Behnoush Parviz

Factor Seven Activating Protease (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.

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.

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.

Increased monocyte tissue factor activity in women following cerebral venous thrombosis.

Sirs: Oral contraceptives (OC), pregnancy, and puerperium are well recognised risk factors for cerebral venous thrombosis (CVT) [1, 2]. Several changes of concentrations of coagulation proteins have been reported in this context [3, 4]. Although clotting factors circulate in excessive amounts, an imbalance would only result in clotting activation, if activating factors and phospholipid surfaces are present. Tissue factor (TF) is expressed on endothelium and monocytes upon various stimuli and is not dependent on proteolytical cleavage and can initiate the coagulation cascade without prior activation [5, 6]. TF has been implicated in the activation of intravascular coagulation in several clinical disorders [5]. This study was carried out to test the hypothesis, that female CVT patients may have a predisposition to an increased expression of TF on blood monocytes. TF activities of fifteen consecutive female patients with radiologically confirmed CVT were compared with those of fifteen controls (non-smokers, non-users of OCs). Demographic data are given in Table 1. Prior examination acute infection was excluded by medical history, leukocyte count, and measurement of the C-reactive protein. Activation of the coagulation system was assessed by measurements of D-dimer and prothrombin fragments F1 + 2. Peripheral blood mononuclear cells were sampled by gradient isolation, incubated with or without endotoxin stimulation (LPS; 10 μg/ml) and assayed for TF activity by use of a one-stage clotting assay [7]. Clotting times were converted to milliunits (mU) of TF activity with reference to a standard curve. Values are given as mU/106 cells. Comparisons of monocyte TF expression among the different groups were performed with a one-way ANOVA and a pair-wise contrast by Scheffé. A possible association of TF activity and the time after CVT, age, OC use, smoking, anticoagulation, and coagulopathies was tested by a Spearman rank correlation. Patients with CVT showed a significant increase in monocyte TF activity compared with healthy controls (P < 0.01, Fig. 1). Similarly, the induction of TF activity in monocytes by LPS stimulation was significantly enhanced in women with CVT (P < 0.05, Fig. 1). Significant differences between patients and controls were still observed when patients, who used OCs or smoked at the time of examination, had been excluded (basal: P < 0.01; stimulated: P < 0.05 respectively). The plasma concentrations of F1 + 2 (median 1.1 nmol/l) and Ddimer (median 142 ng/ml) were found to be normal. No correlation of TF activity with other factors was found. As monocyte TF expression is known to show high interindividual but low intraindividual variability [8], it seems conceivable that the enhanced monocyte TF expression observed after CVT might have existed already prior to the development of thrombosis, although we can not fully exclude that our observation is the effect of CVT. No correlation of TF activity and other factors was found, nor did we detect any on-going lowgrade coagulation. This finding LETTER TO THE EDITORS

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.

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.

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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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.