Heterozygous midnolin knockout attenuates severity of nonalcoholic fatty liver disease in mice fed a Western-style diet high in fat, cholesterol, and fructose.

Although midnolin has been studied for over 20 years, its biological roles in vivo remain largely unknown, especially due to the lack of a functional animal model. Indeed, given our recent discovery that the knockdown of midnolin suppresses liver cancer cell tumorigenicity and that this antitumorigenic effect is associated with modulation of lipid metabolism, we hypothesized that knockout of midnolin in vivo could potentially protect from nonalcoholic fatty liver disease (NAFLD) which has become the most common cause of chronic liver disease in the Western world. Accordingly, in the present study, we have developed and now report on the first functional global midnolin knockout mouse model. Although the overwhelming majority of global homozygous midnolin knockout mice demonstrated embryonic lethality, heterozygous knockout mice were observed to be similar to wild-type mice in their viability and were used to determine the effect of reduced midnolin expression on NAFLD. We found that global heterozygous midnolin knockout attenuated the severity of NAFLD in mice fed a Western-style diet, high in fat, cholesterol, and fructose, and this attenuation in disease was associated with significantly reduced levels of large lipid droplets, hepatic free cholesterol, and serum LDL, with significantly differential gene expression involved in cholesterol/lipid metabolism. Collectively, our results support a role for midnolin in regulating cholesterol/lipid metabolism in the liver. Thus, midnolin may represent a novel therapeutic target for NAFLD. Finally, our observation that midnolin was essential for survival underscores the broad importance of this gene beyond its role in liver biology.NEW & NOTEWORTHY We have developed and now report on the first functional global midnolin knockout mouse model. We found that global heterozygous midnolin knockout attenuated the severity of nonalcoholic fatty liver disease (NAFLD) in mice fed a Western-style diet, high in fat, cholesterol, and fructose, and this attenuation in disease was associated with significantly reduced levels of large lipid droplets, hepatic free cholesterol, and serum LDL, with significantly differential gene expression involved in cholesterol/lipid metabolism.

TWEAK/FN14 promotes profibrogenic pathway activation in Prominin-1-expressing hepatic progenitor cells in biliary atresia.

BACKGROUND AND AIMS: Biliary atresia (BA), a congenital cholestatic liver disease, commonly culminates in end-stage liver disease. We previously demonstrated in BA that Prominin-1 ( Prom1 )-expressing hepatic progenitor cells (HPCs) expand within regions of developing fibrosis, giving rise to cholangiocytes within biliary ductular reactions. Null mutation of Prom1 or ablation of cells expressing Prom1 significantly diminishes fibrogenesis. FN14, the receptor for TNF-like weak inducer of apoptosis (TWEAK), is expressed by HPCs. TWEAK/FN14 signaling promotes fibrosis in multiple organ systems. Therefore, we hypothesized that TWEAK/FN14 signaling mediates Prom1 -expressing HPC proliferation leading to profibrogenic ductular reactions in BA. APPROACH AND RESULTS: The experimental mouse model of BA mediated by perinatal rhesus rotavirus (RRV) infection resulted in increased co-expression of Fn14 in Prom1 -expressing HPCs within regions of ductular reactions. FN14 antagonist L524-0366 decreased ductular reactions, biliary fibrosis and periportal fibroblast activation in RRV injury. L524-0366 inhibition also demonstrated loss of downstream noncanonical NF-kB signaling expression in RRV injury. Murine HPC organoids demonstrated accelerated organoid growth and proliferation when treated with recombinant TWEAK. Increased organoid proliferation with recombinant TWEAK was lost when also treated with L524-0366. Analysis of a large publicly available RNA sequencing database of BA and normal control patients revealed significant increases in expression of PROM1 , FN14 , and genes downstream of TNF signaling and noncanonical NF-κB signaling pathways in BA infants. Infants who failed to achieve bile drainage after hepatoportoenterostomy had higher relative levels of FN14 expression. CONCLUSION: TWEAK/FN14 signaling activation in Prom1 -expressing HPCs contributes to proliferation of profibrogenic ductular reactions in BA.

