Dipyridamole inhibits PDGF-stimulated human peritoneal mesothelial cell proliferation

Abstract

Background. It has been proposed that proliferation of human peritoneal mesothelial cells (HPMCs) accompanied by collagen synthesis may contribute to the development of peritoneal fibrosis (PF) in patients of long-term continuous ambulatory peritoneal dialysis (CAPD). However, the precise molecular mechanism regulating HPMC proliferation has never been reported. Dipyridamole has been reported to have potential as an antiproliferative and antifibrotic agent. We investigated the mechanism and effect of dipyridamole in regulation of HPMC proliferation. Methods. HPMCs were cultured from human omentum by an enzyme digestion method. Cell proliferation was measured by the methyltetrazolium assay and intracellular cAMP was measured using an enzyme immunoassay kit. Cell-cycle distribution of HPMC was analyzed by flow cytometry. Extracellular signal-regulated protein kinase (p44/p42 ERK) activity and expressions of cell-cycle proteins (cyclin D1, CDK4, pRB and p27Kip1) were determined by Western blotting. Results. The addition of DP suppressed PDGF-stimulated HPMC proliferation by cell-cycle arrest at the G1 phase. The antimitogenic effect of dipyridamole was mediated through the cAMP pathway. PDGF (25 ng/mL) increased the ERK1/2 activity of HPMC within 15 minutes, which maximized at 30 minutes, and the pretreatment with dipyridamole (17 μg/mL) substantially reduced the ERK response to PDGF by approximately 78.5%. PDGF induced elevated protein levels of cyclin D1, but the CDK4 protein level did not change. Dipyridamole and DBcAMP had no effect on the levels of cyclin D1 and CDK4 in PDGF- stimulated HPMC. PDGF decreased p27Kip1 and induced pRB phosphorylation of HPMC. In contrast, dipyridamole prevented PDGF-induced p27Kip1 degradation and attenuated PDGF-stimulated pRB phosphorylation. Conclusion. Dipyridamole appears to inhibit PDGF-stimulated HPMC proliferation through attenuated ERK activity, preservation of p27Kip1, and decreased pRB phosphorylation. Thus, dipyridamole may have therapeutic efficacy to prevent or alleviate PF.