Pharmacological modulation of vascular inflammation in atherothrombosis

Vascular inflammation, especially at the level of endothelial cells, has been shown to play a pivotal role in the inception, progression, and clinical complications of atherosclerosis. The common denominators for the activation of inflammatory genes appear to be a small subset of transcription factors-among which include nuclear factor-κB, activator protein-1 (AP-1), and GATA-that function as the central hub of vascular inflammation. Strategies directed to inhibit both the secondary mediators and the primary triggers (atherosclerosis risk factors) appear viable to inhibit atherosclerosis. 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

Nutraceuticals and prevention of atherosclerosis: focus on omega-3 polyunsaturated fatty acids and Mediterranean diet polyphenols

Nutraceuticals are potentially healthful foods that play a role in maintaining human well being, enhancing health and preventing, or even treating, specific diseases. More than for any other diseases, cardiovascular diseases occur in association with risk factors that are amenable to prevention or treatment by nutraceutical interventions. Several ingredients marketed for use in dietary supplements address such risk factors. The ability of nutraceuticals to favorably influence cardiovascular risk factors and atherosclerotic vascular disease should be recognized as an enormous opportunity for the prevention or treatment of this common condition. In this review, we attempt at summarizing some of the recent research findings on omega-3-polyunsaturated fatty acids and antioxidant polyphenols that have beneficial cardiovascular effects to update the practicing clinicians on the potential benefits of nutraceuticals in this area

Homocysteine induces VCAM-1 gene expression through NF-kB and NAD(P)H oxidase activation: protective role of Mediterranean diet polyphenolic antioxidants

Introduction: Hyperhomocysteinemia is a recognized risk factor for atherosclerotic vascular disease, but molecular mechanisms are no completely understood. Because VCAM-1 expression is crucial in monocyte adhesiveness, we evaluated the NF-κB-related induction of VCAM-1 by homocysteine (Hcy) and the possible inhibitory effect of specific dietetic polyphenolic antioxidants, such as trans-resveratrol (RSV) and hydroxytyrosol (HT). Methods and Results: We found that in human umbilical vein endothelial cells (HUVEC), Hcy, at ≥50 μmol/L, but not cysteine, induced VCAM-1 expression at protein and mRNA levels, at enzyme immunoassay and Northern blot, respectively. Transfection studies with deletional VCAM-1 promoter constructs demonstrated that two tandem NF-κB motives in VCAM-1 promoter are necessary for Hcy induced VCAM-1 gene expression. This was confirmed by Hcy induced NF-kB activation at EMSA and the nuclear translocation of its p65 (Rel A) subunit and the degradation of both inhibitors(I)kB-α and -b at Western analysis. Hcy increased intracellular ROS, measured with dichlorofluoresceine fluorescence assays, in HUVEC, by NAD(P)H oxidase activation, determined through the membrane translocation of its p47phox subunit. Antioxidants, NF-κB and NAD(P)H oxidase inhibitors decreased the Hcy inducted intracellular ROS and VCAM-1 expression. Previously, we found that antioxidants RSV and HT inhibited NF-κB mediated VCAM-1 induction by LPS or TNFα. Here we shown that nutritionally relevant concentrations of RSV and HT, but not folate and B vitamins, reduced (>60% inhibition at 10-6 mol/L) Hcy-induced VCAM-1 expression and monocytoid cell adhesion to the endothelium. Conclusions: These data point out that pathophysiologically relevant Hcy concentrations induce VCAM-1 expression through NF-κB-mediated pro-oxidant mechanism. This can be inhibited by natural Mediterranean diet antioxidants, suggesting a their possible therapeutic role in Hcy-induced vascular damage

OMEGA-3 fatty acids contribute to plaque stability differentially affecting the release of matrix metalloproteinases and tissue inhibitors of metalloproteinases by human monocytes/macrophages in culture

