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Aims T-lymphocytes plays an important role in the pathophysiology of acute coronary syndromes. T-cell activation in vitro by pro-inflammatory cytokines may lead to functional tissue factor (TF) expression, indicating a possible contribution of immunity to thrombosis. Oxidized low-density lipoproteins (oxLDLs) are found abundantly in atherosclerotic plaques. We aimed at evaluating the effects of oxLDLs on TF expression in T cells and the role of the lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1). Methods and results CD3+ cells were isolated from healthy volunteers. Gene, protein, and surface expression of TF, as well as of LOX-1, were assessed at different time-points after oxLDL stimulation. To determine whether oxLDL-induced TF was LOX-1 dependent, T cells were pre-incubated with an LOX-1 inhibiting peptide (L-RBP) or with an anti-LOX-1 blocking antibody. To exclude that TF expression was mediated by reactive oxygen species (ROS) generation, oxLDL-stimulated T cells were pre-incubated with superoxide dismutase + catalase or with 4-Hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (Tempol), an intracellular free radical scavenger. Finally, to determine if the observed findings in vitro may have a biological relevance, the presence of CD3+/TF+/LOX-1+ cells was evaluated by immunofluorescence in human carotid atherosclerotic lesions. oxLDLs induced functionally active TF expression in T cells in a dose- and time-dependent manner, independently on ROS generation. No effect was observed in native LDL-treated T cells. LOX-1 expression was also induced by oxLDLs in a time- and dose-dependent manner. Pre-incubation with L-RBP or anti-LOX-1 antibody almost completely inhibited oxLDL-mediated TF expression. Interestingly, human carotid plaques showed significant infiltration of CD3+ cells (mainly CD8+ cells), some of which were positive for both TF and LOX-1. Conclusion oxLDLs induce functional TF expression in T-lymphocytes in vitro via interaction of oxLDLs with LOX-1. Human carotid atherosclerotic plaques contain CD3+/CD8+cells that express both TF and LOX-1, indicating that also in patients these mechanisms may play an important role.

Epidemiologic studies have clearly demonstrated the correlation existing between Vitamin D (Vit. D) deficiency and increased risk of developing cardiovascular disease, suggesting that it might have a protective role in this clinical setting. Although many experimental studies have investigated the molecular mechanisms by which Vit. D might exert these effects, its potential role in protecting against athero-thrombosis is still partially unknown. We have investigated whether Vit. D might exert anti athero-thombotic effects by preventing expression of adhesion molecules (CAMs) and Tissue Factor (TF), molecules involved in atherothrombotic pathophysiology, in oxLDL stimulated endothelial cells (HUVEC). Moreover, we have investigated whether Vit. D effects might be due to the NF-kB modulation. HUVEC cultivated in medium enriched with Vit. D (10 nM) were stimulated with oxLDL (50 μg/ml). TF gene (RT-PCR), protein (Western blot), surface expression (FACS) and procoagulant activity (FXa generation assay) were measured. Similarly, CAMs gene (RT-PCR), surface expression (FACS) and soluble values (ELISA) were measured. NF-kB translocation was also investigated. Vit. D significantly reduced TF gene as well protein expression and procoagulant activity in oxLDL-treated HUVEC. Similar effects were observed for CAMs. These effects were associated with Vit. D modulation of NF-κB pathway. This study, although in vitro, indicate that Vit. D has protective effect on endothelial cells by inhibiting expression of TF and CAMs, proteins involved in atherothrombotic pathophysiology. Further studies will be necessary to translate these findings to a clinical scenario to better define the potential therapeutical role of Vit. D supplementation in the management of cardiovascular disease in patients with Vit. D deficiency.

Grittani, Mario, Grazia Pellegrino, Stefano Conte, Andrea Morello, Alberto Autore, Giovanni Cimmino, Bruno Trimarco, Fabio Morgagni, and Plinio Cirillo. Effects of hypobaric hypoxia on endothelial function and adiponectin levels in airforce aviators. High Alt Med Biol 00:000-000, 2019. Background: Hypobaric hypoxia (HH) increases the risk of high altitude-related illnesses (HARI). The pathophysiological mechanism(s) involved are still partially unknown. Altered vascular reactivity as consequence of endothelial dysfunction during HH might play a role in this phenomenon. Adiponectin exerts protective effect on cardiovascular system since it modulates NO release, antagonizing endothelial dysfunction. Aims of this study, performed in a selected population of airforce aviators, were (1) to investigate whether exposure to acute HH might be associated with endothelial dysfunction and (2) to evaluate whether adiponectin might be involved in modulating this phenomenon. Methods: Twenty aviators were exposed to acute HH in a hypobaric chamber by simulating altitude of 8000 and then 6000 m for 2 hours. Vascular reactivity was evaluated by the EndoPAT test immediately before and after the HH; salivary and blood adiponectin levels were measured. Results: EndoPAT performed immediately after HH divided pilots in two groups: 12 pilots with preserved vascular reactivity and 8 pilots with reduction of vascular reactivity, indicating that HH exposure might cause endothelial dysfunction. Salivary and blood adiponectin levels increased post-HH in a time-dependent manner in all aviators, but the significant increase was observed only in those with preserved vascular reactivity suggesting that HH stimulated release of adiponectin that, in turn, by exerting a protective effect, might reduce endothelial dysfunction. Conclusions: Acute HH may cause endothelial dysfunction due, at least in part, to reduced release of adiponectin. This phenomenon might be involved in pathophysiology of HARI..

The key role of platelets in pathophysiology of Acute Coronary Syndromes (ACS) has been well recognized. Platelet activation and aggregation, together with tissue factor-pathway activation, lead to acute thrombus formation in the coronary vessels at sites of plaque rupture. Thus, antiplatelet therapy with drugs able to interfere with platelet activation/aggregation represents a cornerstone of ACS treatment in intensive care units and catheterisation labs. Several observational studies have described that residual high platelet reactivity, despite antiplatelet therapy, is associated with increased risk of nonfatal Myocardial Infarction (MI), definite/probable stent thrombosis and cardiovascular mortality. Thus, assessment of platelet function with reliable and reproducible platelet function tests might be crucial to identify patients at high risk of thrombosis or not responding to ongoing antiplatelet strategies. However, despite this promising background, some randomized clinical trials have failed to demonstrate improvement in outcomes when using platelet function tests for clinical decision-making. This review, after describing platelet biology and pathophysiology of ACS, briefly considers the drugs currently approved for use in patients with ACS or treated by the percutaneous coronary intervention (PCI). Finally, we provide an updated overview of the current methods to evaluate platelet reactivity in the clinical setting of ACS illustrating their potential advantages/limitations in current clinical practice.

It is generally accepted that the initial event in coagulation and intravascular thrombus formation is the exposure of the flowing blood to cell-surface protein, such as tissue factor (TF). Vascular injury and/or atherosclerotic plaque complication is responsible for this exposure, leading to clinical manifestations such as acute coronary syndromes. For many years, TF has been considered one of the major determinants of plaque-related thrombosis. However, the discoveries of different pools of TF that circulates in the blood as cell-associated TF, microparticles bound, and as soluble form have changed this dogma. Recent evidence suggests that an increased circulating TF activity may potentiate the initial thrombogenic stimulus related to vessel wall-associated TF, leading to the formation of larger and/or more stable thrombus and thus more severe clinical manifestations. Different pathological conditions, such as inflammatory status, enhance TF expression and activity. Conversely, TF upregulation may facilitate inflammation by formation of proinflammatory fibrin fragments and coagulation proteases generation, including FVIIa, FXa, and thrombin. Furthermore, the biology of TF has become more complex by the demonstration that, apart from its known effects on blood coagulation, it is involved in intracellular signaling, proliferation, angiogenesis, and tumor metastasis.

The role of low-density lipoproteins (LDLs) as a major risk factor for cardiovascular disease has been demonstrated by several epidemiological studies. The molecular basis for LDLs in atherosclerotic plaque formation and progression is not completely unraveled yet. Pharmacological modulation of plasma LDL-C concentrations and randomized clinical trials addressing the impact of lipid-lowering interventions on cardiovascular outcome have clearly shown that reducing plasma LDL-C concentrations results in a significant decrease in major cardiovascular events. For many years, statins have represented the most powerful pharmacological agents available to lower plasma LDL-C concentrations. In clinical trials, it has been shown that the greater the reduction in plasma LDL-C concentrations, the lower the rate of major cardiovascular events, especially in high-risk patients, because of multiple risk factors and recurrent events. However, in a substantial number of patients, the recommended LDL target is difficult to achieve because of different factors: genetic background (familial hypercholesterolemia), side effects (statin intolerance), or high baseline plasma LDL-C concentrations. In the last decade, our understanding of the molecular mechanisms involved in LDL metabolism has progressed significantly and the key role of proprotein convertase subtilisin/kexin type 9 (PCSK9) has emerged. This protein is an enzyme able to bind the LDL receptors (LDL-R) on hepatocytes, favoring their degradation. Blocking PCSK9 represents an intriguing new therapeutic approach to decrease plasma LDL-C concentrations, which in recent studies has been demonstrated to also result in a significant reduction in major cardiovascular events.

