Exendin-4, a glucagon-like peptide 1 receptor agonist, protects cholangiocytes from apoptosis.

BACKGROUND AND AIMS: Progression of chronic cholestatic disorders towards ductopenia results from the dysregulation of cholangiocyte survival, with cell death by apoptosis prevailing over compensatory proliferation. Currently, no therapy is available to sustain cholangiocyte survival in the course of those disorders. It was recently shown that cholangiocytes express the glucagon-like peptide-1 receptor (GLP-1R); its activation results in enhanced proliferative reaction to cholestasis. The GLP-1R selective agonist exendin-4 sustains pancreatic beta cell proliferation and prevents cell death by apoptosis. Exendin-4 is now employed in humans as a novel therapy for diabetes. The aim of the present study was to verify whether exendin-4 is effective in preventing cholangiocyte apoptosis. METHODS: In vitro, tests were carried out to determine if exendin-4 is able to prevent apoptosis of cholangiocytes isolated from normal rats induced by glycochenodeoxycholic acid (GCDCA); in vivo, animals subjected to 1 week of bile duct ligation and to a single intraperitoneal injection of CCl(4) were treated with exendin-4 for 3 days. RESULTS: Exendin-4 prevented GCDCA-induced Bax mitochondrial translocation, cytochrome c release and an increase in caspase 3 activity. Phosphatidylinositol 3-kinase, but not cAMP/protein kinase A or Ca(2+)/calmodulin-dependent protein kinase inhibitors, neutralised the effects of exendin-4. In vivo, exendin-4 administration prevented the increase in TUNEL (terminal deoxynucleotidyl transferase-mediated triphosphate end-labelling)-positive cholangiocytes and the loss of bile ducts observed in bile duct-ligated rats treated with CCl(4). CONCLUSION: Exendin-4 prevents cholangiocyte apoptosis both in vitro and in vivo; such an effect is due to the ability of exendin-4 to counteract the activation of the mitochondrial pathway of apoptosis. These findings support the hypothesis that exendin-4 may be effective in slowing down the progression of cholangiopathies to ductopenia.

Human cholangiocarcinoma development is associated with dysregulation of opioidergic modulation of cholangiocyte growth.

BACKGROUND/AIMS: Incidence of cholangiocarcinoma is increasing worldwide, yet remaining highly aggressive and with poor prognosis. The mechanisms that drive cholangiocyte transition towards malignant phenotype are obscure. Cholangiocyte benign proliferation is subjected to a self-limiting mechanism based on the autocrine release of endogenous opioid peptides. Despite the presence of both, ligands interact with delta opioid receptor (OR), but not with microOR, with the consequent inhibition of cell growth. We aimed to verify whether cholangiocarcinoma growth is associated with failure of opioidergic regulation of growth control. METHODS: We evaluated the effects of OR selective agonists on cholangiocarcinoma cell proliferation, migration and apoptosis. Intracellular signals were also characterised. RESULTS: Activation of microOR, but not deltaOR, increases cholangiocarcinoma cell growth. Such an effect is mediated by ERK1/2, PI3K and Ca(2+)-CamKIIalpha cascades, but not by cAMP/PKA and PKCalpha. microOR activation also enhances cholangiocarcinoma cell migration and reduces death by apoptosis. The anti-apoptotic effect of microOR was PI3K dependent. CONCLUSIONS: Our data indicate that cholangiocarcinoma growth is associated with altered opioidergic regulation of cholangiocyte biology, thus opening new scenarios for future surveillance or early diagnostic strategies for cholangiocarcinoma.

Role of endogenous opioids in modulating HSC activity in vitro and liver fibrosis in vivo.

BACKGROUND: Endogenous opioids modulate the growth of nervous and non-nervous cells. Hepatic stellate cells (HSCs) are the main cell phenotype involved in liver fibrogenesis, display molecular markers of neuronal cells and respond to neurotransmitters. AIM: To evaluate the role of endogenous opioids on liver fibrogenesis. METHODS: Activated rat HSCs (passage 1-3) were used to evaluate cell proliferation and intracellular signalling pathway activation. Liver fibrosis was induced in rats by dimethylnitrosamine (DMN) administration. RESULTS: Opioid receptors showed a different pattern of expression when measured in quiescent and activated (in vitro and in vivo) HSCs. The activation of opioid receptors increased HSC proliferation and collagen accumulation. Opioid receptor stimulation induced a calcium-dependent protein kinase C alpha (PKC alpha)/extracellular regulated kinase (ERK)/phosphatidylinositol 3-kinase (PI3K) pathway activation that mediated the effect of endogenous opioids on HSC proliferation and collagen synthesis. In DMN-treated rats, the opioid antagonist naloxone reduced alpha-smooth muscle actin expression (as a marker of HSC activation) and collagen deposition, both measured by morphometry after 5 weeks of treatment. In both DMN-treated rats and human liver biopsies from chronic liver diseases, opioid receptors were observed in HSCs in area of active fibrogenesis. The endogenous opioid met-enkephalin increased its expression in zone 3 hepatocytes close to the area of necrosis after DMN administration and in the cellular target of chronic liver injury in human biopsies, and stimulated HSC proliferation and collagen synthesis. CONCLUSIONS: Endogenous opioids released during chronic liver injury participate in the process of liver fibrogenesis by stimulating HSC proliferation and collagen production in a paracrine manner.

