Biliary Proliferation Research Articles

Hepatic lymphangiogenesis regulates biliary cell proliferation during liver regeneration after partial hepatectomy

Hepatic lymphangiogenesis regulates biliary cell proliferation during liver regeneration after partial hepatectomy

Investigation of cytogenetic, electrophoretic, histopathological and biochemical effects of walnut (juglans regia l.) Leaf extract in rats experimentally induced diabetes by streptozotocin

In this study, the effects of Juglans regia L. (JR) leaf extract on histological damage and cytogenetic, electrophoretic, and biochemical parameters in the liver and kidney tissues of diabetic rats were investigated. In the study, 60 male rats (Sprague-Dawley) were separated into six groups. According to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) data, it was determined that there were increases and decreases in different serum protein expressions because of the treatment of JR leaf extract in the diabetes group. In the kidney tissue of the diabetes group, an increase in volume in the glomeruli and narrowing of the Bowman's space as well as thickening of the walls of tubules were detected. Vacuolization and shedding were observed in the epithelial cells of tubules in the cortical regions in kidney. In diabetic + JR extract groups administered JR leaf extract at doses of 250 mg/kg and 500 mg/kg, serum AST and ALT levels were reduced compared to the diabetic group. Diabetic rats' livers showed spotted necrosis and fibrosis in the portal area, biliary tract proliferation, mild inflammation, increased vascularization, unicellular necrosis in hepatocytes, and sinusoidal dilatation. The JR leaf extract group did not exhibit these problems. JR leaf extract influences reducing hepatotoxic and oxidative damage due to diabetes and increasing the level of antioxidant enzymes. As a result, it was concluded that the application of JR leaf extract may have a protective effect against damage caused by diabetes.

CsHscB Derived from a Liver Fluke Clonorchis sinensis Ameliorates Cholestatic Hepatic Fibrosis in a Mouse Model of Sclerosing Cholangitis.

Primary sclerosing cholangitis (PSC) is a chronic cholestatic liver disease characterized by inflammatory fibrosis usually involving the whole biliary tree. However, there are very limited treatment options to treat this disease. Our previous study found a lipid-protein rCsHscB from a liver fluke - Clonorchis sinensis, which had full capacities of immune regulation. Therefore, we investigated the role of rCsHscB in a mouse model of sclerosing cholangitis induced by xenobiotic 3,5- diethoxycarbonyl-1,4-dihydrocollidine (DDC) to explore whether this protein had potential therapeutic value for PSC. Mice were fed 0.1% DDC for 4 weeks and treated with CsHscB (30 μg/mouse, intraperitoneal injection, once every 3 days); the control group was given an equal amount of PBS or CsHscB under normal diet conditions. All the mice were sacrificed at 4 weeks for the evaluation of biliary proliferation, fibrosis, and inflammation. rCsHscB treatment attenuated DDC-induced liver congestion and enlargement and significantly decreased the upregulation of serum AST and ALT levels. The administration of rCsHscB to DDC-fed mice significantly decreased cholangiocyte proliferation and pro-inflammatory cytokine production compared to mice fed with DDC alone. Also, rCsHscB treatment showed a decreased expression of α-SMA in the liver and other markers of liver fibrosis (Masson staining, Hydroxyproline content, and collagen deposit). More interestingly, DDC-fed mice treated with rCsHscB showed a significant up-regulation of PPAR-γ expression, which was similar to control mice, indicating the involvement of PPAR-γ signaling in the protective action of rCsHscB. Overall, our data show that rCsHscB attenuates the progression of cholestatic fibrosis induced by DDC and supports the potential for manipulating the parasite-derived molecule to treat certain immune-mediated disorders.

