Choline-deficient Ethionine-supplemented Diet Research Articles

YAP affects the efficacy of liver progenitor cells transplantation in CCl4-induced acute liver injury

The liver is a highly regenerative organ. During acute liver injury, the remaining hepatocytes rapidly proliferate to restore liver parenchyma and liver function. However, hepatocytes-driven regeneration is compromised in severe liver injury; instead, liver progenitor cells (LPCs) proliferate and differentiate into hepatocytes or cholangiocytes to restore mass and function of liver. The Hippo signaling pathway is of vital importance in liver regeneration, and Yes-associated protein (YAP) is the key component of the Hippo pathway. The therapeutic role of YAP has been well studied in hepatocytes-driven liver regeneration. However, the role of LPCs transplantation in acute liver injury has not been defined. Here, we investigated the therapeutic effect of splenic-transplantation of LPCs in CCl4-induced acute liver injury and explored the role of YAP during the procedure. LPCs isolated from choline-deficient, ethionine-supplemented diet (CDE) model were infected with GFP-YAP cDNA lentiviral vector, GFP-YAP shRNA lentiviral vector, and GFP lentiviral vector as control, respectively. At 48 h after CCl4 injection, PBS (control group), GFP lentiviral vector-infected LPCs (GFP-LPC group), GFP-YAP cDNA lentiviral vector-infected LPCs (YAP-LPC group) and GFP-YAP shRNA lentiviral vector-infected LPCs (sh-YAP-LPC group) were injected into spleens in CCl4-treated mice. Histological and serological analyses were performed to evaluate pathology and liver function. The effect of LPCs on the proliferation of hepatocytes and inflammation was investigated. We demonstrated that intra-splenic transplantation of LPCs alleviates CCl4-induced acute liver injury and YAP signaling acts a key role during the procedure. Further studies suggested that LPCs alleviate acute liver injury by promoting pre-existing hepatocytes proliferation rather than differentiating into hepatocytes. Furthermore, intra-splenic transplantation of LPCs attenuates inflammation, which facilitates tissue repair in acute liver injury. In conclusion, LPCs transplantation is a potential treatment for acute liver injury and YAP is a prospective therapeutic target in acute liver injury.

Loss of YB-1 alleviates liver fibrosis by suppressing epithelial-mesenchymal transition in hepatic progenitor cells

Previously, we reported that the nuclear translocation of Y-box binding protein 1 (YB-1) is induced by transforming growth factor-β (TGF-β) and promotes hepatic progenitor cells (HPCs) expansion. Here, we explored the mechanisms underlying YB-1 translocation and the impact of YB-1 on the epithelial–mesenchymal transition (EMT) in HPCs. YB-1flox/floxcre+/− (YB-1f/fcre+/−) mice and YB-1f/fcre−/− mice were fed with a 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) or a choline-deficient, ethionine-supplemented (CDE) diet. Liver injury and fibrosis were assessed by performing hematoxylin and eosin (HE) and Masson staining. The expression of collagen and EMT-related markers (E-cadherin, N-cadherin, and Snail) was detected by reverse transcription-polymerase chain reaction (RT-PCR), western blotting, and immunofluorescence analyses. Protein kinase B (AKT) expression in HPCs was silenced via RNA interference. Nuclear YB-1 expression in HPCs was detected via western blotting and immunofluorescence analyses. HPC proliferation was detected by immunofluorescence. Our results indicate that YB-1 transcriptionally regulated the biological behavior of HPCs. HPC-specific YB-1 knockout alleviated liver fibrosis in mice fed with DDC or CDE diet. YB-1 nuclear translocation promoted matrix metallopeptidase 9 transcription. YB-1 depletion in HPCs significantly dampened the EMT and inhibited AKT phosphorylation in vitro and in vivo. AKT knockdown compromised TGF-β-induced YB-1 nuclear translocation, thereby inhibiting the EMT and HPC proliferation. EMT and AKT were highly activated in HPCs in cirrhotic livers. Collectively, our findings indicate that the loss of YB-1 suppressed EMT in HPCs and alleviated liver fibrosis in mice, and that AKT was essential for TGF-β-induced YB-1 nuclear translocation and HPC proliferation.

USP25 Deficiency Exacerbates Acute Pancreatitis via Up-Regulating TBK1-NF-κB Signaling in Macrophages.

