Cell Surface Platelet Tissue Factor Expression: Regulation by P2Y12 and Link to Residual Platelet Reactivity.

BACKGROUND: ADP-induced platelet activation leads to cell surface expression of several proteins, including TF (tissue factor). The role of ADP receptors in platelet TF modulation is still unknown. We aimed to assess the (1) involvement of P2Y1 and P2Y12 receptors in ADP-induced TF exposure; (2) modulation of TFpos-platelets in anti-P2Y12-treated patients with coronary artery disease. Based on the obtained results, we revisited the intracellular localization of TF in platelets. METHODS: The effects of P2Y1 or P2Y12 antagonists on ADP-induced TF expression and activity were analyzed in vitro by flow cytometry and thrombin generation assay in blood from healthy subjects, P2Y12-/-, and patients with gray platelet syndrome. Ex vivo, P2Y12 inhibition of TF expression by clopidogrel/prasugrel/ticagrelor, assessed by VASP (vasodilator-stimulated phosphoprotein) platelet reactivity index, was investigated in coronary artery disease (n=238). Inhibition of open canalicular system externalization and electron microscopy (TEM) were used for TF localization. RESULTS: In blood from healthy subjects, stimulated in vitro by ADP, the percentage of TFpos-platelets (17.3±5.5%) was significantly reduced in a concentration-dependent manner by P2Y12 inhibition only (-81.7±9.5% with 100 nM AR-C69931MX). In coronary artery disease, inhibition of P2Y12 is paralleled by reduction of ADP-induced platelet TF expression (VASP platelet reactivity index: 17.9±11%, 20.9±11.3%, 40.3±13%; TFpos-platelets: 10.5±4.8%, 9.8±5.9%, 13.6±6.3%, in prasugrel/ticagrelor/clopidogrel-treated patients, respectively). Despite this, 15% of clopidogrel good responders had a level of TFpos-platelets similar to the poor-responder group. Indeed, a stronger P2Y12 inhibition (130-fold) is required to inhibit TF than VASP. Thus, a VASP platelet reactivity index <20% (as in prasugrel/ticagrelor-treated patients) identifies patients with TFpos-platelets <20% (92% sensitivity). Finally, colchicine impaired in vitro ADP-induced TF expression but not α-granule release, suggesting that TF is open canalicular system stored as confirmed by TEM and platelet analysis of patients with gray platelet syndrome. CONCLUSIONS: Data show that TF expression is regulated by P2Y12 and not P2Y1; P2Y12 antagonists downregulate the percentage of TFpos-platelets. In clopidogrel good-responder patients, assessment of TFpos-platelets highlights those with residual platelet reactivity. TF is stored in open canalicular system, and its membrane exposure upon activation is prevented by colchicine.

Multicenter evaluation of light transmission platelet aggregation reagents: communication from the ISTH SSC Subcommittee on Platelet Physiology.

BACKGROUND: Light transmission aggregation (LTA) is used widely by the clinical and research communities. Although it is a gold standard, there is a lack of interlaboratory harmonization. OBJECTIVES: The primary objective was to assess whether sources of activators (mainly adenosine diphosphate [ADP], collagen, arachidonic acid, epinephrine, and thrombin receptor activating peptide6) and ristocetin contribute to poor LTA reproducibility. The secondary objective was to evaluate interindividual variability of results to appreciate the distribution of normal values and consequently better interpret pathologic results. METHODS: An international multicenter study involving 28 laboratories in which we compared LTA results obtained with center-specific activators and a comparator that we supplied. RESULTS: We report variability in the potency (P) of activators in comparison with the comparator. Thrombin receptor activating peptide 6 (P, 1.32-2.68), arachidonic acid (P, 0.87-1.43), and epinephrine (P, 0.97-1.34) showed the greatest variability. ADP (P, 1.04-1.20) and ristocetin (P, 0.98-1.07) were the most consistent. The data highlighted clear interindividual variability, notably for ADP and epinephrine. Four profiles of responses were observed with ADP from high-responders, intermediate-responders, and low-responders. A fifth profile corresponding to nonresponders (5% of the individuals) was observed with epinephrine. CONCLUSION: Based on these data, the establishment and adoption of simple standardization principles should mitigate variability due to activator sources. The observation of huge interindividual variability for certain concentrations of activators should lead to a cautious interpretation before reporting a result as abnormal. Confidence can be taken from the fact that difference between sources is not exacerbated in patients treated with antiplatelet agents.

