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Background: Tissue factor (TF) exposure is the key trigger of thrombus formation. Oxidized low-density lipoproteins (oxLDL) induce TF in endothelial cells (ECs), suggesting that oxLDL may act locally promoting thrombosis in atherosclerotic lesions. Colchicine (COL) is an anti-inflammatory agent that

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 pathological process affecting arterial elasticity. The rabbit is one of the most widely used animal models for atherosclerosis research. The aim of this study was to evaluate the feasibility of a method for the automatic assessment of local vascular stiffness in rabbits. An off-line algorithm for the evaluation of arterial diameter (D) and distension (ΔD) from ultrasound image sequences was developed in Matlab and tested in 3 atherosclerotic and 2 control rabbits. Longitudinal scans of the aortic and carotid arteries of the rabbits were recorded in DICOM format. High frame-rate (330Hz) image sequences (3 seconds) were acquired to track the rapid movement of the vessel (heart rate ∼ 240bpm) using iE33 Philips ultrasound system. The probe was hold by a clamp. The measurement variability was assessed by performing two scans for each subject. The high frame-rate provided a temporal resolution of 3 msec allowing the instantaneous tracking of the diameter curve for both carotid and aorta. As regards the variability, the coefficients of variation were: 3%±2% (D) and 7%±3% (ΔD) for the aorta (mean D=3.2mm) and 3%±2% (D) and 5%±4% (ΔD) for the carotid (mean D=1.99mm). Our data demonstrate that the implemented ultrasound image processing algorithm is able to non-invasively assess the vascular instantaneous diameter both in control and atherosclerotic rabbits. The method, together with intra-central ear artery blood pressure measurement, could evaluate the effects of new therapeutic interventions on vascular stiffness in those pathological conditions characterized by arterial dysfunction, such as atherosclerosis.

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.

Background: The aim of this study was to evaluate 2D Speckle Tracking radial strain by ultrasound in stunned and scarred myocardium as defined by cardiac magnetic resonance (CMR) in a porcine reperfusion model of acute MI. Methods: Seven pigs underwent MI induction by 90′ mid-LAD balloon occlusion. CMR was performed in a 1.5 T magnet 4 days post-MI. For edema imaging a T2-weighted sequence was employed. Delayed enhancement (DE) images were acquired 10–15 minutes after the administration of 0.2 mmol/kg of Gd-DTPA using an inversion recovery gradient-echo sequence. Before CMR, echocardiographic short axis images of the LV were obtained at the basal, mid and apical levels. Echo and CMR images were matched and divided into 6 segments per slice. DE and edema were defined as those regions with signal intensity >3 SD of the mean signal of remote normal myocardium, and quantified as % of the LV. Stunned myocardium was defined as edema- DE. Radial strain was compared in segments containing predominantly normal myocardium (25% (edema - DE); n=12] or predominantly scar (>75 % DE; n=19). Results: A total of 126 segments were analyzed. Radial strain was negatively correlated with the amount of edema (r=−0.41) and scar (r=−0.39, p ) Conclusions: Our study suggests that 2D Speckle Tracking radial strain can be a simple tool to differentiate stunned from scarred segments. This could be particularly useful in unstable patients after acute MI.

Non-invasive assessment of plaque burden by Multislice Computed Tomography (MDCT) has been recently shown to be feasible. However, to date, there is no data on its accuracy to assess changes in atheroma burden We and others have previously shown that apoA-IMilano (apoA-IM) infusion induces a large plaque regression in a very short period of time The aim of our study was to assess the feasibility of MDCT to accurately quantify the changes in atheroma volume induced by apoA-IM infusion Methods Aortic atherosclerotic lesions were induced in New-Zealand-White Rabbits (n=7) by 9-month atherogenic diet and aortic denudation, as previously reported. Animals were then randomized to receive 2 infusions, 4 days apart, of apoA-IM (ETC-216, Pfizer. n=4) or equal volume of placebo (n=3). All animals underwent 2 imaging studies in a 64-Slice MDCT scanner (Sensation 64, Siemens), pre- and post-treatment (Rx). After last MDCT study, animals were sacrificed and the aorta processed for histological atherosclerotic lesions characterization and volume quantification . Axial MDCT images were reconstructed (3 mm thick, no overlap). Display settings were manipulated according to previous validated studies. For lumen delineation: window width (WW) 1 and level (WL) 65% of lumen attenuation . For outer vessel boundaries: WW 155% and WL 65%. Results MDCT showed that plaque burden regressed by 29% in apoA-IM group (418mm 3 pre-Rx vs. 302mm 3 post-Rx, p=0.031). Conversely, no significant change was observed by MDCT studies done pre- and post-placebo administration. MDCT performed post-Rx showed that mean plaque volume in apoA-IM animals was 11.5% smaller than that of placebo animals (p=0.018). Mean plaque volume in histology showed similar results:plaque volume was 10.6% smaller in the apoA-IM group (p=0.005 vs. placebo). The interobserver variability for plaque volume quantification in MDCT was 10±4%. Conclusion MDCT detects the changes in atherosclerotic plaque volume induced by apoA-IM infusion. Furthermore, an excellent correlation was found between MDCT and histological plaque volumes. These results, combined to the short time of scan and the accessibility to the coronary territory suggest that MDCT could be applied in human studies testing changes in coronary plaque volume.

