Zinc Finger Protein A20 Promotes Regeneration of Small-for-Sized Liver Allograft and Suppresses Rejection in Rats_CHINESE JOURNAL OF BASES AND CLINICS IN GENERAL SURGERY

Authors：

XU Mingqing ¹ , YAN Lnan ¹ , GOU Xinghua ² , PENG Fangxing ¹ , LI Dehua ² , HAN Lei ² , HUANG Lanying ² , HUANG Yingchun ² , HU Haiyang ²

1. Liver Transplantation Division, West China Hospital, Sichuan University, Chengdu 610041, China;;
2.The New Drug Research Division, Diao Drug Group Ltd., Chengdu 610041, China Corresponding Author： YAN Lü-nan, E-mail： yanlunan688@163.com;

Corresponding author：

Keywords：

Allograft liver transplantation; Small-for-sized liver graft; Zinc finger protein A20; Liver regeneration; Liver sinusoidal endothelial cell

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Objective 　 To examine the effect of zinc finger protein A20 on regeneration of small-for-sized liver allograft, graft rejection and recipient rat survival time.
Methods 　Small-for-sized liver transplantation with 30% partial liver allograft was performed by using a b-rejection combination rat model of DA (RT1a) to Lewis (RT1l) rats. The rats were grouped into rAdEasy-A20 treatment group （A20 group）, the control empty Ad vector rAdEasy treatment group (rAdEasy group) and PS control treatment group (PS group). Ex vivo gene transfer in donor liver graft was performed through portal vein infusion. Animals were assessed for survival days, expression of A20 in liver graft, liver graft regeneration, hepatocyte apoptosis, graft rejection, NF-κB activation and ICAM-1 mRNA expression in liver graft sinusoidal endothelial cells (LSECs), number of liver graft infiltrating mononuclear cells (LIMCs) and the subproportion of NK/NKT cells, and serum IFN-γ level.
Results 　Survival day of A20 group rats was prominently longer than that of PS group rats and rAdEasy group rats (P=0.001 8), whereas survival day of rAdEasy group rats was remarkably shorter than that of PS group rats (P=0.001 8). Regeneration of the small-for-sized liver allograft was markedly augmented by A20, BrdU labelling index of hepatocyte on postoperative day 4 was significantly increased in the A20 group compared with the PS group and rAdEasy group (P＜0.01). Hepatocyte apoptosis on postoperative day 4 was significantly inhibited by A20 (P＜0.01). On postoperative day 4, histologic examination revealed a mild rejection in the A20 group but a more severe rejection in the PS and rAdEasy groups. NF-κB activity and ICAM-1 mRNA expression in LSECs on postoperative day 1 were notably suppressed by A20 overexpression. Flow cytometry analysis showed a marked downregulation of LIMCs number by A20, including more prominent decrease in the subproportion of NK/NKT cells on postoperative day 1 and 4, respectively (P＜0.05). Serum IFN-γ level on postoperative day 4 was also significantly suppressed by A20 overexpression (P＜0.05).
Conclusion 　These data suggest that A20 could effectively promote small-for-sized liver allograft regeneration, suppresses rejection and prolong survival days of recipient rats. These effects of A20 could be related to an inhibition of LSECs activation, suppression of infiltration of LIMCs and the subpopulations such as NK cells and NKT cells into liver graft, and inhibition of hepatocyte apoptosis.

Citation： XU Mingqing,YAN Lnan,GOU Xinghua,PENG Fangxing,LI Dehua,HAN Lei,HUANG Lanying,HUANG Yingchun,HU Haiyang. Zinc Finger Protein A20 Promotes Regeneration of Small-for-Sized Liver Allograft and Suppresses Rejection in Rats. CHINESE JOURNAL OF BASES AND CLINICS IN GENERAL SURGERY, 2008, 15(8): 572-578. doi: Copy

