Sawa H, Sobel B E, Fujii S
Cardiovascular Division, Washington University, St. Louis, Missouri 63110.
J Biol Chem. 1994 May 13;269(19):14149-52.
We have hypothesized that type-1 plasminogen activator inhibitor (PAI-1) may exacerbate accumulation of extracellular matrix in atheroma by inhibiting local generation of plasmin and intramural proteolysis. Thus, suppression of PAI-1 expression would decrease atherogenesis. To inhibit expression of PAI-1 in cultured human umbilical vein endothelial and aortic smooth muscle cells, a 20-base antisense phosphorothioate oligonucleotide targeting specific sequences in the 3'-untranslated region of the PAI-1 gene was used. Studies with 32P-labeled oligomers verified stability in media. Secretion of PAI-1 protein assayed by enzyme-linked immunosorbent assay declined specifically and dose-dependently in cells exposed to the antisense oligonucleotide treated under basal conditions and after stimulation of PAI-1 expression with transforming growth factor beta (0.5 ng/ml for endothelial cell, 5 ng/ml for smooth muscle cell). Inhibition of expression was confirmed by immunoprecipitation of 35S-labeled PAI-1 and was paralleled by decreased steady-state levels of PAI-1 mRNA (Northern blots). Decreased PAI-1 synthesis was accompanied by augmentation of cell-associated plasmin activity. Thus, the antisense oligonucleotide down-regulated PAI-1 elaboration, an approach that may be useful in limiting obstructive vascular lesions.
我们推测，1型纤溶酶原激活物抑制剂（PAI-1）可能通过抑制纤溶酶的局部生成和壁内蛋白水解作用，加剧动脉粥样硬化斑块中细胞外基质的积聚。因此，抑制PAI-1的表达会减少动脉粥样硬化的发生。为了抑制培养的人脐静脉内皮细胞和主动脉平滑肌细胞中PAI-1的表达，使用了一种20个碱基的硫代磷酸反义寡核苷酸，该寡核苷酸靶向PAI-1基因3'-非翻译区的特定序列。对32P标记的寡聚物的研究证实了其在培养基中的稳定性。在基础条件下以及用转化生长因子β（内皮细胞为0.5 ng/ml，平滑肌细胞为5 ng/ml）刺激PAI-1表达后，通过酶联免疫吸附测定法检测，暴露于反义寡核苷酸的细胞中PAI-1蛋白的分泌特异性且剂量依赖性地下降。通过对35S标记的PAI-1进行免疫沉淀证实了表达受到抑制，同时PAI-1 mRNA的稳态水平也下降（Northern印迹法）。PAI-1合成的减少伴随着细胞相关纤溶酶活性的增强。因此，反义寡核苷酸下调了PAI-1的产生，这种方法可能有助于限制阻塞性血管病变。
