Cellular Homeostasis and Repair in the Biliary Tree.

During biliary tree homeostasis, BECs are largely in a quiescent state and their turnover is slow for maintaining normal tissue homeostasis. BTSCs continually replenish new BECs in the luminal surface of EHBDs. In response to various types of biliary injuries, distinct cellular sources, including HPCs, BTSCs, hepatocytes, and BECs, repair or regenerate the injured bile duct. BEC, biliary epithelial cell; BTSC, biliary tree stem/progenitor cell; EHBD, extrahepatic bile ducts; HPC, hepatic progenitor cell.The biliary tree comprises intrahepatic bile ducts and extrahepatic bile ducts lined with epithelial cells known as biliary epithelial cells (BECs). BECs are a common target of various cholangiopathies for which there is an unmet therapeutic need in clinical hepatology. The repair and regeneration of biliary tissue may potentially restore the normal architecture and function of the biliary tree. Hence, the repair and regeneration process in detail, including the replication of existing BECs, expansion and differentiation of the hepatic progenitor cells and biliary tree stem/progenitor cells, and transdifferentiation of the hepatocytes, should be understood. In this paper, we review biliary tree homeostasis, repair, and regeneration and discuss the feasibility of regenerative therapy strategies for cholangiopathy treatment.

Compensatory hepatic adaptation accompanies permanent absence of intrahepatic biliary network due to YAP1 loss in liver progenitors.

Yes-associated protein 1 (YAP1) regulates cell plasticity during liver injury, regeneration, and cancer, but its role in liver development is unknown. We detect YAP1 activity in biliary cells and in cells at the hepatobiliary bifurcation in single-cell RNA sequencing analysis of developing livers. Deletion of Yap1 in hepatoblasts does not impair Notch-driven SOX9+ ductal plate formation but does prevent the formation of the abutting second layer of SOX9+ ductal cells, blocking the formation of a patent intrahepatic biliary tree. Intriguingly, these mice survive for 8 months with severe cholestatic injury and without hepatocyte-to-biliary transdifferentiation. Ductular reaction in the perihilar region suggests extrahepatic biliary proliferation, likely seeking the missing intrahepatic biliary network. Long-term survival of these mice occurs through hepatocyte adaptation via reduced metabolic and synthetic function, including altered bile acid metabolism and transport. Overall, we show YAP1 as a key regulator of bile duct development while highlighting a profound adaptive capability of hepatocytes.

Metformin Preconditioning Improves Hepatobiliary Function and Reduces Injury in a Rat Model of Normothermic Machine Perfusion and Orthotopic Transplantation.

BACKGROUND: Preconditioning of donor livers before organ retrieval may improve organ quality after transplantation. We investigated whether preconditioning with metformin reduces preservation injury and improves hepatobiliary function in rat donor livers during ex situ normothermic machine perfusion (NMP) and after orthotopic liver transplantation. METHODS: Lewis rats were administered metformin via oral gavage, after which a donor hepatectomy was performed followed by a standardized cold storage period of 4 hours. Graft assessment was performed using NMP via double perfusion of the hepatic artery and portal vein. In an additional experiment, rat donor livers preconditioned with metformin were stored on ice for 4 hours and transplanted to confirm postoperative liver function and survival. Data were analyzed and compared with sham-fed controls. RESULTS: Graft assessment using NMP confirmed that preconditioning significantly improved ATP production, markers for hepatobiliary function (total bile production, biliary bilirubin, and bicarbonate), and significantly lowered levels of lactate, glucose, and apoptosis. After orthotopic liver transplantation, metformin preconditioning significantly reduced transaminase levels. CONCLUSIONS: Preconditioning with metformin lowers hepatobiliary injury and improves hepatobiliary function in an in situ and ex situ model of rat donor liver transplantation.

Meta-Analysis of Human and Mouse Biliary Epithelial Cell Gene Profiles.

