependent on enhanced production of the tumor-promoting cytokines IL-6 and TNF, which cause hepatic inflammation and activation of the oncogenic transcription factor STAT3. The chronic inflammatory response caused by obesity and enhanced production of IL-6 and TNF may also increase the risk of other cancers.

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.

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.

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.

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.

TLR4 enhances TGF-beta signaling and hepatic fibrosis.

Hepatic injury is associated with a defective intestinal barrier and increased hepatic exposure to bacterial products. Here we report that the intestinal bacterial microflora and a functional Toll-like receptor 4 (TLR4), but not TLR2, are required for hepatic fibrogenesis. Using Tlr4-chimeric mice and in vivo lipopolysaccharide (LPS) challenge, we demonstrate that quiescent hepatic stellate cells (HSCs), the main precursors for myofibroblasts in the liver, are the predominant target through which TLR4 ligands promote fibrogenesis. In quiescent HSCs, TLR4 activation not only upregulates chemokine secretion and induces chemotaxis of Kupffer cells, but also downregulates the transforming growth factor (TGF)-beta pseudoreceptor Bambi to sensitize HSCs to TGF-beta-induced signals and allow for unrestricted activation by Kupffer cells. LPS-induced Bambi downregulation and sensitization to TGF-beta is mediated by a MyD88-NF-kappaB-dependent pathway. Accordingly, Myd88-deficient mice have decreased hepatic fibrosis. Thus, modulation of TGF-beta signaling by a TLR4-MyD88-NF-kappaB axis provides a novel link between proinflammatory and profibrogenic signals.

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.

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.

Disruption of TAK1 in hepatocytes causes hepatic injury, inflammation, fibrosis, and carcinogenesis.

TGF-beta-activated kinase 1 (TAK1) is a MAP3K family member that activates NF-kappaB and JNK via Toll-like receptors and the receptors for IL-1, TNF-alpha, and TGF-beta. Because the TAK1 downstream molecules NF-kappaB and JNK have opposite effects on cell death and carcinogenesis, the role of TAK1 in the liver is unpredictable. To address this issue, we generated hepatocyte-specific Tak1-deficient (Tak1DeltaHEP) mice. The Tak1DeltaHEP mice displayed spontaneous hepatocyte death, compensatory proliferation, inflammatory cell infiltration, and perisinusoidal fibrosis at age 1 month. Older Tak1DeltaHEP mice developed multiple cancer nodules characterized by increased expression of fetal liver genes including alpha-fetoprotein. Cultures of primary hepatocytes deficient in Tak1 exhibited spontaneous cell death that was further increased in response to TNF-alpha. TNF-alpha increased caspase-3 activity but activated neither NF-kappaB nor JNK in Tak1-deficient hepatocytes. Genetic abrogation of TNF receptor type I (TNFRI) in Tak1DeltaHEP mice reduced liver damage, inflammation, and fibrosis compared with unmodified Tak1DeltaHEP mice. In conclusion, hepatocyte-specific deletion of TAK1 in mice resulted in spontaneous hepatocyte death, inflammation, fibrosis, and carcinogenesis that was partially mediated by TNFR signaling, indicating that TAK1 is an essential component for cellular homeostasis in the liver.

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.

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.

Genetic labeling does not detect epithelial-to-mesenchymal transition of cholangiocytes in liver fibrosis in mice.

