Sonia Eligini

Vastatins Inhibit Tissue Factor in Cultured Human Macrophages A Novel Mechanism of Protection Against Atherothrombosis

We examined the effect of fluvastatin, the first entirely synthetic 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor that is structurally different from other vastatins, on tissue factor (TF) expression in human macrophages spontaneously differentiated in culture from blood monocytes. Fluvastatin decreased TF activity in a dose-dependent manner (1 to 5 mumol/L) in both unstimulated and lipopolysaccharide-stimulated macrophages, and this reduction paralleled the decrease in immunologically recognized TF protein. The same results were obtained with another lipophilic vastatin, simvastatin, but not with hydrophilic pravastatin. The reduction in TF expression was also observed in macrophages enriched in cholesterol after exposure to 50 micrograms/mL acetylated low density lipoprotein. The inhibitory effect of fluvastatin on TF activity and antigen was fully reversible by coincubation with 100 mumol/L mevalonate or 10 mumol/L all-trans-geranylgeraniol but not with dolichol, farnesol, or geraniol. Suppression of TF antigen and activity was accompanied by a diminution in TF mRNA levels, which was completely prevented by mevalonate. Furthermore, fluvastatin impaired bacterial lipopolysaccharide-induced binding of c-Rel/p65 heterodimers to a kappa B site in the TF promoter, indicating that this drug influences induction of the TF gene. We conclude that lipophilic vastatins inhibit TF expression in macrophages, and because this effect is prevented by mevalonate and geranylgeraniol, a geranylgeranylated protein plays a crucial role in the regulation of TF biosynthesis. The suppression of TF in macrophages by vastatins indicates a potential mechanism by which these drugs interfere with the formation and progression of atherosclerotic plaque as well as thrombotic events in hyperlipidemic patients.

Changes of n − 3 and n − 6 fatty acids in plasma and circulating cells of normal subjects, after prolonged administration of 20:5 (EPA) and 22:6 (DHA) ethyl esters and prolonged washout

Eight normal volunteers (four men and four women) were treated with 3 x 1 g capsules of n-3 fatty acid ethyl esters for a period of 18 weeks, followed by a 24 week washout. Fatty acids of plasma, platelets, monocytes and red blood cells were analyzed at 0, 6, 12 and 18 weeks of treatment and at 4, 14 and 24 weeks of washout. During treatment, accumulation of EPA in plasma and cells was almost maximal at 6 weeks, whereas that of DHA reached a peak at 18 weeks. Arachidonic acid declined somewhat at 12 weeks in plasma and more markedly at 18 weeks in red blood cells and monocytes. During washout, EPA returned rapidly toward pretreatment values in all compartments, but it remained significantly higher in plasma and platelets at the end of washout. DHA declined more slowly, maintaining higher than basal values in plasma and platelets and lower than basal in red blood cells, at the end of washout. Rebound increments of AA occurred in plasma. Finally, the plasma levels of AA, but not those of the n-3 fatty acids, were more markedly modified in males than in females. The presented results suggest interactions between circulating fatty acids in the different compartment after n-3 FA administration, and indicate that very long washouts are necessary for a complete recovery from the induced fatty acid modifications.

Reactive oxygen species mediate cyclooxygenase-2 induction during monocyte to macrophage differentiation: critical role of NADPH oxidase.

OBJECTIVEnThe objective of this study was to explore the relationship between monocyte differentiation into macrophages and cyclooxygenase-2 (Cox-2) expression, based upon the observation that high amounts of this enzyme, colocalizing mainly with macrophages, have been found in human atherosclerotic lesions. Moreover, the hypothesis that reactive oxygen species (ROS) could be important as mediators of Cox-2 expression during monocyte differentiation was verified. Although ROS are known as modulators of gene expression profile, their involvement in monocyte differentiation has not been explored previously.nnnMETHODSnHuman adherent monocytes and the promonocytic cell line U937 were differentiated into macrophages by phorbol ester (PMA). Cox-2 was evaluated in terms of protein, mRNA and activity. Intracellular ROS formation was measured by the oxidant sensitive dye 2,7-dichlorofluorescein diacetate. NADPH oxidase subunit p47(phox) was evaluated by Western blot analysis.nnnRESULTSnFunctionally active Cox-2 is expressed during PMA-induced monocyte transition into macrophages and ROS driven by the NADPH oxidase play a critical role in this event.nnnCONCLUSIONnMonocyte differentiation into macrophages, possibly triggered by unquenched ROS, may contribute to the increased inflammatory response within atheromata.