Background & AimsSevere acute pancreatitis can easily lead to systemic inflammatory response syndrome and death. Macrophages are known to be involved in the pathophysiology of acute pancreatitis (AP), and macrophage activation correlates with disease severity. In this study, we examined the role of ubiquitin-specific protease 25, a deubiquitinating enzyme and known regulator of macrophages, in the pathogenesis of AP.MethodsWe used L-arginine, cerulein, and choline-deficient ethionine-supplemented diet–induced models of AP in Usp25-/- mice and wild-type mice. We also generated bone marrow Usp25-/- chimeric mice and initiated L-arginine–mediated AP. Primary acinar cells and bone marrow–derived macrophages were isolated from wild-type and Usp25-/- mice to dissect molecular mechanisms.ResultsOur results show that Usp25 deficiency exacerbates pancreatic and lung injury, neutrophil and macrophage infiltration, and systemic inflammatory responses in L-arginine, cerulein, and choline-deficient ethionine-supplemented diet–induced models of AP. Bone marrow Usp25-/- chimeric mice challenged with L-arginine show that Usp25 deficiency in macrophages exaggerates AP by up-regulating the TANK-binding kinase 1 (TBK1)–nuclear factor-κB (NF-κB) signaling pathway. Similarly, in vitro data confirm that Usp25 deficiency enhances the TBK1–NF-κB pathway, leading to increased expression of inflammatory cytokines in bone marrow–derived macrophages.ConclusionsUsp25 deficiency in macrophages enhances TBK1–NF-κB signaling, and the induction of inflammatory chemokines and type I interferon-related genes exacerbates pancreatic and lung injury in AP.

β-Catenin-NF-κB-CFTR interactions in cholangiocytes regulate inflammation and fibrosis during ductular reaction.

Expansion of biliary epithelial cells (BECs) during ductular reaction (DR) is observed in liver diseases including cystic fibrosis (CF), and associated with inflammation and fibrosis, albeit without complete understanding of underlying mechanism. Using two different genetic mouse knockouts of β-catenin, one with β-catenin loss is hepatocytes and BECs (KO1), and another with loss in only hepatocytes (KO2), we demonstrate disparate long-term repair after an initial injury by 2-week choline-deficient ethionine-supplemented diet. KO2 show gradual liver repopulation with BEC-derived β-catenin-positive hepatocytes and resolution of injury. KO1 showed persistent loss of β-catenin, NF-κB activation in BECs, progressive DR and fibrosis, reminiscent of CF histology. We identify interactions of β-catenin, NFκB, and CF transmembranous conductance regulator (CFTR) in BECs. Loss of CFTR or β-catenin led to NF-κB activation, DR, and inflammation. Thus, we report a novel β-catenin-NFκB-CFTR interactome in BECs, and its disruption may contribute to hepatic pathology of CF.

Maraviroc Prevents HCC Development by Suppressing Macrophages and the Liver Progenitor Cell Response in a Murine Chronic Liver Disease Model.

Simple SummaryLiver stem cells and activated macrophages have been implicated as contributors to liver cancer; hence, reducing their abundance is a potential avenue for therapy. In this article, we demonstrate that Maraviroc, a drug approved for human use, reduces the liver stem cell response and macrophage activation in a mouse model of liver cancer. These findings underline the preventive potential of this drug in liver cancer, a deadly disease for which there are few effective treatments.Maraviroc (MVC), a CCR5 antagonist, reduces liver fibrosis, injury and tumour burden in mice fed a hepatocarcinogenic diet, suggesting it has potential as a cancer therapeutic. We investigated the effect of MVC on liver progenitor cells (LPCs) and macrophages as both have a role in hepatocarcinogenesis. Mice were fed the hepatocarcinogenic choline-deficient, ethionine-supplemented diet (CDE) ± MVC, and immunohistochemistry, RNA and protein expression were used to determine LPC and macrophage abundance, migration and related molecular mechanisms. MVC reduced LPC numbers in CDE mice by 54%, with a smaller reduction seen in macrophages. Transcript and protein abundance of LPC-associated markers correlated with this reduction. The CDE diet activated phosphorylation of AKT and STAT3 and was inhibited by MVC. LPCs did not express Ccr5 in our model; in contrast, macrophages expressed high levels of this receptor, suggesting the effect of MVC is mediated by targeting macrophages. MVC reduced CD45+ cells and macrophage migration in liver and blocked the CDE-induced transition of liver macrophages from an M1- to M2-tumour-associated macrophage (TAM) phenotype. These findings suggest MVC has potential as a re-purposed therapeutic agent for treating chronic liver diseases where M2-TAM and LPC numbers are increased, and the incidence of HCC is enhanced.