Induction of Cell Death in Pancreatic Tumors by Zinc and Its Fluorescence Chelator TSQ.

Pancreatic ductal adenocarcinoma is a devastating cancer and is the fourth-leading cause of cancer death in the USA. Zinc is abundant in the pancreas, but its role in pancreatic cancer remains elusive. The aim of this study is to determine effects of zinc chelators in pancreatic cancer. Pdx1Cre and LSL-KrasG12D mice expressing an oncogenic mutation of KRAS develop pancreatic intraepithelial neoplasia in the pancreas. We found that EPCAM + tumors developed in the mouse pancreas store zinc that is detectable by fluorescence-activated cell sorting using N-(6-methoxy-8-quinolyl)-p-toluenesulfonamide (TSQ), a fluorescence chelator. EPCAM + TSQ + tumor cells isolated from the mouse pancreas formed organoids in matrigel. Upon treatment with N,N,N',N'-tetrakis(2-pyridinylmethyl)-1,2-ethanediamine (TPEN), a zinc chelator, the organoids degenerated and its negative effect was rescued by co-treatment with zinc, indicating that zinc is necessary for the growth and survival of tumor organoids. Different from TPEN, TSQ treatment did not affect the organoid growth and survival. Interestingly, co-treatment with TSQ and zinc resulted in strong emission of TSQ fluorescence in the organoid and its degeneration. The combination of zinc with TSQ, but not with TPEN, also induced cell death in PANC-1, a human pancreatic cancer cell line. These results suggest that a TSQ-zinc complex formed in pancreatic tumors induces cell death if zinc is overloaded.

Intraperitoneal microbial contamination drives post-surgical peritoneal adhesions by mesothelial EGFR-signaling.

Abdominal surgeries are lifesaving procedures but can be complicated by the formation of peritoneal adhesions, intra-abdominal scars that cause intestinal obstruction, pain, infertility, and significant health costs. Despite this burden, the mechanisms underlying adhesion formation remain unclear and no cure exists. Here, we show that contamination of gut microbes increases post-surgical adhesion formation. Using genetic lineage tracing we show that adhesion myofibroblasts arise from the mesothelium. This transformation is driven by epidermal growth factor receptor (EGFR) signaling. The EGFR ligands amphiregulin and heparin-binding epidermal growth factor, are sufficient to induce these changes. Correspondingly, EGFR inhibition leads to a significant reduction of adhesion formation in mice. Adhesions isolated from human patients are enriched in EGFR positive cells of mesothelial origin and human mesothelium shows an increase of mesothelial EGFR expression during bacterial peritonitis. In conclusion, bacterial contamination drives adhesion formation through mesothelial EGFR signaling. This mechanism may represent a therapeutic target for the prevention of adhesions after intra-abdominal surgery.

Loss of lysophosphatidic acid receptor 1 in hepatocytes reduces steatosis via down-regulation of CD36.

Nonalcoholic steatohepatitis is a major public health concern and is characterized by the accumulation of triglyceride in hepatocytes and inflammation in the liver. Steatosis is caused by dysregulation of the influx and efflux of lipids, lipogenesis, and mitochondrial ß-oxidation. Extracellular lysophosphatidic acid (LPA) regulates a broad range of cellular processes in development, tissue injury, and cancer. In the present study, we examined the roles of LPA in steatohepatitis induced by a methionine-choline-deficient (MCD) diet in mice. Hepatocytes express LPA receptor (Lpar) 1-3 mRNAs. Steatosis developed in mice fed the MCD diet was reduced by treatment with inhibitors for pan-LPAR or LPAR1. Hepatocyte-specific deletion of the Lpar1 gene also reduced the steatosis in the MCD model. Deletion of the Lpar1 gene in hepatocytes reduced expression of Cd36, a gene encoding a fatty acid transporter. Although LPA/LPAR1 signaling induces expression of Srebp1 mRNA in hepatocytes, LPA does not fully induce expression of SREBP1-target genes involved in lipogenesis. Human hepatocytes repopulated in chimeric mice are known to develop steatosis and treatment with an LPAR1 inhibitor reduces expression of CD36 mRNA and steatosis. Our data indicate that antagonism of LPAR1 reduces steatosis in mouse and human hepatocytes by down-regulation of Cd36.

