- Email: [email protected]
845 HINT1 IS A PROTEIN POTENTIALLY IMPLICATED IN LIVER REGENERATION
S308
Poster Session − Saturday, April 25
glass microscope slide. Primary screening identified 6 polymers (three polyacrylates 2AE7, 7G7, 3AA7 and three polyurethanes 134, 212, 223) that supported HLC attachment and identity. We further characterised hepatocytic function in detail on these six matrices. Using this strategy, polymer 134 was identified as the most effective cellular support associated with enhanced expression of Fibrinogen, transthyretin (TTR) and soluble fibronectin. Key cytochrome p450 (CYP) activities on the different extracellular matrices were also characterised. CYP1A2 activity was increased ~6 fold and CYP3A4 ~2 fold on polymer 134 as compared to standard matrigel conditions or the other polymers tested. Conclusion: In conclusion, the high through-put screening of a polymer microarray allowed the identification of a new polymer matrix that promotes long-term hepatocellular differentiated function before and after passaging. These attributes bypass current limitations associated adult human hepatocytes, can be manufactured to GMP standards and will play important roles in developing in vitro models of drug toxicicology, provide a resource for the construction of extra-corporeal devices and facilitate novel studies of human liver development and disease. 845 HINT1 IS A PROTEIN POTENTIALLY IMPLICATED IN LIVER REGENERATION C. Hora1 , O. Maurhofer1 , M. Ledermann1 , G. Ferrand2 , H. Li3 , I.B. Weinstein3,4 , J.-F. Dufour1 , J. Martin1 . 1 Institute of Clinical Pharmacology and Visceral Research, University of Berne, Berne, 2 Swiss Institute for Experimental Cancer Research, Epalinges, Switzerland; 3 Herbert Irving Comprehensive Cancer Center, Columbia University, 4 Department of Medicine, Columbia University, New York, NY, USA E-mail: [email protected] Introduction: Liver regeneration plays an important role in liver transplantation and liver cancer treatment. HINT1, a nucleotide binding protein belonging to the HIT protein family functions as a tumour suppressor and participates in the cellular response to DNA damage. The aim of our study was to investigate whether liver regeneration is affected by the lack of Hint1. Methods: 2/3 partial hepatectomy (PH) was performed in male Hint−/ − and C57BL/6 mice (9−10 weeks-old, n = 7−10). Animals were sacrificed at 3, 6, 24, 48, 72 and 96 hours post PH and the remaining livers weighed. Restoration of liver mass was calculated. Cell proliferation was determined by immunohistochemistry using BrdU and Ki67 as markers. Liver histology and apoptosis were evaluated by H&E and cleaved lamin A (cLA) staining. Gene and protein expression of CyclinD1, p21, IL6 and TNFa were analysed by QPCR and immunobloting. Data are means±SD, p0.05. Results: In Hint1−/ − mice, the liver mass did not increase until 24 h after surgery. Increases of 30±18%, 82±20% and 106±10% were recorded at 48 h, 72 h and 96 h after PH, respectively. In C57BL/6 the liver mass increased without delay by 19±7, 36±10, 43±3.5, 59±3%, 119±13.6% and 144±12% at 3 h, 6 h, 24 h, 48 h, 72 h and 96 h after PH, respectively. Proliferation assessed by BrdU and Ki67 and apoptosis assessed with cLA were not significantly different between the 2 groups. At the time of surgery, there was less IL6 and TNFa mRNA mRNA in Hint1−/ − mice vs C57BL6 mice. After PH, levels of IL6 and TNFa mRNA followed a similar pattern in both mice but remained lower in Hint1−/ − . Earlier and higher augmentation of p21 mRNA at 24 h was observed in Hint1−/ − . CyclinD1 mRNA expression was enhanced after 24 h and 72 h PH in Hint1−/ − whereas this expression peaked after 48 h in C57BL6 mice. A higher peak of p21 and CyclinD1 protein expression was measured after 24 h and 48 h in Hint1−/ − in comparison to C57BL6. Conclusion: Liver regeneration is impaired in the case of Hint1 ablation. Our results suggest that Hint1 plays a role in the initiation of liver regeneration by means of a mechanism that affects cytokines production.
