Adenovirus mediated gene transfer of antiapoptotic protein in hepatic ischemia-reperfusion injury: the paradoxical effect of Bcl-2 expression in the reperfused liver.
BACKGROUND
Although Bcl-2 is well known to have antiapoptotic activities in vitro and in vivo, the role of Bcl-2 remains controversial. In the present study, we evaluated whether the adenovirus mediated gene transfer of hBcl-2 could exert an antiapoptotic effect in a rat model of hepatic ischemia-reperfusion (I/R) injury.


METHODS
Each 6 x 10(9) plaque forming unit adenovirus vector encoding LacZ (AxCAilacZ) or hBcl-2 (AxCAhbcl2) was intravenously administered 48 h before I/R injury, in groups 1 and 2, respectively. In group 3, 1 ml of normal saline was injected instead of the virus vectors. Hepatic I/R injury was induced by the temporal occlusion of all hepatic influent vessels for 30 min under a portosystemic shunt. The animals were sacrificed at 6 h, 1, 3, and 14 days after reperfusion (each n = 12 in groups 1 and 2, and n = 8 in group 3). The expressions of hBcl-2 and Bax were evaluated at both the mRNA level by reverse transcription polymerase chain reaction and the protein level by immunohistochemistry. To assess the hepatocyte and sinusoidal endothelial cell damage after I/R injury, the serum asparate aminotransferase (AST), alanine aminotransferase, and hyaluronic acid were all evaluated. The number of apoptotic cells was evaluated by terminal deoxynucleotidyl transferase-mediated dUTP biotin nick end-labeling (TUNEL).


RESULTS
To evaluate the antiapoptotic activities of the hBcl-2 sequence encoded into AxCAhbcl2, rat hepatocarcinoma cells were transfected with AxCAhbcl2 (10(3) moi) or AxCAilacZ (10(3) moi) and then challenged with TGF-beta1 protein (5 ng/ml) to induce apoptosis. Apoptotic cells were counted by TUNEL staining in about 2500 cells, and it was found that adenovirus mediated gene transfer of hBcl-2 significantly protected rat hepatocarcinoma cells from TGF-beta1 induced apoptosis (14.2 +/- 1.2%) in comparison to those of LacZ (21.9 +/- 1.4%). In the reperfused liver in vivo, the mRNA expression of hBcl-2 was detected only in the hBcl-2 transfected group 2. In group 2, a strong degree of Bcl-2 immunoreactivity was recognized as early as 6 h after reperfusion, while it was not recognized in groups 1 and 3 at 6 h after reperfusion. The AST levels were significantly higher in group 2 (AST: 356 +/- 100.1 IU/L) than those in group 1 (AST: 102.7 +/- 15 IU/L) at 1 day after reperfusion (P < 0.05). The number of TUNEL positive cells was significantly higher in group 2 than in groups 1 and 3 at 1 day after reperfusion.


CONCLUSIONS
These results indicated that an overexpression of antiapoptotic protein Bcl-2 paradoxically exerted a proapoptotic effect in the reperfused liver. The in vivo role of Bcl-2 should thus be carefully evaluated, depending on the levels of expression and the target organ.
Oshiro
Takashi
T
First Department of Surgery, University of the Ryukyus, Okinawa, 903-0125, Japan. t-oshiro@geocities.co.jp
Shiraishi
Masayuki
M
Muto
Yoshihiro
Y
D000256:Adenoviridae / Q000235:genetics*; D000410:Alanine Transaminase / Q000097:blood; D000818:Animals; D017209:Apoptosis / Q000502:physiology; D001219:Aspartate Aminotransferases / Q000097:blood; D004195:Disease Models, Animal; D005697:Galactosides; D015870:Gene Expression; D018014:Gene Transfer Techniques; D007150:Immunohistochemistry; D020287:In Situ Nick-End Labeling; D066298:In Vitro Techniques; D007211:Indoles; D008099:Liver / Q000737:chemistry / Q000502:physiology; D008107:Liver Diseases / Q000503:physiopathology / Q000628:therapy*; D008297:Male; D011518:Proto-Oncogene Proteins / Q000032:analysis / Q000235:genetics; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000032:analysis / Q000235:genetics*; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D017208:Rats, Wistar; D015427:Reperfusion Injury / Q000503:physiopathology / Q000628:therapy*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D051028:bcl-2-Associated X Protein
C494828:Bax protein, rat; D005697:Galactosides; D007211:Indoles; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D012333:RNA, Messenger; D051028:bcl-2-Associated X Protein; D001219:Aspartate Aminotransferases; D000410:Alanine Transaminase; C044888:5-bromo-4-chloro-3-indolyl beta-galactoside