n-3 fatty acid ethyl ester administration to healthy subjects and to hypertriglyceridemic patients reduces tissue factor activity in adherent monocytes.

n-3 Fatty acids are known to influence several functions of monocytes, including adhesion, cytokine synthesis, and superoxide generation. Monocytes express tissue factor, a membrane-bound glycoprotein, that acts as a catalyst in the coagulation cascade. In this study we evaluated the effects of administration of n-3 fatty acid ethyl esters to healthy volunteers and to hypertriglyceridemic patients on tissue factor activity (TF activity) in adherent monocytes. n-3 Fatty acids containing 75% eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) (ratio of EPA to DHA, 1.34) were administered (3 g/d) to normal volunteers for 18 weeks. In addition, the effects of this treatment were evaluated in 30 hypertriglyceridemic patients for 24 weeks by using a double-blind, placebo-controlled study. TF activity in adherent monocytes was evaluated with a one-stage clotting assay. Plasma and monocyte fatty acid compositions were determined by gas-liquid chromatography. In healthy volunteers, n-3 fatty acids significantly reduced TF activity in adherent monocytes either in the unstimulated condition or after exposure to endotoxin. The inhibitory effect was observed after 12 weeks of treatment and was more pronounced after 18 weeks (> 70%, P < .001 versus baseline). Concomitantly, levels of EPA and DHA increased in plasma and monocyte lipids. Interestingly, after stopping treatment, monocyte TF activity remained inhibited for at least 14 weeks. Treatment with n-3 fatty acids for 24 weeks also resulted in a significant reduction of TF activity in adherent monocytes from hypertriglyceridemic patients (-31% and -40% in unstimulated and endotoxin-stimulated cells; P < .05 versus baseline).(ABSTRACT TRUNCATED AT 250 WORDS)

Mitochondrial reactive oxygen species: a common pathway for PAR1- and PAR2-mediated tissue factor induction in human endothelial cells

Summary.u2002 Background:u2002Protease‐activated receptors (PARs) comprise a family of G‐protein‐coupled receptors with a unique proteolytic activation mechanism. PARs regulate a broad range of cellular functions and are involved in the pathogenesis of inflammatory disorders. Moreover, PAR1 and PAR2 activation in the endothelium shifts it toward a prothrombotic condition. Objectives:u2002To assess the relevance of intracellular reactive oxygen species (ROS) in the signaling events underlying tissue factor (TF) expression elicited by PAR1 and PAR2 occupancy in endothelial cells, and to investigate their source. Methods:u2002Human umbilical vein endothelial cells (HUVEC) were exposed to specific PAR1 and PAR2 agonist peptides. TF expression was determined by real‐time reverse transcription polymerase chain reaction analysis and measurement of procoagulant activity. ROS generation was determined by a fluorometric assay after cell loading with 2′‐7′‐dichlorofluorescein diacetate. Results:u2002ROS generated by the mitochondrial chain, mostly from complex III, provide a pathway through which PAR1 and PAR2 occupancy induces TF. Other sources of ROS do not participate in TF induction. Activation of both ERK1/2 and p38 MAPK is critical for mitochondrial ROS generation. In addition to these pathways shared by the two PARs, mechanisms downstream from PAR1 and PAR2 activation, different for the two receptors, also induced TF. A module that sensitively regulates PAR1 signaling and ultimately involves NF‐κB activation has been identified. Conclusions:u2002Our data identify ROS originating in mitochondria as key mediators of the signaling pathways triggered by PAR1 and PAR2 engagement in endothelial cells and show that downstream from receptor activation occur cascades that are mechanistically coupled to procoagulant activity.