4119 Cholecystokinin (CCK) Receptor Antagonist Reverses Nonalcoholic Steatohepatitis (NASH) by Reducing Hepatic Macrophages and Inflammatory Cytokines

OBJECTIVES/GOALS: NASH increases the risk of cirrhosis and liver cancer. High-fat diets increase CCK levels and CCK receptors have been identified on fibroblasts and immune cells. We hypothesized that CCK receptor blockade could prevent NASH by altering the hepatic microenvironment and macrophage activation. METHODS/STUDY POPULATION: Female mice were fed a Choline Deficient Ethionine supplemented (CDE) saturated fat diet or control high-fat diet for 18 weeks. Mice in each group were treated with a CCK receptor antagonist, proglumide (0.1 mg/ml) in the drinking water or regular water. Resected livers were stained for H&E for features of NASH and F4/80 for macrophages analysis. Liver RNA was evaluated for the expression of cytokines and chemokines using an 84-gene Profiler array (Qiagen). Oxidative stress was analyzed by qRT-PCR for heat shock proteins (HSPs) 27, 60, 70 and 90 and for glutathione by a fluorometric assay. Differences in CDE fed and CDE/proglumide-treated mouse livers were evaluated. RESULTS/ANTICIPATED RESULTS: Livers from mice on the CDE diet displayed histologic features of NASH that were prevented by proglumide. Cytokines and chemokines expression, especially CCL20 and CCL2, were increased in the CDE fed mice and these levels were reduced greater than 20-fold with proglumide. Infiltration of F4/80+ macrophages was markedly increased in the CDE livers and these were reduced by > 50% (p < 0.0001) with proglumide. RNA expression of HSP70 (p = 0.006) and HSP27 (p = 0.011) were reduced with proglumide. Hepatic glutathione concentration more than doubled in the CDE/proglumide treated mice compared to CDE mice. CCK-B receptor expression increased in the CDE-fed mouse livers compared to controls. DISCUSSION/SIGNIFICANCE OF IMPACT: CCK receptor blockade decreases NASH by reducing hepatic macrophages, oxidative stress, and blocking inflammatory cytokines and chemokines. This data supports our novel hypothesis that CCK receptors play a role in NASH and proglumide may provide an innovative treatment for this condition.

Human Amnion Epithelial Cells Reduce Liver Fibrosis Via Effects On Macrophages Phenotype And Liver Progenitor Cells In Murine Liver Injury

Background & Aim Background: Chronic liver disease is characterised by progressive hepatocyte injury, inflammation and accumulation of extracellular matrix. Liver progenitor cells (LPC) are bipotent cells induced during liver injury that can regenerate both hepatocytes and cholangiocytes. In addition, macrophages play a pivotal role in fibrogenesis and fibrosis resolution. Classically, macrophages are classified into M1 (pro-inflammatory macrophages) and M2 (pro-resolution macrophages). Recently, Ly6C expression has been used to identify distinct macrophage populations, Ly6Chi (inflammatory) and Ly6Clow (resolution). We are developing a therapy for chronic liver fibrosis using human amnion epithelial cells (hAECs). The choline-deficient ethionine-supplemented (CDE) diet is a non-invasive and quick method to induce liver injury and LPC proliferation. Here, we aimed to investigate the effect of hAECs on macrophages and LPC during liver injury induced by a CDE diet. Aim Methods, Results & Conclusion Methods: C57/B16J mice were commenced on a CDE diet, then 2 or 4 million hAECs were infused intraperitoneally 24h later and the CDE diet continued. Outcomes were assessed at days 1, 3, 5, 14, 21 and 42 following hAEC administrations. Sirius red staining and immunohistochemistry were used to determine fibrosis and identify LPC in liver sections. Macrophages and LPC were further identified and characterised using flow cytometery sorting and PCR. Results In CDE treated mice, hAEC significantly reduced liver fibrosis compared to untreated mice. hAEC decreased the number of proinflammatory (Ly6Chi) monocyte-derived macrophages (MDM) and increased the number of anti-inflammatory and tissue remodelling (Ly6Clow) MDM. hAEC also enhanced the expression of matrix-metalloproteinase (MMP)12 and MMP13 in Ly6CLow sorted cells. LPC numbers were reduced significantly in hAEC treated mice. Conclusion Our data confirm the potential efficacy of hAECs as an effective therapy in liver fibrosis.

The role of Caspase‐1 in diet induced chronic liver injury and blood bile barrier regulation