Objectives. High intakes of omega-3 fatty acids has been associated with protection from plaque rupture. The secretion of metalloproteinases (MMPs) by macrophages is believed to play a key role in matrix degradation underlying plaque instability. Conversely, tissue inhibitors of metalloproteinases (TIMPs) would contribute to plaque stability. We therefore studied the effects of omega-3 fatty acids on the release and activity of MMPs and TIMPs in cultured human monocytoid cells. Methods. Human U937 monocytoid cells were differentiated into macrophages by exposure for 24 h to 30 ng/mL phorbol myristate acetate (PMA) and 10 ng/mL tumor necrosis factor(TNF)-α. Both monocytes and macrophages were treated for 48 h with the DHA (22:6 n-3) or EPA (22:6 n-3) (25-100 μmol/L) before stimulation for 24 h with 10 ng/ml TNFα. Cell supernatates were used to test the release of gelatinase A (MMP-2), gelatinase-B (MMP-9), collagenase-1 (MMP-1), TIMP-1 and TIMP-2, by ELISAs, and total gelatinase and anti-gelatinase activities by zymography and retro-zymography techniques, respectively. Results. The long term exposure to 50 μmol/L EPA and DHA, but not to arachidonic acid (20:4 n-6), significantly reduced MMP-9 protein release without affecting the release of MMP-1, MMP-2 and TIMP-1. Conversely, TIMP-2 protein release was significantly increased by EPA and DHA (Table). Zymography for MMP-9 and retro-zymography for TIMP-1 and -2 reproduced the same results. Conclusions. The long term exposure to omega-3 fatty acids significantly reduces MMP-9 release without affecting the release of MMP-1 and -2. This effect, associated with the increase of TIMP-2 protein production and activity, may contribute to explaining the plaque-stabilizing effect by omega-3 fatty acid observed in humans

Hydroxytyrosol, an olive oil phenolic compound, inhibits cyclooxygenase-2 transcription in phorbol--ester-treated human mocytes

Introduction: Inflammation plays a critical role in atherosclerotic plaque development and instability. Cyclooxygenase(COX)-2 is a key mediator of inflammation. It is up-regulated in activated monocytes and macrophages of human atherosclerotic lesions. The extra-virgin olive oil is a source of bioactive compounds which can be responsible of Mediterranean diet athero-protective effect. Aim of the study was to examine the effects of hydroxytyrosol (HT), a phenolic compound from olives and extra-virgin olive oil, on COX-2 expression in phorbol-ester stimulated monocytic-macrophagic cells and to explore the mechanisms involved. Methods: U937 monocytoid cells were pre-treated with HT (0-10 micromol/L) for 60 min before stimulation with 20 nmol/L phorbol myristate acetate (PMA) in RPMI 1640 medium with 5%SFB for 20 h. Cell supernatants were then tested for the release of PGE2 by EIA kit and cell extracts were analysed for COX-2 expression by Western blot analysis. COX-2 mRNA was investigated by semi-quantitative RT-PCR and COX-2 promoter activity was assessed by transient transfection of full length and partially deleted or mutagenized COX-2 promoter constructs. Results: Stimulation of U937 cells with 20 nmol/L PMA for 20 h caused a marked increase in the production of prostaglandin(PG) E2. This effect was inhibited by 1 micromol/L HT both in PMA-treated U937 (by about 25%) and even more in U937 derived-macrophages (> 40%). Pre-treatment of U937 cells with 0.1-10 micromol/L HT reduced PMA-induced COX-2 protein and mRNA in a concentration dependent manner with a 30% reduction already at 1 mmol/L (p<0.01). HT inhibitory effect was isoform specific given that the constitutive COX-1 expression was not affected. In transient transfection assay, HT reduced PMA-induced COX-2 promoter activity of full length construct. Transient transfections utilizing COX-2 promoter deletion constructs and COX-2 promoter constructs, in which specific enhancer elements were mutagenized, indicated that the effects of HT were, at least in part, mediated via NF-kB elements. Conclusions: HT inhibited PGE2 release and COX-2 expression in phorbol-ester-stimulated monocytes and U937-derived macrophages. These data provide new insight into the anti-inflammatory properties of HT and may contribute to explain the cardiovascular protection by specific components of Mediterranean diets

Hydroxytyrosol suppresses MMP-9 activity and expression in human monocytes. A mechanism for plaque stabilization by an olive oil component of Mediterranean diets

