Hepatic stellate cell-expressed endosialin balances fibrogenesis and hepatocyte proliferation during liver damage.
ABSTRACT: Liver fibrosis is a reversible wound-healing response to injury reflecting the critical balance between liver repair and scar formation. Chronic damage leads to progressive substitution of liver parenchyma by scar tissue and ultimately results in liver cirrhosis. Stromal cells (hepatic stellate cells [HSC] and endothelial cells) have been proposed to control the balance between liver fibrosis and regeneration. Here, we show that endosialin, a C-type lectin, expressed in the liver exclusively by HSC and portal fibroblasts, is upregulated in liver fibrosis in mouse and man. Chronic chemically induced liver damage resulted in reduced fibrosis and enhanced hepatocyte proliferation in endosialin-deficient (EN(KO)) mice. Correspondingly, acute-liver-damage-induced hepatocyte proliferation (partial hepatectomy) was increased in EN(KO) mice. A candidate-based screen of known regulators of hepatocyte proliferation identified insulin-like growth factor 2 (IGF2) as selectively endosialin-dependent hepatocyte mitogen. Collectively, the study establishes a critical role of HSC in the reciprocal regulation of fibrogenesis vs. hepatocyte proliferation and identifies endosialin as a therapeutic target in non-neoplastic settings.
Project description:Hepatocellular carcinoma (HCC) is among the most common and deadliest cancers worldwide. A major contributor to HCC progression is the cross talk between tumor cells and the surrounding stroma including activated hepatic stellate cells (HSC). Activation of HSC during liver damage leads to upregulation of the orphan receptor endosialin (CD248), which contributes to regulating the balance of liver regeneration and fibrosis. Based on the established role of endosialin in regulating HSC/hepatocyte cross talk, we hypothesized that HSC-expressed endosialin might similarly affect cell proliferation during hepatocarcinogenesis. Indeed, the histological analysis of human HCC samples revealed an inverse correlation between tumor cell proliferation and stromal endosialin expression. Correspondingly, global genetic inactivation of endosialin resulted in accelerated tumor growth in an inducible mouse HCC model. A candidate-based screen of tumor lysates and differential protein arrays of cultured HSC identified several established hepatotropic cytokines, including IGF2, RBP4, DKK1, and CCL5 as being negatively regulated by endosialin. Taken together, the experiments identify endosialin-expressing HSC as a negative regulator of HCC progression.
Project description:Liver fibrosis is a global health problem and previous studies have demonstrated that reactive oxygen species (ROS) play important roles in fibrogenesis. Parkinson disease (autosomal recessive, early onset) 7 (Park7) also called DJ-1 has an essential role in modulating cellular ROS levels. DJ-1 therefore may play functions in liver fibrogenesis and modulation of DJ-1 may be a promising therapeutic approach. Here, wild-type (WT) and DJ-1 knockout (DJ-1 KO) mice were administrated with carbon tetrachloride (CCl4) to induce liver fibrosis or acute liver injury. Results showed that DJ-1 depletion significantly blunted liver fibrosis, accompanied by marked reductions in liver injury and ROS production. In the acute CCl4 model, deficiency of DJ-1 showed hepatic protective functions as evidenced by decreased hepatic damage, reduced ROS levels, diminished hepatic inflammation and hepatocyte proliferation compared to WT mice. In vitro hepatic stellate cells (HSCs) activation assays indicated that DJ-1 has no direct effect on the activation of HSCs in the context of with or without TGF? treatment. Thus our present study demonstrates that in CCl4-induced liver fibrosis, DJ-1 deficiency attenuates mice fibrosis by inhibiting ROS production and liver injury, and further indirectly affecting the activation of HSCs. These results are in line with previous studies that ROS promote HSC activation and fibrosis development, and suggest the therapeutic value of DJ-1 in treatment of liver fibrosis.
