S. Colli

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.

Tissue factor in atherosclerosis

Thrombosis is a key feature of the initiation and progression of atherosclerosis and its clinical sequelae. Acute thrombosis can lead to arterial occlusion and consequently provoke myocardial infarction, unstable angina, stroke and sudden death. Acute thrombosis can also be a complication of arterial bypass surgery, balloon angioplasty, atherectomy, or coronary artery stenting. The thrombotic response is influenced by several factors, among them the thrombogenicity of the vessel wall and of certain blood components as well as their interaction with the lipid pool. Tissue factor (TF) is considered to be the primary cofactor of cellular origin that is involved in activation of the coagulation pathway. The active form of TF has been shown to be present in specimens of human coronary artery in association both with acellular lipid areas and with macrophages and smooth muscle cells, which suggests that TF plays a major role in determining plaque thrombogenicity. We discuss here what is currently known about the role of tissue factor in atherogenesis, and focus attention on pharmacological approaches in this area.

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.

Increased platelet sensitivity and thromboxane B2 formation in type-II hyperlipoproteinaemic patients

Abstract. Platelet aggregation induced by collagen, ADP and epinephrine, was monitored in 150 type‐II patients (115 type IIA and 35 type IIB) and compared with a reference group of normolipidaemic controls; in addition, malondialdehyde formation and thromboxane B2 were examined in a subsample of the type‐IIA patients. Threshold aggregatory concentrations were significantly lower in the whole group of type‐II patients for all three aggregating agents; no difference in terms of aggregatory response was detected between platelets from type‐IIA and ‐IIB patients. Only 56% of type‐II patients, however, exceeded the 95th percentile of the threshold aggregatory concentrations in controls. The formation of malondialdehyde in platelet‐rich plasma stimulated with thrombin and collagen, was significantly higher in platelets from type‐IIA patients. The production of thromboxane B2 by platelets, from endogenous arachidonic acid in type‐IIA patients, was significantly higher and exceeded the highest level found in controls.

Increased synthesis of plasminogen activator inhibitor-1 by cultured human endothelial cells exposed to native and modified LDLs : an LDL receptor-independent phenomenon

The effects of native and acetylated low density lipoproteins (LDLs and acetyl-LDLs, respectively) on the release of plasminogen activator inhibitor type 1 (PAI-1) by cultured human umbilical vein endothelial cells (ECs) were evaluated. LDL and acetyl-LDL incubated with ECs for 16-18 hours increased the PAI-1 antigen levels in conditioned medium. At a concentration of 100 micrograms/mL, LDL and acetyl-LDL increased PAI-1 by 10.8 and 12.0 ng/mL, respectively (p < 0.05 and p < 0.01 versus control). The increases in PAI-1 antigen levels exerted by the lipoproteins paralleled the changes in PAI-1 activity. The effect of LDL and acetyl-LDL was concentration dependent and specific for PAI-1 because tissue-type plasminogen activator and expression of procoagulant activity were not affected by either lipoprotein. In addition, total protein synthesis evaluated in [35S] methionine-labeled ECs was not affected, and studies with cycloheximide showed that the effect of LDL and acetyl-LDL on PAI-1 release was due to de novo protein synthesis. Experiments using the C7 monoclonal antibody against the LDL receptor and binding-defective LDL indicated that the effect of LDL on the synthesis of PAI-1 was not dependent on the interaction of the LDLs with their specific receptors. Finally, extensive oxidation of LDL prevented and even reversed the effect of LDL on PAI-1 release by ECs. It is concluded that LDL specifically increases the synthesis of PAI-1 by ECs with mechanisms that are not receptor mediated.

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.  Background: Protease‐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: To 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: Human 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: ROS 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: Our 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.

Human monocyte-derived macrophages spontaneously differentiated in vitro show distinct phenotypes.

Tissue macrophages are resident phagocytes that acquire specific phenotypes according to the microenvironment. Morphological and functional heterogeneity has been evidenced in different homeostatic and pathological conditions. Indeed, the nature of macrophage subsets may have either harmful or beneficial functions in disease progression/resolution. Therefore the possibility to pharmacologically manipulate heterogeneity represents a relevant challenge. Since human tissue macrophages are not easily obtained, various in vitro models are currently used that do not adequately reflect the heterogeneity and plasticity of tissue macrophages. We had previously reported that two dominant and distinct macrophage morphotypes co‐exist in the same culture of human monocytes spontaneously differentiated for 7 days in autologous serum. The present study was aimed to the phenotypic characterization of these morphotypes, that is, round‐ and spindle‐shaped. We observed that, besides substantial differences in cytoskeleton architecture, round monocyte‐derived macrophages (MDMs) showed higher lipid content, increased macropinocytosis/efferocytosis capacity, and overexpression of CD163, interleukin (IL)‐10, and transforming growth factor (TGF) β2. Conversely, spindle MDMs exhibited enhanced respiratory burst and higher expression of the chemokine (C‐C motif) ligands 18 and 24 (CCL18 and CCL24). Overall, round MDMs show functional traits reminiscent of the non‐inflammatory and reparative M2 phenotype, whereas spindle MDMs exhibit a pro‐inflammatory profile and express genes driving lymphocyte activation and eosinophil recruitment. MDMs obtained in the culture condition herein described represent a valuable model to disentangle and manipulate the functional heterogeneity of tissue macrophages that has been disclosed in scenarios spanning from inflammatory and wounding responses to atherosclerotic lesions. J. Cell. Physiol. 228: 1464–1472, 2013.

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.

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.