Calycosin Promotes Angiogenesis Involving Estrogen Receptor and Mitogen-Activated Protein Kinase (MAPK) Signaling Pathway in Zebrafish and HUVEC

Abstract

BackgroundAngiogenesis plays an important role in a wide range of physiological processes, and many diseases are associated with the dysregulation of angiogenesis. Radix Astragali is a Chinese medicinal herb commonly used for treating cardiovascular disorders and has been shown to possess angiogenic effect in previous studies but its active constituent and underlying mechanism remain unclear. The present study investigates the angiogenic effects of calycosin, a major isoflavonoid isolated from Radix Astragali, in vitro and in vivo.Methodology Tg(fli1:EGFP) and Tg(fli1:nEGFP) transgenic zebrafish embryos were treated with different concentrations of calycosin (10, 30, 100 µM) from 72 hpf to 96 hpf prior morphological observation and angiogenesis phenotypes assessment. Zebrafish embryos were exposed to calycosin (10, 100 µM) from 72 hpf to 78 hpf before gene-expression analysis. The effects of VEGFR tyrosine kinase inhibitor on calycosin-induced angiogenesis were studied using 72 hpf Tg(fli1:EGFP) and Tg(fli1:nEGFP) zebrafish embryos. The pro-angiogenic effects of calycosin were compared with raloxifene and tamoxifen in 72 hpf Tg(fli1:EGFP) zebrafish embryos. The binding affinities of calycosin to estrogen receptors (ERs) were evaluated by cell-free and cell-based estrogen receptor binding assays. Human umbilical vein endothelial cell cultures (HUVEC) were pretreated with different concentrations of calycosin (3, 10, 30, 100 µM) for 48 h then tested for cell viability and tube formation. The role of MAPK signaling in calycosin-induced angiogenesis was evaluated using western blotting.ConclusionCalycosin was shown to induce angiogenesis in human umbilical vein endothelial cell cultures (HUVEC) in vitro and zebrafish embryos in vivo via the up-regulation of vascular endothelial growth factor (VEGF), VEGFR1 and VEGFR2 mRNA expression. It was demonstrated that calycosin acted similar to other selective estrogen receptor modulators (SERMs), such as raloxifene and tamoxifen, by displaying selective potency and affinity to estrogen receptors ERα and ERβ. Our results further indicated that calycosin promotes angiogenesis via activation of MAPK with the involvement of ERK1/2 and ER. Together, this study revealed, for the first time, that calycosin acts as a selective estrogen receptor modulator (SERM) to promote angiogenesis, at least in part through VEGF-VEGFR2 and MAPK signaling pathways.

Isoindolone derivative QSN-10c induces leukemic cell apoptosis and suppresses angiogenesis via PI3K/AKT signaling pathway inhibition

“…After 8 h of incubation, the network-like structures of endothelial cells were examined using an inverted microscope (Olympus, Tokyo) at 100×magnification. The tube-like structures were defined as endothelial cord formations connected at both ends [22] . The average number of branching points in five random fields per well was quantified using Image Pro-Plus 6.0 software.…”

Pro-angiogenic activity of astragaloside IV in HUVECs in vitro and zebrafish in vivo

“…Radix Astragali is a potential angiogenic modulator that contains a considerable number of effective constituents (18). The angiogenic activity of the ethanol extract of Radix Astragali was further proved in multiple HUVEC angiogenesis assays in our previous study (9), and one active constituent, calycosin, was recently shown to be pro-angiogenic and to promote endothelial cell proliferation in both HUVECs and zebrafish larvae (19). In the present study, we identified another angiogenic compound, AS-IV, which acts differently in zebrafish in the angiogenesis assay in vivo compared with the effect of calycosin, since AS-IV promotes angiogenesis only in zebrafish that experienced vessel damage, rather than in healthy animals.…”

Ethanol Extract of Sambucus formosana Stimulates HUVEC Proliferation and Tube Formation through MEK1/2, ERK1/2 Pathway

“…Globally accepted herbal drugs are thought to be safe and effective, however, more evidence-based confirmation studies are needed. Among the most frequently studied proangiogenic phytochemicals are ginsenosides from Panax ginseng [1], beta-sitosterol from Aloe vera [2], calycosin from Radix Astragali [4], saponins from Panax notoginseng [5] and extracts from Hippophae rhamnoides L. [2], Radix Astragali [6], Puerariae flos [7], Stewartia koreana [8] and Angelica sinensis [9]. Those previous studies are suggested Chinese medicines as formula for treating angiogenesis disease and wound healing.…”

“…A survey of the literatures indicated that some plants and phytochemicals have been used to treat angiogenesis disease [1][2][3][4]. Natural products, such as certain Chinese medicines, contain a variety of angiogenic compounds.…”