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)-?. We used human Simpson-Golabi-Behmel syndrome (SGBS) adipocytes and murine 3T3-L1 adipocytes as cell model systems, and pretreated them with 1-100 ?mol/L OA, 0.1-20 ?mol/L HT or OA plus HT combination before stimulation with 10 ng/mL TNF-?. OA or HT significantly (P<0.05) prevented TNF-?-induced suppression of total adiponectin secretion (by 42% compared with TNF-? alone) as well as mRNA levels (by 30% compared with TNF-? alone). HT and OA also prevented-by 35%-TNF-?-induced downregulation of peroxisome proliferator-activated receptor PPAR?. Co-treatment with HT and OA restored adiponectin and PPAR? expression in an additive manner compared with single treatments. Exploring the activation of JNK, which is crucial for both adiponectin and PPAR? suppression by TNF-?, we found that HT and OA additively attenuated TNF-?-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 PPAR? suppression.

Introduction: Polyphenols of wine have been extensively studied in relation to their health promoting properties. Red wine polyphenols consisted of different class of compounds belonging to flavonoid pathway, such as the resveratrol, quercetin, and anthocyanins which have long been considered to reduce the incidence of mortality and morbidity from cardiovascular diseases (CVD). Endothelial and monocyte activation is a pivotal event in atherosclerotic CVD, in this study was examined the anti-inflammatory effects of Italian red wines polyphenols in human vascular cells, such as endothelial and monocytoid cells. Since the wine polyhenols depend on different factors such as grape cultivar, and wine-making practices, aim of this study was to examine the anti-atherogenic effects of polyphenolic extracts from Italian red wines obtained by two Apulian grape cultivar: Primitivo and Negroamaro. Methods: Six Apulian red wines produced by the cv Negramaro and Primitivo grapes from the 2007-2008 vintage were analyzed. For each wine, triplicate lipophilic fraction analyses were carried out as per Giovinazzo et al., 2005. HPLC wine anthocyanin analysis was performed by direct injection of wine samples as reported by De Villiers et al. 2004. Human Umbilical Vein Endothelial Cells (HUVEC) and human monocytoid cells (U937) were pre-treated with chemically synthesised polyphenols, or with Primitivo and Negroamaro polyphenolic extracts and the corresponding de-alcoholised wines, at 0,2-2 % (v/v), before stimulation with 20 nmol/L phorbol myristate acetate (PMA) or 2 mg/mL lypopolysaccaride (LPS) for 24 h. Then, HUVEC were tested for the expression of Vascular Cell Adhesion Molecule(VCAM)-1 by enzyme immunoassays (EIA), and U937 supernatants were tested for the release of matrix metalloproteinases (MMP)-9 by gelatine zymography. Results: All chemically synthesized polyphenol tested reduced, in a concentration dependent manner, the stimulated expression of VCAM-1 in LPS triggered HUVEC. In the same conditions, lipophilic fraction extracted from both Primitivo and Negroamaro wines as well as the corresponding de-alcoholised wines exhibited an even higher inhibition of VCAM-1 expression (up to 50-70 fold higher than those expected on the base of individual polyphenol content). Similarly, either Primitivo and Negroamaro polyphenolic extracts reduced the PMA stimulated release of MMP-9 by PMA triggered U937.Conclusions: In this study different class of polyphenols from Primitivo and Negramaro red wines were characterised. These fractions, significantly inhibited the monocyte-derived extracellular matrix proteases release as well as the endothelial expression of athero-adhesion molecules. These inhibitory effects could explain, at least in part, the anti-inflammatory and cardio-protective properties of red wine polyphenols.

