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Peroxynitrite induces enterocyte p38 map kinase regulation of GADD45 expression
ASSOCIATION FOR ACADEMIC SURGERY AND SOCIETY OF UNIVERSITY SURGEONS—ABSTRACTS Table 1. Lung morphometric data. Control 3.79 ⫾ 0.35 LW/BW (%) n⫽17 Branch Iterations 11.5 ⫾ 1.4 (#) n⫽8
TO
CDH
CDH & TO
5.44 ⫾ 0.82* 3.07 ⫾ 0.52* 3.61 ⫾ 0.21 n⫽8 n⫽15 n⫽3 11.2 ⫾ 1.3 n⫽4
9.3 ⫾ 1.3* n⫽5
9.4 ⫾ 0.8* n⫽2
All data presented as mean ⫾ SD *Denotes p⬍0.001 as compared to controls 340. THE ROLE OF MESENCHYMAL REGULATORY GENES IN THE PATHOGENESIS OF CCAMS REQUIRING IN UTERO RESECTION. Peranteau WH, Badillo AT, Hedrick HL, Flake AW, Adzick NS, Liechty KW; The Children’s Hospital of Philadelphia Introduction: Congenital cystic adenomatoid malformations (CCAM) are characterized by abnormal airway branching morphogenesis and increased mesenchymal proliferation. In utero, lesions can rapidly expand resulting in fetal hydrops. FGF-10 and Hoxb-5 are mesenchymally expressed genes which regulate branching morphogenesis. We hypothesized that the rapid expansion of mesenchyme in CCAMs that result in fetal hydrops requiring in utero resection may be due to increased expression of mesenchymal regulatory genes compared to CCAMs that are resected after birth. Methods: Tissue was obtained from two CCAMs requiring fetal resection (21 weeks), three term CCAMs and four normal fetal lungs (16-22 weeks). Hoxb-5 and FGF-10 mRNA production was compared after standardization to beta-actin using RT-PCR and densitometric analysis. Results: Expression of Hoxb-5 by fetal CCAMs was the same as normal fetal lung (79.57⫾2.75% vs. 77.27⫾6.80%; p⫽0.29). Postnatal CCAMs demonstrated persistent Hoxb-5 expression but at lower levels than fetal CCAMs (55.59⫾3.7% vs. 79.57⫾2.75%; p⫽0.002) and normal fetal lung (55.59⫾3.7% vs. 77.28⫾6.8%; p⫽0.002). FGF-10 production, like Hoxb-5, was not significantly different in fetal CCAMs and normal fetal lung (58.60⫾9.6% vs. 66.94⫾2.66%; p⫽0.22). However, FGF-10 production by postnatal CCAMs remained elevated and similar to levels produced by fetal CCAMs (58.10⫾1.4% vs. 58.60⫾9.6%; p⫽0.48). Conclusion: Similar expression of Hoxb-5 and FGF-10 by fetal CCAMs and normal fetal lung argue against a pivotal role in the development and expansion of CCAMs requiring fetal resection. However, persistent expression of FGF-10 and Hoxb-5 in postnatal CCAMs highlights their potential role in the pathogenesis of CCAMs. 341. ADULT PANCREATIC EXOCRINE TISSUE IS MAINTAINED BY SELF-DUPLICATION OF THE EXISTING CELLS. M. Horiguchi 1, Y. Kawaguchi 1, S. Kodama 1, K. Furuyama 1, A. Fukuda 1, T. Kuhara 1, K. Fujimoto 1, Y. Dor 2, C. V. Wright 3, R. Doi 1; 1Kyoto University Hospital, Kyoto, JAPAN, 2The Hebrew University-Hadassah Medical School, Jerusalem, ISRAEL, 3Vanderbilt University, Nashville, TN. Introduction: Most of adult tissues are maintained by stem cell differentiation. Recently, Dor et al. reported that adult pancreatic beta-cells are formed by self-duplication rather than stem cell differentiation. However, it is not elucidated how adult pancreatic exocrine tissue is maintained. Using cre-mediated inducible gene inactivation combined with cell tracking, we investigated whether exocrine cells self duplicate or not. In addition, we analyzed if pdx1, a pivotal gene in pancreas organogenesis, is necessary for the maintenance of adult exocrine pancreas. Furthermore, the exocrine cell replacement in the recovery process after acute pancreatitis was analyzed to check if self-duplication of the jeopardized cell or transdifferentiation from other cell type has occurred in this process.
