Action of dipyridamole and warfarin on growth of human endothelial cells cultured in serum-free media

Objective: To study the anti-proliferative effect of Dipyridamole (anti-platelet), Dipyridamole with Warfarin, and Warfarin (anticoagulant) alone, in human endothelial cells in vitro. Design and methods: Human endothelial cells were harvested from umbilical cords. Primary cultures were usually successful. However, subculture yields were usually contaminated with smooth muscle cells. We have developed an improved method for the isolation of endothelial cells by using Collagenase II, coating the culture flask with fibronectin and using serum-free media. The endothelial cells were characterised by anti-PECAM-1/PE (CD31) using Flow Cytometry with viability of 95% after trypsinization with 0.05% Trypsin and 1 mM EDTA. Growth and proliferation studies were performed in vitro in the presence of 5μM Dipyridamole, 5μM Dipyridamole with 5μM Warfarin, 5μM Warfarin alone by cell counts, 3H-thymidine and 3H-leucine incorporation. Results: The incorporation of 3H-Leucine at day 6 in each test condition revealed no significant change. Control 12678 ± 2968 CPM, Dipyridamole 8698 ± 189 CPM, Dipyridamole and Warfarin 7541 ± 413 CPM, and Warfarin alone 10711 ± 732 CPM. With the incorporation of 3H-Thymidine, Dipyridamole alone as well as Dipyridamole with Warfarin reduced the basal proliferation rates significantly when compared to controls. Control 14355 ± 4441 CPM, Dipyridamole 1100 ± 152 CPM (p<0.05), Dipyridamole with Warfarin 1092 ± 272 CPM (p<0.05). Warfarin alone did not reduce proliferation significantly 12870 ± 2677 CPM (NS). Conclusions: We have developed a method to isolate pure endothelial cells from human umbilical cords using Serum-Free Media (SFM). EC with high purity was characterised by anti-PECAM-1/PE (CD31) using Flow Cytometry. Dipyridamole at a concentration of 5 μM inhibited the proliferation of endothelial cells at day 6 by 93%. These techniques can be used for routine analysis and proliferation studies.