The effect of oligonucleotides to c-myb on vascular smooth muscle cell proliferation and neointima formation after porcine coronary angioplasty.
Proto-oncogenes, including c-myb, are expressed early after vascular injury. The application of antisense oligodeoxynucleotides (AS-ODNs) against these genes inhibits cell proliferation and neointima formation in small animals and in peripheral arteries. The aim of this study was to investigate the specificity of action of AS-ODN-c-myb in vitro and to assess its effect, when delivered locally, on neointima formation after percutaneous transluminal coronary angioplasty (PTCA) in porcine coronary arteries. AS-ODN-c-myb inhibited the proliferation of vascular smooth muscle cells (VSMCs) in vitro in a dose-dependent manner. There was a corresponding reduction in steady state levels of c-myb mRNA and protein. Expression of another early gene, c-fos, was unaffected. S1 nuclease analysis demonstrated intact full-length AS-ODN-c-myb retrieved from VSMCs in culture after 12 hours. A range of ODNs, related and unrelated to c-myb, with and without a GGGG sequence, inhibited VSMC proliferation. Phosphorothioated AS-ODN-c-myb was 30 times less potent than unphosphorothioated AS-ODN-c-myb. PTCA induced porcine coronary artery neointima formation. c-myb mRNA was maximally induced 18 hours after injury. Unmodified AS-ODN-c-myb, sense-ODN-c-myb, saline, or nothing was delivered immediately after balloon dilatation via a double-skinned porous balloon (Transport, SciMed). Fluorescence-labeled AS-ODN-c-myb was deposited throughout the vessel wall. Mean maximum intima/media cross-sectional area 4 weeks after PTCA was reduced with AS-ODN-c-myb by 79% compared with saline (P < .05), 82% compared with sense-ODN-c-myb, and 63% compared with nothing (P < .10). Conclusions are as follows: (1) c-myb is expressed in VSMCs after vascular injury. (2) AS-ODN-c-myb is retained intact in VSMCs, reducing their proliferation in vitro in dose-dependent fashion, with reduction in c-myb mRNA and protein, whereas sense-ODN-c-myb is not. (3) A range of ODNs can reduce VSMC proliferation by a non-sequence-specific mechanism. (4) Phosphorothioate protection of antisense molecules may reduce their efficacy. (5) Local delivery of unmodified AS-ODN-c-myb via the Transport catheter reduces neointima formation after porcine PTCA. (6) Local delivery of fluid may exacerbate neointimal thickening.
Gunn
J
J
Section of Cardiology, University of Sheffield, Northern General Hospital, UK. J.Gunn@Sheffield.ac.uk
Holt
C M
CM
Francis
S E
SE
Shepherd
L
L
Grohmann
M
M
Newman
C M
CM
Crossman
D C
DC
Cumberland
D C
DC
D000800:Angioplasty, Balloon / Q000009:adverse effects*; D015906:Angioplasty, Balloon, Coronary / Q000379:methods; D000818:Animals; D002417:Cattle; D002455:Cell Division; D002478:Cells, Cultured; D003331:Coronary Vessels / Q000378:metabolism / Q000502:physiology*; D019085:Fluorescent Antibody Technique, Direct; D016762:Genes, fos / Q000502:physiology; D006801:Humans; D009131:Muscle, Smooth, Vascular / Q000378:metabolism / Q000502:physiology*; D016376:Oligonucleotides, Antisense / Q000494:pharmacology; D009857:Oncogenes / Q000502:physiology*; D012333:RNA, Messenger / Q000378:metabolism; D016601:RNA-Binding Proteins; D013552:Swine
D016376:Oligonucleotides, Antisense; D012333:RNA, Messenger; D016601:RNA-Binding Proteins