SUMMARY Stem/progenitors have been identified intrahepatically in canals of Hering and extrahepatically in glands of the biliary tree. Glands of the biliary tree (peribiliary glands: PBGs) 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 PBGs near the fibromuscular layer. They are phenotypically heterogeneous expressing transcription factors and 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. They give rise to descendents undergoing a maturational lineage process from the interior to the surface of ducts and varying in mature cells generated: pancreatic cells in hepato-pancreatic 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.

Cholangiocytes secrete VEGF and express VEGFR-2 and VEGFR-3, all of which are amplified in BDL cholangiocytes. VEGF induces cholangiocyte proliferation by activation of inositol 1,4,5-triphosphate/[Ca2+]i/protein kinase C alpha and phosphorylation of Src/ERK1/2. VEGF mediates the adaptive proliferative response of cholangiocytes to cholestasis.

Cholangiopathies are characterized by the heterogeneous proliferation of different-sized cholangiocytes. Large cholangiocytes proliferate by a cAMP-dependent mechanism. The function of small cholangiocytes may depend on the activation of inositol trisphosphate (IP(3))/Ca(2+)-dependent signaling pathways; however, data supporting this speculation are lacking. Four histamine receptors exist (HRH1, HRH2, HRH3, and HRH4). In several cells: 1) activation of HRH1 increases intracellular Ca(2+) concentration levels; and 2) increased [Ca(2+)](i) levels are coupled with calmodulin-dependent stimulation of calmodulin-dependent protein kinase (CaMK) and activation of cAMP-response element binding protein (CREB). HRH1 agonists modulate small cholangiocyte proliferation by activation of IP(3)/Ca(2+)-dependent CaMK/CREB. We evaluated HRH1 expression in cholangiocytes. Small and large cholangiocytes were stimulated with histamine trifluoromethyl toluidide (HTMT dimaleate; HRH1 agonist) for 24-48 h with/without terfenadine, BAPTA/AM, or W7 before measuring proliferation. Expression of CaMK I, II, and IV was evaluated in small and large cholangiocytes. We measured IP(3), Ca(2+) and cAMP levels, phosphorylation of CaMK I, and activation of CREB (in the absence/presence of W7) in small cholangiocytes treated with HTMT dimaleate. CaMK I knockdown was performed in small cholangiocytes stimulated with HTMT dimaleate before measurement of proliferation and CREB activity. Small and large cholangiocytes express HRH1, CaMK I, and CaMK II. Small (but not large) cholangiocytes proliferate in response to HTMT dimaleate and are blocked by terfenadine (HRH1 antagonist), BAPTA/AM, and W7. In small cholangiocytes, HTMT dimaleate increased IP(3)/Ca(2+) levels, CaMK I phosphorylation, and CREB activity. Gene knockdown of CaMK I ablated the effects of HTMT dimaleate on small cholangiocyte proliferation and CREB activation. The IP(3)/Ca(2+)/CaMK I/CREB pathway is important in the regulation of small cholangiocyte function.

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Background & Aims Proliferating cholangiocytes secrete and respond to neuroendocrine hormones including secretin. We investigated whether secretin secreted by S cells and cholangiocytes stimulates biliary proliferation in mice. Methods Cholestasis was induced in secretin knockout (Sct−/−) and wild-type (control) mice by bile-duct ligation (BDL). At days 3 and 7 after BDL, control and Sct−/− mice received tail-vein injections of morpholinos against microRNA 125b or let7a. One week later, liver tissues and cholangiocytes were collected; immunohistochemical, immnoblot, luciferase reporter and real-time PCR assays were performed. Intrahepatic bile duct mass (IBDM) and proliferation were measured. Secretin secretion was measured in conditioned media from cholangiocytes and S cells, and in serum and bile. Results Secretin secretion was increased in supernatants from cholangiocytes and S cells and in serum and bile following BDL in control mice. BDL Sct−/− mice had lower IBDM, reduced proliferation, and reduced production of vascular endothelial growth factor A (VEGFA) and nerve growth factor (NGF) compared with BDL control. BDL and control mice given morpholinos against microRNA 125b or let7a had increased IBDM. Livers of mice given morpholinos against microRNA 125b had increased expression of VEGFA while those treated with morpholinos against microRNA let7a had increased expression of NGF. Secretin regulated VEGF and NGF expression that negatively correlated with microRNA 125b and let7a levels in liver tissue. Conclusions Following liver injury, secretin produced by cholangiocytes and S cells reduces microRNA 125b and let7a levels, resulting in upregulation of VEGF and NGF. Modulation of cholangiocyte expression of secretin could be a therapeutic approach for biliary diseases.

