Vasopressin regulates the growth of the biliary epithelium in polycystic liver disease

The neurohypophysial hormone arginine vasopressin (AVP) acts by three distinct receptor subtypes: V1a, V1b, and V2. In the liver, AVP is involved in ureogenesis, glycogenolysis, neoglucogenesis and regeneration. No data exist about the presence of AVP in the biliary epithelium. Cholangiocytes are the target cells in a number of animal models of cholestasis, including bile duct ligation (BDL), and in several human pathologies, such as polycystic liver disease characterized by the presence of cysts that bud from the biliary epithelium. In vivo, liver fragments from normal and BDL mice and rats as well as liver samples from normal and ADPKD patients were collected to evaluate: (i) intrahepatic bile duct mass by immunohistochemistry for cytokeratin-19; and (ii) expression of V1a, V1b and V2 by immunohistochemistry, immunofluorescence and real-time PCR. In vitro, small and large mouse cholangiocytes, H69 (non-malignant human cholangiocytes) and LCDE (human cholangiocytes from the cystic epithelium) were stimulated with vasopressin in the absence/presence of AVP antagonists such as OPC-31260 and Tolvaptan, before assessing cellular growth by MTT assay and cAMP levels. Cholangiocytes express V2 receptor that was upregulated following BDL and in ADPKD liver samples. Administration of AVP increased proliferation and cAMP levels of small cholangiocytes and LCDE cells. We found no effect in the proliferation of large mouse cholangiocytes and H69 cells. Increases were blocked by preincubation with the AVP antagonists. These results showed that AVP and its receptors may be important in the modulation of the proliferation rate of the biliary epithelium

Histamine stimulates the proliferation of small and large cholangiocytes by activation of both IP3/Ca2+ and cAMP-dependent signaling mechanisms

Although large cholangiocytes exert their functions by activation of cyclic adenosine 3',5'-monophosphate (cAMP), Ca(2+)-dependent signaling regulates the function of small cholangiocytes. Histamine interacts with four receptors, H1-H4HRs. H1HR acts by Gαq activating IP(3)/Ca(2+), whereas H2HR activates Gα(s) stimulating cAMP. We hypothesize that histamine increases biliary growth by activating H1HR on small and H2HR on large cholangiocytes. The expression of H1-H4HRs was evaluated in liver sections, isolated and cultured (normal rat intrahepatic cholangiocyte culture (NRIC)) cholangiocytes. In vivo, normal rats were treated with histamine or H1-H4HR agonists for 1 week. We evaluated: (1) intrahepatic bile duct mass (IBDM); (2) the effects of histamine, H1HR or H2HR agonists on NRIC proliferation, IP(3) and cAMP levels and PKCα and protein kinase A (PKA) phosphorylation; and (3) PKCα silencing on H1HR-stimulated NRIC proliferation. Small and large cholangiocytes express H1-H4HRs. Histamine and the H1HR agonist increased small IBDM, whereas histamine and the H2HR agonist increased large IBDM. H1HR agonists stimulated IP(3) levels, as well as PKCα phosphorylation and NRIC proliferation, whereas H2HR agonists increased cAMP levels, as well as PKA phosphorylation and NRIC proliferation. The H1HR agonist did not increase proliferation in PKCα siRNA-transfected NRICs. The activation of differential signaling mechanisms targeting small and large cholangiocytes is important for repopulation of the biliary epithelium during pathologies affecting different-sized bile ducts

Gonadotropin-releasing hormone induces biliary proliferation by both paracrine and autocrine mechanisms

During cholestatic liver disease, there is dysregulation in the balance between biliary growth and loss. In bile ductligated (BDL) rats, this balance is modulated by neuroendocrine peptides via autocrine/paracrine pathways (1). Gonadotropinreleasing hormone (GnRH) is a trophic peptide hormone that modulates reproduc- tive function and proliferation in many cell types (2). We evaluated the autocrine role of GnRH in the regulation of cholangiocyte growth. The expression of GnRH receptors was evaluated in a normal mouse cholangiocyte cell line (NMC), sham and BDL rats. The effect of GnRH administration was evaluated in normal rats and in NMC. GnRH-induced biliary proliferation was evaluated by changes in intrahepatic bile duct mass (IBDM) by CK19 specific staining, the expression of proliferation and function markers. The expression and secretion of GnRH in NMC and isolated cholangiocytes was assessed. GnRH receptor subtypes GnRHR1 and GnRHR2 were expressed in biliary epithelium. Treatment with GnRH increased IBDM as well as proliferation and function markers in cholangiocytes. Transient knockdown and pharmacologic inhibition of GnRHR1 in NMC decreased proliferation. BDL cholangiocytes had increased expression of GnRH compared with normal rats, accompanied by increased GnRH secretion. In vivo and in vitro knockdown of GnRH decreased intrahepatic bile duct mass/cholangiocyte proliferation and fibrosis. GnRH secreted by cholangiocytes promotes biliary proliferation via an autocrine pathway. Disruption of GnRH/ GnRHR signaling may be important for the management of cholestatic liver diseases

