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Expression of E-selectin and its transcripts during intestinal ischemia-reperfusion injury in pigs
Expression of E-Selectin and Its Transcripts During Intestinal Ischemia-Reperfusion Injury in Pigs F. Braun, M. Hosseini, E. Wieland, B. Sattler, S. Laabs, T. Lorf, A.R. Mu¨ller, F. Fa¨ndrich, B. Kremer, and B. Ringe ABSTRACT Background. Ischemia-reperfusion injury (IRI) can result in severe organ dys- or nonfunction. Interaction of leukocytes and endothelial cells mediated by E-selectin appears to be a key step for disturbed microcirculation. Therefore we studied gene and protein expression as well as localization of E-selectin during intestinal IRI. Methods. Intestinal tissue samples were obtained from extracorporeal perfused intestines (cold ischemia time [CIT] 2 or 20 hours, each n ⫽ 5) and additionally in intestinal transplanted pigs (CIT 2 or 20 hours, each n ⫽ 1). Mucosal damage was graded according to the Chiu classification. E-selectin mRNA was determined by PCR and quantitative RT-PCR. Localization of E-selectin mRNA was performed by in situ hybridization and of the protein by immunohistochemistry. Results. Histologically, mucosal damage occurred during reperfusion and was earlier and more severe after 20 hours of CIT. E-selectin mRNA expression was detected by PCR already after laparotomy and was elevated after reperfusion. Interestingly, mRNA expression was already increased after 20 hours of CIT. E-selectin mRNA was localized to the luminal surface of muscular, submucosal, and mucosal endothelial cells and the protein was detected on submucosal arterial endothelium as early as 2 hours after reperfusion. Conclusion. Prolongation of CIT results in more severe mucosal damage during reperfusion, which is associated with protein expression of E-selection that might be used as a marker for activated endothelial cells. Increased E-selectin mRNA at end of 20 hours of CIT might indicate a preactivated state of endothelial cells potentially triggered by bacterial translocation or products.
S
EVERE ISCHEMIA-REPERFUSION injury (IRI) can result in organ dys- or nonfunction. The morphological characteristics of severe reperfusion injury are disturbed microcirculation and cellular damage. The pathophysiology of IRI is complex and the activation of endothelial cells is implicated as a key step.1 E-selectin supports initial rolling of leukocytes on activated endothelium and mediates stable arrest of leukocytes. E-selectin is not detectable on quiescent endothelial cells and can be used as a marker of activated endothelial cells.2 Little is known about the kinetics and localization of E-selectin during intestinal IRI. Therefore, we studied expression and localization of E-selectin during intestinal IRI. METHODS Intestinal tissue samples were obtained from extracorporeal perfused pig intestines after a cold ischemia time (CIT) of 2 or 20 © 2004 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710 Transplantation Proceedings, 36, 265–266 (2004)
hours (each n ⫽ 5) and additionally in intestinal transplanted pigs after a CIT of 2 or 20 hours (each n ⫽ 1). The extracorporeal perfusion system was described previously.3 Transplantation was performed orthotopically. Histidine-tryptophane-ketoglutarate From the Klinik fu¨r Allgemeine Chirurgie und Thoraxchirurgie (F.B., A.R.M., F.F., B.K.), Universita¨t Schleswig-Holstein, Campus Kiel, Kiel, Germany; Zentrum Innere Medizin (M.H., E.W.), Pathologie (B.S.), and Chirurgie (S.L., T.L.), Universita¨t Go¨ttingen, Go¨ttingen, Germany; and Center for Liver, Pancreas and Biliary Diseases (B.R.), MCP Hahnemann University, Philadelphia, Pa, USA. This study was supported by grant SFB 402 D1 and G.I.F. 2013-1079.2/2000. Address reprint requests to PD Dr Andrea Mueller, Klinik fu¨r Allgemeine Chirurgie und Thoraxchirurgie, Universita¨t SchleswigHolstein, Campus Kiel, Arnold-Heller-Strasse 7, 24105 Kiel, Germany. E-mail: [email protected] 0041-1345/04/$–see front matter doi:10.1016/j.transproceed.2004.01.081 265
266 (HTK) solution was used for preservation. Mucosal damage was graded according to the classification of Chiu et al.4 E-selectin mRNA was determined by PCR and quantitative RT-PCR. Localization of E-selectin mRNA was performed by in situ hybridization and E-selectin protein by immunohistochemistry.