Different Contribution of Monocyte- and Platelet-Derived Microvesicles to Endothelial Behavior.

Several contributions of circulating microvesicles (MVs) to the endothelial dysfunction have been reported in the past; a head-to-head comparison of platelet- and monocyte-derived MVs has however never been performed. To this aim, we assessed the involvement of these MVs in vessel damage related processes, i.e., oxidative stress, inflammation, and leukocyte-endothelial adhesion. Platelets and monocytes isolated from healthy subjects (HS, n = 15) were stimulated with TRAP-6 and LPS to release MVs that were added to human vascular endothelial cell (hECV) culture to evaluate superoxide anion production, inflammatory markers (IL-6, TNFα, NF-κB mRNA expression), and hECV adhesiveness. The effects of the MVs-induced from HS were compared to those induced by MVs spontaneously released from cells of patients with ST-segment elevation myocardial infarction (STEMI, n = 7). MVs released by HS-activated cells triggered a threefold increase in oxidative burst in a concentration-dependent manner. Only MVs released from monocytes doubled IL-6, TNFα, and NF-κB mRNA expression and monocyte-endothelial adhesion. Interestingly, the effects of the MVs isolated from STEMI-monocytes were not superimposable to previous ones except for adhesion to hECV. Conversely, MVs released from STEMI-platelets sustained both redox state and inflammatory phenotype. These data provide evidence that MVs released from activated and/or pathologic platelets and monocytes differently affect endothelial behavior, highlighting platelet-MVs as causative factors of impaired endothelial function in the acute phase of STEMI.

Montelukast Inhibits Platelet Activation Induced by Plasma From COVID-19 Patients.

Leukotrienes are important pro-inflammatory lipid mediators derived from the arachidonic acid metabolism. In particular, cysteinyl leukotrienes, namely LTC4, LTD4, and LTE4 are involved in many of the principal features of asthma, while more recently they have also been implicated in cardiovascular diseases. COVID-19 is characterized by an overwhelming state of inflammation, sometimes resulting in an acute respiratory distress syndrome. Furthermore, severe COVID-19 patients present an endothelial cell damage characterized by a hyperinflammatory/procoagulant state and a widespread thrombotic disease. Leukotriene receptor antagonists, such as montelukast, have long been proven to have an efficacy in asthma, while more recently they have been suggested to have a protective role also in cardiovascular diseases. As elevated levels of LTE4 have been detected in bronchoalveolar lavage of COVID-19 patients, and montelukast, in addition to its anti-inflammatory properties, has been suggested to have a protective role in cardiovascular diseases, we decided to investigate whether this drug could also affect the platelet activation characteristic of COVID-19 syndrome. In this contribution, we demonstrate that montelukast inhibits platelet activation induced by plasma from COVID-19 patients by preventing the surface expression of tissue factor (TF) and P-selectin, reducing the formation of circulating monocyte- and granulocyte-platelet aggregates, and, finally, in completely inhibiting the release of TFpos-circulating microvesicles. These data suggest the repurposing of montelukast as a possible auxiliary treatment for COVID-19 syndrome.

Oxidized LDL-dependent pathway as new pathogenic trigger in arrhythmogenic cardiomyopathy.

Arrhythmogenic cardiomyopathy (ACM) is hallmarked by ventricular fibro-adipogenic alterations, contributing to cardiac dysfunctions and arrhythmias. Although genetically determined (e.g., PKP2 mutations), ACM phenotypes are highly variable. More data on phenotype modulators, clinical prognosticators, and etiological therapies are awaited. We hypothesized that oxidized low-density lipoprotein (oxLDL)-dependent activation of PPARγ, a recognized effector of ACM adipogenesis, contributes to disease pathogenesis. ACM patients showing high plasma concentration of oxLDL display severe clinical phenotypes in terms of fat infiltration, ventricular dysfunction, and major arrhythmic event risk. In ACM patient-derived cardiac cells, we demonstrated that oxLDLs are major cofactors of adipogenesis. Mechanistically, the increased lipid accumulation is mediated by oxLDL cell internalization through CD36, ultimately resulting in PPARγ upregulation. By boosting oxLDL in a Pkp2 heterozygous knock-out mice through high-fat diet feeding, we confirmed in vivo the oxidized lipid dependency of cardiac adipogenesis and right ventricle systolic impairment, which are counteracted by atorvastatin treatment. The modulatory role of oxidized lipids on ACM adipogenesis, demonstrated at cellular, mouse, and patient levels, represents a novel risk stratification tool and a target for ACM pharmacological strategies.

