Fetal hepatic artery ligation suppresses liver hematopoiesis in utero

ASSOCIATION FOR ACADEMIC SURGERY AND SOCIETY OF UNIVERSITY SURGEONS—ABSTRACTS the purpose of this study was to determine whether treatment with IGF-1 protects intestinal epithelial cells from oxidative stressinduced apoptosis. Methods: Rat intestinal epithelial (RIE-1) cells were plated and pretreated with varying dosages of IGF-1 (25 to 100 nM) for 30 minutes. Oxidative stress was induced using hydrogen peroxide (H 2O 2; 500 ␮M) for 3 h. Total protein was extracted for Western blot analysis to determine phosphorylation of Akt (p-Akt), a downstream effector for PI3-K pathway. Intestinal cell apoptosis was determined by ELISA assay. Results: Consistent with previous findings, H 2O 2 treatment resulted in increased intestinal epithelial cell apoptosis when compared to cells treated with vehicle alone (control). This increase in H 2O 2-mediated apoptosis was significantly attenuated by nearly 40% when cells were pretreated with IGF-1 (50 nM). Similar decreases in H 2O 2-mediated apoptosis were noted with all dosages of IGF-1. IGF-1 treatment alone did not affect intestinal cell apoptosis. The increased expression of p-Akt was noted with IGF-1 treatment, confirming activation of the PI3-K pathway. H 2O 2 treatment also increased intestinal epithelial cell p-Akt protein levels. Conclusions: The PI3-K, a key cell survival pathway, is activated during oxidative stress-induced intestinal epithelial cell injury. IGF-1, as an inducer of the PI3-K pathway, demonstrates an important protective effect in intestinal epithelial cells against oxidative stress-induced apoptosis. A better understanding of PI3-K as a key anti-apoptotic pathway during oxidative stress-induced gut injury will facilitate the development of novel therapy against NEC. 191. FETAL HEPATIC ARTERY LIGATION SUPPRESSES LIVER HEMATOPOIESIS IN UTERO. S. Kunisaki, H. Azpurua, J. Fuchs, S. Graves, D. Zurakowski, D. Fauza; Children’s Hospital Boston, Boston, MA. Introduction: Fetal hematopoiesis involves a successive interchange of progenitor cell activity among different anatomical sites, with the liver playing a pivotal role in the sequence. The mechanisms that regulate this unique process may hold the clues to the treatment and possibly cure of numerous hematological and non-hematological diseases, both before and after birth. We hypothesized that fetal hepatic hematopoiesis is controlled by arterial blood flow to the liver. This study was aimed at determining the effects of changes in hepatic artery flow upon liver hematopoiesis in utero. Methods: The experimental design was validated by previous data demonstrating (1) the negligible role of arterial blood flow on fetal hepatic oxygenation and (2) an increase in hepatic arterial flow as a consequence of biliary obstruction. Fetal lambs (n⫽15) were divided into 3 groups at 106-113 days gestation (term⫽145 days). Group I (n⫽5) underwent ligation and division of the right and left hepatic arteries; group II (n⫽4) underwent ligation and division of the common bile duct; and group III (n⫽6) were sham-operated controls. Euthanasia was performed at comparable gestational ages near term, 23.7⫹/-6.4 days postoperatively. Animals were blindly assessed for hepatic hematopoietic cell/island density by both standard histology and transferrin receptor (CD71) immunohistochemistry. Additional analyses included hepatic angiography, peripheral blood hemoglobin (Hb) content, and colony-forming unit (CFU) assays of hematopoietic progenitors. Statistical comparisons were by non-parametric and mixed model regression analyses, as appropriate (P⬍0.05). Results: Overall fetal survival was 93%. In group I, post-mortem arteriograms confirmed complete occlusion of the hepatic arteries, albeit with variable local collateralization. There was a significant reduction in the density of hematopoietic progenitors in group I, when compared to groups II and III (Figure). This was further confirmed by a significant reduction of CFU in group I. Group II had a significantly higher density of hematopoietic progenitors than the other two groups. Fetal Hb levels were lower in group I when compared to groups II and III, but this did not reach statistical significance in this series. Conclusions: Fetal hepatic hematopoeisis is dependent on arterial blood flow to the liver. These data demonstrate a unique and previously unrecognized role of the hepatic artery in fetal development.