End‐stage chronic liver disease is a major cause of mortality worldwide. Irrespective of the underlying cause, most chronic liver diseases are characterized by fibrosis, hepatocellular necrosis, inflammation, ductular reactions and proliferation of the liver progenitor cells. Vast differences exist between experimental models that mimic these processes, and their identification is fundamental for translational research. However, the underlying differences in the molecular mechanism driving liver injury in different models remains largely unknown due to our inability to visualize the initiation and progression of liver injury in vivo in mice. Recently we have introduced quantitative Liver Intravital Microscopy (qLIM) that enables real‐time assessment of bile transport and blood‐bile barrier (BBlB) integrity in the intact liver of live mice (Pradhan‐Sundd et al., Gastroenterology, 2018) to visualize injury progression in two widely used experimental models of chronic liver injury ‐the choline‐deficient ethionine‐supplemented (CDE) or 3, 5‐diethoxycarbonyl 1, 4‐dihydrocollidine (DDC) diet, respectively. qLIM analysis revealed that chronic liver injury involves physical breach of BBlB, mixing of blood with bile and loss of bile transport across hepatocytes in both CDE and DDC diet fed mice. Mechanistically we showed an association of disruption of the BBlB and loss of various tight junctional proteins with sustained inflammation and apoptotic cell death (Pradhan‐Sundd et al., Gastroenterology, 2018). Thus, we hypothesized that blocking Caspase ‐1 induced inflammatory response can inhibit the loss of BBlB and thus postpone the chronic liver injury initiation in these diet fed mice. Indeed, mice with deletion of caspase‐1 displayed reduced liver injury, ductular reaction, fibrosis, inflammation, and apoptosis after two weeks of CDE and DDC diet. Remarkably, qLIM analysis showed intact blood biliary barrier at day 4 and 6 of CDE and DDC fed mice suggestive of delay in injury initiation. Furthermore, we demonstrate that the recovery from CDE and DDC diet induced liver injury is significantly accelerated in caspase‐1 KO mouse. We also found upregulation several tight junctional claudins in the liver upon caspase ‐1 knockdown. Current investigation is underway to analyse the regulation of tight junctional claudins by Caspase‐1. These findings may lead insight to disease progression and possible treatment of diet induced chronic liver injury.

A Cholecystokinin Receptor Antagonist Halts Nonalcoholic Steatohepatitis and Prevents Hepatocellular Carcinoma

Background and AimsNonalcoholic steatohepatitis (NASH) is a common inflammatory liver condition that may lead to cirrhosis and hepatocellular carcinoma (HCC). Risk factors for NASH include a saturated fat diet, altered lipid metabolism, and genetic and epigenetic factors, including microRNAs. Serum levels of cholecystokinin (CCK) are elevated in mice and humans that consume a high-saturated fat diet. CCK receptors (CCK-Rs) have been reported on fibroblasts which when activated can induce fibrosis; however, their role in hepatic fibrosis remains unknown. We hypothesized that elevated levels of CCK acting on the CCK-Rs play a role in the development of NASH and in NASH-associated HCC.MethodsWe performed a NASH Prevention study and Reversal study in mice fed a saturated fat 75% choline-deficient–ethionine-supplemented (CDE) diet for 12 or 18 weeks. In each study, half of the mice received untreated drinking water, while the other half received water supplemented with the CCK-R antagonist proglumide. CCK-R expression was evaluated in mouse liver and murine HCC cells.ResultsCCK receptor antagonist treatment not only prevented NASH but also reversed hepatic inflammation, fibrosis, and steatosis and normalized hepatic transaminases after NASH was established. Thirty-five percent of the mice on the CDE diet developed HCC compared with none in the proglumide-treated group. We found that CCK-BR expression was markedly upregulated in mouse CDE liver and HCC cells compared with normal hepatic parenchymal cells, and this expression was epigenetically regulated by microRNA-148a.ConclusionThese results support the novel role of CCK receptors in the pathogenesis of NASH and HCC.

Abstract 2728: Cholecystokinin receptor antagonist proglumide reverses nonalcoholic steatohepatitis and prevents hepatocellular carcinoma

Abstract Background: The fastest growing cause of cancer related death is hepatocellular carcinoma (HCC), in part due to the rising prevalence of nonalcoholic steatohepatitis (NASH). HCC cases are expected to increase during 2015-2030. Cholecystokinin (CCK), blood levels are increased in those consuming high fat diets and CCK receptors have been identified in normal liver, fibroblasts, and immune cells. Hypothesis: We hypothesize that CCK receptor blockade could decrease NASH and prevent HCC in a mouse model.Methods: We examined whether CCK receptor blockade with proglumide could decrease NASH in the choline deficient ethionine supplemented (CDE)-diet mouse model. Two cohorts of Female C57BL/6 mice (N=65) mice were examined: (1) a preventive study in which mice were treated for 12 and 18 weeks to determine if proglumide could prevent NASH and HCC and (2) a ‘reversal study’ in which histologic and biochemical NASH was induced on a 75% CDE high fat diet for 12 weeks followed by treatment with proglumide for 6 weeks (until week 18) while remaining on the CDE diet to determine if CCK-receptor blockade could reverse NASH once established. Control mice received the same high fat diet with choline and half received proglumide therapy. Results: Alanine aminotransferase, aspartate aminotransferases, and bilirubin were increased in mice on the CDE/Regular (untreated water) diet and these values returned to near normal with proglumide therapy in the prevention study and also reversed after NASH was established. Histologic scoring showed significant decreased inflammation, fibrosis and steatosis in mice on the high fat CDE diet when treated with proglumide. 35% of the mice (N=23) at week 18 developed HCC or dysplastic nodules on the CDE diet while none of the mice in the CDE/proglumide prevention (N=15) or reversal therapy groups (N=15) had HCC. Mice on the lard-control diet had fatty livers at week 18 but no evidence of NASH histologically or biochemically and no HCC. Fibrosis was decreased in mice treated with proglumide as evidence by Masson’s trichrome stain and Western blot for FAP. Gene expression of the liver’s collagen-4, TGFβR11, PDGH-Rβ, and collagen-1α1 as determined by qRT-PCR decreased with proglumide treatment. Control diet fed mice had normal serum tests and fibrosis biomarkers. Conclusion: This data supports our novel hypothesis that CCK receptors play a role in NASH-associated HCC. CCK receptor blockade decreased histologic and biochemical evidence of NASH and prevented HCC. Since proglumide has already been tested in human subjects for peptic ulcer disease decades ago and is known to be orally bioavailable, results of our study could easily be translated into a clinical trial in human subjects to improve NASH to prevent HCC. Supported by AGA Research Foundation, Ruesch Foundation, and a grant from NIH, TL1TR001431 Citation Format: Martha D. Gay, Victor Ciofoaia, Sandeep Nadella, Hong Cao, Matthew Huber, Robin D. Tucker, Narayan Shivapurkar, Bhaskar Kallakury, Alexander H. Kroemer, Jill P. Smith. Cholecystokinin receptor antagonist proglumide reverses nonalcoholic steatohepatitis and prevents hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2728.

