Figure 6

Involvement of the VEGF in NGF-stimulated angiogenesis. (A) ELISA quantification of VEGF in conditioned media from HUVEC and MDA-MB-231 cells. Cells were treated with NGF (100 ng/ml) for 6 h or 24 h, conditioned media were concentrated before ELISA assay, as described in materials and methods. (B) Invasion assay using Transwells. HUVEC were treated with NGF (100 ng/ml) or VEGF (10 ng/ml) in the presence of isotype control or anti-VEGF neutralizing antibodies (1 μg/ml) for 24 h. (C) In vivo angiogenesis assay. Matrigel containing a mixture of NGF and isotype control or anti-VEGF neutralizing antibodies (37.5 μg/ml) was subcutaneously injected into SCID mice (five mice per group) as described in materials and methods. Hemoglobin in Matrigel plugs was quantified by Drabkin method 7 days after injection. Results are the mean of three independent experiments. Student's t-test, *p < 0.01; ^#p < 0.05 versus control.