BACKGROUND: Chronic liver diseases are frequently accompanied with activation of biliary epithelial cells (BECs) that can differentiate into hepatocytes and cholangiocytes, providing an endogenous back-up system. Functional studies on BECs often rely on isolations of an BEC cell population from healthy and/or injured livers. However, a consensus on the characterization of these cells has not yet been reached. The aim of this study was to compare the publicly available transcriptome profiles of human and mouse BECs and to establish gene signatures that can identify quiescent and activated human and mouse BECs. METHODS: We used publicly available transcriptome data sets of human and mouse BECs, compared their profiles and analyzed co-expressed genes and pathways. By merging both human and mouse BEC-enriched genes, we obtained a quiescent and activation gene signature and tested them on BEC-like cells and different liver diseases using gene set enrichment analysis. In addition, we identified several genes from both gene signatures to identify BECs in a scRNA sequencing data set. RESULTS: Comparison of mouse BEC transcriptome data sets showed that the isolation method and array platform strongly influences their general profile, still most populations are highly enriched in most genes currently associated with BECs. Pathway analysis on human and mouse BECs revealed the KRAS signaling as a new potential pathway in BEC activation. We established a quiescent and activated BEC gene signature that can be used to identify BEC-like cells and detect BEC enrichment in alcoholic hepatitis, non-alcoholic steatohepatitis (NASH) and peribiliary sclerotic livers. Finally, we identified a gene set that can distinguish BECs from other liver cells in mouse and human scRNAseq data. CONCLUSIONS: Through a meta-analysis of human and mouse BEC gene profiles we identified new potential pathways in BEC activation and created unique gene signatures for quiescent and activated BECs. These signatures and pathways will help in the further characterization of this progenitor cell type in mouse and human liver development and disease.

STAT3 deficiency prevents hepatocarcinogenesis and promotes biliary proliferation in thioacetamide-induced liver injury.

AIM: To elucidate the role of STAT3 in hepatocarcinogenesis and biliary ductular proliferation following chronic liver injury. METHODS: We investigated thioacetamide (TAA)-induced liver injury, compensatory hepatocyte proliferation, and hepatocellular carcinoma (HCC) development in hepatic STAT3-deficient mice. In addition, we evaluated TAA-induced biliary ductular proliferation and analyzed the activation of sex determining region Y-box9 (SOX9) and Yes-associated protein (YAP), which regulate the transdifferentiation of hepatocytes to cholangiocytes. RESULTS: Both compensatory hepatocyte proliferation and HCC formation were significantly decreased in hepatic STAT3-deficient mice as compared with control mice. STAT3 deficiency resulted in augmentation of hepatic necrosis and fibrosis. On the other hand, biliary ductular proliferation increased in hepatic STAT3-deficient livers as compared with control livers. SOX9 and YAP were upregulated in hepatic STAT3-deficient hepatocytes. CONCLUSION: STAT3 may regulate hepatocyte proliferation as well as transdifferentiation into cholangiocytes and serve as a therapeutic target for HCC inhibition and biliary regeneration.

Reconstruction of the mouse extrahepatic biliary tree using primary human extrahepatic cholangiocyte organoids.

The treatment of common bile duct (CBD) disorders, such as biliary atresia or ischemic strictures, is restricted by the lack of biliary tissue from healthy donors suitable for surgical reconstruction. Here we report a new method for the isolation and propagation of human cholangiocytes from the extrahepatic biliary tree in the form of extrahepatic cholangiocyte organoids (ECOs) for regenerative medicine applications. The resulting ECOs closely resemble primary cholangiocytes in terms of their transcriptomic profile and functional properties. We explore the regenerative potential of these organoids in vivo and demonstrate that ECOs self-organize into bile duct-like tubes expressing biliary markers following transplantation under the kidney capsule of immunocompromised mice. In addition, when seeded on biodegradable scaffolds, ECOs form tissue-like structures retaining biliary characteristics. The resulting bioengineered tissue can reconstruct the gallbladder wall and repair the biliary epithelium following transplantation into a mouse model of injury. Furthermore, bioengineered artificial ducts can replace the native CBD, with no evidence of cholestasis or occlusion of the lumen. In conclusion, ECOs can successfully reconstruct the biliary tree, providing proof of principle for organ regeneration using human primary cholangiocytes expanded in vitro.

