Fibromodulin, an oxidative stress-sensitive proteoglycan, regulates the fibrogenic response to liver injury in mice.

Collagen I deposition contributes to liver fibrosis, yet little is known about other factors that mediate this process. Fibromodulin is a liver proteoglycan that regulates extracellular matrix organization and is induced by fibrogenic stimuli. We propose that fibromodulin contributes to the pathogenesis of fibrosis by regulating the fibrogenic phenotype of hepatic stellate cells (HSCs). We analyzed liver samples from patients with hepatitis C-associated cirrhosis and healthy individuals (controls). We used a coculture model to study interactions among rat HSCs, hepatocytes, and sinusoidal endothelial cells. We induced fibrosis in livers of wild-type and Fmod(-/-) mice by bile duct ligation, injection of CCl(4), or administration of thioacetamide. Liver samples from patients with cirrhosis had higher levels of fibromodulin messenger RNA and protein than controls. Bile duct ligation, CCl(4), and thioacetamide each increased levels of fibromodulin protein in wild-type mice. HSCs, hepatocytes, and sinusoidal endothelial cells produced and secreted fibromodulin. Infection of HSCs with an adenovirus that expressed fibromodulin increased expression of collagen I and α-smooth muscle actin, indicating increased activation of HSCs and fibrogenic potential. Recombinant fibromodulin promoted proliferation, migration, and invasion of HSCs, contributing to their fibrogenic activity. Fibromodulin was sensitive to reactive oxygen species. HepG2 cells that express cytochrome P450 2E1 produced fibromodulin, and HSCs increased fibromodulin production in response to pro-oxidants. In mice, administration of an antioxidant prevented the increase in fibromodulin in response to CCl(4). Coculture of hepatocytes or sinusoidal endothelial cells with HSCs increased the levels of reactive oxygen species in the culture medium, along with collagen I and fibromodulin proteins; this increase was prevented by catalase. Fibromodulin bound to collagen I, but the binding did not prevent collagen I degradation by matrix metalloproteinase 13. Bile duct ligation caused liver fibrosis in wild-type but not Fmod(-/-) mice. Fibromodulin levels are increased in livers of patients with cirrhosis. Hepatic fibromodulin activates HSCs and promotes collagen I deposition, which leads to liver fibrosis in mice.

Mormone E ，Lu Y ，Ge X ，Fiel MI ，Nieto N ... -  《-》

Osteopontin, an oxidant stress sensitive cytokine, up-regulates collagen-I via integrin α(V)β(3) engagement and PI3K/pAkt/NFκB signaling.

A key feature in the pathogenesis of liver fibrosis is fibrillar Collagen-I deposition; yet, mediators that could be key therapeutic targets remain elusive. We hypothesized that osteopontin (OPN), an extracellular matrix (ECM) cytokine expressed in hepatic stellate cells (HSCs), could drive fibrogenesis by modulating the HSC pro-fibrogenic phenotype and Collagen-I expression. Recombinant OPN (rOPN) up-regulated Collagen-I protein in primary HSCs in a transforming growth factor beta (TGFβ)-independent fashion, whereas it down-regulated matrix metalloprotease-13 (MMP13), thus favoring scarring. rOPN activated primary HSCs, confirmed by increased α-smooth muscle actin (αSMA) expression and enhanced their invasive and wound-healing potential. HSCs isolated from wild-type (WT) mice were more profibrogenic than those from OPN knockout (Opn(-/-)) mice and infection of primary HSCs with an Ad-OPN increased Collagen-I, indicating correlation between both proteins. OPN induction of Collagen-I occurred via integrin α(v)β(3) engagement and activation of the phosphoinositide 3-kinase/phosphorylated Akt/nuclear factor kappa B (PI3K/pAkt/NFκB)-signaling pathway, whereas cluster of differentiation 44 (CD44) binding and mammalian target of rapamycin/70-kDa ribosomal protein S6 kinase (mTOR/p70S6K) were not involved. Neutralization of integrin α(v) β(3) prevented the OPN-mediated activation of the PI3K/pAkt/NFκB-signaling cascade and Collagen-I up-regulation. Likewise, inhibition of PI3K and NFκB blocked the OPN-mediated Collagen-I increase. Hepatitis C Virus (HCV) cirrhotic patients showed coinduction of Collagen-I and cleaved OPN compared to healthy individuals. Acute and chronic liver injury by CCl(4) injection or thioacetamide (TAA) treatment elevated OPN expression. Reactive oxygen species up-regulated OPN in vitro and in vivo and antioxidants prevented this effect. Transgenic mice overexpressing OPN in hepatocytes (Opn(HEP) Tg) mice developed spontaneous liver fibrosis compared to WT mice. Last, chronic CCl(4) injection and TAA treatment caused more liver fibrosis to WT than to Opn(-/-) mice and the reverse occurred in Opn(HEP) Tg mice. OPN emerges as a key cytokine within the ECM protein network driving the increase in Collagen-I protein contributing to scarring and liver fibrosis.