RESULTS
Mean (⫾ SD) perfusion time was 546 ⫾ 55 after a CIT of 2 hours and 426 ⫾ 69 minutes after a CIT of 20 hours. Mucosal damage was minimal at the end of cold preservation irrespective of the length of CIT. At 1 hour of extracorporeal reperfusion, intestines exposed to 20 hours of CIT showed more severe damage of intestinal mucosa compared to intestines with CIT of 2 hours. At the end of reperfusion, severe mucosal damage was seen in all intestines. In intestinal tissue, E-selectin-mRNA expression was detected before and after perfusion with HTK solution, at the end of cold preservation, and after reperfusion. At the end of CIT, E-selectin-mRNA expression was higher after 20 hours than after 2 hours of CIT. Immediately after start of reperfusion, E-selectin mRNA expression increased in the intestinal tissue exposed to CIT for 2 hours, whereas E-selectin mRNA expression decreased in intestinal tissue exposed to CIT for 20 hours. Thereafter, E-selectin mRNA expression increased continuously during the reperfusion phase. In extracorporeal perfused intestinal specimens, E-selectin-mRNA was detected on the luminal surface of muscular, submucosal, and mucosal endothelial cells. Immunohistochemically, E-selectin was not detected prior to reperfusion. At 2 hours after reperfusion, endothelial cells of small submucosal arteries stained positive for E-selectin and intensive staining was detectable thereafter. Immunohistochemically, kinetics of semiquantitative staining intensity of E-selectin in extracorporeal perfused intestine was similar after 2 and 20 hours of cold preservation. DISCUSSION
Enhanced biosynthesis of adhesion molecules that mediate leukocyte-endothelial cell adhesion results in response to ischemia-reperfusion injury.1 In our model, E-selectinmRNA expression was detected already after laparotomy and increased after reperfusion. Endothelial cells stained positive for E-selection and E-selectin-mRNA in all intestinal layers. Immunohistochemically, arterial endothelium stained positive for E-selectin at 2 hours after start of reperfusion. Wyble et al reported expression of E-selectin
BRAUN, HOSSEINI, WIELAND ET AL
on human endothelial cells as early as 2 hours with maximum effects after 4 hours after addition of TNF-␣ and IL-1.5 In mice intestine exposed to 45 minutes of ischemia, increasing levels of E-selectin expression were found after 5 hours of reperfusion.6 Several mechanisms are suggested in the pathway of E-selectin expression including TNF-␣induced NF-B activation and exogenous lipopolysaccharide (LPS) application.7,8 The enhanced expression of E-selectin-mRNA during cold preservation in our model reflects transcription at the state of hypoxia. In vitro, E-selectin-mRNA is not expressed in response to hypoxia alone, but additional stimulation with TNF-␣ or LPS results in up-regulation of E-selectin-mRNA.9 In conclusion, prolonged cold preservation predisposes to enhanced transcription of E-selectin and is associated with earlier and more severe mucosal damage after reperfusion. Considering E-selectin as a marker of activated endothelial cells, they become activated earliest after reperfusion. Increased E-selectin mRNA at end of 20 hours of CIT might indicate a preactivated state of endothelial cells potentially triggered by bacterial translocation or products. Therefore, it appears beneficial to keep the cold ischemia time as short as possible in intestinal transplantation. REFERENCES 1. Carden DL, Granger DN: Pathophysiology of ischemia-reperfusion injury. J