Prunus mahaleb L. is a marginal fruit crop producing cherry-like dark purple drupes with avery bitter taste, rich in polyphenolic compounds.A mahaleb fruit concentrated extract (mfce)has been assayed for its biological activities. In this work, we report results on the in vitroeffects of mfce including: i) anti-proliferative, gap junction intercellular communication (GJIC)modulation and pro-apoptotic properties in a breast cancer cell line (MCF-7), ii) antiinflammatoryproperties in human vascular endothelial cells (HUVEC), iii) anti-mutageniceffect on yeast Saccharomyces cerevisiae strain D7. Mfce exerted a dose-dependent antiproliferativeeffect on MCF-7 cells together with a significant time-dependent increase inGJIC and a pro-apoptotic effect. Furthermore, mfce significantly reduced levels of endothelialinflammatory antigens in a concentration-dependent manner. Finally, mfce protectedS. cerevisiae cells against H2O2 cytotoxicity by direct radical scavenging activity. This studydemonstrated multiple healthful biological effects of mfce, highlighting it as a potentialnutraceutical product.

Introduction: Wine grape pomace (WGP), the byproduct of wine making, is a good source of polyphenols, dietary fiber, metals, and may be incorporated into various food products as a functional ingredient. This study investigated the effects of different drying methods and storage up to 6 months on the retention of polyphenols and essential metals in two types of red WGP skin. The biological properties of WGP polyphenol extracts were also analysed in inflamed human cultured endothelial cells.Material & Methods: Antioxidant activity, total phenols content, polyphenol and anthocyanin analyses were carried out (Gerardi et al., 2015; D'Introno et al. 2009). Metal concentrations were measured using the ICP-AES technique (Scortichini et al., 2018). Biological activity of WGP polyphenol extracts were evaluated through the expression of endothelial adhesion molecules (VCAM-1, ICAM-1 and E-Selectin) by EIA (Calabriso et al., 2016).Results: The different drying methods tested showed similar polyphenol contents and antioxidant activity of two WGP skin. The WGP polyphenol extracts significantly reduced the endothelial adhesion molecules expression in endothelial cells stimulated by lipopolysaccharide.Conclusion: Our findings appreciate the grape skins pomace as rich source of bioactive compounds with antioxidant and anti-inflammatory activity and suggest their exploitation as functional ingredients.

Matrix metalloproteinase (MMP)-9 plays an important role in stroke by accelerating matrix degradation, disrupting the blood-brain barrier and increasing infarct size. Dipyridamole is an antiplatelet agent with recognised benefits in ischaemic stroke prevention. In addition to its antiplatelet properties, recent studies have reported that dipyridamole also features anti-inflammatory and anti-oxidant properties. We therefore investigated whether dipyridamole can ameliorate the proinflammatory profile of human monocytes, a source of MMP-9 in stroke, in terms of regulation of MMP-9 activity and expression, and explored underlying mechanisms. Human peripheral blood mononuclear cells (PBMC) and U937 cells were treated with increasing concentrations of dipyridamole (up to 10 mu g/ml) for 60 minutes before stimulation with tumour necrosis factor (TNF)-alpha or phorbol myristate acetate (PMA). Exposure of PBMC and U937 to dipyridamole reduced TNF-alpha- and PMA-induced MMP-9 activity and protein release as well as MMP-9 mRNA, without significantly affecting the release of TIMP-1. This inhibitory effect was independent of dipyridamole-induced cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) increase. Correspondingly, dipyridamole also significantly inhibited TNF-alpha-induced nuclear factor (NF)-kappa B activation and nuclear translocation of the p65 NF-kappa B subunit through a mechanism involving the inhibition of IkB alpha degradation and p38 MAPK activation. In conclusion, dipyridamole, at therapeutically achievable concentrations, reduces the expression and release of MMP-9 through a mechanism involving p38 MAPK and NF-kappa B inhibition. These results indicate that dipyridamole exerts anti-inflammatory properties in human monocytes that may favourably contribute to its actions in the secondary prevention of stroke, independent of its antiplatelet properties.

