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reperfusion injury via oxygen radicals derived from Kupffer cells
HEPATOLOGY, Vol. 38, No. 4, Suppl. 1, 2003
AASLD ABSTRACTS
41
ISCHEMIC PRECONDITIONING PROTECTS HEPATOCYTES AGAINST WARM ISCHEMIA/REPERFUSION INJURY VIA OXYGEN RADICALS DERIVED FROM KUPFFER CELLS. Kazuaki Tejima, Masahiro Arai, Hitoshi Ikeda, Tomoaki Tomiya, Mikio Yanase, Yukiko Inoue, Kayo Nagashima, Naoko Watanabe, Masao Omata, University of Tokyo, Tokyo, Japan; Kenji Fujiwara, Saitama Medical School, Saitama, Japan Background A brief period of tissue ischemia followed by reperfusion renders tissue resistant against subsequent prolonged ischemia and reperfusion, a p h e n o m e n o n called ischemic preconditioning. Hepatic ischemic preconditioning protects sinusoidal endothelial cells against subsequent cold storage/warm reperfusion injury and improves graft survival after liver transplantation in rats. Aim Ischemic preconditioning also reduces liver injury after warm ischemia and reperfusion, in which hepatocyte damage and Kupffer cell activation are dominant features. However, it is incompletely elucidated which cell type ischemic preconditioning affects and how it works. Therefore, our aim was to investigate the mechanisms of ischemic preconditioning against w a r m ischemia and reperfusion injury. Method Male Sprague-Dawley rats were injected intravenously with 20 mg/kg gadolinium chloride (GdCI3), 10000 U/kg superoxide dismutase (SOD) or 300 mg/kg N-acethyl-L-cyctein (NAC) to suppress Kupffer cells, superoxide formation or oxygen radical formation, respectively. Livers were then preconditioned by clamping the hepatic artery and portal vein to the median and left lobes for 10 min followed by 10 min reperfusion. Subsequently, livers were subjected to warm ischemia/reperfusion injury in in vivo experiments and in liver perfusion experiments as follows. In vivo experiments: The blood flow to the median and left lobes was occluded with a vascular clamp for 40 min. One hour after removal of the clamp, blood was collected for determination of ALT activity and hyaluronic acid (HA) concentration. Liver perfusion experiments: Livers were perfused through the portal vein for 10 min with physiological buffer and stored at 37°C in the same buffer. After 40 min, livers were reperfused with the buffer containing 500 ng/mL HA for 60 min in a recirculating system for determination of ALT activity in the perfusate and hepatic HA uptake as a marker of sinusoidal endothelial cell injury. Livers of other non-treated rats were preconditioned by perfusion with the buffer containing H202 instead of ischemic preconditioning, and t h e n subjected to w a r m ischemia/reperfusion similarly. In other experiments, livers were subjected to ischemic preconditioning and perfused with the buffer containing 0.5 mg/mL nitro blue tetrazolium (NBT) for 10 min and fixed with formalin. Since NBT reacts with superoxide to form insoluble blue formazan, Kupffer cell superoxide formation was evaluated in histological sections. Results Ischemic preconditioning decreased serum ALT activity compared to control (168 ± 34 [mean ± SEM] vs 333 ± 52, p < 0.05), but did not change serum HA concentration. Pretreatment with GdCI3 reversed the effect of ischemic preconditioning (262 ± 41), though GdCI3 itself did not change ALT activity after ischemia/reperfusion (318 ± 33). Similar results were obtained in liver perfusion experiments, suggesting that ischemic preconditioning protected hepatocytes, b u t did not change sinusoidal endothelial cell injury. It is also suggested that Kupffer cells mediate hepatocytes protection by ischemic preconditioning. In rats treated with SOD or NAC, decrease in ALT activity in the perfusate by ischemic preconditioning was reversed (38 ± 3.5 [sham] vs 48 ± 1.7 [ischemic preconditioning] and 43 ± 4.8 vs 48 ± 4.1, respectively). Liver perfusion with 0.1 m M H202 decreased ALT activity after warm ischemia/reperfusion compared to control (22 ± 3.4 vs 37 ± 2.8, p < 0.01). Liver perfusion with NBT revealed superoxide formation in Kupffer cells even after 10 min ischemia. Conclusion Ischemic preconditioning reduced warm ischemia/reperfusion injury by protection of hepatocyte, not by suppression of sinusoidal endothelial cell injury in contrast with the effect against cold ischemia/reperfusion injury. This effect may be mediated by oxygen radical formation in Kupffer cells during ischemic preconditioning. Disclosures: Masahiro Arai - No relationships to disclose Kenji Fujiwara - No relationships to disclose