Hepatic ferroptosis plays an important role as the trigger for initiating inflammation in nonalcoholic steatohepatitis

Nonalcoholic steatohepatitis (NASH) is a metabolic liver disease that progresses from simple steatosis to the disease state of inflammation and fibrosis. Previous studies suggest that apoptosis and necroptosis may contribute to the pathogenesis of NASH, based on several murine models. However, the mechanisms underlying the transition of simple steatosis to steatohepatitis remain unclear, because it is difficult to identify when and where such cell deaths begin to occur in the pathophysiological process of NASH. In the present study, our aim is to investigate which type of cell death plays a role as the trigger for initiating inflammation in fatty liver. By establishing a simple method of discriminating between apoptosis and necrosis in the liver, we found that necrosis occurred prior to apoptosis at the onset of steatohepatitis in the choline-deficient, ethionine-supplemented (CDE) diet model. To further investigate what type of necrosis is involved in the initial necrotic cell death, we examined the effect of necroptosis and ferroptosis inhibition by administering inhibitors to wild-type mice in the CDE diet model. In addition, necroptosis was evaluated using mixed lineage kinase domain-like protein (MLKL) knockout mice, which is lacking in a terminal executor of necroptosis. Consequently, necroptosis inhibition failed to block the onset of necrotic cell death, while ferroptosis inhibition protected hepatocytes from necrotic death almost completely, and suppressed the subsequent infiltration of immune cells and inflammatory reaction. Furthermore, the amount of oxidized phosphatidylethanolamine, which is involved in ferroptosis pathway, was increased in the liver sample of the CDE diet-fed mice. These findings suggest that hepatic ferroptosis plays an important role as the trigger for initiating inflammation in steatohepatitis and may be a therapeutic target for preventing the onset of steatohepatitis.

Roles for Notch‐Sox9 in biliary epithelial cells‐to‐hepatocytes differentiation during liver regeneration

Liver regeneration after most forms of injury is mediated through proliferation of hepatocytes. However, when hepatocyte proliferation is impaired, such as during chronic liver disease, liver progenitor cells (LPCs) from the biliary epithelial cell (BEC) compartment can give rise to hepatocytes to mediate hepatic repair. Promotion of LPC‐to‐hepatocyte differentiation in patients with chronic liver disease could serve as a potential new therapeutic option, but first requires the identification of the molecular mechanisms driving this process. Notch signaling has been identified as an important signaling pathway promoting the BEC fate during development, and has also been implicated in regulating LPC differentiation during regeneration. SRY‐related HMG box transcription factor 9 (Sox9) is a direct target of Notch signaling in the liver, and Sox9 has also been shown to promote the BEC fate during development. We have recently shown that inhibition of Hdac1 activity during liver regeneration in both zebrafish and mouse models enhances sox9 expression in LPCs and impairs LPC‐to‐hepatocyte differentiation. Therefore, we hypothesized that inhibition of Notch signaling would promote LPC‐to‐hepatocyte differentiation by repressing sox9 expression. To this end, we blocked Notch activation during liver regeneration through treatment with γ‐secretase inhibitor LY411575 in a zebrafish LPC‐driven regeneration model and demonstrated enhanced induction of Hnf4a in LPCs. Alternatively, enhancing Notch signaling via Notch3 intracellular domain (N3ICD) overexpression impaired Hnf4a induction. Hepatocyte ablation in sox9b heterozygous mutant embryos enhanced Hnf4a induction, while BEC‐specific Sox9b overexpression impaired LPC‐to‐hepatocyte differentiation. Moreover, LY411575 treatment promoted the differentiation of murine LPCs into hepatocytes in vitro. Lastly, we administrated LY411575 to mice with hepatocyte‐specific deletion of β‐catenin, which induces differentiation of LPCs into hepatocytes following a choline‐deficient, ethionine‐supplemented (CDE) diet. We also examined the effect of liver‐specific Sox9 ablation upon CDE diet liver injury. Our results will elucidate the detailed roles for Notch‐Sox9 signaling in liver regeneration and LPC‐to‐hepatocyte differentiation.Support or Funding InformationThe work was supported by NIH grants to D.S. (DK101426), S.P.M. (DK62277, DK100287, CA204586), and J.O.R. (T32EB0010216, 1F31DK115017). S.P.M. is an Endowed Chair for Experimental Pathology.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Hepatocyte‐Specific β‐Catenin Deletion During Severe Liver Injury Provokes Cholangiocytes to Differentiate Into Hepatocytes