285
Materials and Methods: Elastase-CreERTM; ROSA26 mice were injected with tamoxifen. Mice just after injection (pulse group) and 2 ,4, 6 month later(chase group) were analyzed. Elastase-CreERTM; ROSA26;PDX-1loxp/- mice, injected as above and analyzed. After the tamoxifen pulse, acute severe pancreatitis is made by cerulein injection. Analysis was performed at 1, 3, 7, 14 days after last injection. Results: Proportion of labeled cells is not different between pulse group and chase group, indicating that adult pancreatic exocrine cells are formed by self-duplication. Conclusion: Preliminary results indicate that pdx1 plays some role in this self-duplication. 342. PEROXYNITRITE INDUCES ENTEROCYTE P38 MAP KINASE REGULATION OF GADD45 EXPRESSION. A. Young 1, A. Grishin 2, C. Leaphart 1, C. Wong 1, X. Zhang 2, H. R. Ford 2, J. S. Upperman 1; 1University of Pittsburgh, Pittsburgh, PA, 2University of Southern California, Los Angeles, CA. Introduction: Necrotizing enterocolitis (NEC) is characterized by intestinal inflammation and barrier disruption. We have previously shown that nitric oxide and toxic nitrogen intermediates such as peroxynitrite (PN) are expressed in NEC. Furthermore, PN induces P38 map kinases and leads to apoptosis in enterocytes. We therefore hypothesized that PN activation in enterocytes impairs growth arrest and DNA damage-inducible (GADD) 45 gene and explored the relative contributions of known mitogen-activated protein kinase (MAPK) pathways. Methods: IEC-6 enterocytes were treated with PN (50uM) or decomposed control(DPN) and GADD45 RNA levels were assayed with rtPCR and protein levels were assayed by immunoblots. Activation of MAPK and MAPK phosphatase-1 (MKP1) were examined by immunoblots with phospho-MAPK antibodies. MAPK activity was blocked by treatment with pharmacological inhibitors or transfection with dominant-negative MAPK constructs. Results: PN treatment caused a transient time-dependent increase in GADD45 mRNA followed by a subsequent decrease in GADD45 protein expression. This was preceded by rapid and sustained activation of p38. SB203580, a specific inhibitor of p38, but not SP600125 (JNK inhibitor) blocked PN-induced apoptosis; dominant-negative p38 MAPK constructs also inhibited apoptosis. GADD45 expression was not inhibited by DPN. Surprisingly, MKP-1 was induced by 2h in PN group but not in DPN. Conclusions: These data support the hypothesis that p38 is a key mediator in PN-induced enterocyte apoptosis by impairing the expression of an important DNA repair protein. P38 may be an important therapeutic target in treating NEC.