Follicle-Stimulating Hormone influences biliary epithelium growth in Autosomal Dominant Polycystic Kidney Disease

Autosomal Dominant Polycystic Kidney Disease (ADPKD) is a genetic disorder characterized by the development of renal cysts and various extrarenal manifestations, such as hepatic cysts that bud from biliary epithelium (Alvaro et al., 2008; Onori et al., 2010). We previously showed that Follicle-Stimulating Hormone (FSH) is a trophic factor for the biliary cells in normal rat and in experimental model of bile duct ligation. (Mancinelli et al, 2009). From these data, we aimed to investigate the role of FSH on biliary epithelium in ADPKD. In vivo evaluation of FSH receptor, FSH, p-ERK and c-myc expression in liver fragments from normal and ADPKD patients and in vitro PCNA and cAMP levels in normal human cholangiocytes (H69) and in a cell line obtained from the epithelium lining the hepatic cysts (LCDE) was performed. We found that FSH induces the proliferation of the cystic epithelium and co-localize with p-ERK and c-myc, proteins activated in cAMP signalling mechanism. In vitro FSH sustains cellular growth by the activation of cAMP/ERK signalling pathway with or without the transient silencing of the FSH gene in LCDE by siRNA. These results indicate that FSH has an important role in cystic growth via the cAMP pathway. FSH candidates as a possible target for medical therapy of hepatic cysts during ADPKD

Knockdown of hepatic GnRH reduces liver fibrosis in a mouse model of PSC

Primary sclerosing cholangitis (PSC) is a chronic cholestatic liver disease characterized by progressive inflammation and fibrosis of intrahepatic and extrahepatic bile ducts. [1]. Cholangiocyte proliferation occurs in all pathologic conditions of liver injury where it is associated with inflammation and regeneration. During these processes, biliary cells start to secrete different cytokines, growth factors, neuropeptides and hormones which represent potential mechanisms for cross talk with other liver cells [2].Gonadotropin-releasing hormone (GnRH) is a trophic peptide hormone synthesized by hypothalamic neurons and biliary epithelium and exerts its biological effects on cholangiocytes by interaction with the receptor subtype, GnRHR1, expressed by both cholangiocytes and HSCs[3]. Studies have shown that microRNA-200b is associated with the progression of hepatic fibrosis. However, the role of GnRH/GnRHR1/miR-200b axis in the progression of hepatic fibrosis in PSC is unknown. Using a mouse model of PSC (Mdr2-/-), we found that hepatic knockdown of GnRH decreased IBDM and liver fibrosis. In vivo, treatment with GnRH increases the expression of miR-200b and markers of fibrosis with an upregulation of the GnRH/GnRHR1 axis and miR-200b in human PSC samples. In vitro, inhibition of miR-200b decreases fibrotic gene expression in cultured murine cholangiocyte and HSC lines. In conclusion, these findings provides novel insights that the modulation of the GnRH/GnRHR1/miR-200b axis may regulates the progression of liver fibrosis in PSC

Lactoferrin regulate biliary epithelium growth and the activation of hepatic progenitor cell niche

Lactoferin (Lf) is an iron-binding glycoprotein belonging to the transferrin family and it is present at high levels in breast milk and colostrum. This protein has many known functions and it is a potential antibacterial, antiviral, immunostimulatory, antioxidant, and cancer preventive agent.It has been seen that a 105 kDa Lf receptor (LfR) specifically mediates the effects of Lf in several different cell types (1). In human cholangiopathies, cholangiocytes are able to proliferate and replace the cell loss restoring the integrity of damaged biliary epithelium. However, when cholangiocyte proliferation is severely impaired, the activation of facultative hepatic progenitor cells (HPCs) takes place (2). The aims of our study have been i) to investigate the expression of Lf and LfR in cholangiocytes and in HPCs both in rats and in humans; and ii) to evaluate the in vitro effects of bLf on cholangiocyte proliferation and on HPC activation. Liver specimens have been obtained from normal (N=5) and bile duct ligated (BDL) (N=5) rats; from normal patients (N=5) and from patients with primary biliary cirrhosis (PBC, N=5); Specimens were processed for histology, immunohistochemistry and immunofluorescence. Moreover for the in vitro study small and large cholangioytes from mouse, human non malignant cholangiocytes (H69), and HPCs treated or not with lactoferrin were used. Our results showed that: i) cholangiocytes and hepatic progenitor cells express lactoferrin and its receptor, ii) cholangiocytes and HPC proliferation is enhanced by lactoferrin; iii) the treatment with lactoferrin determine the commitment of HPCs towards cholangiocyte fate; this commitment is characterized by HPC morphological and phenotypical changes. Our current findings suggest that modulation of lactoferrin may be an important therapeutic tool for managing the proliferation of cholangiocyte and the activation of progenitor cell compartment in biliary disorders