BACKGROUND: Antithrombotic management of patients with atrial fibrillation (AF) requiring percutaneous coronary intervention (PCI) is highly variable; limited evidence‐based guidelines exist to influence practice. HYPOTHESIS: Patient characteristics and availability of novel drugs may have contributed to practice variability. METHODS: We undertook an international multicenter retrospective registry of AF patients treated with PCI. The primary measures of interest were antiplatelet and OAC prescriptions at discharge. We compared temporal trends between Prior (2010–2012) and Recent (2013–2015) cohorts and investigated variables associated with OAC prescription. RESULTS: We identified 488 cases (140 Prior, 348 Recent). Median CHADS(2) and HAS‐BLED scores were 2 (IQR, 1–3) and 2 (IQR, 2–3). Clinical characteristics were similar between cohorts, with high (85%) prevalence of ACS. More patients in the Recent cohort, compared with Prior, received OAC (56.9% vs 44.3%; P = 0.01) and NOAC (27.3% vs 3.6%; P

Introduction Platelets activation/aggregation with subsequent thrombus formation is the main event in the pathophysiology of acute coronary syndrome. Once activated, platelets show an extensive cytoskeleton rearrangement that leads to recruitment of additional platelets to finally cause haemostatic plug formation. Thus, the cytoskeleton plays a pivotal role in this phenomenon. Colchicine (COLC) is an anti-inflammatory drug proven to reduce major cardiovascular events in patients with coronary artery disease. The molecular mechanisms by which COLC exerts these protective effects remain partially still unknown. Since COLC causes disruption of tubulin, a component of cell cytoskeleton, we investigated whether this drug might interfere with platelet aggregation by acting on cytoskeleton rearrangement. Methods and results Platelets isolated from healthy volunteers were activated with Adenosine Diphosphate (ADP, 20 μM) Collagen (COLL, 60 μg/ml) and Thrombin Activating Receptor Peptide (TRAP 25 μM) with/without COLC 10 μM pretreatment. After stimulus, aggregation was measured by light aggregometry overtime. Microtubules structure was assessed by immunohistochemistry and key proteins involved in regulation of actin-filament assembly and contractility such as Myosin Phosphatase Targeting subunit (MYPT), LIM domain kinase 1(LIMK1) and cofilin were evaluated by Western Blot analysis. Colchicine pretreatment significantly blunted ADP/COLL/TRAP-induced platelet aggregation (up to 40%). COLC effects appeared mediated by microtubules depolymerization and cytoskeleton disarrangement associated to inactivation of MYPT and LIMK1 that finally interfered with cofilin activity. Conclusions Our data indicate that colchicine exerts anti-platelet effects in vitro via inhibition of key proteins involved in cytoskeleton rearrangement, suggesting that its beneficial cardiovascular properties may be due, at least in part, to an inhibitory effect of platelet activity.

In recent years, Pregnancy-associated Plasma Protein-A (PAPP-A), a metalloproteinase originally identified in the plasma of pregnant women and then found in both men and women, has been studied for its potential involvement in cardiovascular disease. Several studies have indicated that PAPP-A might be considered as a marker of Acute Coronary Syndromes (ACS) able to predict outcome of patients with this clinical presentation. We have recently demonstrated that PAPP-A is able to induce TF expression in endothelial cells in vitro, suggesting an active involvement of this metalloproteinase in thrombotic events. We found that transcoronary PAPP-A levels were significantly higher in ACS-NSTEMI patients as compared to patients with SCAD and that elevated PAPP-A concentrations were associated with higher rate of TF consumption. results of this study permit for the first time to speculate that this metalloproteinase, does not represent only an independent risk factor for adverse cardiovascular or a marker of disease to obtain and early diagnosis of ACS, but it rather might play an “active” role in the pathophysiology of ACS as an effector molecule able to induce thrombus formation in the coronary circulation.

Platelet activation triggers thrombus formation in physiological and pathological conditions, such as acute coronary syndromes. Current therapies still fail to prevent thrombotic events in numerous patients, indicating that the mechanisms modulating platelet response during activation need to be clarified. The evidence that platelets are capable of de novo protein synthesis in response to stimuli raised the issue of how megakaryocyte-derived mRNAs are regulated in these anucleate cell fragments. Proteogenomics was applied here to investigate this phenomeon in platelets activated in vitro with Collagen or Thrombin Receptor Activating Peptide. Combining proteomics and transcriptomics allowed in depth platelet proteome characterization, revealing a significant effect of either stimulus on proteome composition. In silico analysis revealed the presence of resident immature RNAs in resting platelets, characterized by retained introns, while unbiased proteogenomics correlated intron removal by RNA splicing with changes on proteome composition upon activation. This allowed identification of a set of transcripts undergoing maturation by intron removal during activation and resulting in accumulation of the corresponding peptides at exon-exon junctions. These results indicate that RNA splicing events occur in platelets during activation and that maturation of specific pre-mRNAs is part of the activation cascade, contributing to a dynamic fine-tuning of the transcriptome.

Tissue factor pathway inhibitor (TFPI) is a serine-protease inhibitor, which modulates coagulation tissue factor-dependent (TF). It binds directly and inhibits the TF-FVII/FVIIa complex as well as FXa. Time to reperfusion of acute ischemic myocardium is essential for tissue salvage. However, reperfusion also results in a unique form of myocardial damage, such as contractile dysfunction, decreased coronary flow and altered vascular reactivity. Oxidants and reactive oxygen species (ROS) formation is increased in the post-ischemic heart and is responsible of post-ischemic injury. It has been reported that ROS promote a procoagulant state via TF expression while no data are available on the effect on TFPI. Endothelial cells were incubated with two different ROS generating systems, xanthine (X)/xanthine oxidase (XO) for 5 min, or H2O2 (500 μM) for 24 h. TFPI activity was measured in supernatants by chromogenic assay and TFPI-mRNA analyzed by RT-PCR 2 h after ROS exposure. Unstimulated cells and cells exposed to either X or XO served as controls. Western blot and ligand dot blot was performed to evaluate ROS effect on TFPI structure and binding to FXa. ROS generated by X/XO as well as H2O2 system resulted in decreased TFPI activity compared to unstimulated cells while X or XO alone had no effect. No differences in TFPI mRNA levels versus controls was observed. A significant degradation of TFPI was induced by ROS exposure, resulting in a decreased ability to bind FXa. ROS induce a procoagulant state in endothelial cells by altering TFPI structure, resulting in inhibition of TFPI binding to Factor Xa and loss of activity. This phenomenon might have important consequences during reperfusion of post-ischemic myocardium.

Background The RNA-binding protein Argonaute 2 (AGO2) is a key effector of RNA-silencing pathways It exerts a pivotal role in microRNA maturation and activity and can modulate chromatin remodeling, transcriptional gene regulation and RNA splicing. Estrogen receptor beta (ERβ) is endowed with oncosuppressive activities, antagonizing hormone-induced carcinogenesis and inhibiting growth and oncogenic functions in luminal-like breast cancers (BCs), where its expression correlates with a better prognosis of the disease. Results Applying interaction proteomics coupled to mass spectrometry to characterize nuclear factors cooperating with ERβ in gene regulation, we identify AGO2 as a novel partner of ERβ in human BC cells. ERβ–AGO2 association was confirmed in vitro and in vivo in both the nucleus and cytoplasm and is shown to be RNA-mediated. ChIP-Seq demonstrates AGO2 association with a large number of ERβ binding sites, and total and nascent RNA-Seq in ERβ + vs ERβ − cells, and before and after AGO2 knock-down in ERβ + cells, reveals a widespread involvement of this factor in ERβ-mediated regulation of gene transcription rate and RNA splicing. Moreover, isolation and sequencing by RIP-Seq of ERβ-associated long and small RNAs in the cytoplasm suggests involvement of the nuclear receptor in RISC loading, indicating that it may also be able to directly control mRNA translation efficiency and stability. Conclusions These results demonstrate that AGO2 can act as a pleiotropic functional partner of ERβ, indicating that both factors are endowed with multiple roles in the control of key cellular functions. Electronic supplementary material The online version of this article (doi:10.1186/s13059-017-1321-0) contains supplementary material, which is available to authorized users.

Background Matrix metalloproteases (MMPs) have been implicated in the pathogenesis of acute coronary syndromes (ACS). However, little is known about the mechanisms responsible for MMP expression in ACS. C-reactive protein (CRP) not only is an independent risk factor for cardiovascular events, but also may exert direct pro-atherosclerotic effects. Therefore, we aimed at determining whether CRP might induce MMP-9 in two different experimental conditions: 1) smooth muscle cells (SMCs) in vitro, and 2) patients with ACS. Methods and results Effects of increasing concentrations of CRP on MMP-9 expression were evaluated in vitro in human SMCs. TIMP-1 protein expression, the selective inhibitor of MMP-9, was also evaluated. CRP dose-dependently induced MMP-9 expression in SMCs by promoting MMP-mRNA transcription, as well as MMP-9 secretion. In contrast, no differences were found for TIMP-1 protein expression. In vivo, MMP-9 and CRP levels were measured in blood samples obtained from the aorta (Ao) and the coronary sinus (Cs) of patients with normal coronary arteries (controls, n=21), stable angina (n=24), and ACS (n=30). Both MMP-9 and CRP plasma levels were significantly increased across the coronary circulation only in patients with ACS. Interestingly, a significant correlation between MMP-9 and CRP plasma levels was found. Conclusions CRP induced MMP-9 expression and activity in human SMCs in culture; patients presenting with ACS have increased transcoronary plasma levels of MMP-9 and CRP with a significant correlation between these two markers. This may explain the heightened risk of coronary events in subjects with elevated levels of CRP.

