title: Processing of chimeric antisense oligonucleotides by human vascular smooth muscle cells and human atherosclerotic plaque. Implications for antisense therapy of restenosis after angioplasty.
abstract: 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.
  
  
  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.
authors.affiliation: John. P. Robarts Research Institute, University of Western Ontario, London, Canada. gpickrng@rri.uwo.ca
authors.forename: J G
authors.initials: JG
authors.lastname: Pickering
authors.forename: J M
authors.initials: JM
authors.lastname: Isner
authors.forename: C M
authors.initials: CM
authors.lastname: Ford
authors.forename: L
authors.initials: L
authors.lastname: Weir
authors.forename: A
authors.initials: A
authors.lastname: Lazarovits
authors.forename: E F
authors.initials: EF
authors.lastname: Rocnik
authors.forename: L H
authors.initials: LH
authors.lastname: Chow
mesh_terms: D017130:Angioplasty / Q000009:adverse effects; D000818:Animals; D001161:Arteriosclerosis / Q000235:genetics / Q000378:metabolism* / Q000628:therapy; D001483:Base Sequence; D002478:Cells, Cultured; D002678:Chimera; D004247:DNA / Q000096:biosynthesis; D004355:Drug Stability; D015316:Genetic Therapy; D006801:Humans; D008081:Liposomes; D008969:Molecular Sequence Data; D009131:Muscle, Smooth, Vascular / Q000378:metabolism*; D016376:Oligonucleotides, Antisense / Q000235:genetics / Q000378:metabolism* / Q000627:therapeutic use; D018809:Proliferating Cell Nuclear Antigen / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D012008:Recurrence; D013347:Subcellular Fractions / Q000378:metabolism
chemical_list: D008081:Liposomes; D016376:Oligonucleotides, Antisense; D018809:Proliferating Cell Nuclear Antigen; D012333:RNA, Messenger; D004247:DNA