Tabuchi Noriyuki, Shichiri Masayoshi, Shibamiya Aya, Koyama Takatoshi, Nakazawa Fumie, Chung Jihwa, Hirosawa Shinsaku, Sunamori Makoto
Department of Cardio-Thoracic Surgery, Tokyo Medical and Dental University Graduate School, Tokyo, Japan.
Eur J Cardiothorac Surg. 2004 Nov;26(5):995-1001. doi: 10.1016/j.ejcts.2004.07.028.
Immediate loss of thrombomodulin activity in the endothelium of vein grafts has been demonstrated during 90 min 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.
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.
The trial of gene transfection using variable doses of DNAs confirmed that 7.5 microg 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.
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.
静脉移植物内皮中的血栓调节蛋白活性在暴露于动脉循环90分钟期间会立即丧失；这种活性丧失被认为是早期血栓形成的一个重要原因。传统的静脉采集后体外基因转染无法覆盖植入后紧接着的这一急性期。我们建立了一种高效的非病毒基因治疗方案，利用修饰的转铁蛋白受体促进基因转移。使用该技术，我们研究了在移植前两天对大鼠静脉内皮进行体内血栓调节蛋白基因治疗是否可预防模拟动脉循环下静脉移植物的抗血栓形成能力受损。
打开SD大鼠的腹部，在通过近端和远端夹闭减少血流的情况下，将阳离子脂质体：转铁蛋白：血栓调节蛋白基因复合物或无DNA的载体应用于大鼠的下腔静脉。2天后，收获转染的静脉，并在暴露于人工循环之前和之后测定血栓调节蛋白表达和抗血栓形成特性。
使用不同剂量DNA进行基因转染的试验证实，尽管会伴随其他下游器官基因表达增加，但7.5微克的总DNA是向下腔静脉转染血栓调节蛋白基因的最有效量。通过向下腔静脉转染血栓调节蛋白基因，静脉内皮中活化蛋白C的生成能力与仅用载体处理的静脉相比增加了三倍（P<0.01）。在模拟动脉循环下，仅用载体处理的静脉灌注后血栓调节蛋白活性降至灌注前水平的36%（P<0.01），而转染的移植物即使在灌注后仍将活性维持在正常静脉内皮水平。因此，转染的静脉中由模拟动脉循环诱导的内皮凝血酶活性增加明显减弱（P<0.01），而免疫组织化学证实内皮细胞层得以保留。
转铁蛋白受体促进的体内基因转移至下腔静脉可在移植物植入后立即导致足够的血栓调节蛋白基因表达，并在暴露于动脉压力后维持抗血栓形成能力。尽管需要进一步研究，但目前的结果提示了针对静脉移植物疾病急性期进行基因治疗的可能性。
