1. In the present study we examined the effects of
PCA-4230, a novel
antithrombotic agent, on the growth of cultured
A10 vascular smooth muscle cells (rat'aorta). 2. The action of
PCA-4230 on cell proliferation and on serum-induced
DNA synthesis was determined by measuring the cell number and the incorporation of the
thymidine analogue
5-bromo-2'-deoxyuridine (
BrdU), respectively. 3.
PCA-4230 reversibly inhibited vascular smooth muscle cell proliferation. The increase in cell number was significantly reduced in the presence of 1 and 50 microM
PCA-4230. 4.
DNA synthesis was concentration-dependently inhibited by
PCA-4230 (0.5 to 50 microM) in
A10 cells that were synchronized by 48 h serum
starvation and then re-stimulated by serum repletion, with an IC50 value of 13 microM. However, serum-induced
DNA synthesis in bovine aortic endothelial cells was not significantly affected by
PCA-4230. In addition,
PCA-4230 (50 microM) caused a significant drop in
PDGF-BB-mediated
BrdU incorporation in
A10 cells. 5. The effect of
PCA-4230 on serum-induced
DNA synthesis was compared to that elicited by
nifedipine, another
dihydropyridine-class inhibitor of vascular smooth muscle proliferation.
PCA-4230 (10 microM) elicited a degree of inhibition similar to that of
nifedipine at equimolar concentration. 6. To define the nature of the cell proliferation inhibition, an evaluation of cell cycle progression was undertaken. Flow cytometry studies of
DNA content in synchronized cells revealed a block of the serum-inducible cell cycle progression. This inhibitory effect was markedly reduced when
PCA-4230 was added 2 h after serum repletion. 7. Accordingly,
PCA-4230 (50 microM) caused a 95 and 90% decrease in the elevation of c-fos and c-jun proto-oncogenes expression as evaluated by Northern blot analysis of
mRNA induced early after serum addition. 8. The present results indicate that
PCA-4230 inhibits vascular smooth muscle cell proliferation, in culture, by altering the cell cycle progression. Flow cytometric studies of
DNA content and the down regulation of c-fos and c-jun proto-oncogenes, suggest that the
drug is acting at the early G0/G1 transition phase.
PCA-4230 may hold promising potential for the prevention of structural abnormalities of blood vessels associated with
atherosclerosis and
vascular diseases.