Mimuro J, Muramatsu S, Hakamada Y, Mori K, Kikuchi J, Urabe M, Madoiwa S, Ozawa K, Sakata Y
Division of Cell and Molecular Medicine, Center for Molecular Medicine, Jichi Medical School, Tochigi-ken Japan.
Gene Ther. 2001 Nov;8(22):1690-7. doi: 10.1038/sj.gt.3301579.
We were able to facilitate plasminogen activator inhibitor 1 (PAI-1) promoter activity approximately by 14-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 TGFbeta 1 and TNFalpha stimulation. The characteristics of the enhanced PAI-1 promoter are 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 between 600- and 1000-fold higher than constitutive thrombomodulin gene expression in cultured human umbilical vein endothelial cells and was up-regulated by TGFbeta1 and TNFalpha stimulation which may down-regulate endogenous thrombomodulin gene expression in endothelial cells. The brain vascular endothelial cells of Mongolian gerbils could also be transduced by the same rAAV vector in vivo. Transduction of endothelial cells by rAAV vectors to express enhanced PAI-1 promoter-driven transgenes may be a useful gene therapy approach for vascular diseases.
我们能够使用一种增强子元件使纤溶酶原激活物抑制剂1（PAI-1）启动子活性提高约14倍。这种增强的PAI-1启动子具有很强的基础活性，与CAG启动子活性相当，并且在转化生长因子β1（TGFβ1）和肿瘤坏死因子α（TNFα）刺激方面具有与PAI-1启动子相似的反应。增强的PAI-1启动子的特性被认为适合血管细胞中抗凝分子的及时和组织特异性表达。因此，我们使用增强的PAI-1启动子开发了重组腺相关病毒（rAAV）载体，并成功地在体外将血管内皮细胞转导，使其在增强的PAI-1启动子调控下表达血栓调节蛋白转基因。在rAAV载体转导的培养内皮细胞中，由增强的PAI-1启动子驱动的血栓调节蛋白转基因表达比培养的人脐静脉内皮细胞中组成型血栓调节蛋白基因表达高600至1000倍，并且在TGFβ1和TNFα刺激下上调，而这可能会下调内皮细胞中的内源性血栓调节蛋白基因表达。蒙古沙鼠的脑血管内皮细胞在体内也可被相同的rAAV载体转导。通过rAAV载体转导内皮细胞以表达增强的PAI-1启动子驱动的转基因可能是一种用于血管疾病的有用基因治疗方法。
