Maria Annunziata Carluccio

Olive Oil and Red Wine Antioxidant Polyphenols Inhibit Endothelial Activation. Antiatherogenic Properties of Mediterranean Diet Phytochemicals

Objective—Epidemiology suggests that Mediterranean diets are associated with reduced risk of cardiovascular disease. Because monocyte adhesion to the endothelium is crucial in early atherogenesis, we evaluated whether typical olive oil and red wine polyphenols affect endothelial–leukocyte adhesion molecule expression and monocyte adhesion. Methods and Results—Phytochemicals in olive oil and red wine, including oleuropein, hydroxytyrosol, tyrosol, elenolic acid, and resveratrol, with or without antioxidant activity, were incubated with human umbilical vein endothelial cells for 30 minutes, followed by co-incubation with bacterial lipopolysaccharide or cytokines to trigger adhesion molecule expression. At nutritionally relevant concentrations, only oleuropein, hydroxytyrosol, and resveratrol, possessing a marked antioxidant activity, reduced monocytoid cell adhesion to stimulated endothelium, as well as vascular cell adhesion molecule-1 (VCAM-1) mRNA and protein by Northern analysis and cell surface enzyme immunoassay. Reporter gene assays with deletional VCAM-1 promoter constructs indicated the relevance of nuclear factor-&kgr;B, activator protein-1, and possibly GATA binding sites in mediating VCAM-1 transcriptional inhibition. The involvement of nuclear factor-&kgr;B and activator protein-1 was finally demonstrated at electrophoretic mobility shift assays. Conclusions—Olive oil and red wine antioxidant polyphenols at nutritionally relevant concentrations transcriptionally inhibit endothelial adhesion molecule expression, thus partially explaining atheroprotection from Mediterranean diets.

Oleic Acid Inhibits Endothelial Activation: A Direct Vascular Antiatherogenic Mechanism of a Nutritional Component in the Mediterranean Diet

Because oleic acid is implicated in the antiatherogenic effects attributed to the Mediterranean diet, we investigated whether this fatty acid can modulate endothelial activation, ie, the concerted expression of gene products involved in leukocyte recruitment and early atherogenesis. We incubated sodium oleate with human umbilical vein endothelial cells for 0 to 72 hours, followed by coincubation of oleate with human recombinant tumor necrosis factor, interleukin (IL)-1alpha, IL-1beta, IL-4, Escherichia coli lipopolysaccharide (LPS), or phorbol 12-myristate 13-acetate for a further 6 to 24 hours. The endothelial expression of vascular cell adhesion molecule-1 (VCAM-1), E-selectin, and intercellular adhesion molecule-1 was monitored by cell surface enzyme immunoassays or flow cytometry, and steady-state levels of VCAM-1 mRNA were assessed by Northern blot analysis. At 10 to 100 micromol/L for >24 hours, oleate inhibited the expression of all adhesion molecules tested. After a 72-hour incubation with oleate and a further 16-hour incubation with oleate plus 1 microg/mL LPS, VCAM-1 expression was reduced by >40% compared with control. Adhesion of monocytoid U937 cells to LPS-treated endothelial cells was reduced concomitantly. Oleate also produced a quantitatively similar reduction of VCAM-1 mRNA levels on Northern blot analysis and inhibited nuclear factor-kappaB activation on electrophoretic mobility shift assays. Incubation of endothelial cells with oleate for 72 hours decreased the relative proportions of saturated (palmitic and stearic) acids in total cell lipids and increased the proportions of oleate in total cell lipids without significantly changing the relative proportions of polyunsaturated fatty acids. Although less potent than polyunsaturated fatty acids in inhibiting endothelial activation, oleic acid may contribute to the prevention of atherogenesis through selective displacement of saturated fatty acids in cell membrane phospholipids and a consequent modulation of gene expression for molecules involved in monocyte recruitment.

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.