Inhibition of Stearoyl-CoA Desaturase Induces the Unfolded Protein Response in Pancreatic Tumors and Suppresses Their Growth.

OBJECTIVE: Pancreatic ductal adenocarcinoma is the fourth-leading cause of cancer death in the United States, and there is an urgent need for effective therapies. Stearoyl-CoA desaturase (SCD) is an enzyme localized in the endoplasmic reticulum and generates monounsaturated fatty acid from saturated fatty acid. In this study, we examined the role of SCD in pancreatic cancer. METHODS: We isolated epithelial cell adhesion molecule-positive pancreatic tumors from the Pdx1Cre;LSL-KrasG12D mouse and formed organoids in Matrigel. Using a SCD inhibitor, A939572, we tested its effects on growth and cell death in tumor organoids, tumors developed in the Pdx1Cre;LSL-KrasG12D mouse, and a human pancreatic ductal adenocarcinoma cell line, PANC-1. RESULTS: A939572 treatment rapidly induced degeneration of mouse tumor organoids and activated the unfolded protein response (UPR). Cotreatment of oleic acid, but not stearic acid, reduced the UPR in the organoids and rescued the inhibitory effect of the SCD inhibitor on their growth. Administration of A939572 to Pdx1Cre;LSL-KrasG12D mice caused cell death in early pancreatic tumors, but not in acini or islets. The SCD inhibitor induced the UPR in PANC-1 and suppressed their growth but did not induce cell death. CONCLUSIONS: The inhibition of the SCD enzyme causes an UPR and cell death in early pancreatic tumors.

Moderate alcohol intake promotes pancreatic ductal adenocarcinoma development in mice expressing oncogenic Kras.

Kras mutations are associated with pancreatic ductal adenocarcinoma (PDAC). Although tobacco smoking, pancreatitis, and obesity are known environmental risk factors for PDAC, the contribution of moderate alcohol intake to PDAC remains elusive. In the present study, we tested whether a combination of risk factors or moderate alcohol intake induces PDAC development in mice. Control Pdx1Cre and Pdx1Cre;LSL-KrasG12D mutant mice were fed a Western alcohol diet containing high levels of cholesterol and saturated fat, 3.5% alcohol, and lipopolysaccharide for 5 mo. In addition, mice were treated with cerulein, for induction of pancreatitis, and nicotine every month. Treatment with all of these risk factors promoted development of advanced pancreatic neoplasia and PDAC in the Pdx1Cre;LSL-KrasG12D mice but not in the control Pdx1Cre mice. Moderate alcohol intake or Western diet feeding also significantly promoted advanced neoplasia and PDAC development in Pdx1Cre;LSL-KrasG12D mice compared with mice fed a regular chow. Alcohol, but not Western diet, increased tumor development in the liver in the Pdx1Cre;LSL-KrasG12D mice, but its origin remained elusive due to leakiness of Pdx1Cre in hepatocytes. RNA-seq analysis revealed that alcohol feeding increases expression of markers for tumors (Epcam, Krt19, Prom1, Wt1, and Wwtr1), stroma (Dcn, Fn1, and Tnc), and cytokines (Tgfb1 and Tnf) and decreases expression of Fgf21 and Il6 in the pancreatic tumor tissues. Immunostaining showed heterogeneous expression of nephronectin, S100 calcium-binding protein A6, and vascular cell adhesion molecule 1 in pancreatic tumors surrounded by podoplanin-positive stromal cells. Our data indicate that moderate alcohol drinking is a risk factor for development of PDAC.NEW & NOTEWORTHY Heavy alcohol intake has been suspected to be a risk factor of pancreatic ductal adenocarcinoma (PDAC) in humans. However, the contribution of moderate alcohol intake to PDAC development remains elusive. In the present study, we experimentally show that moderate alcohol feeding significantly induces advanced stages of pancreatic intraepithelial neoplasia development and invasive PDAC in Pdx1Cre;LSL-KrasG12D mutant mice. Our data indicate that moderate alcohol drinking is a risk factor for PDAC.

