Development of gene therapy with a cyclic adenosine monophosphate response element decoy oligodeoxynucleotide to prevent vascular intimal hyperplasia

Abstract Objective Intimal hyperplasia(IH) is the main cause of therapeutic failure after vascular and endovascular surgery. However, there is currently no targeted therapy for the treatment of IH. We recently reported that the inhibition of cyclic adenosine monophosphateresponse element (CRE) binding protein ( CREB) activation is important in vein graft IH. We focused on a decoyoligodeoxynucleotide (ODN) therapeutic strategy for suppressing IH as a clinical application. The objective of this study was to confirm the therapeutic effect of a CRE decoyODN in an animal model as a novel therapy for preventing intimal hyperplasiaas the first step of the preclinical study of our strategy. Methods We designed two phosphorothioate CREs and two scramble decoyODNs and screened them using a CREBtranscription assay to check their ability to bind to a CRE sequence. We chose a CRE decoyODN with high first-binding ability and transfected it into vascular smooth muscle cells (VSMCs) in vitro. Proliferation and migration were assessed using MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) assays and modified Boyden chamber assays. We examined CRE activity using a luciferase reporter gene assay. We assessed the expression of messenger RNAs by quantitative real-time polymerase chain reaction. In a wire-injury mouse model (C57BL6, n = 6), CRE decoyODN was transfected into the injured vessel wall using an ultrasound- sonoporationmethod in vivo. Mitogen-activated protein kinase-activated protein kinase 3 ( MAPKAPK3 ) and four and a half LIM domains5 ( FHL5 ) expression of pregrafting vein remnants were assessed by immunohistologic analyses. Results Compared with scramble decoyODN, the selected CRE decoyODN could significantly decrease CRE activity (mean ± standard error of the mean: 0.20 ± 0.03 vs 1.00 ± 0.16, n = 6; P P P CRE decoyODN significantly suppressed the formation of IH at injured vessel walls in an animal model, as analyzed by pathologic staining (0.20 ± 0.02 vs 0.56 ± 0.08, area of the intima/area of the artery vs the control after 21 days’ transfection, n = 6; P Conclusions CREB-CRE signaling is an important mechanism of IH formation, and CRE decoytherapy can help preventing IH. This study is the first part of the preclinical study of our strategy.