Endogenous AMPKα2 Mediates the Inhibition of Biliary Fibroblasts Proliferation

Background: Although it has been established that activating adenosine monophosphateactivated protein kinase (AMPK) inhibits cell proliferation in several cells, it is unknown whether AMPK is involved in inhibiting biliary fibroblast growth. Objective: The objective of this study is to specifically investigate the influence of AMPK isoforms on proliferation. Methods: To further address its underlying molecular mechanisms, primary cultured rat biliary fibroblasts were transfected with sequence-specific AMPK1 or AMPK2 siRNA. Results: Our findings show that knocking down AMPK2 greatly increased the proliferation of primary cultured biliary fibroblasts, accompanied by the activation of mTOR, an increase in S-phase kinaseassociated protein 2 (Skp2) expression, and a decrease in p27 protein levels. AMPK2 inhibition-triggered Skp2 overexpression and concomitant p27 decrease, as well as biliary fibroblast proliferation, were reversed by rapamycin inhibition or previous silencing of Skp2 production by targeted small interfering RNA (siRNA) transfection. Conclusion: We concluded that AMPK2 regulates the mTOR/Skp2/p27 signaling pathway and causes endogenous suppression of primary cultured biliary fibroblast growth. The reduction of biliary fibroblast proliferation by AMPK2 could be a potential method in treating benign biliary stricture (BBS).

Smad4 restricts injury-provoked biliary proliferation and carcinogenesis.

Cholangiocarcinoma (CCA) is a deadly and heterogeneous type of cancer characterized by a spectrum of epidemiologic associations as well as genetic and epigenetic alterations. We seek to understand how these features inter-relate in the earliest phase of cancer development and through the course of disease progression. For this, we studied murine models of liver injury integrating the most commonly occurring gene mutations of CCA - including Kras, Tp53, Arid1a and Smad4 - as well as murine hepatobiliary cancer models and derived primary cell lines based on these mutations. Among commonly mutated genes in CCA, we found that Smad4 functions uniquely to restrict reactive cholangiocyte expansion to liver injury through restraint of the proliferative response. Inactivation of Smad4 accelerates carcinogenesis, provoking pre-neoplastic biliary lesions and CCA development in an injury setting. Expression analyses of Smad4-perturbed reactive cholangiocytes and CCA lines demonstrated shared enriched pathways, including cell-cycle regulation, MYC signaling and oxidative phosphorylation, suggesting that Smad4 may act via these mechanisms to regulate cholangiocyte proliferation and progression to CCA. Overall, we showed that TGFβ/SMAD4 signaling serves as a critical barrier restraining cholangiocyte expansion and malignant transformation in states of biliary injury.

Nicotine promotes development of bile duct ligation-induced liver fibrosis by increasing expression of nicotinic acetylcholine receptors in rats.

Liver fibrosis and cigarette smoking seem to be directly linked. Nicotine, as an agonist of nicotinic acetylcholine receptors (nAChRs), induces many downstream signaling pathways. The pathways through which nicotine affects the process of liver fibrosis have not been clarified. The present study aimed to investigate the nicotine-induced effects on fibrosis progression in cholestatic rats. First, the Wistar rats were subjected to sham or bile duct ligation (BDL) surgery. The rats were treated with low and high doses of nicotine (1 or 10 mg/kg) for three weeks. They were monitored for their body weights before and 21 days after BDL. Also, spleens were weighed to calculate the spleen/body weight ratio. Ductular proliferation and fibrosis were evaluated using hematoxylin and eosin (H&E) as well as Masson's trichrome staining. The mRNA expression of α4nAChR, α7nAChR, and fibrosis gene α-smooth muscle actin (α-SMA) was measured by real-time PCR. The findings showed that nicotine promotes the development of BDL-induced liver fibrosis. The ratio of spleen/body weight was significantly affected by nicotine exposure. H&E and Masson's trichrome staining showed that the level of liver fibrosis was higher in the cholestatic BDL groups, and this effect was significantly augmented in the nicotine-treated rats. Also, α4nAChR, α7nAChR, and α-SMA expression was observed in the BDL rats and increased following nicotine treatment. The activation of nAChR triggers biliary proliferation and liver fibrosis. Studying the intracellular mechanism of nicotine and alteration in the expression of nicotinic receptors following nicotine exposure can be useful both in diagnosing nicotine-related diseases and finding new treatment strategies.

Erratum: Modulation of the tryptophan hydroxylase 1/monoamine oxidase-A/5-hydroxytryptamine/5-hydroxytryptamine receptor 2A/2B/2C axis regulates biliary proliferation and liver fibrosis during cholestasis.

Erratum: Modulation of the tryptophan hydroxylase 1/monoamine oxidase-A/5-hydroxytryptamine/5-hydroxytryptamine receptor 2A/2B/2C axis regulates biliary proliferation and liver fibrosis during cholestasis.

