Cardiovascular pharmacology Inhibitory effects of quercetin on angiogenesis in larval zebrafish and human umbilical vein endothelial cells $ Daxian Zhao a,c,n , Chuanjie Qin b , Xiaohui Fan c , Yuncong Li c , Binhe Gu c a State Key Laboratory of Food Science and Technology, School of Life Science and Food Engineering, Nanchang University, Nanchang 330047, PR China b College of Life Science, Neijiang Normal University, Neijiang 641112, PR China c Department of Soil and Water Science, Tropical Research and Education Center, University of Florida, Homestead, FL 33031, USA article info Article history: Received 14 September 2013 Received in revised form 25 October 2013 Accepted 31 October 2013 Available online 13 November 2013 Keywords: Quercetin Flavonoids Angiogenesis Zebrafish Human umbilical vein endothelial cells Anti-cancer abstract Angiogenesis plays an essential role in many physiological and pathological processes. Quercetin, a plant pigment and traditional Chinese medicinal herb, is an important flavonoid that has anti-cancer activity. However, the function of quercetin in blood vessel development in vivo and in vitro is still unclear. In this study, we investigated the anti-angiogenic activity of quercetin in zebrafish embryos and in human umbilical vein endothelial cells (HUVECs). Our results showed that quercetin disrupted the formation of intersegmental vessels, the dorsal aorta and the posterior cardinal vein in transgenic zebrafish embryos. In HUVECs, quercetin inhibited cell viability, the expression of vascular endothelial growth factor receptor 2 and tube formation in a dose-dependent manner. In inhibiting angiogenesis, quercetin was found to be involved in suppressing the extracellular signal-regulated kinase signaling pathway in vivo and in vitro. This study has shown that quercetin has potent anti-angiogenic activity and may be a candidate anti-cancer agent for future research. & 2013 Elsevier B.V. All rights reserved. 1. Introduction The circulatory system in vertebrates is a network of arteries, veins and capillaries, and the formation of the vascular system includes vasculogenesis and angiogenesis (Larrivee et al., 2009; Risau, 1997). Angiogenesis, the emergence of new blood vessels via branching from an existing vascular system, plays an important role in embryonic vascular formation and development (Potente et al., 2011; Risau, 1997). Studies have shown that tumors promote the angiogenic process, including the proliferation and migration of endothelial cells. One novel strategy to suppress tumor devel- opment is the inhibition of angiogenesis (McMahon, 2000; Pratheeshkumar et al., 2012), and there is increasing evidence demonstrating that angiogenesis is involved in increased cellular infiltration and proliferation (Jackson et al., 1997). Vascular endothelial growth factor (VEGF) is the most important angiogenic factor that increases mitogenic activity and the survival of vascular endothelial cells (Roberts and Palade, 1997; Yu et al., 2010). VEGF exerts its activity on endothelial cells through two types of receptor tyrosine kinases (RTKs): vascular endothelial growth factor receptor 1 (VEGFR-1) and vascular endothelial growth factor receptor 2 (VEGFR-2). VEGFR-2 plays a critical role in mediating the mitogenesis and proliferation of endothelial cells (Pratheeshkumar et al., 2012), and activation of VEGFR-2 enhances proliferation, migration and tube formation of endothelial cells by activating the phosphorylation of multiple signaling pathways, including the extracellular signal-regulated kinase (ERK), c-Jun amino-terminal kinase (JNK), phosphatidylinositide 3-kinase (PI3K), protein kinase B (AKT) and p38 mitogen-activated protein kinase (p38MAPK) pathways (Ferrara et al., 2003). Quercetin, a bioactive flavonoid with a molecular weight of 302.24 g mol À1 is a Chinese herbal medicine found in various edible plants, such as red onions, apples, tea, broccoli, red grapes and a number of berries (Bischoff, 2008). It exhibits a broad range of pharmacological activities, and is considered to be an anti- inflammatory, anti-oxidant, anti-tumor and anti-ulcer agent, as well as exerting immunomodulatory and vasodilatory effects (Ajay et al., 2006; Alvarez et al., 1999; Shoskes and Nickel, 2011). In our study, we chose transgenic zebrafish (Danio rerio; fli1: EGFP), which are a useful model for the high-throughput screening of drugs and compounds (Cheng et al., 2001; Peterson et al., 2001), to investigate the effect of quercetin on angiogenesis. We Contents lists available at ScienceDirect journal homepage: www.elsevier.com/locate/ejphar European Journal of Pharmacology 0014-2999/$ - see front matter & 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ejphar.2013.10.069 Abbreviations: HUVECs, human umbilical vein endothelial cells; ISV, interseg- mental vessel; DA, dorsa aorta; PCV, posterior cardinal vein; VEGF, vascular endothelial growth factor; RTKs, receptor tyrosine kinases; DMSO, dimethylsulfoxide; hpf, hours postfertilization; PVDF, polyvinylidene difluoride; ERK, extracellular signal-regulated kinase ☆ Chemical compounds studied in this article. Quercetin (PubChem CID: 5280343). n Corresponding author. Tel./fax: þ86 791 83969531. E-mail address: zhaodaxian@ncu.edu.cn (D. Zhao). European Journal of Pharmacology 723 (2014) 360–367