Buczko W, Cierniewski C, Kobylańska A, Koziołkiewicz M, Okruszek A, Pawłowska Z, Pluskota E, Stec W J
Medical University of Białystok, Department of Pharmacodynamics, Poland.
Pharmacol Ther. 1997 Oct-Dec;76(1-3):161-75. doi: 10.1016/s0163-7258(97)00091-0.
Oligonucleotides with a nucleotide sequence complementary to various regions of human plasminogen activator inhibitor type-1 (PAI-1) mRNA have been studied as antisense inhibitors of expression of PAI-1 protein in cultured cells [human umbilical vein endothelial cells (HUVEC), human aortic smooth muscle cells, human hybrid endothelial cells]. Hexadeca(deoxyribonucleoside phosphorothioate) 13 complementary to a fragment of a signal peptide PAI-1 mRNA was found to be most active, giving ca. 70% inhibition of PAI-1 release in a time- and dose-dependent way. The stereo-regular All-S(P) and All-R(P) diastereomers of 13 were studied and found to inhibit PAI-1 synthesis in HUVEC in a stereo-dependent manner, with the All-S(P) diastereomer considerably more active than the stereo-random construct and All-R(P) isomer. The observed stereo-dependent activity of oligonucleotide phosphorothioate constructs is presumably governed by their resistance to nucleases. The corresponding phosphodiester analogue of 13 was not active unless covalently bound at its 5'-end to a lipophilic alcohol residue (menthol, heptadecanol). The observed antisense activity of phosphodiester oligonucleotide bioconjugates in cultured human hybrid endothelial cells was paralleled by their increased stability in human plasma with respect to unconjugated oligonucleotide. The oligo(deoxyribonucleoside phosphorothioate) complementary to the same signal peptide region of rat PAI-1 mRNA was found to reduce the PAI-1 level in blood plasma of rats after intravenous administration into the tail vein. The effect was both time- and dose-dependent. The same oligonucleotide was found to protect against arterial thrombus formation in the rat (lower incidence of venous thrombosis, lower thrombus weight, and increased occlusion time in experimentally induced thrombosis). An anti-PAI-1 inhibitory activity has been independently reported for a 20-mer oligo(2'-O-methyl-ribonucleoside phosphorothioate) complementary to a 3'-untranslated region of human PAI-1 mRNA in cultured HUVEC and human aortic smooth muscle cells.
与人类1型纤溶酶原激活物抑制剂（PAI-1）mRNA不同区域具有互补核苷酸序列的寡核苷酸，已作为PAI-1蛋白在培养细胞（人脐静脉内皮细胞（HUVEC）、人主动脉平滑肌细胞、人杂交内皮细胞）中表达的反义抑制剂进行了研究。发现与PAI-1 mRNA信号肽片段互补的十六（脱氧核糖核苷硫代磷酸酯）13活性最高，能以时间和剂量依赖的方式抑制约70%的PAI-1释放。对13的立体规则全S(P)和全R(P)非对映异构体进行了研究，发现它们以立体依赖的方式抑制HUVEC中的PAI-1合成，全S(P)非对映异构体的活性明显高于立体随机构建体和全R(P)异构体。寡核苷酸硫代磷酸酯构建体观察到的立体依赖活性可能受其对核酸酶的抗性支配。13的相应磷酸二酯类似物没有活性，除非在其5'-末端与亲脂性醇残基（薄荷醇、十七烷醇）共价结合。磷酸二酯寡核苷酸生物共轭物在培养的人杂交内皮细胞中观察到的反义活性，与其在人血浆中相对于未共轭寡核苷酸的稳定性增加相平行。发现与大鼠PAI-1 mRNA相同信号肽区域互补的寡（脱氧核糖核苷硫代磷酸酯）在尾静脉内注射后可降低大鼠血浆中的PAI-1水平。该效应具有时间和剂量依赖性。发现相同的寡核苷酸可预防大鼠动脉血栓形成（静脉血栓形成发生率降低、血栓重量降低以及实验性诱导血栓形成的阻塞时间增加）。已独立报道了一种与培养的HUVEC和人主动脉平滑肌细胞中人类PAI-1 mRNA的3'-非翻译区域互补的20聚体寡（2'-O-甲基-核糖核苷硫代磷酸酯）具有抗PAI-1抑制活性。