Alpha Lipoic Acid (ALA) Alleviates Hepatocytes Toxicity of Titanium Dioxide Nanoparticles in Rats

BACKGROUND: Titanium dioxide nanoparticles (TiO2 NPs) uptake may primarily cause adverse effects by inducing oxidative stress, resulting in cell damage, genotoxicity, inflammation, and immune response. To date, there are limited studies investigating the adverse effect of TiO2 NPs on liver health and no studies found a naturally occurring compound able to ameliorate such effect. Thus, this study investigated alpha lipoic acid (ALA) potential for reversing the biochemical and histopathological changes that TiO2 NPs exposure causes in rat liver.METHODS: Thirty adult male albino rats were divided into: control rats received distilled water, control rats treated with 50 mg/kg ALA, rats intoxicated with TiO2 NPs, and TiO2 NPs-intoxicated rats treated 50 mg/kg ALA. Rats were sacrificed before blood samples were collected to assess the liver function using parameters of alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin, albumin and total protein. Liver tissue homogenates were prepared to assess hepatic antioxidant and oxidative stress using parameters of superoxide dismutase (SOD), catalase (CAT), glutathione (GSH), and malondialdehyde (MDA). Liver tissue sections were used for histopathological analysis and caspase-3 immunohistochemical analysis.RESULTS: TiO2 NPs produced deleterious effects on rat liver tissue, as confirmed through biochemical results, caspase-3 immunohistochemistry, and histological alterations. TiO2 NPs intoxication induced hepatocyte vacuolation, blood vessels congestion, biliary proliferation, apoptosis, and fibrosis. However, ALA treatment of TiO2 NPs-intoxicated rats significantly alleviated deleterious impact on the liver.CONCLUSION: Administration of 600 mg/kg TiO2 NPs to rats resulted in hepatic degenerative lesions, depletion of GSH, oxidative stress, and apoptosis. However, these changes were mitigated by ALA administration. Therefore, ALA offers protection against deleterious effects of TiO2 NPs intoxication.KEYWORDS: titanium dioxide nanoparticles, TiO2 NPs, hepatotoxicity, alpha-lipoic acid (ALA), rat

Ductular reaction-associated neutrophils promote biliary epithelium proliferation in chronic liver disease

Ductular reaction expansion is associated with poor prognosis in patients with advanced liver disease. However, the mechanisms promoting biliary cell proliferation are largely unknown. Here, we identify neutrophils as drivers of biliary cell proliferation and the defective wound-healing response. The intrahepatic localization of neutrophils was evaluated in patients with chronic liver disease. Neutrophil dynamics were analyzed by intravital microscopy and neutrophil-labeling assays in DDC-treated mice. Neutrophil depletion or inhibition of recruitment was achieved using a Ly6g antibody or a CXCR1/2 inhibitor, respectively. Mice deficient in PAD4 (peptidyl arginine deiminase 4) and ELANE/NE (neutrophil elastase) were used to investigate the mechanisms underlying ductular reaction expansion. In this study we describe a population of ductular reaction-associated neutrophils (DRANs), which are in direct contact with biliary epithelial cells in chronic liver diseases and whose numbers increased in parallel with disease progression. We show that DRANs are immobilized at the site of ductular reaction for a prolonged period of time. In addition, liver neutrophils display a unique phenotypic and transcriptomic profile, showing a decreased phagocytic capacity and increased oxidative burst. Depletion of neutrophils or inhibition of their recruitment reduces DRANs and the expansion of ductular reaction, while mitigating liver fibrosis and angiogenesis. Mechanistically, neutrophils deficient in PAD4 and ELANE abrogate neutrophil-induced biliary cell proliferation, thus indicating the role of neutrophil extracellular traps and elastase release in ductular reaction expansion. Overall, our study reveals the accumulation of DRANs as a hallmark of advanced liver disease and a potential therapeutic target to mitigate ductular reaction and the maladaptive wound-healing response. Our results indicate that neutrophils are highly plastic and can have an extended lifespan. Moreover, we identify a new role of neutrophils as triggers of expansion of the biliary epithelium. Overall, the results of this study indicate that ductular reaction-associated neutrophils (or DRANs) are new players in the maladaptive tissue-healing response in chronic liver injury and may be a potential target for therapeutic interventions to reduce ductular reaction expansion and promote tissue repair in advanced liver disease.