Liver regeneration after injury is normally mediated by proliferation of hepatocytes, although recent studies have suggested biliary epithelial cells (BECs) can differentiate into hepatocytes during severe liver injury when hepatocyte proliferation is impaired. We investigated the effect of hepatocyte‐specific β‐catenin deletion in recovery from severe liver injury and BEC‐to‐hepatocyte differentiation. To induce liver injury, we administered choline‐deficient, ethionine‐supplemented (CDE) diet to three different mouse models, the first being mice with deletion of β‐catenin in both BECs and hepatocytes (Albumin‐Cre; Ctnnb1flox/flox mice). In our second model, we performed hepatocyte lineage tracing by injecting Ctnnb1flox/flox; Rosa‐stopflox/flox‐EYFP mice with the adeno‐associated virus serotype 8 encoding Cre recombinase under the control of the thyroid binding globulin promoter, a virus that infects only hepatocytes. Finally, we performed BEC lineage tracing via Krt19‐CreERT; Rosa‐stopflox/flox‐tdTomato mice. To observe BEC‐to‐hepatocyte differentiation, mice were allowed to recover on normal diet following CDE diet–induced liver injury. Livers were collected from all mice and analyzed by quantitative real‐time polymerase chain reaction, western blotting, immunohistochemistry, and immunofluorescence. We show that mice with lack of β‐catenin in hepatocytes placed on the CDE diet develop severe liver injury with impaired hepatocyte proliferation, creating a stimulus for BECs to differentiate into hepatocytes. In particular, we use both hepatocyte and BEC lineage tracing to show that BECs differentiate into hepatocytes, which go on to repopulate the liver during long‐term recovery. Conclusion: β‐catenin is important for liver regeneration after CDE diet–induced liver injury, and BEC‐derived hepatocytes can permanently incorporate into the liver parenchyma to mediate liver regeneration.

Loss of trefoil factor 1 inhibits biliary regeneration but accelerates the hepatic differentiation of progenitor cells in mice

Although the regeneration of the adult liver depends on hepatic progenitor cells (HPCs), many uncertainties regarding hepatic regeneration in the injured liver remain. Trefoil factor family 1 (TFF1), a secretory protein predominantly expressed in the gastrointestinal tract, is responsible for mucosal restitution. Here, we investigated the role of TFF1 in liver regeneration using a mouse model of hepatic injury (choline-deficient ethionine-supplemented diet and carbon tetrachloride administration) and genetically engineered mice (TFF1 knockout (TFF1−/−)). Immunohistochemistry analysis of human liver samples revealed TFF1 expression in the hepatocytes close to ductular reaction and the regenerating biliary epithelium in injured liver. The number of cytokeratin 19 (CK19)-positive bile ducts was significantly decreased in the TFF1−/− mice after liver injury. Notch pathway in the TFF1−/− mice was also downregulated. HPCs in the control mice differentiated into biliary cells (CK19+/SRY HMG box 9 (SOX9)+) more frequently. In contrast, HPCs in the TFF1−/− mice more frequently differentiated into a hepatic lineage (alpha fetoprotein+/SOX9+) after acute liver damage. Hepatocyte proliferation was upregulated, and the liver weight was increased in TFF1−/− mice in response to chronic liver damage. Thus, TFF1 is responsible for liver regeneration after liver injury by promoting HPC differentiation into a biliary lineage and inhibiting HPC differentiation into a hepatic lineage.

