Ribozyme oligonucleotides against transforming growth factor-beta inhibited neointimal formation after vascular injury in rat model: potential application of ribozyme strategy to treat cardiovascular disease.
BACKGROUND
Because the mechanisms of atherosclerosis or restenosis after angioplasty have been postulated to involve an increase in transforming growth factor (TGF)-beta, a selective decrease in TGF-beta may have therapeutic value. Thus, we used the ribozyme strategy to actively cleave the targeted gene to selectively inhibit TGF-beta expression.


RESULTS
We constructed ribozyme oligonucleotides (ONs) targeted to the sequence of the TGF-beta gene that shows 100% homology among the human, rat, and mouse species. The specificity of ribozyme against TGF-beta gene was confirmed by selective inhibition of TGF-beta mRNA in cultured vascular smooth muscle cells as well as balloon-injured blood vessels in vivo. Importantly, the marked decrease in TGF-beta resulted in significant inhibition of neointimal formation after vascular injury in a rat carotid artery model (P:<0.01), whereas DNA-based control ONs and mismatched ribozyme ONs did not have any inhibitory effect on neointimal formation. Inhibition of neointimal formation was accompanied by (1) a reduction in collagen synthesis and mRNA expression of collagen I and III and (2) a significant decrease in DNA synthesis as assessed by proliferating cell nuclear antigen staining. Moreover, we modified ribozyme ONs containing phosphorothioate DNA and RNA targeted to the TGF-beta gene. Of importance, modified ribozyme ONs showed a further reduction in TGF-beta expression.


CONCLUSIONS
Overall, this study provides the first evidence that selective blockade of TGF-beta resulted in inhibition of neointimal formation, accompanied by a reduction in collagen synthesis and DNA synthesis in a rat model. We anticipate that modification of ribozyme ON pharmacokinetics will facilitate the potential clinical utility of the ribozyme strategy.
Yamamoto
K
K
Department of Geriatric Medicine, Osaka University Medical School, Suita, Japan.
Morishita
R
R
Tomita
N
N
Shimozato
T
T
Nakagami
H
H
Kikuchi
A
A
Aoki
M
M
Higaki
J
J
Kaneda
Y
Y
Ogihara
T
T
D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D002340:Carotid Artery Diseases / Q000378:metabolism / Q000473:pathology / Q000628:therapy*; D002404:Catheterization / Q000009:adverse effects; D018014:Gene Transfer Techniques; D006801:Humans; D007150:Immunohistochemistry; D066298:In Vitro Techniques; D008297:Male; D051379:Mice; D008856:Microscopy, Fluorescence; D009841:Oligonucleotides / Q000737:chemistry / Q000235:genetics*; D016337:RNA, Catalytic / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000037:antagonists & inhibitors / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D014162:Transfection; D016212:Transforming Growth Factor beta / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D017540:Tunica Media / Q000378:metabolism* / Q000473:pathology
D009841:Oligonucleotides; D016337:RNA, Catalytic; D012333:RNA, Messenger; D016212:Transforming Growth Factor beta