Epidemiological studies associate whole-grain consumption with several health benefits and increasing evidence suggests whole-grain wheat polyphenols as healthy agents with anti-inflammatory properties (1). However, many studies demonstrated the impact, usually negative, of wheat bran, rich in polyphenols, on bread quality. We have previously evaluated the bread making ability of meals composed of re-milled semolina biofortified with selected durum wheat milling by-products (200 g/kg) that were: i) residuals of the second and third debranning steps of durum wheat (DB), ii) the micronized and air-classified thin fraction obtained from the same residuals (MB), or iii) coarse bran obtained from conventional roller milling of non-debranned durum wheat (B) (2). We showed that total soluble phenolic compounds and antioxidant activity were significantly higher in MB and DB than in B (2), with acceptable bread quality in particular for MB. However, their biological anti-inflammatory potential was unknown. The aim of this study was to analyse the vascular anti-inflammatory properties of the phenolic extracts obtained from different biofortified bread by evaluating endothelium-monocyte adhesion and endothelial and monocytic inflammatory gene expression. Cultured human endothelial and monocytic cells were incubated with increasing concentrations (1, 5 or 10 ?g/mL) of phenolic acids extracts from biofortified bread (B, DB, MB) before stimulation with inflammatory challenge lipopolysaccharide (LPS 1 ?g/mL). All phenolic acids extracts inhibited, in a concentration-dependent manner, the stimulated endothelial leukocyte adhesion, and the protein expression of endothelial adhesion molecules. By real time PCR, we found that B, DB and MB phenolic extracts down-regulated the mRNA levels of adhesion molecules as well as pro-inflammatory cytokines and chemoattractants in stimulated endothelial cells and monocytes. Our findings appreciate the bread biofortified with selected durum wheat milling by-products as a source of phenolic acids with multiple anti-inflammatory properties.

Early atherosclerosis features functional and structural changes in the endothelial barrier function that affect the traffic of molecules and solutes between the vessel lumen and the vascular wall. Such changes are mechanistically related to the development of atherosclerosis. Proatherogenic stimuli and cardiovascular risk factors, such as dyslipidaemias, diabetes, obesity, and smoking, all increase endothelial permeability sharing a common signalling denominator: An imbalance in the production/disposal of reactive oxygen species (ROS), broadly termed oxidative stress. Mostly as a consequence of the activation of enzymatic systems leading to ROS overproduction, proatherogenic factors lead to a pro-inflammatory status that translates in changes in gene expression and functional rearrangements, including changes in the transendothelial transport of molecules, leading to the deposition of low-density lipoproteins (LDL) and the subsequent infiltration of circulating leucocytes in the intima. In this review, we focus on such early changes in atherogenesis and on the concept that proatherogenic stimuli and risk factors for cardiovascular disease, by altering the endothelial barrier properties, co-ordinately trigger the accumulation of LDL in the intima and ultimately plaque formation.

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, DOPE) 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 DOPE (1-10 mu g/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. DOPE 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. (C) 2015 Elsevier Inc. All rights reserved.

Mitochondria are fundamental organelles producing energy and reactive oxygen species (ROS); their impaired functions play a key role in endothelial dysfunction. Hydroxytyrosol (HT), a well-known olive oil antioxidant, exerts health benefits against vascular diseases by improving endothelial function. However, the HT role in mitochondrial oxidative stress in endothelial dysfunction is not clear yet. To investigate the HT effects on mitochondrial ROS production in the inflamed endothelium, we used an in vitro model of endothelial dysfunction represented by cultured endothelial cells, challenged with phorbol myristate acetate (PMA), an inflammatory, prooxidant, and proangiogenic agent. We found that the pretreatment of endothelial cells with HT (1-30 ?mol/L) suppressed inflammatory angiogenesis, a crucial aspect of endothelial dysfunction. The HT inhibitory effect is related to reduced mitochondrial superoxide production and lipid peroxidation and to increased superoxide dismutase activity. HT, in a concentration-dependent manner, improved endothelial mitochondrial function by reverting the PMA-induced reduction of mitochondrial membrane potential, ATP synthesis, and ATP5ß expression. In PMA-challenged endothelial cells, HT also promoted mitochondrial biogenesis through increased mitochondrial DNA content and expression of peroxisome proliferatoractivated receptor gamma coactivator 1-alpha, nuclear respiratory factor-1, and mitochondrial transcription factor A. These results highlight that HT blunts endothelial dysfunction and pathological angiogenesis by ameliorating mitochondrial function, thus suggesting HT as a potential mitochondria-targeting antioxidant in the inflamed endothelium.