846 ATP RELEASE AFTER PARTIAL HEPATECTOMY REGULATES LIVER REGENERATION E. Gonzales1 , B. Julien1 , V. Serri`ere-Lanneau1 , A. Nicou1 , I. Doignon1 , L. Lagoudakis1 , I. Garcin1 , D. Azoulay2 , J.-C. Duclos-Vall´ee2 , D. Castaing2 , D. Samuel2 , A. Hernandez-Garcia3 , S. Awad3 , L. Combettes1 , S. Thevananther3 , T. Tordjmann1 . 1 INSERM U757, Universit´e Paris-Sud, Orsay, 2 Centre H´epatobiliaire, Hopital Paul Brousse, Villejuif, France; 3 Baylor College of Medicine, Houston, Texas, USA E-mail: [email protected] Background and Aims: Paracrine interactions are critical to liver physiology, particularly during regeneration, although physiological involvement of extracellular ATP, a crucial intercellular messenger in epithelia, remains unclear. The physiological release of ATP and its impact on regeneration after partial hepatectomy, which have never been explored, were investigated in this study. Methods: Hepatic ATP release before and after partial hepatectomy was examined in plasma of rats and in human living donors for liver transplantation. The involvement of Kupffer cells was evaluated by pretreating rats with clodronate liposomes. In rat liver sections and isolated hepatocytes, quinacrine was used for in vivo staining of ATP-enriched intracellular compartments, and lysotracker was used to stain lysosomes. Rats were treated with an antagonist for P2 purinergic receptors [phosphate-6azo(benzene-2,4-disulfonic) acid, PPADS], and liver regeneration after hepatectomy was analyzed. Results: A robust and transient ATP release was observed immediately after hepatectomy in the rat and in humans. Experiments after clodronate liposomes treatment suggested a partial contribution of Kupffer cells to the ATP release in the rat. Quinacrine-stained vesicles, predominantly detected in periportal hepatocytes before, significantly disappeared after partial hepatectomy, in parallel with a decrease in ATP liver content. These vesicles were colabelled with lysotracker in liver sections and isolated hepatocytes. PPADS treatment dampened hepatocyte cell cycle progression after hepatectomy, as revealed by a reduction in BrdU incorporation, phosphorylated histone 3 immunostaining, cyclin D1 and A expression and immediate early genes induction. Conclusion: Extracellular ATP is released immediately after partial hepatectomy from hepatocytes and Kupffer cells and promotes liver regeneration in the rat. We suggest that in hepatocytes, ATP may be released from a lysosomal compartment. Finally, observations made in living donors suggest that purinergic signalling could be critical for human liver regeneration. 847 ADULT-DERIVED HUMAN LIVER MESENCHYMALLIKE STEM/PROGENITOR CELLS INCREASE HEPATIC METABOLIC FUNCTION AFTER IN VITRO DIFFERENTIATION D.N. Khuu1 , M. Najimi1 , P.B. Calderon2 , E.M. Sokal1 . 1 Laboratory of Pediatric Hepatology and Cell Therapy, 2 Pharmacocinetic, Metabolism, Nutrition and Toxicology Department, Universit´e Catholique de Louvain, Brussel, Belgium E-mail: [email protected] Background and Aims: Adult liver derived progenitor cells (LDPC) were recently isolated and characterized from healthy human livers. Their ability to differentiate into hepatocyte-like cells led to propose them as potential alternative cell to mature hepatocyte for cell therapy of human metabolic liver diseases. However, beside genotypic and phenotypic differentiation, these hepatic progenitor cells should exhibit some, if not all, metabolic liver functions. Such properties were investigated in the current in vitro study. Methods: LDPCs were isolated from 5 different healthy cadaveric donor livers and in vitro differentiated (P3−6) after sequential incubation with growth factors and cytokines. Three important functions were investigated: gluconeogenesis, urea synthesis and cytochrome 3A4 activity in differentiated LDPCs, with primary mature hepatocytes and hepG2 cells as controls.