The P2X4 purinergic receptor impacts liver regeneration after partial hepatectomy in mice through the regulation of biliary homeostasis.

UNLABELLED: Many regulatory pathways are involved in liver regeneration after partial hepatectomy (PH), to initiate growth, protect liver cells, and sustain remnant liver functions. Extracellular adenosine triphosphate rises in blood and bile after PH and contributes to liver regeneration, although purinergic receptors and mechanisms remain to be precisely explored. In this work we analyzed during regeneration after PH the involvement of P2X4 purinergic receptors, highly expressed in the liver. P2X4 receptor expression in the liver, liver histology, hepatocyte proliferation, plasma bile acid concentration, bile flow and composition, and lysosome distribution in hepatocytes were studied in wild-type and P2X4 knockout (KO) mice, before and after PH. P2X4 receptors were expressed in hepatocytes and Kupffer cells; in hepatocytes, P2X4 was concentrated in subcanalicular areas closely costained with lysosomal markers. After PH, delayed regeneration, hepatocyte necrosis, and cholestasis were observed in P2X4-KO mice. In P2X4-KO mice, post-PH biliary adaptation was impaired with a smaller increase in bile flow and HCO3 (-) biliary output, as well as altered biliary composition with reduced adenosine triphosphate and lysosomal enzyme release. In line with these data, lysosome distribution and biogenesis were altered in P2X4-KO compared with wild-type mice. CONCLUSION: During liver regeneration after PH, P2X4 contributes to the complex control of biliary homeostasis through mechanisms involving pericanalicular lysosomes, with a resulting impact on hepatocyte protection and proliferation. (Hepatology 2016;64:941-953).

Physical Activity and the Biliary Tract in Health and Disease.

BACKGROUND: Cholecystitis and gallstones affect a large segment of the population in developed nations, and a small proportion of affected individuals subsequently develop cancer of the gallbladder. However, little is known about the possible beneficial effects of physical activity. OBJECTIVE: Accordingly, a systematic review examined the influence of both acute and chronic exercise on gallbladder motility, and relationships were examined between habitual physical activity, gallbladder disease, and gallbladder cancer. METHODS: A search of Ovid/MEDLINE from 1996 to November 2014 yielded 67 articles relating to physical activity and gallbladder function or disease; 18 of these relevant to the objectives of the review were supplemented by 22 papers from personal files and other sources. Because of the limited volume of material, all were considered, although note was taken of the quality of activity measurement, care in excluding covariates, and experimental design (cross-sectional, case-control or randomized controlled trial). RESULTS: The impact of physical activity upon gallbladder function remains unclear; acute activity could augment emptying by stimulating cholecystokinin release, and one of two training experiments found a small increase in gallbladder motility. The largest and most recent cross-sectional and case-control trials show a reduced risk of gallbladder disease in active individuals. A small number of randomized controlled trials in humans and one animal study generally support these trends, although the number of cases of gallstones are too few for statistical significance. Three studies of gallbladder cancer also show a non-significant trend to benefit from physical activity. CONCLUSIONS: Although there remains a need for further research, regular physical activity seems likely to reduce the risk of both gallstones and gallbladder cancer. A substantial number of individuals must be persuaded to exercise in order to avoid one case of gallbladder disease, but the attempt appears warranted because of the other health benefits of regular physical activity.

Tissue-specific regulation of the mouse Pkhd1 (ARPKD) gene promoter.