Isolation of a unique hepatic stellate cell population expressing integrin α8 from embryonic mouse livers.

BACKGROUND: Hepatic stellate cells (HSCs) play an important role in liver fibrogenesis. However, little is known about their phenotype and role in liver development. The aim of this study is to identify specific markers for embryonic HSCs. RESULTS: Using antibodies against ALCAM and PDPN, we separated mesothelial cells (MCs) and HSCs from developing livers and identified integrin α8 (ITGA8) as a marker for embryonic desmin+ HSCs that are preferentially localized near the developing liver surface and α-smooth muscle actin+ perivascular mesenchymal cells around the vein. A cell lineage-tracing study revealed that upon differentiation, MC-derived HSCs or perivascular mesenchymal cells express ITGA8 during liver development. Using anti-ITGA8 antibodies, we succeeded in isolating MC-derived HSCs and perivascular mesenchymal cells from embryonic livers. In direct co-culture, ITGA8+ mesenchymal cells promoted the expression of hepatocyte and cholangiocyte markers in hepatoblasts. In the normal adult liver, expression of ITGA8 was restricted to portal fibroblasts in the portal triad. Upon liver injury, myofibroblasts increased the expression of ITGA8. CONCLUSIONS: ITGA8 is a specific cell surface marker of MC-derived HSCs and perivascular mesenchymal cells in the developing liver. Our data suggest that ITGA8+ mesenchymal cells maintain the phenotype of hepatoblast in liver development. Developmental Dynamics 247:867-881, 2018. © 2018 Wiley Periodicals, Inc.

Hepatic Prominin-1 expression is associated with biliary fibrosis.

BACKGROUND: Intrahepatic biliary fibrosis, as seen with cholestatic liver injuries such as biliary atresia, is mechanistically distinct from fibrosis caused by hepatocyte toxicity. We previously demonstrated the expansion of cells expressing the stem/progenitor cell marker Prominin-1, within regions of developing fibrosis in biliary atresia. Thus, we hypothesized that Prominin-1 expression is biliary fibrosis-specific. METHODS: Gene expression of Prominin-1 was analyzed in adult mice undergoing either cholestatic bile duct ligation or hepatotoxic carbon tetrachloride administration by quantitative polymerase chair reaction. Lineage tracing of Prominin-1-expressing cells and Collagen-1α-expressing cells was performed after bile duct ligation in Prominin-1cre-ert2-lacz;Gfplsl and Collagen-1αGfp transgenic mice, respectively. RESULTS: Prominin-1 expression increased significantly after bile duct ligation compared with sham (6.6 ± 0.9-fold change at 2 weeks, P < .05) but not with carbon tetrachloride (-0.7 ± 0.5-fold change, not significant). Upregulation of Prominin-1 was observed histologically throughout the liver as early as 5 days after bile duct ligation in Prominin-1cre-ert2-lacz mice by LacZ staining in nonhepatocyte cells. Lineage tracing of Prominin-1-expressing cells labeled prior to bile duct ligation in Prominin-1cre-ert2-lacz;Gfplsl mice, demonstrated increasing colocalization of GREEN FLUORESCENT PROTEIN with biliary marker CYTOKERATIN-19 within ductular reactions up to 5 weeks after bile duct ligation consistent with biliary transdifferentiation. In contrast, rare colocalization of GREEN FLUORESCENT PROTEIN with mesenchymal marker α-SMOOTH MUSCLE ACTIN in Prominin-1cre-ert2-lacz;Gfplsl mice and some colocalization of GREEN FLUORESCENT PROTEIN with PROMININ-1 in Collagen-1αGfp mice, indicate minimal contribution of Prominin-1 progenitor cells to the pool of collagen-producing myofibroblasts. CONCLUSION: During biliary fibrosis Prominin-1-expressing progenitor cells transdifferentiate into cells within ductular reactions. This transdifferentiation may promote fibrosis.

Activation of hepatic stellate cell in Pten null liver injury model.