Purpose: Mediterranean diets, of which olive oil is an important component, are associated with low prevalence of cardiovascular diseases. The production of inflammatory mediators, such as prostaglandin (PG) E2, the overexpression of the inducible cyclooxygenase (COX)- 2 isoform and the activation of matrix metalloproteinase(MMP)-9 by macrophages likely contributes to plaque instability leading to acute coronary events. We studied the effects of the olive oil phenolic antioxidant hydroxytyrosol (HT) on MMP-9 and COX-2 activity and expression in human monocytes and explored underlying mechanisms. Methods: Human monocytes were treated either with 1-50 &#956;mol/L HT for 60 min or with selective inhibitors of PKC or COX isoenzymes for 30 min before stimulation with 30 nmol/L phorbol myristate acetate (PMA) for 24 h. Cell supernatants were tested for the release of MMP-9, PGE2 and TIMP-1 and -2 by ELISA and MMP-9 activity by zymography. Cell protein extracts were analyzed by Western analysis for COX-2 expression and for membrane translocation of PKCs and the NADPH oxidase p47phox subunit. We analyzed the activity of COX-2 promoter by transient transfection experiments and the.activation of the transcription factor Nuclear Factor(NF)-kappaB by EMSA. Results: PMA and, to a lesser extent, PGE2, induced the release of MMP-9 in monocytes. Cell exposure to HT before PMA stimulation reduced MMP-9 activity and expression (IC50 for HT of 10 micromol/L p < 0.01) without affecting the release of TIMP-1 and -2. Correspondingly, HT inhibited PMA-induced PGE2 production (by 54 ? 7%) and COX-2 expression (by 43 ? 5%) without affecting COX-1. Inhibition by HT was mediated by the suppression of NF-kappaB and the NADPH oxidase p47phox and PKC&#945;/&#946;1 activation. Conclusions: Our findings show that HT, at concentrations nutritionally achievable, inhibits the expression and the release of MMP-9 at least in part by the suppression of COX-2 dependent PGE2 pathway. Such effect occurs through the attenuation of PKC&#945;/&#946;1 and NADPH oxidase activation. Overall, such results contribute to explaining the vascular protective effects exerted by olive oil in Mediterranean diets

Peroxisome proliferator-activated receptory inhibits angiogenesis by suppressing creb-mediated cyclooxygenase-2 expression in human endothelium

Objetives. Neoangiogenesis contributes to diabetic vasculopathy and intraplaque hemorrhage in atherosclerosis. The activation of Peroxisome Proliferator-Activated Receptor(PPAR)&#947; is known to inhibit angiogenesis. We therefore examined the effects of PPAR&#947; agonists on the pro-angiogenic enzyme cyclooxygenase(COX)-2 in human umbilical vein endothelial cells challenged with vascular endothelial growth factor (VEGF) and phorbol 12-myristate 13-acetate (PMA). Methods and Results.A 24 h exposure of HUVEC to the PPAR&#947; agonists rosiglitazone (RSG) and GW1929 significantly attenuated VEGF- and PMA-stimulated COX-2 activity (by 30%, immunoassay for 6-keto-PGF1&#945;), as well as protein (by 50%, Western analysis) and mRNA expression (by 50%, RT-PCR). This effect was abolished by the PPAR&#947; antagonists bisphenol A diglycidyl ether and GW9662. COX-2 promoter activity 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 knockdown of the transcription factor CRE binding protein (CREB) confirmed the role of CREB in mediating COX-2 transcription. Correspondingly, PPAR&#947; agonists also attenuated CREB phosphorylation/activation. Since Protein Kinase(PK)C is involved in VEGF-induced COX-2 expression and CREB activation, we also investigated which isoforms of PKC were affected by RSG. While the inhibition of both conventional PKC&#945; and &#946; suppressed VEGF- and PMA-stimulated CREB activation and COX-2 expression, RGS only reduced VEGF- and PMA-stimulated PKC&#945; membrane translocation. Conclusions. The anti-angiogenic effect of PPAR&#947; agonists is due, at least in part, to their interference with the PKC&#945;-mediated activation of CREB and the related expression of COX-2. PKC&#945; may therefore be a novel therapeutic target for antidiabetic drugs in atherosclerosis

The Olive oil antioxidant hydroxytyrosol (HT) reduces the matrix metalloproteinase-9 activity and expression in human mocytoid cells through a prostaglandin (PG) E2-dependent mechanisms

