title: 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.
abstract: 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.
authors.affiliation: Department of Geriatric Medicine, Osaka University Medical School, Suita, Japan.
authors.forename: K
authors.initials: K
authors.lastname: Yamamoto
authors.forename: R
authors.initials: R
authors.lastname: Morishita
authors.forename: N
authors.initials: N
authors.lastname: Tomita
authors.forename: T
authors.initials: T
authors.lastname: Shimozato
authors.forename: H
authors.initials: H
authors.lastname: Nakagami
authors.forename: A
authors.initials: A
authors.lastname: Kikuchi
authors.forename: M
authors.initials: M
authors.lastname: Aoki
authors.forename: J
authors.initials: J
authors.lastname: Higaki
authors.forename: Y
authors.initials: Y
authors.lastname: Kaneda
authors.forename: T
authors.initials: T
authors.lastname: Ogihara
mesh_terms: 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
chemical_list: D009841:Oligonucleotides; D016337:RNA, Catalytic; D012333:RNA, Messenger; D016212:Transforming Growth Factor beta