1.	Omura T, Nakagawa T, Randall HB, et al. Increased immune responses to regenerating partial liver grafts in the rat [Ｊ]. J Surg Res, 1997; 70(1)∶ 34.
2.	Shiraishi M, Csete ME, Yasunaga C, et al. Regeneration-induced accelerated rejection in reduced-size liver grafts [Ｊ]. Transplantation, 1994； 57(3)∶ 336.
3.	Huda KA, Guo L, Haga S, et al. Ex vivo adenoviral gene transfer of constitutively activated STAT3 reduces post-transplant liver injury and promotes regeneration in a 20% rat partial liver transplant model [Ｊ]. Transplant Int， 2006; 19(5)∶ 415.
4.	Braet F, De Zanger R, Sasaoki T, et al. Assessment of a method of isolation, purification and cultivation of rat liver sinusoidal endothelial cells [Ｊ]. Lab Invest, 1994; 70(6)∶ 944.
5.	Ogura Y, Martinez OM, Villanueva JC, et al. Apoptosis and allograft rejection in the absence of CD8+ T cells [Ｊ]. Transplantation, 2001; 71(12)∶ 1827.
6.	Xu MQ, Suo YP, Gong JP, et al. Augmented regeneration of partial liver allograft induced by nuclear factor-κB decoy oligodeoxynucleotides-modified dendritic cells [Ｊ]. World J Gastroentrol, 2004； 10(4)∶ 573.
7.	Luo D, Vanderkerken K, Bouwens L, et al. The role of adhesion molecules in the recruitment of hepatic natural killer cells (pit cells) in rat liver [Ｊ]. Hepatology, 1996; 24(6)∶ 1475.
8.	Adams DH， Hubscher SG， Shaw J, et al. Intercellular adhesion molecule-1 on liver allografts during rejection [Ｊ]. Lancet， 1989; 2(8672)∶ 1122.
9.	Steinhoff G, Behrend M, Schrader B, et al. Intercellular immune adhesion molecules in human liver transplants: Overview on expression patterns of leukocyte receptor and ligand molecules [Ｊ]. Hepatology, 1993; 18(2)∶ 440.
10.	Omura T, Ishikura H, Nakajima Y, et al. The expression of LFA-1/ICAM-1 in liver transplantation in rats [Ｊ]. Transplant Proc, 1992; 24(4)∶ 1618.
11.	Opipari AW Jr, Boguski MS, Dixit VM. The A20 cDNA induced by tumor necrosis factor alpha encodes a novel type of zinc finger protein [Ｊ]. J Biol Chem, 1990; 265(25)∶ 14705.
12.	Cooper JT, Stroka DM, Brostjan C, et al. A20 blocks endothelial cell activation through a NF-κB-dependent mechanism [Ｊ]. J Biol Chem, 1996; 271(30)∶ 18068.
13.	Ferran C, Stroka DM, Badrichani AZ, et al. A20 inhibits NF-κB activation in endothelial cells without sensitizing to tumor necrosis factor-mediated apoptosis [Ｊ]. Blood, 1998; 91(7)∶ 2249.
14.	Longo CR, Arvelo MB, Patel VI, et al. A20 protects from CD40-CD40 ligand-mediated endothelial cell activation and apoptosis [Ｊ]. Circulation, 2003; 108(9)∶ 1113.
15.	Arvelo MB, Cooper JT, Longo C, et al. A20 protects mice from galactosmine/lipopolysaccharide acute toxic lethal hepatitis [Ｊ]. Hepatology, 2002; 35(3)∶ 535.
16.	Hoffmann MW， Wonigeit K, Steinhoff G, et al. Production of cytokines (TNF-alpha, IL-1-beta) and endothelial cell activation in human liver allograft rejection [Ｊ]. Transplantation, 1993; 55(2)∶ 329.
17.	Tamura F, Masuhara A, Sakaida I, et al. FK506 promotes liver regeneration by suppressing natural killer cell activity [Ｊ]. J Gastroenterol Hepatol， 1998; 13(7)∶ 703.
18.	Huang W, Dong Z, Wei H, et al. Selective elimination of hepatic natural killer T cells with concanavalin A improves liver regeneration in mice [Ｊ]. Liver Int， 2006; 26(3)∶ 339.
19.	Sato Y, Tsukada K, Matsumoto Y, et al. Interferon-gamma inhibits liver regeneration by stimulating major histocompatibility complex class II antigen expression by regenerating liver [Ｊ]. Hepatology, 1993; 18(2)∶ 340.
20.	Obara H, Nagasaki K, Hsieh CL, et al. IFN-γ, produced by NK cells that infiltrate liver allografts early after transplantation, links the innate and adaptive immune responses [Ｊ]. Am J Transplant, 2005; 5(9)∶ 2094.
21.	Navarro F, Portalès P, Candon S, et al. Natural killer cell and αβ and γδ lymphocyte traffic into the liver graft immediately after liver transplantation [Ｊ]. Transplantation, 2000; 69(4)∶〖KG-8〗 633.
22.	Longo CR, Patel VI, Shrikhande GV, et al. A20 protects mice from lethal radical hepatectomy by promoting hepatocyte proliferation via a p21waf1-dependent mechanism [Ｊ]. Hepatology, 2005; 42(1)∶ 156.
23.	Yamamoto H, Ohdan H, Shintaku S, et al. Role of the bcl-2/bax pathway in hepatocyte apoptosis during acute rejection after rat liver transplantation [Ｊ]. Transpl Int， 1998; 11 (Suppl 1)∶ S179.
24.	Krams SM, Egawa H, Quinn MB, et al. Apoptosis as a mechanism of cell death in liver allograft rejection [Ｊ]. Transplantation, 1995; 59(4)∶ 621.
25.	Hsieh CL, Obara H, Ogura Y, et al. NK cells and transplantation [Ｊ]. Transpl Immunol， 2002; 9(2-4)∶ 111.
26.	Kojima Y, Kawasaki-Koyanagi A, Sueyoshi N, et al. Localization of Fas ligand in cytoplasmic granules of CD8+ cytotoxic T lymphocytes and natural killer cells： participation of Fas ligand in granule exocytosis model of cytotoxicity [Ｊ]. Biochem Biophys Res Commun， 2002; 296(2)∶ 328.
27.	Ariki N, Morimoto Y, Yagi T, et al. Activated T cells and soluble molecules in the portal venous blood of patients with cholestatic and hepatitis C virus-positive liver cirrhosis. Possible promotion of Fas/FasL-mediated apoptosis in the bile-duct cells and hepatocyte injury [Ｊ]. J Int Med Res， 2003; 31(3)∶ 170.
28.	Wang J, Li W, Min J, et al. Fas siRNA reduces apoptotic cell death of allogeneic-transplanted hepatocytes in mouse spleen [Ｊ]. Transplant Proc， 2003; 35(4)∶ 1594.