Worldwide increase in life expectancy is a major contributor to the epidemic of chronic degenerative diseases. Aging, indeed, simultaneously affects multiple organ systems, and it has been hypothesized that systemic alterations in regulators of tissue physiology may regulate this process. Cardiac aging itself is a major risk factor for cardiovascular diseases and, because of the intimate relationship with the brain, may contribute to increase the risk of neurodegenerative disorders. Blood-borne factors may play a major role in this complex and still elusive process. A number of studies, mainly based on the revival of parabiosis, a surgical technique very popular during the 70s of the 20 th century to study the effect of a shared circulation in two animals, have indeed shown the potential that humoral factors can control the aging process in different tissues. In this article we review the role of circulating factors in cardiovascular aging. A better understanding of these mechanisms may provide new insights in the aging process and provide novel therapeutic opportunities for chronic age-related disorders.

Acute thrombus formation is the pathophysiological substrate underlying several clinical conditions, such as acute coronary syndrome (ACS) and stroke. Activation of coagulation cascade is a key step of the thrombotic process: vessel injury results in exposure of the glycoprotein tissue factor (TF) to the flowing blood. Once exposed, TF binds factor VII/VIIa (FVII/FVIIa) and in presence of calcium ions, it forms atertiary complex able to activate FX to FXa, FIX to FIXa, and FVIIa itself. The final step is thrombin formation at the site of vessel injury with subsequent platelet activation, fibrinogen to fibrin conversion and ultimately thrombus formation. Platelets are the key cells in primary hemostasis. For years they have been considered only as cell fragments participating to primary hemostasis and onto which coagulation factors are assembled in the process of thrombus formation. However, recent advances in platelets pathophysiology have shown that these cells are able to regulate their gene and protein expression, make de novo protein synthesis, and release different mediators with paracrine effects that may interfere with different cell function. Pharmacological modulation of both side of thrombosis, coagulation cascade and platelet activation, is of great clinical importance. Several clinical trials have clearly shown the efficacy of anticoagulation and/or anti platelet aggregation in different thrombotic disorders. This article aims at reviewing the recent advancements on the two faces of thrombosis focusing on the emerging role of platelets not only as clot-forming components, but highlighting their involvement in the inflammatory-immune system, as well as in modulation of different cell function.

Background Paroxysmal atrial tachyarrhythmias frequently occur in beta-thalassemia major (beta-TM) patients. The aim of the current study was to evaluate the atrial electromechanical delay (AEMD) in a large beta-TM population with normal cardiac function and its relationship to atrial fibrillation (AF) onset.Methods Eighty beta-TM patients ( 44 men, 36 women), with a mean age of 36.2 +/- 11.1 years, and 80 healthy subjects used as controls, matched for age and gender, were studied for the occurrence of AF during a 5-year follow-up, through 30-day external loop recorder (ELR) monitoring performed every 6 months. Intra-AEMD and inter-AEMD of both atria were measured through tissue Doppler echocardiography. P-wave dispersion (PD) was carefully measured using 12-lead electrocardiogram ( ECG).Results Compared to the healthy control group, the beta-TM patients showed a statistically significant increase in inter-AEMD, intra-left AEMD, maximum P-wave duration, and PD. Dividing the beta-TM group into two subgroups (patients with or without AF), the inter-AEMD, intra-left AEMD, maximum P-wave duration, and PD were significantly higher in the subgroup with AF compared to the subgroup without AF. There were significant good correlations of intra-left AEMD and inter-AEMD with PD. A cut-off value of 40.1 ms for intra-left AEMD had a sensitivity of 76.2% and a specificity of 97.5% in identifying beta-TM patients with AF risk. A cut-off value of 44.8 ms for inter-AEMD had a sensitivity of 81.2% and a specificity of 98.7% in identifying this category of patients.Conclusions Our results showed that the echocardiographic atrial electromechanical delay indices (intra-left and inter-AEMD) and the PD were significantly increased in beta-TM subjects with normal cardiac function. PD and AEMD represent non-invasive, inexpensive, useful, and simple parameters to assess the AF risk in beta-TM patients.

In the last twenty years, our comprehension of the molecular mechanisms involved in formation, progression and complication of atherosclerotic plaque has advanced significantly and the main role of inflammation and immunity in this phenomenon is now largely accepted. Accumulating evidence highlight the crucial role of different inflammatory and immune cells, such as monocytes and T-lymphocytes, in the pathophysiology of atherosclerotic lesion, particularly in contributing to its complications, such as rupture or ulceration. According to the new terminology, "vulnerable plaque" identifies an inflamed atherosclerotic lesion that is particularly prone to rupture. Once disrupted, prothrombotic material is exposed to the flowing blood, thus activating coagulation cascade and platelet aggregation, ultimately leading to acute thrombus formation within the coronary vessel. To date this is the key event underlying the clinical manifestation of acute coronary syndromes (ACS). The degree of vessel occlusion (complete vs. incomplete) and the time of blood flow cessation will define the severity of clinical picture. This phenomenon seems to be the final effect of a complex interaction between different local and systemic factors, involving the degree of inflammation, type of cells infiltration and the rheological characteristics of blood flow at the site of plaque rupture, thrombogenic substrates within the atherosclerotic lesion and different soluble mediators, already present or acutely released in the circulating blood. This article will review currently available data on the pathophysiology of ACS, emphasizing the immunological and inflammatory aspects of vulnerable plaque. We may postulate that intraplaque antigens and local microenvironment will define the immune-inflammatory response and cells phenotype, thus determining the severity of clinical manifestations. In the last twenty years, our comprehension of the molecular mechanisms involved in the formation, progression and complication of atherosclerotic plaque has advanced significantly and the main role of inflammation and immunity in this phenomenon is now largely accepted. Accumulating evidence highlight the crucial role of different inflammatory and immune cells, such as monocytes and T-lymphocytes, in the pathophysiology of atherosclerotic lesion, particularly in contributing to its complications, such as rupture or ulceration. According to the new terminology, “vulnerable plaque” identifies an inflamed atherosclerotic lesion that is particularly prone to rupture. Once disrupted, prothrombotic material is exposed to the flowing blood, thus activating coagulation cascade and platelet aggregation, ultimately leading to acute thrombus formation within the coronary vessel. To date this is the key event underlying the clinical manifestations of acute coronary syndromes (ACS). The degree of vessel occlusion (complete vs. incomplete) and the time of blood flow cessation will define the severity of clinical picture. This phenomenon seems to be the final effect of a complex interaction between different local and systemic factors, involving the degree of inflammation, type of cells infiltration and the rheological characteristics of blood flow at the site of plaque rupture, thrombogenic substrates within the atherosclerotic lesion and different soluble mediators, already present or acutely released in the circulating blood. This article will review currently available data on the pathophysiology of ACS, emphasizing the immunological and inflammatory aspects of vulnerable plaque. We may postulate that intraplaque antigens and local microenvironment will define the immune-inflammatory response and cells phenotype, thus determining the severity of clinical manifestations.

Background. Inflammation plays an essential role in the development and complications of atherosclerotic plaques, including acute coronary syndromes (ACS). Indeed, previous reports have shown that within the coronary circulation of ACS patients, several soluble mediators are released. Moreover, it has been demonstrated that endothelial dysfunction might play an important role in atherosclerosis as well as ACS pathophysiology. However, the mechanisms by which these soluble mediators might affect endothelial functions are still largely unknown. We have evaluated whether soluble mediators contained in serum from coronary circulation of ACS patients might promote changes of gene profile in human coronary endothelial cells (HCAECs). Methods. HCAECs were stimulated in vitro for 12 hours with serum obtained from the coronary sinus (CS) and the aorta (Ao) of ACS patients; stable angina (SA) patients served as controls. Gene profiles of stimulated cells were evaluated by microarray gene expression profiling and Real-time PCR. Results. HCAECs stimulated with serum from CS of ACS patients showed a significant change (up-regulation and down-regulation) in gene expression profile as compared with cells stimulated with serum form CS of SA patients. Moreover, ad-hoc subanalysis indicated the upregulation of Th-17/IL-17 pathway-related genes. Conclusions. This study demonstrates that, in ACS patients,the chemical mediators released in the coronary circulation might be able to perturb coronary endothelial cells modifying their gene profile. These modified endothelial cells, through down-regulation of protective gene and, mainly, through up-regulation of gene able to modulate the Th-17/IL-17 pathway, might play a key role in progression of coronary atherosclerosis and in developing future acute events.

