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Formononetin promotes angiogenesis through the estrogen receptor alpha-enhanced ROCK pathway.

Li S, Dang Y, Zhou X, Huang B, Huang X, Zhang Z, Kwan YW, Chan SW, Leung GP, Lee SM, Hoi MP - Sci Rep (2015)

Bottom Line: In addition, results from co-immunoprecipitation suggested formononetin induced cell migration via recruiting of ERα/ROCK-II activated complex formation.More interestingly, in zebrafish embryo we observed that formononetin significantly promoted angiogenic sproutings in the subintestinal vessels (SIVs) that could be completely abolished by ROCK inhibitor.In this study, we elucidated the underlying mechanisms that formononetin produced proangiogenesis effects through an ERα-enhanced ROCK-II signaling pathways.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Quality Research in Chinese Medicine and Institute of Chinese Medical Sciences, University of Macau, Macao, China.

ABSTRACT
Formononetin is an isoflavone that has been shown to display estrogenic properties and induce angiogenesis activities. However, the interrelationship between the estrogenic properties and angiogenesis activities of formononetin are not well defined. In the present study, docking and enzymatic assay demonstrated that formononetin displayed direct binding to the ligand-binding domain (LBD) of estrogen receptor alpha (ERα) with an agonistic property. Results from Human Umbilical Vein Endothelial Cells (HUVEC) by using real-time migration xCELLigence system, immunofluorescence and western blotting provided strong evidences of formononetin induced endothelial cell migration and dramatic actin cytoskeleton spatial modification through ERα-enhanced-ROCK-II/MMP2/9 signaling pathways. In addition, results from co-immunoprecipitation suggested formononetin induced cell migration via recruiting of ERα/ROCK-II activated complex formation. More interestingly, in zebrafish embryo we observed that formononetin significantly promoted angiogenic sproutings in the subintestinal vessels (SIVs) that could be completely abolished by ROCK inhibitor. In this study, we elucidated the underlying mechanisms that formononetin produced proangiogenesis effects through an ERα-enhanced ROCK-II signaling pathways. Results from the present study also expand our knowledge about the enigmatic underlying mechanisms of phytoestrogenic compounds in the promotion of angiogenesis in relation to ERα and ROCK interaction in endothelial cells and their relationship with actin assembly and cell migration.

No MeSH data available.


Related in: MedlinePlus

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.
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f11: 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.

Mentions: The bars chart in Fig. 11J represent the gene expression of angiogenesis factors after treatment with 50 μM formononetin for 6 h. There was a significant increase of the mRNA expressions of VEGF-A (1.6-fold at 50 μM; p < 0.01) and Flt1 (or VEGFR1) (1.25-fold at 50 μM; p < 0.05) but not Kdr (or VEGFR2) (1.18-fold at 50 μM; p > 0.05) compared to the control.


Formononetin promotes angiogenesis through the estrogen receptor alpha-enhanced ROCK pathway.

Li S, Dang Y, Zhou X, Huang B, Huang X, Zhang Z, Kwan YW, Chan SW, Leung GP, Lee SM, Hoi MP - Sci Rep (2015)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4645220&req=5

f11: 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.
Mentions: The bars chart in Fig. 11J represent the gene expression of angiogenesis factors after treatment with 50 μM formononetin for 6 h. There was a significant increase of the mRNA expressions of VEGF-A (1.6-fold at 50 μM; p < 0.01) and Flt1 (or VEGFR1) (1.25-fold at 50 μM; p < 0.05) but not Kdr (or VEGFR2) (1.18-fold at 50 μM; p > 0.05) compared to the control.

Bottom Line: In addition, results from co-immunoprecipitation suggested formononetin induced cell migration via recruiting of ERα/ROCK-II activated complex formation.More interestingly, in zebrafish embryo we observed that formononetin significantly promoted angiogenic sproutings in the subintestinal vessels (SIVs) that could be completely abolished by ROCK inhibitor.In this study, we elucidated the underlying mechanisms that formononetin produced proangiogenesis effects through an ERα-enhanced ROCK-II signaling pathways.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Quality Research in Chinese Medicine and Institute of Chinese Medical Sciences, University of Macau, Macao, China.

ABSTRACT
Formononetin is an isoflavone that has been shown to display estrogenic properties and induce angiogenesis activities. However, the interrelationship between the estrogenic properties and angiogenesis activities of formononetin are not well defined. In the present study, docking and enzymatic assay demonstrated that formononetin displayed direct binding to the ligand-binding domain (LBD) of estrogen receptor alpha (ERα) with an agonistic property. Results from Human Umbilical Vein Endothelial Cells (HUVEC) by using real-time migration xCELLigence system, immunofluorescence and western blotting provided strong evidences of formononetin induced endothelial cell migration and dramatic actin cytoskeleton spatial modification through ERα-enhanced-ROCK-II/MMP2/9 signaling pathways. In addition, results from co-immunoprecipitation suggested formononetin induced cell migration via recruiting of ERα/ROCK-II activated complex formation. More interestingly, in zebrafish embryo we observed that formononetin significantly promoted angiogenic sproutings in the subintestinal vessels (SIVs) that could be completely abolished by ROCK inhibitor. In this study, we elucidated the underlying mechanisms that formononetin produced proangiogenesis effects through an ERα-enhanced ROCK-II signaling pathways. Results from the present study also expand our knowledge about the enigmatic underlying mechanisms of phytoestrogenic compounds in the promotion of angiogenesis in relation to ERα and ROCK interaction in endothelial cells and their relationship with actin assembly and cell migration.

No MeSH data available.


Related in: MedlinePlus