EMERGENT TECHNOLOGIES I 343. PERFORMANCE OF “SUPERCRYOPRECIPITATE” AND OTHER HEMOSTATIC AGENTS IN A RAT LIVER TRAUMA MODEL. Hayashi MS, Ji P, Owens WJ, Edward S, Steward E, Imagawa DK; University of California at Irvine The purpose of this study was to compare “Supercryoprecipitate,” a novel agent developed at our institution, against other commercial products to obtain hemostasis in a rat liver trauma model. Ordinarily, cryoprecipitate contains ⬍40% of the plasma proteins from a unit of blood as determined by protein electrophoresis. By adding a concentrated citrate solution to the standard method of obtaining cryoprecipitate, we developed “Supercryoprecipitate,” a novel agent with retrieval of nearly 100% of the plasma proteins including factor VIII, fibrinogen, and von Willebrand factor. 52 Sprague-Dawley rats
TO
CDH
CDH & TO
5.44 ⫾ 0.82* 3.07 ⫾ 0.52* 3.61 ⫾ 0.21 n⫽8 n⫽15 n⫽3 11.2 ⫾ 1.3 n⫽4
9.3 ⫾ 1.3* n⫽5
9.4 ⫾ 0.8* n⫽2
All data presented as mean ⫾ SD *Denotes p⬍0.001 as compared to controls 340. THE ROLE OF MESENCHYMAL REGULATORY GENES IN THE PATHOGENESIS OF CCAMS REQUIRING IN UTERO RESECTION. Peranteau WH, Badillo AT, Hedrick HL, Flake AW, Adzick NS, Liechty KW; The Children’s Hospital of Philadelphia Introduction: Congenital cystic adenomatoid malformations (CCAM) are characterized by abnormal airway branching morphogenesis and increased mesenchymal proliferation. In utero, lesions can rapidly expand resulting in fetal hydrops. FGF-10 and Hoxb-5 are mesenchymally expressed genes which regulate branching morphogenesis. We hypothesized that the rapid expansion of mesenchyme in CCAMs that result in fetal hydrops requiring in utero resection may be due to increased expression of mesenchymal regulatory genes compared to CCAMs that are resected after birth. Methods: Tissue was obtained from two CCAMs requiring fetal resection (21 weeks), three term CCAMs and four normal fetal lungs (16-22 weeks). Hoxb-5 and FGF-10 mRNA production was compared after standardization to beta-actin using RT-PCR and densitometric analysis. Results: Expression of Hoxb-5 by fetal CCAMs was the same as normal fetal lung (79.57⫾2.75% vs. 77.27⫾6.80%; p⫽0.29). Postnatal CCAMs demonstrated persistent Hoxb-5 expression but at lower levels than fetal CCAMs (55.59⫾3.7% vs. 79.57⫾2.75%; p⫽0.002) and normal fetal lung (55.59⫾3.7% vs. 77.28⫾6.8%; p⫽0.002). FGF-10 production, like Hoxb-5, was not significantly different in fetal CCAMs and normal fetal lung (58.60⫾9.6% vs. 66.94⫾2.66%; p⫽0.22). However, FGF-10 production by postnatal CCAMs remained elevated and similar to levels produced by fetal CCAMs (58.10⫾1.4% vs. 58.60⫾9.6%; p⫽0.48). Conclusion: Similar expression of Hoxb-5 and FGF-10 by fetal CCAMs and normal fetal lung argue against a pivotal role in the development and expansion of CCAMs requiring fetal resection. However, persistent expression of FGF-10 and Hoxb-5 in postnatal CCAMs highlights their potential role in the pathogenesis of CCAMs. 341. ADULT PANCREATIC EXOCRINE TISSUE IS MAINTAINED BY SELF-DUPLICATION OF THE EXISTING CELLS. M. Horiguchi 1, Y. Kawaguchi 1, S. Kodama 1, K. Furuyama 1, A. Fukuda 1, T. Kuhara 1, K. Fujimoto 1, Y. Dor 2, C. V. Wright 3, R. Doi 1; 1Kyoto University Hospital, Kyoto, JAPAN, 2The Hebrew University-Hadassah Medical School, Jerusalem, ISRAEL, 3Vanderbilt University, Nashville, TN. Introduction: Most of adult tissues are maintained by stem cell differentiation. Recently, Dor et al. reported that adult pancreatic beta-cells are formed by self-duplication rather than stem cell differentiation. However, it is not elucidated how adult pancreatic exocrine tissue is maintained. Using cre-mediated inducible gene inactivation combined with cell tracking, we investigated whether exocrine cells self duplicate or not. In addition, we analyzed if pdx1, a pivotal gene in pancreas organogenesis, is necessary for the maintenance of adult exocrine pancreas. Furthermore, the exocrine cell replacement in the recovery process after acute pancreatitis was analyzed to check if self-duplication of the jeopardized cell or transdifferentiation from other cell type has occurred in this process.
