Ribozyme Oligonucleotides Against Transforming Growth Factor-β Inhibited Neointimal Formation After Vascular Injury in Rat Model

Abstract

Background —Because the mechanisms of atherosclerosis or restenosis after angioplasty have been postulated to involve an increase in transforming growth factor (TGF)-β, a selective decrease in TGF-β may have therapeutic value. Thus, we used the ribozyme strategy to actively cleave the targeted gene to selectively inhibit TGF-β expression. Methods and Results —We constructed ribozyme oligonucleotides (ONs) targeted to the sequence of the TGF-β gene that shows 100% homology among the human, rat, and mouse species. The specificity of ribozyme against TGF-β gene was confirmed by selective inhibition of TGF-β mRNA in cultured vascular smooth muscle cells as well as balloon-injured blood vessels in vivo. Importantly, the marked decrease in TGF-β 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-β gene. Of importance, modified ribozyme ONs showed a further reduction in TGF-β expression. Conclusions —Overall, this study provides the first evidence that selective blockade of TGF-β 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.