Growth inhibitory activity of indapamide on vascular smooth muscle cells

Abnormal vascular smooth muscle cell proliferation has a fundamental role in the pathogenesis of vascular diseases. Indapamide is an oral diuretic antihypertensive drug effective for patients with mild or moderate essential hypertension. We now investigated the effects of indapamide on the growth of aortic vascular smooth muscle cells (A10 cell line). Indapamide inhibited cell proliferation as measured by the tetrazolium salt XTT (sodium 3'-[1-(phenylamino-carbonyl)-3,4-tetrazolium]-bis(4-methoxy-6-nitro)benzene sulfonic acid hydrate) test. The increase in cell number was significantly reduced in the presence of indapamide 10(-6) and 5 x 10(-4) M (P < 0.05 n = 3 and P < 0.01, n = 3, respectively). Serum-induced DNA synthesis, determined as the incorporation of 5-bromo-2'-deoxyuridine (BrdU), was concentration-dependently inhibited by indapamide. BrdU incorporation was 47.2+/-1.6% (10% foetal calf serum). Indapamide treatment markedly prevented BrdU incorporation (37.2+/-2.1%, 29.2+/-4.8%, 15.0+/-1.8%, 8.7+/-2.1%) indapamide 10(-6), 10(-5), 5 x 10(-5) and 5 x 10(-4) M, respectively. Cell-cycle progression was also evaluated. Flow cytometry analysis of DNA content in synchronised cells revealed blocking of the serum-inducible cell-cycle progression by indapamide. This inhibition was abolished when the drug was added 2 h after serum repletion, indicating that indapamide must act at the early events of a cell cycle to be fully effective against DNA synthesis. In addition, serum-induced intracellular Ca2+ movements and also p44/p42 mitogen-activated protein kinase (MAPK) phosphorylation were studied in the presence or absence of indapamide. Indapamide 10(-5) and 5 x 10(-5) M decreased significantly cytosolic free calcium, and the p44/p42 mitogen-activated protein kinase phosphorylation (5 x 10(-5) M) stimulated by 10% foetal calf serum. In accordance with this finding, indapamide (5 x 10(-4) M) caused a 95% to 99% decrease in the early elevation of c-fos expression as evaluated by northern blot analysis of mRNA induced after serum addition. In conclusion, our results indicate that indapamide reduces vascular smooth muscle cell proliferation by a mechanism which involves a decrease in the intracellular Ca2+ movements that might link with the mitogen-activated protein kinase (MAPK) pathway, altering cell-cycle progression.