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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

Formononetin induced cell migration and stress fiber formation in HUVECs.Formononetin enhanced the migration of HUVECs following scrape injury. (A,B) Serum-starved HUVEC monolayers were wounded (0 h) by micro-tips and washed with PBS, followed by addition of (A,a) 0.1% DMSO medium or (B,b) 25 μM formononetin for 8 h. (C) Statistical analysis of cell migration recorded by phase contrast microscopy following each scratch. The extent of migration was determined by averaging the mean length of the scraped area (gap width) of the migration pattern of endothelial cells. Formononetin also induced the rearrangement of actin cytoskeleton in HUVECs. Serum-starved HUVECs were treated with (D,d) 0.1% DMSO as control, and formononetin at (E,e) 12.5, (F,f) 25 and (G,g) 50 μM for 8 h. Cell nuclei were labeled with Hochest 33342 and F-actin was labeled with tetramethyl rhodamine isothiocyanate (TRITC)-phalloidin. Formononetin increased the formation of stress fibers that terminated at pointed edges (yellow arrowhead) and growth lamellipodia (white asterisk). White and yellow scale bars represent 50 and 20 μm, respectively. (H) HUVECs migration in response to different concentrations of formononetin was analyzed using the xCELLigence system. Real-time cell migrations of serum-starved HUVECs following the treatment of 12.5, 25 and 50 μM formononetin for 24 h were recorded. (I) Statistical analysis of the migration index of the xCELLigence system following formononetin treatments for 20 h. 40 ng/ml of VEGF was served as a positive control. Values are given as the increment of migration ± SD (n = 3), for three independent experiments. **p < 0.01, **p < 0.001 vs. control.
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f2: Formononetin induced cell migration and stress fiber formation in HUVECs.Formononetin enhanced the migration of HUVECs following scrape injury. (A,B) Serum-starved HUVEC monolayers were wounded (0 h) by micro-tips and washed with PBS, followed by addition of (A,a) 0.1% DMSO medium or (B,b) 25 μM formononetin for 8 h. (C) Statistical analysis of cell migration recorded by phase contrast microscopy following each scratch. The extent of migration was determined by averaging the mean length of the scraped area (gap width) of the migration pattern of endothelial cells. Formononetin also induced the rearrangement of actin cytoskeleton in HUVECs. Serum-starved HUVECs were treated with (D,d) 0.1% DMSO as control, and formononetin at (E,e) 12.5, (F,f) 25 and (G,g) 50 μM for 8 h. Cell nuclei were labeled with Hochest 33342 and F-actin was labeled with tetramethyl rhodamine isothiocyanate (TRITC)-phalloidin. Formononetin increased the formation of stress fibers that terminated at pointed edges (yellow arrowhead) and growth lamellipodia (white asterisk). White and yellow scale bars represent 50 and 20 μm, respectively. (H) HUVECs migration in response to different concentrations of formononetin was analyzed using the xCELLigence system. Real-time cell migrations of serum-starved HUVECs following the treatment of 12.5, 25 and 50 μM formononetin for 24 h were recorded. (I) Statistical analysis of the migration index of the xCELLigence system following formononetin treatments for 20 h. 40 ng/ml of VEGF was served as a positive control. Values are given as the increment of migration ± SD (n = 3), for three independent experiments. **p < 0.01, **p < 0.001 vs. control.

Mentions: In angiogenesis, endothelial cells proliferation and migration contribute to the dissemination from pre-existing vessels to form new vessels. The effects of formononetin on endothelial cells proliferation and migration were investigated in human umbilical vein endothelial cells (HUVECs). Cell proliferation and cytotoxicity were first evaluated by using the XTT and LDH assays. It was observed that formononetin (12.5, 25, 50 or 100 μM for 48 h) did not induce HUVECs cell proliferation, and 100 μM formononetin caused cytotoxicity by inducing significant LDH release (Supplementary Fig. 1). We then further investigated the effect of formononetin on cell migration. It was observed that formononetin enhanced HUVECs migration following scrape injury (Fig. 2A–C). Quantitative analysis of the real-time migration of HUVECs in response to formononetin with the xCELLigence system showed that formononetin enhanced cell migration in a dose-dependent manner (Fig. 2H). More interestingly, it was found that formononetin induced actin cytoskeleton rearrangement in HUVECs and enhanced the formation of stress fibers (Fig. 2E,e–G,g). These stress fibers terminated at pointed edges and were typical morphological feature in migrating cells. These results indicated that formononetin stimulated HUVECs migration by inducing stress fiber formation.