Elevated Serum Alpha-fetoprotein Levels in Non-alcoholic Steatohepatitis: Possible Molecular Mechanisms and Potential Clinical Significance

With the improvement of living standards in recent years, up to 90% of obese patients have nonalcoholic fatty liver disease (NAFLD). The number of nonalcoholic steatohepatitis (NASH)-related deaths will gradually increase, and NASH is expected to be the most common cause of liver-related deaths in the future. Therefore, there is an urgent need to find effective and reliable serum biomarkers to distinguish simple hepatic steatosis (SS) from NASH. Liver cell regeneration, oxidative stress-induced DNA methylation, and biliary epithelial cell proliferation were reported to increase serum alpha-fetoprotein (AFP) levels. AFP has long been used as a tool to monitor liver cancer. However, the function of AFP in NAFLD, especially NASH, has not been clarified. Moreover, whether an elevated AFP level indicates the occurrence of NASH or serves as a serum biomarker remains to be elucidated. The miRNA-122 pathway, DNA methylation and DNA damage, and activation of resident stem cells and/or progenitor cells in the liver, as well as necrosis, regeneration, and repair of liver cells, may contribute to slight increases in AFP levels in the development of NASH in patients with NAFLD. Furthermore, mildly elevated AFP levels may indicate the development of NASH. This review explores the role of elevated AFP levels in the development of NASH, with a specific focus on the underlying molecular mechanisms and the clinical significance.

Lantana camara poisoning in cattle that took refuge during a storm in a forest invaded by this plant

An outbreak of poisoning by Lantana camara occurred in cattle when a herd sought refuge in a Eucalyptus forest heavily infested by this plant. The animals showed apathy, elevated serum activities of hepatic enzymes, severe photosensitivity, jaundice, hepatomegaly and nephrosis. After a clinical manifestation period of 2–15 days, 74 out of 170 heifers died. The main histological changes were random hepatocellular necrosis, cholestasis, biliary proliferation and, in one animal, centrilobular necrosis. Immunostaining for Caspase 3 detected scattered apoptotic hepatocytes.

Open hepatic artery flow with portal vein clamping protects against bile duct injury compared to pringles maneuver

Background Conventional hepatic artery and portal vein clamping strategies can prevent blood loss and ischemia-reperfusion liver injury, and such preventative measures are the key to successful liver surgery. However, ischemic-induced damage to cholangiocytes is rarely considered. Here, we aimed to investigate the effect of different hepatic inflow interruption methods on bile duct injury. Methods Forty rats were randomly grouped as sham, Pringle maneuver (PM) and hepatic arterial blood flow open (HAFO) groups. We evaluated liver histology and function in liver sections, and biliary histology, cholangiocyte apoptosis and proliferation, cytokine production, and bile composition. RNA sequencing is performed to explore possible molecular mechanisms. The Blood-biliary barrier permeability and tight junctions were analyzed by HRP injection, immunofluorescence staining and analysis of ZO-1 expression by immunoblotting. Results HAFO significantly attenuated ischemia-induced liver injury and decreased ALT, ALP, TBIL, and DBIL levels in serum. The histopathological observations showed that bile duct injury in the PM group was more serious than that in the HAFO group. The numbers of apoptotic biliary epithelial cells in HAFO-treated rat bile ducta were lower than those in the PM group. RNA-seq showed that tight junctions may be related to the mechanism underlying the protective effect of HAFO, as shown by the reduced HRP levels and increased ZO-1 and claudin-1/3 expression in the HAFO group compared to the PM group. Conclusion Compared with PM, HAFO alleviated the ischemic injury to the biliary system, which was characterized by decreased biliary epithelial cell apoptosis, reduced inflammatory responses and decreased blood-biliary-barrier permeability.

Modeling bile duct ischemia and reoxygenation injury inhuman cholangiocyte organoids for screening of novel cholangio-protective agents.