Background— Recent studies indicate that T-cell activation may play an important role in the pathophysiology of acute coronary syndromes (ACS). However, although those studies detected T-cell expansion in peripheral blood cells, demonstration of specific T-cell expansion within the plaque of patients with ACS is lacking. The present study aims to address whether a specific, immune-driven T-lymphocyte recruitment occurs within the unstable plaque of patients with ACS. Methods and Results— We simultaneously examined the T-cell repertoire using CDR3 size analysis both in coronary plaques (obtained by directional atherectomy) and in peripheral blood of patients with either ACS (n=11) or chronic stable angina (n=10). Unstable plaques showed a 10-fold increase in T-cell content by quantitative PCR. Using spectratyping analysis, we found several specific T-cell clonotype expansions only in unstable plaque from each patient with ACS, indicating a specific, antigen-driven recruitment of T cells within unstable lesions. Conclusions— For the first time, T-cell repertoire was investigated directly into coronary plaques; using this approach, we demonstrate that coronary plaque instability in the setting of ACS is associated with immune-driven T-cell recruitment, specifically within the plaque.

Objective: Inflammation plays a pivotal role in atherothrombosis. In addition to being a prognostic marker for major cardiovascular events, recent data indicate that C-reactive protein (CRP) might directly promote atherothrombosis by exerting direct effects on vascular cells. The aim of the present study was to determine whether CRP might affect the prothrombotic and proliferative characteristics of endothelial (ECs) and smooth muscle cells (SMCs). Methods and results: Incubation of ECs and SMCs with CRP resulted in a dose-dependent activation of cell proliferation, which was mediated by activation of the p44/42 MAP Kinase (ERK 1/2) pathway. In addition, CRP also induced tissue factor (TF) expression in both cell types in a dose-dependent fashion, exerting its effect at the transcriptional level, as demonstrated by semiquantitative and by real time PCR. Activation of the transcription factor, NF-kappa B, by CRP was demonstrated by EMSA and by suppression of TF expression by the NF-kappa B inhibitor, pyrrolidine-dithio-carbamate ammonium. Conclusions: These data indicate that CRP exerts direct effects on ECs and SMCs by promoting proliferation and TF expression and support the notion that CRP, besides representing a marker of inflammation, is an effector molecule able to induce a pro-atherothrombotic phenotype in cells of the vessel wall. (c) 2005 European Society of Cardiology. Published by Elsevier B.V. All rights reserved.

T cells have a central role in atherosclerosis and Acute Coronary Syndromes (ACS). Th17 cells have a key role in chronic inflammation since they secrete a wide array of cytokines, including IL-17, having pro-inflammatory effects by recruiting other inflammatory cells and acting directly on target cells. So far, Th17 role in atherosclerosis is still a matter of debate. To study the role of Th17 cells in the pathophysiology of ACS, we studied 38 patients (ACS,n=23; Stable Angina,n=15. Cytokine production was measured by Luminex™ technology in sera obtained from the aorta(Ao)and the coronary sinus(CS)during diagnostic coronary angiography. Th17 cells were measured by flow cytometry after staining with MoAb for CD3,CD4,CCR6 and CD161. 5 out of 23 patients with ACS had a high number of Th17 cells (>15% of total T cells) in the coronary sinus and high amounts of IL-17 (>50pg/ml). No correlations were found with the presence of Th1 or Th2 or related cytokines. Only one patient with Stable Angina had a high number of Th17 cells in the coronary sinus. After a 18 months follow-up we found that patients with ACS having high number of Th17 presented a worse disease course, in terms of successive needs for hospitalization, stroke and/ or exitus. Further exploitation of these data could lead to a better understanding of processes leading to atherosclerosis and ACS, and to the identification of new prognostic biomarkers in atherosclerosis and to the set up of new therapeutic strategies.

