Colesevelam attenuates cholestatic liver and bile duct injury in Mdr2-/- mice by modulating composition, signalling and excretion of faecal bile acids.

BACKGROUND AND AIMS: Interruption of the enterohepatic circulation of bile acids (BAs) may protect against BA-mediated cholestatic liver and bile duct injury. BA sequestrants are established to treat cholestatic pruritus, but their impact on the underlying cholestasis is still unclear. We aimed to explore the therapeutic effects and mechanisms of the BA sequestrant colesevelam in a mouse model of sclerosing cholangitis. METHODS: Mdr2-/- mice received colesevelam for 8 weeks. Gene expression profiles of BA homeostasis, inflammation and fibrosis were explored in liver, intestine and colon. Hepatic and faecal BA profiles and gut microbiome were analysed. Glucagon-like peptide 1 (GLP-1) levels in portal blood were measured by ELISA. Furthermore, Mdr2-/- mice as well as wild-type 3,5-diethoxy-carbonyl-1,4-dihydrocollidine-fed mice were treated with GLP-1-receptor agonist exendin-4 for 2 weeks prior to analysis. RESULTS: Colesevelam reduced serum liver enzymes, BAs and expression of proinflammatory and profibrogenic markers. Faecal BA profiling revealed increased levels of secondary BAs after resin treatment, while hepatic and biliary BA composition showed a shift towards more hydrophilic BAs. Colonic GLP-1 secretion, portal venous GLP-1 levels and intestinal messenger RNA expression of gut hormone Proglucagon were increased, while ileal Fgf15 expression was abolished by colesevelam. Exendin-4 treatment increased bile duct mass without promoting a reactive cholangiocyte phenotype in mouse models of sclerosing cholangitis. Microbiota analysis showed an increase of the phylum δ-Proteobacteria after colesevelam treatment and a shift within the phyla Firmicutes from Clostridiales to Lactobacillus. CONCLUSION: Colesevelam increases faecal BA excretion and enhances BA conversion towards secondary BAs, thereby stimulating secretion of GLP-1 from enteroendocrine L-cells and attenuates liver and bile duct injury in Mdr2-/- mice.

Metabolic preconditioning protects BSEP/ABCB11-/- mice against cholestatic liver injury.

BACKGROUND & AIMS: Cholestasis is characterized by intrahepatic accumulation of potentially cytotoxic bile acids (BAs) subsequently leading to liver injury with disruption of hepatocellular integrity, inflammation, fibrosis and ultimately liver cirrhosis. Bile salt export pump (BSEP/ABCB11) is the main canalicular BA transporter and therefore the rate limiting step for hepatobiliary BA excretion. In this study we aimed to investigate the role of BSEP/ABCB11 in the development of acquired cholestatic liver and bile duct injury. METHODS: Wild-type (WT) and BSEP knockout (BSEP-/-) mice were subjected to common bile duct ligation (CBDL) or 3.5-diethoxycarbonyl-1.4-dihydrocollidine (DDC) feeding as models for cholestasis with biliary obstruction and bile duct injury. mRNA expression profile, serum biochemistry, liver histology, immunohistochemistry, hepatic hydroxyproline levels and BA composition as well as biliary pressure were assessed. RESULTS: BSEP-/- mice were protected against acquired cholestatic liver injury induced by 7days of CBDL or 4weeks of DDC feeding, as reflected by unchanged serum levels of liver transaminases, alkaline phosphatase and BAs. Notably, BSEP-/- mice were also protected from cholestasis-induced hepatic inflammation and biliary fibrosis. In line with induced BA detoxification/hydroxylation pathways in BSEP-/- mice, polyhydroxylated BAs were increased 4-fold after CBDL and 6-fold after DDC feeding in comparison with cholestatic WT mice. Finally, following CBDL, biliary pressure in WT mice increased up to 47mmH2O but remained below 11mmH2O in BSEP-/- mice. CONCLUSION: Metabolic preconditioning with subsequent changes in BA metabolism favors detoxification of potentially toxic BAs and thereby protects BSEP-/- mice from cholestatic liver and bile duct injury. LAY SUMMARY: Reduced hepatobiliary bile acid transport due to loss of BSEP function leads to increased hydroxylation of bile acids in the liver. Metabolic preconditioning with a hydrophilic bile pool protects the BSEP-/- mice from acquired cholestatic liver disease.

Raf-1 levels determine the migration rate of primary endometrial stromal cells of patients with endometriosis.

Endometriosis is a disease characterized by the localization of endometrial tissue outside the uterine cavity. The differences observed in migration of human endometrial stromal cells (hESC) obtained from patients with endometriosis versus healthy controls were proposed to correlate with the abnormal activation of Raf-1/ROCKII signalling pathway. To evaluate the mechanism by which Raf-1 regulates cytoskeleton reorganization and motility, we used primary eutopic (Eu-, n = 16) and ectopic (Ec-, n = 8; isolated from ovarian cysts) hESC of patients with endometriosis and endometriosis-free controls (Co-hESC, n = 14). Raf-1 siRNA knockdown in Co- and Eu-hESC resulted in contraction and decreased migration versus siRNA controls. This phenotype was reversed following the re-expression of Raf-1 in these cells. Lowest Raf-1 levels in Ec-hESC were associated with hyperactivated ROCKII and ezrin/radixin/moesin (E/R/M), impaired migration and a contracted phenotype similar to Raf-1 knockdown in Co- and Eu-hESC. We further show that the mechanism by which Raf-1 mediates migration in hESC includes direct myosin light chain phosphatase (MYPT1) phosphorylation and regulation of the levels of E/R/M, paxillin, MYPT1 and myosin light chain (MLC) phosphorylation indirectly via the hyperactivation of ROCKII kinase. Furthermore, we suggest that in contrast to Co-and Eu-hESC, where the cellular Raf-1 levels regulate the rate of migration, the low cellular Raf-1 content in Ec-hESC, might ensure their restricted migration by preserving the contracted cellular phenotype. In conclusion, our findings suggest that cellular levels of Raf-1 adjust the threshold of hESC migration in endometriosis.

Testosterone induced downregulation of migration and proliferation in human Umbilical Vein Endothelial Cells by Androgen Receptor dependent and independent mechanisms.

Recent research has emphasized the potential unfavorable effects of declining testosterone (T) levels in men and the putative beneficial effect of androgen therapy in select women. Some controversy surrounding the mechanism of action and the effects of T on endothelium remains. In this study, we evaluated the mechanism of T action on pooled primary Human Umbilical Vein Endothelial Cells (HUVEC) of mixed gender by focusing on two important processes, proliferation and migration. In our in vitro model system, we found that only the supra-physiological dose of T affected these two processes irrespective of the ratio of male to female cells in the pools. At a concentration of 1 µM, T downregulated the proliferation of HUVEC by inducing arrest in the G1 cell cycle phase in an Androgen Receptor (AR)-independent manner. We show that treatment with 1 µM T also induced downregulation of HUVEC migration. This process was AR-dependent and was associated with persistent phosphorylation of ezrin, radixin and moesin. Regardless of the mechanism of action, the treatment of HUVEC with both supra- and physiological doses of T was associated with posttranscriptional stabilization of the AR upon ligand binding.