Autosomal recessive polycystic kidney disease, an inherited disorder characterized by the formation of cysts in renal collecting ducts and biliary dysgenesis, is caused by mutations of the polycystic kidney and hepatic disease 1 (PKHD1) gene. Expression of PKHD1 is tissue specific and developmentally regulated. Here, we show that a 2.0-kb genomic fragment containing the proximal promoter of mouse Pkhd1 directs tissue-specific expression of a lacZ reporter gene in transgenic mice. LacZ is expressed in renal collecting ducts beginning during embryonic development but is not expressed in extrarenal tissues. The Pkhd1 promoter contains a binding site for the transcription factor hepatocyte nuclear factor (HNF)-1ß, which is required for activity in transfected cells. Mutation of the HNF-1ß-binding site abolishes the expression of the lacZ reporter gene in renal collecting ducts. Transgenes containing the 2.0-kb promoter and 2.7 kb of additional genomic sequence extending downstream to the second exon are expressed in the kidney, intrahepatic bile ducts, and male reproductive tract. This pattern overlaps with the endogenous expression of Pkhd1 and coincides with sites of expression of HNF-1ß. We conclude that the proximal 2.0-kb promoter is sufficient for tissue-specific expression of Pkhd1 in renal collecting ducts in vivo and that HNF-1ß is required for Pkhd1 promoter activity in collecting ducts. Additional genomic sequences located from exons 1-2 or elsewhere in the gene locus are required for expression in extrarenal tissues.

Functional magnetic resonance cholangiography with Gd-EOB-DTPA: a study in healthy volunteers.

PURPOSE: To describe the patterns of bile distribution in the biliary tree, duodenum, jejunum, and stomach, and to determine the gallbladder ejection fraction (GBEF) by using functional magnetic resonance cholangiography (MRC) with gadolinium-ethoxybenzyl-diethylenetriamine penta-acetic acid (Gd-EOB-DTPA) in healthy volunteers. MATERIALS AND METHODS: Forty subjects were included in this study. After conventional MRC, pre-fatty meal MRC (PRFM) was obtained at 30, 40, 50, and 60min after contrast agent injection. Then, post-fatty meal MRC (POFM) was obtained every 10min for 1h. We assessed the PRFM and POFM for opacification of contrast agent in the first- and second-order intrahepatic ducts (IHDs) and the common bile duct (CBD). Contrast agent opacification in the cystic duct was assessed, and the percentage volume of contrast agent filling in the gallbladder (GB) was calculated on PRFM. We calculated the GBEF and assessed the presence of contrast agent in the GB, duodenum, jejunum, and stomach. RESULTS: Thirty-six (90%) subjects showed grade 3 CBD opacification (visible contrast and well-defined bile duct border) on 60-min PRFM. Thirty-four (85%) subjects showed grade 3 first-order IHD opacification on 60-min PRFM. All (100%) subjects showed cystic duct opacification of contrast agent, and the average percentage volume of contrast agent filling in the GB was 68.81%±16.84% on 60-min PRFM. The GBEF at 30-min POFM was 35.00%±18.26%. Ten (25%) subjects had no contrast agent in the stomach and small bowel on all PRFMs. Twelve (30%) subjects had contrast medium in the stomach on PRFM and/or POFM. CONCLUSIONS: Functional MRC with Gd-EOB-DTPA can allow determining the distribution of bile in the biliary tree and small intestine, as well as the GBEF.

Melatonin in the biliary tract and liver: health implications.

Melatonin is a widely-produced and ubiquitously-distributed molecule with multiple critical functions in all organs and organisms. These functions are mediated by both receptor-mediated and receptor-independent actions of the indole. This survey reviews the reports documenting the presence and function of melatonin in the hepatobiliary system. The published data document the exceptionally high concentrations of melatonin in the bile; herein, we speculate on the significance of these high melatonin levels to the function of the biliary tree. Moreover, we suggest that the elevated concentrations of melatonin in the bile fluid may be a consequence of its recirculation in what is referred to as the enterohepatic circulation. The article also examines the published reports related to melatonin levels in hepatocytes, which appear to be independent of pineal-derived melatonin. In both the biliary system and liver, melatonin provides protection against free radicals in cells of these organs. This is particularly important in these organs since they are under constant assault by highly toxic agents/processes that could compromise their critical physiology. As in other tissues, melatonin provides hepatocytes and cholangiocytes with a buffer against free radicals that are persistently produced and thereby this indole protects against oxidative molecular damage and metabolic dysfunction. Melatonin achieves this protection via the diverse free radical scavenging mechanisms of it and its metabolites (known as the antioxidant cascade), due to its ability to reduce electron leakage from the respiratory complexes in the inner mitochondrial membrane (radical avoidance) and as a result of the stimulation of antioxidative enzymes.