Project description:Interleukin-15 (IL-15) and its high affinity receptor interleukin-15 receptor alpha (IL-15R?) are widely expressed in immune cells and hepatic resident cells. IL-15 signaling has important functions in homeostasis of natural killer (NK), natural killer T (NKT) and cytotoxic T (CD8(+) T) cells, and in liver regeneration. We hypothesized that IL-15 has a protective role in liver fibrosis progression by maintaining NK cell homeostasis.Fibrosis was induced using two mechanistically distinct models. Congenic bone marrow transplantation was used to evaluate the contribution of IL-15 signaling from various compartments to NK, CD8(+) T and NKT cell homeostasis and fibrogenesis. The gene expression profile of hepatic stellate cell (HSC) from IL-15R? knockout (IL-15R?KO) mice and wild-type mice were captured using microarray analysis and validated in isolated HSC. Quantitative real-time PCR was used to assess repressors of collagen transcription.IL-15R?KO mice exhibited more fibrosis in both models. IL-15 signaling from specific types of hepatic cells had divergent roles in maintaining liver NK, CD8(+) T and NKT cells, with a direct and protective role on radio-resistant non-parenchymal cells beyond the control of NK homeostasis. HSCs isolated from IL-15R?KO mice demonstrated upregulation of collagen production. Finally, IL-15R?KO HSC with or without transforming growth factor beta (TGF-?) stimulation exhibited increased expression of fibrosis markers and decreased collagen transcription repressors expression.IL-15R? signaling has a direct anti-fibrotic effect independent of preserving NK homeostasis. These findings establish a rationale to further explore the anti-fibrotic potential of enhancing IL-15 signaling in HSCs.We investigated how a cellular protein, Interleukin-15 (IL-15), decreases the amount of scar tissue that is formed upon liver injury. We found that IL-15 and its receptor decrease the amount of scar tissue that is created by specialized liver cells (called stellate cells) and increase the number of a specific subgroup of immune cells (natural killer cells) that are known to eliminate stellate cells.GSE45612, GSE 68001 and GSE 25097.
Project description:INTRODUCTION:CD248 (endosialin) is a stromal cell marker expressed on fibroblasts and pericytes. During liver injury, myofibroblasts are the main source of fibrotic matrix. OBJECTIVE:To determine the role of CD248 in the development of liver fibrosis in the rodent and human setting. DESIGN:CD248 expression was studied by immunostaining and quantitative PCR in both normal and diseased human and murine liver tissue and isolated hepatic stellate cells (HSCs). Hepatic fibrosis was induced in CD248(-/-) and wild-type controls with carbon tetrachloride (CCl4) treatment. RESULTS:Expression of CD248 was seen in normal liver of humans and mice but was significantly increased in liver injury using both immunostaining and gene expression assays. CD248 was co-expressed with a range of fibroblast/HSC markers including desmin, vimentin and ?-smooth muscle actin (?-SMA) in murine and human liver sections. CD248 expression was restricted to isolated primary murine and human HSC. Collagen deposition and ?-SMA expression, but not inflammation and neoangiogenesis, was reduced in CD248(-/-) mice compared with wild-type mice after CCl4 treatment. Isolated HSC from wild-type and CD248(-/-) mice expressed platelet-derived growth factor receptor ? (PDGFR-?) and PDGFR-? at similar levels. As expected, PDGF-BB stimulation induced proliferation of wild-type HSC, whereas CD248(-/-) HSC did not demonstrate a proliferative response to PDGF-BB. Abrogated PDGF signalling in CD248(-/-) HSC was confirmed by significantly reduced c-fos expression in CD248(-/-) HSC compared with wild-type HSC. CONCLUSIONS:Our data show that deletion of CD248 reduces susceptibility to liver fibrosis via an effect on PDGF signalling, making it an attractive clinical target for the treatment of liver injury.
Project description:Liver fibrosis is a reversible wound-healing process that is protective in the short term, but prolonged fibrotic responses lead to excessive accumulation of extracellular matrix components that suppresses hepatocyte regeneration, resulting in permanent liver damage. Upon liver damage, nonparenchymal cells including immune cells and hepatic stellate cells (HSCs) have crucial roles in the progression and regression of liver fibrosis. Here, we report differential roles of the glucocorticoid receptor (GR), acting in immune cells and HSCs, in liver fibrosis. In the carbon tetrachloride hepatotoxin-induced fibrosis model, both steroidal and nonsteroidal GR ligands suppressed expression of fibrotic genes and decreased extracellular matrix deposition but also inhibited immune cell infiltration and exacerbated liver injury. These counteracting effects of GR ligands were dissociated in mice with conditional GR knockout in immune cells (GR(LysM)) or HSC (GR(hGFAP)): the impacts of dexamethasone on immune cell infiltration and liver injury were totally blunted in GR(LysM) mice, whereas the suppression of fibrotic gene expression was diminished in GR(hGFAP) mice. The effect of GR activation in HSC was further confirmed in the LX-2 HSC cell line, in which antifibrotic effects were mediated by GR ligand inhibition of Sma and mad-related protein 3 (SMAD3) expression. We conclude that GR has differential roles in immune cells and HSCs to modulate liver injury and liver fibrosis. Specific activation of HSC-GR without alteration of GR activity in immune cells provides a potential therapeutic approach to treatment of hepatic fibrosis.