Oxidized Low Density Lipoprotein Suppresses Expression of Inducible Cyclooxygenase in Human Macrophages

Atherogenesis involves several aspects of chronic inflammation and wound healing. Indeed, the atheroma is considered a special case of tissue response to injury. Injurious stimuli may include lipoproteins trapped within lesions where protein and lipid moieties have undergone chemical modifications. We have studied the effect of oxidized low density lipoproteins (ox-LDL) on inducible cyclooxygenase (Cox-2) in human monocyte-derived macrophages exposed to bacterial lipopolysaccharide (LPS). Levels of both Cox-2 and constitutive cyclooxygenase (Cox-1) were assessed using Western blot analysis. Prior incubation of macrophages with ox-LDL resulted in a strong inhibition of Cox-2 induced by LPS, without effect on Cox-1. The inhibitory effect was dependent on ox-LDL concentration and its onset was early in time (already detectable 1 hour after macrophage exposure to ox-LDL). Native LDL, and other forms of modified LDL, were without effect. The inhibition was dependent on endocytosis of ox-LDL and could be reproduced using the lipid extract from ox-LDL. Lysophosphatidylcholine, 7beta-hydroxycholesterol, and 7-oxocholesterol failed to mimic the inhibition, but oxidized arachidonic acid-containing phospholipids, produced by autoxidation of 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine, markedly inhibited Cox-2. The observation that ox-LDL downregulates Cox-2 in human macrophages may explain the fact that, within atheromata, the transformation of macrophages into foam cells results in attenuation of the inflammatory response, thus contributing to progression of atherogenesis.

Normal Vascular Function Despite Low Levels of High-Density Lipoprotein Cholesterol in Carriers of the Apolipoprotein A-I Milano Mutant

Background— Carriers of the apolipoprotein A-IMilano (apoA-IM) mutant have very low plasma high-density lipoprotein cholesterol (HDL-C) levels but do not show any history of premature cardiovascular disease or any evidence of preclinical vascular disease. HDL is believed to prevent the development of vascular dysfunction, which may well contribute to HDL-mediated atheroprotection. Whether the low HDL level of apoA-IM carriers is associated with impaired vascular function is presently unknown. Methods and Results— The vascular response to reactive hyperemia, assessed by measuring postischemic increase in forearm arterial compliance, and the plasma concentration of soluble cell adhesion molecules were evaluated in 21 adult apoA-IM carriers, 21 age- and gender-matched nonaffected relatives (control subjects), and 21 healthy subjects with low HDL-C (low-HDL subjects). The average plasma HDL-C and apoA-I levels of apoA-IM carriers were remarkably lower than those of control subjects and significantly lower than those of low-HDL subjects. The postischemic increase in forearm arterial compliance in the apoA-IM carriers was 2-fold greater than in low-HDL subjects and remarkably similar to that of control subjects. Plasma soluble cell adhesion molecule levels were similar in apoA-IM carriers and control subjects but were greater in low-HDL subjects. When incubated with endothelial cells, HDL isolated from apoA-IM carriers was more effective than HDL from control and low-HDL subjects in stimulating endothelial nitric oxide synthase expression and activation and in downregulating tumor necrosis factor-&agr;–induced expression of vascular cell adhesion molecule-1. Conclusions— Despite their very low HDL levels, apoA-IM carriers do not display typical features of impaired vascular function because of an improved activity of apoA-IM HDL in maintaining endothelial cell homeostasis.

