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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 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.
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f10: 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.

Mentions: In order to determine if formononetin exerted pro-angiogenic effects in vivo, we employed the transgenic zebrafish models Tg (fli1: EGFP)y1 and Tg (fli1: nEGFP)y7. Tg (fli1: EGFP)y1 zebrafish were engineered to express green fluorescent protein (GFP) in the entire vasculature and Tg (fli1: nEGFP)y7 harbored nuclear-localized GFP expression in the endothelial cells permitting real time in vivo analysis of individual endothelial cells. Figure 10A showed that under normal condition, zebrafish embryos developed a smooth, basket-like structure in the region of the subintestinal vessels (SIVs) at 72 hour post fertilization (hpf). The treatments of formononetin at 25 μM (Fig. 10B,b) or 50 μM (Fig. 10C,c) increased the sprouting in the SIVs in a dose-dependent manner. The increase in sprouts formation induced by formononetin was similar to the effect of zebrafish VEGF-A (ZF-VEGF-A) (1 ng/embryo) (Fig. 10D,d). We further evaluated the phenotype and the behavior of individual endothelial cells to determine if the pro-angiogenic effects of formononetin were caused by endothelial cell proliferation and migration. Quantitative analysis indicated that formononetin (50 μM) significantly induced an increase in the length of sprouting vessels (Fig. 10E) as well as a slightly increase in the endothelial cell populations throughout the region of the SIVs (Fig. 10F), similar to the effects induced by ZF-VEGF-A injection.


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

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

f10: 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.
Mentions: In order to determine if formononetin exerted pro-angiogenic effects in vivo, we employed the transgenic zebrafish models Tg (fli1: EGFP)y1 and Tg (fli1: nEGFP)y7. Tg (fli1: EGFP)y1 zebrafish were engineered to express green fluorescent protein (GFP) in the entire vasculature and Tg (fli1: nEGFP)y7 harbored nuclear-localized GFP expression in the endothelial cells permitting real time in vivo analysis of individual endothelial cells. Figure 10A showed that under normal condition, zebrafish embryos developed a smooth, basket-like structure in the region of the subintestinal vessels (SIVs) at 72 hour post fertilization (hpf). The treatments of formononetin at 25 μM (Fig. 10B,b) or 50 μM (Fig. 10C,c) increased the sprouting in the SIVs in a dose-dependent manner. The increase in sprouts formation induced by formononetin was similar to the effect of zebrafish VEGF-A (ZF-VEGF-A) (1 ng/embryo) (Fig. 10D,d). We further evaluated the phenotype and the behavior of individual endothelial cells to determine if the pro-angiogenic effects of formononetin were caused by endothelial cell proliferation and migration. Quantitative analysis indicated that formononetin (50 μM) significantly induced an increase in the length of sprouting vessels (Fig. 10E) as well as a slightly increase in the endothelial cell populations throughout the region of the SIVs (Fig. 10F), similar to the effects induced by ZF-VEGF-A injection.

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