Flavonoids are nutrients usually included in human diet with several significant biological activities. Nobiletin is a flavonoid that, besides having anti-inflammatory and anti-tumoral activity, seems to exert protective effects on cardiovascular system. Several studies investigated nobiletin as a natural drug to antagonize the atherosclerotic disease. On the contrary, literature about its potential role in modulating the main acute complication of atherosclerosis, thrombosis, is still scanty. Several studies have indicated that Tissue Factor (TF) plays a pivotal role in the pathophysiology of cardiovascular thrombotic events by triggering the formation of intracoronary thrombi. Oxidized-LDL have an important role in promoting athero-thrombotic events. This study investigates whether nobiletin might exert protective cardiovascular effects by preventing the oxidized-LDL mediated expression of TF in human endothelial cells in vitro. Moreover, we have studied whether the nobiletin effects might be modulated by the inhibition of the NF-kB pathway. Introduction: Flavonoids are nutrients usually included in human diet with several significant biological activities. Nobiletin is a flavonoid that, besides having anti-inflammatory and anti-tumoral activity, seems to exert protective effects on cardiovascular system. Several studies investigated nobiletin as a natural drug to antagonize the atherosclerotic disease. On the contrary, literature about its potential role in modulating the main acute complication of atherosclerosis, thrombosis, is still scanty.Several studies have indicated that Tissue Factor (TF) plays a pivotal role in the pathophysiology of cardiovascular thrombotic events by triggering the formation of intracoronary thrombi. Oxidized-LDL have an important role in promoting athero-thrombotic events. This study investigates whether nobiletin might exert protective cardiovascular effects by preventing the oxidized-LDL mediated expression of TF in human endothelial cells in vitro. Moreover, we have studied whether the nobiletin effects might be modulated by the inhibition of the NF-kappa B pathway.Methods and results: In HUVEC, ox-LDL induced TF-mRNA transcription as demonstrated by real time PCR and expression of functionally active TF as demonstrated by Western-blot, FACS analysis and pro coagulant activity assay. Nobiletin prevented these ox-LDL-mediated effects by exerting antioxidant effects, finally leading to inhibition of the transcription factor NF-kappa B.Conclusions: These data suggest that nobiletin might be a potential antithrombotic agent of dietary origin. This flavonoid, through its antioxidant proprieties, might potentially exert an antithrombotic activity by inhibiting TF expression/activity in a cell population never investigated before in this context and that is normally represented in vessel wall such as endothelial cells. (C) 2016 Elsevier Inc. All rights reserved.

Acute myocardial infarction is a clinical cardiac emergency associated with potential and substantial morbidity and mortality. This disorder is triggered by an acute coronary syndrome caused by episodes of plaque ulceration, fissuration, or rupture with subsequent production of thrombogenic material and intravascular thrombus formation. The presence or absence of ST-segment elevation on the ECG defines the two main clinical spectrum of ST-segment elevation or non-ST-segment elevation myocardial infarction. In the last three decades, pharmacological and interventional management as well as in-hospital care have dramatically improved. Recent advances in antithrombotic strategies, percutaneous access (radial versus femoral), timing of revascularization, new generation coronary stents, lipid profile management, and cardiac rehabilitation programs at discharge have lead the European Task Force on ST-Elevation Myocardial Infarction to revise the 2012 guidelines and to release, in the current year, an updated version. Acute myocardial infarction is a clinical cardiac emergency associated with potential and substantial morbidity and mortality. This disorder is triggered by an acute coronary syndrome caused by episodes of plaque ulceration, fissuration, or rupture with subsequent production of thrombogenic material and intravascular thrombus formation. The presence or absence of ST-segment elevation on the ECG defines the two main clinical spectrum of ST-segment elevation or non-ST-segment elevation myocardial infarction. In the last three decades, pharmacological and interventional management as well as in-hospital care have dramatically improved. Recent advances in antithrombotic strategies, percutaneous access (radial versus femoral), timing of revascularization, new generation coronary stents, lipid profile management, and cardiac rehabilitation programs at discharge have lead the European Task Force on ST-Elevation Myocardial Infarction to revise the 2012 guidelines and to release, in the current year, an updated version. Acute myocardial infarction is a clinical cardiac emergency associated with potential and substantial morbidity and mortality. This disorder is triggered by an acute coronary syndrome caused by episodes of plaque ulceration, fissuration, or rupture with subsequent production of thrombogenic material and intravascular thrombus formation. The presence or absence of ST-segment elevation on the ECG defines the two main clinical spectrum of ST-segment elevation or non-ST-segment elevation myocardial infarction. In the last three decades, pharmacological and interventional management as well as in-hospital care have dramatically improved. Recent advances in antithrombotic strategies, percutaneous access (radial versus femoral), timing of revascularization, new generation coronary stents, lipid profile management, and cardiac rehabilitation programs at discharge have lead the European Task Force on ST-Elevation Myocardial Infarction to revise the 2012 guidelines and to release, in the current year, an updated version.

The incidence of ST-segment elevation myocardial infarction (STEMI) has significantly decreased. Conversely, the rate of non-STEMI (NSTEMI) has increased. Patients with NSTEMI have lower short-term mortality compared to patients with STEMI, whereas at long-term follow-up, the mortality becomes comparable. This might be due to the differences in baseline characteristics, including older age and a greater prevalence of comorbidities in the NSTEMI population. Although antithrombotic strategies used in patients with NSTEMI have been well studied in clinical trials and updated guidelines are available, patterns of use and outcomes in clinical practice are less well described. Thus, a panel of Italian cardiology experts assembled under the auspices of the "Campania NSTEMI Study Group" for comprehensive discussion and consensus development to provide practical recommendations, for both clinical and interventional cardiologists, regarding optimal management of antithrombotic therapy in patients with NSTEMI. This position article presents and discusses various clinical scenarios in patients with NSTEMI or unstable angina, including special subsets (eg, patients aged ≥85 years, patients with chronic renal disease or previous cerebrovascular events, and patients requiring triple therapy or long-term antithrombotic therapy), with the panel recommendations being provided for each scenario. The incidence of ST-segment elevation myocardial infarction (STEMI) has significantly decreased. Conversely, the rate of non-STEMI (NSTEMI) has increased. Patients with NSTEMI have lower short-term mortality compared to patients with STEMI, whereas at long-term follow-up, the mortality becomes comparable. This might be due to the differences in baseline characteristics, including older age and a greater prevalence of comorbidities in the NSTEMI population. Although antithrombotic strategies used in patients with NSTEMI have been well studied in clinical trials and updated guidelines are available, patterns of use and outcomes in clinical practice are less well described. Thus, a panel of Italian cardiology experts assembled under the auspices of the "Campania NSTEMI Study Group" for comprehensive discussion and consensus development to provide practical recommendations, for both clinical and interventional cardiologists, regarding optimal management of antithrombotic therapy in patients with NSTEMI. This position article presents and discusses various clinical scenarios in patients with NSTEMI or unstable angina, including special subsets (eg, patients aged ≥85 years, patients with chronic renal disease or previous cerebrovascular events, and patients requiring triple therapy or long-term antithrombotic therapy), with the panel recommendations being provided for each scenario.

Pregnancy-Associated Plasma Protein-A (PAPP-A) is a zinc-binding metalloproteinase protein produced by placental syncytio-trophoblasts and secreted into the maternal circulation where its concentration progressively increases until term. In recent years, PAPP-A has been studied for its potential involvement in cardiovascular (CV) disease. However, all those studies did not provide a clear view to identify the pathophysiological links between PAPP-A plasma levels and the occurrence of CV events. In this review, starting from a complete description of PAPP-A structure and biology, we present an updated overview of experimental as well as clinical evidence on the role of this metalloproteinase in CV disease. Finally, we discuss possible therapeutic approaches to antagonize its potential detrimental CV effects. Pregnancy-Associated Plasma Protein-A (PAPP-A) is a zinc-binding metalloproteinase protein produced by placental syncytio-trophoblasts and secreted into the maternal circulation where its concentration progressively increases until term. In recent years, PAPP-A has been studied for its potential involvement in cardiovascular (CV) disease. However, all those studies did not provide a clear view to identify the pathophysiological links between PAPP-A plasma levels and the occurrence of CV events. In this review, starting from a complete description of PAPP-A structure and biology, we present an updated overview of experimental as well as clinical evidence on the role of this metalloproteinase in CV disease. Finally, we discuss possible therapeutic approaches to antagonize its potential detrimental CV effects.

Objective: To verify the existence of association between plasma levels of pro- or anti-inflammatory mediators and atherosclerotic burden at coronary and carotid arteries in individuals aged of 80 or more years old. Methods: Healthy individuals aged between 80 and 102 years old (n = 178) underwent evaluation of plasma cytokines and acute phase proteins, intima-media thickness (IMT) and presence of plaques in carotid arteries by ultrasound and coronary artery calcification (CAC) by cardiac computed tomography. Results: There was no association between CAC and carotid plaques (p = 0.8), maximum (p = 0.06) or mean IMT (p = 0.2). No association was found between the presence of carotid plaques and CRP (p = 0.4), TNF-α (p = 0.8) or IL-10 (p = 0.2). Likewise, individuals in the first three quartiles for CRP, TNF-α or IL-10 had similar values of CAC, mean and maximum IMT. In contrast, individuals above the 75th percentile for CRP or for TNF-α had enhanced maximum IMT (p = 0.017 and p < 0.0001) and CAC (p = 0.026 and p = 0.01) and subjects with IL-10 levels above the 75th percentile had lower maximum IMT (p = 0.027) and CAC (p = 0.006) as compared with those below this percentile. There was no difference in mean IMT for individuals above or below the 75th percentile for CRP, TNF-α or IL-10. Conclusion: In very old individuals, CAC and maximum IMT were positively associated with systemic inflammatory activity only in those above the 75th percentile. The markers of atherosclerotic burden at coronary and carotid arteries were not related to each other and were distinctly associated with pro- and anti-inflammatory mediators, suggesting that atherosclerosis development is different in these vascular beds.

