The antiangiogenic activity of the various rPEDF forms on bFGF-induced vessel sprouting in the ex vivo aortic ring assay. (A) Aortic rings from BALB/C mice embedded in collagen matrix were exposed to rPEDF, plPEDF, S24, 114A mutant, S24, 114E mutant, S227A mutant, or S227E mutant (10 nM) in the presence or absence of bFGF (50 ng/mL) in the serum-free BIO-MPM-1 medium. Control rings were treated with serum-free BIO-MPM-1 medium or with bFGF (50 ng/mL). Following 10 days of incubation, rings were fixed and stained with crystal violet (0.02%) to illustrate sprouting and vessel formation. Representative micrographs of ring of each arm of the experiment are shown. Micrographs were taken using inverted microscope (Nikon TE 2000U) connected to DUC camera under × 4 and × 10 objective. (B) Quantitative analysis of the assay described in panel A is presented as a mean ± SD of 6 distinct experiments. Student t test was used to analyze statistical significance of the differences between rings treated with bFGF and rings treated with the combination of bFGF and the various PEDF forms (^*P < .01).