Poster Session − Saturday, April 25
glass microscope slide. Primary screening identified 6 polymers (three polyacrylates 2AE7, 7G7, 3AA7 and three polyurethanes 134, 212, 223) that supported HLC attachment and identity. We further characterised hepatocytic function in detail on these six matrices. Using this strategy, polymer 134 was identified as the most effective cellular support associated with enhanced expression of Fibrinogen, transthyretin (TTR) and soluble fibronectin. Key cytochrome p450 (CYP) activities on the different extracellular matrices were also characterised. CYP1A2 activity was increased ~6 fold and CYP3A4 ~2 fold on polymer 134 as compared to standard matrigel conditions or the other polymers tested. Conclusion: In conclusion, the high through-put screening of a polymer microarray allowed the identification of a new polymer matrix that promotes long-term hepatocellular differentiated function before and after passaging. These attributes bypass current limitations associated adult human hepatocytes, can be manufactured to GMP standards and will play important roles in developing in vitro models of drug toxicicology, provide a resource for the construction of extra-corporeal devices and facilitate novel studies of human liver development and disease. 845 HINT1 IS A PROTEIN POTENTIALLY IMPLICATED IN LIVER REGENERATION C. Hora1 , O. Maurhofer1 , M. Ledermann1 , G. Ferrand2 , H. Li3 , I.B. Weinstein3,4 , J.-F. Dufour1 , J. Martin1 . 1 Institute of Clinical Pharmacology and Visceral Research, University of Berne, Berne, 2 Swiss Institute for Experimental Cancer Research, Epalinges, Switzerland; 3 Herbert Irving Comprehensive Cancer Center, Columbia University, 4 Department of Medicine, Columbia University, New York, NY, USA E-mail: [email protected] Introduction: Liver regeneration plays an important role in liver transplantation and liver cancer treatment. HINT1, a nucleotide binding protein belonging to the HIT protein family functions as a tumour suppressor and participates in the cellular response to DNA damage. The aim of our study was to investigate whether liver regeneration is affected by the lack of Hint1. Methods: 2/3 partial hepatectomy (PH) was performed in male Hint−/ − and C57BL/6 mice (9−10 weeks-old, n = 7−10). Animals were sacrificed at 3, 6, 24, 48, 72 and 96 hours post PH and the remaining livers weighed. Restoration of liver mass was calculated. Cell proliferation was determined by immunohistochemistry using BrdU and Ki67 as markers. Liver histology and apoptosis were evaluated by H&E and cleaved lamin A (cLA) staining. Gene and protein expression of CyclinD1, p21, IL6 and TNFa were analysed by QPCR and immunobloting. Data are means±SD, p0.05. Results: In Hint1−/ − mice, the liver mass did not increase until 24 h after surgery. Increases of 30±18%, 82±20% and 106±10% were recorded at 48 h, 72 h and 96 h after PH, respectively. In C57BL/6 the liver mass increased without delay by 19±7, 36±10, 43±3.5, 59±3%, 119±13.6% and 144±12% at 3 h, 6 h, 24 h, 48 h, 72 h and 96 h after PH, respectively. Proliferation assessed by BrdU and Ki67 and apoptosis assessed with cLA were not significantly different between the 2 groups. At the time of surgery, there was less IL6 and TNFa mRNA mRNA in Hint1−/ − mice vs C57BL6 mice. After PH, levels of IL6 and TNFa mRNA followed a similar pattern in both mice but remained lower in Hint1−/ − . Earlier and higher augmentation of p21 mRNA at 24 h was observed in Hint1−/ − . CyclinD1 mRNA expression was enhanced after 24 h and 72 h PH in Hint1−/ − whereas this expression peaked after 48 h in C57BL6 mice. A higher peak of p21 and CyclinD1 protein expression was measured after 24 h and 48 h in Hint1−/ − in comparison to C57BL6. Conclusion: Liver regeneration is impaired in the case of Hint1 ablation. Our results suggest that Hint1 plays a role in the initiation of liver regeneration by means of a mechanism that affects cytokines production.