Background The structural secuelae of acute myocardial infarction (AMI) is mostly dictated by left ventricular (LV) remodelling, leading to heart failure. Monocyte chemoattractant protein-1 (MCP-1), matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) play a critical role in LV remodelling. β-blockers are first line therapy for AMI and heart failure; however, the mechanisms responsible for their benefits remain poorly understood. Different β-blocker agents have been shown to exert beneficial activities both in AMI and heart failure, however, their role in early remodelling after ischemia/reperfusion is to be fully elucidated. We sought to compare the effect of 2 of the most prescribed β-blocker agents in early markers of LV remodelling after AMI. Methods A reperfused AMI was induced in Yorshire pigs, being randomized to early intravenous carvedilol, metoprolol or placebo. Twenty-four hours after reperfusion markers of early remodelling were addressed in the LV. Results The early administration of both β-blockers is able to significantly reduce macrophage infiltration as well as the expression and activity of MCP-1 and MMP-2 compared to placebo. The effects of carvedilol were much stronger than those of metoprolol. Conversely, carvedilol upregulated the expression TIMP-2 to a greater extent than metoprolol. Conclusions In an AMI model closely mimicking human pathophysiology, the early administration of carvedilol reduced the expression of markers associated with early LV remodelling to greater extent than metoprolol. These findings may explain the superior clinical benefits exerted by carvedilol in heart failure.

Aims The aim of this study was to investigate the effects of liver X receptors (LXRs)-β preferential activation by LXR-623 (WAY-252623), a novel LXRs agonist, on plaque progression/regression in a rabbit model of advanced atherosclerosis. Methods and results Advanced atherosclerosis was induced in New Zealand White Rabbits ( n = 41). At the end of atherosclerosis induction, animals underwent a baseline magnetic resonance imaging (MRI) and were randomized to receive LXR-623 (1.5, 5, or 15 mg/kg/day), simvastatin (5 mg/kg/day), or placebo. The combination of LXR-1.5/simvastatin was also tested. After a final MRI, animals were euthanized and their aortas processed for further analysis. Simvastatin significantly reduced lesion progression (−25%; P < 0.01) in comparison with the placebo group. A similar effect was observed in the LXR-1.5 and -5 groups. A significant regression (16.5%; P < 0.01) of existing atherosclerosis was observed in the LXR-1.5/simvastatin group. Histological and molecular analysis showed plaque stabilization in the animals treated with the LXR-1.5 and -5, and LXR-1.5/simvastatin. The effects of LXR-623 were observed in the presence of a non-significant effect on total-cholesterol, low-density lipoproteins-cholesterol, and triglyceride levels. Conclusion The results of the present study show that LXR-623 significantly reduces the progression of atherosclerosis and induces plaque regression in combination with simvastatin. These observations could drive future development of novel anti-atherosclerotic therapeutic approaches.

Acute thrombus formation on disrupted atherosclerotic plaques plays a key role during the onset of acute coronary syndromes. Lesion disruption facilitates the interaction between circulating blood and prothrombotic substances, such as tissue factor (TF) present within the atherosclerotic lesion. For a long period of time, vessel-wall TF has been considered the major determinant of thrombosis. However, this old dogma has been recently changed owing to the discovery of a different pool of TF that circulates in flowing blood (blood-borne TF). Several studies have shown that blood-borne TF circulates in different pools that are associated with selected blood cells, such as monocytes, granulocytes and platelets in cell-derived microparticles, and as a soluble protein generated by alternative splicing of its full-length mRNA. Recent studies have identified a hypercoagulable state associated with an increased circulating TF activity, leading to the concept of 'vulnerable blood'. Part of the blood-borne TF circulates in an 'inactive' form and it is required to be 'activated' to exert its thrombogenic potential. Certain pathological conditions, such as smoking, hyperlipidemia and diabetes, show a higher incidence of thrombotic complications. These conditions are also characterized by the presence of high levels of circulating TF activity. Recent evidence may also suggest that an increased circulating TF activity may potentiate the initial thrombogenic stimulus represented by vessel wall-associated TF, leading to the formation of larger and/or more stable thrombus, and thus more severe acute coronary syndromes. It has been reported that inflammation increases TF expression and activity by different cell types. On the other hand, TF upregulation may facilitate inflammation by enhancing intravascular fibrin deposition, formation of proinflammatory fragments of fibrin, and by generating coagulation proteases, including FVIIa, FXa and thrombin, that activate protease-activated receptors. Furthermore, the biology of TF is know known to be more complex than previously thought by the demonstration that this protein, apart from its known effects on blood coagulation, can also function as a signaling receptor.

Objectives The aims of this study were to investigate the feasibility of contrast-enhanced ultrasound (CEU) imaging for in vivo visualization of intraplaque neovascularization and to correlate the in vivo observations with histological assessment of neovessel density and plaque composition in an experimental animal model of advanced atherosclerosis. Background Recent evidence has linked plaque angiogenesis with enhanced atherosclerotic plaque progression and vulnerability. Increased neovascularization has been detected in ruptured human lesions and is associated with clinical manifestations of plaque rupture. Methods Advanced aortic atherosclerosis was induced in New Zealand white rabbits (n = 21; high cholesterol–rich diet/double-balloon aortic denudation). Animals underwent standard and CEU imaging at the end of the atherosclerosis induction period. Six age-matched animals served as control subjects. Within 24 h, animals were euthanized and aortas processed for histopathological evaluation of plaque composition and neovascularization. Imaged plaques were classified as contrast enhanced (CE) positive or CE negative, according to their contrast enhancement on CEU imaging. The lesions were also classified as class III (predominantly echogenic) or class II (predominantly echolucent), according to their echogenicity on non-CEU images. Results No contrast enhancement was observed in control animals. In atherosclerotic animals, class III lesions showed an increased contrast enhancement compared with class II lesions and CE-positive lesions showed greater neovascularization than CE-negative plaques. Macrophage density, but not smooth muscle cell density, was significantly higher in CE-positive than CE-negative lesions. As expected, class III lesions showed increased macrophage density compared with class II plaques. Intraplaque neovessel density at histology was significantly higher in CE-positive than in CE-negative lesions. Class III plaques showed a significantly higher neovessel density compared with class II lesions. A strong correlation between intraplaque neovessels and contrast enhancement was found. Conclusions CEU imaging is a feasible noninvasive imaging modality to evaluate intraplaque neovascularization. A noninvasive imaging modality to assess lesion neovascularization could be of great importance to identify vascularized, “high-risk” lesions before rupture.

High levels of high-density lipoprotein (HDL) have protective effects against atherosclerosis and cardiovascular diseases. The postulated mechanism of action for these benefits is an enhanced reverse cholesterol transport. Apolipoprotein A-I (ApoA-I) is the major protein of HDL. The clinical benefits of raising ApoA-I/HDL have been clearly established by clinical and epidemiological studies. Despite these observations, there are not very effective pharmacological means for raising HDL. ApoA-I gene delivery by viral vectors seems a promising strategy to raise ApoA-I/HDL levels. Sustained gene expression in animals and humans has been attained using adeno-associated viral (AAV) vectors. The aim of the present study was to determine the efficiency, safety, and biological activity of human ApoA-I intramuscularly delivered using an AAV vector in mice. AAV serotype 8 vectors encoding for human ApoA-I transgene were administered intraportally and intramuscularly in ApoA-I- deficient animals. ApoA-I levels were measured every 2 weeks post administration. The effectiveness of the generated HDL was tested in vitro in cholesterol-loaded macrophages. The administration of the vectors resulted in a significant and sustained increase in ApoA-I and HDL plasma levels for up to 16 weeks at similar extent by both routes of administration. Activity of the generated HDL in removal of cholesterol from cholesterol-loaded macrophages was similar in both groups. Our data suggest that intramuscular AAV8-mediated gene transfer of human ApoA-I results in a significant and maintained increase in ApoA-I and functional HDL.

Recombinant-ApoA-I(M) (rApoA-I(M)) administration has been shown to regress and stabilize atherosclerotic plaques. However, the mechanisms responsible for these beneficial effects are not fully understood. The aims of the present study were to define whether the benefits of rApoA-I(M) treatment were mediated via an enhanced reverse cholesterol transport (RCT) and/or anti-inflammation-related mechanisms. Advanced aortic lesions were induced in New Zealand White rabbits (n= 16). Animals were randomized to placebo or rApoA-I(M) (rApoA-I(M)/phospholipids; ETC-216), two infusions 4 days apart. Four days after last dose, aortas and livers were processed for cholesterol content, expression of RCT-related receptors (ATP-binding cassette A-1 [ABCA-1] and scavenger receptor BI [SR-BI]), and inflammation-related markers (inducible nitric oxide synthase [iNOS] and capase-3). Oxidative stress was assessed in the vessel wall and in plasma. rApoA-I(M) administration resulted in a significant reduction in the hepatic and aortic cholesterol content without differences in plasma levels. This effect was associated with an up-regulation of vessel wall ABCA-1, as well as a hepatic and arterial-wall SR-BI up-regulation. Systemic and atherosclerotic-plaque inflammation markers were significantly reduced by the rApoA-I(M) administration, as demonstrated by a reduction in circulating oxidative stress markers and prostaglandin F1-alpha levels, and the down-regulation of the iNOS and caspase 3 in the aortic lesions. rApoA-IM up-regulated the ABCA-1 and SR-BI levels to a greater extent than the wild-type form of apoA-I in in vitro studies done with lipid-rich macrophages. Our data suggest that rApoA-I(M) administration enhances RCT and induces a 'rescue' from the global inflammatory status associated with atherosclerotic disease. The Milano form of apoA-I seems to be more efficient in RCT than the apoA-I wild-type.