Urtasun R ，Lopategi A ，George J ，Leung TM ，Lu Y ，Wang X ，Ge X ，Fiel MI ，Nieto N ... -  《-》

ECM1 Prevents Activation of Transforming Growth Factor β, Hepatic Stellate Cells, and Fibrogenesis in Mice.

Activation of TGFB (transforming growth factor β) promotes liver fibrosis by activating hepatic stellate cells (HSCs), but the mechanisms of TGFB activation are not clear. We investigated the role of ECM1 (extracellular matrix protein 1), which interacts with extracellular and structural proteins, in TGFB activation in mouse livers. We performed studies with C57BL/6J mice (controls), ECM1-knockout (ECM1-KO) mice, and mice with hepatocyte-specific knockout of EMC1 (ECM1Δhep). ECM1 or soluble TGFBR2 (TGFB receptor 2) were expressed in livers of mice after injection of an adeno-associated virus vector. Liver fibrosis was induced by carbon tetrachloride (CCl4) administration. Livers were collected from mice and analyzed by histology, immunohistochemistry, in situ hybridization, and immunofluorescence analyses. Hepatocytes and HSCs were isolated from livers of mice and incubated with ECM1; production of cytokines and activation of reporter genes were quantified. Liver tissues from patients with viral or alcohol-induced hepatitis (with different stages of fibrosis) and individuals with healthy livers were analyzed by immunohistochemistry and in situ hybridization. ECM1-KO mice spontaneously developed liver fibrosis and died by 2 months of age without significant hepatocyte damage or inflammation. In liver tissues of mice, we found that ECM1 stabilized extracellular matrix-deposited TGFB in its inactive form by interacting with αv integrins to prevent activation of HSCs. In liver tissues from patients and in mice with CCl4-induced liver fibrosis, we found an inverse correlation between level of ECM1 and severity of fibrosis. CCl4-induced liver fibrosis was accelerated in ECM1Δhep mice compared with control mice. Hepatocytes produced the highest levels of ECM1 in livers of mice. Ectopic expression of ECM1 or soluble TGFBR2 in liver prevented fibrogenesis in ECM1-KO mice and prolonged their survival. Ectopic expression of ECM1 in liver also reduced the severity of CCl4-induced fibrosis in mice. ECM1, produced by hepatocytes, inhibits activation of TGFB and its activation of HSCs to prevent fibrogenesis in mouse liver. Strategies to increase levels of ECM1 in liver might be developed for treatment of fibrosis.

Fan W ，Liu T ，Chen W ，Hammad S ，Longerich T ，Hausser I ，Fu Y ，Li N ，He Y ，Liu C ，Zhang Y ，Lian Q ，Zhao X ，Yan C ，Li L ，Yi C ，Ling Z ，Ma L ，Zhao X ，Xu H ，Wang P ，Cong M ，You H ，Liu Z ，Wang Y ，Chen J ，Li D ，Hui L ，Dooley S ，Hou J ，Jia J ，Sun B ... -  《-》

Fibronectin extra domain-A promotes hepatic stellate cell motility but not differentiation into myofibroblasts.

