Factor VII Activating Protease Expression in Human Platelets and Accumulation in Symptomatic Carotid Plaque.

Background Factor VII activating protease (FSAP) is of interest as a marker for vascular inflammation and plaque destabilization. The aim of this study was to analyze the expression profile of FSAP in endarterectomy specimens that were taken from patients with asymptomatic and symptomatic carotid atherosclerotic plaques and to compare them with circulating FSAP levels. Methods and Results Plasma FSAP concentration, activity, and mRNA expression were measured in endarterectomy specimens and in monocytes and platelets. Plaque and plasma FSAP levels were higher in symptomatic patients (n=10) than in asymptomatic patients (n=14). Stronger FSAP immunostaining was observed in advanced symptomatic lesions, in intraplaque hemorrhage-related structures, and in lipid-rich areas within the necrotic core. FSAP was also colocalized with monocytes and macrophages (CD11b/CD68-positive cells) and platelets (CD41-positive cells) of the plaques. Moreover, human platelets expressed FSAP in vitro, at both the mRNA and protein levels. Expression is stimulated by thrombin receptor-activating peptide and ADP and reduced by acetylsalicylic acid. Conclusions Plasma FSAP levels were significantly increased in patients with symptomatic carotid stenosis and thus may be involved in plaque development This plaque-associated FSAP may be produced by platelets or macrophages or may be taken up from the circulation. To establish FSAP's utility as a circulating or plaque biomarker in patients with symptomatic carotid atherosclerotic plaques, further studies are needed.

Expression of the Marburg I Single Nucleotide Polymorphism (MI-SNP) and the Marburg II Single Nucleotide Polymorphism (MII-SNP) of the Factor VII-Activating Protease (FSAP) Gene and Risk of Coronary Artery Disease (CAD): A Pilot Study in a Single Population.

BACKGROUND Factor VII-activating protease (FSAP) has a role in vascular inflammation and may have a role coronary artery disease (CAD). The aim of this study was to investigate the association between two naturally occurring single nucleotide polymorphisms (SNPs) in the FSAP gene and the risk of coronary artery disease (CAD). MATERIAL AND METHODS Of 733 patients, 173 patients had symptoms of angina, and 560 patients had CAD confirmed by coronary angiography. All patients were genotyped for SNPs of the FSAP gene, Marburg I (MI-SNP) and Marburg II (MII-SNP), using 5' exonuclease TaqMan assays. Logistic regression analysis was used to evaluate the association between two gene polymorphisms, metabolic and other cardiovascular risk factors in patients with CAD. RESULTS The presence of MI-SNP and MII-SNP FSAP gene polymorphisms were not associated with the presence of CAD. However, the MII-SNP polymorphism was significantly associated with a reduced risk of developing CAD (OR=0.422; 95% CI, 0.194-0.920; P=0.035); the MI-SNP polymorphism was associated with absence of hyperlipoproteinemia (OR=0.601; 95% CI, 0.344-1.051; P=0.074). There was no significant association between expression of the MI-SNP and MII-SNP FSAP gene polymorphisms and the incidence of myocardial infarction, or of a history of diabetes mellitus, arterial hypertension, obesity, or smoking. CONCLUSIONS The MI-SNP and MII-SNP FSAP gene polymorphisms were not predictive or prognostic biomarkers for CAD or its main risk factors. However, the presence of the MII-SNP polymorphism was associated with a reduced risk of developing CAD.

MicroRNA expression profile of human advanced coronary atherosclerotic plaques.

MicroRNA (miR) is reported to be involved in vascular inflammation and may represent a novel class of diagnostic biomarkers in cardiovascular disease. We aimed to identify the miR expression profile in human advanced coronary atherosclerotic plaques (CAP) and to connect this expression to the processes in atherosclerosis. Microarray techniques and TaqMan polymerase chain reaction were used to analyse the global expression of 352 miRs in CAP obtained during ACS MULTI-LINK study. 11 miRs were selected on the basis of their implication in atherosclerosis, endothelial activation, and inflammation. 6 miRs were found to be differently expressed in CAP when compared to non-atherosclerotic internal mammary arteries (IMA, p < 0.05). The expression of miR-21, -92a, and -99a was verified and found to be significantly up-regulated in CAP versus IMA (p < 0.001). We also performed bioinformatic analysis and found several potential target genes of miR-92a and -99a as well as several pathways with impact on atherosclerosis which could be differently expressed due to this miRNA profile. The most up-regulated miRs are involved in processes known to be connected to atherosclerosis. Interfering with the miR expression in the artery wall is a potential way to affect atherosclerotic plaque and cardiovascular disease development.