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Fetal hepatic artery ligation is a novel surgical model for the study of fetal hematopoiesis.

192. HYPOXIA DISRUPTS INTESTINAL GAP JUNCTIONS THROUGH ACTIVATION OF A PP1-DEPENDENT PHOSPHATASE AND THE DE-PHOSPHORYLATION OF CONNEXIN43 IN THE PATHOGENESIS OF NECROTIZING ENTEROCOLITIS. Leaphart CL, Qureshi FG, Levy RM, Mollen KP, Prince JM, Hackam DJ; Children’s Hospital of Pittsburgh Introduction: Necrotizing enterocolitis (NEC) develops after a period of global hypoxia, and is characterized by a disruption in the intestinal barrier. The mechanisms by which hypoxia leads to barrier dysfunction remain largely unknown. We have shown that intestinal barrier function requires gap junctions comprised of connexin43 (Cx43) that must be phosphorylated to function. We now hypothesize that hypoxia leads to an impairment in intestinal gap junction function in vitro and in vivo, and sought to determine the mechanisms involved. Methods: IEC-6 cells were subjected to hypoxia in a flow regulated, vacuum sealed hypoxia chamber (1% O 2, 37 oC) for 1-12h. The expression and phosphorylation of Cx43 were assessed by SDS-PAGE and confocal immunofluorescence. To induce NEC, newborn rats were subjected to global hypoxia (10mins 5% O 2) and formula gavage, and mucosal scrapings were obtained from the terminal ileum and subjected to SDS-PAGE. To restore Cx43 phosphorylation, IEC-6 cells were pre-treated with a variety of tyrosine phosphatase inhibitors, including pervanadate(10uM,1h) and calyculin A(2nM,30nM). Results: Hypoxic treatment caused a timedependent decrease in the phosphorylation of Cx43, which correlated with a decrease in gap junction communication between adjacent enterocytes. There were no effects of hypoxia on the expression of total Cx43, or on cell viability. To verify the in vivo significance of these findings, phosphorylation of Cx43 was also significantly decreased in ileal mucosal scrapings from newborn rats subjected to global hypoxia and formula gavage as compared to control, breast fed animals. Strikingly, pre-treatment of IEC-6 cells with the protein phosphatase 1 (PP1) dependent phosphatase inhibitor calyculin A led to the restoration of phosphorylation of Cx43 and enhanced gap junction connectivity, indicating that a hypoxia-induced phosphatase mediated the effect. Conclusion: Hypoxia inhibits gap junction function in enterocytes through the activation of a protein phosphatase 1 dependent phosphatase resulting in the de-phosphorylation of Cx43. These findings suggest that further targeting of this hypoxiainduced phosphatase may provide a novel therapeutic strategy in the management of diseases of intestinal inflammation like NEC. 193. EPIDERMAL GROWTH FACTOR ENHANCES INTESTINAL GAP JUNCTION FUNCTION THROUGH POSTTRANSLATIONAL MODIFICATION OF CONNEXIN 43 IN VITRO AND IN VIVO. C. L. Leaphart, J. A. Cavallo, J. Li, C. Wong, D. J. Hackam; Children’s Hospital of Pittsburgh, Pittsburgh, PA. Introduction: Necrotizing enterocolitis is characterized by impaired mucosal barrier function, in part through alterations in gap junctions comprised of Connexin43 (Cx43) which must be phosphorylated in order to function. Cx43 half-life is tightly regulated by ubiquitination, leading to its degradation over time. Epidermal