Myofibroblasts are the primary cell type involved in physiologic wound healing and its pathologic counterpart, fibrosis. Cellular fibronectin that contains the alternatively spliced extra domain A (EIIIA) is up-regulated during these processes and is believed to promote myofibroblast differentiation. We sought to determine the requirement for EIIIA in fibrosis and differentiation of myofibroblasts in rodent livers. We used a mechanically tunable hydrogel cell culture system to study differentiation of primary hepatic stellate cells and portal fibroblasts from rats into myofibroblasts. Liver fibrosis was induced in mice by bile duct ligation or administration of thioacetamide. EIIIA was not required for differentiation of rat hepatic stellate cells or portal fibroblasts into fibrogenic myofibroblasts. Instead, hepatic stellate cells cultured on EIIIA-containing cellular fibronectin formed increased numbers of lamellipodia; their random motility and chemotaxis also increased. These increases required the receptor for EIIIA, the integrin α(9)β(1). In contrast, the motility of portal fibroblasts did not increase on EIIIA, and these cells expressed little α(9)β(1). Male EIIIA(-/-) mice were modestly protected from thioacetamide-induced fibrosis, which requires motile hepatic stellate cells, but not from bile duct ligation-induced fibrosis, in which portal fibroblasts are more important. Notably, myofibroblasts developed during induction of fibrosis with either thioacetamide or bile duct ligation in EIIIA(-/-) mice. EIIIA is dispensable for differentiation of hepatic stellate cells and portal fibroblasts to myofibroblasts, both in culture and in mouse models of fibrosis. Our findings, however, indicate a role for EIIIA in promoting stellate cell motility and parenchymal liver fibrosis.

Olsen AL ，Sackey BK ，Marcinkiewicz C ，Boettiger D ，Wells RG ... -  《-》

Aryl Hydrocarbon Receptor Signaling Prevents Activation of Hepatic Stellate Cells and Liver Fibrogenesis in Mice.

The role of aryl hydrocarbon receptor (AhR) in liver fibrosis is controversial because loss and gain of AhR activity both lead to liver fibrosis. The goal of this study was to investigate how the expression of AhR by different liver cell types, hepatic stellate cells (HSCs) in particular, affects liver fibrosis in mice. We studied the effects of AhR on primary mouse and human HSCs, measuring their activation and stimulation of fibrogenesis using RNA-sequencing analysis. C57BL/6J mice were given the AhR agonists 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or 2-(1'H-indole-3'-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE); were given carbon tetrachloride (CCl4); or underwent bile duct ligation. We also performed studies in mice with disruption of Ahr specifically in HSCs, hepatocytes, or Kupffer cells. Liver tissues were collected from mice and analyzed by histology, immunohistochemistry, and immunoblotting. AhR was expressed at high levels in quiescent HSCs, but the expression decreased with HSC activation. Activation of HSCs from AhR-knockout mice was accelerated compared with HSCs from wild-type mice. In contrast, TCDD or ITE inhibited spontaneous and transforming growth factor β-induced activation of HSCs. Mice with disruption of Ahr in HSCs, but not hepatocytes or Kupffer cells, developed more severe fibrosis after administration of CCl4 or bile duct ligation. C57BL/6J mice given ITE did not develop CCl4-induced liver fibrosis, whereas mice without HSC AhR given ITE did develop CCl4-induced liver fibrosis. In studies of mouse and human HSCs, we found that AhR prevents transforming growth factor β-induced fibrogenesis by disrupting the interaction of Smad3 with β-catenin, which prevents the expression of genes that mediate fibrogenesis. In studies of human and mouse HSCs, we found that AhR prevents HSC activation and expression of genes required for liver fibrogenesis. Development of nontoxic AhR agonists or strategies to activate AhR signaling in HSCs might be developed to prevent or treat liver fibrosis.

Yan J ，Tung HC ，Li S ，Niu Y ，Garbacz WG ，Lu P ，Bi Y ，Li Y ，He J ，Xu M ，Ren S ，Monga SP ，Schwabe RF ，Yang D ，Xie W ... -  《-》