High plasma levels of low density lipoproteins (LDLs) represent one of the major risk factors for cardiovascular disease, as shown by many epidemiological studies. On the other hand, randomized trials designed to address the clinical impact of lipid lowering interventions, have clearly shown that reduction in LDL plasma levels lead to a significant decrease in major cardiovascular events. Based on these observations, pharmacological modulation of LDLs has been highly investigated. Statins, alone or in combination, represent the most powerful agents to date available to reach the LDLs levels suggested by the current guidelines. However, in some patients the recommended LDL reduction is difficult to be achieved because of genetic background (familial hypercholesterolemia), side effects (statin intolerance), or simply because of a non-sufficient response. In the last few years, our understanding of the basic mechanisms involved in the lipoprotein metabolism has progressed significantly. The crucial role of proprotein convertase subtilisin/kexin type 9 (PCSK9) has emerged. The main characterized function of PCSK9 relates to the binding to LDL-C receptors (LDLR) in hepatocytes. However, PCSK9 does not interfere with the binding between LDL and its own receptor, but with the ability of the latest to return to the surface of the hepatocyte and bind new LDL molecules. Based on these observations, blocking PCSK-9 may reduce the LDLR clearance, thus increasing the ability of LDLR to remove circulating LDLs. Pharmacological inhibition of this protein has been proposed as new therapeutic approach. The clinical evidence available to date seem to fully support this hypothesis.

Background— β-Blockers improve clinical outcome when administered early after acute myocardial infarction. However, whether β-blockers actually reduce the myocardial infarction size is still in dispute. Cardiac magnetic resonance imaging can accurately depict the left ventricular (LV) ischemic myocardium at risk (T2-weighted hyperintense region) early after myocardial infarction, as well as the extent of necrosis (delayed gadolinium enhancement). The aim of this study was to determine whether early administration of metoprolol could increase myocardial salvage, measured as the difference between the extent of myocardium at risk and myocardial necrosis. Methods and Results— Twelve Yorkshire pigs underwent a 90-minute left anterior descending coronary occlusion, followed by reperfusion. They were randomized to metoprolol (7.5 mg during myocardial infarction) or placebo. Global and regional LV function, extent of myocardium at risk, and myocardial necrosis were quantified by cardiac magnetic resonance imaging studies performed 4 and 22 days after reperfusion in 10 survivors. Despite similar extent of myocardium at risk in metoprolol- and placebo-treated pigs (30.9% of LV versus 30.6%; P =NS), metoprolol resulted in 5-fold-larger salvaged myocardium (32.4% versus 6.2% of myocardium at risk; P =0.015). The LV ejection fraction significantly improved in metoprolol-treated pigs between days 4 and 22 (37.2% versus 43.0%; P =0.037), whereas it remained unchanged in pigs treated with placebo (35.1% versus 35.0%; P =NS). The extent of myocardial salvage was related directly to LV ejection fraction improvement ( P =0.031) and regional LV wall motion recovery ( P =0.039) at day 22. Conclusions— Early metoprolol administration during acute coronary occlusion increases myocardial salvage. The extent of myocardial salvage, measured as the difference between myocardium at risk and myocardial necrosis, was associated with regional and global LV motion improvement.

Tissue factor pathway inhibitor (TFPI) is a multivalent, Kunitz-type plasma proteinase inhibitor that modulates tissue factor-dependent coagulation in vivo. TFPI possesses a peculiar two-step mechanism of action; it directly inhibits activated factor X and subsequently produces feedback inhibition of the factor VIIa/tissue factor catalytic complex in a factor Xa-dependent fashion. TFPI biochemistry and physiology have been extensively studied during the last decade. Its pathophysiologic role in thrombotic disorders has, however, only recently started to be unraveled. In particular, circulating plasma TFPI levels have been found to modulate the activity of the tissue factor-dependent coagulation cascade. In animal models, neutralization of circulating TFPI activity results in restoration of intravascular thrombus formation previously abolished by aspirin. In patients with acute myocardial infarction, TFPI plasma levels measured in blood samples obtained from the coronary sinus were significantly lower than those measured in blood obtained from the ascending aorta, indicating acute consumption of TFPI within the coronary circulation of patients with intracoronary thrombosis. Finally, recent data indicate that transfection of the arterial wall with the gene coding for TFPI is an effective therapeutic intervention to prevent intravascular thrombus fort-nation. Taken together, these observations underline the pathophysiologic importance of TFPI in regulating the procoagulant activity of tissue factor and open new potential therapeutic approaches for the treatment of thrombotic disorders.

It is generally accepted that the initial event in coagulation and intravascular thrombus formation is the exposure of cell-surface protein, such as tissue factor (TF). TF is exposed to the flowing blood as a consequence of vascular injury induced, for instance, by PTCA, or by spontaneous rupture of an atherosclerotic plaque. Expression of TF may also be induced in monocytes and endothelial cells in several conditions such as sepsis and cancer, causing a more generalized activation of clotting. In addition to its essential role in hemostasis, TF may be also implicated in several pathophysiological processes, such as intracellular signaling, cell proliferation, and inflammation. For all these reasons, TF has been the subject of intense research focus. Many experimental studies have demonstrated that inhibition of TF:factor VIIa procoagulant activity is a powerful inhibitor of in vivo thrombosis and that this approach usually results in a less-pronounced bleeding tendency compared with other "more classical" antithrombotic interventions. Alternative approaches may be represented by antibodies directed against TF, by transfection of the arterial wall with natural inhibitors of the TF:factor VIIa complex, such as the TF pathway inhibitor, or with catalytic RNA (ribozyme), which could inhibit the expression of the TF protein by the disruption of cellular TF mRNA. All these approaches seem particularly attractive because they may result in complete inhibition of local thrombosis without incurring potentially harmful systemic effects. Further studies are warranted to determine the efficacy and safety of such approaches in patients.

Acute ApoA-I Milano administration induces plaque regression and stabilisation in the long term

SummaryAdipocytes are cells able to produce and secrete several active substances (adipokines) with direct effects on vascular cells. Apelin, one of the most recently identified adipokines has been studied in cardiovascular system physiology in regard to vessel vasodilation and myocardial contraction, but it has not yet completely characterised for its pathophysiological role in cardiovascular disease and especially in acute coronary syndromes (ACS). Several studies have indicated that tissue factor (TF) plays a pivotal role in the pathophysiology of ACS by triggering the formation of intracoronary thrombi following endothelial injury. This study investigates the effects of apelin 12 and apelin 13 on TF in human umbilical endothelial cells (HUVECs) and monocytes. Cells were stimulated with increasing concentrations of apelin 12 or apelin 13 and then processed to evaluate TF-mRNA levels by real-time PCR as well as TF expression/activity by FACS analysis and pro-coagulant activity. Finally, a potential molecular pathway involved in modulating this phenomenon was investigated. We demonstrate that apelin 13 but not apelin 12 induces transcription of mRNA for TF. In addition, we show that this adipokine promotes surface expression of TF that is functionally active. Apelin 13 effects on TF appear modulated by the activation of the G-protein-transcription factor nuclear factor (NF)-ΚB axis since G-protein inhibitors suppressed NF-ΚB mediated TF expression. Data of the present study, although in vitro, indicate that apelin-13, induces a procoagulant phenotype in HUVECs and monocytes by promoting TF expression. These observations support the hypothesis that this adipokine might play a relevant role as an active partaker in athero-thrombotic disease.

During the past few years, a new and attractive approach--transcatheter aortic valve implantation (TAVI)--has been developed for patients who are symptomatic of aortic stenosis and, due to the high expected operative risk, would not be otherwise treated. Unfortunately, TAVI can result in endocarditis of the percutaneously implanted valve that may present atypically and cause delays in diagnosis and treatment. Herein, the case is described of a 79-year-old female affected by endocarditis on aortic valve percutaneously implanted caused by Enterococcus faecium, complicated by iatrogenic pancytopenia and subacute disseminated intravascular coagulation, that proved fatal at six months after TAVI. During the past few years, a new and attractive approach--transcatheter aortic valve implantation (TAVI)--has been developed for patients who are symptomatic of aortic stenosis and, due to the high expected operative risk, would not be otherwise treated. Unfortunately, TAVI can result in endocarditis of the percutaneously implanted valve that may present atypically and cause delays in diagnosis and treatment. Herein, the case is described of a 79-year-old female affected by endocarditis on aortic valve percutaneously implanted caused by Enterococcus faecium, complicated by iatrogenic pancytopenia and subacute disseminated intravascular coagulation, that proved fatal at six months after TAVI.