Ischemia of the bile duct is a common feature in liver disease and transplantation, which represents a major cause of morbidity and mortality, especially after liver transplantation. Detailed knowledge of its pathogenesis remains incomplete due to the lack of appropriate invitro models. To recapitulate biliary damage induced by ischemia and reperfusion invitro, human intrahepatic cholangiocyte organoids (ICOs) were grown at low oxygen levels of 1% up to 72h, followed by re-oxygenation at normal levels. ICOs stressed by ischemia and subsequent re-oxygenation represented the dynamic change in biliary cell proliferation, upregulation of epithelial-mesenchymal transition (EMT)-associated markers, and the evocation of phase-dependent cell death programs similar to what is described in patients. Clinical-grade alpha-1 antitrypsin was identified as a potent inhibitor of both ischemia-induced apoptosis and necroptosis. These findings demonstrate that ICOs recapitulate ischemic cholangiopathy invitro and enable drug assessment studies for the discovery of new therapeutics for ischemic cholangiopathies. Dutch Digestive FoundationMLDS D16-26; TKI-LSH (Topconsortium Kennis en Innovatie-Life Sciences & Health) grant RELOAD, EMC-LSH19002; Medical Delta program "Regenerative Medicine 4D"; China Scholarship Council No. 201706230252.

Update on Etiology and Pathogenesis of Biliary Atresia.

Biliary atresia is a rare inflammatory sclerosing obstructive cholangiopathy that initiates in infancy as complete choledochal blockage and progresses to the involvement of intrahepatic biliary epithelium. Growing evidence shows that biliary atresia is not a single entity with a single etiology but a phenotype resulting from multifactorial events whose common path is obliterative cholangiopathy. The etiology of biliary atresia has been explained as resulting from genetic variants, toxins, viral infection, chronic inflammation or bile duct lesions mediated by autoimmunity, abnormalities in the development of the bile ducts, and defects in embryogenesis, abnormal fetal or prenatal circulation and susceptibility factors. It is increasingly evident that the genetic and epigenetic predisposition combined with the environmental factors to which the mother is exposed are potential triggers for biliary atresia. There is also an indication that a progressive thickening of the arterial middle layer occurs in this disease, suggestive of vascular remodeling and disappearance of the interlobular bile ducts. It is suggested that the hypoxia/ischemia process can affect portal structures in biliary atresia and is associated with both the extent of biliary proliferation and the thickening of the medial layer.

Biliary epithelial cells are facultative liver stem cells during liver regeneration in adult zebrafish.

The liver is a highly regenerative organ, yet the presence of a dedicated stem cell population remains controversial. Here, we interrogate a severe hepatocyte injury model in adult zebrafish to define that regeneration involves a stem cell population. After near-total hepatocyte ablation, single-cell transcriptomic and high-resolution imaging analyses throughout the entire regenerative timeline reveal that biliary epithelial cells undergo transcriptional and morphological changes to become hepatocytes. As a population, biliary epithelial cells give rise to both hepatocytes and biliary epithelial cells. Biliary epithelial cells proliferate and dedifferentiate to express hepatoblast transcription factors prior to hepatocyte differentiation. This process is characterized by increased MAPK, PI3K, and mTOR signaling, and chemical inhibition of these pathways impairs biliary epithelial cell proliferation and fate conversion. We conclude that, upon severe hepatocyte ablation in the adult liver, biliary epithelial cells act as facultative liver stem cells in an EGFR-PI3K-mTOR-dependent manner.

Secretin alleviates biliary and liver injury during late-stage primary biliary cholangitis via restoration of secretory processes.

