Figure 4.

Chordin-like 1 modulates the antiangiogenic effect of bone morphogenetic protein-4. Human umbilical vascular endothelial cells(HUVECs) and human diploid fibroblasts were obtained (day 1) as cocultures in 24 well plates. Medium, with treatments or vehicle, was replenished on days 1, 4, 7, and 9. The assay was treated with VEGF, Suramin, recombinant human BMP-2, BMP-4, and CHL-1. Tubule formation was examined at day 11. Cells were fixed, quantitated, and visualized using a combined ELISA and histology kit. A: VEGF (2 ng/ml) and Suramin (20 mM) were used as positive and negative angiogenesis controls, respectively. B: Cells were treated with rhBMP-2 and rhBMP-4. BMP-4 significantly inhibited angiogenesis at 10 ng/ml. C: CHL-1 inhibited BMP-4; CHL-1 alone had no significant effect on angiogenesis, however it inhibited BMP-4s anti-angiogenic effects. Images A-C are shown at magnification 10X. Representative images are shown in A-C. D: Angiogenesis was quantitated by using anti-CD31 antibody coupled to a soluble substrate, ρ-nitrophenol phosphate (ρ-NPP), which permits quantitation by an optical density measurement. The asterisk indicates a significance at p<0.05 and the double asterisk indicates a significance at p<0.001.