Inhibition of sphingosine-1-phosphate- and vascular endothelial growth factor-induced endothelial cell chemotaxis by red grape skin polyphenols correlates with a decrease in early platelet-activating factor synthesis

Abstract

Vascular endothelial growth factor (VEGF) and platelet-derived lipid sphingosine-1-phosphate (S1P) are two proinflammatory mediators which contribute to angiogenesis, in part through the synthesis of platelet-activating factor (PAF). The red grape skin polyphenolic extract (SGE) both prevents and inhibits angiogenesis in the Matrigel model, decreases the basal motility of endothelial and cancer cells, and reverses the chemotactic effect of S1P and VEGF on bovine aortic endothelial cells (BAECs) as well as the chemotactic effect of conditioned medium on human HT-1080 fibrosarcoma, human U-87 glioblastoma, and human DAOY medulloblastoma cells. Inhibition of VEGF- and S1P-mediated chemotaxis by SGE is associated with a down-regulation of ERK and p38/MAPK phosphorylation and a decreased in acute PAF synthesis. Notably, as do extracellular inhibitors of PAF receptor, SGE prevents S1P-induced PAF synthesis and the resulting activation of the S1P/endothelial differentiation gene-1 cascade. Given the key role of VEGF and S1P in inflammation, angiogenesis, and tumor invasion, SGE may therefore contribute to prevent (or to delay) the development of diseases associated with angiogenesis dysregulation, including cancer. The dual inhibition of S1P- and VEGF-mediated migration of endothelial cell and of serum-stimulated migration of U-87 cells suggests a usefulness of SGE against highly invasive human glioblastoma.

Introduction

Vascular endothelial growth factor (VEGF) has first been identified as a platelet-derived growth factor [1]. It is already established that free radicals markedly increase the expression and the release of VEGF in a variety of cell types [2], [3]. VEGF contributes to inflammation through the synthesis by endothelial cells (EC) of platelet-activating factor (PAF), a potent inflammatory mediator capable of promoting EC migration [4]. Compelling evidence has revealed that VEGF expressed by cancer cells deprived from oxygen or nutrients is capable of inducing neovascularization (neoangiogenesis), the process by which tumors grow and invade surrounding host tissues [1], [5], [6]. Thereby, in addition to its proinflammatory effects, VEGF, through binding with high affinity on VEGF receptor-2 (VEGFR-2, Flk-1/KDR) at the endothelial cell surface, contributes to switch tumors from a dormant toward an invasive state [5]. Of note, VEGF is the proangiogenic cytotokine most closely associated with aggressive tumor proliferation [5], [6], [7], [8], [9]. As a result, inhibition of VEGF-mediated signaling in EC has become a primary target for antiangiogenic strategies [8]. Interestingly, in clinical practice, VEGF signaling may serve both as a prognosis factor of tumor aggressivity and as a marker for the effectiveness of antiangiogenic inhibitors used in cancer therapy [9], [10].

In man, various agents present in serum contribute to amplify VEGF signaling and, consequently, aggressive cancer growth [11]. One of the most effective is the sphingolipid derivative: sphingosine 1-phosphate (S1P), which has recently been identified as the major chemoattractant of serum [12]. This bioactive lipid is released by stimulated platelets during activation of clotting cascade and by several cell types in response to various extracellular stimuli [13]. It is involved in a variety of physiological processes, including thrombosis and hemostasis [12], and plays an important role in the regulation of EC migration and proliferation which are prerequisite of the angiogenic process [12], [14]. It is already established that in vivo, S1P contributes to neovascularization [12], to inflammation [14], and to the metastastic cascade [15]. Recently, S1P-driven EC inflammation has been associated with the synthesis of platelet-activating factor, a potent inflammatory mediator which takes part in the S1P chemotactic effect, and S1P-induced EC migration has been demonstrated to be PAF dependent [16].

All these data support a close link between increased S1P and VEGF signaling, inflammation, neoangiogenesis, and cancer invasion and metastasis. By inhibiting events sensitive to redox regulations, such as the activity of key enzymes involved in inflammation and angiogenesis, radical scavenging agents may thereby positively affect cancer proliferation. Until the discovery of the “French paradox” [17], much attention has been focused on the health properties of polyphenols from wine and grape berries (see [18], [19] as reviews). These properties depend in large part to their antioxidant and free radical scavenging properties (OK). Because these are dose dependent and structure related [20], small qualitative and quantitative differences in the polyphenolic pattern of grape extracts are likely to have profound repercussions on the antiangiogenic, anti-inflammatory, and anticancer activity of these extracts. Agarwal and co-workers have reported that grape seed anthocyanins inhibit angiogenesis in vitro [21], [22] while another team has reported the opposite effects [23], [24]. It cannot be excluded that these different results are due to differences in the chemical composition of the extracts tested by each team. The polyphenolic composition of red grape skin polyphenols significantly differs from that of grape seeds [25]. To our knowledge, neither the antiangiogenic activity of red grape skin extract nor, more generally, the effects of grape polyphenols on S1P-induced EC migration have been examined. In the present study, we show for the first time that in vitro, red grape skin polyphenols issued from Cabernet Sauvignon berries (SGE) decreases the basal motility of endothelial and cancer cells, and antagonizes the chemoattractant effect of VEGF and S1P on BAEC, in part by down-regulating PAF synthesis. We demonstrated that SGE not only inhibits and prevents angiogenesis in a dose-dependent manner but also reduces the migration of cancer cells under basal and stimulated conditions. Taken together, the present findings suggest a chemopreventive and a therapeutic value of SGE against cancer and diseases characterized by alterations in the physiological mechanisms controlling angiogenesis. Considering its capacity to antagonize both S1P- and VEGF-induced EC motility, and the motility of U-87 human glioblastoma cells induced by conditioned medium, SGE may, in particular, be helpful against highly invasive glioblastoma.