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
Introduction
Angiogenesis, the development of new capillaries from pre-existing microvessels, plays a crucial role in normal physiological processes [1]. However, when the dynamic balance between stimulators and inhibitors is tilted, excessive angiogenesis ensues and represent a major contributor to a number of pathologies, including inflammatory diseases and cancer. A critical role has been attributed to angiogenic factor-induced production of reactive oxygen species (ROS)1 in stimulating redox-signaling pathways leading to gene induction involved in angiogenesis [1]. Crucial players in the angiogenic process also include matrix metalloproteinases (MMPs), zinc-dependent endopeptidases, which degrade extracellular matrix components allowing endothelial cells to penetrate and reshape connective tissue. In particular, endothelial cells have been described to produce, among others, MMP-9, which plays a major role in initiating and sustaining angiogenesis [2], [3]. MMP-9 contributes to tumor growth and metastasis [4], as well as to atherosclerotic lesion formation and vulnerability [5]. Another important pathway regulating physiological/pathological angiogenic processes involves the cyclooxygenase (COX) enzymes, that catalyse the committed step in prostanoid synthesis, converting free arachidonic acid into the prostaglandin (PG) precursors PGG2 and PGH2 [6]. The actions of tissue-specific terminal PG synthases then catalyse the formation of prostanoids, including prostacyclin (PGI2) and PGE2. While COX-1 is constitutively expressed by most cell types, COX-2 is induced by pro-inflammatory and mitogenic stimuli and mediates inflammation, atherosclerosis [7], and cancer promotion [7]. There is also growing evidence for the involvement of COX-2-derived mediators in angiogenic processes [8], and in keeping with this role nonsteroidal anti-inflammatory drugs that selectively block COX-2 activity have both anti-angiogenic and anti-carcinogenic actions [7]. Furthermore, both COX-2 and MMP-9 expression is regulated by ROS-sensitive pathways [9], [10], and a functional link between COX-2 activity and MMP-9 production has been described in different cell types, including endothelial cells [11], suggesting COX-2 and related MMP-9 as strategic therapeutic targets for angiogenesis inhibition.
Diet is a cornerstone in cancer and cardiovascular disease prevention. In particular, growing evidence has demonstrated the beneficial and preventive role of the Mediterranean diet in the occurrence of diseases associated with inflammation, oxidative damage and angiogenesis, including coronary heart disease, cancer, metabolic and neurodegenerative diseases [12]. Although generally considered non-nutritive agents, plant polyphenols have drawn much attention as potential contributors to the Mediterranean diet beneficial properties [13], [14]. Antioxidant polyphenols from olives and virgin olive oil [15], [16], [17] and from red wine [17], [18] have been shown to exert important anti-inflammatory, cardioprotective and anti-cancer activities both in vitro and in vivo. In particular, some anti-angiogenic properties in endothelial cells have been described for resveratrol [19], [20] and quercetin [21], but the molecular mechanisms remain largely unclear. On the contrary, the effect of olive oil antioxidants on angiogenesis have not been explored.
Aim of the study was to investigate and compare the effects of dietary polyphenols from virgin olive oil, oleuropein (OE), hydroxytyrosol (HT), and from red wine, resveratrol (RSV) and quercetin (Q), on endothelial cell angiogenic responses in inflammatory condition induced by the pro-angiogenic factor phorbol 12-myristate 13-acetate (PMA) [22], and to explore potential mechanisms of action, investigating their effects on COX-2 and MMP-9 expression.
Section snippets
Materials
HT (2-(3,4-dihydroxyphenyl)ethanol) and trans-RSV (3,4′5-trihydroxy-trans-stilbene) were obtained from Cayman Chemical (Ann Arbor, MI), glycoside OE (2-(3,4-dihydroxyphenyl)ethyl(2S-(2alpha,3E,4beta))-3-ethyllidene-2-(beta-d-glucopyranosyloxy)-3,4-dihydro-5-(methoxycarbonyl)-2H-pyran-4-acetate) from Extrasynthese (Genay Cedex, France), Q (3,5,7,3′,4′-pentahydroxylflavone) and PMA were obtained from Sigma–Aldrich (St. Louis, MO). The collagenase inhibitor GM6001 was obtained from Chemicon
Antioxidant polyphenols from virgin olive oil and red wine inhibit angiogenic responses in endothelial cells
We first evaluated the effects of four dietary polyphenols, OE, HT, RSV and Q (Fig. 1A), on angiogenesis in vitro by performing Matrigel and scratch wound healing assays, which measure endothelial tube formation and migration, respectively (Fig. 1). As shown in Fig. 1B and C, a 1 h pretreatment with polyphenols reduced the stimulated tube-like differentiation of HUVEC in Matrigel in a concentration-dependent manner, with a significant effect for OE and HT starting from 10 μmol/L, whereas a
Discussion
In the present study we demonstrated that the polyphenols OE, HT, Q and RSV suppressed inflammatory angiogenesis in cultured human vascular endothelial cells via the attenuation of stimulated COX-2 expression and prostanoid production as well as MMP-9 release.
Inhibition of pathological angiogenesis has become a promising target for preventive and therapeutic purposes in cancer as well as in many inflammatory disorders, including atherosclerotic vascular diseases [31], [32]. During the last
Conflict of Interest
None to declare.
Acknowledgments
This work was supported by Grants from Apulian Region (Italy), POR Strategic Projects [Grant numbers CIP PS_101 and CIP PS_008]. We 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.
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