846 ATP RELEASE AFTER PARTIAL HEPATECTOMY REGULATES LIVER REGENERATION E. Gonzales1 , B. Julien1 , V. Serri`ere-Lanneau1 , A. Nicou1 , I. Doignon1 , L. Lagoudakis1 , I. Garcin1 , D. Azoulay2 , J.-C. Duclos-Vall´ee2 , D. Castaing2 , D. Samuel2 , A. Hernandez-Garcia3 , S. Awad3 , L. Combettes1 , S. Thevananther3 , T. Tordjmann1 . 1 INSERM U757, Universit´e Paris-Sud, Orsay, 2 Centre H´epatobiliaire, Hopital Paul Brousse, Villejuif, France; 3 Baylor College of Medicine, Houston, Texas, USA E-mail: [email protected] Background and Aims: Paracrine interactions are critical to liver physiology, particularly during regeneration, although physiological involvement of extracellular ATP, a crucial intercellular messenger in epithelia, remains unclear. The physiological release of ATP and its impact on regeneration after partial hepatectomy, which have never been explored, were investigated in this study. Methods: Hepatic ATP release before and after partial hepatectomy was examined in plasma of rats and in human living donors for liver transplantation. The involvement of Kupffer cells was evaluated by pretreating rats with clodronate liposomes. In rat liver sections and isolated hepatocytes, quinacrine was used for in vivo staining of ATP-enriched intracellular compartments, and lysotracker was used to stain lysosomes. Rats were treated with an antagonist for P2 purinergic receptors [phosphate-6azo(benzene-2,4-disulfonic) acid, PPADS], and liver regeneration after hepatectomy was analyzed. Results: A robust and transient ATP release was observed immediately after hepatectomy in the rat and in humans. Experiments after clodronate liposomes treatment suggested a partial contribution of Kupffer cells to the ATP release in the rat. Quinacrine-stained vesicles, predominantly detected in periportal hepatocytes before, significantly disappeared after partial hepatectomy, in parallel with a decrease in ATP liver content. These vesicles were colabelled with lysotracker in liver sections and isolated hepatocytes. PPADS treatment dampened hepatocyte cell cycle progression after hepatectomy, as revealed by a reduction in BrdU incorporation, phosphorylated histone 3 immunostaining, cyclin D1 and A expression and immediate early genes induction. Conclusion: Extracellular ATP is released immediately after partial hepatectomy from hepatocytes and Kupffer cells and promotes liver regeneration in the rat. We suggest that in hepatocytes, ATP may be released from a lysosomal compartment. Finally, observations made in living donors suggest that purinergic signalling could be critical for human liver regeneration. 847 ADULT-DERIVED HUMAN LIVER MESENCHYMALLIKE STEM/PROGENITOR CELLS INCREASE HEPATIC METABOLIC FUNCTION AFTER IN VITRO DIFFERENTIATION D.N. Khuu1 , M. Najimi1 , P.B. Calderon2 , E.M. Sokal1 . 1 Laboratory of Pediatric Hepatology and Cell Therapy, 2 Pharmacocinetic, Metabolism, Nutrition and Toxicology Department, Universit´e Catholique de Louvain, Brussel, Belgium E-mail: [email protected] Background and Aims: Adult liver derived progenitor cells (LDPC) were recently isolated and characterized from healthy human livers. Their ability to differentiate into hepatocyte-like cells led to propose them as potential alternative cell to mature hepatocyte for cell therapy of human metabolic liver diseases. However, beside genotypic and phenotypic differentiation, these hepatic progenitor cells should exhibit some, if not all, metabolic liver functions. Such properties were investigated in the current in vitro study. Methods: LDPCs were isolated from 5 different healthy cadaveric donor livers and in vitro differentiated (P3−6) after sequential incubation with growth factors and cytokines. Three important functions were investigated: gluconeogenesis, urea synthesis and cytochrome 3A4 activity in differentiated LDPCs, with primary mature hepatocytes and hepG2 cells as controls.