Project description:SerpinB3 is a hypoxia- and hypoxia-inducible factor-2?-dependent cystein protease inhibitor that is up-regulated in hepatocellular carcinoma and in parenchymal cells during chronic liver diseases (CLD). SerpinB3 up-regulation in CLD patients has been reported to correlate with the extent of liver fibrosis and the production of transforming growth factor-?1, but the actual role of SerpinB3 in hepatic fibrogenesis is still poorly characterized. In the present study we analyzed the pro-fibrogenic action of SerpinB3 in cell cultures and in two different murine models of liver fibrosis. "In vitro" experiments revealed that SerpinB3 addition to either primary cultures of human activated myofibroblast-like hepatic stellate cells (HSC/MFs) or human stellate cell line (LX2 cells) strongly up-regulated the expression of genes involved in fibrogenesis and promoted oriented migration, but not cell proliferation. Chronic liver injury by CCl4 administration or by feeding a methionine/choline deficient diet to transgenic mice over-expressing human SerpinB3 in hepatocytes confirmed that SerpinB3 over-expression significantly increased the mRNA levels of pro-fibrogenic genes, collagen deposition and ?SMA-positive HSC/MFs as compared to wild-type mice, without affecting parenchymal damage. The present study provides for the first time evidence that hepatocyte release of SerpinB3 during CLD can contribute to liver fibrogenesis by acting on HSC/MFs.
Project description:Prolonged exposure of mice to diet containing 0.1% 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) results in hepatobiliary injury, atypical ductular proliferation, oval cell appearance, and limited fibrosis. Previously, we reported that short-term ingestion of DDC diet by hepatocyte-specific ?-catenin conditional knockout (KO) mice led to fewer A6-positive oval cells than wildtype (WT) littermates. To examine the role of ?-catenin in chronic hepatic injury and repair, we exposed WT and KO mice to DDC for 80 and 150 days. Paradoxically, long-term DDC exposure led to significantly more A6-positive cells, indicating greater atypical ductular proliferation in KO, which coincided with increased fibrosis and cholestasis. Surprisingly, at 80 and 150 days in KO we observed a significant amelioration of hepatocyte injury. This coincided with extensive repopulation of ?-catenin null livers with ?-catenin-positive hepatocytes at 150 days, which was preceded by appearance of ?-catenin-positive hepatocyte clusters at 80 days and a few ?-catenin-positive hepatocytes at earlier times. Intriguingly, occasional ?-catenin-positive hepatocytes that were negative for progenitor markers were also observed at baseline in the KO livers, suggesting spontaneous escape from cre-mediated recombination. These cells with hepatocyte morphology expressed mature hepatocyte markers but lacked markers of hepatic progenitors. The gradual repopulation of KO livers with ?-catenin-positive hepatocytes occurred only following DDC injury and coincided with a progressive loss of hepatic cre-recombinase expression. A few ?-catenin-positive cholangiocytes were observed albeit only after long-term DDC exposure and trailed the appearance of ?-catenin-positive hepatocytes.In a chronic liver injury model, ?-catenin-positive hepatocytes exhibit growth and survival advantages and repopulate KO livers, eventually limiting hepatic injury and dysfunction despite increased fibrosis and intrahepatic cholestasis.
Project description:HCV infection typically induces liver injury and inflammation, which appears to be responsible for the associated fibrogenesis. To date, the mechanism underlying the different rates of disease progression remains unclear. The aim of the study is to understand the possible role of the HCV non-structural (NS) 3/4A protein in the fibrosis progression. We used NS3/4A-expressing transgenic mice (NS3/4A-Tg) to accomplish the goals of the study. Different stages of liver fibrosis were induced in wild-type and NS3/4A-Tg mice by single carbon tetrachloride (acute) or multiple injections for 4 (intermediate) or 8 (chronic) weeks. Fibrotic parameters, inflammatory responses and hepatocyte turnover were extensively examined. Hepatic expression of HCV NS3/4A did not induce spontaneous liver damage. However, NS3/4A expression exerted contrasting effects during acute and chronic liver damage. During early fibrogenesis and intermediate fibrosis (4 weeks), NS3/4A-Tg mice exhibited enhanced liver damage whereas reduced fibrosis was observed in NS3/4A-Tg during chronic liver fibrosis (8 weeks). Furthermore, attenuated inflammation was observed in NS3/4A-Tg during chronic fibrosis with increase in M2 macrophages, hepatocyte proliferation, decreased hepatocyte apoptosis and decreased ductular reaction. In conclusion, during early fibrogenesis, HCV NS3/4A contributes to liver damage. While, during chronic liver fibrosis, NS3/4A dampens inflammation and induces hepatocyte regeneration thereby contributing to slow fibrosis progression to promote its survival or persistence.