Publisher Correction: Identification of microRNAs as potential cellular monocytic biomarkers in the early phase of myocardial infarction: a pilot study.

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Identification of microRNAs as potential cellular monocytic biomarkers in the early phase of myocardial infarction: a pilot study.

MicroRNA has been increasingly suggested to be involved in vascular inflammation. The aim of this study was to assess the expression profile of miRs as possible novel cellular biomarkers in circulating monocytes in patients with ST-segment elevation myocardial infarction (STEMI). Microarray techniques and TaqMan polymerase chain reaction were used to analyse the global expression of 352 miRNAs in peripheral blood monocytes from healthy donors (n = 20) and patients (n = 24) with acute STEMI. The expression level of miR-143 in monocytes from STEMI patients compared to healthy controls was increased, whereas the expression of miR-1, -92a, -99a, and -223 was reduced significantly. During 3.5 ± 1.5 months of follow-up miR-1 and -223 were back to baseline, whereas miR-92a and -99a return to normal levels over 3 months, but remained lower than healthy controls. Furthermore, monocytic expression of miR-143 was positively correlated with hs-CRP (R2 = 0.338; P < 0.031), but not with cTnT. Importantly, treatment of monocytes isolated from healthy individuals with INFγ, but not LPS or TNFα caused an upregulation of miR-143 and downregulation of miR-1. Our findings identify circulating monocytes as putative biomarkers and as novel carriers for the cell-specific transfer of miRs in the early phase of myocardial infarction.

Differential Expression of MicroRNAs in Endarterectomy Specimens Taken from Patients with Asymptomatic and Symptomatic Carotid Plaques.

OBJECTIVE: Stroke and transient ischemic attacks are considered as clinical manifestations of atherosclerotic disease due to on-going vascular inflammation and finally atherothrombosis of the carotid arteries. MicroRNAs (miRNA/miR) are known to be involved in vascular inflammation and plaque destabilization. The aim of this study was to analyze the expression profile of selected miRNAs in endarterectomy specimen from carotid arteries that were taken from patients with asymptomatic and symptomatic atherosclerotic plaques. METHODS AND RESULTS: 11 miRNAs were selected and their expression was analyzed using real-time RT-PCR. Therefore, samples were divided into three different groups. On the one hand we investigated the expression patterns from patients in asymptomatic (n = 14) and symptomatic (n = 10) plaques; on the other hand we took samples from normal configurated internal mammary arteries (n = 15). Out of these 11 targets we identified some miRNAs, which were up- or down-regulated in either one of the two groups. Interestingly, the expression of two miRNAs was significantly different between asymptomatic and symptomatic samples, namely miR-21 (P<0.01) and miR-143 (P<0.05). CONCLUSION: In the present study, we identified miRNA subtypes which showed different expression in endarterectomy specimen from patients with asymptomatic and symptomatic plaques, suggesting that these miRNAs correlated with advanced vascular inflammation and plaque stability. They may represent new therapeutic targets for vascular proliferative diseases such as atherosclerosis.

Monocyte CD40 expression in young healthy female smokers and/or oral contraceptives users without additional risk factors for atherosclerosis.

OBJECTIVE: Atherosclerosis, as an inflammatory disease, is characterized by pathologically altered levels of cytokines. We investigated whether smoking and/or oral contraceptives (OCs) affect the CD40/CD40L plasma levels and expression in young females without other risk factors for atherosclerosis. PATIENTS AND METHODS: A case-control single-center design was used. Expression levels of CD40/CD40L were analyzed in healthy non-pregnant, pre-menopausal, non-smoking women who did not take OCs (n=49), women who currently smoke and take OCs (n=40), and women who are only smokers (n=40) or currently take OCs (n=42). RESULTS: In OC users, there was a significant increase in CD40 mRNA expression in circulating monocytes as compared with smokers and control group. However, there were no significant differences in CD40 mRNA expression in monocytes between smokers and non-smokers. Interestingly, CD40 mRNA expression in women taking OCs and currently smoking was significantly decreased compared to only OC users (p<0.001). With regard to plasma CD40 levels there were significant differences between OC-users and control group. However, contrary to our expectations, there were no significant differences in expression levels of CD40L between four groups. In vitro experiments demonstrated enhanced CD40 mRNA and surface expression in human monocyte-derived macrophages stimulated with estrogens. Furthermore, nicotine pretreatment led to a suppression of estrogens stimulated CD40 induction. CONCLUSIONS: In young healthy females without additional risk factors for atherosclerosis, OCs, but not smoking, are associated with dramatic changes in CD40 gene and plasma levels. These findings may be providing an important link between OCs and enhancement of pro-inflammatory and atherothrombotic conditions in healthy women.