Role of secretin in the growth of the biliary epithelium in normal and cholestatic mice

Background: Secretin stimulates bicarbonate secretion in large ducts by interaction with receptors by a cAMP-dependent pathway [1]. In BDL rats, the growth of large (but not small) cholangiocytes is activated by the cAMP/ERK1/2/Elk-1 pathway; and is associated with increased secretin receptor (SR) expression [2]. The aim of our study was to define the role of secretin in experimental models of cholestatic mice. Methods: We used (SEC+/+) (wild-type, WT) and SEC-/- male mice that underwent sham surgery or BDL for 7 days treated with saline or secretin. Then, we used: (i) liver sections to evaluate secretin protein expression, cholangiocyte growth, apoptosis, the presence of VEGF-A/C and NGF; (ii) purified and immortalized small and large BDL cholangiocytes to measure PCNA protein expression and the same previous growth factors. Results: In vivo, we demonstrated that: (i) secretin is expressed in normal liver by large bile ducts; and (ii) secretin expression increases in large bile ducts following BDL. Isolated large cholangiocytes from BDL WT mice express higher levels of the mRNA for secretin and secrete greater amounts of this hormone compared to normal large cholangiocytes. Knockout of the secretin gene reduced BDL-induced increase in large IBDM compared to WT BDL mice, an event that was associated with enhanced apoptosis of large cholangiocytes and decreased expression of VEGF-A/C and NGF. Concomitant with reduced large IBDM there was increased small IBDM in SEC-/- KO BDL mice compared to WT BDL mice. After prolonged treatment, secretin increased the biliary expression of PCNA, VEGF-A/C and NGF. Conclusions: The study introduces the novel concept that cholangiocytes express and secrete secretin and that manipulation of this gene may be an important target for the management of biliary disorders

Amelioration of Ductular Reaction by Stem Cell Derived Extracellular Vesicles in MDR2 Knockout Mice via Lethal-7 microRNA

Cholangiopathies are diseases that affect cholangiocytes, the cells lining the biliary tract. Liver stem cells (LSCs) are able to differentiate into all cells of the liver and possibly influence the surrounding liver tissue by secretion of signaling molecules. One way in which cells can interact is through secretion of extracellular vesicles (EVs), which are small membrane-bound vesicles that contain proteins, microRNAs (miRNAs), and cytokines. We evaluated the contents of liver stem cell–derived EVs (LSCEVs), compared their miRNA contents to those of EVs isolated from hepatocytes, and evaluated the downstream targets of these miRNAs. We finally evaluated the crosstalk among LSCs, cholangiocytes, and human hepatic stellate cells (HSCs). We showed that LSCEVs were able to reduce ductular reaction and biliary fibrosis in multidrug resistance protein 2 (MDR2)−/− mice. Additionally, we showed that cholangiocyte growth was reduced and HSCs were deactivated in LSCEV-treated mice. Evaluation of LSCEV contents compared with EVs derived from hepatocytes showed a large increase in the miRNA, lethal-7 (let-7). Further evaluation of let-7 in MDR2−/− mice and human primary sclerosing cholangitis samples showed reduced levels of let-7 compared with controls. In liver tissues and isolated cholangiocytes, downstream targets of let-7 (identified by ingenuity pathway analysis), Lin28a (Lin28 homolog A), Lin28b (Lin28 homolog B), IL-13 (interleukin 13), NR1H4 (nuclear receptor subfamily 1 group H member 4) and NF-κB (nuclear factor kappa B), are elevated in MDR2−/− mice, but treatment with LSCEVs reduced levels of these mediators of ductular reaction and biliary fibrosis through the inhibition of NF-κB and IL-13 signaling pathways. Evaluation of crosstalk using cholangiocyte supernatants from LSCEV-treated cells on cultured HSCs showed that HSCs had reduced levels of fibrosis and increased senescence. Conclusion: Our studies indicate that LSCEVs could be a possible treatment for cholangiopathies or could be used for target validation for future therapies