Inhibition of vascular smooth muscle cell proliferation by DNA-RNA chimeric hammerhead ribozyme targeting to rat platelet-derived growth factor A-chain mRNA.
BACKGROUND
Spontaneously hypertensive rats (SHR)-derived vascular smooth muscle cells (VSMC) show exaggerated growth and increasingly express platelet-derived growth factor (PDGF) A-chain mRNA compared to VSMC from normotensive Wistar-Kyoto (WKY) rats.


OBJECTIVE
To investigate the effects of designed DNA-RNA chimeric hammerhead ribozyme to rat PDGF A-chain on exaggerated growth of VSMC from SHR.


METHODS
We designed and synthesized a 38-base DNA-RNA chimeric hammerhead ribozyme with two phosphorothioate linkages at the 3' terminal to cleave rat PDGF A-chain mRNA at the GUC sequence at nucleotide 921. We confirmed the cleavage activity of designed ribozyme by in vitro cleavage reaction and by lipofectin-mediated transfection of ribozyme into VSMC.


RESULTS
Doses of 0.1 and 1 micromol/l DNA-RNA chimeric ribozyme dose-dependently inhibited basal DNA synthesis in VSMC from SHR. A dose of 1 micromol/l DNA-RNA chimeric ribozyme time-dependently inhibited basal DNA synthesis in VSMC from SHR. However, the same doses of all-RNA ribozyme had no effects on DNA synthesis in VSMC from SHR. Fluorescein isothiocyanate-labeled DNA-RNA chimeric ribozyme was recognized in cytosol at 30 min, and in nucleus at 60 min after lipofectin transfection. A dose of 1 micromol/l DNA-RNA chimeric ribozyme significantly inhibited expressions of both PDGF A-chain mRNA and PDGF-AA protein in VSMC from SHR, but not from WKY rats.


CONCLUSIONS
These results indicated that the designed DNA-RNA chimeric ribozyme to PDGF A-chain mRNA effectively and specifically inhibited the exaggerated growth of VSMC from SHR at low concentrations, which were mediated by the reduction of PDGF A-chain mRNA and PDGF-AA protein expressions.
Hu
W Y
WY
Second Department of Internal Medicine, Nihon University School of Medicine, Tokyo, Japan.
Fukuda
N
N
Nakayama
M
M
Kishioka
H
H
Kanmatsuse
K
K
D000818:Animals; D001483:Base Sequence; D002455:Cell Division / Q000187:drug effects; D002478:Cells, Cultured; D004247:DNA / Q000096:biosynthesis; D006973:Hypertension / Q000628:therapy*; D008969:Molecular Sequence Data; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000187:drug effects*; D010982:Platelet-Derived Growth Factor / Q000096:biosynthesis / Q000235:genetics*; D016337:RNA, Catalytic / Q000494:pharmacology*; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D011918:Rats, Inbred SHR; D011921:Rats, Inbred WKY
D010982:Platelet-Derived Growth Factor; D016337:RNA, Catalytic; D012333:RNA, Messenger; D004247:DNA