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 ?mol/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 ?mol/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)-?B and protein kinase C (PKC)? and PKC?1 activation.CONCLUSION: HT, at nutritionally relevant concentrations, reduces MMP-9 and COX-2 induction in activated human monocytes via PKC? and PKC?1 inhibition, thus featuring novel anti-inflammatory properties. Overall, such results contribute to explaining the vascular protective effects by olive oil polyphenols in Mediterranean diets.

Abnormal angiogenesis is implicated in a number of human diseases and endothelial growth inhibition represents a common approach in tumor therapy. Recently itraconazole, frequently used in humans as antifungal drug, which blocks the biosynthesis of cholesterol, has been found to be antiangiogenic in primary umbilical vein endothelial cells. However, the exact antiangiogenic mechanisms remain largely unknown. In this paper, we studied the effect of itraconazole in human dermal microvascular endothelial cells (HMEC-1), an immortalized cell line to study adult angiogenesis. A 50% reduction of microtubule formation was observed after itraconazole treatment which was partially rescued by cholesterol addition. We found that itraconazole inhibits angiogenesis markers such as VEGF, AAMP and e-NOS. mTOR and ERK1/2 phosphorylation as well as the expression of Gli1, one of the main controllers of the Shh pathway, were also inhibited by itraconazole. Cholesterol addition did not completely rescue inhibition of these pathways, suggesting that the itraconazole antiangiogenic activity could be due to multiple mechanisms. Our results may contribute to novel approaches to block angiogenesis with therapeutic application

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 mu mol/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)-kappa B. 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. (C) 2012 Elsevier Inc. All rights reserved.

Purpose: The aim of the study was to evaluate the vascular anti-inflammatory effects of polyphenolic extracts from two typical South Italy red wines, the specific contribution of individual polyphenols and the underlying mechanisms of action.Methods: Human endothelial cells were incubated with increasing concentrations (1-50 ?g/mL) of Primitivo and Negroamaro polyphenolic extracts (PWPE and NWPE, respectively) or pure polyphenols (1-25 ?mol/L), including hydroxycinnamic acids (p-coumaric, caffeic and caftaric acids), flavonols (kaempferol, quercetin, myricetin) or stilbenes (trans-resveratrol, trans-piceid) before stimulation withlipopolysaccharide. Through multiple assays, we analyzed the endothelial-monocyte adhesion, theendothelial expression of adhesion molecules (ICAM-1, VCAM-1 and E-Selectin), monocytechemoattractant protein-1 (MCP-1) and macrophage colony-stimulating factor (M-CSF), as well as ROSintracellular levels and the activation of NF-?B and AP-1.Results: Both PWPE and NWPE, already at 1 ?g/mL, inhibited monocyte adhesion to stimulated endothelial cells, a key event in triggering vascular inflammation. They down-regulated the expression of adhesion molecules, ICAM-1, VCAM-1, E-Selectin, as well as MCP-1 and M-CSF, at mRNA and protein levels. All polyphenols reduced intracellular ROS, and everything, except caftaric acid, inhibited the endothelial expression of adhesion molecules and MCP-1, although with different potency. Flavonols and resveratrol significantly reduced also the endothelial expression and release of M-CSF. The decrease in endothelial inflammatory gene expression was related to the inhibition of NF-?B and AP-1 activation but not to intracellular oxidative stress.Conclusions: This study showed multiple anti-inflammatory and anti-atherosclerotic properties of red wine polyphenolic extracts and indentified specific bioactive polyphenols which could counteract inflammatory diseases including atherosclerosis.

