Abstract 692: Development of the Gene Therapy With CRE Decoy ODN to Prevent Vascular Intimal Hyperplasia

Abstract

Background: Intimal hyperplasia (IH) is the main cause of vein graft stenosis or failure after bypass surgery. However, in the previous study, no therapeutic targets for the treatment of IH have been identified. Our recent research have been reported that the inhibition of Cyclic adenosine monophosphate response-element (CRE) binding protein (CREB) activation is a key role for vein graft IH. Objective: In consideration of future clinical application, we focused on decoy oligodeoxynucleotide (ODN) transfection as gene therapy strategy for suppressing IH. The goal of the present study is to identify whether the CRE decoy ODN had the therapeutic efficacy for suppressing IH. Methods: We developed phosphorothioate CRE decoy ODN and checked binding ability to a CRE sequence using CREB transcription assay . We chose a decoy ODN having high first-binding ability and transfected it to vascular smooth muscle cell(VSMC) in vitro. Proliferation was assessed using MTS assay and migration was assessed using a modified Boyden-chamber assay. We examined CRE activity using Luciferase reporter gene assay. We checked expression of mRNAs using qRT-PCR. In wire-injury mouse model(C57BL6,n=6), CRE decoy ODN was transfected to injured vessel wall using ultrasound-sonoporation method in vivo. Results: CREB transcription factor assay showed CRE decoy had about 80 folds binding ability of control decoy in IC50 value (vs.control,n=5). Transfer of CRE decoy ODN resulted in significantly downregulated CRE activity(vs.control,P<.01,n=12) and inhibited VSMC proliferation (vs.control,P<.01,n=12) and migration (vs.control,P<.01,n=5). In a wire-injury mouse model, histopathological analysis revealed that transfer of the CRE decoy ODN significantly repressed IH whereas intensive IH was observed in the control group. In this vessel tissue, Levels of mRNA CCNA2 and Bcl2 were significantly lower in CRE decoy group than in control group(n=6,P<.01,respectively). CRE-activated gene expression was strongly repressed. Conclusions: The present result suggested that CRE decoy ODN inhibiting CREB function provide an effective therapeutic approach to suppressing IH.