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. METHODS AND 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.
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. METHODS AND 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: Taichi Sakaguchi; Shi Fang Yan; Shi Du Yan; Dmitri Belov; Ling Ling Rong; Monica Sousa; Martin Andrassy; Steven P Marso; Stephan Duda; Bernd Arnold; Birgit Liliensiek; Peter P Nawroth; David M Stern; Ann Marie Schmidt; Yoshifumi Naka Journal: J Clin Invest Date: 2003-04 Impact factor: 14.808
Authors: Mingyi Wang; Jing Zhang; Gaia Spinetti; Li-Qun Jiang; Robert Monticone; Di Zhao; Linda Cheng; Melissa Krawczyk; Mark Talan; Gianfranco Pintus; Edward G Lakatta Journal: Am J Pathol Date: 2005-11 Impact factor: 4.307