Association of circulating factor seven activating protease (FSAP) and of oral Omega-3 fatty acids supplements with clinical outcome in patients with atrial fibrillation: the OMEGA-AF study.

Factor VII Activating Protease (FSAP) activates factor VII (FVII) as well as pro-urokinase (uPA). Our goal was to evaluate the relation between plasma levels of FSAP and clinical instability in atrial fibrillation (AF) and possible effects of oral omega-3 fatty acids (FA) supplements. 101 patients with persistent AF were analyzed in the OMEGA-AF Study. Plasma FSAP levels were measured at baseline and after 12 weeks of treatment with omega-3 FA. The median FSAP antigen concentration, in contrast to FSAP activity, was higher in patients with persistent AF. The maintenance of SR after successful cardioversion (CV) did not lead to a normalization of FSAP concentration. Supplementation with omega-3 FA but not placebo significantly reduced elevated FSAP concentration. Furthermore, elevated FSAP levels did not indicate a significantly increased risk of recurrence of AF after electrical CV or cardiovascular clinical events during 1 year of follow-up. Plasma FSAP concentration was increased in patients with AF and may be involved in the pathogenesis of this condition. The possible effects of omega-3 FA on clinical AF potential could be linked with modulation of circulating FSAP levels.

Regulation of monocyte/macrophage function by factor VII activating protease (FSAP).

OBJECTIVE: Factor VII activating protease (FSAP) is a novel regulator of vascular inflammation and hemostasis. However, the molecular mechanism by which circulating FSAP influences inflammatory events and progression of atherosclerosis is not yet entirely understood. Here we have investigated the influence of FSAP on monocyte/macrophage functions. METHODS: We stimulated human monocyte-derived macrophages with FSAP and analyzed their cellular responses. RESULTS: FSAP induced IκB-dependent NF-κB activation in a time- and concentration-dependent fashion. FSAP also activated the phosphorylation and proteolytic degradation of the inhibitor protein IκBα. The phosphorylation of the p65 subunit of NF-κB was induced by FSAP, which is known to contribute to the enhancement of DNA-binding activity of NF-κB. Concomitantly, FSAP up-regulated the expression of pro-inflammatory cytokines, matrix metalloproteinases, cell adhesion molecules and tissue factor. In the presence of FSAP there was increased monocytes adhesion and transendothelial migration in a beta2 integrin dependent manner. CONCLUSIONS: Our findings suggest that FSAP activates the NF-κB pathway and the associated downstream pro-inflammatory factors in monocytic cells. This adds to a spectrum of FSAP effects on the vascular system that may explain its association with cardiovascular diseases.

Factor seven activating protease (FSAP) expression in human placenta and its role in trophoblast migration.

OBJECTIVES: Factor seven activating protease (FSAP) is a plasma serine protease known to play a critical role in hemostasis and remodeling processes: FSAP levels increase markedly during normal pregnancy. In order to define the role of FSAP in vascular pathophysiology in pregnant women and particularly in the placenta, we performed this study (i) to evaluate the FSAP expression in human placenta and (ii) to identify the role of FSAP in human trophoblast migration. STUDY DESIGN: FSAP expression in placental tissues was analyzed by using immunohistochemistry and reverse transcriptase-polymerase chain reaction (RT-PCR). To determine whether FSAP plays any role in trophoblast migration, we used human trophoblast cells in transwell migration assays. RESULTS: Immunohistochemistry showed that FSAP protein was expressed by syncytiotrophoblast and in the cytoplasma of invasive extravillous trophoblasts (EVT) within the maternal decidua (DC) in implantation sites of human first trimester placenta. Furthermore, FSAP mRNA and protein decreased with gestational age (p<0.05, 1st vs 3rd trimester). FSAP (10µg/ml) had a significant stimulatory effect on the migration of human trophoblast cells. This effect was abolished by addition of aprotinin to block the enzymatic activity of FSAP. CONCLUSIONS: The high expression level of FSAP in the placenta supports a relevant role of this protease in trophoblast migration and vascular remodeling, identifies a new concept of coagulation/fibrinolysis at the feto-maternal interface and may be essential for the maintenance of pregnancy.