Selective inhibition of ion transport mechanisms regulating intracellular pH reduces proliferation and induces apoptosis in cholangiocarcinoma cells.

BACKGROUND: Cells within the acidic extracellular environment of solid tumours maintain their intracellular pH through the activity of the Na(+)/H(+) exchanger and the Na(+) dependent Cl(-)/HCO(3)(-) exchanger. The inhibition of these mechanisms could therefore inhibit cancer cell growth. AIM: We evaluated the effect of two selective inhibitors of these transporters (cariporide and S3705) on proliferation and apoptosis of human cholangiocarcinoma cells (HUH-28 and Mz-ChA-1 cells) as a function of external pH (7.4 and 6.8). METHODS/RESULTS: HUH-28 cells incubated for 24h at external pH 7.4 or 6.8 without inhibitors maintained intracellular pH at physiological level, whereas incubation with cariporide and/or S3705 caused the intracellular pH of cells to drop. Incubation of HUH-28 cells with cariporide and/or S3705 was able to reduce proliferation, evaluated by a colorimetric ELISA method, and to induce apoptosis, evaluated by measuring caspase-3 activity and Annexin-V staining, and these effects were more evident at external pH 6.8. S3705 but not cariporide was able to inhibit serum-induced phosphorylation of ERK1/2, AKT and BAD, intracellular molecules involved in cancer cell proliferation and survival. Similar results were obtained in Mz-ChA-1 cells. CONCLUSIONS: (1) Inhibition of intracellular pH regulatory mechanisms by cariporide and S3705 reduces proliferation and induces apoptosis in cholangiocarcinoma cells; and (2) these drugs might have potential therapeutic value against cholangiocarcinoma.

Hepatoprotective and antifibrotic effect of a new silybin-phosphatidylcholine-Vitamin E complex in rats.

BACKGROUND: Silybin, the main active component of silymarin, has been reported to reduce hepatic fibrosis by 30% in bile duct ligated rats, whereas Vitamin E alone does not significantly modify liver damage and collagen deposition in chronic liver injury. AIM: The aim of the present study was to evaluate the hepatoprotective and the antifibrotic properties of a new silybin-phosphatidylcholine-Vitamin E complex, characterised by elevated oral bioavailability and lipophilicity, on rat hepatic fibrosis induced by dimethylnitrosamine administration and by bile duct ligation. METHODS/RESULTS: The complex was administered by gastric gavage at a dose of 250 and 75 mg/kg (as silybin and Vitamin E, respectively). Treatment with the complex was able to prevent the dimethylnitrosamine-induced loss in body and liver weight, as well as to reduce the degree of liver injury, as determined by alanine aminotransferase values and necroinflammatory score. This was associated with reduced hepatic stellate cells proliferation both after 1 and 5 weeks of treatment. Treatment with the complex reduced also hepatic stellate cells activation and collagen deposition. Treatment with dimethylnitrosamine induced an increase in alpha1(I) procollagen, TGF(beta1), tissue inhibitor of metalloproteinase 1 and metalloproteinase 2 mRNA expression, which were significantly reduced by administration of the complex. In the bile duct ligation model, the administration of the complex was able to reduce hepatic stellate cells proliferation and activation, as well as collagen deposition and alpha1(I) procollagen mRNA expression. CONCLUSIONS: These results suggest that this new silybin-phosphatidylcholine-Vitamin E complex could be an interesting drug to be tested in patients with chronic liver disease.

Effect of cyanidin 3-O-beta-glucopyranoside on hepatic stellate cell proliferation and collagen synthesis induced by oxidative stress.