Nutraceuticals are potentially healthful foods that play a role in maintaining human wellbeing, enhancing health and preventing, or even treating, specific diseases. More than forany other diseases, cardiovascular diseases occur in association with risk factors that areamenable to prevention or treatment by nutraceutical interventions. Several ingredientsmarketed for use in dietary supplements address such risk factors. The ability of nutraceu-ticals to favorably influence cardiovascular risk factors and atherosclerotic vascular diseaseshould be recognized as an enormous opportunity for the prevention or treatment of thiscommon condition. In this review, we attempt at summarizing some of the recent researchfindings on ? -3-polyunsaturated fatty acids and antioxidant polyphenols that have bene-ficial cardiovascular effects to update the practicing clinicians on the potential benefits ofnutraceuticals in this area

Atherosclerosis is now widely accepted to be an inflammatory disease, characterized by degenerative as well as proliferative changes and extracellular accumulation of lipid and cholesterol, in which an ongoing inflammatory reaction plays an important role both in initiation and progression/destabilization, converting a chronic process into an acute disorder. Neovascularization has also been recognized as an important process for the progression/destabilization of atherosclerotic plaques. In fact, vulnerable atherosclerotic plaques prone to rupture are characterized by an enlarged necrotic core, containing an increased number of vasa vasorum, apoptotic macrophages, and more frequent intraplaque haemorrhage. Various functional roles have been assigned to intimal microvessels, however the relationship between the process of angiogenesis and its causal association with the progression and complications of atherosclerosis are still challenging and controversial. 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, including plaque angiogenesis. 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 an attenuation of activation of the nuclear factor-?B 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.

Vascular inflammation, especially at the level of endothelial cells, has been shown to play a pivotal role in theinception, progression, and clinical complications of atherosclerosis. The common denominators for the activationof inflammatory genes appear to be a small subset of transcription factors--among which include nuclear factor-?B,activator protein-1 (AP-1), and GATA--that function as the central hub of vascular inflammation. Strategies directedto inhibit both the secondary mediators and the primary triggers (atherosclerosis risk factors) appear viable to inhibitatherosclerosis. However, attempts have now been made to address the central hub of vascular inflammation. "Old"drugs, such as dipyridamole, can also now be revisited for properties related to inhibition of vascular inflammation,probably by acting on the common hub of inflammation.

Red wine is a treasured source of polyphenols, such as stilbenes, flavonols and hydroxycinnamic acids, depending on different factors, such as grape cultivar and wine-making practices. The red wine polyphenols (RWPs) have long been associated to reduced mortality and morbidity from cardiovascular diseases and cancer. Since matrix metalloproteinase (MMP) activity is involved in extracellular matrix degradation, a crucial step both in inflammation and cell migration, aim of this study was to examine the effects of specific classes of polyphenols from Primitivo and Negroamaro grapes and wines produced in the Apulian region (Southern Italy), on the release and activity of MMP-9 in human monocytoid cells.U937 monocitoid cells were pre-treated with increasing concentrations of polyphenolic extracts (PE) of Primitivo and Negramaro grape and wine, before stimulation with 30 nM phorbol myristate acetate (PMA) for 24 h. The release and activity of MMP-9 in culture medium was tested by ELISA and zymography, respectively. Both Primitivo and Negroamaro PE exhibited a concentration-dependent inhibition of MMP-9 release and activity, without any reduction in cell viability. The inhibitory effect was higher than that produced by individual chemically synthesized polyphenols.. In this study, the PE from Primitivo and Negramaro grapes and red wines were characterized, and shown to significantly and synergistically inhibit the monocyte-derived release and activity of extracellular matrix proteases. These inhibitory effects could explain, at least in part, the anti-inflammatory and anti-cancerogenic properties of RWP.

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

The activation of peroxisome proliferator-activated receptor (PPAR)? is known to inhibit angiogenesis. As a potential mechanism for this, we aimed at examining the effects of PPAR? agonists on the pro-angiogenic enzyme cyclooxygenase (COX)-2 in human endothelium.Methods and results Cultured endothelial cells were pre-incubated with the PPAR? 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 PPAR? antagonists bisphenol A diglycidyl ether and GW9662 as well as by PPAR? 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, PPAR? agonists attenuated CREB activation. As both protein kinase C (PKC)? and ? 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 PKC? membrane translocation.Conclusion VEGF induces CREB-mediated COX-2 expression through a PKC?-dependent pathway in human endothelium. The anti-angiogenic effect of PPAR? agonists is due, at least in part, to an interference with the VEGF-stimulated PKC?-mediated activation of CREB and the related expression of COX-2.