Primary biliary cholangitis (PBC) is characterised by ductopenia, ductular reaction, impairment of anion exchanger 2 (AE2) and the 'bicarbonate umbrella'. Ductulo-canalicular junction (DCJ) derangement is hypothesised to promote PBC progression. The secretin (Sct)/secretin receptor (SR) axis regulates cystic fibrosis transmembrane receptor (CFTR) and AE2, thus promoting choleresis. We evaluated the role of Sct/SR signalling on biliary secretory processes and subsequent injury in a late-stage PBC mouse model and human samples. At 32 weeks of age, female and male wild-type and dominant-negative transforming growth factor beta receptor II (late-stage PBC model) mice were treated with Sct for 1 or 8 weeks. Bulk RNA-sequencing was performed in isolated cholangiocytes from mouse models. Biliary Sct/SR/CFTR/AE2 expression and bile bicarbonate levels were reduced in late-stage PBC mouse models and human samples. Sct treatment decreased bile duct loss, ductular reaction, inflammation, and fibrosis in late-stage PBC models. Sct reduced hepatic bile acid levels, modified bile acid composition, and restored the DCJ and 'bicarbonate umbrella'. RNA-sequencing identified that Sct promoted mature epithelial marker expression, specifically anterior grade protein 2 (Agr2). Late-stage PBC models and human samples exhibited reduced biliary mucin 1 levels, which were enhanced by Sct treatment. Loss of Sct/SR signalling in late-stage PBC results in a faulty 'bicarbonate umbrella' and reduced Agr2-mediated mucin production. Sct restores cholangiocyte secretory processes and DCJ formation through enhanced mature cholangiocyte phenotypes and bile duct growth. Sct treatment may be beneficial for individuals with late-stage PBC. Secretin (Sct) regulates biliary proliferation and bicarbonate secretion in cholangiocytes via its receptor, SR, and in mouse models and human samples of late-stage primary biliary cholangitis (PBC), the Sct/SR axis is blunted along with loss of the protective 'bicarbonate umbrella'. We found that both short- and long-term Sct treatment ameliorated ductular reaction, immune cell influx, and liver fibrosis in late-stage PBC mouse models. Importantly, Sct treatment promoted bicarbonate and mucin secretion and hepatic bile acid efflux, thus reducing cholestatic and toxic bile acid-associated injury in late-stage PBC mouse models. Our work perpetuates the hypothesis that PBC pathogenesis hinges on secretory defects, and restoration of secretory processes that promote the 'bicarbonate umbrella' may be important for amelioration of PBC-associated damage.

Interleukin-33 deficiency prevents biliary injuries and repairments caused by Clonorchis sinensis via restraining type 2 cytokines

BackgroundClonorchiasis caused by Clonorchis sinensis is a zoonotic parasitic disease characterized by cholangitis, biliary proliferation, biliary fibrosis, and even cholangiocarcinoma. Our previous study showed that the expression of interleukin (IL)-33 is increased in both humans and mice infected by C. sinensis, suggesting that IL-33 is potentially involved in the pathogenesis of clonorchiasis. However, the roles and potential mechanism of IL-33 underlying remain unknown.MethodsWild-type (WT) and IL-33 knockout (KO) mice (BALB/c female mice) were orally infected with 45 metacercariae of C. sinensis for 8 weeks. Biliary injuries and fibrosis were extensively evaluated. Hepatic type II cytokines (IL-4, IL-13, and IL-10) were detected by ELISA.ResultsFor wild-type mice, we found that the mice infected with C. sinensis showed severe biliary injuries and fibrosis compared with the normal mice that were free from worm infection. In addition, the levels of type II cytokines such as IL-4, IL-13, and IL-10 in infected wild-type mice were significantly higher than in the control mice without infection (P < 0.05). However, IL-33 deficiency (IL-33 KO) prevents the augmentation of biliary injuries and fibrosis caused by C. sinensis infection. Furthermore, the increased levels of these type II cytokines induced by worm infection were also reversed in IL-33 KO mice.ConclusionOur present study demonstrates that IL-33 contributes to the pathogenesis of C. sinensis-induced biliary injuries and repair, which can potentially orchestrate type 2 responses. These findings highlight the pathophysiological role of IL-33 in the progression of clonorchiasis.

Knockout of liver fluke granulin, Ov-grn-1, impedes malignant transformation during chronic infection with Opisthorchis viverrini.

Infection with the food-borne liver fluke Opisthorchis viverrini is the principal risk factor for cholangiocarcinoma (CCA) in the Mekong Basin countries of Thailand, Lao PDR, Vietnam, Myanmar and Cambodia. Using a novel model of CCA, involving infection with gene-edited liver flukes in the hamster during concurrent exposure to dietary nitrosamine, we explored the role of the fluke granulin-like growth factor Ov-GRN-1 in malignancy. We derived RNA-guided gene knockout flukes (ΔOv-grn-1) using CRISPR/Cas9/gRNA materials delivered by electroporation. Genome sequencing confirmed programmed Cas9-catalyzed mutations of the targeted genes, which was accompanied by rapid depletion of transcripts and the proteins they encode. Gene-edited parasites colonized the biliary tract of hamsters and developed into adult flukes. However, less hepatobiliary tract disease manifested during chronic infection with ΔOv-grn-1 worms in comparison to hamsters infected with control gene-edited and mock-edited parasites. Specifically, immuno- and colorimetric-histochemical analysis of livers revealed markedly less periductal fibrosis surrounding the flukes and less fibrosis globally within the hepatobiliary tract during infection with ΔOv-grn-1 genotype worms, minimal biliary epithelial cell proliferation, and significantly fewer mutations of TP53 in biliary epithelial cells. Moreover, fewer hamsters developed high-grade CCA compared to controls. The clinically relevant, pathophysiological phenotype of the hepatobiliary tract confirmed a role for this secreted growth factor in malignancy and morbidity during opisthorchiasis.