Platelet and Endothelial Activation as Potential Mechanisms Behind the Thrombotic Complications of COVID-19 Patients.

The authors hypothesized that the cytokine storm described in COVID-19 patients may lead to consistent cell-based tissue factor (TF)-mediated activation of coagulation, procoagulant microvesicles (MVs) release, and massive platelet activation. COVID-19 patients have higher levels of TF+ platelets, TF+ granulocytes, and TF+ MVs than healthy subjects and coronary artery disease patients. Plasma MV-associated thrombin generation is present in prophylactic anticoagulated patients. A sustained platelet activation in terms of P-selectin expression and platelet-leukocyte aggregate formation, and altered nitric oxide/prostacyclin synthesis are also observed. COVID-19 plasma, added to the blood of healthy subjects, induces platelet activation similar to that observed in vivo. This effect was blunted by pre-incubation with tocilizumab, aspirin, or a P2Y12 inhibitor.

MicroRNA-222 Regulates Melanoma Plasticity.

Melanoma is one of the most aggressive and highly resistant tumors. Cell plasticity in melanoma is one of the main culprits behind its metastatic capabilities. The detailed molecular mechanisms controlling melanoma plasticity are still not completely understood. Here we combine mathematical models of phenotypic switching with experiments on IgR39 human melanoma cells to identify possible key targets to impair phenotypic switching. Our mathematical model shows that a cancer stem cell subpopulation within the tumor prevents phenotypic switching of the other cancer cells. Experiments reveal that hsa-mir-222 is a key factor enabling this process. Our results shed new light on melanoma plasticity, providing a potential target and guidance for therapeutic studies.

Association of Microvesicles With Graft Patency in Patients Undergoing CABG Surgery.

BACKGROUND: Graft patency is one of the major determinants of long-term outcome following coronary artery bypass graft surgery (CABG). Biomarkers, if indicative of the underlying pathophysiological mechanisms, would suggest strategies to limit graft failure. The prognostic value of microvesicles (MVs) for midterm graft patency has never been tested. OBJECTIVES: The aim of this study was to evaluate whether MV pre-operative signature (number, cellular origin, procoagulant phenotype) could predict midterm graft failure and to investigate potential functional role of MVs in graft occlusion. METHODS: This was a nested case-control substudy of the CAGE (CoronAry bypass grafting: factors related to late events and Graft patency) study that enrolled 330 patients undergoing elective CABG. Of these, 179 underwent coronary computed tomography angiography 18 months post-surgery showing 24% graft occlusion. Flow cytometry MV analysis was performed in 60 patients (30 per group with occluded [cases] and patent [control subjects] grafts) on plasma samples collected the day before surgery and at follow-up. RESULTS: Before surgery, cases had 2- and 4-fold more activated platelet-derived and tissue-factor positive MVs respectively than control subjects. The MV procoagulant capacity was also significantly greater. Altogether this MV signature properly classified graft occlusion (area under the curve 0.897 [95% confidence interval: 0.81 to 0.98]; p < 0.0001). By using an MV score (0 to 6), the odds ratio for occlusion for a score above 3 was 16.3 (95% confidence interval: 4.1 to 65.3; p < 0.0001). CONCLUSIONS: The pre-operative signature of MVs is independently associated with midterm graft occlusion in CABG patients and a cumulative MV score stratifies patients' risk. Because the MV signature mirrors platelet activation, patients with a high MV score could benefit from a personalized antiplatelet therapy.

Repurposing of drugs approved for cardiovascular diseases: Opportunity or mirage?