175A
Hitoshi Ikeda - No relationships to disclose Yukiko Inoue - No relationships to disclose Kayo Nagashima - No relationships to disclose Masao Omata - No relationships to disclose Kazuaki Tejima - No relationships to disclose Tomoaki Tomiya - No relationships to disclose Naoko Watanabe - No relationships to disclose Mikio Yanase - No relationships to disclose
42
A20 CONFERS A PROLIFERATIVE ADVANTAGE TO HEPATOCYTES AND PROMOTES LIVER REGENERATION. Christopher R Longo, Virendra I Patel, Maria B Arvelo, Soizic Daniel, Shane T Grey, Chrish'ane Ferran, Beth Israel Deaconess Medical Center, Boston, MA
Background: Living donor liver transplantation (LDLT) and split livers have emerged as a solution to ease the shortage of liver transplants and have achieved remarkable success in pediatric populations. In adults, LDLT is limited by the size of the graft that can be safely harvested and transplanted. Similar limitations apply w h e n extensive liver resections are n e e d e d for the treatment of hepatic tumors. We have previously demonstrated that the anti-apoptotic gene A20 is part of the physiologic response of hepatocytes to injury, protecting t h e m from apoptosis and limiting inflammation by inhibiting NF-KB activation. Our objective was to check whether A20 expression would help decrease the minimal liver mass required for mice survival. Methods and Results: Two-thirds partial hepatectomy is well tolerated and is an accepted model of liver regeneration in rodents. We performed an extended (78%) liver resection ( n - 1 2 / g r o u p ) in non-infected (NI) mice (10-12 week BALB/c) and mice infected with recombinant A20 adenovirus (rAd.A20) or a control adenovirus (rAd./3-gal). Surviving mice suffer a delay in regeneration as assessed by the n u m b e r of proliferation cell nuclear antigen (PCNA) positive nuclei 48 h following resection (5-10 positive cells/HPF in mice with 78% liver resection vs. 60-70 positive cells/ HPF in mice with a 2/3 liver resection). Interestingly, such a delay was not noted in mice expressing A20 (70-80 PCNA positive cells/ HPF were detected 48 h following 78% resection). A20 also prevented the development of coagulopathy as assessed by p r o t h r o m b i n time. We extended our resection to a radical hepatectomy comprising 87% of the liver (n-12/group). We observed 100% lethality in NI and rAd./3-gal infected mice. Expression of A20 resulted in the survival of 60% of the animals and correlated with increased PCNA positive hepatocytes as an indicator of increased cell proliferation. A20 mediated increase in hepatocyte proliferation was not merely d e p e n d e n t upon its anti-apoptotic function. Expression of A20 in hepatocyte cultures, in a nonapoptotic milieu, increased their proliferative response to serum and HGF as compared to NI and rAd./3-gal infected cells. Conclusion: These results demonstrate a novel pro-proliferatif function for A20 in hepatocytes and argue for its critical role in accelerating liver regeneration and promoting hepatocyte survival and function, even w h e n facing extreme metabolic demands. Gene therapy with A20 may allow for successful transplantation with smaller LDLT, split liver allografts and even hepatocellular grafts. Additionally, A20 based therapy to the liver may allow for extensive liver resections in the setting of cancer therapy. Disclosures: Maria B Arvelo - No relationships to disclose Soizic Daniel - No relationships to disclose Christiane Ferran - No relationships to disclose Shane T Grey - No relationships to disclose Christopher R Longo - No relationships to disclose Virendra I Patel - No relationships to disclose