Hdac1 Regulates Differentiation of Bipotent Liver Progenitor Cells During Regeneration via Sox9b and Cdk8

Upon liver injury in which hepatocyte proliferation is compromised, liver progenitor cells (LPCs), derived from biliary epithelial cells (BECs), differentiate into hepatocytes. Little is known about the mechanisms of LPC differentiation. We used zebrafish and mouse models of liver injury to study the mechanisms. We used transgenic zebrafish, Tg(fabp10a:CFP-NTR), to study the effects of compounds that alter epigenetic factors on BEC-mediated liver regeneration. We analyzed zebrafish with disruptions of the histone deacetylase 1 gene (hdac1) or exposed to MS-275 (an inhibitor of Hdac1, Hdac2, and Hdac3). We also analyzed zebrafish with mutations in sox9b, fbxw7, kdm1a, and notch3. Zebrafish larvae were collected and analyzed by whole-mount immunostaining and in situ hybridization; their liver tissues were collected for quantitative reverse transcription polymerase chain reaction. We studied mice in which hepatocyte-specific deletion of β-catenin (Ctnnb1flox/flox mice injected withAdeno-associated virus serotype 8 [AAV8]-TBG-Cre) induces differentiation of LPCs into hepatocytes after a choline-deficient, ethionine-supplemented (CDE) diet. Liver tissues were collected and analyzed by immunohistochemistry and immunoblots. We performed immunohistochemical analyses of liver tissues from patients with compensated or decompensated cirrhosis or acute on chronic liver failure (n= 15). Loss of Hdac1 activity in zebrafish blocked differentiation of LPCs into hepatocytes by increasing levels of sox9b mRNA and reduced differentiation of LPCs into BECs by increasing levels of cdk8 mRNA, which encodes a negative regulator gene of Notch signaling. We identified Notch3 as the receptor that regulates differentiation of LPCs into BECs. Loss of activity of Kdm1a, a lysine demethylase that forms repressive complexes with Hdac1, produced the same defects in differentiation of LPCs into hepatocytes and BECs as observed in zebrafish with loss of Hdac1 activity. Administration of MS-275 to mice with hepatocyte-specific loss of β-catenin impaired differentiation of LPCs into hepatocytes after the CDE diet. HDAC1 was expressed in reactive ducts and hepatocyte buds of liver tissues from patients with cirrhosis. Hdac1 regulates differentiation of LPCs into hepatocytes via Sox9b and differentiation of LPCs into BECs via Cdk8, Fbxw7, and Notch3 in zebrafish with severe hepatocyte loss. HDAC1 activity was also required for differentiation of LPCs into hepatocytes in mice with liver injury after the CDE diet. These pathways might be manipulated to induce LPC differentiation for treatment of patients with advanced liver diseases.

A pivotal role of BEX1 in liver progenitor cell expansion in mice

BackgroundThe activation and expansion of bipotent liver progenitor cells (LPCs) are indispensable for liver regeneration after severe or chronic liver injury. However, the underlying molecular mechanisms regulating LPCs and LPC-mediated liver regeneration remain elusive.MethodsHepatic brain-expressed X-linked 1 (BEX1) expression was evaluated using microarray screening, real-time polymerase chain reaction, immunoblotting and immunofluorescence. LPC activation and liver injury were studied following a choline-deficient, ethionine-supplemented (CDE) diet in wild-type (WT) and Bex1−/− mice. Proliferation, apoptosis, colony formation and hepatic differentiation were examined in LPCs from WT and Bex1−/− mice. Peroxisome proliferator-activated receptor gamma was detected in Bex1-deficient LPCs and mouse livers, and was silenced to analyse the expansion of LPCs from WT and Bex1−/− mice.ResultsHepatic BEX1 expression was increased during CDE diet-induced liver injury and was highly elevated primarily in LPCs. Bex1−/− mice fed a CDE diet displayed impaired LPC expansion and liver regeneration. Bex1 deficiency inhibited LPC proliferation and enhanced LPC apoptosis in vitro. Additionally, Bex1 deficiency inhibited the colony formation of LPCs but had no effect on their hepatic differentiation. Mechanistically, BEX1 inhibited peroxisome proliferator-activated receptor gamma to promote LPC expansion.ConclusionOur findings indicate that BEX1 plays a pivotal role in LPC activation and expansion during liver regeneration, potentially providing novel targets for liver regeneration and chronic liver disease therapies.

Dysregulated bile transporters and impaired tight junctions promote chronic liver injury in mice

Liver fibrosis, hepatocellular necrosis, inflammation and proliferation of liver progenitor cells are the hallmarks of chronic liver injury. Several murine models have been used to mimic the pathophysiology of chronic liver injury. However, the underlying differences in the molecular mechanism driving liver injury in different models remains largely unknown due to our inability to visualize the progression of liver injury in vivo in mice. Here, we use intravital imaging of bile transport and blood‐bile barrier integrity in the intact liver of live mice. We show for the first time that breach of blood‐bile barrier and impairment of bile secretion promotes choline‐deficient‐ethionine‐supplemented (CDE) and 3, 5‐diethoxycarbonyl‐1, 4‐dihydrocollidine (DDC) diets induced chronic liver injury in mice. The impairment in bile secretion was associated with the disruption of the blood‐bile barrier, differential expression of several tight junction adhesion molecules and loss of bile transporters. Surprisingly, the prolonged use of CDE diet led to reappearance of tight junction adhesion molecules as well as bile transporters, which was concomitant to the reestablishment of blood‐bile barrier integrity and rescue of bile secretion. Our findings demonstrate the potential of intravital imaging in elucidating the underlying pathophysiology in mice models of chronic liver injury and identify the loss of bile transporters or tight junction adhesion molecules as two distinct mechanisms contributing to progression of chronic liver injury.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Effects of Berberine on Acute Necrotizing Pancreatitis and Associated Lung Injury.