BACKGROUND: Hepatic fibrosis is a prominent pathological feature associated with chronic liver disease including non-alcoholic hepatosteatosis (NASH), and a precursor for liver cancer development. We previously reported that PTEN loss in the liver, which leads to hyperactivated liver insulin signaling results in NASH development. Here we used the same mouse model to study the progression from steatosis to fibrosis. RESULTS: The Pten null livers develop progressive liver fibrosis as indicated by Sirius Red staining and increased expression of collagen I, Timp 1, SMAα, and p75NTR. Consistently, hepatic stellate cells (HSCs) isolated from Pten null livers are readily activated when compared with that from mice with intact PTEN. Deletion of AKT2, the downstream target of PTEN signal, blocked NASH development, and alleviated fibrosis. HSCs from the Pten/Akt2 double null mice are quiescent like those isolated from the control livers. Our analysis shows that the activation of HSCs does not depend on the intrinsic signals regulated by PI3K/AKT, the target of PTEN, but does depend on steatosis and injury to the liver. During the progression of liver fibrosis in the Pten null model, Wnt ligands and signaling receptor are induced, concurrent with the reduction of sFRP5, a Wnt antagonist. We showed that treatment of HSCs with Wnt receptor antagonist blocks the observed morphological changes when HSCs undergo activation in culture. This signal appears to be mediated by ß-catenin, as manipulating ß-catenin signaling alters marker gene expressions of HSC activation. CONCLUSIONS: Wnt/ß-catenin activation serves as an important mediator for fibrosis development resulting from NASH using a mouse model where NASH is mimicked by PTEN loss.

The Role of Mesothelial Cells in Liver Development, Injury, and Regeneration.

Mesothelial cells (MCs) cover the surface of visceral organs and the parietal walls of cavities, and they synthesize lubricating fluids to create a slippery surface that facilitates movement between organs without friction. Recent studies have indicated that MCs play active roles in liver development, fibrosis, and regeneration. During liver development, the mesoderm produces MCs that form a single epithelial layer of the mesothelium. MCs exhibit an intermediate phenotype between epithelial cells and mesenchymal cells. Lineage tracing studies have indicated that during liver development, MCs act as mesenchymal progenitor cells that produce hepatic stellate cells, fibroblasts around blood vessels, and smooth muscle cells. Upon liver injury, MCs migrate inward from the liver surface and produce hepatic stellate cells or myofibroblast depending on the etiology, suggesting that MCs are the source of myofibroblasts in capsular fibrosis. Similar to the activation of hepatic stellate cells, transforming growth factor ß induces the conversion of MCs into myofibroblasts. Further elucidation of the biological and molecular changes involved in MC activation and fibrogenesis will contribute to the development of novel approaches for the prevention and therapy of liver fibrosis.

Myofibroblastic Conversion and Regeneration of Mesothelial Cells in Peritoneal and Liver Fibrosis.

Mesothelial cells (MCs) form a single epithelial layer and line the surface of body cavities and internal organs. Patients who undergo peritoneal dialysis often develop peritoneal fibrosis that is characterized by the accumulation of myofibroblasts in connective tissue. Although MCs are believed to be the source of myofibroblasts, their contribution has remained obscure. We determined the contribution of peritoneal MCs to myofibroblasts in chlorhexidine gluconate (CG)-induced fibrosis compared with that of phenotypic changes of liver MCs. CG injections resulted in disappearance of MCs from the body wall and the accumulation of myofibroblasts in the connective tissue. Conditional linage tracing with Wilms tumor 1 (Wt1)-CreERT2 and Rosa26 reporter mice found that 17% of myofibroblasts were derived from MCs in peritoneal fibrosis. Conditional deletion of transforming growth factor-ß type II receptor in Wt1(+) MCs substantially reduced peritoneal fibrosis. The CG treatment also induced myofibroblastic conversion of MCs in the liver. Lineage tracing with Mesp1-Cre mice revealed that Mesp1(+) mesoderm gave rise to liver MCs but not peritoneal MCs. During recovery from peritoneal fibrosis, peritoneal MCs, but not liver MCs, contribute to the regeneration of the peritoneal mesothelium, indicating an inherent difference between parietal and visceral MCs. In conclusion, MCs partially contribute to myofibroblasts in peritoneal and liver fibrosis, and protection of the MC layer leads to reduced development of fibrous tissue.