Circulating factor VII activating protease (FSAP) is associated with clinical outcome in acute coronary syndrome.

BACKGROUND: Factor VII activating protease (FSAP) is a circulating serine protease strongly expressed in unstable plaques and may serve as a marker of plaque destabilization. The aim of this study was to examine the relation between plasma concentrations of FSAP and clinical instability and outcome in coronary artery disease (CAD). METHODS AND RESULTS: Circulating FSAP concentration and activity, as well as FSAP mRNA expression in monocytes, were measured in 231 sequential patients who underwent coronary angiography because of stable angina pectoris (n=50), unstable angina pectoris (n=43), or acute myocardial infarction (n=87). FSAP activity, but not FSAP antigen concentration, was elevated in patients with CAD compared with a control group. Elevated FSAP activity (≥1.035 plasma equivalent units [PEU]/ml) indicated a significantly increased risk of death or non-fatal myocardial infarction during 1 year of follow-up as compared with patients with low activity of FSAP (odds ratio 1.895 [95% confidence interval 1.093-3.283]; P=0.023). Furthermore, there were no significant changes in the FSAP expression in monocytes from CAD and control subjects in the basal state but there were differences after stimulation with proinflammatory factors. CONCLUSIONS: Plasma FSAP activity was significantly increased in patients with acute coronary syndrome and may be involved in the pathogenesis of these conditions. High levels of FSAP activity were predictive of adverse events during follow-up, suggesting its potential role in risk stratification and clinical management of CAD patients.

Nicotine modulation of factor VII activating protease (FSAP) expression in human monocytes.

AIM: Factor VII activating protease (FSAP) is a plasma serine protease involved in hemostasis and remodeling processes. Increased levels of circulating FSAP during pregnancy and in women using oral contraceptives (OCs) indicate that the hormonal status critically influences FSAP expression. In this respect, the aim of this study was to quantify nicotine modulation of FSAP expression in human monocytes/macrophages isolated from healthy female smokers and non-smokers, and from women who use OCs and smoke. METHODS: FSAP concentration and activity were measured in plasma samples obtained from healthy non-pregnant, pre-menopausal, non-smoking women who did not use OCs (n=69), non-pregnant, pre-menopausal women who currently smoke and use OCs (n=43), and women who are only smokers (n=40) or currently use OCs (n=48). Expressions of FSAP mRNA and protein in monocytes isolated from healthy non-pregnant female or healthy male donors were analyzed. RESULTS: Strongest circulating FSAP concentration and activity occurred in women with combined smoking and use of OCs compared to the control group. Enhanced FSAP levels were also observed in smoking women when compared to non-smokers. Ex vivo experiments demonstrated enhanced FSAP expression in monocytes isolated from women using OCs and currently smoking. Nicotine enhanced FSAP mRNA and protein levels in monocytes. CONCLUSIONS: Monocytes from healthy female smokers show a constitutively enhanced FSAP expression and this effect could be replicated in vitro by stimulating monocytes with nicotine. The upregulation of FSAP due to nicotine and OC usage may be linked to a higher incidence of arteriothromboembolic diseases related to their usage.

Factor seven ativating potease (FSAP) levels during normal pregnancy and in women using oral contraceptives.

