Research ArticleExtra virgin olive oil rich in polyphenols modulates VEGF-induced angiogenic responses by preventing NADPH oxidase activity and expression☆
Graphical abstract
Introduction
Angiogenesis, the process by which new vascular networks develop from preexisting vessels, is a hallmark feature of many physiological processes. However, excessive or insufficient angiogenesis contributes to a number of pathologies, ranging from cancer, atherosclerosis, macular degeneration and retinopathy to impaired repair of ischemic tissues [1], [2]. Angiogenesis is a complex process comprising endothelial cell proliferation, migration, invasion, extracellular proteolysis, tube formation and vessel remodelling [3], [4]. The angiogenic process is controlled by many proangiogenic factors including matrix metalloproteinases (MMPs), such as the gelatinases MMP-2 and MMP-9, which degrade extracellular matrices [5], and vascular endothelial growth factor (VEGF), which strongly stimulates endothelial cell migration and proliferation and new blood vessels formation, through mechanism including MMPs induction [6]. The angiogenic activity of VEGF is mainly mediated by reactive oxygen species (ROS) produced via the activation of the reduced β-nicotinamide adenine dinucleotide phosphate (NADPH) oxidase [7], [8].
The biological function of NADPH oxidase complex is the generation of ROS, via the transfer of NADPH-derived electrons to molecular oxygen, resulting in superoxide formation. NADPH oxidase, originally identified in phagocytes, where it contributes to host defence, consists of two membrane-bound components, gp91phox (also known as Nox2) and p22phox, and several cytosolic regulatory subunits, including p40phox, p47phox, p67phox and the small GTPase Rac. Activation of NADPH oxidase requires the phosphorylation and subsequent translocation of cytosolic subunits to membrane and assembly with p22phox and Nox2. Each of these NADPH oxidase components has been identified in endothelial cells [9], which express also Nox4, a homologue of Nox2 [10]. The endothelial NADPH oxidase, differently from its leukocyte counterpart, is preassembled, displays constitutive low-level activity and produces an intracellular influx of ROS which are known to serve as second messengers activating multiple intracellular proinflammatory pathways [11], [12], [13]. There is evidence that the Nox2-type NADPH oxidase and the p47phox activation have an important role in mediating angiogenic responses both in vitro [14] and in vivo [15]. However, Nox4 also exhibits a critical role in mediating angiogenic responses [16], and its upregulation has been implicated in the development of cardiovascular pathologies [17], [18] and tumor growth [19].
Oxidative stress can be modulated by diet which, in turn, influences the development of noncommunicable diseases including metabolic and vascular diseases and cancer [20], [21]. The low rate of all-cause and cardiovascular mortality in Mediterranean countries can be partially explained on the basis of the antioxidative and antiinflammatory effects associated with consumption of the Mediterranean diet [22]. In particular, extra virgin olive oil, the major fatty component of the Mediterranean diet, exhibits beneficial effects which are mainly attributed to its minor components such as phenolic compounds [21]. Previous studies have shown that the consumption of extra virgin olive oil with a high content of phenolic compounds improves endothelial dysfunction and reduces oxidative stress plasma parameters [23], [24], [25]. The phenolic composition of olive oil varies in quantity (50–800 mg/kg) and quality depending on the olive variety, the degree of ripeness, soil composition, climate, agricultural and processing techniques and storage [26]. Four major classes of polyphenols can be found in extra virgin olive oil including flavonoids, lignans, simple phenols and secoiridoids. The last two groups can be found exclusively in olive oils [27]. In addition to their direct antioxidant effects, olive oil polyphenols counteract the cellular response induced by oxidative stress and improve endothelial function [28], [29].
In our earlier study, pure olive oil polyphenols, such as hydroxytyrosol and oleuropein, have been shown to reduce the inflammatory angiogenesis in human vascular endothelial cells [30] through reduced intracellular oxidative stress. However, the olive oil polyphenols effects on NADPH oxidase have not yet been investigated. Since food extracts are often more effective than their isolated constituents [31], in the present study, we evaluated the effects of total polyphenols extracted from Apulian extra virgin olive oil on the VEGF-induced angiogenic responses and the potential interference with NADPH oxidase. Since most of the polyphenolic compounds are modified after olive oil intake and their derived metabolites are found in plasma [32], [33], we also evaluated the effects of the consumption of extra virgin olive oil on the vascular endothelium, by treating endothelial cells with serum obtained after the intake of olive oil-based breakfasts with high polyphenol content.
Section snippets
Materials
The extra virgin olive oil employed in this study was from the cultivar Coratina obtained from the Apulian region (Italy). The materials for cell cultures were obtained from Gibco/BRL. The ROS-sensitive probe carboxy-2’,7’-dichlorofluorescein diacetate (carboxy-H2DCFDA) was purchased from Molecular Probes, while lucigenin and VEGF-A165 were obtained from Sigma-Aldrich. Primary antibodies against p47phox, Nox4, Na+/K+ATPase and peroxidase-conjugated secondary antibody were purchased from Santa
OOPE inhibits VEGF-induced angiogenic response in endothelial cells
Seeing as the intake of olive oil polyphenols in the Mediterranean countries has been estimated to be about 20–30 mg/die and the circulating levels of olive oil phenols in the range of μg/ml [41], we decided to perform our investigations using OOPE concentrations from 0.1 to 10 μg/ml also in accordance with the range used by Dell’Agli et al. [42]. Quantitative and qualitative analysis of olive oil polyphenols showed that total phenols accounted for about 590 mg/kg of oil (Table 1). The main
Discussion
Our findings show that OOPE was able to reduce VEGF-induced angiogenic responses in a concentration-dependent manner, through inhibition of cell migration, invasiveness and tube-like structure formation. Previous studies showed the antiangiogenic effects of pure olive oil polyphenols in endothelial and tumor cells [30], [44], [45], [46], [47]. For the first time, we report here the antiangiogenic effects of total extra virgin olive oil polyphenolic extract in human vascular endothelial cells
Conflict of interest
The authors have no conflict of interest.
Acknowledgements
The authors are grateful to the Division of Obstetrics and Gynecology at the “Vito Fazzi” Hospital in Lecce (Italy) and at the “Ignazio Veris Delli Ponti” Hospital in Scorrano (Lecce, Italy) for providing umbilical cords. This work was partially supported by Grants from Apulia Region (Italy), POR Strategic Projects, CIP PS_101 and by Grants from National Operational Programme for Research and Competitiveness 2007–2013, PON01_01958, PIVOLIO.
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Grants and funding sources: This work was partially supported by grants from Apulia Region (Italy), POR Strategic Projects, CIP PS_101 and by Grants from National Operational Programme for Research and Competitiveness 2007–2013, PON01_01958, PIVOLIO.