We set out to examine whether berberine (BBR) might affect the severity of pancreatitis and pancreatitis-associated lung injury in choline-deficient ethionine-supplemented (CDE) diet-induced severe acute pancreatitis. Severe acute pancreatitis was induced by feeding a CDE diet for 3 days. Berberine was administered intraperitoneally during CDE diet. Mice were killed on days 1, 2, and 3 after the onset of CDE diet. The severity of pancreatitis was assessed by evaluating changes to the pancreas and lung and survival rate. Blood, pancreas, and lung were harvested for further examination. Furthermore, the regulating mechanisms of BBR were evaluated on the pancreas. Administration of BBR significantly inhibited histological damage to the pancreas and lung and decreased serum level of amylase and lipase, myeloperoxidase activity, cytokine production, and the mortality rate. Furthermore, administration of BBR inhibited activation of nuclear factor kappa B, c-Jun N-terminal kinases, and p38 in the pancreas during CDE diet. These findings suggest that BBR attenuates the severity of pancreatitis by inhibiting activation of nuclear factor kappa B, c-Jun N-terminal kinase, and p38 and that BBR could be used as a beneficial agent to regulate AP.

Imatinib accelerates progenitor cell-mediated liver regeneration in choline-deficient ethionine-supplemented diet-fed mice.

Severe chronic hepatic injury can induce complex reparative processes. Ductular reaction and the appearance of small hepatocytes are standard components of this response, which is thought to have both adverse (e.g. fibrosis, carcinogenesis) and beneficial (regeneration) consequences. This complex tissue reaction is regulated by orchestrated cytokine action. We have investigated the influence of the tyrosine kinase inhibitor imatinib on a regenerative process. Ductular reaction was induced in mice by the widely used choline-deficient ethionine-supplemented diet (CDE). Test animals were treated daily with imatinib. After 6weeks of treatment, imatinib successfully reduced the extent of ductular reaction and fibrosis in the CDE model. Furthermore, the number of small hepatocytes increased, and these cells had high proliferative activity, were positive for hepatocyte nuclear factor 4 and expressed high levels of albumin and peroxisome proliferator-activated receptor alpha. The overall functional zonality of the hepatic parenchyma (cytochrome P450 2E1 and glucose 6 phosphatase activity; endogenous biotin content) was maintained. The expression of platelet-derived growth factor receptor beta, which is the major target of imatinib, was downregulated. The anti-fibrotic activity of imatinib has already been reported in several experimental models. Additionally, in the CDE model imatinib was able to enhance regeneration and preserve the functional arrangement of hepatic lobules. These results suggest that imatinib might promote the recovery of the liver following parenchymal injury through the inhibition of platelet-derived growth factor receptor beta.

Carbon monoxide-bound hemoglobin vesicles ameliorate multiorgan injuries induced by severe acute pancreatitis in mice by their anti-inflammatory and antioxidant properties.

Carbon monoxide (CO) has attracted attention as a possible therapeutic agent for affecting anti-inflammatory and antioxidant activities. Previously, CO-bound hemoglobin vesicle (CO-HbV) was developed as a nanotechnology-based CO donor, and its safety profile and therapeutic potential as a clinically applicable carrier of CO were examined in vitro and in vivo. In the present study, the therapeutic efficacy of CO-HbV against severe acute pancreatitis was examined with secondary distal organ-injured model mice that were fed with a choline-deficient ethionine-supplemented diet. A CO-HbV treatment significantly reduced the mortality of the acute pancreatitis model mice compared to saline and HbV. Biochemical and histological evaluations clearly showed that CO-HbV suppressed acute pancreatitis by inhibiting the production of systemic proinflammatory cytokines, neutrophil infiltration, and oxidative injuries in pancreatic tissue. Interestingly, CO-HbV also diminished the subsequent damage to distal organs including liver, kidneys, and lungs. This could be due to the suppression of neutrophil infiltration into tissues and the subsequently enhanced oxidative injuries. In contrast, O2-bound HbV, the inactive form of CO-HbV, was ineffective against both pancreatitis and distal organ injuries, confirming that CO was directly responsible for the protective effects of CO-HbV in acute pancreatitis. These findings suggest that CO-HbV has anti-inflammatory and antioxidant characteristics of CO and consequently exerts a superior protective effect against acute pancreatitis-induced multiorgan damage.