Background Anti-proliferative drugs released from endo-vascular stents
have substantially contributed to reduce in-stent restenosis rates in coronary
arteries bearing single primary lesions by down-regulating coronary smooth
muscle cell (CSMC) growth. However, the considerably lower drug efficacy
shown in treatment of more complex coronary lesions suggests that alternative
anti-proliferative approaches can be beneficial. Thus, we explored the use of
hammerhead ribozymes as tools to knock down cyclin E and E2F1, two potent
activators of cell proliferation which cooperate to promote the G1 to S phase
transition.
Methods Two ribozymes, one directed against cyclin E and the other against
E2F1 mRNAs, were delivered by liposomes to cultured human CSMCs. The
influences on cell proliferation were measured evaluating BrdU incorporation
into newly synthesised DNA. The effects on cell cycle phase distribution were
determined by BrdU and 7-aminoactinomycin D incorporation into DNA.
Results Both ribozymes exhibited a sequence-specific and dose-dependent
reduction in BrdU incorporation, which, at a concentration of 280 nM,
persisted up to 4 days after transfection of CSMCs. A combined administration
of the two ribozymes (210 + 210 nM) resulted in a more pronounced decrease
in BrdU incorporation compared to the administration of an equimolar
amount (420 nM) of each of them. Finally, both ribozymes induced a
significant (P < 0.05) reduction in S phase cells with a concomitant increase
of G1/G0 and G2-M phase cells, compared to controls.
Conclusions The ribozymes selected represent potent tools to prevent CSMC
proliferation, especially when administered together, and thus are ideal
candidates for in vivo application.
