Silvia S. Barbieri

Eicosanoids and Their Drugs in Cardiovascular Diseases: Focus on Atherosclerosis and Stroke

Eicosanoids are biologically active lipids in both physiologic and pathophysiologic situations. These mediators rapidly generate at sites of inflammation and act through specific receptors that following the generation of a signal transduction cascade, lead to coordinated cellular responses to specific stimuli. Prostanoids, that is, prostaglandins and thromboxane A2, are active products of the cyclooxygenase pathway, while leukotrienes and lipoxins derive from the lipoxygenase pathway. In addition, a complex family of prostaglandin isomers called isoprostanes is derived as free‐radical products of oxidative metabolism. While there is a wide consensus on the importance of the balance between proaggregating (thromboxane A2) and antiaggregating (prostacyclin) cyclooxygenase products in cardiovascular homeostasis, an increasing body of evidence suggests a key role also for other eicosanoids generated by lipoxygenases, epoxygenases, and nonenzymatic pathways in cardiovascular diseases. This intricate network of lipid mediators is unique considering that from a single precursor, arachidonic acid, may derive an array of bioproducts that interact within each other synergizing or, more often, behaving as functional antagonists.

Suppressing PTEN Activity by Tobacco Smoke Plus Interleukin-1β Modulates Dissociation of VE-Cadherin/β-Catenin Complexes in Endothelium

Objectives—Tobacco smoke (TS) interacts with inflammatory cytokines to produce endothelial dysfunction. We hypothesized that interleukin-1&bgr; (IL-1&bgr;) plus TS (TS/IL-1&bgr;) induces disassembly of endothelial junctional complexes of VE-cadherin/&bgr;-catenin by suppression of PTEN activity and investigated molecular mechanisms that modulate PTEN-deactivation in this situation. Methods and Results—TS/IL-1&bgr; exposure, which disrupted adherens junctions and induced nuclear &bgr;-catenin accumulation, increased tyrosine phosphorylation (p-Tyr) of VE-cadherin and &bgr;-catenin, and reduced PTEN activity. Overexpression or silencing of PTEN modulated p-Tyr of both VE-cadherin and &bgr;-catenin, changed assembly of adherens junction complexes, and altered nuclear &bgr;-catenin accumulation. In addition, inhibiting ROS production stimulated by TS/IL-1&bgr; decreased activation of Src, EGFR and p38MAPK, phosphorylation of PTEN, VE-cadherin and &bgr;-catenin, and abrogated the effect of TS/IL-1&bgr; to disorganize adherens junctions, resulting in reduced endothelial permeability and decreased nuclear &bgr;-catenin accumulation. Finally, exposure of ApoE−/− mice to cigarette smoke–induced phosphorylation of Src, EGFR, p-38MAPK, PTEN, and &bgr;-catenin, and disrupted VE-cadherin/&bgr;-catenin complexes in cardiovascular tissue. Conclusions—TS interaction with IL-1&bgr; modulates PTEN activity though the ROS/Src/EGFR-p38MAPK pathway. PTEN deactivation is essential to increase VE-cadherin and &bgr;-catenin p-Tyr and to disassemble VE-cadherin/&bgr;-catenin membrane complexes, events that lead to accumulation of &bgr;-catenin within the nucleus.

Effects of Transforming Growth Factor-β1 on Human Vocal Fold Fibroblasts

Objectives: We studied the effect of transforming growth factor (TGF)–β on immortalized human vocal fold fibroblasts. Methods: Normal human vocal fold fibroblasts were subjected to sequential lentiviral transduction with genes for human telomerase (hTERT) and SV40 large T antigen in order to produce an “immortalized” cell line of normal phenotype. After confirmation of vocal fold fibroblast transfection, these cells, referred to as HVOX, were treated with various concentrations of exogenous TGF-β1 and assayed for collagen secretion, migration, and proliferation. In addition, components of the TGF-β signaling pathway were examined in this cell line. Results: TGF-β stimulated collagen secretion and migration without altering proliferation of HVOX. HVOX constitutively expressed type I and II TGF-β receptors, as well as messenger RNA for the Smad signaling proteins and for all TGF-β isoforms. Exogenous TGF-β1 induced temporally dependent alterations in Smad2 and Smad3 gene expression. TGF-β increased Smad7 expression at both 4 and 24 hours. Prolonged exposure to TGF-β decreased TGF-β1 gene expression. Conclusions: Insight into the underlying pathophysiology of vocal fold fibrosis is likely to yield improved therapeutic strategies to mitigate vocal fold scarring. Our data suggest that TGF-β signaling may be both paracrine and autocrine in this vocal fold fibroblast cell line, and we therefore propose that TGF-β may be a reasonable target for therapies to prevent and/or treat vocal fold fibrosis, given its putative role in both acute and chronic vocal fold injury, as well as its effects on vocal fold fibroblasts.

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.

