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(Circulation. 1996;93:772-780.)
© 1996 American Heart Association, Inc.

Articles

Processing of Chimeric Antisense Oligonucleotides by Human Vascular Smooth Muscle Cells and Human Atherosclerotic Plaque

Implications for Antisense Therapy of Restenosis After Angioplasty

J. Geoffrey Pickering, MD, PhD; Jeffrey M. Isner, MD; Carol M. Ford, MSc; Lawrence Weir, PhD; Andrew Lazarovits, MD; Edward F. Rocnik, BSc; Lawrence H. Chow, MD

From the John. P. Robarts Research Institute and University Hospital, Department of Medicine (Cardiology) and Biochemistry, University of Western Ontario, London, Canada (J.G.P., C.M.F., A.L., E.F.R., L.H.C.), and the Departments of Medicine (Cardiology), Pathology, and Biomedical Research, St Elizabeth's Medical Center, Tufts University School of Medicine, Boston, Mass (J.M.I., L.W.).

Correspondence to J. Geoffrey Pickering, University Hospital, 339 Windermere Rd, London, Ontario N6A 5A5, Canada. E-mail gpickrng@rri.uwo.ca.

Background Antisense oligonucleotides have been used in animals to inhibit the accumulation of vascular smooth muscle cells (VSMCs) after arterial injury. This has raised prospects for an oligonucleotide-mediated approach to prevent restenosis in patients undergoing angioplasty. However, little is known about the processing of oligonucleotides by human VSMCs or their bioavailability in human atherosclerotic tissue.

Methods and Results Oligonucleotides were synthesized with a mixture of unmodified and sulfur-modified linkages (S-chimeric oligonucleotides). These were more stable than unmodified oligonucleotides and could be recovered from within human VSMCs after 36 hours. Oligonucleotide antisense to human proliferating cell nuclear antigen mRNA specifically reduced DNA synthesis (P<.01) and proliferating cell nuclear antigen protein content (P<.05) in human VSMCs. Confocal microscopy of both live and fixed cells showed modest oligonucleotide uptake that was primarily nuclear. Surprisingly, cationic liposomes did not enhance nuclear uptake but led to extensive, punctated cytoplasmic loading without an enhanced antisense effect. Oligonucleotides incubated with human coronary atherosclerosis fragments associated with cells within 1 hour, despite the presence of abundant extracellular matrix.

Conclusions S-chimeric oligonucleotides are stable and can specifically inhibit gene expression in human VSMCs. Nuclear transport is a feature of oligonucleotide processing by human VSMCs, indicating a potential influence at the nuclear level rather than with cytoplasmic mRNA. Cationic liposomes increased oligonucleotide uptake but not intracellular bioavailability, and S-chimeric oligonucleotides can be incorporated into cells within human atherosclerotic plaque, despite the presence of a dense extracellular matrix.

Key Words: muscle, smooth • genes • angioplasty

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