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