1. 　Omura T, Nakagawa T, Randall HB, et al. Increased immune responses to regenerating partial liver grafts in the rat [Ｊ]. J Surg Res, 1997; 70(1)∶ 34.
2. 　Shiraishi M, Csete ME, Yasunaga C, et al. Regeneration-induced accelerated rejection in reduced-size liver grafts [Ｊ]. Transplantation, 1994； 57(3)∶ 336.
3. 　Huda KA, Guo L, Haga S, et al. Ex vivo adenoviral gene transfer of constitutively activated STAT3 reduces post-transplant liver injury and promotes regeneration in a 20% rat partial liver transplant model [Ｊ]. Transplant Int， 2006; 19(5)∶ 415.
4. 　Braet F, De Zanger R, Sasaoki T, et al. Assessment of a method of isolation, purification and cultivation of rat liver sinusoidal endothelial cells [Ｊ]. Lab Invest, 1994; 70(6)∶ 944.
5. 　Ogura Y, Martinez OM, Villanueva JC, et al. Apoptosis and allograft rejection in the absence of CD8+ T cells [Ｊ]. Transplantation, 2001; 71(12)∶ 1827.
6. 　Xu MQ, Suo YP, Gong JP, et al. Augmented regeneration of partial liver allograft induced by nuclear factor-κB decoy oligodeoxynucleotides-modified dendritic cells [Ｊ]. World J Gastroentrol, 2004； 10(4)∶ 573.
7. 　Luo D, Vanderkerken K, Bouwens L, et al. The role of adhesion molecules in the recruitment of hepatic natural killer cells (pit cells) in rat liver [Ｊ]. Hepatology, 1996; 24(6)∶ 1475.
8. 　Adams DH， Hubscher SG， Shaw J, et al. Intercellular adhesion molecule-1 on liver allografts during rejection [Ｊ]. Lancet， 1989; 2(8672)∶ 1122.
9. 　Steinhoff G, Behrend M, Schrader B, et al. Intercellular immune adhesion molecules in human liver transplants: Overview on expression patterns of leukocyte receptor and ligand molecules [Ｊ]. Hepatology, 1993; 18(2)∶ 440.
10. 　Omura T, Ishikura H, Nakajima Y, et al. The expression of LFA-1/ICAM-1 in liver transplantation in rats [Ｊ]. Transplant Proc, 1992; 24(4)∶ 1618.
11. 　Opipari AW Jr, Boguski MS, Dixit VM. The A20 cDNA induced by tumor necrosis factor alpha encodes a novel type of zinc finger protein [Ｊ]. J Biol Chem, 1990; 265(25)∶ 14705.
12. 　Cooper JT, Stroka DM, Brostjan C, et al. A20 blocks endothelial cell activation through a NF-κB-dependent mechanism [Ｊ]. J Biol Chem, 1996; 271(30)∶ 18068.
13. 　Ferran C, Stroka DM, Badrichani AZ, et al. A20 inhibits NF-κB activation in endothelial cells without sensitizing to tumor necrosis factor-mediated apoptosis [Ｊ]. Blood, 1998; 91(7)∶ 2249.
14. 　Longo CR, Arvelo MB, Patel VI, et al. A20 protects from CD40-CD40 ligand-mediated endothelial cell activation and apoptosis [Ｊ]. Circulation, 2003; 108(9)∶ 1113.
15. 　Arvelo MB, Cooper JT, Longo C, et al. A20 protects mice from galactosmine/lipopolysaccharide acute toxic lethal hepatitis [Ｊ]. Hepatology, 2002; 35(3)∶ 535.
