Chimeric DNA-RNA hammerhead ribozyme targeting PDGF A-chain mRNA specifically inhibits neointima formation in rat carotid artery after balloon injury.
OBJECTIVE
Restenosis of the coronary artery after percutaneous transluminal coronary angioplasty (PTCA) occurs in 30-50% of patients and remains a major clinical problem. We developed ribozyme that targets platelet-derived growth factor (PDGF) A-chain mRNA as a gene therapy for restenosis after PTCA. Thus, we examined the effects of a chimeric DNA-RNA ribozyme targeting PDGF A-chain mRNA on neointima formation in rat carotid artery after balloon injury and evaluated its specificity for PDGF A-chain mRNA by microarray analysis.


METHODS
Rat carotid artery was injured with a 2F Fogarty catheter, and PDGF A-chain specific ribozyme was delivered to the injured artery with polyethylenimine. Two weeks after injury, the artery was removed, and the intima/media (I/M) ratio was evaluated. Six hours after injury, mRNA was extracted with oligo dT cellulose, and expression of PDGF A-chain mRNA was evaluated by reverse transcription-polymerase chain reaction. Expression of PDGF-AA protein was evaluated by Western blot analysis. Expression of 970 genes was evaluated by microarray (GeneChip, Affimetrix Inc).


RESULTS
FITC-labeled ribozyme was taken up into the midlayer smooth muscle of the carotid artery until 24 h after balloon injury. Two and 5 microg of ribozyme significantly reduced neointima formation by 44 and 55% of control levels, respectively, in a dose-dependent manner. Ribozyme markedly inhibited expression of PDGF A-chain mRNA as well as production of PDGF-AA protein in injured vessels. Microarray analysis revealed that expression of 525 genes was increased after balloon injury. These genes included FLK-1, interleukin-1 receptor, retinoic acid receptor alpha2 isoform, heat shock protein, MAP kinase kinase, Fas antigen, G6Pase, PI-5-P-kinase, p38 MAP kinase, proliferating cell nuclear antigen, transforming growth factor-beta, extracellular signal-related kinase, and fibroblast growth factor receptor. With respect to expression of cytokine and growth factor mRNAs, the best ribozyme specifically inhibited expression of PDGF A-chain mRNA.


CONCLUSIONS
Our chimeric DNA-RNA hammerhead ribozyme targeting PDGF A-chain mRNA inhibited neointima formation in rat carotid artery after balloon injury with specific inhibition of expression of PDGF A-chain mRNA, suggesting that this ribozyme may be useful for therapy of restenosis of coronary artery after PTCA.
Kotani
Motoko
M
Second Department of Internal Medicine, Nihon University School of Medicine, Tokyo 173-8610, Japan.
Fukuda
Noboru
N
Ando
Hideyuki
H
Hu
Wen-Yang
WY
Kunimoto
Satoshi
S
Saito
Satoshi
S
Kanmatsuse
Katsuo
K
D000818:Animals; D002339:Carotid Arteries / Q000473:pathology*; D016893:Carotid Stenosis / Q000209:etiology* / Q000473:pathology / Q000628:therapy; D002404:Catheterization / Q000009:adverse effects*; D015870:Gene Expression; D015316:Genetic Therapy / Q000379:methods*; D006133:Growth Substances / Q000235:genetics; D008297:Male; D020411:Oligonucleotide Array Sequence Analysis; D010982:Platelet-Derived Growth Factor / Q000032:analysis / Q000235:genetics*; D016337:RNA, Catalytic / Q000494:pharmacology*; D051381:Rats; D017208:Rats, Wistar; D014162:Transfection / Q000379:methods; D017539:Tunica Intima / Q000473:pathology*
D006133:Growth Substances; D010982:Platelet-Derived Growth Factor; D016337:RNA, Catalytic; C075278:platelet-derived growth factor A