Drug repurposing is a promising way in drug discovery to identify new therapeutic uses -different from the original medical indication- for existing drugs. It has many advantages over traditional approaches to de novo drug discovery, since it can significantly reduce healthcare costs and development timeline. In this review, we discuss the possible repurposing of drugs approved for cardiovascular diseases, such as ß-blockers, angiotensin converting enzyme inhibitors (ACE-Is), angiotensin II receptor blockers (ARBs), statins, aspirin, cardiac glycosides and low-molecular-weight heparins (LMWHs). Indeed, numerous experimental and epidemiological studies have reported promising anti-cancer activities for these drugs. It is worth mentioning, however, that the results of these studies are often controversial and very few data were obtained by controlled prospective clinical trials. Therefore, no final conclusion has yet been reached in this area and no final recommendations can be made. Moreover, ß-blockers, ARBs and statins showed promising results in randomised controlled trials (RCTs) where pathological conditions other than cancer were considered. The results obtained have led or may lead to new indications for these drugs. For each drug or class of drugs, the potential molecular mechanisms of action justifying repurposing, results obtained in vitro and in animal models and data from epidemiological and randomized studies are described.

Biological profile of monocyte-derived macrophages in coronary heart disease patients: implications for plaque morphology.

The prevalence of a macrophage phenotype in atherosclerotic plaque may drive its progression and/or instability. Macrophages from coronary plaques are not available, and monocyte-derived macrophages (MDMs) are usually considered as a surrogate. We compared the MDM profile obtained from coronary artery disease (CAD) patients and healthy subjects, and we evaluated the association between CAD MDM profile and in vivo coronary plaque characteristics assessed by optical coherence tomography (OCT). At morphological analysis, MDMs of CAD patients had a higher prevalence of round than spindle cells, whereas in healthy subjects the prevalence of the two morphotypes was similar. Compared to healthy subjects, MDMs of CAD patients had reduced efferocytosis, lower transglutaminase-2, CD206 and CD163 receptor levels, and higher tissue factor (TF) levels. At OCT, patients with a higher prevalence of round MDMs showed more frequently a lipid-rich plaque, a thin-cap fibroatheroma, a greater intra-plaque macrophage accumulation, and a ruptured plaque. The MDM efferocytosis correlated with minimal lumen area, and TF levels in MDMs correlated with the presence of ruptured plaque. MDMs obtained from CAD patients are characterized by a morpho-phenotypic heterogeneity with a prevalence of round cells, showing pro-inflammatory and pro-thrombotic properties. The MDM profile allows identifying CAD patients at high risk.

Endothelial Dysfunction in Patients with Severe Mitral Regurgitation.

Mitral valve prolapse (MVP) is the most common cause of severe mitral regurgitation. It has been reported that MVP patients-candidates for mitral valve repair (MVRep)-showed an alteration in the antioxidant defense systems as well as in the L-arginine metabolic pathway. In this study, we investigate if oxidative stress and endothelial dysfunction are an MVP consequence or driving factors. Forty-five patients undergoing MVRep were evaluated before and 6 months post surgery and compared to 29 controls. Oxidized (GSSG) and reduced (GSH) forms of glutathione, and L-arginine metabolic pathway were analyzed using liquid chromatography-tandem mass spectrometry methods while osteoprotegerin (OPG) through the ELISA kit and circulating endothelial microparticles (EMP) by flow cytometry. Six-month post surgery, in MVP patients, the GSSG/GSH ratio decreased while symmetric and asymmetric dimethylarginines levels remained comparable to the baseline. Conversely, OPG levels significantly increased when compared to their baseline. Finally, pre-MVRep EMP levels were significantly higher in patients than in controls and did not change post surgery. Overall, these results highlight that MVRep completely restores the increased oxidative stress levels, as evidenced in MVP patients. Conversely, no amelioration of endothelial dysfunction was evidenced after surgery. Thus, therapies aimed to restore a proper endothelial function before and after surgical repair could benefit MVP patients.

PCSK9 induces a pro-inflammatory response in macrophages.