BACKGROUND & AIMS: Chronic injury changes the fate of certain cellular populations, inducing epithelial cells to generate fibroblasts by epithelial-to-mesenchymal transition (EMT) and mesenchymal cells to generate epithelial cells by mesenchymal-to-epithelial transition (MET). Although contribution of EMT/MET to embryogenesis, renal fibrosis, and lung fibrosis is well documented, role of EMT/MET in liver fibrosis is unclear. We determined whether cytokeratin-19 positive (K19(+)) cholangiocytes give rise to myofibroblasts (EMT) and/or whether glial fibrillary acidic protein positive (GFAP(+)) hepatic stellate cells (HSCs) can express epithelial markers (MET) in response to experimental liver injury. METHODS: EMT was studied with Cre-loxP system to map cell fate of K19(+) cholangiocytes in K19(YFP) or fibroblast-specific protein-1 (FSP-1)(YFP) mice, generated by crossing tamoxifen-inducible K19(CreERT) mice or FSP-1(Cre) mice with Rosa26(f/f-YFP) mice. MET of GFAP(+) HSCs was studied in GFAP(GFP) mice. Mice were subjected to bile duct ligation or CCl(4)-liver injury, and livers were analyzed for expression of mesodermal and epithelial markers. RESULTS: On Cre-loxP recombination, >40% of genetically labeled K19(+) cholangiocytes expressed yellow fluorescent protein (YFP). All mice developed liver fibrosis. However, specific immunostaining of K19(YFP) cholangiocytes showed no expression of EMT markers alpha-smooth muscle actin, desmin, or FSP-1. Moreover, cells genetically labeled by FSP-1(YFP) expression did not coexpress cholangiocyte markers K19 or E-cadherin. Genetically labeled GFAP(GFP) HSCs did not express epithelial or liver progenitor markers in response to liver injury. CONCLUSION: EMT of cholangiocytes identified by genetic labeling does not contribute to hepatic fibrosis in mice. Likewise, GFAP(Cre)-labeled HSCs showed no coexpression of epithelial markers, providing no evidence for MET in HSCs in response to fibrogenic liver injury.

Modulation of hepatic fibrosis by c-Jun-N-terminal kinase inhibition.

BACKGROUND & AIMS: c-Jun N-terminal kinase (JNK) is activated by multiple profibrogenic mediators; JNK activation occurs during toxic, metabolic, and autoimmune liver injury. However, its role in hepatic fibrogenesis is unknown. METHODS: JNK phosphorylation was detected by immunoblot analysis and confocal immunofluorescent microscopy in fibrotic livers from mice after bile duct ligation (BDL) or CCl(4) administration and in liver samples from patients with chronic hepatitis C and non-alcoholic steatohepatitis. Fibrogenesis was investigated in mice given the JNK inhibitor SP600125 and in JNK1- and JNK2-deficient mice following BDL or CCl(4) administration. Hepatic stellate cell (HSC) activation was determined in primary mouse HSCs incubated with pan-JNK inhibitors SP600125 and VIII. RESULTS: JNK phosphorylation was strongly increased in livers of mice following BDL or CCl(4) administration as well as in human fibrotic livers, occurring predominantly in myofibroblasts. In vitro, pan-JNK inhibitors prevented transforming growth factor (TGF) beta-, platelet-derived growth factor-, and angiotensin II-induced murine HSC activation and decreased platelet-derived growth factor and TGF-beta signaling in human HSCs. In vivo, pan-JNK inhibition did not affect liver injury but significantly reduced fibrosis after BDL or CCl(4). JNK1-deficient mice had decreased fibrosis after BDL or CCl(4), whereas JNK2-deficient mice displayed increased fibrosis after BDL but fibrosis was not changed after CCl(4). Moreover, patients with chronic hepatitis C who displayed decreased fibrosis in response to the angiotensin receptor type 1 blocker losartan showed decreased JNK phosphorylation. CONCLUSIONS: JNK is involved in HSC activation and fibrogenesis and represents a potential target for antifibrotic treatment approaches.

Hepatocytes do not undergo epithelial-mesenchymal transition in liver fibrosis in mice.

UNLABELLED: The origin of fibrogenic cells in liver fibrosis remains controversial. We assessed the emerging concept that hepatocytes contribute to production of extracellular matrix (ECM) in liver fibrosis through epithelial-mesenchymal transition (EMT). We bred triple transgenic mice expressing ROSA26 stop beta-galactosidase (beta-gal), albumin Cre, and collagen alpha1(I) green fluorescent protein (GFP), in which hepatocyte-derived cells are permanently labeled by beta-gal and type I collagen-expressing cells are labeled by GFP. We induced liver fibrosis by repetitive carbon tetrachloride (CCl(4)) injections. Liver sections and isolated cells were evaluated for GFP and beta-gal as well as expression of alpha-smooth muscle actin (alpha-SMA) and fibroblast-specific protein 1 (FSP-1). Upon stimulation with transforming growth factor beta-1, cultured hepatocytes isolated from untreated liver expressed both GFP and beta-gal with a fibroblast-like morphological change but lacked expression of other mesenchymal markers. Cells from CCl(4)-treated livers never showed double-positivity for GFP and beta-gal. All beta-gal-positive cells exhibited abundant cytoplasm, a typical morphology of hepatocytes, and expressed none of the mesenchymal markers including alpha-SMA, FSP-1, desmin, and vimentin. In liver sections of CCl(4)-treated mice, GFP-positive areas were coincident with fibrotic septa and never overlapped X-gal-positive areas. CONCLUSION: Type I collagen-producing cells do not originate from hepatocytes. Hepatocytes in vivo neither acquire mesenchymal marker expression nor exhibit a morphological change clearly distinguishable from normal hepatocytes. Our results strongly challenge the concept that hepatocytes in vivo acquire a mesenchymal phenotype through EMT to produce the ECM in liver fibrosis.

