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.

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.

Iodinated radiocontrast media have been the most widely used pharmaceuticals for intravascular administration in diagnostic and interventional angiographic procedures. Although they are regarded as relatively safe drugs and vascular biocompatibility of contrast media has been progressively improved, severe adverse reactions may occur, among which acute nephropathy is one of the most clinically significant complications after intravascular administration of contrast media and a powerful predictor of poor early and long-term outcomes. Since radiocontrast media are given through the arterial or the venous circulation in vascular procedures, morphological and functional changes of the microvascular and macrovascular endothelial cells substantially contribute to the pathogenesis of organ-specific and systemic adverse reactions of contrast media. Endothelial toxicity of contrast media seems to be the result of both direct proapoptotic effects and morphological derangements, as well as endothelial dysfunction and induction of inflammation, oxidative stress, thrombosis, and altered vasomotor balance, with predominant vasoconstrictive response in atherosclerotic coronary arteries and kidney microcirculation. Further understanding of pathogenetic mechanisms underlying contrast media-induced adverse reactions in cellular targets, including endothelial cells, will hopefully lead to the development of novel preventive strategies appropriately curbing the pathogenesis of contrast media vasotoxicity. © 2012 Elsevier Inc.

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.

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.

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.

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.

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

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.

The lower occurrence of cardiovascular disease and cancer in populations around the Mediterranean basin as detected in the 1950s was correctly attributed to the peculiar dietary habits of those populations. Essentially, until the mid-20th century, typical Mediterranean diets were rich in fruits, vegetables, legumes, whole-wheat bread, nuts, fish, and, as a common culinary trait, the routine use of extra-virgin olive oil. Nowadays, the regular adoption of such dietary patterns is still thought to result in healthful benefits. Such patterns ensure the assumption of molecules with antioxidant and anti-inflammatory actions, among which ?-3 polyunsaturated fatty acids (PUFAs), ?-9 monounsaturated fatty acids (oleic acid), and phenolic compounds. The aim of this review is to provide an update of the vasculo-protective pathways mediated by ?-3 PUFAs and polyphenols in the context of the modern Mediterranean dietary habits, including the possible cross-talk and synergy between these typical components. This review complements a parallel one focusing on the role of dietary nitrates and alimentary fats.

This review summarizes available evidence on the beneficial effects of inorganic nitrates and the monounsaturated fatty acid (MUFA) oleic acid, largely contained in Mediterranean diet, on blood pressure and coagulation activity.Inorganic nitrate. Normal vascular function requires NO production from the 1-arginine-NO synthase (NOS) pathway. This process is defective in conditions of local hypoxia, and here nitrite can substitute for 1-arginine-NOS derived NO. In this context, NO generation from the nitrate-nitrite-NO pathway mostly derived from green leafy vegetables appears to be an alternative source for NOS-dependent NO production, ensuring NO bioavailability also in situations when the endogenous 1-arginine/NO synthase pathway is dysfunctional or physiologically reduced in local hypoxic conditions.Olive oil and oleic acid. In addition to effects on lipoprotein metabolism and oxidation, the beneficial effects of oleic acid occur also on coagulation activity, namely on coagulation factor VII (FVII). Normally, a substantial increase of FVII coagulant activity (FVIIc) occurs within 2-3. h after a fatty meal and persists for several hours thereafter. When a background diet high in MUFA is consumed, a lower post-prandial increase of FVIIc takes place.