285
Materials and Methods: Elastase-CreERTM; ROSA26 mice were injected with tamoxifen. Mice just after injection (pulse group) and 2 ,4, 6 month later(chase group) were analyzed. Elastase-CreERTM; ROSA26;PDX-1loxp/- mice, injected as above and analyzed. After the tamoxifen pulse, acute severe pancreatitis is made by cerulein injection. Analysis was performed at 1, 3, 7, 14 days after last injection. Results: Proportion of labeled cells is not different between pulse group and chase group, indicating that adult pancreatic exocrine cells are formed by self-duplication. Conclusion: Preliminary results indicate that pdx1 plays some role in this self-duplication. 342. PEROXYNITRITE INDUCES ENTEROCYTE P38 MAP KINASE REGULATION OF GADD45 EXPRESSION. A. Young 1, A. Grishin 2, C. Leaphart 1, C. Wong 1, X. Zhang 2, H. R. Ford 2, J. S. Upperman 1; 1University of Pittsburgh, Pittsburgh, PA, 2University of Southern California, Los Angeles, CA. Introduction: Necrotizing enterocolitis (NEC) is characterized by intestinal inflammation and barrier disruption. We have previously shown that nitric oxide and toxic nitrogen intermediates such as peroxynitrite (PN) are expressed in NEC. Furthermore, PN induces P38 map kinases and leads to apoptosis in enterocytes. We therefore hypothesized that PN activation in enterocytes impairs growth arrest and DNA damage-inducible (GADD) 45 gene and explored the relative contributions of known mitogen-activated protein kinase (MAPK) pathways. Methods: IEC-6 enterocytes were treated with PN (50uM) or decomposed control(DPN) and GADD45 RNA levels were assayed with rtPCR and protein levels were assayed by immunoblots. Activation of MAPK and MAPK phosphatase-1 (MKP1) were examined by immunoblots with phospho-MAPK antibodies. MAPK activity was blocked by treatment with pharmacological inhibitors or transfection with dominant-negative MAPK constructs. Results: PN treatment caused a transient time-dependent increase in GADD45 mRNA followed by a subsequent decrease in GADD45 protein expression. This was preceded by rapid and sustained activation of p38. SB203580, a specific inhibitor of p38, but not SP600125 (JNK inhibitor) blocked PN-induced apoptosis; dominant-negative p38 MAPK constructs also inhibited apoptosis. GADD45 expression was not inhibited by DPN. Surprisingly, MKP-1 was induced by 2h in PN group but not in DPN. Conclusions: These data support the hypothesis that p38 is a key mediator in PN-induced enterocyte apoptosis by impairing the expression of an important DNA repair protein. P38 may be an important therapeutic target in treating NEC.
EMERGENT TECHNOLOGIES I 343. PERFORMANCE OF “SUPERCRYOPRECIPITATE” AND OTHER HEMOSTATIC AGENTS IN A RAT LIVER TRAUMA MODEL. Hayashi MS, Ji P, Owens WJ, Edward S, Steward E, Imagawa DK; University of California at Irvine The purpose of this study was to compare “Supercryoprecipitate,” a novel agent developed at our institution, against other commercial products to obtain hemostasis in a rat liver trauma model. Ordinarily, cryoprecipitate contains ⬍40% of the plasma proteins from a unit of blood as determined by protein electrophoresis. By adding a concentrated citrate solution to the standard method of obtaining cryoprecipitate, we developed “Supercryoprecipitate,” a novel agent with retrieval of nearly 100% of the plasma proteins including factor VIII, fibrinogen, and von Willebrand factor. 52 Sprague-Dawley rats