Intraplaque release of inflammatory cytokines from macrophages is implicated in atherogenesis by inducing the proliferation and migration of media smooth muscle cells (SMCs). PCSK9 is present and released by SMCs within the atherosclerotic plaque but its function is still unknown. In the present study, we tested the hypothesis that PCSK9 could elicit a pro-inflammatory effect on macrophages. THP-1-derived macrophages and human primary macrophages were exposed to different concentrations (0.250 ÷ 2.5 µg/ml) of human recombinant PCSK9 (hPCSK9). After 24 h incubation with 2.5 µg/ml PCSK9, a significant induction of IL-1ß, IL-6, TNF-α, CXCL2, and MCP1 mRNA, were observed in both cell types. Co-culture of THP-1 macrophages with HepG2 overexpressing hPCSK9 also showed the induction of TNF-α (2.4 ± 0.5 fold) and IL-1ß (8.6 ± 1.8 fold) mRNA in macrophages. The effect of hPCSK9 on TNF-α mRNA in murine LDLR-/- bone marrow macrophages (BMM) was significantly impaired as compared to wild-type BMM (4.3 ± 1.6 fold vs 31.1 ± 6.1 fold for LDLR-/- and LDLR+/+, respectively). Finally, a positive correlation between PCSK9 and TNF-α plasma levels of healthy adult subjects (males 533, females 537) was observed (B = 8.73, 95%CI 7.54 ÷ 9.93, p < 0.001). Taken together, the present study provides evidence of a pro-inflammatory action of PCSK9 on macrophages, mainly dependent by the LDLR.

D-dimer is associated with arterial and venous coronary artery bypass graft occlusion.

OBJECTIVE: In this observational prospective study, we assessed the role of clinical variables and circulating biomarkers in graft occlusion at 18 months to identify a signature for graft occlusion. METHODS: A total of 330 patients undergoing primary elective coronary artery bypass grafting were enrolled. Blood collection for biomarker assessment was performed before surgery and discharge. Patients were then scheduled to undergo coronary computed tomography angiography at 18 months follow-up, and 179 patients underwent coronary computed tomography angiography 18 ± 2 months postoperatively. RESULTS: There were 46 of 503 (9.1%) occluded grafts; of these, 29 (63%) were venous and 17 (37%) were arterial grafts; overall, 43 of 179 patients (24%) had at least 1 occluded graft. Logistic mixed effects model assessing independent factors associated with graft occlusion identified that lower D-dimer levels at baseline (odds ratio [OR], 2.58; 95% confidence interval [CI], 1.36-4.89; P = .00) and total protein content at discharge (OR, 1.09; 95% CI, 1.01-1.19; P = .028) were related to overall graft occlusion at follow-up, along with an arterial graft other than the left internal thoracic artery (OR, 2.92; 95% CI, 1.24-6.9; P = .078); moreover, a venous graft emerged was possibly associated with graft occlusion (OR, 1.51; 95% CI, 0.95-2.39; P = .078). By separately analyzing saphenous vein and arterial grafts, D-dimer levels (OR, 2.67; 95% CI, 1.15-6.2; P = .022 and OR, 2.5; 95% CI, 1.01-7.0; P = .05 for venous and arterial graft, respectively) were still associated with arterial and venous graft occlusion at follow-up. CONCLUSIONS: We identified D-dimer as a biomarker associated with arterial and venous grafts occlusion. This may help stratify patients at risk of graft failure and identify new molecular targets to prevent this complication.

ACE-Inhibition Benefit on Lung Function in Heart Failure is Modulated by ACE Insertion/Deletion Polymorphism.

PURPOSE: The benefit of angiotensin converting enzyme (ACE) inhibition in chronic heart failure (HF) is partially due to its effects on pulmonary function and particularly on lung diffusion, the latter being counteracted by acetylsalicylic acid (ASA). Tissue ACE activity is largely determined by an insertion/deletion (I/D) polymorphism resulting in three possible genotypes (DD, ID and II). It is not clear if ACE inhibitor therapy could exert different effects in these genotypes. The aim of the study was to understand whether I/D polymorphism interferes with ACE inhibitor's protection of the lungs in HF during acute fluid overload. METHODS: 100 HF patients (left ventricular ejection fraction ≤40 %) in stable clinical conditions, treated with enalapril but without ASA performed pulmonary function tests including lung diffusion (DLco) and its subcomponents, membrane diffusion (Dm) and capillary volume (Vcap), and a cardiopulmonary exercise test before and immediately after rapid infusion of 500 cc saline. RESULTS: ACE I/D genotype prevalence was: DD = 28, ID =55 and II = 17 cases. No significant differences in major pulmonary function and exercise parameters were observed before saline infusion among ACE genotypes. After fluid challenge, DD patients presented a higher DLco and Dm reduction than ID and II (DLco -2.3 ± 1.3 vs. -0.8 ± 1.9 and -0.6 ± 1 mL/mmHg/min, p < 0.0001 and p < 0.01; Dm -7 ± 5 vs. -3.2 ± 7.4 and -1.3 ± 5 mL/mmHg/min, p < 0.05, respectively) and a higher increase in VE/VCO2 slope than II (1.8 ± 1.9 vs. -0.8 ± 2.3, p = 0.01). CONCLUSIONS: ACE DD genotype is associated with higher vulnerability of the alveolar-capillary membrane to acute fluid overload in HF patients treated with ACE inhibitors.