Objectives: Olive oil HT is believed to be among the most active cardio-protective components of Mediterranean diets. Since the PGE2-dependent secretion of metalloproteinase(MMP)-9 by macrophages plays a key role in matrix degradation underlying plaque instability, we studied the effects of HT on the release and activity of MMP-9 in cultured human monocytoid cells as a possible explanation to the olive oil vascular protective effect. Methods: Human monocytoid cells were treated with 1-50 &#956;mol/L HT or, alternatively, with selective inhibitors of PKC isoenzymes and cyclooxygenase(COX)-2 for 60 min before stimulation with phorbol myristate acetate (PMA) or PGE2 for 24 h. Cell supernatants were then tested for the release of MMP-9, PGE2 and tissue inhibitor of metalloproteinases (TIMP)-1 and -2, while cell extracts were analysed by Western blot for COX-2 expression and PKCs membrane translocation (as an index of PKCs activation). Results: PMA and, to a lesser extent, PGE2, induced the cell release of MMP-9. Cell exposure to HT (as well as to NS-398 and G?6976, inhibitors of COX-2 and PKC&#945; and &#946; respectively) before PMA stimulation reduced MMP-9 release (IC50 for HT of 10 &#956;mol/L p<0.01) without affecting the release of TIMP-1 and -2. Correspondingly, HT inhibited PMA-induced PGE2 production (by 54?7%) and the expression of COX-2 (by 43?5%) without affecting COX-1. Furthermore, HT also reduced the membrane transloction of PKC&#945;, which is critically involved in the expression of COX-2. Conclusions: Our findings support the involvement of COX-2 derived PGE2 in the expression of MMP-9 by monocyte-like cells and demonstrate that HT inhibits the expression and the release of MMP-9. This effect appears to be mediated by the interference by HT with the stimulated expression of COX-2 and, upstream of this, with the activation of PKC&#945;. Overall, such results contribute to explaining the vascular protective effects exerted by olive oil in Mediterranean diets

Hydroxytyrosol suppresses phorbol ester-induced matrix metalloproteinase-9 expression by inhibiting both PKCalpha/beta1 and NF-kB activation in human monocytoid cells

Objectives: Mediterranean diets, of which olive oil is an important component, are associated with low prevalence of cardiovascular diseases, but active dietary components and their mechanisms of action are incompletely understood. The local production of active matrix metalloproteinase (MMP)-9 by macrophages likely contributes to plaque matrix degradation and plaque instability, leading to acute coronary events. We sought to examine the effect of the olive oil phenolic antioxidant hydroxytyrosol (HT) on MMP-9 expression and activity in monocytoid cells and to explore mechanisms of action involved. Methods: U937 human monocytoid cells were pre-treated with HT (0-100 &#956;mol/L) for 30 min before stimulation with 50 nmol/L phorbol myristate acetate (PMA) in a serum-free medium for 24 h or alternatively with inhibitors of PKC iso-enzymes or of the NF-&#954;B pathway. Cell supernatants were then tested for gelatinase activity by zymography. MMP-9 protein and mRNA expression was assayed by ELISA and RT-PCR. We assessed the activation of transcription factor Nuclear Factor (NF)-&#954;B by EMSA and Western analysis of nuclear extracts, and the activation of PKC iso-enzymes by membrane translocation analysis. Results: HT (1-100 &#956;mol/L) reduced PMA-induced MMP-9 activity at zymography analysis in a concentration-dependent manner, with inhibitory concentration producing 50% of the effect (IC50) at 10 &#956;mol/L (p<0.01). In addition, 10 &#956;mol/L HT reduced MMP-9 protein release and mRNA levels by about (by 60?5% and 40?7%, respectively, p<0.01), without significantly affecting TIMP-1 and -2 release. HT (10 &#956;mol/L) also significantly inhibited NF-&#954;B activation (by 57?8%) and PMA-induced membrane translocation of PKC&#945; and &#946;1 (by 50?8% and 35?5%, respectively, for all, p<0.05), suggesting a plausible mechanism for the downregulation of MMP-9 expression by HT. Conclusions: HT, the major olive and olive oil phenolic antioxidant, inhibits MMP-9 expression and release, interfering with PKC&#945;/&#946;1 and NF-&#954;B activation in human monocytoid cells. This may contribute to plaque stabilization, explaining at least in part the cardiovascular protection by specific components of Mediterranean diets