HIF-1alpha-pathway activation in cholangiocytes of patients with biliary atresia: An immunohistochemical/molecular exploratory study

BackgroundBiliary atresia is a neonatal disease characterized by choledochal obstruction and progressive cholangiopathy requiring liver transplantation in most patients. Hypoxia-ischemia affecting the biliary epithelium may lead to biliary obstruction. We hypothesized that ischemic cholangiopathy involving disruption of the peribiliary vascular plexus could act as a triggering event in biliary atresia pathogenesis. MethodsLiver and porta hepatis paraffin-embedded samples of patients with biliary atresia or intrahepatic neonatal cholestasis (controls) were immunohistochemically evaluated for HIF-1alpha-nuclear signals. Frozen histological samples were analyzed for gene expression in molecular profiles associated with hypoxia-ischemia. Prospective clinical-laboratory and histopathological data of biliary atresia patients and controls were reviewed. ResultsImmunohistochemical HIF-1alpha signals localized to cholangiocytes were detected exclusively in liver specimens from biliary atresia patients. In 37.5% of liver specimens, HIF-1alpha signals were observed in biliary structures involving progenitor cell niches and peribiliary vascular plexus. HIF-1alpha signals were also detected in biliary remnants of 81.8% of porta hepatis specimens. Increased gene expression of molecules linked to REDOX status, biliary proliferation, and angiogenesis was identified in biliary atresia liver specimens. In addition, there was a trend towards decreased GSR expression levels in the HIF-1alpha-positive group compared to the HIF-1alpha-negative group. ConclusionActivation of the HIF-1alpha pathway may be associated with the pathogenesis of biliary atresia, and additional studies are necessary to confirm the significance of this finding. Ischemic cholangiopathy and REDOX status disturbance are putative explanations for HIF-1alpha activation. These findings may give rise to novel lines of clinical and therapeutic investigation in the BA field.

A missense mutation in the proprotein convertase gene furinb causes hepatic cystogenesis during liver development in zebrafish.

Hepatic cysts are fluid‐filled lesions in the liver that are estimated to occur in 5% of the population. They may cause hepatomegaly and abdominal pain. Progression to secondary fibrosis, cirrhosis, or cholangiocarcinoma can lead to morbidity and mortality. Previous studies of patients and rodent models have associated hepatic cyst formation with increased proliferation and fluid secretion in cholangiocytes, which are partially due to impaired primary cilia. Congenital hepatic cysts are thought to originate from faulty bile duct development, but the underlying mechanisms are not fully understood. In a forward genetic screen, we identified a zebrafish mutant that developed hepatic cysts during larval stages. The cyst formation was not due to changes in biliary cell proliferation, bile secretion, or impairment of primary cilia. Instead, time‐lapse live imaging data showed that the mutant biliary cells failed to form interconnecting bile ducts because of defects in motility and protrusive activity. Accordingly, immunostaining revealed a disorganized actin and microtubule cytoskeleton in the mutant biliary cells. By whole‐genome sequencing, we determined that the cystic phenotype in the mutant was caused by a missense mutation in the furinb gene, which encodes a proprotein convertase. The mutation altered Furinb localization and caused endoplasmic reticulum (ER) stress. The cystic phenotype could be suppressed by treatment with the ER stress inhibitor 4‐phenylbutyric acid and exacerbated by treatment with the ER stress inducer tunicamycin. The mutant liver also exhibited increased mammalian target of rapamycin (mTOR) signaling. Treatment with mTOR inhibitors halted cyst formation at least partially through reducing ER stress. Conclusion: Our study has established a vertebrate model for studying hepatic cystogenesis and illustrated the contribution of ER stress in the disease pathogenesis.