The effects of formononetin on angiogenic sprouting in the SIVs of Tg (fli1: EGFP)y1 and Tg (fli1: nEGFP)y7 zebrafish embryos.

(A,a) Tg (fli1: EGFP)y1 and Tg (fli1: nEGFP)y7 zebrafish embryos (48 hpf) were treated with 0.1% DMSO for 24 h to serve as negative control. The SIVs of zebrafish embryos at 72 hpf developed into a smooth basket-like structure. Zebrafish embryos (48 hpf) treated with formononetin for 24 h at (B,b) 25 µM or (C,c) 50 µM showed that there was an increase of sprouting in the subintestinal vessels (SIVs) baskets stretching into the posterior yolk extension. (D,d) Zebrafish embryos were injected with zebrafish VEGF-A (ZF-VEGF-A) (1 ng/embryo) at 1 hpf and incubated in embryo medium until 72 hpf to serve as positive control. Yellow arrows indicated sprouting vessel formation; white asterisks indicated intersection branch formation. Statistical analysis showed that formononetin increased (E) the length of vascular sprouting in the SIVs and (F) the number of endothelial cells in the SIVs in a dose-dependent manner. The number of endothelial cells was measured using Image J software package. Data are plotted as mean ± SD, (n > 3), *p < 0.05, **p < 0.01 vs. control.

The effects of ICI 182,780 (ER antagonist) and vegfa morpholino on formononetin-induced angiogenesis in zebrafish embryos in vivo.

Tg (fli1: EGFP)y1 zebrafish embryos (48 hpf) were treated with (A) 0.1% DMSO (negative control), (B) formononetin (50 µM) (positive control), (C) co-treatment of ICI 182,780 (50 µM) and formononetin (50 µM), (D) ICI 182, 780 (50 µM) alone. All treatments were for 24 h. Tg (fli1: EGFP)y1 zebrafish embryos (1 hpf) were injected with (E) Vegfa MO (2 ng) alone and (F) Vegfa MO (2 ng) injection + formononetin treatment (50 µM, at 48 hpf for 24 h) and were observed at 72 hpf. (G,H) Control images of zebrafish embryos at 1–4 cells stage and 24 hpf after Vegfa MO injection. (I) Data were analyzed by using the Image J software package. Quantitative analysis indicated the total length of SIVs for each group. (J) Evaluation of gene expressions in formononetin-treated zebrafish embryos by using real-time PCR. Data are plotted as means ± SD from three individual experiments. *p < 0.05, **p < 0.01 vs. control group.

The effects of ROCK inhibitor in formononetin-induced angiogenesis in zebrafish embryos in vivo.

Tg (fli1: EGFP)y1 zebrafish embryos (48 hpf) were treated with (A) 0.1% DMSO (negative control), (B) formononetin (25 µM), (C) formononetin (50 µM), (D) H1152 (10 µM, ROCK inhibitor) alone, (E) co-treatment of H1152 (10 µM) +formononetin (25 µM), (F) co-treatment of H1152 (10 µM) +formononetin (50 µM). All treatments were for 24 h. (G) Quantitative analysis indicated the length of sprouting in SIVs for each group. Data are plotted as means ± SD, from three individual experiments. *p < 0.05, ***p < 0.001 vs. control group.