Notch signalling beyond liver development: emerging concepts in liver repair and oncogenesis.

Notch signalling is an evolutionarily conserved intercellular pathway involved in many aspects of development and tissue renewal in several organs. The importance of Notch signalling in liver development and morphogenesis is well established. However, the post-natal role of Notch in liver repair/regeneration is only now beginning to be unveiled. Despite the simplicity of the pathway activation, a fine spatial-temporal regulation of Notch signalling is required to avoid pathologic effects. This review highlights recent advances in the field indicating that Notch signalling is involved in the reparative morphogenesis of the biliary tree and in liver carcinogenesis. Defective Notch signalling leads to impaired ability of the liver to repair liver damage, while excessive activation may be involved in liver cancer. Even though much remains to be understood about these mechanisms, including the cross-talk between Notch signalling and other liver morphogens, current evidence suggests that the modulation of the Notch pathway may represent a therapeutic target in chronic liver disease.

Physiology of cholangiocytes.

Cholangiocytes are epithelial cells that line the intra- and extrahepatic ducts of the biliary tree. The main physiologic function of cholangiocytes is modification of hepatocyte-derived bile, an intricate process regulated by hormones, peptides, nucleotides, neurotransmitters, and other molecules through intracellular signaling pathways and cascades. The mechanisms and regulation of bile modification are reviewed herein.

Role of melatonin in the oxidative damage prevention at different times of hepatic regeneration.

The process of regenerating liver is the result of a balance between stimulating factors and inhibitors of hepatocyte proliferation. Melatonin and its metabolites have been found to protect tissues against oxidative damage generated by a variety of toxic agents and metabolic processes. Furthermore, studies in liver of rats showed a decrease in the liver mitochondrial hydroxylation of drugs returning to the normal state after the administration of antioxidants. This study was designed to determine, in experimental animals, whether the administration of an antioxidant agent such as melatonin could prevent cells events leading to tissue injury and hepatic dysfunction after partial hepatectomy (PH). Biliary flow (BF), oxidative stress in hepatic tissue and Na⁺/K⁺ ATPase activities in whole plasma membrane were determined. PH decreased the Na⁺/K⁺ ATPase activity. PH significantly reduced the BF (36%) and promoted oxidative stress with an increase of lipoperoxidation and decrease of glutathione peroxidase and catalase activities. Treatment with melatonin prevented the decrease of BF in rats with hepatectomy and normalized the Na⁺/K⁺ ATPase activity. Moreover, melatonin markedly attenuated oxidative stress produced by PH. This may be the results of the higher efficacy of melatonin in scavenging various free radicals and also because of its ability in stimulating the antioxidant enzymes. We suggest that oxidative stress before and during liver regeneration has a crucial role in cholestasis, apoptotic/necrotic hepatocellular damage and the impairment in liver transport function induced by PH and that melatonin could modulate the degree of oxidative stress and through it prevent the alterations in liver function carrier.

Biliary tree stem/progenitor cells in glands of extrahepatic and intraheptic bile ducts: an anatomical in situ study yielding evidence of maturational lineages.

Stem/progenitors have been identified intrahepatically in the canals of Hering and extrahepatically in glands of the biliary tree. Glands of the biliary tree (peribiliary glands) are tubulo-alveolar glands with mucinous and serous acini, located deep within intrahepatic and extrahepatic bile ducts. We have shown that biliary tree stem/progenitors (BTSCs) are multipotent, giving rise in vitro and in vivo to hepatocytes, cholangiocytes or pancreatic islets. Cells with the phenotype of BTSCs are located at the bottom of the peribiliary glands near the fibromuscular layer. They are phenotypically heterogeneous, expressing transcription factors as well as surface and cytoplasmic markers for stem/progenitors of liver (e.g. SOX9/17), pancreas (e.g. PDX1) and endoderm (e.g. SOX17, EpCAM, NCAM, CXCR4, Lgr5, OCT4) but not for mature markers (e.g. albumin, secretin receptor or insulin). Subpopulations co-expressing liver and pancreatic markers (e.g. PDX1(+)/SOX17(+)) are EpCAM(+/-), and are assumed to be the most primitive of the BTSC subpopulations. Their descendants undergo a maturational lineage process from the interior to the surface of ducts and vary in the mature cells generated: pancreatic cells in hepatopancreatic ducts, liver cells in large intrahepatic bile ducts, and bile duct cells along most of the biliary tree. We hypothesize that there is ongoing organogenesis throughout life, with BTSCs giving rise to hepatic stem cells in the canals of Hering and to committed progenitors within the pancreas. The BTSCs are likely to be central to normal tissue turnover and injury repair and to be key elements in the pathophysiology of liver, pancreas and biliary tree diseases, including oncogenesis.

