Novel Therapeutic Strategy for Restenosis Using Ribozyme Oligonucleotides Against Transforming Growth Factor-β
Background Since the mechanisms of atherosclerosis or restenosis after angioplasty have been postulated to involve an increase in TGF-β, a selective decrease in TGF-βmay have therapeutic value. Thus, we employed the ribozyme a unique class of RNA molecules that not only store information but also process catalytic activity, to selectively inhibit TGF-βexpression. Methods & Results We constructed ribozyme oligonucleotides targeted to the sequence of TGF-βgene which shows 100 % homology among human, rat and mouse. The specificity of ribozyme against TGF-βgene was confirmed by selective inhibition of TGF-βmRNA in cultured human VSMC as well as balloon-injured blood vessels in vivo. In vitro study Transfection of TGF-βribozyme ON into human VSMC by cationic liposome significantly decreased mRNA expression and protein of TGF-βinduced by Ang II as compared to control In vivo study Transfection of FITC-labeled ribozyme ON resulted in fluorescence in the balloon-injured vessels at 1 day after transfection. The fluorescence was localized primarily in cell nuclei , and persisted for up to 2 weeks after transfection.And then,transfection of TGF-βribozyme ON into rat cartid balloon injry model significantly decreased mRNA expression of TGF-βinduced by balloon injry ,and have significant inhibitory effects on neointimal formation after vascular injury(P<0.01), whereas DNA-based control oligonucleotides and mismatched ribozyme oligonucleotides 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 PCNA staining. Conclusion Overall, this study provides the first evidence that selective blockade of TGF-βby ribozyme resulted in inhibition of neointimal formation, accompanied by a reduction in collagen synthesis and DNA synthesis in a rat model. We anticipate that ribozyme ON pharmacokinetics will facilitate the potential clinical utility of the ribozyme strategy.