Endocrine cells distribution in human proximal small intestine: an immunohistochemical and morphometrical study

Atrophy of the pancreatic remnant after pancreaticoduodenectomy might be consequent to deregulation of pancreatic endocrine stimuli after duodenal removal. Relative technical surgical solution could be the anastomosis of the 1st jejunal loop to the stomach and the 2nd to the pancreatic stump. Data on the distribution of endocrine cells within the proximal intestine might represent the lacking tile of the problem. Our aims were to investigate the distribution pattern of serotonin, cholecystokinin and secretin cells in the duodenum, the 1st and 2nd jejunal loops of humans. Bowel specimens of ten patients submitted to pancreaticoduodenectomy were collected; immunohistochemical reactions and morphometric analyses were performed. A general ab-oral decrease of enteroendocrine cells was found. The rate of serotonin cells showed a significant 30.67±8.13% reduction starting from the 1st jejunal loop versus duodenum. The rate of both cholecystokinin and secretin cells in the duodenum was superimposable to that in the 1st jejunal loop, with a significant 62.88±4.80% loss of cholecystokinin and 39.5±9.31% of secretin cells in the 2nd loop. After removal of duodenum, preservation of the 1st jejunal loop could impact the function of pancreatic remnant maintaining the physiological enteroendocrine stimulus for pancreatic secretion that can compensate, at least in part for the abolished duodenal hormonal release

Recent advances on the mechanisms regulating cholangiocyte proliferation and the significance of the neuroendocrine regulation of cholangiocyte pathophysiology

Cholangiocytes are epithelial cells lining the biliary epithelium. Cholangiocytes play several key roles in the modification of ductal bile and are also the target cells in chronic cholestatic liver diseases (i.e., cholangiopathies) such as PSC, PBC, polycystic liver disease (PCLD) and cholangiocarcinoma (CCA). During these pathologies, cholangiocytes (which in normal condition are in a quiescent state) begin to proliferate acquiring phenotypes of neuroendocrine cells, and start secreting different cytokines, growth factors, neuropeptides, and hormones to modulate cholangiocytes proliferation and interaction with the surrounding environment, trying to reestablish the balance between proliferation/loss of cholangiocytes for the maintenance of biliary homeostasis. The purpose of this review is to summarize the recent findings on the mechanisms regulating cholangiocyte proliferation and the significance of the neuroendocrine regulation of cholangiocyte pathophysiology. To clarify the mechanisms of action of these factors we will provide new potential strategies for the management of chronic liver diseases

The Hepatic Microcirculation in Experimental Cirrhosis a Scanning Electron Microscopy Study of Microcorrosion Casts

The experimental model of liver cirrhosis induced by intragastric administration of CCl4 reproduces not only the histological picture of the postnecrotic cirrhosis but also its pathophysiological features. Corrosion casts of livers affected by CCl4-induced cirrhosis show the loss of the lobular pattern. Once the cirrhosis has completely developed, the whole microvascular bed appears to be composed of groups of sinusoid nodules of diameters varying between 0.3 and 1.5 mm.. Pre- and post-sinusoidal vessels and anastomoses between the former and the latter are mainly located at the perinodular spaces. This microvascular situation modifies the normal perfusion gradient within the parenchyma. Nevertheless, it can allow a still viable function

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

H3 histamine receptor-mediated activation of protein kinase calpha inhibits the growth of cholangiocarcinoma in vitro and in vivo

Histamine regulates functions via four receptors (HRH1, HRH2, HRH3, and HRH4). The D-myo-inositol 1,4,5-trisphosphate (IP(3))/Ca(2+)/protein kinase C (PKC)/mitogen-activated protein kinase pathway regulates cholangiocarcinoma growth. We evaluated the role of HRH3 in the regulation of cholangiocarcinoma growth. Expression of HRH3 in intrahepatic and extrahepatic cell lines, normal cholangiocytes, and human tissue arrays was measured. In Mz-ChA-1 cells stimulated with (R)-(alpha)-(-)-methylhistamine dihydrobromide (RAMH), we measured (a) cell growth, (b) IP(3) and cyclic AMP levels, and (c) phosphorylation of PKC and mitogen-activated protein kinase isoforms. Localization of PKC alpha was visualized by immunofluorescence in cell smears and immunoblotting for PKC alpha in cytosol and membrane fractions. Following knockdown of PKC alpha, Mz-ChA-1 cells were stimulated with RAMH before evaluating cell growth and extracellular signal-regulated kinase (ERK)-1/2 phosphorylation. In vivo experiments were done in BALB/c nude mice. Mice were treated with saline or RAMH for 44 days and tumor volume was measured. Tumors were excised and evaluated for proliferation, apoptosis, and expression of PKC alpha, vascular endothelial growth factor (VEGF)-A, VEGF-C, VEGF receptor 2, and VEGF receptor 3. HRH3 expression was found in all cells. RAMH inhibited the growth of cholangiocarcinoma cells. RAMH increased IP(3) levels and PKC alpha phosphorylation and decreased ERK1/2 phosphorylation. RAMH induced a shift in the localization of PKC alpha expression from the cytosolic domain into the membrane region of Mz-ChA-1 cells. Silencing of PKC alpha prevented RAMH inhibition of Mz-ChA-1 cell growth and ablated RAMH effects on ERK1/2 phosphorylation. In vivo, RAMH decreased tumor growth and expression of VEGF and its receptors; PKC alpha expression was increased. RAMH inhibits cholangiocarcinoma growth by PKC alpha-dependent ERK1/2 dephosphorylation. Modulation of PKC alpha by histamine receptors may be important in regulating cholangiocarcinoma growth. (Mol Cancer Res 2009;7(10):1704-13

