Elsevier

Biochemical Pharmacology

Volume 80, Issue 9, 1 November 2010, Pages 1386-1395
Biochemical Pharmacology

Resveratrol modulates angiogenesis through the GSK3β/β-catenin/TCF-dependent pathway in human endothelial cells

https://doi.org/10.1016/j.bcp.2010.07.034Get rights and content

Abstract

Vascular endothelial growth factor (VEGF) plays a critical role in angiogenesis due to its potent and specific ability to promote the proliferation and migration of endothelial cells. Resveratrol has been shown to have many health-benefiting effects, including the protection of cardiovascular system. In this study we examined the effect of resveratrol on angiogenesis in human umbilical vein endothelial cells (HUVECs). We observed that resveratrol was able to modulate the expression of VEGF and the formation of vascular network in a biphasic pattern. While resveratrol at low concentrations, from 1 to 10 μM, up-regulated the expression of VEGF and promoted angiogenesis, it had opposite effect at high concentrations (20 μM and higher). The biphasic effect of resveratrol on angiogenesis was confirmed by chick chorioallantoic membrane assay. Up-regulation of VEGF expression depended on the nuclear accumulation and transcriptional activity of β-catenin. Correspondingly, GSK3β, a negative regulator of β-catenin, turned into a less active state (phosphorylated at Ser9) in cells exposed to 5 μM of resveratrol, but became more active at 20 μM. We demonstrated that both Akt and ERK signaling pathways, which are known to be critical for angiogenesis, became activated in response to 5 μM of resveratrol and functioned to inactivate GSK3β. Our findings may have implications in the management of cardiovascular diseases and other conditions such as cancer by the use of resveratrol.

Introduction

Angiogenesis, the sprouting of new capillaries from the preexisting vasculature, is integral to many physiological and pathological processes, including embryonic vascular development, wound healing, organ regeneration, diabetic retinopathy, rheumatoid arthritis, cardiovascular diseases, tumor growth and metastasis [1]. During angiogenesis, endothelial cells proliferate, migrate, and form tube-like structures. This cascade of events is tightly controlled by angiogenic factors. Vascular endothelial growth factor (VEGF) has potent and specific ability to regulate key steps in angiogenesis, including proliferation and migration of endothelial cells [1]. Over-expression of VEGF and its receptors promotes blood vessel formation, whereas the inhibition of VEGF function blocks angiogenesis [1], [2].

Glycogen-synthase kinase 3β (GSK3β) serves as a nodal point of convergent signaling pathways in endothelial cells in regulating angiogenic responses. Inhibition of GSK3β induces an angiogenic phenotype in endothelial cells [3]. GSK3β is known to be regulated by several signaling pathways, including PI3K/Akt [4], [5], MEK/ERK [3], [6], and Wnt signaling pathway [7]. Among them the Wnt/GSK3β pathway is most extensively studied. In the presence of Wnt signaling, CKI and GSK3β become inactivated, leading to cytoplasmic, and subsequently nuclear, accumulation of β-catenin. β-catenin in the nucleus forms complexes with members of the lymphoid enhancer factor/T-cell factor (LEF/TCF) family of transcription factors to activate transcription [8], [9]. In the absence of Wnt signaling, β-catenin is constitutively phosphorylated, by GSK3β and CKI, at serine and threonine residues in the N-terminal region, resulting in ubiquitination and subsequent proteosomal degradation [9], [10]. Increasing evidence has implicated the Wnt/β-catenin signaling pathway in vascular development in normal and pathological conditions [11], [12]. Wnt ligands and their receptors are expressed in vascular cells [11]. Endothelial cell-specific inactivation of the β-catenin gene in mice results in defective vascularization and embryonic lethality [12].

Several studies showed that the human VEGF promoter contains β-catenin-TCF binding motifs and that VEGF promoter activity can be increased by stabilized β-catenin [5], [13], [14]. Moreover, β-catenin is sufficient to promote vessel growth in vivo and confer a pro-angiogenic phenotype to endothelial cells in vitro through the transcriptional activation of VEGF [14]. In addition, VEGF expression can also been stimulated by hypoxia inducible factor (HIF-1) [1]. Therefore, VEGF serves as an important target molecule in treating diseases resulting from insufficient or excessive blood vessel formation.

Increasing evidence has shown that resveratrol (trans-3,5,4′-trihydroxystilbene) possesses anti-oxidant, anti-cancer, anti-aging, and anti-inflammatory function. In particular, the cardioprotective effect of resveratrol has been intensively investigated [15]. Resveratrol has been found to protect the vessels from atherosclerosis [16], to reduce myocardial damage during ischemia-reperfusion [17], [18], and to modulate vascular cell functions [19]. Nitric oxide (NO) [20], thioredoxin-1 [19], adenosine receptors [21], [22], PI3K and mitogen-activated protein kinase (MAPK) [17], [23], and mTOR [18] have all been proposed to mediate the cardioprotective effects of resveratrol.