TAK1-mediated autophagy and fatty acid oxidation prevent hepatosteatosis and tumorigenesis.

The MAP kinase kinase kinase TGFß-activated kinase 1 (TAK1) is activated by TLRs, IL-1, TNF, and TGFß and in turn activates IKK-NF-κB and JNK, which regulate cell survival, growth, tumorigenesis, and metabolism. TAK1 signaling also upregulates AMPK activity and autophagy. Here, we investigated TAK1-dependent regulation of autophagy, lipid metabolism, and tumorigenesis in the liver. Fasted mice with hepatocyte-specific deletion of Tak1 exhibited severe hepatosteatosis with increased mTORC1 activity and suppression of autophagy compared with their WT counterparts. TAK1-deficient hepatocytes exhibited suppressed AMPK activity and autophagy in response to starvation or metformin treatment; however, ectopic activation of AMPK restored autophagy in these cells. Peroxisome proliferator-activated receptor α (PPARα) target genes and ß-oxidation, which regulate hepatic lipid degradation, were also suppressed in hepatocytes lacking TAK1. Due to suppression of autophagy and ß-oxidation, a high-fat diet challenge aggravated steatohepatitis in mice with hepatocyte-specific deletion of Tak1. Notably, inhibition of mTORC1 restored autophagy and PPARα target gene expression in TAK1-deficient livers, indicating that TAK1 acts upstream of mTORC1. mTORC1 inhibition also suppressed spontaneous liver fibrosis and hepatocarcinogenesis in animals with hepatocyte-specific deletion of Tak1. These data indicate that TAK1 regulates hepatic lipid metabolism and tumorigenesis via the AMPK/mTORC1 axis, affecting both autophagy and PPARα activity.

Expansion of prominin-1-expressing cells in association with fibrosis of biliary atresia.

UNLABELLED: Biliary atresia (BA), the most common cause of end-stage liver disease and the leading indication for pediatric liver transplantation, is associated with intrahepatic ductular reactions within regions of rapidly expanding periportal biliary fibrosis. Whereas the extent of such biliary fibrosis is a negative predictor of long-term transplant-free survival, the cellular phenotypes involved in the fibrosis are not well established. Using a rhesus rotavirus-induced mouse model of BA, we demonstrate significant expansion of a cell population expressing the putative stem/progenitor cell marker, PROMININ-1 (PROM1), adjacent to ductular reactions within regions of periportal fibrosis. PROM1positive (pos) cells express Collagen-1α1. Subsets of PROM1pos cells coexpress progenitor cell marker CD49f, epithelial marker E-CADHERIN, biliary marker CYTOKERATIN-19, and mesenchymal markers VIMENTIN and alpha-SMOOTH MUSCLE ACTIN (αSMA). Expansion of the PROM1pos cell population is associated with activation of Fibroblast Growth Factor (FGF) and Transforming Growth Factor-beta (TGFß) signaling. In vitro cotreatment of PROM1-expressing Mat1a-/- hepatic progenitor cells with recombinant human FGF10 and TGFß1 promotes morphologic transformation toward a myofibroblastic cell phenotype with increased expression of myofibroblastic genes Collagen-1α1, Fibronectin, and α-Sma. Infants with BA demonstrate similar expansion of periportal PROM1pos cells with activated Mothers Against Decapentaplegic Homolog 3 (SMAD3) signaling in association with increased hepatic expression of FGF10, FGFR1, and FGFR2 as well as mesenchymal genes SLUG and SNAIL. Infants with perinatal subtype of BA have higher tissue levels of PROM1 expression than those with embryonic subtype. CONCLUSION: Expansion of collagen-producing PROM1pos cells within regions of periportal fibrosis is associated with activated FGF and TGFß pathways in both experimental and human BA. PROM1pos cells may therefore play an important role in the biliary fibrosis of BA.