Anti-TNFα agents curb platelet activation in patients with rheumatoid arthritis.

BACKGROUND: Cardiovascular disease is important in rheumatoid arthritis (RA). Tissue factor (TF) is expressed upon platelet activation and initiates coagulation. Anti-tumour necrosis factor-α (TNFα) agents seem to decrease RA-associated cardiovascular events. We investigated whether (1) TNFα activates human platelets and (2) TNFα pharmacological blockade modulates the platelet-leucocyte reciprocal activation in RA. DESIGN: The expression of platelet TNFα receptors has been assessed by flow cytometry and immunogold electron microscopy. Platelet and leucocyte activation has been assessed also in the presence of antibodies against the TNFα receptors 1 and 2 and of infliximab. TF expression, binding to fibrinogen and phosphatidylserine exposure, has been assessed by flow cytometry, TF activity by coagulation time and by endogenous thrombin generation. Markers of platelet and leucocyte activation have been assessed in 161 subjects: 42 patients with RA, 12 with osteoarthritis, 37 age-matched and sex-matched patients with chronic stable angina and 70 age-matched and sex-matched healthy subjects. RESULTS: TNFα elicited the platelet activation and the expression of TF, which in turn prompted thrombin generation and clot formation. Inhibition of the TNFα-induced activation restricted platelet ability to activate leucocytes and to induce leucocyte TF. TNFα inhibition did not influence platelet activation induced by collagen, ADP or thrombin receptor activating peptide-6. Platelets of patients with RA were more activated than those of controls. Activation was reduced in patients treated with TNFα inhibitors. CONCLUSIONS: TNFα-dependent pathways control platelet activation and TF expression in RA. Further studies will verify whether the protective effect of TNFα inhibitors on cardiovascular events involves their ability to modulate platelet function.

Direct anticoagulant drugs to overcome limitations of vitamin K antagonists. A critical appraisal of data in atrial fibrillation patients.

INTRODUCTION: The usefulness of anticoagulation in patients with atrial fibrillation (AF) is well known. However, the inherent limitations of vitamin K antagonists (VKAs) have made the development of new oral anticoagulants necessary. Drugs directed against thrombin or the factor Xa are currently available. AREAS COVERED: These molecules, being administered at fixed doses and not requiring laboratory monitoring, overcome one crucial problem associated with the use of VKAs. However, data about the bleeding risk related to the use of these molecules should be further analyzed. EXPERT OPINION: The efficacy of direct anticoagulants (DACs) in AF-related stroke prevention has been considered the primary outcome in all Phase III published trials. On the other hand, the reduction of the bleeding risk is an important goal achieved by the DACs as compared with VKAs. Besides data deriving from randomized trials, when talking about new drugs, the need of evidences from the 'everyday clinical practice' are often requested. The aim of this literature revision is to report and analyze data from specific subgroups about which little is known. In particular, information about the use of DACs in oncologic patients, in patients receiving concomitant antiplatelet drugs and in the perioperative period is currently lacking. The parallel evaluation of all these data may lead to the identification of clinical and demographical criteria to choose when to switch to DACs.

Chemotactic effect of prorenin on human aortic smooth muscle cells: a novel function of the (pro)renin receptor.

