Effects of Antisense c- myb Oligonucleotides on Vascular Smooth Muscle Cell Proliferation and Response to Vessel Wall Injury

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Abstract
The process of restenosis after arterial balloon dilatation has been demonstrated to involve smooth muscle cell hyperplasia. Initial reports with antisense oligonucleotides directed against the proto-oncogene c- myb suggest marked in vitro specificity and in vivo efficacy. In the present study, we sought to confirm and extend the hypothesis that antisense to c- myb results in a specific antiproliferative effect with a comprehensive assessment by using different oligonucleotide preparations, different species, and tissue and cellular uptake experiments. Phosphorothioate-protected oligonucleotides representing the appropriate sequence for antisense to c- myb and multiple controls were used to inhibit proliferation of platelet-derived growth factor– and fetal bovine serum–stimulated rat, dog, and human aortic smooth muscle cells in vitro and neointimal proliferation in the rat carotid injury model. In vitro experiments using identical culture conditions in rat, dog, and human aortic smooth muscle cells failed to show specificity as well as consistency in growth inhibitory effects that could be attributed to an antisense mechanism. Proliferation of smooth muscle cell growth in culture was consistently inhibited with oligomers containing a contiguous 4-guanosine residue motif. In vivo, the rat carotid injury neointimal hyperplasia was similar for antisense c- myb (0.095±0.009 mm 2 ) and sense c- myb (0.090±0.009 mm 2 ). Fluorescent-labeled oligonucleotides were present in tissue after local delivery via pluronic gel, and their activity rapidly declined over a 72-hour period. Our findings point to the potential nonspecificity and lack of consistency of the antisense oligonucleotide to c- myb in vitro and in vivo. An alternative nonantisense mechanism for the inhibition of smooth muscle cell proliferation, involving contiguous 4-guanosine residues, is proposed.

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