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.

c-Jun N-terminal kinase-1 from hematopoietic cells mediates progression from hepatic steatosis to steatohepatitis and fibrosis in mice.

BACKGROUND & AIMS: c-Jun N-terminal kinase (JNK) plays a pivotal role in the development of the metabolic syndrome including nonalcoholic fatty liver disease. However, the mechanism underlying the contribution of JNK to the progression from simple steatosis to steatohepatitis and liver fibrosis is unresolved. METHODS: Hepatic steatosis, inflammation, and fibrosis were examined in wild-type, jnk1(-/-), or jnk2(-/-) mice fed a choline-deficient L-amino acid-defined (CDAA) diet for 20 weeks. The functional contribution of JNK isoforms in Kupffer cells was assessed in vitro and in vivo using chimeric mice in which the hematopoietic compartment including Kupffer cells was replaced by wild-type, jnk1(-/-), or jnk2(-/-) cells. RESULTS: CDAA diet induced significantly less hepatic inflammation and less liver fibrosis despite a similar level of hepatic steatosis in jnk1(-/-) mice as compared with wild-type or jnk2(-/-) mice. CDAA diet-induced hepatic inflammation was chronic and mediated by Kupffer cells. Pharmacologic inhibition of JNK or gene deletion of jnk1 but not jnk2 repressed the expression of inflammatory and fibrogenic mediators in primary Kupffer cells. In vivo, CDAA diet induced less hepatic inflammation and liver fibrosis despite an equivalent level of hepatic steatosis in chimeric mice with jnk1(-/-) hematopoietic cells as compared with chimeric mice with wild-type or jnk2(-/-) hematopoietic cells. CONCLUSIONS: jnk1(-/-) mice are resistant to diet-induced steatohepatitis and liver fibrosis. JNK1 in hematopoietic cells, especially in Kupffer cells, contributes to the development of liver fibrosis by inducing chronic inflammation.

Angiotensin-converting-enzyme 2 inhibits liver fibrosis in mice.

UNLABELLED: The renin-angiotensin system (RAS) plays a major role in liver fibrosis. Recently, a homolog of angiotensin-converting-enzyme 1 (ACE1), termed ACE2, has been identified that appears to be a negative regulator of the RAS by degrading Ang II to Ang(1-7). The aim of this study was to characterize the long-term effects of gene deletion of ACE2 in the liver, to define the role of ACE2 in acute and chronic liver disease, and to characterize the role of Ang(1-7) in hepatic stellate cell (HSC) activation. Ace2 knockout (KO) mice and wild-type (wt) littermates underwent different models of acute and chronic liver injury. Liver pathology was analyzed by histology, immunohistochemistry, alpha smooth muscle actin (alpha-SMA) immunoblotting, and quantitative polymerase chain reaction (qPCR). Murine HSCs were isolated by collagenase-pronase-perfusion, and density gradient centrifugation. One-year-old ace2 KO mice spontaneously developed an inflammatory cell infiltration and mild hepatic fibrosis that was prevented by treatment with irbesartan. Ace2 KO mice showed increased liver fibrosis following bile duct ligation for 21 days or chronic carbon tetrachloride (CCl(4)) treatment. In contrast, ace2 KO mice subjected to acute liver injury models did not differ from wt littermates. Treatment with recombinant ACE2 attenuated experimental fibrosis in the course of cholestatic and toxic liver injury. HSCs express the Ang(1-7) receptor Mas and Ang(1-7) inhibited Ang II-induced phosphorylation of extracellular signal-regulated kinase (ERK)-1/2 in cultured HSCs. CONCLUSION: ACE2 is a key negative regulator of the RAS and functions to limit fibrosis through the degradation of Ang II and the formation of Ang(1-7). Whereas loss of ACE2 activity worsens liver fibrosis in chronic liver injury models, administration of recombinant ACE2 shows therapeutic potential.

