Research Article
Extra virgin olive oil rich in polyphenols modulates VEGF-induced angiogenic responses by preventing NADPH oxidase activity and expression

https://doi.org/10.1016/j.jnutbio.2015.09.026Get rights and content

Abstract

Previous studies have shown the antiinflammatory, antioxidant and antiangiogenic properties by pure olive oil polyphenols; however, the effects of olive oil phenolic fraction on the inflammatory angiogenesis are unknown. In this study, we investigated the effects of the phenolic fraction (olive oil polyphenolic extract, OOPE) from extra virgin olive oil and related circulating metabolites on the VEGF-induced angiogenic responses and NADPH oxidase activity and expression in human cultured endothelial cells. We found that OOPE (1–10 μg/ml), at concentrations achievable nutritionally, significantly reduced, in a concentration-dependent manner, the VEGF-induced cell migration, invasiveness and tube-like structure formation through the inhibition of MMP-2 and MMP-9. OOPE significantly (P<0.05) reduced VEGF-induced intracellular reactive oxygen species by modulating NADPH oxidase activity, p47phox membrane translocation and the expression of Nox2 and Nox4. Moreover, the treatment of endothelial cells with serum obtained 4 h after acute intake of extra virgin olive oil, with high polyphenol content, decreased VEGF-induced NADPH oxidase activity and Nox4 expression, as well as, MMP-9 expression, as compared with fasting control serum. Overall, native polyphenols and serum metabolites of extra virgin olive oil rich in polyphenols are able to lower the VEGF-induced angiogenic responses by preventing endothelial NADPH oxidase activity and decreasing the expression of selective NADPH oxidase subunits. Our results provide an alternative mechanism by which the consumption of olive oil rich in polyphenols may account for a reduction of oxidative stress inflammatory-related sequelae associated with chronic degenerative diseases.

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.

References (66)

  • H. Zrelli et al.

    Hydroxytyrosol reduces intracellular reactive oxygen species levels in vascular endothelial cells by upregulating catalase expression through the AMPK-FOXO3a pathway

    Eur J Pharmacol

    (2011)
  • E. Scoditti et al.

    Mediterranean diet polyphenols reduce inflammatory angiogenesis through MMP-9 and COX-2 inhibition in human vascular endothelial cells: a potentially protective mechanism in atherosclerotic vascular disease and cancer

    Arch Biochem Biophys

    (2012)
  • V.L. Singleton et al.

    Analysis of total phenols and other oxidation substrates and antioxidants by means of Folin-Ciocalteu reagent

    Methods Enzymol

    (1999)
  • E. Scoditti et al.

    Hydroxytyrosol suppresses MMP-9 and COX-2 activity and expression in activated human monocytes via PKCalpha and PKCbeta1 inhibition

    Atherosclerosis

    (2014)
  • S. Lamy et al.

    Diet-derived polyphenols inhibit angiogenesis by modulating the interleukin-6/STAT3 pathway

    Exp Cell Res

    (2012)
  • M. Ushio-Fukai et al.

    Reactive oxygen species and angiogenesis: NADPH oxidase as target for cancer therapy

    Cancer Lett

    (2008)
  • R.F. Wu et al.

    Vascular endothelial growth factor causes translocation of p47phox to membrane ruffles through WAVE1

    J Biol Chem

    (2003)
  • R. Carnevale et al.

    Extra virgin olive oil blunt post-prandial oxidative stress via NOX2 down-regulation

    Atherosclerosis

    (2014)
  • H. Xu et al.

    Differential roles of PKCalpha and PKCepsilon in controlling the gene expression of Nox4 in human endothelial cells

    Free Radic Biol Med

    (2008)
  • P. Rosignoli et al.

    Effect of olive oil phenols on the production of inflammatory mediators in freshly isolated human monocytes

    J Nutr Biochem

    (2013)
  • Z. Jia et al.

    Luteolin protects against vascular inflammation in mice and TNF-alpha-induced monocyte adhesion to endothelial cells via suppressing IKappaBalpha/NF-kappaB signaling pathway

    J Nutr Biochem

    (2015)
  • S. Lamy et al.

    Olive oil compounds inhibit vascular endothelial growth factor receptor-2 phosphorylation

    Exp Cell Res

    (2014)
  • K.L. Tuck et al.

    Major phenolic compounds in olive oil: metabolism and health effects

    J Nutr Biochem

    (2002)
  • P. Carmeliet et al.

    Molecular mechanisms and clinical applications of angiogenesis

    Nature

    (2011)
  • P. Carmeliet et al.

    Angiogenesis in cancer and other diseases

    Nature

    (2000)
  • J. Folkman

    Angiogenesis

    Annu Rev Med

    (2006)
  • S.M. Weis et al.

    Tumor angiogenesis: molecular pathways and therapeutic targets

    Nat Med

    (2011)
  • J.E. Rundhaug

    Matrix metalloproteinases and angiogenesis

    J Cell Mol Med

    (2005)
  • A.S. Chung et al.

    Developmental and pathological angiogenesis

    Annu Rev Cell Dev Biol

    (2011)
  • K.K. Griendling et al.

    NAD(P)H oxidase: role in cardiovascular biology and disease

    Circ Res

    (2000)
  • A. Gorlach et al.

    A gp91phox containing NADPH oxidase selectively expressed in endothelial cells is a major source of oxygen radical generation in the arterial wall

    Circ Res

    (2000)
  • T. Ago et al.

    Nox4 as the major catalytic component of an endothelial NAD(P)H oxidase

    Circulation

    (2004)
  • C. Kunsch et al.

    Oxidative stress as a regulator of gene expression in the vasculature

    Circ Res

    (1999)
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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.

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