INTRODUCTION: Factor seven activating protease (FSAP) is a plasma serine protease involved in haemostasis and remodeling processes. We have investigated whether pregnancy or the use of oral contraceptives (OCs) influences circulating FSAP levels. The effect of female sex hormones on FSAP expression in cultured cells was also determined. MATERIALS AND METHODS: FSAP levels and activity was measured in plasma samples obtained at different gestation stages from healthy pregnant women (n=101), from non-pregnant women, pre-menopausal women who currently use OCs (n=48), and non-pregnant women who did not use OCs (n=69). RESULTS: In late pregnancy the plasma FSAP antigen (median 2.28 PEU/ml [range 1.11 to 2.62 PEU/ml]; p<0.001 vs control group) and activity (median 2.98 PEU/ml [range 1.05 to 4.24 PEU/ml]; p<0.001 vs control group) was significantly higher compared with levels in non-pregnant women and remained elevated after delivery. Plasma FSAP levels in women using OCs was also significantly elevated compared to the control group. Ex vivo experiments demonstrated enhanced FSAP expression in monocytes isolated from women using OCs. In vitro experiments showed that FSAP mRNA levels were strongly induced by estradiol in monocytes but not in hepatocytes. CONCLUSIONS: Increased levels of circulating FSAP in pregnancy and in women using OCs indicate that hormonal status critically influences FSAP expression. Hormonal influences could be observed in monocytes in vivo and ex-vivo but not in hepatocytes indicating cell-specific regulation. Future studies designed to investigate the role of FSAP in haemostasis and remodeling processes should consider the role of female sex hormones on FSAP expression.

Factor Seven Activating Protease (FSAP) expression in human monocytes and accumulation in unstable coronary atherosclerotic plaques.

OBJECTIVE: The Factor Seven Activating Protease (FSAP) is known to influence fibrinolysis and to play a critical role in the inhibition of vascular smooth muscle cell (VSMC) proliferation and migration as well as neointima formation. In order to define the role of FSAP in vascular pathophysiology we have investigated the expression of FSAP protein and mRNA in human vascular cells and coronary atherosclerotic plaques with defined clinical features. METHODS AND RESULTS: Directional coronary atherectomy (DCA) specimens from 40 lesions were analyzed for FSAP antigen and mRNA expression. Higher level of FSAP mRNA (p<0.001) as well as FSAP immunostaining (p<0.005) was observed in patients with acute coronary syndromes compared to patients with stable angina pectoris. FSAP antigen was found to be focally accumulated in hypocellular and lipid-rich areas within the necrotic core of atherosclerotic plaques. FSAP was also co-localized with CD11b/CD68 expressing cells in macrophage-rich shoulder regions of the plaques. Monocyte-derived macrophages expressed FSAP in vitro and this was further induced by pro-inflammatory mediators. CONCLUSIONS: FSAP accumulation in coronary atherosclerotic lesions is due to either local synthesis by monocytes/macrophages, or uptake from the plasma due to plaque hemorrhage. The higher expression of FSAP in unstable plaques suggests that it may destabilize plaque through reducing VSMC proliferation/migration and altering the hemostatic balance.

Statins prevent NF-kappaB transactivation independently of the IKK-pathway in human endothelial cells.

Statins have been linked to a wide range of vascular benefits, many of them are likely to be due to attenuation of chronic vascular inflammation. Nuclear factor kappaB (NF-kappaB) is one of the key regulators of transcription of a variety of genes involved in immune and inflammatory responses. Therefore, we investigated the effect of statins on TNF-alpha-induced NF-kappaB signaling in human endothelial cells (EC). ECs were pre-incubated for 16 h with cerivastatin (10(-9) to 10(-7) M) or vehicle in the presence or absence of mevalonate, followed by stimulation with 20 ng/ml TNF-alpha. Statin-treatment prevented TNF-alpha-induced NF-kappaB binding activity, nuclear translocation of the NF-kappaB p65 subunit, as well as NF-kappaB controlled tissue factor (TF) gene transcription in cultured EC. IkappaBalpha phosphorylation and IkappaBalpha degradation, however, still occurred in statin-treated cells. TNF-alpha also activated phosphatidylinositol (PI)3-kinase, as reflected by phosphorylation of Akt. Statin treatment of cells abrogated TNF-alpha-induced Akt phosphorylation and p65 nuclear translocation. As observed with statins, inhibition of PI3-kinase activity by Ly294002 also blocked TNF-alpha-induced p65 translocation, but did not prevent IkappaBalpha phosphorylation nor IkappaBalpha degradation. These studies demonstrate that TNF-alpha-induced NF-kappaB activation is abrogated by statin treatment in HUVEC independently of the classical IKK-pathway but via inhibition of PI3-kinase/Akt signaling.