BACKGROUND: Reactive oxygen species play a role in the pathogenesis of hepatic fibrosis, mainly through the activation of hepatic stellate cells. Cyanidin-3-O-beta-glucopyranoside is a natural antioxidant compound distributed in several fruits and vegetables. AIM: To evaluate the effect of cyanidin-3-O-beta-glucopyranoside on hepatic stellate cells proliferation and collagen synthesis induced by a pro-oxidant agent. METHODS/RESULTS: Oxidative stress was induced by incubation of hepatic stellate cells with a ferric nitrilotriacetate complex (100 micromol/L). Incubation with ferric nitrilotriacetate induced an increased intracellular hydroperoxide formation, which was completely inhibited by cyanidin-3-O-beta-glucopyranoside at a concentration of 50mumol/L. Similarly, cyanidin-3-O-beta-glucopyranoside was able to inhibit ferric nitrilotriacetate-induced hepatic stellate cells proliferation, evaluated by an ELISA method, with a maximal effect at 50mumol/L. Incubation of hepatic stellate cells with cyanidin-3-O-beta-glucopyranoside inhibited ferric nitrilotriacetate-induced extracellular signal-regulated kinase 1/2 activation, evaluated by western blot, whereas it did not affect p70S6 kinase and AKT expression. Finally, cyanidin-3-O-beta-glucopyranoside reduced ferric nitrilotriacetate-induced Na(+)/H(+) exchange activation, evaluated by a spectrofluorimetric method, and collagen type I synthesis, evaluated by northern blot. CONCLUSION: Cyanidin-3-O-beta-glucopyranoside is able to modulate hepatic stellate cells proliferation and type I collagen synthesis induced by a pro-oxidant agent, thus suggesting a potential role for this antioxidant compound in the prevention of fibrosis in chronic liver diseases.

The anti-fibrotic effect of pirfenidone in rat liver fibrosis is mediated by downregulation of procollagen alpha1(I), TIMP-1 and MMP-2.

BACKGROUND: Pirfenidone (5 methyl-1-phenyl-2(1H)-pyridone) is a novel anti-fibrotic agent, which has been shown to decrease collagen deposition in a variety of animal models in vivo, and recently in hepatic fibrosis also. At cellular level, we have recently demonstrated that pirfenidone is able to inhibit proliferation of hepatic stellate cells induced by platelet-derived growth factor, as well as collagen type I accumulation and alpha1(I) procollagen mRNA expression. AIMS: To evaluate if pirfenidone maintains its anti-fibrotic properties also when administered after the induction of hepatic damage and to further investigate the molecular mechanisms leading to the anti-fibrotic effect of pirfenidone. METHODS AND RESULTS: Rats treated with dimethylnitrosamine (10 mg/kg) for 5 weeks received a liquid diet containing 0.5% pirfenidone starting from the third week. Pirfenidone treatment reduced the degree of liver injury, as determined by alanine aminotransferase values and necro-inflammatory score, which was associated with reduced hepatic stellate cells proliferation and collagen deposition. Treatment with dimethylnitrosamine increased transcripts levels for transforming growth factorbeta1, procollagen alpha1(I), tissue inhibitors of metalloproteinase-1 and matrix metalloproteinase-2 by 7-, 7-, 4- and 15-fold, respectively. Pirfenidone administration downregulated elevated levels of those transcripts by 50-60%, and this was associated with a 70% reduction in collagen deposition. CONCLUSIONS: (1) Pirfenidone is effective also if administered after the induction of the hepatic damage; (2) the anti-fibrotic effect of pirfenidone is mainly due to the reduced expression of profibrogenic procollagen alpha1(I) and TIMP-1, most likely through the downregulation of transforming growth factorbeta1 mRNA, and of matrix metalloproteinase-2, which is mainly implicated in the degradation of the normal extracellular matrix.

Absence of somatic mutations in natriuretic peptide receptor type A gene in human aldosterone-secreting adenomas.

Somatic mutations of genes codifying for key regulatory proteins are the cause of different types of hormone-secreting adenomas. Natriuretic peptides (NP) are the strongest inhibitors of aldosterone secretion but aldosterone-secreting adenomas (aldosteronomas) are resistant to this inhibition and have reduced binding sites for NPs. The objective of this study was to sequence the entire coding region of the NP receptor type A (NPRA, codified by the Npr1 gene) to find loss-of-function somatic mutations. Total RNA was extracted from eight aldosteronomas and cDNA was synthesized. NPRA mRNA expression was evaluated by Northern blot analysis and compared with beta-actin mRNA as the housekeeping gene. Twelve primer couples were designed on the basis of the Npr1 gene organization to amplify, by PCR, all 22 coding exons of the gene. The two strands of amplified DNAs were purified and directly sequenced by automated capillary sequencer. NPRA mRNA expression did not differ among aldosteronomas. Npr1 open reading frame sequences obtained from eight aldosteronomas did not contain any mutation. The coding sequences of all 22 exons were identical in all samples and identical to published sequences. In the 3'-untranslated region (3'-UTR) a new length difference 3C/4C polymorphism was found at position 15 129 (three adenomas were 3C/4C and two were 3C/3C). Such a 3C/4C polymorphism was present in genomic DNA from 80 control subjects (25, 4C/4C; 40, 3C/4C; 15, 3C/3C). Mutations in the coding exons of the Npr1 gene do not appear to be a common cause of aldosteronomas. Moreover, the exons of Npr1 encoding for the translated portion of mRNA do not appear to be prone to polymorphisms. The polymorphism identified in the 3'-UTR might affect mRNA stability resulting in lower receptor synthesis, but it is not likely to confer a predisposition to the development of aldosteronomas.