Kopp C W, Grey S T, Siegel J B, McShea A, Vetr H, Wrighton C J, Schulte am Esch J, Bach F H, Robson S C
Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts 02215, USA.
Transplantation. 1998 Jul 27;66(2):244-51. doi: 10.1097/00007890-199807270-00019.
Xenograft rejection may predispose to vascular thrombosis because of putative cross-species' functional incompatibilities between natural anticoagulants present on the donor endothelium and host activated coagulation factors. For example, porcine thrombomodulin expressed on porcine aortic endothelial cells (PAEC) does not provide the expected thrombomodulin (TM)-cofactor activity for human protein C in the presence of human thrombin. In addition, TM may be down-regulated after cellular activation. Our aim was to express human TM cofactor activity in PAEC and to study the proinflammatory effect of tumor necrosis factor-alpha (TNF-alpha) on stable expressed human thrombomodulin in vitro.
Retroviral transduction of PAEC with the gene encoding for human thrombomodulin (hTM) resulted in expression of high levels of specific TM cofactor activity on PAEC (0.62 microg/ml activated protein C/10(5) cells). High-level expression of hTM resulted in a 620-fold higher activation of human protein C in the presence of human thrombin when compared with mock-transduced PAEC (0.0001 microg/ml/10(5) cells; P<0.001). Transduced PAEC expressing hTM also bound more human thrombin than control PAEC, as determined by inhibition of thrombin-induced platelet activation (P<0.05). We noted that exposure to TNF-alpha significantly reduced exogenous hTM cofactor activity on transduced PAEC in a time- and dose-dependent fashion; this occurred despite the relatively stable expression of hTM mRNA and hTM antigen in these cells. Treatment of transduced PAEC with selected antioxidants could protect against the loss of hTM cofactor activity directly associated with the oxidative stress induced by TNF-alpha activation responses.
Our data show that the functional deficiency of the anticoagulant protein C pathway in PAEC may be corrected by viral transduction of these cells. As analysis of the hTM function showed modulation under conditions of cellular activation, we suggest that expression of hTM mutants resistant to oxidation may have greater therapeutic utility in the genetic modification of porcine xenografts.
由于供体内皮细胞上的天然抗凝剂与宿主活化凝血因子之间存在假定的跨物种功能不相容性，异种移植排斥可能易导致血管血栓形成。例如，猪主动脉内皮细胞（PAEC）上表达的猪血栓调节蛋白在人凝血酶存在的情况下，不能为人类蛋白C提供预期的血栓调节蛋白（TM）辅因子活性。此外，细胞活化后TM可能会下调。我们的目的是在PAEC中表达人TM辅因子活性，并研究肿瘤坏死因子-α（TNF-α）对体外稳定表达的人血栓调节蛋白的促炎作用。
用编码人血栓调节蛋白（hTM）的基因对PAEC进行逆转录病毒转导，导致PAEC上高水平表达特异性TM辅因子活性（0.62微克/毫升活化蛋白C/10⁵个细胞）。与mock转导的PAEC相比（0.0001微克/毫升/10⁵个细胞；P<0.001），hTM的高水平表达在人凝血酶存在的情况下使人蛋白C的活化增加了620倍。通过抑制凝血酶诱导的血小板活化测定，表达hTM的转导PAEC也比对照PAEC结合更多的人凝血酶（P<0.05）。我们注意到，暴露于TNF-α以时间和剂量依赖的方式显著降低了转导PAEC上的外源性hTM辅因子活性；尽管这些细胞中hTM mRNA和hTM抗原表达相对稳定，但仍出现这种情况。用选定的抗氧化剂处理转导PAEC可以防止与TNF-α激活反应诱导的氧化应激直接相关的hTM辅因子活性丧失。
我们的数据表明，PAEC中抗凝蛋白C途径的功能缺陷可通过这些细胞的病毒转导得到纠正。由于对hTM功能的分析表明在细胞活化条件下其受到调节，我们建议表达抗氧化的hTM突变体在猪异种移植的基因改造中可能具有更大的治疗效用。