Background. Tissue Factor (TF), the regulatory cofactor of FVII(a), triggers blood coagulation as it converts FX and FIX to their active forms FXa and FIXa, respectively. In 2003 we showed that level of decryptable circulating TF activity in diabetic, smoker, and hyperlipidemic populations is a functionally labile entity. Existence of such form of circulating TF has generated the concept of vulnerable or hyperreactive blood. Recently, heightened TF activity was noted by many groups in various types of clinical samples, including human plasma, via utilization of a commercial TF activity assay termed ACTICHROME® whose approach to measure plasma TF activity is direct and distinct from that developed by us. Objective. To assess levels of plasma TF activity in patients with coronary artery disease (CAD), by the ACTICHROME® as well as our assay. Results. Suitability of the ACTICHROME® assay for direct assessment of TF activity in plasma was compared with our technique of plasma TF immunocapture followed by TF relipidation. We assessed TF activity in plasma aliquots pre-incubated with FVIIai, an inactive form of FVIIa that retains binding affinity to TF yet lacks protease activity, thereby serving as a highly effective and specific TF inhibitor. The ACTICHROME® assay was not able to detect bona fide TF activity: the tested plasma exhibited activity that could not be inhibited by pre-incubation with FVIIai. Thus, under the conditions of the ACTICHROME® assay, FX to FXa conversion in human platelet rich plasma is achieved by FVIIa in a TF independent manner. In contrast to the ACTICHROME® assay, in our TF assay pre-incubation of samples with FVIIai fully precluded FX activation. We therefore refer to plasma TF activity levels obtained using our TF assay as tTFp (total TF potential), and to the FVIIa activity levels obtained using the ACTICHROME® assay – ACTI-FVIIa. Assessment of tTFp in CAD plasma samples (n=28) revealed that in a subset of patients, tTFp was lower than that found in normal plasma (58.5%-69.6%, p=0.0001 NL, n=6 vs CAD tTFp 130%, n=7). The tTFp values in age matched healthy subjects (n=6) were in the close range of 75.2%-109.5%. We also assessed each healthy control and CAD patient sample for ACTI-FVIIa. To our surprise, ACTI-FVIIa values in all CAD samples were high, ranging between 15.8–132.1 pM of the TF standard, whereas ACTI-FVIIa values in the healthy subject group were much lower, in the close range of 3.2–8.8 pM. Interestingly, ACTI-VIIa of the three CAD samples exhibiting the highest tTFp was significantly lower than that of the three CAD samples exhibiting the lowest tTFp (highest tTFp: ACTI-VIIa = 39.2 ± 17.3, lowest tTFp: ACTI-VIIa = 97.4 ± 30.1, p=0.043). Heightened ACTI-FVIIa values in CAD samples prompted us to ascertain whether circulating MP and/or platelets may account for this phenomenon. Conventional size MP and platelets were removed from plasma by ultracentrifugation. Approximately 50% of ACTI-FVIIa in normal plasma and ≥95% of ACTI-FVIIa in CAD plasma was attributable to conventional size MP; in contrast, tTFp values in plasma samples pre- and post-spin were fairly similar: ultracentrifugation of CAD plasma resulted in only ~24.5% reduction of TF activity whereas in normal plasma, it resulted in ~42% reduction in TF activity, indicating that similarly to CAD plasma, the majority of functional TF species in normal plasma are not physically associated with conventional size MP, yet a somewhat larger proportion of functional TF is indeed carried by them compared to CAD plasma. Conclusions. We show that in CAD plasma, circulating MP of the conventional size do not carry most of functional TF yet, interestingly, CAD plasma strongly potentiates FVIIa activity in a TF-independent manner. We also show that the ACTICHROME® assay, when used to measure TF activity in human plasma, yields results that do not reflect bona fide TF activity. The majority of functional TF in human plasma appears to circulate in very small MP and in true solution; lastly, all examined plasma samples carried TF activity, underscoring the fact that potentially active TF protein is always present in circulation. These observations highlight the therapeutic possibilities of TF pathway inhibitors for treatment and prevention of atherothrombotic diseases.