Figure 6. Effect of EPO on HUVEC proliferation and tube formation on matrigel.

HUVECs were treated with either 20 IU/ml or 100 IU/ml and either 100 IU/ml EPO or 200 ng/ 100μl VEGF in the absence or presence of 0.25 mg/ml bevacizumab for 48 hours after plating the cells on matrigel. The extent of HUVEC tube formation on matrigel was assessed after 24 hours under a light microscope at 10× magnification. The tubular structures were graded semiquantitatively on a scale of 0 to 5 by evaluation of the relative presence and stages of formation of tubes on the matrigel: 0 =  well separated individual cells, 1 =  cells had begun to migrate and align themselves, 2 =  visible capillary tubes and no sprouting, 3 =  visible sprouting of new capillary tubes, 4 =  early formation of closed polygons, 5 =  development of complex mesh-like structures. (A) Each bar represents the mean grade of tube formation ± SD in the matrigel. *P<0.05, **P<0.01 and ***P<0.001. (B) The white bars are the mean grade of tube formation ± SD in the matrigel of untreated HUVECs, VEGF- and EPO-treated HUVECs. The black bars represent the mean grade of tube formation ± SD in the matrigel of untreated HUVECs VEGF- and EPO-treated HUVECs that were exposed to bevacizumab. *P<0.05 and ***P<0.001, for the difference between HUVECs that were or were not exposed to bevacizumab. NS  =  not significantly different. (C) From top to bottom: representative micrograph of untreated HUVECs on matrigel, VEGF- and EPO-treated HUVECs after 24 hours of plating with (+) or without (−) bevacizumab. (D) Cultured HUVECs were treated with or without 100 IU/ml EPO in the presence of either bevacizumab, PD173074, or tyrphostin, a combination of bevacizumab, PD173074 and tyrphostin, or in the presence of wortmannin. Proliferation of HUVECs was measured by incorporation of [³H]-thymidine to DNA. Duplicate cell counts were averaged for 3 experiments and the data were expressed as the percentage of control. *P<0.05, **P<0.01 and ***P<0.001 for the difference between untreated, or EPO- treated HUVECs that were exposed to bevacizumab, PD173074, tyrphostin or wortmannin. NS  =  not significantly different.