Diets with high content of antioxidant polyphenols are associated with low prevalence of cardiovascular diseases and cancer. Inflammatory angiogenesis is a key pathogenic process both in cancer and atherosclerosis, and is tightly regulated by the proinflammatory enzyme cyclooxygenase (COX)-2 and the matrix degrading enzymes matrix metalloproteinases (MMPs). We studied the effects of antioxidant polyphenols from virgin olive oil (oleuropein and hydroxytyrosol) and red wine (resveratrol and quercetin) on endothelial cell angiogenic response in vitro, and explored underlying mechanisms. Cultured endothelial cells were pre-incubated with 0.1-50 μmol/L polyphenols before stimulation with phorbol myristate acetate (PMA). All tested polyphenols reduced endothelial cell tube formation on matrigel and migration in wound healing assays. The reduced angiogenesis was associated with the inhibition of PMA-induced COX-2 protein expression and prostanoid production, as well as MMP-9 protein release and gelatinolytic activity. These effects were accompanied by a significant reduction in the stimulated intracellular reactive oxygen species levels and in the activation of the redox-sensitive transcription factor nuclear factor (NF)-κB. Our findings reveal that olive oil and red wine polyphenols reduce inflammatory angiogenesis in cultured endothelial cells, through MMP-9 and COX-2 inhibition, supporting a potential protective role for dietary polyphenols in atherosclerotic vascular disease and cancer.

Basic mechanisms behind the effects of n-3 fatty acids on cardiovascular disease

The epidemiological association between high intakes of n-3 fatty acids (FA) and decreased morbidity and mortality from cardiovascular disease (CVD) can be explained by two main basic mechanisms: (a) an effect on atherothrombosis, and (b) an effect on cardiac arrhythmias. These mechanisms probably reflect different beneficial influences of n-3 FA on cardiovascular biology. Effects on atherothrombosis include the modulation of the expression of pro-atherogenic genes (e.g., endothelial leukocyte adhesion molecules, inflammatory cytokines and cyclooxygenase (COX)-2) and the hepatic synthesis of very low density lipoproteins (VLDL), and are slow in onset, requiring incorporation into cell membrane phospholipids, and usually doses in humans in the order of 3g/day or higher. Effects on cardiac arrhythmias include complex interactions with ion channels (sodium, potassium and calcium channels), typically requiring the presence of free FA in extracellular fluids and usually occurring with lower doses (around 1g/day) of nutritional or pharmacological intake. We have focused most of our research effort in unraveling the pathophysiological background of protection by n-3 FA from atherothrombosis. As the result of incorporation of n-3 FA in the sn-2 position predominantly of the phosphatidyl ethanolamine pool in the inner leaflet of the plasma membrane, n-3 FA appear on the one hand to increase the production of bioactive lipid mediators (protectins and resolvins) affecting cytokine-induced signal transduction; and on the other hand to directly interfere with the generation of reactive oxygen species (mostly hydrogen peroxide), directly responsible for the activation of the transcription factor nuclear factor (NF)-kappaB, which controls the expression of a variety of pro-inflammatory and pro-atherogenic genes, including those encoding for interleukin (IL)-1, IL-6, IL-8, tumor necrosis factor (TNF)alpha, vascular cell adhesion molecule-1 (VCAM-1), E-selectin, and COX-2. The upstream-direct or indirect-inhibition of cytokine- and other atherogenic trigger-induced signaling pathway may involve interference with the activation of protein kinase (PK) C isoforms and NADP(H) oxidase. Such interference may also explain the blunt anti-inflammatory effect of n-3 FA in many experimental models and clinical conditions of inflammation. All together, these mechanisms may provide an integrated view of how n-3 FA may affect CVD.