AASLD ABSTRACTS
41
ISCHEMIC PRECONDITIONING PROTECTS HEPATOCYTES AGAINST WARM ISCHEMIA/REPERFUSION INJURY VIA OXYGEN RADICALS DERIVED FROM KUPFFER CELLS. Kazuaki Tejima, Masahiro Arai, Hitoshi Ikeda, Tomoaki Tomiya, Mikio Yanase, Yukiko Inoue, Kayo Nagashima, Naoko Watanabe, Masao Omata, University of Tokyo, Tokyo, Japan; Kenji Fujiwara, Saitama Medical School, Saitama, Japan Background A brief period of tissue ischemia followed by reperfusion renders tissue resistant against subsequent prolonged ischemia and reperfusion, a p h e n o m e n o n called ischemic preconditioning. Hepatic ischemic preconditioning protects sinusoidal endothelial cells against subsequent cold storage/warm reperfusion injury and improves graft survival after liver transplantation in rats. Aim Ischemic preconditioning also reduces liver injury after warm ischemia and reperfusion, in which hepatocyte damage and Kupffer cell activation are dominant features. However, it is incompletely elucidated which cell type ischemic preconditioning affects and how it works. Therefore, our aim was to investigate the mechanisms of ischemic preconditioning against w a r m ischemia and reperfusion injury. Method Male Sprague-Dawley rats were injected intravenously with 20 mg/kg gadolinium chloride (GdCI3), 10000 U/kg superoxide dismutase (SOD) or 300 mg/kg N-acethyl-L-cyctein (NAC) to suppress Kupffer cells, superoxide formation or oxygen radical formation, respectively. Livers were then preconditioned by clamping the hepatic artery and portal vein to the median and left lobes for 10 min followed by 10 min reperfusion. Subsequently, livers were subjected to warm ischemia/reperfusion injury in in vivo experiments and in liver perfusion experiments as follows. In vivo experiments: The blood flow to the median and left lobes was occluded with a vascular clamp for 40 min. One hour after removal of the clamp, blood was collected for determination of ALT activity and hyaluronic acid (HA) concentration. Liver perfusion experiments: Livers were perfused through the portal vein for 10 min with physiological buffer and stored at 37°C in the same buffer. After 40 min, livers were reperfused with the buffer containing 500 ng/mL HA for 60 min in a recirculating system for determination of ALT activity in the perfusate and hepatic HA uptake as a marker of sinusoidal endothelial cell injury. Livers of other non-treated rats were preconditioned by perfusion with the buffer containing H202 instead of ischemic preconditioning, and t h e n subjected to w a r m ischemia/reperfusion similarly. In other experiments, livers were subjected to ischemic preconditioning and perfused with the buffer containing 0.5 mg/mL nitro blue tetrazolium (NBT) for 10 min and fixed with formalin. Since NBT reacts with superoxide to form insoluble blue formazan, Kupffer cell superoxide formation was evaluated in histological sections. Results Ischemic preconditioning decreased serum ALT activity compared to control (168 ± 34 [mean ± SEM] vs 333 ± 52, p < 0.05), but did not change serum HA concentration. Pretreatment with GdCI3 reversed the effect of ischemic preconditioning (262 ± 41), though GdCI3 itself did not change ALT activity after ischemia/reperfusion (318 ± 33). Similar results were obtained in liver perfusion experiments, suggesting that ischemic preconditioning protected hepatocytes, b u t did not change sinusoidal endothelial cell injury. It is also suggested that Kupffer cells mediate hepatocytes protection by ischemic preconditioning. In rats treated with SOD or NAC, decrease in ALT activity in the perfusate by ischemic preconditioning was reversed (38 ± 3.5 [sham] vs 48 ± 1.7 [ischemic preconditioning] and 43 ± 4.8 vs 48 ± 4.1, respectively). Liver perfusion with 0.1 m M H202 decreased ALT activity after warm ischemia/reperfusion compared to control (22 ± 3.4 vs 37 ± 2.8, p < 0.01). Liver perfusion with NBT revealed superoxide formation in Kupffer cells even after 10 min ischemia. Conclusion Ischemic preconditioning reduced warm ischemia/reperfusion injury by protection of hepatocyte, not by suppression of sinusoidal endothelial cell injury in contrast with the effect against cold ischemia/reperfusion injury. This effect may be mediated by oxygen radical formation in Kupffer cells during ischemic preconditioning. Disclosures: Masahiro Arai - No relationships to disclose Kenji Fujiwara - No relationships to disclose
