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172 ASSOCIATION FOR ACADEMIC SURGERY AND SOCIETY OF UNIVERSITY SURGEONS—ABSTRACTS ingestion manifested a 43% reduction in Fgf10 expression after 24 ...

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172

ASSOCIATION FOR ACADEMIC SURGERY AND SOCIETY OF UNIVERSITY SURGEONS—ABSTRACTS

ingestion manifested a 43% reduction in Fgf10 expression after 24 hours compared to a 20-fold increase in Fgf10 expression in PH only mice. Conclusion: Liver regeneration is dependent on FGFR2b activation, for which FGF10 is the most common ligand. An increase in expression of Fgf10 expression after PH is dependent on hepatic stellate cell activation. Impaired liver regeneration caused by ethanol ingestion may be in part due to the abrogation of Fgf10 upregulation after PH.

51. CONVERSION OF PANCREATIC PROGENITORS TO DUODENAL AND BILIARY FATES BY PTF1A-CREMEDIATED INACTIVATION OF PDX1. K. Furuyama 1, Y. Kawaguchi 1, S. Kodama 1, M. Horiguchi 1, A. Fukuda 1, T. Kuhara 1, M. Kawaguchi 1, S. Uemoto 1, C. V. Wright 2, R. Doi 1; 1Kyoto University, Kyoto, Japan, 2Vanderbilt University, Nashville, TN Introduction: Pancreas organogenesis begins in formation of dorsal and ventral pancreatic buds at the specific sites in the primitive Pdx1-expressing endoderm at Embryonic day 9.5 (e9.5) where Pdx1 and Ptf1a, the master genes in pancreatogenesis, are co-expressed. Previously reported, Ptf1a functions as a transcriptional switch between pancreatic and duodenal cell fates, thus Ptf1a expression indicates specification of pancreatic lineage. On the other hand, global pdx1 inactivation resulted in pancreatic agenesis, and cell type-specific- (e.g. beta-cells, acinar cells) or time-specific- (e.g. using Tet-on/off system) pdx1 inactivation revealed the various requirement of Pdx1 at each developmental stages and in each cell types. Aim and Methods: To address the role of Pdx1 in pancreatic progenitors, we performed Ptf1a-cre-mediated pdx1 inactivation. At the same time, genetic lineage tracing using ROSA26 reporter allele enabled us to trace the cell fate after pdx1 inactivation. Results: Developmentaly, there is no phenotypic difference at e11.5. Around e14.5, ductal brunching and primitive acinar tissue begin to be formed in control mice but no brunching morphogenesis or no acinar tissue formation were observed in Ptf1a-cre-mediated pdx1inactivated mice. At birth, Ptf1a-cre-mediated pdx1 inactivation resulted in ballooned duct phenotype which has a remarkably dilated duct-like structure in dorsal pancreatic region with complete lack of acinar tissue. Histologically, lineage-labeled cells form singlelayered epithelial duct-like structure with no brunching. In this epithelium and nearby, glucagon-producing cells and rare insulinproducing cell are detected but they do not form normal islet structure Surprisingly, small subsets of pdx1-inactivated cells are found in the duodenum (successive to the pancreatic duct) and bile duct, indicating that pdx1 inactivation at the very early stages of pancreatogenesis switches the character of pancreatic progenitors such that their progeny proliferate in and adopt the normal fates of duodenal and biliary epithelia. Conclusion: Unlike global Pdx1 KO phenotype, Ptf1a-cre-mediated pdx1 mutant revealed a dilated duct-like structure. Moreover, a subset of Ptf1a-cre-mediated pdx1 inactivated cells converts their fate from pancreas to duodenum and biliary system. Although Ptf1a defines pancreatic progenitor status, progenitor cells could not initiate subsequent pancreatic differentiation

program without Pdx1 function. This finding supports the possible interconversion / plasticity between the progenitors of different organs in the endodermal tissue.

52. SYSTEMIC HYPOXIA INCREASES PHAGOCYTOSIS BY PERITONEAL MACROPHAGES IN A P38-DEPENDENT MANNER IN THE PATHOGENESIS OF NECROTIZING ENTEROCOLITIS. R. J. Anand, S. C. Gribar, J. W. Kohler, T. D. Dubowski, J. Li, D. J. Hackam; Children’s Hospital of Pittsburgh, Pittsburgh, PA Introduction: Necrotizing enterocolitis (NEC) - the leading cause of gastrointestinal death in newborns - develops after a hypoxic insult to a pre-term infant, although the precise mechanisms by which hypoxia leads to the development of NEC remain incompletely known. NEC is characterized by enhanced pro-inflammatory activity of immune cells including macrophages within the peritoneal cavity. Macrophages contribute to both bacterial clearance and tissue injury by phagocytosis, which requires RhoA-GTPases and p38MAPKinase signaling. We now hypothesize that hypoxia can enhance phagocytosis by macrophages in the pathogenesis of NEC, and sought to determine the molecular mechanisms involved. Methods: Peritoneal macrophages were obtained from newborn C3H/HeOUJ mice by lavage. The extent of phagocytosis was quantified by confocal microscopy after feeding peritoneal macrophages or murine macrophage RAW 264.7 cells a meal of IgG-opsonized sheep erythrocytes (20 minutes, 37°C) and removing unbound particles by hypotonic lysis. Hypoxia was induced in C3H/HeOUJ mice or isolated macrophages using a modular hypoxic chamber. Signaling by RhoA-GTPase was assessed by pull-down assay, and by p38MAPK by SDS-PAGE with phospho-specific antibodies in the presence or absence of the inhibitor SB202190, (10␮M, 1h). Experimental NEC was induced using a combination of enteric formula and hypoxia treatment to newborn C3H/HeOUJ mice. Data are mean⫾SEM, significance for p⬍0.05. Results: Phagocytosis by peritoneal macrophages was significantly increased in mice that were subjected to hypoxia as compared with untreated animals (29⫾8% increase over normoxic control, p⬍0.05). This effect was also observed in vitro, as RAW 264.7 murine macrophages demonstrated an increase in phagocytosis after hypoxic treatment as compared with normoxic cells (see Figure A), while reoxygenation restored the rates of phagocytosis to control levels. In understanding the mechanisms involved, hypoxia caused no changes in RhoA-GTPase signaling within RAW 264.7 macrophages, but caused a significant increase in the phosphorylation of p38MAPK, suggesting a role for p38 in mediating the effect of hypoxia (see figure B). Inhibition of p38MAPK signaling led to a significant reversal of the effects of hypoxia on phagocytosis by RAW 264.7 cells, suggesting that p38MAPK activation is required to induce this effect (see Figure A). Strikingly, the