Anthocyanins, the naturally occurring pigments responsible for most red to blue colours of flowers, fruits and vegetables, have also attracted interest because of their potential health effects. With the aim of contributing to major insights into their structure-activity relationship (SAR), we have evaluated the radical scavenging and biological activities of selected purified anthocyanin samples (PASs) from various anthocyanin-rich plant materials: two fruits (mahaleb cherry and blackcurrant) and two vegetables (black carrot and "Sun Black" tomato), differing in anthocyanin content (ranging from 4.9 to 38.5 mg/g DW) and molecular structure of the predominant anthocyanins. PASs from the abovementioned plant materials have been evaluated for their antioxidant capacity using Trolox Equivalent Antioxidant Capacity (TEAC) and Oxygen Radical Absorbance Capacity (ORAC) assays. In human endothelial cells, we analysed the anti-inflammatory activity of different PASs by measuring their effects on the expression of endothelial adhesion molecules VCAM-1 and ICAM-1. We demonstrated that all the different PASs showed biological activity. They exhibited antioxidant capacity of different magnitude, higher for samples containing non-acylated anthocyanins (typical for fruits) compared to samples containing more complex anthocyanins acylated with cinnamic acid derivatives (typical for vegetables), even though this order was slightly reversed when ORAC assay values were expressed on a molar basis. Concordantly, PASs containing non-acylated anthocyanins reduced the expression of endothelial inflammatory antigens more than samples with aromatic acylated anthocyanins, suggesting the potential beneficial effect of structurally diverse anthocyanins in cardiovascular protection.

Red wine polyphenols have long been associated to a reduced morbidity and mortality from atherosclerotic disease in part by reducing inflammation, however the effects of different polyphenolic compounds are not clear. The aim of this study was to evaluate the anti-inflammatory and anti-oxidant effects of polyphenolic extracts from two red wines typical of Southern Italy, and to dissect the specific contribute of hydroxycinnamic acids, flavonols and stilbenes and to investigate underlying mechanisms of action in endothelial cells.Methodology and Results: Through multiple assays, we showed that both Primitivo and Negroamaro Wine Polyphenolic Extracts significantly inhibited monocyte adhesion to endothelium, endothelial expression of adhesion molecules, monocyte chemoattractant protein-1 and macrophage colony-stimulating factor as well as intracellular oxidative stress and the activation of NF-?B and AP-1, without any reduction in cell viability. We also showed that all tested polyphenols exhibited a strong antioxidant action in stimulated human endothelial cells but not all significantly exhibited anti-inflammatory activity which in turn was related to the inhibition of NF-?B and AP-1 activation.Conclusions: The present study provides new evidences on specifc anti-inflammatory and anti-atherosclerotic role of red wine polyphenols including hydroxycinnamic acids, flavonols and stilbenes.

Matrix metalloproteinases (MMPs) are endopeptidases responsible for the hydrolysis of various components of extracellular matrix. MMPs, namely gelatinases MMP-2 and MMP-9, contribute to the progression of chronic and degenerative diseases. Since gelatinases' activity and expression are regulated by oxidative stress, we sought to evaluate whether supplementation with polyphenol-rich red grape skin extracts modulated the matrix-degrading capacity in cell models of vascular inflammation. Human endothelial and monocytic cells were incubated with increasing concentrations (0.5-25 ?g/mL) of Negroamaro and Primitivo red grape skin polyphenolic extracts (NSPE and PSPE, respectively) or their specific components (0.5-25 ?mol/L), before stimulation with inflammatory challenge. NSPE and PSPE inhibited, in a concentration-dependent manner, endothelial invasion as well as the MMP-9 and MMP-2 release in stimulated endothelial cells, and MMP-9 production in inflamed monocytes, without affecting tissue inhibitor of metalloproteinases (TIMP)-1 and TIMP-2. The matrix degrading inhibitory capacity was the same for both NSPE and PSPE, despite their different polyphenolic profiles. Among the main polyphenols of grape skin extracts, trans-resveratrol, trans-piceid, kaempferol and quercetin exhibited the most significant inhibitory effects on matrix-degrading enzyme activities. Our findings appreciate the grape skins as rich source of polyphenols able to prevent the dysregulation of vascular remodelling affecting degenerative and inflammatory diseases.

