the purpose of this study was to determine whether treatment withIGF-1 protects intestinal epithelial cells from oxidative stress-induced apoptosis. Methods: Rat intestinal epithelial (RIE-1) cellswere plated and pretreated with varying dosages of IGF-1 (25 to 100nM) for 30 minutes. Oxidative stress was induced using hydrogenperoxide (H2O2; 500 �M) for 3 h. Total protein was extracted forWestern blot analysis to determine phosphorylation of Akt (p-Akt), adownstream effector for PI3-K pathway. Intestinal cell apoptosis wasdetermined by ELISA assay. Results: Consistent with previous find-ings, H2O2 treatment resulted in increased intestinal epithelial cellapoptosis when compared to cells treated with vehicle alone (con-trol). This increase in H2O2-mediated apoptosis was significantlyattenuated by nearly 40% when cells were pretreated with IGF-1 (50nM). Similar decreases in H2O2-mediated apoptosis were noted withall dosages of IGF-1. IGF-1 treatment alone did not affect intestinalcell apoptosis. The increased expression of p-Akt was noted withIGF-1 treatment, confirming activation of the PI3-K pathway. H2O2

treatment also increased intestinal epithelial cell p-Akt protein lev-els. Conclusions: The PI3-K, a key cell survival pathway, is acti-vated during oxidative stress-induced intestinal epithelial cell in-jury. IGF-1, as an inducer of the PI3-K pathway, demonstrates animportant protective effect in intestinal epithelial cells against oxi-dative stress-induced apoptosis. A better understanding of PI3-K asa key anti-apoptotic pathway during oxidative stress-induced gutinjury will facilitate the development of novel therapy against NEC.

191. FETAL HEPATIC ARTERY LIGATION SUPPRESSESLIVER 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 inter-change of progenitor cell activity among different anatomical sites,with the liver playing a pivotal role in the sequence. The mechanismsthat regulate this unique process may hold the clues to the treatmentand possibly cure of numerous hematological and non-hematologicaldiseases, both before and after birth. We hypothesized that fetalhepatic hematopoiesis is controlled by arterial blood flow to the liver.This study was aimed at determining the effects of changes in he-patic artery flow upon liver hematopoiesis in utero. Methods: Theexperimental design was validated by previous data demonstrating(1) the negligible role of arterial blood flow on fetal hepatic oxygen-ation and (2) an increase in hepatic arterial flow as a consequence ofbiliary obstruction. Fetal lambs (n�15) were divided into 3 groups at106-113 days gestation (term�145 days). Group I (n�5) underwentligation and division of the right and left hepatic arteries; group II(n�4) underwent ligation and division of the common bile duct; andgroup III (n�6) were sham-operated controls. Euthanasia was per-formed at comparable gestational ages near term, 23.7�/-6.4 dayspostoperatively. Animals were blindly assessed for hepatic hemato-poietic cell/island density by both standard histology and transferrinreceptor (CD71) immunohistochemistry. Additional analyses in-cluded hepatic angiography, peripheral blood hemoglobin (Hb) con-tent, and colony-forming unit (CFU) assays of hematopoietic progen-itors. Statistical comparisons were by non-parametric and mixedmodel regression analyses, as appropriate (P�0.05). Results: Over-all fetal survival was 93%. In group I, post-mortem arteriogramsconfirmed complete occlusion of the hepatic arteries, albeit withvariable local collateralization. There was a significant reduction inthe density of hematopoietic progenitors in group I, when comparedto groups II and III (Figure). This was further confirmed by a signif-icant reduction of CFU in group I. Group II had a significantly higherdensity of hematopoietic progenitors than the other two groups.Fetal Hb levels were lower in group I when compared to groups IIand III, but this did not reach statistical significance in this series.Conclusions: Fetal hepatic hematopoeisis is dependent on arterialblood flow to the liver. These data demonstrate a unique and previ-ously unrecognized role of the hepatic artery in fetal development.

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

192. HYPOXIA DISRUPTS INTESTINAL GAP JUNCTIONSTHROUGH ACTIVATION OF A PP1-DEPENDENTPHOSPHATASE AND THE DE-PHOSPHORYLATION OFCONNEXIN43 IN THE PATHOGENESIS OF NECROTIZ-ING ENTEROCOLITIS. Leaphart CL, Qureshi FG, Levy RM,Mollen KP, Prince JM, Hackam DJ; Children’s Hospital ofPittsburgh

Introduction: Necrotizing enterocolitis (NEC) develops after aperiod of global hypoxia, and is characterized by a disruption in theintestinal barrier. The mechanisms by which hypoxia leads to bar-rier dysfunction remain largely unknown. We have shown that in-testinal barrier function requires gap junctions comprised of con-nexin43 (Cx43) that must be phosphorylated to function. We nowhypothesize that hypoxia leads to an impairment in intestinal gapjunction function in vitro and in vivo, and sought to determine themechanisms involved. Methods: IEC-6 cells were subjected to hyp-oxia in a flow regulated, vacuum sealed hypoxia chamber (1% O2,37oC) for 1-12h. The expression and phosphorylation of Cx43 wereassessed by SDS-PAGE and confocal immunofluorescence. To induceNEC, newborn rats were subjected to global hypoxia (10mins 5% O2)and formula gavage, and mucosal scrapings were obtained from theterminal ileum and subjected to SDS-PAGE. To restore Cx43 phos-phorylation, IEC-6 cells were pre-treated with a variety of tyrosinephosphatase inhibitors, including pervanadate(10uM,1h) and caly-culin A(2nM,30nM). Results: Hypoxic treatment caused a time-dependent decrease in the phosphorylation of Cx43, which correlatedwith a decrease in gap junction communication between adjacententerocytes. There were no effects of hypoxia on the expression oftotal Cx43, or on cell viability. To verify the in vivo significance ofthese findings, phosphorylation of Cx43 was also significantly de-creased in ileal mucosal scrapings from newborn rats subjected toglobal hypoxia and formula gavage as compared to control, breast fedanimals. Strikingly, pre-treatment of IEC-6 cells with the proteinphosphatase 1 (PP1) dependent phosphatase inhibitor calyculin Aled to the restoration of phosphorylation of Cx43 and enhanced gapjunction connectivity, indicating that a hypoxia-induced phospha-tase mediated the effect. Conclusion: Hypoxia inhibits gap junctionfunction in enterocytes through the activation of a protein phospha-tase 1 dependent phosphatase resulting in the de-phosphorylation ofCx43. These findings suggest that further targeting of this hypoxia-induced phosphatase may provide a novel therapeutic strategy in themanagement of diseases of intestinal inflammation like NEC.

193. EPIDERMAL GROWTH FACTOR ENHANCES INTESTI-NAL GAP JUNCTION FUNCTION THROUGH POST-TRANSLATIONAL MODIFICATION OF CONNEXIN 43IN 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 im-paired mucosal barrier function, in part through alterations in gapjunctions comprised of Connexin43 (Cx43) which must be phosphor-ylated in order to function. Cx43 half-life is tightly regulated byubiquitination, leading to its degradation over time. Epidermal

233ASSOCIATION FOR ACADEMIC SURGERY AND SOCIETY OF UNIVERSITY SURGEONS—ABSTRACTS