Overexpression of interleukin-1beta in the murine pancreas results in chronic pancreatitis.

BACKGROUND & AIMS: Chronic pancreatitis is a significant cause of morbidity and a known risk factor for pancreatic adenocarcinoma. Interleukin-1beta is a proinflammatory cytokine involved in pancreatic inflammation. We sought to determine whether targeted overexpression of interleukin-1beta in the pancreas could elicit localized inflammatory responses and chronic pancreatitis. METHODS: We created a transgenic mouse model (elastase sshIL-1beta) in which the rat elastase promoter drives the expression of human interleukin-1beta. Mice were followed up for up to 2 years. Pancreata of elastase sshIL-1beta mice were analyzed for chronic pancreatitis-associated histologic and molecular changes. To study the potential effect of p53 mutation in chronic pancreatitis, elastase sshIL-1beta mice were crossed with p53(R172H) mice. RESULTS: Three transgenic lines were generated, and in each line the pancreas was atrophic and occasionally showed dilation of pancreatic and biliary ducts secondary to proximal fibrotic stenosis. Pancreatic histology showed typical features of chronic pancreatitis. There was evidence for increased acinar proliferation and apoptosis, along with prominent expression of tumor necrosis factor-alpha; chemokine (C-X-C motif) ligand 1; stromal cell-derived factor 1; transforming growth factor-beta1; matrix metallopeptidase 2, 7, and 9; inhibitor of metalloproteinase 1; and cyclooxygenase 2. The severity of the lesions correlated well with the level of human interleukin-1beta expression. Older mice displayed acinar-ductal metaplasia but did not develop mouse pancreatic intraepithelial neoplasia or tumors. Elastase sshIL-1beta*p53(R172H/+) mice had increased frequency of tubular complexes, some of which were acinar-ductal metaplasia. CONCLUSIONS: Overexpression of interleukin-1beta in the murine pancreas induces chronic pancreatitis. Elastase sshIL-1beta mice consistently develop severe chronic pancreatitis and constitute a promising model for studying chronic pancreatitis and its relationship with pancreatic adenocarcinoma.

Hepatic stellate cells secrete angiopoietin 1 that induces angiogenesis in liver fibrosis.

BACKGROUND & AIMS: Although angiogenesis is closely associated with liver fibrosis, the angiogenic factors involved in liver fibrosis are not well characterized. Angiopoietin 1 is an angiogenic cytokine indispensable for vascular development and remodeling. It functions as an agonist for the receptor tyrosine kinase with immunoglobulin G-like and endothelial growth factor-like domains 2 (Tie2) and counteracts apoptosis, promotes vascular sprouting or branching, and stabilizes vessels. METHODS: Liver samples from patients with liver fibrosis were evaluated for mRNA expression of angiogenic cytokines. Liver fibrosis was induced in BALB/c mice by either carbon tetrachloride (CCl(4)) or bile duct ligation (BDL). Hepatic stellate cells (HSCs) were isolated from BALB/c mice. We used an adenovirus expressing the extracellular domain of Tie2 (AdsTie2) to block angiopoietin signaling in mice and evaluated its effect on liver fibrosis. RESULTS: mRNA expression level of angiopoietin 1 was increased in human fibrotic livers and correlated with the expression level of CD31, an endothelial cell marker. During experimental models of murine liver fibrosis, angiopoietin 1 was expressed by activated HSCs. In primary cultures, activated HSCs express and secrete angiopoietin 1 more abundantly than quiescent HSCs, and the inflammatory cytokine tumor necrosis factor-alpha stimulates its expression in an nuclear factor-kappaB-dependent manner. AdsTie2 inhibits angiogenesis and liver fibrosis induced by either CCl(4) or BDL. CONCLUSIONS: These results reveal an angiogenic role of HSCs mediated by angiopoietin 1, which contributes to development of liver fibrosis. Thus, angiogenesis and hepatic fibrosis are mutually stimulatory, such that fibrosis requires angiogenesis and angiogenesis requires angiopoietin 1 from activated HSCs.