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

This study investigates the effects of tomato puree fortification (ftp) with several anthocyanin-rich food colorants on bioactive compound content (phenolics, isoprenoids), antioxidant capacity, in vitro biological activities and consumer acceptance. Tomato puree (tp) was added with different anthocyanin extracts from Daucus carota L. var. atrorubens (Anthocarrot), Vitis vinifera L. fruit skins (Enocolor), Sambucus nigra L. (Elderberry) or Prunus mahaleb L. (Mahaleb) fruits. The consumer acceptance (colour, flavor, taste, visual appearance) was at high level, except for Mahaleb-added extracts. Compared to the control (tp), the addition of colouring extracts increased significantly the total phenolic content, before pasteurization, in addition to the expected anthocyanin content. However, after pasteurization, only Anthocarrot-ftp preserved increased phenolic (+53%) content, as well as higher antioxidant capacity (50%). Consistently, against tp, Anthocarrot-ftp exhibited an increased anti-inflammatory capacity as showed by the reduced expression of vascular cell adhesion molecule (VCAM)-1 in human cultured endothelial cells, under inflammatory conditions.

Adipose tissue inflammation is a mechanistic link between obesity and its related sequelae, including insulin resistance and type 2 diabetes. Dual ligands of peroxisome proliferator activated receptor (PPAR)? and ?, combining in a single molecule the metabolic and inflammatory-regulatory properties of ? and ? agonists, have been proposed as a promising therapeutic strategy to antagonize adipose tissue inflammation. Here we investigated the effects of the dual PPAR?/? agonist aleglitazar on human adipocytes challenged with inflammatory stimuli. Human Simpson-Golabi-Behmel syndrome (SGBS) adipocytes were treated with aleglitazar or - for comparison - the selective agonists for PPAR? or ? fenofibrate or rosiglitazone, respectively, for 24 h before stimulation with TNF-?. Aleglitazar, at concentrations as low as 10 nmol/L, providing the half-maximal transcriptional activation of both PPAR? and PPAR?, reduced the stimulated expression of several pro-inflammatory mediators including interleukin (IL)-6, the chemokine CXC-L10, and monocyte chemoattractant protein (MCP)-1. Correspondingly, media from adipocytes treated with aleglitazar reduced monocyte migration, consistent with suppression of MCP-1 secretion. Under the same conditions, aleglitazar also reversed the TNF-?-mediated suppression of insulin-stimulated ser473 Akt phosphorylation and decreased the TNF-?-induced ser312 IRS1 phosphorylation, two major switches in insulin-mediated metabolic activities, restoring glucose uptake in insulin-resistant adipocytes. Such effects were similar to those obtainable with a combination of single PPAR? and ? agonists. In conclusion, aleglitazar reduces inflammatory activation and dysfunction in insulin signaling in activated adipocytes, properties that may benefit diabetic and obese patients. The effect of aleglitazar was consistent with dual PPAR? and ? agonism, but with no evidence of synergism.

