A novel chimeric ribozyme vector produces potent inhibition of ICAM-1 expression on ischemic vascular endothelium.
BACKGROUND
Inhibition of intercellular adhesion molecule-1 (ICAM-1) expression can ameliorate the inflammation induced by ischemia-reperfusion injury (IRI) in animal models. However, current strategies to reduce ICAM-1 expression have been limited by the lack of stability, poor specificity, and the transient nature of synthesized regulatory molecules (antisense/ribozyme).


METHODS
A chimeric expression vector was generated by fusing a ribozyme targeting sequence against ICAM-1 to stabilizing stem-loop structures and nuclear localization signals that are components of endogenous U1 small nuclear RNA. Oligonucleotide scanning was used to predict accessible sites for targeting within the rat ICAM-1 transcript. Efficacy of the chimeric ribozyme vector was tested by transfection of rat aortic endothelial (RAE) cells (in vitro) and intraportal delivery in a rat hepatic IRI model (in vivo).


RESULTS
Transfection of RAE cells with the chimeric ribozyme vector produced potent and specific inhibition of ICAM-1 mRNA and protein levels by >65%. This reduction in ICAM-1 expression was accompanied by a proportional decrease in neutrophil adhesion to RAE cells. In vivo intraportal delivery of the chimeric targeting vector to rats sustaining hepatic IRI produced a marked reduction in ICAM-1 expression on liver endothelium after reperfusion.


CONCLUSIONS
A chimeric ribozyme vector effectively inhibited ICAM-1 expression in vascular endothelial cells and in rat liver following IRI, demonstrating a novel gene targeting technique that may be ideally suited to clinical applications aimed at ameliorating IRI.
Sonnenday
Christopher J
CJ
Department of Surgery, The Johns Hopkins University School of Medicine, Baltimore, MA, USA.
Warren
Daniel S
DS
Cooke
Sara K
SK
Dietz
Harry C
HC
Montgomery
Robert A
RA
D000818:Animals; D001011:Aorta / Q000166:cytology; D002448:Cell Adhesion; D004730:Endothelium, Vascular; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors; D006499:Hepatic Artery; D007249:Inflammation; D018799:Intercellular Adhesion Molecule-1 / Q000096:biosynthesis*; D008099:Liver / Q000166:cytology; D009504:Neutrophils; D016337:RNA, Catalytic; D012333:RNA, Messenger / Q000096:biosynthesis; D051381:Rats; D015427:Reperfusion Injury / Q000235:genetics* / Q000517:prevention & control* / Q000662:veterinary; D014158:Transcription, Genetic; D014162:Transfection; D019076:Transgenes
D016337:RNA, Catalytic; D012333:RNA, Messenger; D018799:Intercellular Adhesion Molecule-1