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–Derived Prostacyclin Regulates Arterial Thrombus Formation by Suppressing Tissue Factor in a Sirtuin-1–Dependent-Manner

Background— Selective inhibitors of cyclooxygenase (COX)-2 increase the risk of myocardial infarction and thrombotic events, but the responsible mechanisms are not fully understood. Methods and Results— We found that ferric chloride–induced arterial thrombus formation was significantly greater in COX-2 knockout compared with wild-type mice. Cross-transfusion experiments excluded the likelihood that COX-2 knockout platelets, despite enhanced aggregation responses to collagen and thrombin, are responsible for increased arterial thrombus formation in COX-2 knockout mice. Importantly, we observed that COX-2 deletion decreased prostacyclin synthase and production and peroxisome proliferator-activated receptor- and sirtuin-1 (SIRT1) expression, with consequent increased upregulation of tissue factor (TF), the primary initiator of blood coagulation. Treatment of wild-type mice with a prostacyclin receptor antagonist or a peroxisome proliferator-activated receptor-&dgr; antagonist, which predisposes to arterial thrombosis, decreased SIRT1 expression and increased TF activity. Conversely, exogenous prostacyclin or peroxisome proliferator-activated receptor-&dgr; agonist completely reversed the thrombotic phenotype in COX-2 knockout mice, restoring normal SIRT1 levels and reducing TF activity. Furthermore, inhibition of SIRT1 increased TF expression and activity and promoted generation of occlusive thrombi in wild-type mice, whereas SIRT1 activation was sufficient to decrease abnormal TF activity and prothrombotic status in COX-2 knockout mice. Conclusions— Modulation of SIRT1 and hence TF by prostacyclin/peroxisome proliferator-activated receptor-&dgr; pathways not only represents a new mechanism in controlling arterial thrombus formation but also might be a useful target for therapeutic intervention in the atherothrombotic complications associated with COX-2 inhibitors.

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 (6 g/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.

Vascular pentraxin 3 controls arterial thrombosis by targeting collagen and fibrinogen induced platelets aggregation

Aim The long pentraxin PTX3 plays a non-redundant role during acute myocardial infarction, atherosclerosis and in the orchestration of tissue repair and remodeling during vascular injury, clotting and fibrin deposition. The aim of this work is to investigate the molecular mechanisms underlying the protective role of PTX3 during arterial thrombosis. Methods and results PTX3 KO mice transplanted with bone marrow from WT or PTX3 KO mice presented a significant reduction in carotid artery blood flow following FeCl3 induced arterial thrombosis (− 80.36 ± 11.5% and − 95.53 ± 4.46%), while in WT mice transplanted with bone marrow from either WT or PTX3 KO mice, the reduction was less dramatic (− 45.55 ± 1.37% and − 53.39 ± 9.8%), thus pointing to a protective effect independent of a hematopoietic cells derived PTX3. By using P-selectin/PTX3 double KO mice, we further excluded a role for P-selectin, a target of PTX3 released by neutrophils, in vascular protection played by PTX3. In agreement with a minor role for hematopoietic cell-derived PTX3, platelet activation (assessed by flow cytometric expression of markers of platelet activation) was similar in PTX3 KO and WT mice as were haemostatic properties. Histological analysis indicated that PTX3 localizes within the thrombus and the vessel wall, and specific experiments with the N-terminal and the C-terminal PTX3 domain showed the ability of PTX3 to selectively dampen either fibrinogen or collagen induced platelet adhesion and aggregation. Conclusion PTX3 interacts with fibrinogen and collagen and, by dampening their pro-thrombotic effects, plays a protective role during arterial thrombosis.

BDNFVal66met polymorphism: a potential bridge between depression and thrombosis.

Aims Epidemiological studies strongly suggest a link between stress, depression, and cardiovascular diseases (CVDs); the mechanistic correlation, however, is poorly understood. A single-nucleotide polymorphism in the BDNF gene (BDNFVal66Met), associated with depression and anxiety, has been proposed as a genetic risk factor for CVD. Using a knock-in mouse carrying the BDNFVal66Met human polymorphism, which phenocopies psychiatric-related symptoms found in humans, we investigated the impact of this SNP on thrombosis. Methods and results BDNFMet/Met mice displayed a depressive-like phenotype concomitantly with hypercoagulable state and platelet hyperreactivity. Proteomic analysis of aorta secretome from BDNFMet/Met and wild-type (WT) mice showed differential expression of proteins involved in the coagulation and inflammatory cascades. The BDNF Met allele predisposed to carotid artery thrombosis FeCl3-induced and to death after collagen/epinephrine injection. Interestingly, transfection with BDNFMet construct induced a prothrombotic/proinflammatory phenotype in WT cells. SIRT1 activation, using resveratrol and/or CAY10591, prevented thrombus formation and restored the physiological levels of coagulation and of platelet markers in BDNFMet/Met mice and/or cells transfected with the Met allele. Conversely, inhibition of SIRT1 by sirtinol and/or by specific siRNA induced the prothrombotic/proinflammatory phenotype in WT mice and cells. Finally, we found that BDNF Met homozygosity is associated with increased risk of acute myocardial infarction (AMI) in humans. Conclusion Activation of platelets, alteration in coagulation pathways, and changes in vessel wall protein expression in BDNFMet/Met mice recapitulate well the features occurring in the anxiety/depression condition. Furthermore, our data suggest that the BDNFVal66Met polymorphism contribute to the individual propensity for arterial thrombosis related to AMI.