Scope: High intakes of n-3 fatty acids exert anti-inflammatory effects and cardiovascular protection, but the underlying molecular basis is incompletely defined. By genome-wide analysis we searched for novel effects of docosahexaenoic acid (DHA) on gene expression and pathways in human vascular endothelium under pro-inflammatory conditions. Methods and Results: Human umbilical vein endothelial cells were treated with DHA and then stimulated with interleukin(IL)-1?. Total RNA was extracted, and gene expression examined by DNA microarray. DHA alone altered the expression of 188 genes, decreasing 92 and increasing 96. IL- 1? changed the expression of 2031 genes, decreasing 997 and increasing 1034. Treatment with DHA before stimulation significantly affected the expression of 116 IL-1?-deregulated genes, counter-regulating the expression of 55 genes among those decreased and of 61 among those increased. Functional and network analyses identified immunological, inflammatory and metabolic pathways as the most affected. Newly identified DHA-regulated genes are involved in stemness, cellular growth, cardiovascular system function and cancer, and included cytochrome p450 4F2(CYP4F2), transforming growth factor(TGF)-?2, Cluster of Differentiation (CD)47, caspase recruitment domain(CARD)11 and phosphodiesterase(PDE)5?. Conclusions: Endothelial exposure to DHA regulates novel genes and related pathways. Such unbiased identification should increase our understanding of mechanisms by which n-3 fatty acids affect human diseases.

IntroduzionePrunus mahaleb L. è un arbusto deciduo che cresce in boschi radi anche su terreni marginali e in climi aridi. L'albero è nativo dei paesi del mediterraneo e dell'Asia centrale e produce dei piccoli frutti simili a ciliegie di un colore quasi nero, non commestibili a causa del loro sapore amaro. Studi precedenti hanno dimostrato un elevato contenuto in antocianine in questi frutti, che suggerisce il loro utilizzo come fonte di coloranti alimentari dotati di proprietà salutistiche.ObiettiviCaratterizzazione chimica di un estratto concentrato di frutti di P. mahaleb e studio in vitro dei suoi effetti anticancerogeni, antiinfiammatori e antimutagenici.MetodologiaI frutti di mahaleb sono stati estratti con solventi consentiti per uso alimentare (etanolo + 1% acido citrico 1M) e l'estratto, concentrato sino a 60 °Brix, analizzato utilizzando metodiche HPLC. Sono stati effettuati saggi in vitro per studiare la capacità dell'estratto di inibire la proliferazione di linee cellulari tumorali umane (MCF-7), le sue proprietà antinfiammatorie su cellule endoteliali vascolari umane e infine i suoi effetti antimutagenici su linee di lievito Saccharomyces cerevisiaeD7.Risultati e DiscussioneL'estratto concentrato ha mostrato un elevato contenuto in antocianine, flavonoli e cumarina ed una elevata attività antiossidante simile o superiore a quella di altri noti estratti concentrati di frutti rossi. Lo studio delle attività biologiche dell'estratto ha mostrato: i) un significativo effetto antiproliferativo e capacità di modulare le GJIC in cellule MCF-7; ii) una inibizione dell'espressione di markers infiammatori tipici dell'attivazione endoteliale (ICAM-1, VCAM-1, E-selectine) in cellule endoteliali ottenute da vena ombelicale umana in coltura; iii) una riduzione di circa tre volte dell'effetto mutageno di H2O2 su cellule di S. cerevisiae. Questi risultati suggeriscono l'utilizzo dei frutti di P. mahaleb processati, come fonte di coloranti alimentari naturali e di composti bioattivi con proprietà salutistiche.

Valorization of the neglected Prunus mahaleb L., fruits as a source of functional moleculesGerardi C.1, Frassinetti S.3, Leone A.1, Calabriso N.2, Carluccio M.A.2, F. Blando1 and Mita G. 11 Institute of Sciences of Food Production, CNR, Lecce, Italy2 Institute of Clinical Physiology, CNR, Lecce, Italy3 Institute of Biology and Agricultural Biotechnology, CNR, Pisa, ItalyAbstract: Prunus mahaleb L. is a tree producing dark-red small stone fruits, not used for fresh consumption due to their astringent and sour taste. In this communication will be described a "mahaleb fruit concentrated extract" (mfce) and its chemical characterization. The extract showed high anthocyanin, flavonol and coumarin content. Moreover, the results of the in vitro assays for mfce biological activities will be reported. Mfce showed strong antioxidant capacity and anti-proliferative, pro-apoptotic, anti-inflammatory and anti-mutagenic effects. This study demonstrated several healthful effects of mfce that may be of interest as natural food colorant and as source of molecules for the formulation of functional foods.