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

Shang Li,Xuelin Zhou,Yuanye Dang,Maggie Pui Man Hoi,Zherui Zhang,George Pak Heng Leung,Bin Huang,Shun Wan Chan,Simon Ming Yuen Lee,Xiaohui Huang,Yiu Wa Kwan

doi:10.1038/srep16815

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.

Highlights

Estrogen receptors (ERs) are present in the vascular endothelium and it is observed that estradiol could induce angiogenesis through the activation of both long-term and rapid estrogen receptors (ER) signaling[7,8,9]
The evaluation of formononetin-induced sprouting angiogenesis was evaluated in zebrafish embryo in vivo and the formononetin-induced Rho-associated protein kinase (ROCK) signaling in cytoskeleton reorganization and endothelial cell migration were evaluated in human umbilical vein endothelial cells (HUVECs)
Since our molecular docking and enzymatic transactivation assay showed that formononetin exhibited direct binding and agonistic at ERα, we further investigated the role of ERα activity in the pro-angiogenic endothelial cell migration induced by formononetin in HUVECs

Summary

Introduction

Estrogen receptors (ERs) are present in the vascular endothelium and it is observed that estradiol could induce angiogenesis through the activation of both long-term (genomic) and rapid (non-genomic) ER signaling[7,8,9]. Rho-associated protein kinase (ROCK), plays a crucial role in cytoskeleton regulation by phosphorylating the myosin-binding subunit of myosin light chain (MLC) phosphatase, thereby inhibiting the myosin phosphatase activity and maintaining MLC in a contractile state[12]. This induces F-actin stress fibers formation and focal adhesions that are essential during cell migration[13,14].

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Results

Conclusion