Regulation of endothelial cell functions by gene transfer using viral vectors

我们可以使用增强子元件将纤溶酶原激活物抑制物1(PAI-1)的启动子活性提高约20倍。这种增强的PAI-1启动子具有很强的基础活性，与CAg启动子的活性相当，并且在转化生长因子β1和肿瘤坏死因子α刺激方面的反应类似于PAI-1启动子。增强的PAI-1启动子的特性被认为适合于在血管细胞中及时和组织特异性地表达抗凝剂分子。因此，我们构建了含有增强型PAI-1启动子的重组腺相关病毒载体，并在体外成功地转导血管内皮细胞在增强型PAI-1启动子调控下表达血栓调节蛋白转基因。在重组腺病毒载体转导的培养内皮细胞中，由增强型PAI1启动子驱动的血栓调节蛋白转基因表达是原生型血栓调节蛋白基因表达的6 0 0~10 0 0倍，并被转化生长因子β1和肿瘤坏死因子α刺激上调，从而可能下调内源性血栓调节蛋白基因的表达。同样的rAAV载体也可以在体内转导沙土鼠脑血管内皮细胞。RAAV载体转化内皮细胞表达增强型PAI-1启动子驱动的转基因可能是治疗血管疾病的一种有用的基因治疗方法。

We could facilitate plasminogen activator inhibitor 1 (PAI-1) promoter activity approximately by 20-fold using an enhancer element. This enhanced PAI- 1 promoter has a strong basal activity, comparable to CAG promoter activity, and has a response similar to the PAI-1 promoter with respect to TGF β1 and TNF α stimulation. The characteristic of the enhanced PAI-1 promoter is thought to be suited to timely and tissue specific expression of anticoagulant molecules in the vascular cells. Thus, we developed recombinant adeno-associated virus (rAAV) vectors using the enhanced PAI-1 promoter and were successful in transducing vascular endothelial cells to express the thrombomodulin transgene under the regulation of the enhanced PAI-1 promoter in vitro. Thromobomodulin transgene expression driven by the enhanced PAI- 1 promoter in rAAV vector-transduced cultured endothelial cells was 600〜1000-fold higher than constitutive thrombomodulin gene expression in cultured human umbilical vein endothelial cells and was up-regulated by TGFβ1 and TNFα stimulation which may down-regulate endogenous thrombomodulin gene expression in endothelial cells. The brain vascular endothelial cells of Mongolian gerbils also could be transduced by the same rAAV vector in vivo. Transition of endothelial cells by rAAV vectors to express enhanced PAI- 1 promoter-driven transgenes may be a useful gene therapy approach for vascular diseases.