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)

Formononetin induced cell migration and stress fiber formation in HUVECs.Formononetin enhanced the migration of HUVECs following scrape injury. (A,B) Serum-starved HUVEC monolayers were wounded (0 h) by micro-tips and washed with PBS, followed by addition of (A,a) 0.1% DMSO medium or (B,b) 25 μM formononetin for 8 h. (C) Statistical analysis of cell migration recorded by phase contrast microscopy following each scratch. The extent of migration was determined by averaging the mean length of the scraped area (gap width) of the migration pattern of endothelial cells. Formononetin also induced the rearrangement of actin cytoskeleton in HUVECs. Serum-starved HUVECs were treated with (D,d) 0.1% DMSO as control, and formononetin at (E,e) 12.5, (F,f) 25 and (G,g) 50 μM for 8 h. Cell nuclei were labeled with Hochest 33342 and F-actin was labeled with tetramethyl rhodamine isothiocyanate (TRITC)-phalloidin. Formononetin increased the formation of stress fibers that terminated at pointed edges (yellow arrowhead) and growth lamellipodia (white asterisk). White and yellow scale bars represent 50 and 20 μm, respectively. (H) HUVECs migration in response to different concentrations of formononetin was analyzed using the xCELLigence system. Real-time cell migrations of serum-starved HUVECs following the treatment of 12.5, 25 and 50 μM formononetin for 24 h were recorded. (I) Statistical analysis of the migration index of the xCELLigence system following formononetin treatments for 20 h. 40 ng/ml of VEGF was served as a positive control. Values are given as the increment of migration ± SD (n = 3), for three independent experiments. **p < 0.01, **p < 0.001 vs. control.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f2: Formononetin induced cell migration and stress fiber formation in HUVECs.Formononetin enhanced the migration of HUVECs following scrape injury. (A,B) Serum-starved HUVEC monolayers were wounded (0 h) by micro-tips and washed with PBS, followed by addition of (A,a) 0.1% DMSO medium or (B,b) 25 μM formononetin for 8 h. (C) Statistical analysis of cell migration recorded by phase contrast microscopy following each scratch. The extent of migration was determined by averaging the mean length of the scraped area (gap width) of the migration pattern of endothelial cells. Formononetin also induced the rearrangement of actin cytoskeleton in HUVECs. Serum-starved HUVECs were treated with (D,d) 0.1% DMSO as control, and formononetin at (E,e) 12.5, (F,f) 25 and (G,g) 50 μM for 8 h. Cell nuclei were labeled with Hochest 33342 and F-actin was labeled with tetramethyl rhodamine isothiocyanate (TRITC)-phalloidin. Formononetin increased the formation of stress fibers that terminated at pointed edges (yellow arrowhead) and growth lamellipodia (white asterisk). White and yellow scale bars represent 50 and 20 μm, respectively. (H) HUVECs migration in response to different concentrations of formononetin was analyzed using the xCELLigence system. Real-time cell migrations of serum-starved HUVECs following the treatment of 12.5, 25 and 50 μM formononetin for 24 h were recorded. (I) Statistical analysis of the migration index of the xCELLigence system following formononetin treatments for 20 h. 40 ng/ml of VEGF was served as a positive control. Values are given as the increment of migration ± SD (n = 3), for three independent experiments. **p < 0.01, **p < 0.001 vs. control.
Mentions: In angiogenesis, endothelial cells proliferation and migration contribute to the dissemination from pre-existing vessels to form new vessels. The effects of formononetin on endothelial cells proliferation and migration were investigated in human umbilical vein endothelial cells (HUVECs). Cell proliferation and cytotoxicity were first evaluated by using the XTT and LDH assays. It was observed that formononetin (12.5, 25, 50 or 100 μM for 48 h) did not induce HUVECs cell proliferation, and 100 μM formononetin caused cytotoxicity by inducing significant LDH release (Supplementary Fig. 1). We then further investigated the effect of formononetin on cell migration. It was observed that formononetin enhanced HUVECs migration following scrape injury (Fig. 2A–C). Quantitative analysis of the real-time migration of HUVECs in response to formononetin with the xCELLigence system showed that formononetin enhanced cell migration in a dose-dependent manner (Fig. 2H). More interestingly, it was found that formononetin induced actin cytoskeleton rearrangement in HUVECs and enhanced the formation of stress fibers (Fig. 2E,e–G,g). These stress fibers terminated at pointed edges and were typical morphological feature in migrating cells. These results indicated that formononetin stimulated HUVECs migration by inducing stress fiber formation.

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