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(Circulation. 1998;98:1898-1904.)
© 1998 American Heart Association, Inc.

Basic Science Reports

Novel Therapeutic Strategy for Atherosclerosis

Ribozyme Oligonucleotides Against Apolipoprotein(a) Selectively Inhibit Apolipoprotein(a) But Not Plasminogen Gene Expression

Ryuichi Morishita, MD, PhD; Shingo Yamada, PhD; Kei Yamamoto, MD; Naruya Tomita, MD, PhD; Iwao Kida, BS; Ikunosuke Sakurabayashi, MD, PhD; Akira Kikuchi, PhD; Yasufumi Kaneda, MD, PhD; Richard Lawn, PhD; Jitsuo Higaki, MD, PhD; ; Toshio Ogihara, MD, PhD

From the Department of Geriatric Medicine, Osaka University Medical School (R.M., K.Y., N.T., I.K., J.H., T.O.), Osaka, Japan; Shinotest Co Ltd, Shizuoka, Japan (S.Y.); Omiya Medical Center, Jichi University Medical School, Saitama, Japan (I.S.); Toyohashi Technology University, Toyohashi, Japan (A.K.); Institute for Molecular and Cellular Biology, Osaka University Medical School (Y.K.), Osaka, Japan; and the Division of Cardiovascular Medicine, Falk Cardiovascular Research Center, Stanford University, School of Medicine, Stanford, Calif (R.L.).

Correspondence to Jitsuo Higaki, MD, PhD, Department of Geriatric Medicine, Osaka University Medical School, Suita 565, Japan.

Background—Because mechanisms of atherosclerosis by lipoprotein(a) [Lp(a)] have been postulated in the decrease in active transforming growth factor-ß 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.

Methods and Results—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.

Conclusions—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.

Key Words: atherosclerosis • lipoproteins • vasculature • genes • muscle, smooth

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