838 MODULATION OF MIRNAS BY BILE ACIDS: IMPACT ON RAT LIVER REGENERATION

838 MODULATION OF MIRNAS BY BILE ACIDS: IMPACT ON RAT LIVER REGENERATION

S306 Poster Session − Saturday, April 25 838 MODULATION OF MIRNAS BY BILE ACIDS: IMPACT ON RAT LIVER REGENERATION R.E. Castro1,2 , Y. Zeng3 , C.J. S...

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S306

Poster Session − Saturday, April 25

838 MODULATION OF MIRNAS BY BILE ACIDS: IMPACT ON RAT LIVER REGENERATION R.E. Castro1,2 , Y. Zeng3 , C.J. Steer2,4 , B.T. Kren2 , C.M.P. Rodrigues1 . 1 iMed. UL, Faculty of Pharmacy, University of Lisbon, Lisbon, Portugal, 2 Department of Medicine, 3 Department of Pharmacology, 4 Department of Genetics, Cell Biology and Development, University of Minnesota Medical School, Minneapolis, Minnesota, USA E-mail: [email protected] Background and Aims: Bile acids (BAs) are strong modulators of apoptosis and proliferation in hepatocytes. In addition, BAs modulate mRNA levels during liver regeneration. Here, we used microRNA (miRNA) analysis to gain insight into the mechanisms by which BAs modulate cell fate and how this impacts on liver regeneration. Methods: RNA was isolated from rat livers harvested at 3−72 h, following 70% partial hepatectomy (PH) or sham operations, of either 0.4% UDCA or control diet-fed animals. miRNA expression profiles were determined using a custom microarray platform; real time RT-PCR was used for validation. miRNA-21 and target protein Programmed Cell Death 4 (PDCD-4) were evaluated in isolated primary rat hepatocytes, following BA exposure for 4−48 h. In functional studies, miRNA-21 was overexpressed by transfecting cells with a specific miRNA-21 precursor. Cell death was evaluated by LDH and Hoechst assays and proliferation by the MTS assay. Results: miRNAs involved in regulating cell proliferation, differentiation and death, such as miRNA-21, were significantly modulated after PH. UDCA feeding further increased the number of miRNAs modulated after PH and significantly changed the expression of several miRNAs in sham-operated rats. Real time RT-PCR analysis confirmed the increased expression of miRNA-21 after PH in both animal groups. In rat hepatocytes, miRNA-21 expression slightly increased with time in control and UDCA exposed cells. Interestingly, deoxycholic acid (DCA) was a stronger modulator of miRNA-21, decreasing expression up to 50% at 48 h. Consistently, PDCD-4 was significantly increased in cells incubated with DCA. Finally, miRNA-21 overexpression inhibited PDCD-4 protein expression, increased cell viability, and prevented DCA-induced PDCD-4 expression and cell death. Conclusions: miRNAs may play a significant role in modulating proliferation and cell cycle progression genes after PH. Furthermore, BAs modulate miRNA expression, in particular miRNA-21, in both regenerating rat liver and primary rat hepatocytes. Finally, repression of miRNA-21 by DCA may contribute to its pro-apoptotic role through PDCD-4 modulation. Ultimately, a better knowledge of the mechanisms by which BAs modulate proliferation and apoptosis, as well as the switches that control liver regeneration, may have broad implications for developing novel therapeutic options. (Supported, in part, by PTDC/SAU-FCF/67912/2006). 839 CHARACTERIZATION OF STEM CELL COMPARTMENTS IN ADULT HUMAN LIVER L. Porretti1 , A. Cattaneo1 , F. Colombo1 , R. Lopa1 , S. Mazzucchelli1 , G. Rossi1 , V. Mazzaferro2 , G. Svegliati Baroni3 , F. Bertolini4 , D. Prati1,5 . 1 Fondazione Ospedale Maggiore Policlinico, 2 Istituto Nazionale Tumori, Milan, 3 Ospedale di Ancona, Ancona, 4 Istituto Europeo di Oncologia, Milan, 5 Ospedale A Manzoni, Lecco, Italy E-mail: [email protected] Background and Aim: The human liver is a complex tissue, composed of epithelial, endothelial, hematopoietic, and mesenchymal elements. Therefore, the intrahepatic stem cell population would consist of an heterogeneous pool of cells with various phenotypes and multiple molecular and functional markers. In this study, we identified and characterized intrahepatic stem cells and progenitors committed towards the epithelial, endothelial, hematopoietic, and mesenchymal lineages. Methods: Single-mononuclear cell suspensions obtained after enzymatic digestion of liver specimens from 20 patients with chronic liver disease and 10 multi-organ donors were examined by six-color flow cytometry,