SummaryPlatelets carry megakaryocyte-derived mRNAs whose translation efficiency before and during activation is not known, although this can greatly affect platelet functions, both under basal conditions and in response to physiological and pathological stimuli, such as those involved in acute coronary syndromes. Aim of the present study was to determine whether changes in microRNA (miRNA) expression occur in response to activating stimuli and whether this affects activity and composition of platelet transcriptome and proteome. Purified platelet-rich plasmas from healthy volunteers were collected and activated with ADP, collagen, or thrombin receptor activating peptide. Transcriptome analysis by RNA-Seq revealed that platelet transcriptome remained largely unaffected within the first 2 hours of stimulation. In contrast, quantitative proteomics showed that almost half of > 700 proteins quantified were modulated under the same conditions. Global miRNA analysis indicated that reorganisation of platelet proteome occurring during activation reflected changes in mature miRNA expression, which therefore, appears to be the main driver of the observed discrepancy between transcriptome and proteome changes. Platelet functions significantly affected by modulated miRNAs include, among others, the integrin/cytoskeletal, coagulation and inflammatory-immune response pathways. These results demonstrate a significant reprogramming of the platelet miRNome during activation, with consequent significant changes in platelet proteome and provide for the first time substantial evidence that fine-tuning of resident mRNA translation by miRNAs is a key event in platelet pathophysiology.

Cholesterol deposition plays a central role in atherogenesis. The accumulation of lipid material is the result of an imbalance between the influx and efflux of cholesterol within the arterial wall. High levels of plasma low-density lipoprotein-cholesterol are considered the major mechanism responsible for the influx and accumulation of cholesterol in the arterial wall, while high-density lipoprotein (HDL)- cholesterol seems responsible for its efflux. The mechanism by which cholesterol is removed from extra-hepatic organs and delivered to the liver for its catabolism and excretion is called reverse cholesterol transport (RCT). Epidemiological evidence has associated high levels of HDL-cholesterol/ApoA-I with protection against atherosclerotic disease, but the ultimate mechanism(s) responsible for the beneficial effect is not well established. HDLs are synthesized by the liver and small intestine and released to the circulation as a lipid-poor HDL (nascent HDL), mostly formed by ApoA-I and phospholipids. Through their metabolic maturation, HDLs interact with the ABCA1 receptor in the macrophage surface increasing their lipid content by taking phospholipids and cholesterol from macrophages becoming mature HDL. The cholesterol of the HDLs is transported to the liver, via the scavenger receptor class B, type I, for further metabolization and excretion to the intestines in the form of bile acids and cholesterol, completing the process of RCT. It is clear that an inherited mutation or acquired abnormality in any of the key players in RCT mat affect the atherosclerotic process.

The main function of platelets is to participate in primary hemostasis through four distinct steps: adhesion, activation, secretion, and aggregation. Unraveling the molecular mechanisms underlying these steps has led to a better understanding of the pathophysiology of bleeding disorders, on one hand, and of thrombotic diseases, such as acute coronary syndromes, on the other. Platelets are cytoplasmic fragments of megakaryocytes formed in the bone marrow. They lack nuclei but contain organelles and structures, such as mitochondria, microtubules, and granules. Platelet granules contain different bioactive chemical mediators, many of which have a fundamental role in hemostasis and/or tissue healing. The platelet cytoplasm contains an open canalicular system that increases the effective surface area for the intake of stimulatory agonists and the release of effector substances. The submembrane region contains microfilaments of actin and myosin that mediate morphologic alterations characteristic of shape change. Resting platelets remain in the circulation for an average of approximately 10 days before being removed by macrophages of the reticuloendothelial system. A wide variety of transmembrane receptors cover the platelet membrane, including many integrins, leucine-rich repeat receptors, G protein-coupled receptors, proteins belonging to the immunoglobulin superfamily, C-type lectin receptors, tyrosine kinase receptors, and a variety of other types. In this article, we will review platelet biology under physiological and pathological conditions, with particular emphasis on the function of their membrane receptors.

Apolipoprotein A-I (ApoA-I)/high-density lipoprotein (HDL)-raising treatments are effective antiatherosclerotic strategies. We have compared the antiatherogenic effects of human ApoA-I (hApoA-I) overexpression by intraportal and intramuscular gene transfer in atherosclerotic ApoE-knockout mice. Atherosclerotic lesions were induced by atherogenic diet. After atherosclerosis induction, a group of animals was killed and served as atherosclerosis baseline-control group. The remaining animals were randomized into the following groups: (1) atherosclerosis-progression-control, (2) intraportal/vector administration, and (3) intramuscular/vector administration. Aortas and hearts were processed for atherosclerotic quantification by en face Sudan IV and Oil Red-O, respectively. Liver and muscle specimens were processed for protein/gene expression analysis. A sustained increase in hApoA-I/HDL plasma levels was observed in both transduced groups. hApoA-I overexpression abolished plaque progression versus progression-control group. hApoA-I overexpression significantly reduced lesion macrophage, feature indicative of plaque stabilization. Scavenger receptor class-B type I (SR-BI), but not ATP-binding cassette, sub-family A (ABCA), member 1 (ABCA-1), was significantly upregulated in treated groups versus progression-controls. The results of this study show a similar effect of hApoA-I/HDL overexpression on plaque progression/stabilization by 2 different routes of administration. Our results showing similar effects using either intramuscular administration and intraportal route of administration may have significant clinical implications, given the reduced medical risk to patient and cost of intramuscular injections.

During the last two decades, a new perspective on atherosclerosis has prevailed based on accumulating evidence on the role of inflammatory and immune-competent cells, such as monocytes and T-lymphocytes, in contributing to the development of this disease. It is now generally accepted that a specific T-cell response is crucial in determining not only the development and growth of the atherosclerotic lesion, but also its fate toward a stable or an unstable pattern of evolution (i.e., rupture or ulceration), eventually leading to the clinical occurrence of acute coronary syndromes (ACS). We found that transcoronary IL-17 levels tended to be higher in ACS patients as compared to patients with Stable Angina. We observed a bimodal distribution of IL-17 transcoronary gradients, with 10 out of 23 patients showing elevated IL-17 transcoronary gradients. The ACS patients with elevated transcoronary IL-17 levels, showed also a significant increase in the percentage of Th-17 lymphocytes when samples from the coronary sinus were compared to the ones obtained from the Aorta Finally, patients with a Δ% transcoronary IL-17 gradient > 10 showed troponin I (Tn I) levels at admission significantly higher as compared to the levels measured in the patient population with a Δ% transcoronary IL-17 gradient < 10. Intriguingly, a significant correlation between ∆% transcoronary IL-17 levels and Tn I levels at admission was found only in patients with ∆% transcoronary IL-17 levels > 10 while no correlation was found in the other ACS patients. Our findings highlight that expansion of Th-17 lymphocytes, with the consequent increase in transcoronary IL-17 gradient, might have an important pathophysiological role in a subset of patients with ACS. In addition, data about relationship existing between IL-17 and troponin levels, indicate that this subset of patients presents with a more severe disease and suggest that they may possibly have a poorer prognosis.

Aims To investigate whether selected matrix metalloproteinases (MMPs) are released in the coronary circulation of patients with acute coronary syndrome (ACS), whether this release is related to platelet activation, and whether it contributes to sustained platelet activation. Methods and results Blood from the aorta (Ao) and the coronary sinus (Cs) was obtained from 21 controls (non-cardiac chest pain), 24 stable angina (SA), and 30 ACS patients, before performing percutaneous transluminal coronary angioplasty. Selected MMPs, some platelet activation- and atheroma-related markers, and the platelet activation-potentiating activity of plasma were measured. Total MMP-2, active MMP-2, and MMP-9 were released in the coronary circulation of patients with ACS, but not of those with SA or controls. Similarly, transcoronary gradients of b-thromboglobulin (b-TG) and platelet factor 4, two platelet-specific proteins, and of soluble CD40L and secretory phospholipase A2 (sPLA2), markers of inflammation and platelet activation, were higher in ACS patients than in the other groups. In contrast, plasma monocyte chemoattractant protein-1, a platelet-unrelated marker of atherogenesis, was not increased in the Cs compared with Ao in any of the groups. Transcoronary gradients of both b-TG and sPLA2 correlated with those of total and active MMP-2 in ACS, but not in controls or SA. Plasma from the Cs of ACS patients potentiated platelet activation, an effect suppressed by the specific MMP-2-inhibitor, tissue inhibitor of MMP-2 (TIMP-2). Conclusion Matrix metalloproteinase-2 is released in the coronary circulation of ACS patients, derives in part from activated platelets, and may contribute to sustained intracoronary platelet activation.

Background C-reactive protein (CRP) plasma levels correlate with cardiovascular events. Although a direct role for CRP in atherothrombosis has been suggested, at the moment little is known about its involvement in the pathophysiology of acute coronary syndromes (ACS). Thus, the aim of this study was to determine whether CRP is produced in the culprit lesion and released within the coronary circulation of patients with ACS and whether it may affect coronary endothelial function. Methods Blood samples were simultaneously obtained from the aorta (Ao) and the coronary sinus (CS) of patients with normal coronary artery (n=16), stable angina (n=30), and ACS (n=29) for later measurement of plasma CRP levels. Endothelium-dependent and -independent coronary vasodilation were evaluated by means of a Doppler Flow Wire in response to the increasing intracoronary doses of acetylcholine and adenosine, respectively. Results CRP plasma levels were significantly higher across the coronary circulation only in ACS patients with the culprit lesion located in the left coronary artery, while no differences between CS and Ao CRP plasma levels were observed in all other groups. Transcardiac CRP levels were correlated with impairment in coronary endothelium-dependent vasodilation. In six additional patients (SA=3 and ACS=3), subjected to coronary atherectomy, real-time quantitative PCR revealed presence of CRP mRNA only in unstable plaques. Conclusions Thus, CRP is produced and released within the coronary circulation of patients with ACS; this is associated with impairment of endothelial function, suggesting a new pathophysiological link between CRP and ACS.