Rat and human biliary epithelium is morphologically and functionally heterogeneous. Since no information exists on the heterogeneity of the murine intrahepatic biliary epithelium, and with increased usage of transgenic mouse models to study liver disease pathogenesis, we sought to evaluate the morphological, secretory and proliferative phenotypes of small and large bile ducts and purified cholangiocytes in normal and cholestatic mouse models.MethodsFor morphometry, normal and BDL mouse livers (C57/BL6) were dissected into blocks of 2-4 μm2, embedded in paraffin, sectioned, and stained with H&E. Sizes of bile ducts and cholangiocytes were evaluated by using SigmaScan to measure the diameters of bile ducts and cholangiocytes. In small and large normal and BDL cholangiocytes, we evaluated the expression of cholangiocyte specific markers, keratin-19 (KRT19), secretin receptor (SR), cystic fibrosis transmembrane conductance regulator (CFTR), and chloride bicarbonate anion exchanger 2 (Cl-/HCO-3 AE2) by immunofluorescence and western blot; and intracellular cAMP levels and chloride efflux in response to secretin (100 nM). To evaluate cholangiocyte proliferative responses after bile duct ligation (BDL), small and large cholangiocytes were isolated from BDL mice. The proliferation status was determined by analysis of the cell cycle by FACS and bile duct mass was determined by the number of KRT19-positive bile ducts in liver sections.ResultsIn situ morphometry established that the biliary epithelium of mice is morphologically heterogeneous, which smaller cholangiocyte lining smaller bile ducts and larger cholangiocytes lining larger ducts. Both small and large cholangiocytes express KRT19 and only large cholangiocytes from normal and BDL mice express SR, CFTR, and Cl-/HCO-3 exchanger and respond to secretin with increased cAMP levels and chloride efflux. Following BDL, only large mouse cholangiocytes proliferate.ConclusionSimilar to rats, mouse intrahepatic biliary epithelium is morphologically, and functionally heterogeneous. The mouse is a suitable model for defining the heterogeneity of the biliary tree.

Background and Aims Lipopolysaccharides (LPS) is increased in nonalcoholic fatty liver disease (NAFLD), but its relationship with liver inflammation is not defined. Approach and Results We studied Escherichia coli LPS in patients with biopsy‐proven NAFLD, 25 simple steatosis (nonalcoholic fatty liver) and 25 nonalcoholic steatohepatitis (NASH), and in mice with diet‐induced NASH. NASH patients had higher serum LPS and hepatocytes LPS localization than controls, which was correlated with serum zonulin and phosphorylated nuclear factor‐κB expression. Toll‐like receptor 4 positive (TLR4+) macrophages were higher in NASH than simple steatosis or controls and correlated with serum LPS. NASH biopsies showed a higher CD61+ platelets, and most of them were TLR4+. TLR4+ platelets correlated with serum LPS values. In mice with NASH, LPS serum levels and LPS hepatocyte localization were increased compared with control mice and associated with nuclear factor‐κB activation. Mice on aspirin developed lower fibrosis and extent compared with untreated ones. Treatment with TLR4 inhibitor resulted in lower liver inflammation in mice with NASH. Conclusions In NAFLD, Escherichia coli LPS may increase liver damage by inducing macrophage and platelet activation through the TLR4 pathway.

Cholangiocarcinomas (CCAs) comprise a mucin-secreting form, intrahepatic or perihilar, and a mixed form located peripherally. We characterized cancer stem cells (CSCs) in CCA subtypes and evaluated their cancerogenic potential. CSC markers were investigated in 25 human CCAs in primary cultures and established cell lines. Tumorigenic potential was evaluated in vitro or in xenografted mice after s.c. or intrahepatic injection in normal and cirrhotic (carbon tetrachloride-induced) mice. CSCs comprised more than 30% of the tumor mass. Although the CSC profile was similar between mucin-intrahepatic and mucin-perihilar subtypes, CD13(+) CSCs characterized mixed-intrahepatic, whereas LGR5(+) characterized mucin-CCA subtypes. Many neoplastic cells expressed epithelial-mesenchymal transition markers and coexpressed mesenchymal and epithelial markers. In primary cultures, epithelial-mesenchymal transition markers, mesenchymal markers (vimentin, CD90), and CD13 largely predominated over epithelial markers (CD133, EpCAM, and LGR5). In vitro, CSCs expressing epithelial markers formed a higher number of spheroids than CD13(+) or CD90(+) CSCs. In s.c. tumor xenografts, tumors dominated by stromal markers were formed primarily by CD90(+) and CD13(+) cells. By contrast, in intrahepatic xenografts in cirrhotic livers, tumors were dominated by epithelial traits reproducing the original human CCAs. In conclusion, CSCs were rich in human CCAs, implicating CCAs as stem cell-based diseases. CSC subpopulations generate different types of cancers depending on the microenvironment. Remarkably, CSCs reproduce the original human CCAs when injected into cirrhotic livers.