Statins inhibit cyclooxygenase-2 and matrix metalloproteinase-9 in human endothelial cells: anti-angiogenic actions possibly contributing to plaque stability

AIMS Cyclooxygenase (COX)-2 expression is increased in inflammation and angiogenesis and also in atherosclerotic plaques, where it co-localizes with metalloproteinases (MMPs) involved in the fibrous cap weakening. Insight into the regulation of COX-2 and MMP-9 expression suggests the involvement of a Rho-dependent pathway. Because statins interfere with Rho activation, we investigated the statin effect on COX-2 and MMP expressions in the human endothelium. METHODS AND RESULTS Simvastatin and atorvastatin were incubated with endothelial cells for 12 h before stimulation with phorbol myristate acetate or tumour necrosis factor-alpha, for times suitable to assess the endothelial tube differentiation on Matrigel and COX-2 and MMPs activities, proteins, and mRNA expressions. At 0.1-10 micromol/L, both statins reduced COX-2 expression and activity, without affecting COX-1. The statin effect was reversed by mevalonate and geranylgeranyl-pyrophosphate and mimicked by the Rho inhibitor C3 transferase, indicating the involvement of Rho in the signal transduction pathway leading to COX-2 expression. In parallel, statins, as well as COX-2 inhibitors, reduced the MMP-9 stimulated release and the endothelial tubular differentiation. CONCLUSION In the human vascular endothelium, statins reduce COX-2 and MMP-9 expression and activity. Through this mechanism, statins exert an anti-angiogenic effect possibly contributing to the cholesterol-lowering-unrelated protective effects of statins against plaque inflammatory angiogenesis and rupture.

PPARγ agonists inhibit angiogenesis by suppressing PKCα- and CREB-mediated COX-2 expression in the human endothelium

AIMS The activation of peroxisome proliferator-activated receptor (PPAR)gamma is known to inhibit angiogenesis. As a potential mechanism for this, we aimed at examining the effects of PPARgamma agonists on the pro-angiogenic enzyme cyclooxygenase (COX)-2 in human endothelium. METHODS AND RESULTS Cultured endothelial cells were pre-incubated with the PPARgamma agonists rosiglitazone (RSG) or GW1929 before stimulation with vascular endothelial growth factor (VEGF) or phorbol myristate acetate (PMA). RSG and GW1929 attenuated VEGF- and PMA-stimulated COX-2 activity, as well as protein and mRNA expression. This effect was abolished by the PPARgamma antagonists bisphenol A diglycidyl ether and GW9662 as well as by PPARgamma small-interfering RNAs (siRNAs). Transient transfection experiments revealed that the induction of COX-2 promoter was significantly inhibited by RSG through an interference with the cAMP response element (CRE) site. COX-2 downregulation after siRNA targeting CRE-binding protein (CREB) confirmed the role of CREB in mediating COX-2 transcription. Correspondingly, PPARgamma agonists attenuated CREB activation. As both protein kinase C (PKC)alpha and beta are involved in VEGF-induced COX-2 expression and CREB activation, we investigated which isoform(s) of PKC was affected by RSG. RSG only reduced VEGF- and PMA-stimulated PKCalpha membrane translocation. CONCLUSION VEGF induces CREB-mediated COX-2 expression through a PKCalpha-dependent pathway in human endothelium. The anti-angiogenic effect of PPARgamma agonists is due, at least in part, to an interference with the VEGF-stimulated PKCalpha-mediated activation of CREB and the related expression of COX-2.

Hydroxytyrosol suppresses MMP-9 and COX-2 activity and expression in activated human monocytes via PKCα and PKCβ1 inhibition.