Mesodermal mesenchymal cells give rise to myofibroblasts, but not epithelial cells, in mouse liver injury.

UNLABELLED: Hepatic stellate cells (HSCs) and portal fibroblasts (PFs) are believed to be the major source of myofibroblasts that participate in fibrogenesis by way of synthesis of proinflammatory cytokines and extracellular matrices. Previous lineage tracing studies using MesP1(Cre) and Rosa26lacZ(flox) mice demonstrated that MesP1+ mesoderm gives rise to mesothelial cells (MCs), which differentiate into HSCs and PFs during liver development. In contrast, several in vivo and in vitro studies reported that HSCs can differentiate into other cell types, including hepatocytes, cholangiocytes, and progenitor cell types known as oval cells, thereby acting as stem cells in the liver. To test whether HSCs give rise to epithelial cells in adult liver, we determined the hepatic lineages of HSCs and PFs using MesP1(Cre) and Rosa26mTmG(flox) mice. Genetic cell lineage tracing revealed that the MesP1+ mesoderm gives rise to MCs, HSCs, and PFs, but not to hepatocytes or cholangiocytes, in the adult liver. Upon carbon tetrachloride injection or bile duct ligation surgery-mediated liver injury, mesodermal mesenchymal cells, including HSCs and PFs, differentiate into myofibroblasts but not into hepatocytes or cholangiocytes. Furthermore, differentiation of the mesodermal mesenchymal cells into oval cells was not observed. These results indicate that HSCs are not sufficiently multipotent to produce hepatocytes, cholangiocytes, or oval cells by way of mesenchymal-epithelial transition in vivo. CONCLUSION: Cell lineage tracing demonstrated that mesodermal mesenchymal cells including HSCs are the major source of myofibroblasts but do not differentiate into epithelial cell types such as hepatocytes, cholangiocytes, and oval cells.

Hepatic stellate cells in liver development, regeneration, and cancer.

Hepatic stellate cells are liver-specific mesenchymal cells that play vital roles in liver physiology and fibrogenesis. They are located in the space of Disse and maintain close interactions with sinusoidal endothelial cells and hepatic epithelial cells. It is becoming increasingly clear that hepatic stellate cells have a profound impact on the differentiation, proliferation, and morphogenesis of other hepatic cell types during liver development and regeneration. In this Review, we summarize and evaluate the recent advances in our understanding of the formation and characteristics of hepatic stellate cells, as well as their function in liver development, regeneration, and cancer. We also discuss how improved knowledge of these processes offers new perspectives for the treatment of patients with liver diseases.

Mesothelial cells give rise to hepatic stellate cells and myofibroblasts via mesothelial-mesenchymal transition in liver injury.

In many organs, myofibroblasts play a major role in the scarring process in response to injury. In liver fibrogenesis, hepatic stellate cells (HSCs) are thought to transdifferentiate into myofibroblasts, but the origins of both HSCs and myofibroblasts remain elusive. In the developing liver, lung, and intestine, mesothelial cells (MCs) differentiate into specific mesenchymal cell types; however, the contribution of this differentiation to organ injury is unknown. In the present study, using mouse models, conditional cell lineage analysis has demonstrated that MCs expressing Wilms tumor 1 give rise to HSCs and myofibroblasts during liver fibrogenesis. Primary MCs, isolated from adult mouse liver using antibodies against glycoprotein M6a, undergo myofibroblastic transdifferentiation. Antagonism of TGF-ß signaling suppresses transition of MCs to mesenchymal cells both in vitro and in vivo. These results indicate that MCs undergo mesothelial-mesenchymal transition and participate in liver injury via differentiation to HSCs and myofibroblasts.

Morphogens and hepatic stellate cell fate regulation in chronic liver disease.