analyzing at least 106 cells per sample. Specific culture systems were implemented to confirm the identity and differentiation potential of stem cell lineages. Results: More than 70% of total intrahepatic stem cells and progenitors are committed towards the epithelial lineage (epCAM+/CD49f+/ CD29+/CD45−), while the hematopoietic (CD34+/CD45+ and CD133+/ CD45+) and endothelial precursors (KDR+/CD146+/CD45−) accounted for less than 10%. Mesenchymal stem cells, identified by their adipogenic and osteogenic differentiation potential, represented about 15% of the total hepatic stem cell compartment. Intrahepatic stem cells showed several cultural and immunophenotypic peculiarities when compared to their extrahepatic counterparts, and some differences were observed between donors and patients. After the sequential characterization of intrahepatic progenitors cells with multiple monoclonal antibodies, we developed a single-tube, six-color staining that could simultaneously distinguish the four stem cell lineages. Conclusions: Our data confirm that both the healthy and diseased adult human liver contains stem cells and progenitors of different origins. Most cells belong to the epithelial lineage, but mesenchymal, endothelial, and hematopoietic elements are also represented. This study provides a background for future biological and clinical investigations on intrahepatic stem cells. 840 ISOLATION AND FOUNCTIONAL IDENTIFICATION OF A NOVEL HUMAN HEPATIC GROWTH FACTOR: HEPATOPOIETIN CN C.-P. Cui1 , C.-T. Wu1 , L.-S. Wang1 , D.-J. Zhang1 , Y. Liu2 , J. Chang1 , D.-D. Zhang1 , J. Liu2 , G.-S. Zhong1 , S.-J. Du1 . 1 Beijing Institute of Radiation Medicine, Beijing, 2 Tianjin University, Tianjin, China E-mail: [email protected] Background: Hepatic stimulating substance (HSS) was first isolated from weanling rat liver in 1975 and found to stimulate hepatic DNA synthesis both in vitro and in vivo. Since then, mammalian and human HSS have been investigated for their potential to treat hepatic diseases. However, the essential nature in composition and structure of HSS remain puzzling because HSS has not been completely purified. Aims: To isolate and identify novel hepatic growth factors from new born calf liver and evaluate its protective effect on liver injury and fibrosis induced by carbon tetrachloride (CCl4 ) injection. Methods: Heating, ethanol precipitation, andion-exchange chromatographies had been carried out to isolate the protein with specific stimulating activity from new born calf liver, and [3 H] thymidine deoxyribose (TdR)/bromodeoxyuridine (BrdU) incorporation and carboxyfluorescein diacetate succinimidyl ester (CFSE)-based proliferation assay to determine the bioactivity in vitro and in vivo. Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activity in rat serum were measured using biochemical assay. Hepatic hydroxyproline (Hyp) level was determined in the hydrolysates of liver samples. Immunostaining and Masson’s trichrome staining were conducted to evaluate hepatocyte proliferation and fibrosis. Results: We report the purification of a novel 30-kDa protein from a crude extract of calf liver HSS. This protein is a member of the leucine-rich acidic nuclear protein family (LANP) and has been named Hepatopoietin Cn (HPPCn). Studies of partially hepatectomized mice show that levels of HPPCn messenger RNA increase after liver injury. Furthermore, the recombinant human protein (rhHPPCn) was shown to stimulate hepatic DNA synthesis and activate signaling pathways involved in hepatocyte proliferation in vitro and in vivo. Additionaly exogenous rhHPPCn could reduce ALT/AST levels in rat serum following single and repeated CCl4 injection and alleviate hepatocyte necrosis and protect the liver from the development of fibrotic lesions by proliferation stimulation. Conclusions: HPPCn is a novel hepatic growth factor that plays a role in liver regeneration and could protect hepatocytes from injury induced by CCl4 as a proliferation stimulator.