175A
Hitoshi Ikeda - No relationships to disclose Yukiko Inoue - No relationships to disclose Kayo Nagashima - No relationships to disclose Masao Omata - No relationships to disclose Kazuaki Tejima - No relationships to disclose Tomoaki Tomiya - No relationships to disclose Naoko Watanabe - No relationships to disclose Mikio Yanase - No relationships to disclose
42
A20 CONFERS A PROLIFERATIVE ADVANTAGE TO HEPATOCYTES AND PROMOTES LIVER REGENERATION. Christopher R Longo, Virendra I Patel, Maria B Arvelo, Soizic Daniel, Shane T Grey, Chrish'ane Ferran, Beth Israel Deaconess Medical Center, Boston, MA
Background: Living donor liver transplantation (LDLT) and split livers have emerged as a solution to ease the shortage of liver transplants and have achieved remarkable success in pediatric populations. In adults, LDLT is limited by the size of the graft that can be safely harvested and transplanted. Similar limitations apply w h e n extensive liver resections are n e e d e d for the treatment of hepatic tumors. We have previously demonstrated that the anti-apoptotic gene A20 is part of the physiologic response of hepatocytes to injury, protecting t h e m from apoptosis and limiting inflammation by inhibiting NF-KB activation. Our objective was to check whether A20 expression would help decrease the minimal liver mass required for mice survival. Methods and Results: Two-thirds partial hepatectomy is well tolerated and is an accepted model of liver regeneration in rodents. We performed an extended (78%) liver resection ( n - 1 2 / g r o u p ) in non-infected (NI) mice (10-12 week BALB/c) and mice infected with recombinant A20 adenovirus (rAd.A20) or a control adenovirus (rAd./3-gal). Surviving mice suffer a delay in regeneration as assessed by the n u m b e r of proliferation cell nuclear antigen (PCNA) positive nuclei 48 h following resection (5-10 positive cells/HPF in mice with 78% liver resection vs. 60-70 positive cells/ HPF in mice with a 2/3 liver resection). Interestingly, such a delay was not noted in mice expressing A20 (70-80 PCNA positive cells/ HPF were detected 48 h following 78% resection). A20 also prevented the development of coagulopathy as assessed by p r o t h r o m b i n time. We extended our resection to a radical hepatectomy comprising 87% of the liver (n-12/group). We observed 100% lethality in NI and rAd./3-gal infected mice. Expression of A20 resulted in the survival of 60% of the animals and correlated with increased PCNA positive hepatocytes as an indicator of increased cell proliferation. A20 mediated increase in hepatocyte proliferation was not merely d e p e n d e n t upon its anti-apoptotic function. Expression of A20 in hepatocyte cultures, in a nonapoptotic milieu, increased their proliferative response to serum and HGF as compared to NI and rAd./3-gal infected cells. Conclusion: These results demonstrate a novel pro-proliferatif function for A20 in hepatocytes and argue for its critical role in accelerating liver regeneration and promoting hepatocyte survival and function, even w h e n facing extreme metabolic demands. Gene therapy with A20 may allow for successful transplantation with smaller LDLT, split liver allografts and even hepatocellular grafts. Additionally, A20 based therapy to the liver may allow for extensive liver resections in the setting of cancer therapy. Disclosures: Maria B Arvelo - No relationships to disclose Soizic Daniel - No relationships to disclose Christiane Ferran - No relationships to disclose Shane T Grey - No relationships to disclose Christopher R Longo - No relationships to disclose Virendra I Patel - No relationships to disclose