Pathol 190:255, 2000 2. Bevilacqua MP, Pober JS, Mendrick DL, et al: Identification of an inducible endotehlial-leukocyte adhesion molecule. Proc Natl Acad Sci 82:9238, 1987 3. Braun F, Schu ¨tz E, Laabs S, et al: Development of a porcine small bowel ex vivo perfusion model. Transplant Proc 30:2613, 1998 4. Chiu CJ, McArdle H, Brown R, et al: Intestinal mucosal lesion in low-flow states. Arch Surg 101:478, 1970 5. Wyble CW, Desai TR, Clark ET, et al: Physiologic concentrations of TNF-alpha and IL-1beta released from reperfused intestine upregulate E-selectin and ICAM-1. J Surg Res 63:333, 1996 6. Russell J, Epstein CJ, Grisham MB, et al: Regulation of E-selectin expression in postischemic intestinal microvasculature. Am J Physiol Gastrointest Liver Physiol 278:G878, 2000 7. Rahman A, Kefer J, Bando M, et al: E-selectin expression in human endothelial cells by TNF-a induced oxidant generation and NFkB activation. Am J Physiol 275:L533, 1998 8. Bauer P, Russell JM, Granger DN: Role of endotoxin in intestinal reperfusion induced expression of E-selectin. Am J Physiol 278:R1140, 2000 9. Zund G, Nelson DP, Neufeld EJ, et al: Hypoxia enhances stimulus dependent induction of E-selectin of aortic endothelial cells. Proc Natl Acad Sci USA 93:7075, 1996
S
EVERE ISCHEMIA-REPERFUSION injury (IRI) can result in organ dys- or nonfunction. The morphological characteristics of severe reperfusion injury are disturbed microcirculation and cellular damage. The pathophysiology of IRI is complex and the activation of endothelial cells is implicated as a key step.1 E-selectin supports initial rolling of leukocytes on activated endothelium and mediates stable arrest of leukocytes. E-selectin is not detectable on quiescent endothelial cells and can be used as a marker of activated endothelial cells.2 Little is known about the kinetics and localization of E-selectin during intestinal IRI. Therefore, we studied expression and localization of E-selectin during intestinal IRI. METHODS Intestinal tissue samples were obtained from extracorporeal perfused pig intestines after a cold ischemia time (CIT) of 2 or 20 © 2004 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710 Transplantation Proceedings, 36, 265–266 (2004)
hours (each n ⫽ 5) and additionally in intestinal transplanted pigs after a CIT of 2 or 20 hours (each n ⫽ 1). The extracorporeal perfusion system was described previously.3 Transplantation was performed orthotopically. Histidine-tryptophane-ketoglutarate From the Klinik fu¨r Allgemeine Chirurgie und Thoraxchirurgie (F.B., A.R.M., F.F., B.K.), Universita¨t Schleswig-Holstein, Campus Kiel, Kiel, Germany; Zentrum Innere Medizin (M.H., E.W.), Pathologie (B.S.), and Chirurgie (S.L., T.L.), Universita¨t Go¨ttingen, Go¨ttingen, Germany; and Center for Liver, Pancreas and Biliary Diseases (B.R.), MCP Hahnemann University, Philadelphia, Pa, USA. This study was supported by grant SFB 402 D1 and G.I.F. 2013-1079.2/2000. Address reprint requests to PD Dr Andrea Mueller, Klinik fu¨r Allgemeine Chirurgie und Thoraxchirurgie, Universita¨t SchleswigHolstein, Campus Kiel, Arnold-Heller-Strasse 7, 24105 Kiel, Germany. E-mail: [email protected] 0041-1345/04/$–see front matter doi:10.1016/j.transproceed.2004.01.081 265
266 (HTK) solution was used for preservation. Mucosal damage was graded according to the classification of Chiu et al.4 E-selectin mRNA was determined by PCR and quantitative RT-PCR. Localization of E-selectin mRNA was performed by in situ hybridization and E-selectin protein by immunohistochemistry.