OBJECTIVE Hydroxytyrosol (HT), the major olive oil antioxidant polyphenol in cardioprotective Mediterranean diets, is endowed with anti-inflammatory and anti-atherosclerotic activity. The production of cyclooxygenase (COX)-2-dependent inflammatory eicosanoids and the functionally linked release of matrix metalloproteinase (MMP)-9 by macrophages likely contribute to plaque instability leading to acute coronary events. Objective of the study was to examine the HT effects on inflammatory markers in human activated monocytes, including MMP-9 and COX-2 activity and expression and explore HT underlying mechanisms. METHODS AND RESULTS Human peripheral blood mononuclear cells (PBMC) and U937 monocytes were treated with 1-10 μmol/L HT before activation with phorbol myristate acetate (PMA). HT blunted monocyte matrix invasive potential and reduced MMP-9 release and expression at zymography, ELISA and RT-PCR, with an IC50 = 10 μmol/L ( P< 0.05), without affecting tissue inhibitor of metalloproteinase (TIMP)-1. Moreover, HT inhibited prostaglandin (PG)E2 production and COX-2 expression, without affecting COX-1. These effects were mediated by inhibition of transcription factor nuclear factor (NF)-κB and protein kinase C (PKC)α and PKCβ1 activation. CONCLUSION HT, at nutritionally relevant concentrations, reduces MMP-9 and COX-2 induction in activated human monocytes via PKCα and PKCβ1 inhibition, thus featuring novel anti-inflammatory properties. Overall, such results contribute to explaining the vascular protective effects by olive oil polyphenols in Mediterranean diets.

Omega-3 fatty acids, inflammation and angiogenesis: nutrigenomic effects as an explanation for anti-atherogenic and anti-inflammatory effects of fish and fish oils.

Atherosclerosis is a dynamic process with inflammatory aspects playing a considerable pathogenetic role. In this process, the vascular endothelium is the key regulator of vascular function, promoting the maintenance of vascular homeostasis or the progression towards vascular disease. In the past 30 years, the dietary intake of omega–3 (n–3) polyunsaturated fatty acids – mainly derived from fish – has emerged as an important way to modify cardiovascular risk through beneficial effects on all stages of atherosclerosis. This review specifically focuses on the modulating effects of n–3 fatty acids on molecular events involved in early and late atherogenesis, including effects on endothelial expression of adhesion molecules, as well as pro-inflammatory and pro-angiogenic enzymes. By accumulating in endothelial membrane phospholipids, omega–3 fatty acids have been shown to decrease the transcriptional activation of several genes through a decreased activation of the nuclear factor-ĸB system of transcription factors. This occurs secondary to decreased generation of intracellular reactive oxygen species. This series of investigations configures a clear example of nutrigenomics, i.e. how nutrients may affect gene expression, ultimately affecting a wide spectrum of human diseases.

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

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.

Additive regulation of adiponectin expression by the mediterranean diet olive oil components oleic Acid and hydroxytyrosol in human adipocytes.

Adiponectin, an adipocyte-derived insulin-sensitizing and anti-inflammatory hormone, is suppressed in obesity through mechanisms involving chronic inflammation and oxidative stress. Olive oil consumption is associated with beneficial cardiometabolic actions, with possible contributions from the antioxidant phenol hydroxytyrosol (HT) and the monounsaturated fatty acid oleic acid (OA, 18:1n-9 cis), both possessing anti-inflammatory and vasculo-protective properties. We determined the effects of HT and OA, alone and in combination, on adiponectin expression in human and murine adipocytes under pro-inflammatory conditions induced by the cytokine tumor necrosis factor(TNF)-α. We used human Simpson-Golabi-Behmel syndrome (SGBS) adipocytes and murine 3T3-L1 adipocytes as cell model systems, and pretreated them with 1-100 μmol/L OA, 0.1-20 μmol/L HT or OA plus HT combination before stimulation with 10 ng/mL TNF-α. OA or HT significantly (P<0.05) prevented TNF-α-induced suppression of total adiponectin secretion (by 42% compared with TNF-α alone) as well as mRNA levels (by 30% compared with TNF-α alone). HT and OA also prevented—by 35%—TNF-α-induced downregulation of peroxisome proliferator-activated receptor PPARγ. Co-treatment with HT and OA restored adiponectin and PPARγ expression in an additive manner compared with single treatments. Exploring the activation of JNK, which is crucial for both adiponectin and PPARγ suppression by TNF-α, we found that HT and OA additively attenuated TNF-α-stimulated JNK phosphorylation (up to 55% inhibition). In conclusion, the virgin olive oil components OA and HT, at nutritionally relevant concentrations, have additive effects in preventing adiponectin downregulation in inflamed adipocytes through an attenuation of JNK-mediated PPARγ suppression.