Morishita R, Yamada S, Yamamoto K, Tomita N, Kida I, Sakurabayashi I, Kikuchi A, Kaneda Y, Lawn R, Higaki J, Ogihara T
Department of Geriatric Medicine, Osaka University Medical School Osaka, Japan.
Circulation. 1998 Nov 3;98(18):1898-904. doi: 10.1161/01.cir.98.18.1898.
Because mechanisms of atherosclerosis by lipoprotein(a) [Lp(a)] have been postulated in the decrease in active transforming growth factor-beta conversion by decreased plasmin, selective decrease in apolipoprotein(a) [apo(a)] independent of plasminogen may have therapeutic values. Although antisense can decrease apo(a), its application may be difficult because of very high homology of apo(a) gene to plasminogen. Thus we used ribozyme strategy that actively cleaves targeted genes to selectively inhibit apo(a) expression.
We constructed ribozyme oligonucleotides containing phosphorothioate DNA- and RNA-targeted kringle 4 of the apo(a) gene that showed 80% homology to plasminogen. Transfection of human apo(a) gene produced Lp(a) in medium of HepG2 cells, whereas Lp(a) could not be detected in control cells. Cotransfection of ribozyme and apo(a) gene resulted in the decrease in mRNA of apo(a) but not plasminogen. Moreover, marked decrease in Lp(a) was also observed in the medium transfected with ribozyme and apo(a) gene compared with apo(a) gene alone (P<0.01), whereas there was no significant change in plasminogen level between ribozyme-transfected and control cells. Incubation of human vascular smooth muscle cells (VSMC) with conditioned medium from apo(a)-transfected HepG2 cells resulted in a significant increase in VSMC number, whereas addition of conditioned medium from cells cotransfected with ribozyme oligonucleotides and apo(a) gene resulted in no VSMC growth (P<0.01). DNA-based control oligonucleotides and mismatched ribozyme oligonucleotides did not have an inhibitory effect on Lp(a) production.
Overall, our data revealed that transfection of ribozyme against the apo(a) gene resulted in the selective inhibition of the apo(a) but not the plasminogen gene, providing novel therapeutic strategy for treatment of high Lp(a), a risk factor for atherosclerosis.
由于脂蛋白(a)[Lp(a)]致动脉粥样硬化的机制被认为是通过纤溶酶减少导致活性转化生长因子-β转化减少，因此，独立于纤溶酶原的载脂蛋白(a)[apo(a)]选择性降低可能具有治疗价值。尽管反义技术可降低apo(a)水平，但由于apo(a)基因与纤溶酶原具有很高的同源性，其应用可能存在困难。因此，我们采用核酶策略，通过主动切割靶向基因来选择性抑制apo(a)表达。
我们构建了含有硫代磷酸酯DNA和RNA靶向apo(a)基因kringle 4的核酶寡核苷酸，其与纤溶酶原具有80%的同源性。在HepG2细胞培养基中，转染人apo(a)基因可产生Lp(a)，而对照细胞中未检测到Lp(a)。核酶与apo(a)基因共转染导致apo(a)的mRNA水平降低，但纤溶酶原的mRNA水平未降低。此外，与单独转染apo(a)基因相比，核酶与apo(a)基因共转染的培养基中Lp(a)水平显著降低(P<0.01)，而转染核酶的细胞与对照细胞之间纤溶酶原水平无显著变化。用人apo(a)转染的HepG2细胞的条件培养基孵育人血管平滑肌细胞(VSMC)，可导致VSMC数量显著增加，而添加核酶寡核苷酸与apo(a)基因共转染细胞的条件培养基则不会导致VSMC生长(P<0.01)。基于DNA的对照寡核苷酸和错配核酶寡核苷酸对Lp(a)的产生没有抑制作用。
总体而言，我们的数据表明，转染针对apo(a)基因的核酶可选择性抑制apo(a)基因而非纤溶酶原基因，为治疗动脉粥样硬化危险因素高Lp(a)提供了新的治疗策略。
