In vivo gene transfer of human thrombomodulin improves intrahepatic hypercoagulability and ischemia-reperfusion injury after warm ischemia.

2005 Fiscal Year Final Research Report Summary

• Project/Area Number: 15591419
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Digestive surgery
• Research Institution: University of the Ryukyus
• Principal Investigator: SHIRAISHI Masayuki University of the Ryukyus, Faculty of Medicine, associate professor, 医学部, 助教授 (00264482)
• Co-Investigator(Kenkyū-buntansha): MIYAGUNI Takao University of the Ryukyus, University Hospital, assistant professor, 医学部附属病院, 講師 (00295320)
• Project Period (FY): 2003 – 2005
• Keywords: thrombomodulin / ischemia-reperfusion injury / adenovirus vector / genetherapy / warm ischemia / non-heart beating donor

Research Abstract

Background.
In hepatic ischemia-reperfusion injury (IRI), a massive loss of thrombomodulin (TM) from the sinusoidal endothelial cells is thought to play a central role in the development of intrahepatic hypercoagulability and liver damage. The genetic modification of the liver graft to express exogenous TM might thus help to replace any lost TM, thereby reducing hepatic IRI.
Materials and methods.
Three groups of 12 rats each received an intra-venous injection of marker LacZ adenovirus vector in group 1 (AxCALacZ,1×10^9 cfu/ml), human TM (hTM) adenovirus vector in group 2 (AxCAhTM,1×10^9 cfu/ml), or normal saline in group 3 (1 ml). Forty-eight hrs after gene transfer, the rats were anesthetized by ether and then were subjected to hepatic warm ischemia for 30 min. In all the groups, 3 rats each were sacrificed at 6, 12, 24 hrs, and 7 days after reperfusion, in order to obtain both serum and hepatic tissue samples. The serum samples were used to determine the levels of hepatocyte enzymes (A LT), hyaluronic acid, and others. The hepatic expression of marker LacZ was evaluated by X-gal staining. The protein and mRNA expression of hTM were evaluated by immunohistochemical staining and RT-PCR. The local hepatic blood flow was measured by a laser blood flowmeter. Intrahepatic neutrophil aggregation was also evaluated by Naphthol AS-D chloroacetate staining.
Results.
The expression of marker LacZ (X-gal staining ) and hTM (immunohistochemical staining and RT-PCR) was detected only in the livers from groups 1 and 2, respectively, up until 7 days after reperfusion. Intrahepatic hypercoagulability after reperfusion, as indicated by thrombus in the central venules, only decreased remarkably in the hTM transfected group 2 at 6 to 12 hrs after reperfusion. At 12 hrs after reperfusion, the serum ALT levels, hyaluronic acid, hepatic tissue blood flow, and intrahepatic neutrophil aggregation significantly improved only in the hTM treated group 2 in comparison to those of groups 1 and 3.
Conclusions.
These findings thus suggested that the adenovirus mediated gene transfer of hTM helped to attenuate liver damage in a rat model of warm ischemic liver injury.