AIMS: The discovery of a specific prorenin receptor (PRR) suggests a biological function of prorenin that is independent of angiotensin I production. In the present study, we investigated the role of PRR on smooth muscle cell (SMC) migration. METHODS AND RESULTS: PRR was expressed in human mammary arteries and in cultured human aortic SMCs. Prorenin induced SMC migration in a dose-dependent manner, as assessed by Boyden chamber chemotaxis assay, and increased SMC random motility, as determined by video microscopy. The prorenin decoy peptide inhibited SMC migration in response to prorenin, and knockdown of PRR by small interfering RNA completely inhibited the migratory response to prorenin, demonstrating that the chemotactic action of prorenin is mediated by the PRR. Prorenin induced cytoskeleton reorganization and lamellipodia formation and increased the intracellular levels of both RhoA-GTP and Rac1-GTP through PRR. These effects were required for SMC migration, because the suppression by small interfering RNA of either Rac1 or RhoA GTP-bound forms completely blocked the PRR-mediated chemotactic effect. Prorenin also induced the formation of larger focal adhesions and cleavage of the focal adhesion kinase (pp125(FAK)) into two main fragments with molecular weights of 50 and 90 kDa. The generation of these two fragments of pp125(FAK) was reduced by the calpain inhibitor ALLN, which also inhibited SMC migration in response to prorenin. CONCLUSIONS: These results demonstrate that prorenin is a chemotactic factor for human aortic SMCs expressing PRR. This effect is elicited through reorganization of the cytoskeleton and focal adhesion, activation of RhoA and Rac1, and calpain-mediated cleavage of pp125(FAK).

Transcriptional regulation of the human FPR2/ALX gene: evidence of a heritable genetic variant that impairs promoter activity.

Lipoxin (LX) A(4,) a main endogenous stop-signal of inflammation, activates the G-protein-coupled receptor FPR2/ALX, which triggers potent anti-inflammatory signaling in vivo. Thus, the regulation of FPR2/ALX expression may have pathophysiological and therapeutic relevance. Here, we mapped a nucleotide sequence with strong FPR2/ALX promoter activity. Chromatin immunoprecipitation revealed specificity protein 1 (Sp1) binding to the core promoter. Site-directed mutagenesis of the Sp1 cis-acting element and Sp1 overexpression established that this transcription factor is key for maximal promoter activity, which is instead suppressed by DNA methylation. LXA(4) enhanced FPR2/ALX promoter activity (+74%) and mRNA expression (+87.5%) in MDA-MB231 cells. A single nucleotide mutation (A/G) was detected in the core promoter of one subject with history of cardiovascular disease and of his two daughters. This mutation reduced by ∼35-90% the promoter activity in vitro. Moreover, neutrophils from individuals carrying the A/G variant displayed ∼10- and 3-fold reduction in FPR2/ALX mRNA and protein, respectively, compared with cells from their relatives or healthy volunteers expressing the wild-type allele. These results uncover FPR2/ALX transcriptional regulation and provide the first evidence of mutations that affect FPR2/ALX transcription, thus opening new opportunities for the understanding of the LXA(4)-FPR2/ALX axis in human disease.

Gene expression profiling reveals multiple differences in platelets from patients with stable angina or non-ST elevation acute coronary syndrome.

INTRODUCTION: Platelets play a key role in coronary artery disease. They have the capacity of protein synthesis through translation of megakaryocyte-derived mRNAs, which may influence pathophysiological functions. The present study aimed to prove the concept that platelets from patients with non-ST elevation acute coronary syndrome (NSTE-ACS) have differential mRNA expression profiles, in the hypothesis that this may influence their thrombogenicity. MATERIALS AND METHODS: Gene expression profiles were determined in RNA pools from resting platelets of patients with stable angina (SA, n = 14) or NSTE-ACS (n = 15) using a glass microarray platform. Validation was done by real-time PCR and immunoblot analyses in independent sets of individual samples (26 SA and 17 NSTE-ACS patients, in total). Parallel comparison with healthy subjects was performed to relate the relative abundance of validated genes in CAD patients to a control expression level. RESULTS: Microarray analysis identified 45 transcripts with a significant ≥ ± 2.0-fold difference in expression between NSTE-ACS and SA platelet pools. Thus, gene expression profiles at least partially discriminate unstable from stable CAD. Validation confirmed a significant over-expression of 3 genes in NSTE-ACS at both mRNA and protein level. In particular, the glycoprotein Ib ß-polypeptide (GP1BB) was increased in NSTE-ACS also in comparison with healthy subjects. CONCLUSION: This study provides evidence that NSTE-ACS platelets are potentially preconditioned to a higher degree of reactivity on the transcriptional level. Our data suggest that a different composition of the mRNA pool might mediate an increased platelet prothrombotic potential in NSTE-ACS patients.