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Eur J Cardiothorac Surg 2004;26:995-1001
© 2004 Elsevier Science NL

Non-viral in vivo thrombomodulin gene transfer prevents early loss of thromboresistance of grafted veins

^a Department of Cardio-Thoracic Surgery, Tokyo Medical and Dental University Graduate School, Tokyo, Japan
^b Department of Allied Health Sciences, Tokyo Medical and Dental University Graduate School, Tokyo, Japan
^c Tokyo Medical and Dental University Medical Hospital, 1-5-45, Yushima, Bunkyo-ku, Tokyo 113-8519, Japan

Received 18 February 2004; received in revised form 2 July 2004; accepted 8 July 2004.

^* Corresponding author. Tel.: +81-3-5803-4571; fax: +81-3-5803-0254. (E-mail: mshichiri.cme{at}tmd.ac.jp).

Objective: Immediate loss of thrombomodulin activity in the endothelium of vein grafts has been demonstrated during 90min exposure to arterial circulation; this loss of activity is ascribed as an important cause of early thrombosis. Conventional ex vivo gene transfection after vein harvest cannot cover this acute period immediately after implantation. We have established a highly efficient non-viral gene therapy protocol utilizing modified transferrin receptor-facilitated gene transfer. Using this technique, we examined whether in vivo thrombomodulin gene therapy, directed to the endothelium of rat veins 2 days prior to grafting, may prevent thromboresistance impairment of vein grafts under simulated arterial circulation. Methods: Abdomen of SD rat was opened and cationic liposome:transferrin:thrombomodulin gene complexes or the vector without DNAs were applied to the inferior vena cava of rats while blood flow was reduced by proximal and distal clamping. After 2 days, the transfected veins were harvested and thrombomodulin expression and thromboresistance properties determined before and after exposure to an artificial circuit. Results: The trial of gene transfection using variable doses of DNAs confirmed that 7.5µg of total DNAs was the most efficient quantity for thrombomodulin gene transfection to IVCs, although accompanying an increase of gene expression in other downstream organs. By transfection of the thrombomodulin gene in IVCs, the generation capacity of activated protein C in venous endothelium increased three-fold compared with veins treated with vector alone (P<0.01). Under simulated arterial circulation, perfusion of veins treated with vector alone decreased thrombomodulin activity to 36% of preperfused levels (P<0.01), whereas transfected grafts preserved the activity at normal vein endothelium levels even after perfusion. Consequently, the increase in endothelial thrombin activity induced by simulated arterial circulation was markedly attenuated in transfected veins (P<0.01), while immunohistochemistry confirmed the preservation of endothelial lining. Conclusions: Transferrin receptor-facilitated in vivo gene transfer to the inferior vena cava resulted in sufficient thrombomodulin gene expression immediately after graft implantation and subsequent maintenance of thromboresistance even after exposure to arterial pressure. Although further studies are needed, the present results suggest the possibility of gene therapy targeting acute phases of vein graft disease.