Hepatic stellate cells (HSC) are the liver mesenchymal cell type which responds to hepatocellular damage and participates in wound healing. Although HSC myofibroblastic trans-differentiation (activation) is implicated in excessive extracellular matrix deposition, molecular understanding of this phenotypic switch from the viewpoint of cell fate regulation is limited. Recent studies demonstrate the roles of anti-adipogenic morphogens (Wnt, Necdin, Shh) in epigenetic repression of the HSC differentiation gene Pparγ as a causal event in HSC activation. These morphogens have positive cross-interactions which converge to epigenetic repression of Pparγ involving the methyl-CpG binding protein MeCP2. However, these morphogens expressed by activated HSC may also participate in cross-talk between HSC and hepatoblasts/hepatocytes to support liver regeneration, and their aberrant regulation may contribute to liver tumorigenesis. Implications of HSC-derived morphogens in these possibilities are discussed.

Hepatic stellate cell-derived delta-like homolog 1 (DLK1) protein in liver regeneration.

Hepatic stellate cells (HSCs) undergo myofibroblastic activation in liver fibrosis and regeneration. This phenotypic switch is mechanistically similar to dedifferentiation of adipocytes as such the necdin-Wnt pathway causes epigenetic repression of the master adipogenic gene Pparγ, to activate HSCs. Now we report that delta-like 1 homolog (DLK1) is expressed selectively in HSCs in the adult rodent liver and induced in liver fibrosis and regeneration. Dlk1 knockdown in activated HSCs, causes suppression of necdin and Wnt, epigenetic derepression of Pparγ, and morphologic and functional reversal to quiescent cells. Hepatic Dlk1 expression is induced 40-fold at 24 h after partial hepatectomy (PH) in mice. HSCs and hepatocytes (HCs) isolated from the regenerating liver show Dlk1 induction in both cell types. In HC and HSC co-culture, increased proliferation and Dlk1 expression by HCs from PH are abrogated with anti-DLK1 antibody (Ab). Dlk1 and Wnt10b expression by Sham HCs are increased by co-culture with PH HSCs, and these effects are abolished with anti-DLK Ab. A tail vein injection of anti-DLK1 Ab at 6 h after PH reduces early HC proliferation and liver growth, accompanied by decreased Wnt10b, nonphosphorylated ß-catenin, p-ß-catenin (Ser-552), cyclins (cyclin D and cyclin A), cyclin-dependent kinases (CDK4, and CDK1/2), p-ERK1/2, and p-AKT. In the mouse developing liver, HSC precursors and HSCs express high levels of Dlk1, concomitant with Dlk1 expression by hepatoblasts. These results suggest novel roles of HSC-derived DLK1 in activating HSCs via epigenetic Pparγ repression and participating in liver regeneration and development in a manner involving the mesenchymal-epithelial interaction.

Rosmarinic acid and baicalin epigenetically derepress peroxisomal proliferator-activated receptor γ in hepatic stellate cells for their antifibrotic effect.

UNLABELLED: Hepatic stellate cells (HSCs) undergo myofibroblastic transdifferentiation (activation) to participate in liver fibrosis and identification of molecular targets for this cell fate regulation is essential for development of efficacious therapeutic modalities for the disease. Peroxisomal proliferator-activated receptor γ (PPARγ) is required for differentiation of HSCs and its epigenetic repression underlies HSC activation. The herbal prescription Yang-Gan-Wan (YGW) prevents liver fibrosis, but its active ingredients and molecular mechanisms are unknown. Here we demonstrate YGW prevents and reverses HSC activation by way of epigenetic derepression of Pparγ involving reductions in MeCP2 expression and its recruitment to Pparγ promoter, suppressed expression of PRC2 methyltransferase EZH2, and consequent reduction of H2K27di-methylation at the 3' exon. High-performance liquid chromatography / mass spectrometry (HPLC/MS) and nuclear magnetic resonance (NMR) analyses identify polyphenolic rosmarinic acid (RA) and baicalin (BC) as active phytocompounds. RA and BC suppress the expression and signaling by canonical Wnts, which are implicated in the aforementioned Pparγ epigenetic repression. RA treatment in mice with existing cholestatic liver fibrosis inhibits HSC activation and progression of liver fibrosis. CONCLUSION: These results demonstrate a therapeutic potential of YGW and its active component RA and BC for liver fibrosis by way of Pparγ derepression mediated by suppression of canonical Wnt signaling in HSCs.