Oxidized phospholipids inhibit cyclooxygenase-2 in human macrophages via nuclear factor-κB/IκB- and ERK2-dependent mechanisms

Objective: Oxidized low-density lipoproteins (ox-LDL) or their components suppress macrophage inflammatory response by down-regulating cytokine synthesis, nitric oxide synthase and inducible cyclooxygenase (Cox-2). This event is crucial for the pathophysiological process leading to the formation of atherosclerotic plaque. Our present study focused on the mechanisms through which oxidized phospholipids inhibit LPS-induced Cox-2 expression in human macrophages. Methods: Macrophages were incubated with a mixture of oxidized fragmented phospholipids (ox-PAPC), present in modified LDL, and then exposed to LPS. Cox-2 was evaluated in terms of protein levels, mRNA and activity. Results: Ox-PAPC dose-dependently inhibited Cox-2 protein, mRNA and activity by preventing NF-κB binding to DNA. This effect was consequent to alterations of the degradation pattern of IκBα. Moreover, ox-PAPC markedly prevented extracellular signal-regulated kinase (ERK2) activation, leading to Cox-2 expression, whereas activation of the transcription factor peroxisome proliferator-activated receptors (PPARs) was not influenced. Conclusion: ox-PAPC down-regulates LPS-induced Cox-2 expression in human macrophages by targeting both NF-κB/IκB and ERK2 pathways. An altered inflammatory response by macrophages within atheromata may contribute to the progression of atherosclerosis.

Cyclooxygenase-2 mediates hydrogen peroxide-induced wound repair in human endothelial cells

Cyclooxygenase-2 (Cox-2) metabolites produced by endothelial cells, particularly prostacyclin and prostaglandin E(2), profoundly affect vascular tone, regional blood flow, and angiogenesis. We have previously shown that reactive oxygen species induce Cox-2 expression in human endothelial cells (HUVEC), either on their own or as components of the signaling pathway triggered by TNFalpha, the prototypical inflammatory cytokine. Here we investigated the role of Cox-2 induced by hydrogen peroxide (H(2)O(2)), either exogenous or endogenously generated by TNFalpha, in the repair of a mechanically wounded HUVEC monolayer and probed the sources of H(2)O(2) that are involved in TNFalpha signaling and the pathways through which H(2)O(2) modulates Cox-2 expression. Results indicate that H(2)O(2)-induced Cox-2 activity participates in the repair of wounded monolayers. Both NADPH oxidase and the mitochondrial electron transport chain are involved in H(2)O(2) generation. Signaling triggered by H(2)O(2) for Cox-2 induction acts by increasing the protein tyrosine kinase phosphorylation that follows inhibition of protein phosphatase activity. The activation of p38 MAPK and its interaction in the inhibition of serine/threonine phosphatase activity are both critical steps in this event. We conclude that Cox-2 induced by H(2)O(2) plays an important role in promoting endothelial wound repair after injury, so that the cardioprotective effect of Cox-2 is due at least in part to its power of healing damaged endothelium.

Effect of n‐3 fatty acids on carotid atherosclerosis and haemostasis in patients with combined hyperlipoproteinemia: A double‐blind pilot study in primary prevention

Background. Intake of n‐3 polyunsaturated fatty acids (n‐3 PUFA) either from natural sources or dietary supplementation is inversely associated with atherothrombosis. Aim. A double‐blind pilot study was designed to address the impact of n‐3 PUFA on atherosclerosis, haemostasis and vascular status in patients with combined hyperlipoproteinemia. Methods. Carotid intima‐media thickness (C‐IMT), texture of intima‐media complex (T‐IMC), lipids and platelet function were evaluated in 64 patients with combined hyperlipoproteinemia who received placebo or n‐3 PUFA (6u2005g/day) for 2 years. C‐IMT and T‐IMC were assessed by B‐mode ultrasound. Lipids and platelet function were determined by validated methods. Results. C‐IMT increased in placebo, but not in n‐3 PUFA group with respect to baseline. In contrast T‐IMC decreased in n‐3 PUFA, but not in placebo; in both cases, however, treatment effect did not reach statistical significance. A fall of triglycerides, concomitant to a rise of high‐ and low‐density lipoprotein cholesterol (HDL and LDL), was observed in the active treated group. Platelet function was significantly reduced by n‐3 PUFA. Conclusions. Results show a favourable effectiveness of n‐3 PUFA on IMT progression and T‐IMC that deserves to be confirmed in larger studies. Despite the small sample size, the beneficial effect of n‐3 PUFA on platelet function, triglycerides and HDL‐C is clearly highlighted.