16. 　Hoffmann MW， Wonigeit K, Steinhoff G, et al. Production of cytokines (TNF-alpha, IL-1-beta) and endothelial cell activation in human liver allograft rejection [Ｊ]. Transplantation, 1993; 55(2)∶ 329.
17. 　Tamura F, Masuhara A, Sakaida I, et al. FK506 promotes liver regeneration by suppressing natural killer cell activity [Ｊ]. J Gastroenterol Hepatol， 1998; 13(7)∶ 703.
18. 　Huang W, Dong Z, Wei H, et al. Selective elimination of hepatic natural killer T cells with concanavalin A improves liver regeneration in mice [Ｊ]. Liver Int， 2006; 26(3)∶ 339.
19. 　Sato Y, Tsukada K, Matsumoto Y, et al. Interferon-gamma inhibits liver regeneration by stimulating major histocompatibility complex class II antigen expression by regenerating liver [Ｊ]. Hepatology, 1993; 18(2)∶ 340.
20. 　Obara H, Nagasaki K, Hsieh CL, et al. IFN-γ, produced by NK cells that infiltrate liver allografts early after transplantation, links the innate and adaptive immune responses [Ｊ]. Am J Transplant, 2005; 5(9)∶ 2094.
21. 　Navarro F, Portalès P, Candon S, et al. Natural killer cell and αβ and γδ lymphocyte traffic into the liver graft immediately after liver transplantation [Ｊ]. Transplantation, 2000; 69(4)∶〖KG-8〗 633.
22. 　Longo CR, Patel VI, Shrikhande GV, et al. A20 protects mice from lethal radical hepatectomy by promoting hepatocyte proliferation via a p21waf1-dependent mechanism [Ｊ]. Hepatology, 2005; 42(1)∶ 156.
23. 　Yamamoto H, Ohdan H, Shintaku S, et al. Role of the bcl-2/bax pathway in hepatocyte apoptosis during acute rejection after rat liver transplantation [Ｊ]. Transpl Int， 1998; 11 (Suppl 1)∶ S179.
24. 　Krams SM, Egawa H, Quinn MB, et al. Apoptosis as a mechanism of cell death in liver allograft rejection [Ｊ]. Transplantation, 1995; 59(4)∶ 621.
25. 　Hsieh CL, Obara H, Ogura Y, et al. NK cells and transplantation [Ｊ]. Transpl Immunol， 2002; 9(2-4)∶ 111.
26. 　Kojima Y, Kawasaki-Koyanagi A, Sueyoshi N, et al. Localization of Fas ligand in cytoplasmic granules of CD8+ cytotoxic T lymphocytes and natural killer cells： participation of Fas ligand in granule exocytosis model of cytotoxicity [Ｊ]. Biochem Biophys Res Commun， 2002; 296(2)∶ 328.
27. 　Ariki N, Morimoto Y, Yagi T, et al. Activated T cells and soluble molecules in the portal venous blood of patients with cholestatic and hepatitis C virus-positive liver cirrhosis. Possible promotion of Fas/FasL-mediated apoptosis in the bile-duct cells and hepatocyte injury [Ｊ]. J Int Med Res， 2003; 31(3)∶ 170.
28. 　Wang J, Li W, Min J, et al. Fas siRNA reduces apoptotic cell death of allogeneic-transplanted hepatocytes in mouse spleen [Ｊ]. Transplant Proc， 2003; 35(4)∶ 1594.

CHINESE JOURNAL OF BASES AND CLINICS IN GENERAL SURGERY

Zinc Finger Protein A20 Promotes Regeneration of Small-for-Sized Liver Allograft and Suppresses Rejection in Rats

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