Figure 4
Contribution of purinergic receptor subtypes in angiogenic responses to extracellular ATP in VVEC. (A): Growth-arrested cells were pre-incubated with purinergic receptor antagonists suramin (25 μM), MRS2179 (2 μM), MRS2211 (25 μM) or DIDS (25 μM) for 45 min or remained untreated and then stimulated with ATP (100 μM) in the presence of 0.125 μCi [3H]-thymidine for 24 hrs. Incorporated radioactivity was measured in total cell lysates using a β counter. Data represent means ± SE from four independent experiments conducted on two distinct VVEC populations; ** p < 0.01 vs. control; § p < 0.05, §§ p < 0.01 - platelet extract-stimulated cells vs. platelet extract-stimulated cells treated with antagonists; # p < 0.05, ## p < 0.01 ATP-stimulated cells vs. ATP-stimulated cells treated with antagonists; non-stimulated control; @ p < 0.05, @@ p < 0.01 ATP- and platelet extract-stimulated cells vs. ATP- and platelet extract-stimulated cells treated with antagonists; (B) Growth arrested cells were placed on top of inserts in serum free DMEM and pre-treated with suramin (50 μM), MRS2179 (2 μM), MRS2211 (50 μM) or DIDS (50 μM) for 45 min or remained untreated. Cell migration was stimulated by adding platelet extracts (32 μg/ml), ATP (100 μM) or their combination to the lower transwell compartment. Quantitative data represent the means ± SE from three independent experiments; ** p < 0.01, ***p < 0.001 vs. control; § p < 0.05 - platelet extract-stimulated cells vs. platelet extract-stimulated cells treated with antagonists; # p < 0.05, ## p < 0.01 ATP-stimulated cells vs. ATP-stimulated cells treated with antagonists; (C) Growth-arrested VVEC were plated on growth factor-reduced Matrigel in serum free DMEM and pre-treated with purinergic receptor antagonists as described above in the panel A. Tube formation was stimulated by the addition of platelet extracts (32 μg/ml), ATP (100 μM), or a combination of both. Data show images from one representative experiment. Similar results were obtained in at least three experiments conducted on distinct VVEC populations.