RESULTS
Mean (⫾ SD) perfusion time was 546 ⫾ 55 after a CIT of 2 hours and 426 ⫾ 69 minutes after a CIT of 20 hours. Mucosal damage was minimal at the end of cold preservation irrespective of the length of CIT. At 1 hour of extracorporeal reperfusion, intestines exposed to 20 hours of CIT showed more severe damage of intestinal mucosa compared to intestines with CIT of 2 hours. At the end of reperfusion, severe mucosal damage was seen in all intestines. In intestinal tissue, E-selectin-mRNA expression was detected before and after perfusion with HTK solution, at the end of cold preservation, and after reperfusion. At the end of CIT, E-selectin-mRNA expression was higher after 20 hours than after 2 hours of CIT. Immediately after start of reperfusion, E-selectin mRNA expression increased in the intestinal tissue exposed to CIT for 2 hours, whereas E-selectin mRNA expression decreased in intestinal tissue exposed to CIT for 20 hours. Thereafter, E-selectin mRNA expression increased continuously during the reperfusion phase. In extracorporeal perfused intestinal specimens, E-selectin-mRNA was detected on the luminal surface of muscular, submucosal, and mucosal endothelial cells. Immunohistochemically, E-selectin was not detected prior to reperfusion. At 2 hours after reperfusion, endothelial cells of small submucosal arteries stained positive for E-selectin and intensive staining was detectable thereafter. Immunohistochemically, kinetics of semiquantitative staining intensity of E-selectin in extracorporeal perfused intestine was similar after 2 and 20 hours of cold preservation. DISCUSSION
Enhanced biosynthesis of adhesion molecules that mediate leukocyte-endothelial cell adhesion results in response to ischemia-reperfusion injury.1 In our model, E-selectinmRNA expression was detected already after laparotomy and increased after reperfusion. Endothelial cells stained positive for E-selection and E-selectin-mRNA in all intestinal layers. Immunohistochemically, arterial endothelium stained positive for E-selectin at 2 hours after start of reperfusion. Wyble et al reported expression of E-selectin
BRAUN, HOSSEINI, WIELAND ET AL
on human endothelial cells as early as 2 hours with maximum effects after 4 hours after addition of TNF-␣ and IL-1.5 In mice intestine exposed to 45 minutes of ischemia, increasing levels of E-selectin expression were found after 5 hours of reperfusion.6 Several mechanisms are suggested in the pathway of E-selectin expression including TNF-␣induced NF-B activation and exogenous lipopolysaccharide (LPS) application.7,8 The enhanced expression of E-selectin-mRNA during cold preservation in our model reflects transcription at the state of hypoxia. In vitro, E-selectin-mRNA is not expressed in response to hypoxia alone, but additional stimulation with TNF-␣ or LPS results in up-regulation of E-selectin-mRNA.9 In conclusion, prolonged cold preservation predisposes to enhanced transcription of E-selectin and is associated with earlier and more severe mucosal damage after reperfusion. Considering E-selectin as a marker of activated endothelial cells, they become activated earliest after reperfusion. Increased E-selectin mRNA at end of 20 hours of CIT might indicate a preactivated state of endothelial cells potentially triggered by bacterial translocation or products. Therefore, it appears beneficial to keep the cold ischemia time as short as possible in intestinal transplantation. REFERENCES 1. Carden DL, Granger DN: Pathophysiology of ischemia-reperfusion injury. J Pathol 190:255, 2000 2. Bevilacqua MP, Pober JS, Mendrick DL, et al: Identification of an inducible endotehlial-leukocyte adhesion molecule. Proc Natl Acad Sci 82:9238, 1987 3. Braun F, Schu ¨tz E, Laabs S, et al: Development of a porcine small bowel ex vivo perfusion model. Transplant Proc 30:2613, 1998 4. Chiu CJ, McArdle H, Brown R, et al: Intestinal mucosal lesion in low-flow states. Arch Surg 101:478, 1970 5. Wyble CW, Desai TR, Clark ET, et al: Physiologic concentrations of TNF-alpha and IL-1beta released from reperfused intestine upregulate E-selectin and ICAM-1. J Surg Res 63:333, 1996 6. Russell J, Epstein CJ, Grisham MB, et al: Regulation of E-selectin expression in postischemic intestinal microvasculature. Am J Physiol Gastrointest Liver Physiol 278:G878, 2000 7. Rahman A, Kefer J, Bando M, et al: E-selectin expression in human endothelial cells by TNF-a induced oxidant generation and NFkB activation. Am J Physiol 275:L533, 1998 8. Bauer P, Russell JM, Granger DN: Role of endotoxin in intestinal reperfusion induced expression of E-selectin. Am J Physiol 278:R1140, 2000 9. Zund G, Nelson DP, Neufeld EJ, et al: Hypoxia enhances stimulus dependent induction of E-selectin of aortic endothelial cells. Proc Natl Acad Sci USA 93:7075, 1996