Background: Myocardial infarct size is a strong predictor of cardiovascular events. Intravenous metoprolol before coronary reperfusion has been shown to reduce infarct size; however, it is unknown whether oral metoprolol initiated early after reperfusion, as clinical guidelines recommend, is similarly cardioprotective. We compared the extent of myocardial salvage associated with intravenous pre-reperfusion-metoprolol administration in comparison with oral post-reperfusion-metoprolol or placebo. We also studied the effect on suspected markers of reperfusion injury. Methods: Thirty Yorkshire-pigs underwent a reperfused myocardial infarction, being randomized to pre-reperfusion-metoprolol, post-reperfusion-metoprolol or placebo. Cardiac magnetic resonance imaging was performed in eighteen pigs at day 3 for the quantification of salvaged myocardium. The amounts of at-risk and infarcted myocardium were quantified using T2-weighted and post-contrast delayed enhancement imaging, respectively. Twelve animals were sacrificed after 24 h for reperfusion injury analysis. Results: The pre-reperfusion-metoprolol group had significantly larger salvaged myocardium than the post-reperfusion-metoprolol or the placebo groups (31 +/- 4%, 13 +/- 6%, and 7 +/- 3% of myocardium at-risk respectively). Post-mortem analyses suggest lesser myocardial reperfusion injury in the pre-reperfusion-metoprolol in comparison with the other 2 groups (lower neutrophil infiltration, decreased myocardial apoptosis, and higher activation of the salvage-kinase phospho-Akt). Salvaged myocardium and reperfusion injury pair wise comparisons proved there were significant differences between the pre-reperfusion-metoprolol and the other 2 groups, but not among the latter two. Conclusions: The intravenous administration of metoprolol before coronary reperfusion results in larger myocardial salvage than its oral administration initiated early after reperfusion. If confirmed in the clinical setting, the timing and route of beta-blocker initiation could be revisited. (C) 2009 Elsevier Ireland Ltd. All rights reserved.

Atherosclerosis constitutes the single most important contributor to the increasing problem of cardiovascular disease. Endothelial dysfunction is considered the early pivotal event in atherogenesis and precedes development of clinically detectable atherosclerotic plaques. The term “vulnerable plaque” identifies an atherosclerotic plaque that is particularly susceptible to disruption, with exposure of tissue factor to the blood flow and subsequent activation of coagulation cascade resulting in lumen occlusion by overlying thrombi. Despite the central role of vulnerable plaques in the onset of cardiovascular events, there are still certain situations (e.g. eroded lesions) wherein hyperactive blood (vulnerable blood) takes the central role. The magnitude and severity of arterial thrombosis is a complex phenomenon and depends on several factors: arterial vessel wall substrates, local rheologic characteristics of blood flow, and systemic factors in the circulating blood. Biomarker of “vulnerable blood,” including blood markers reflecting hypercoagulability, is one tool to identify high-risk individuals for accurate diagnosis and stratification. The importance of the coagulation/fibrinolytic system is highlighted by several autopsic studies that show a high prevalence of old plaque disruptions without infarctions. The imbalance between coagulation and anticoagulation systems is likely to result in an acute event. The prolonged presence of residual thrombus over a disrupted or eroded plaque can induce smooth muscle migration and produce new intima, leading to plaque expansion. Therefore, an active fibrinolytic system may be able to prevent luminal thrombosis in some cases of plaque disruption. Its rapid decline is associated with an increasing plaque burden and vulnerability, and it is related to endothelial cell injury. Identification of vulnerable atherosclerotic plaque and improvement of endothelial function represent a primary approach in the management of cardiovascular patients.

Aims Aortic stenosis (AS) is associated with significant morbidity and mortality. Recombinant apolipoprotein A-I Milano (rApoA-IM) induces atherosclerotic plaque regression. The aims of this study were to determine the effects of rApoA-IM on experimental aortic valve degeneration and its mechanisms of action. Methods and results New Zealand White rabbits ( n = 20) were fed an atherogenic diet for 9 months and then randomized to either placebo or rApoA-IM. Echocardiography was used to assess the effect of the treatments on AS. Porcine aortic valve myofibroblasts (PAVMF) treated with oxidized low-density lipoprotein served to define the effects of rApoA-IM on the expression of monocyte chemoattractant protein-1 (MCP-1), nuclear factor (NF)-κB, and alkaline phosphatase (AP). Recombinant apolipoprotein A-I Milano increased aortic valve area (AVA) by 32% (0.25 ± 0.05 to 0.34 ± 0.07 cm2, P < 0.01); whereas AVA remained unchanged in the placebo group (0.24 ± 0.05 to 0.26 ± 0.04 cm2, P = 0.58). Histopathological examination of aortic valves in the rApoA-IM animals showed significantly less leaflet thickening, inflammation, and calcification vs. the placebo group. In vitro , rApoA-IM significantly inhibited MCP-1, AP, and NF-κB and decreased intracellular cholesterol content in PAVMF. Conclusion Recombinant apolipoprotein A-I Milano treatment reverses AS in this experimental rabbit model. The beneficial effects seem to be mediated by enhanced cholesterol removal and by reduced inflammation and calcification.

Atherosclerosis is a diffuse pathological process characterized by the deposition of lipid and other blood-borne material within the arterial wall of almost all vascular territories. Cholesterol accumulation plays a central role in atherogenesis. It is the result of an imbalance between cholesterol influx and efflux. The disease progresses silently with focal clinical manifestation due to plaque rupture with superimposed thrombus: atherothrombosis. It has been shown that the atherosclerotic plaque composition rather than the degree of arterial stenosis can be the determinant of rupture. This has led to the search for new imaging techniques that can provide information about plaque composition. Ultrasound measurements of carotid and aortic wall thickness are an accurate ‘noninvasive’ technique that permits correlation with clinical events. Magnetic resonance imaging can evaluate in detail the arterial anatomy of almost all vascular territories. Multidetector computed tomography recently emerged for measuring luminal stenosis, coronary calcium, and even the extent of non-calcified coronary plaque volume. Next future is the molecular imaging based on the knowledge of molecular mechanisms involved in the genesis and progression of atherosclerotic lesions and the contrast agent able to identify different molecules and/or cells in the target zone.

Assessment of changes in plaque volume is increasingly used as a surrogate-endpoint in clinical trials testing the efficacy of anti-atherosclerotic interventions. Multi-detector computed tomography (MDCT) can detect and quantify non-calcified atherosclerotic plaques, but its ability to monitor changes in plaque volume has not yet been tested. We sought to test the ability of MDCT to detect and quantify serial changes in atheroma burden in comparison with magnetic resonance imaging (MRI). Methods Rabbits ( n =12) with experimentally induced abdominal atherosclerosis were randomized to receive a plaque-regressing agent (recombinant apoA-I Milano , n =8) or placebo ( n =4). All animals underwent two 64-slice MDCT angiography and MRI studies (pre- and post-treatment). The primary endpoint was the change in plaque burden (defined as vessel wall volume in the 5cm distal to the left renal artery) between pre- and post-treatment MDCT in comparison with MRI. Results MDCT detected a significant decrease in plaque burden caused by recombinant apoA-I Milano (464 [423–535] to 405 [363–435]mm 3 , p =0.03) that was confirmed by MRI (324 [286–412] to 298 [282–399]mm 3 , p =0.03). No significant effect was noted in the placebo group either by MDCT or MRI. There were strong correlations between both modalities for the quantification of plaque burden ( r =0.750, p r =0.657, p =0.020). MDCT overestimated plaque burden compared to MRI. On MDCT, the mean interobserver variability for plaque burden was 2.5±0.4%. Conclusions In an animal model of atherosclerosis, MDCT accurately documented serial changes in aortic plaque burden, demonstrating good correlation and agreement with MRI-derived measurements and low interobserver variability.

Atherothrombotic vascular disease is a complex disorder in which inflammation and coagulation play a pivotal role. Rupture of high-risk, vulnerable plaques with the subsequent tissue factor (TF) exposure is responsible for coronary thrombosis, the main cause of unstable angina, acute myocardial infarction, and sudden cardiac death. Tissue factor (TF), the key initiator of coagulation is an important modulator of inflammation. TF is widely expressed in atherosclerotic plaques and found in macrophages, smooth muscle cells, extracellular matrix and acellular lipid-rich core. TF expression can be induced by various stimulants such as C-reactive protein, oxLDL, hyperglycemia and adipocytokines. The blood-born TF encrypted on the circulating microparticles derived from vascular cells is a marker of vascular injury and a source of procoagulant activity. Another form of TF, called alternatively spliced has been recently identified in human and murine. It is soluble, circulates in plasma and initiates coagulation and thrombus propagation. Evidence indicates that elevated levels of blood-borne or circulating TF has been associated with metabolic syndrome, type 2 diabetes and cardiovascular risk factors and is a candidate biomarker for future cardiovascular events. Therapeutic strategies have been developed to specifically interfere with TF activity in the treatment of cardiovascular disease.