Stem/Progenitor Cell Niches Involved in Hepatic and Biliary Regeneration

Niches containing stem/progenitor cells are present in different anatomical locations along the human biliary tree and within liver acini. The most primitive stem/progenitors, biliary tree stem/progenitor cells (BTSCs), reside within peribiliary glands located throughout large extrahepatic and intrahepatic bile ducts. BTSCs are multipotent and can differentiate towards hepatic and pancreatic cell fates. These niches’ matrix chemistry and other characteristics are undefined. Canals of Hering (bile ductules) are found periportally and contain hepatic stem/progenitor cells (HpSCs), participating in the renewal of small intrahepatic bile ducts and being precursors to hepatocytes and cholangiocytes. The niches also contain precursors to hepatic stellate cells and endothelia, macrophages, and have a matrix chemistry rich in hyaluronans, minimally sulfated proteoglycans, fetal collagens, and laminin. The microenvironment furnishes key signals driving HpSC activation and differentiation. Newly discovered third niches are pericentral within hepatic acini, contain Axin

Peribiliary gland damage due to liver transplantation involves peribiliary vascular plexus and vascular endothelial growth factor

Extrahepatic bile ducts are characterized by the presence of peribiliary glands (PBGs), which represent stem cell niches implicated in biliary regeneration. Orthotopic liver transplantation may be complicated by non-anastomotic strictures (NAS) of the bile ducts, which have been associated with ischemic injury of PBGs and occur more frequently in livers obtained from donors after circulatory death than in those from brain-dead donors. The aims of the present study were to investigate the PBG phenotype in bile ducts after transplantation, the integrity of the peribiliary vascular plexus (PVP) around PBGs, and the expression of vascular endothelial growth factor-A (VEGF-A) by PBGs. Transplanted ducts obtained from patients who underwent liver transplantation were studied (N=62). Controls included explanted bile duct samples not used for transplantation (N=10) with normal histology. Samples were processed for histology, immunohistochemistry and immunofluorescence. Surface epithelium is severely injured in transplanted ducts; PBGs are diffusely damaged, particularly in ducts obtained from circulatory-dead compared to brain-dead donors. PVP is reduced in transplanted compared to controls. PBGs in transplanted ducts contain more numerous progenitor and proliferating cells compared to controls, show higher positivity for VEGF-A compared to controls, and express VEGF receptor-2. In conclusion, PBGs and associated PVP are damaged in transplanted extrahepatic bile ducts; however, an activation of the PBG niche takes place and is characterized by proliferation and VEGF-A expression. This response could have a relevant role in reconstituting biliary epithelium and vascular plexus and could be implicated in the genesis of non-anastomotic strictures

Vasopressin induces cholangiocyte proliferation in experimental cholestasis and in Polycystic Liver Disease

The hormone vasopressin (hereafter AVP) is a neuropeptide mainly synthesized in the brain’s hypothalamic paraventricular (PVN) and supraoptic (SON) nuclei, works by three distinct receptor subtypes: V1a, V1b, and V2 [1]. In liver, AVP is involved in glycogenolysis and neoglucogenesis and regenerative processes [2]. Cholangiocytes are the cells that line the biliary ducts and they are the target in a number of animal models of cholestasis including bile duct ligation (BDL) and in several human pathologies such as polycystic liver disease (PLD) characterized by the presence of numerous cysts within the liver that arise from biliary epithelium [3]. Since no data exist about the presence and the role of AVP and receptors in biliary epithelium, we aimed to evaluate the effects of AVP in experimental model of cholestasis and in course of PLD. In vivo, normal and BDL liver fragments from rats, normal and PLD from human patients were collected to evaluate: (i) intrahepatic bile duct mass (IBDM) by immunohistochemistry for citokeratin-19 (CK-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 cystic epithelium) were stimulated with AVP in the absence/presence of antagonists such as OPC-31260 and Tolvaptan, before assessing cellular growth by MTT proliferation assay, cAMP levels by a RIA kit and the expression of some angiogenic factors, such as platelet-derived growth factor (PDGF) and Angiopoietins (Ang-1 and Ang-2). Cholangiocytes express V2 receptor that was upregulated following BDL and in course of polycystic disease. Treatment with AVP of cholangiocyte cultures increased proliferation, cAMP levels and expression of PDGF, Ang-1, Ang-2 in small cholangiocytes and LCDE cells. These increments were blocked by pre-incubation with the AVP antagonists. Our results showed that AVP play an important role in growth of the biliary epithelium during cholestasis and in cystic epithelium in course of PLD acting on the cAMP signalling pathway and increasing angiogenic factors. Additional studies are necessary, but these first results may be considered important in the regulation of the biliary growth/loss in course of cholangiopathies

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