Resveratrol can activate both the PI3K/Akt and the MAPK pathways [17], [23], which negatively regulate GSK3β. We previously showed that resveratrol could promote osteoblastic differentiation by augmenting β-catenin signaling pathway [24]. In this study we examined the effect of resveratrol on angiogenesis. We observed that resveratrol modulates angiogenesis in a biphasic pattern. Whereas low concentration of resveratrol promotes angiogenesis in human umbilical vein endothelial cells (HUVECs), at high concentration it has inhibitory effect. We demonstrated that resveratrol modulates angiogenesis in HUVECs through GSK3β/β-catenin/TCF pathways, which is in turn regulated by the activation of PI3K/Akt and MEK/ERK signaling pathways.

Section snippets

Experimental reagents

Resveratrol (Sigma, St. Louis, MO, USA) was dissolved in dimethyl sulfoxide (DMSO). The final concentration of DMSO in the culture medium was less than 0.05% (v/v). Control cultures received the same amount of DMSO. Dulbecco's Minimum Essential Medium (DMEM), fetal bovine serum, lithium chloride (LiCl) were purchased from Gibco (Carlsbad, CA, USA). The PI3K inhibitor, LY294002, the MEK1 inhibitor, PD98059, the polyclonal phospho-specific antibodies against Akt (Ser473), ERK/1/2, GSK3β (Ser9)

Resveratrol modulates VEGF and VEGFR2 expression in a biphasic manner

Because VEGF is critical in angiogenesis, we first examined the transcription of VEGF in HUVECs in response to resveratrol. HUVECs were treated with increasing concentrations of resveratrol (0.2, 1, 5, 10, 15, 20, and 50 μM) for 24 h, the mRNA level of VEGF-A was determined by using quantitative real-time PCR. As shown in Fig. 1A, resveratrol treatment changed the expression levels of VEGF in a dose-dependent manner. When applied in the range from 1 to 10 μM, resveratrol significantly increased

Discussion

Resveratrol has been shown to have cardioprotective effect [16], [17], [18]. On the other hand, many studies also showed that it possesses antiangiogenic effect [30], [31], [32], [33], [34], [35], [36], [37]. The mechanisms by which resveratrol regulates cardiovascular function were also shown to be multifaceted [17], [18], [19], [20], [21], [22], [23]. In the present study we found that resveratrol had opposite effects on the expression of VEGF and angiogenesis depending on the concentration

Acknowledgements

This work was supported by National Basic Research Program of China; grant number 2007CB512001, National High-tech Research and Development Program of China; grant number: 2006AA02A406, and National Science Foundation Research Grant; grant number: 30901987.

References (45)

  • V. Marin et al.

    Endothelial cell culture: protocol to obtain and cultivate human umbilical endothelial cells

    J Immunol Methods

    (2001)
  • K.H. Hong et al.

    Monocyte chemoattractant protein-1-induced angiogenesis is mediated by vascular endothelial growth factor-A

    Blood

    (2005)
  • S.Y. Park et al.

    Hypoxia enhances LPA-induced HIF-1alpha and VEGF expression: their inhibition by resveratrol

    Cancer Lett

    (2007)
  • J.M. Dann et al.

    Regulation of Vascular Endothelial Growth Factor in endometrial tumour cells by resveratrol and EGCG

    Gynecol Oncol

    (2009)
  • H.P. Gerber et al.

    Vascular endothelial growth factor regulates endothelial cell survival through the phosphatidylinositol 3’-kinase/Akt signal transduction pathway. Requirement for Flk-1/KDR activation

    J Biol Chem

    (1998)
  • Y. Fujio et al.

    Akt mediates cytoprotection of endothelial cells by vascular endothelial growth factor in an anchorage-dependent manner

    J Biol Chem

    (1999)
  • N.M. Mazure et al.

    Induction of vascular endothelial growth factor by hypoxia is modulated by a phosphatidylinositol 3-kinase/Akt signaling pathway in Ha-ras-transformed cells through a hypoxia inducible factor-1 transcriptional element

    Blood

    (1997)
  • S. Frame et al.

    A common phosphate binding site explains the unique substrate specificity of GSK3 and its inactivation by phosphorylation

    Mol Cell

    (2001)
  • N. Ferrara

    Vascular endothelial growth factor: basic science and clinical progress

    Endocrine Rev

    (2004)
  • L. Miquerol et al.

    Embryonic development is disrupted by modest increases in vascular endothelial growth factor gene expression

    Development (Cambridge, England)

    (2000)
  • I. Shiojima et al.

    Role of Akt signaling in vascular homeostasis and angiogenesis

    Circulat Res

    (2002)
  • M. Peifer et al.

    Wnt signaling in oncogenesis and embryogenesis—a look outside the nucleus

    Science (New York, NY)

    (2000)
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