Higher clearance of micafungin in neonates compared with adults: role of age-dependent micafungin serum binding.

Micafungin, a new echinocandin antifungal agent, has been used widely for the treatment of various fungal infections in human populations. Micafungin is predominantly cleared by biliary excretion and it binds extensively to plasma proteins. Micafungin body weight-adjusted clearance is higher in neonates than in adults, but the mechanisms underlying this difference are not understood. Previous work had revealed the roles of sinusoidal uptake (Na(+) -taurocholate co-transporting peptide, NTCP; organic anion transporting polypeptide, OATP) as well as canalicular efflux (bile salt export pump, BSEP; breast cancer resistance protein, BCRP) transporters in micafungin hepatobiliary elimination. In the present study, the relative protein expression of hepatic transporters was compared between liver homogenates from neonates and adults. Also, the extent of micafungin binding to serum from neonates and adults was measured in vitro. The results indicate that relative expression levels of NTCP, OATP1B1/3, BSEP, BCRP and MRP3 were similar in neonates and in adults. However, the micafungin fraction unbound (f(u) ) in neonatal serum was about 8-fold higher than in the adult serum (0.033±0.012 versus 0.004±0.001, respectively). While there was no evidence for different intrinsic hepatobiliary clearance of micafungin between neonates and adults, our data suggest that age-dependent serum protein binding of micafungin is responsible for its higher clearance in neonates compared with adults.

Role of vascular endothelial growth factor in protection of intrahepatic cholangiocytes mediated by hypoxic preconditioning after liver transplantation in rats.

OBJECTIVE: To investigate the effect on intrahepatic cholangiocytes mediated by hypoxic preconditioning (HP) after liver transplantation and the role of vascular endothelial growth factor (VEGF). MATERIALS AND METHODS: This experiment was based on a model of rat orthotopic liver autotransplantation. Sprague-Dawley rats were randomly divided into 3 groups: normal control, autotransplantation (AT), and HP. The HP group was subjected to 8% oxygen atmosphere for 90 minutes before surgery. At 6, 12, 24, and 48 hours after autotransplantation, the rats were killed for testing .Serum total bilirubin, direct bilirubin, and alkaline phosphatase concentrations were determined. The microstructure of cholangiocytes and the ultramicrostructure of cholangioles were determined. Immunohistochemistry was used to detect the expression of VEGF and the proliferation rate of cholangiocytes. RESULTS: Total bilirubin, direct bilirubin, and alkaline phosphatase concentrations in the AT group increased considerably more than in the HP group during the entire interval (P < .05). Light microscopy demonstrated that the microstructure of cholangiocytes in the AT group was damaged more seriously than in the HP group. At transmission electron microscopy, the ultramicrostructure of cholangioles was changed more obviously than in the HP group. The expression of VEGF on cholangiocytes and the proliferation rate of cholangiocytes were higher in the HP group than in the AT group over the entire experiment (P < .05). CONCLUSION: Hypoxic preconditioning has a protective effect on cholangiocytes after liver autotransplantation. The mechanism may be related to HP-induced overexpression of VEGF on cholangiocytes.

[Possibilities of rational choice and application of autotissues for hermetization of the suture connections in biliary surgery].

The results of experimental investigation of peculiarities of morphological transformation course in autotissues under the influence of bile were adduced. There was proposed a choice of peritoneum as an optimal plastic material in conditions of possible deficiency of local tissues during biliary surgery performance.