Effect of PTX3 on FGF2 receptor interaction and mitogenic activity in endothelial cells. (A) GM 7373 cells were treated with FGF2 and PTX3 in the absence (•) or in the presence of anti-PTX3 (□) or irrelevant (▵) antibodies (both at 99 μg/mL). (B) GM 7373 cells were treated with 0.4% FCS with no addition (control), FGF2 (1.66 nM), 10% FCS, DAG (15 μM), TPA (8.0 nM), EGF (0.6 nM), VEGF (0.7 nM), FGF4 (1.66 nM), or FGF8 (1.66 nM) in the absence (▪) or in the presence (□) of PTX3 (66 nM). (C) GM 7373 cells, E1G11 cells, or HUVE cells were incubated with FGF2 (0.55 nM) in the absence (□) or in the presence (▪) of PTX3 (222 nM). All mitogens induced a statistically significant increase in the proliferation rate (Student t test, P < .05). Each point is the mean ± SEM of 3 to 7 determinations in duplicate. (D) PTX3 (66 nM) was added to GM 7373 cells at the indicated periods of time before or after the beginning of FGF2 treatment (T₀). When FGF2 was added after PTX3, cells were washed extensively before addition of the growth factor. Cells were counted 24 hours after the beginning of FGF2 treatment. Two additional independent experiments gave similar results. In panels A, C, and D, data are expressed as percentage of the increase in cell number relative to control cells treated with the different mitogens in the absence of PTX3. (E) GM 7373 cells were treated with ¹²⁵I-FGF2 in the presence of PTX3. Then, ¹²⁵I-FGF2 bound to HSPGs (○) and FGFRs (•) was evaluated and expressed as percentage of the radioactivity measured in the absence of PTX3. The arrow points to the inhibition measured in the presence of a 100-fold molar excess of unlabeled FGF2. Each point is the mean ± SEM of 3 determinations in duplicate.