Background: Angiogenesis occurs within the interstitial matrix. We therefore cultured endothelial cells within collagen gels rather than on the surface of culture dishes. We measured the effect of a human glioblastoma-derived factor, and the combined effect of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF), on both the proliferative and morphologic changes exhibited by endothelial cells.
Experimental design: Bovine capillary endothelial (BCE) cells were cultured in a gel of type I collagen. Effects of growth factors were evaluated by the cell recovery from the gel and the morphologic changes of cells. The glioblastoma-derived factor was characterized by column chromatography, immunoblotting, and immunoadsorption with an anti-VEGF antibody.
Results: After 4 days of culture in the collagen gel with 10% calf serum Dulbecco's minimum essential medium, most of the BCE cells died. Addition of glioblastoma-conditioned medium resulted in a change in morphology from a round shape to an elongated spindle shape; moreover, the number of BCE cells was enhanced. The major activity in the conditioned medium was VEGF. In the 3-dimensional gel, we found a higher activity associated with VEGF, compared with bFGF when the growth factors were added 24 hours after seeding. In this assay, the response to bFGF diminished within 24 hours, but that to VEGF continued for 48 hours. When the two factors were added to the culture simultaneously, the cell number was greater than the sum of those stimulated with either growth factor alone. In the presence of both bFGF and VEGF, cord-like structures became prominent in the BCE cell cultures.
Conclusions: A human glioblastoma cell line produced VEGF, which enhanced the proliferation of BCE cells and induced an elongated shape in collagen gels. VEGF and bFGF increased the rate of proliferation and the number of cord-like structures in a synergistic manner.