Project description:Prohibitin 1 (PHB1) is a highly conserved, ubiquitously expressed protein that participates in diverse processes including mitochondrial chaperone, growth and apoptosis. The role of PHB1 in vivo is unclear and whether it is a tumor suppressor is controversial. Mice lacking methionine adenosyltransferase 1A (MAT1A) have reduced PHB1 expression, impaired mitochondrial function, and spontaneously develop hepatocellular carcinoma (HCC). To see if reduced PHB1 expression contributes to the Mat1a knockout (KO) phenotype, we generated liver-specific Phb1 KO mice. Expression was determined at the messenger RNA and protein levels. PHB1 expression in cells was varied by small interfering RNA or overexpression. At 3 weeks, KO mice exhibit biochemical and histologic liver injury. Immunohistochemistry revealed apoptosis, proliferation, oxidative stress, fibrosis, bile duct epithelial metaplasia, hepatocyte dysplasia, and increased staining for stem cell and preneoplastic markers. Mitochondria are swollen and many have no discernible cristae. Differential gene expression revealed that genes associated with proliferation, malignant transformation, and liver fibrosis are highly up-regulated. From 20 weeks on, KO mice have multiple liver nodules and from 35 to 46 weeks, 38% have multifocal HCC. PHB1 protein levels were higher in normal human hepatocytes compared to human HCC cell lines Huh-7 and HepG2. Knockdown of PHB1 in murine nontransformed AML12 cells (normal mouse hepatocyte cell line) raised cyclin D1 expression, increased E2F transcription factor binding to cyclin D1 promoter, and proliferation. The opposite occurred with PHB1 overexpression. Knockdown or overexpression of PHB1 in Huh-7 cells did not affect proliferation significantly or sensitize cells to sorafenib-induced apoptosis.Hepatocyte-specific PHB1 deficiency results in marked liver injury, oxidative stress, and fibrosis with development of HCC by 8 months. These results support PHB1 as a tumor suppressor in hepatocytes.
Project description:Inflammasome activation plays a central role in the development of drug-induced and obesity-associated liver disease. However, the sources and mechanisms of inflammasome-mediated liver damage remain poorly understood. Our aim was to investigate the effect of NLRP3 inflammasome activation on the liver using novel mouse models. We generated global and myeloid cell-specific conditional mutant Nlrp3 knock-in mice expressing the D301N Nlrp3 mutation (ortholog of D303N in human NLRP3), resulting in a hyperactive NLRP3. To study the presence and significance of NLRP3-initiated pyroptotic cell death, we separated hepatocytes from nonparenchymal cells and developed a novel flow-cytometry-based (fluorescence-activated cell sorting; FACS) strategy to detect and quantify pyroptosis in vivo based on detection of active caspase 1 (Casp1)- and propidium iodide (PI)-positive cells. Liver inflammation was quantified histologically by FACS and gene expression analysis. Liver fibrosis was assessed by Sirius Red staining and quantitative polymerase chain reaction for markers of hepatic stellate cell (HSC) activation. NLRP3 activation resulted in shortened survival, poor growth, and severe liver inflammation; characterized by neutrophilic infiltration and HSC activation with collagen deposition in the liver. These changes were partially attenuated by treatment with anakinra, an interleukin-1 receptor antagonist. Notably, hepatocytes from global Nlrp3-mutant mice showed marked hepatocyte pyroptotic cell death, with more than a 5-fold increase in active Casp1/PI double-positive cells. Myeloid cell-restricted mutant NLRP3 activation resulted in a less-severe liver phenotype in the absence of detectable pyroptotic hepatocyte cell death.Our data demonstrate that global and, to a lesser extent, myeloid-specific NLRP3 inflammasome activation results in severe liver inflammation and fibrosis while identifying hepatocyte pyroptotic cell death as a novel mechanism of NLRP3-mediated liver damage.