thromboplastin and Atherosclerosis

Atherosclerosis is a chronic inflammatory disease that is characterized by the formation of lipid rich plaques in the wall of medium to large sized arteries. Atherothrombosis represents the terminal manifestation of this pathology in which atherosclerotic plaque rupture or erosion triggers the formation of occlusive thrombi. Occlusion of arteries and resultant tissue ischemia in the heart and brain causes myocardial infarction and stroke, respectively. Tissue factor (TF) is the receptor for the coagulation protease factor VIIa, and formation of the TF:factor VIIa complex triggers blood coagulation. TF is expressed at high levels in atherosclerotic plaques by both macrophage-derived foam cells and vascular smooth muscle cells, as well as extracellular vesicles derived from these cells. Importantly, TF mediated activation of coagulation is critically important for arterial thrombosis in the setting of atherosclerotic disease. The major endogenous inhibitor of the TF:factor VIIa complex is TF pathway inhibitor 1 (TFPI-1), which is also present in atherosclerotic plaques. In mouse models, increased or decreased expression of TFPI-1 has been found to alter atherosclerosis. This review highlights the contribution of TF-dependent activation of coagulation to atherthrombotic disease.

Topics: Animals; Atherosclerosis; Blood Coagulation; Mice; Plaque, Atherosclerotic; Thromboplastin; Thrombosis

Tissue factor (TF) is the high-affinity receptor and cofactor for factor (F)VII/VIIa. The TF-FVIIa complex is the primary initiator of blood coagulation and plays an essential role in hemostasis. TF is expressed on perivascular cells and epithelial cells at organ and body surfaces where it forms a hemostatic barrier. TF also provides additional hemostatic protection to vital organs, such as the brain, lung, and heart. Under pathological conditions, TF can trigger both arterial and venous thrombosis. For instance, atherosclerotic plaques contain high levels of TF on macrophage foam cells and microvesicles that drives thrombus formation after plaque rupture. In sepsis, inducible TF expression on monocytes leads to disseminated intravascular coagulation. In cancer patients, tumors release TF-positive microvesicles into the circulation that may contribute to venous thrombosis. TF also has nonhemostatic roles. For instance, TF-dependent activation of the coagulation cascade generates coagulation proteases, such as FVIIa, FXa, and thrombin, which induce signaling in a variety of cells by cleavage of protease-activated receptors. This review will focus on the roles of TF in protective hemostasis and pathological thrombosis.

Topics: Animals; Atherosclerosis; Blood Coagulation; Factor IX; Factor VIIa; Factor X; Fibrinolytic Agents; Gene Expression Regulation; Hemostasis; Humans; Neoplasms; Risk Factors; Sepsis; Signal Transduction; Thromboplastin; Thrombosis

The term microparticle (MP) identifies a heterogeneous population of vesicles playing a relevant role in the pathogenesis of vascular diseases, cancer and metabolic diseases such as diabetes mellitus. MPs are released by virtually all cell types by shedding during cell growth, proliferation, activation, apoptosis or senescence processes. MPs, in particular platelet- and endothelial-derived MPs (PMPs and EMPs), are increased in a wide range of thrombotic disorders, with an interesting relationship between their levels and disease pathophysiology, activity or progression. EMP plasma levels have been associated with several cardiovascular diseases and risk factors. PMPs are also shown to be involved in the progressive formation of atherosclerotic plaque and development of arterial thrombosis, especially in diabetic patients. Indeed, diabetes is characterised by an increased procoagulant state and by a hyperreactive platelet phenotype, with enhanced adhesion, aggregation, and activation. Elevated MP levels, such as TF+ MPs, have been shown to be one of the procoagulant determinants in patients with type 2 diabetes mellitus. Atherosclerotic plaque constitutes an opulent source of sequestered MPs, called "plaque" MPs. Otherwise, circulating MPs represent a TF reservoir, named "blood-borne" TF, challenging the dogma that TF is a constitutive protein expressed in minute amounts. "Blood-borne" TF is mainly harboured by PMPs, and it can be trapped within the developing thrombus. MP detection and enumeration by polychromatic flow cytometry (PFC) have opened interesting perspectives in clinical settings, particularly for the evaluation of MP numbers and phenotypes as independent marker of cardiovascular risk, disease and outcome in diabetic patients.

Topics: Acute Coronary Syndrome; Atherosclerosis; Biomarkers; Blood Platelets; Cardiovascular Diseases; Cell-Derived Microparticles; Diabetes Mellitus; Diabetes Mellitus, Type 2; Diabetic Cardiomyopathies; Endothelial Cells; Humans; Plaque, Atherosclerotic; Risk Factors; Stroke; Thromboplastin

Tissue factor (TF) is known to be the key element in the initiation of the extrinsic pathway of the coagulation cascade and appears to be a critical determinant of atherosclerotic plaque thrombogenicity. TF is needed to produce thrombin from prothrombin. In the extrinsic pathway, TF activates factor Vll. TF is expressed mainly on subendothelial tissues, but TF expression may be induced on endothelial cells by inflammatory mediators such as tumor necrosis factor-alpha (TNF-alpha). Subendothelial TF is responsible for initiating fibrin formation at sites of vascular injury, bloodborne TF may be an important contributor to propagation of the developing thrombus. It has been postulated that the blood-borne TF initiates the thrombogenic stimulus, leading to the formation of larger and more stable thrombus. TF may attach to cellular receptors, which in turn affect the production and release of inflammatory mediators. Baseline plasma TF activity has been demonstrated as an independent predictor for cardiovascular death in patients with acute myocardial infarction. TF is expressed by macrophage-derived foam cells in atherosclerotic plaques. TF levels were higher in atheroma from patients with unstable angina than with stable angina. These results suggest that high levels of TF exposed upon plaque rupture trigger atherothrombosis. Inhibition of TF would be expected to reduce thrombosis associated with a variety of diseases. TF pathway is a potential target for new therapeutic agents that can decrease TF activity, such as active site-inactivated factor VIIa, recombinant TF inhibitor and antibodies against TF or peptides interfering with TF-FVIIa complex activity. Significant clinical forms of atherosclerosis, such as sudden death, myocardial infarction, and stroke have common pathogenesis. The occlusion of the vessel lumen is the result from atherosclerotic plaque rupture/erosion that initiate thrombus formation. This thrombus has complex structure and contains predominantly fibrin in addition to platelets, suggesting an important role for the coagulation cascade in plaque thrombus formation. Tissue factor (TF) is known to be the key element in the initiation of the extrinsic pathway of the coagulation cascade and appears to be a critical determinant of atherosclerotic plaque thrombogenicity.

Topics: Atherosclerosis; Humans; Molecular Targeted Therapy; Plaque, Atherosclerotic; Thromboplastin; Thrombosis

The contribution of vessel wall-derived tissue factor (TF) to atherothrombosis is well established, whereas the pathophysiological relevance of the blood-borne TF is still a matter of debate, and controversies on the presence of platelet-associated TF still exist. In the past 15 years, several studies have documented the presence of TF in human platelets, the capacity of human platelets to use TF mRNA to make de novo protein synthesis, and the increase in the percentage of TF positive platelets in pathological conditions such as coronary artery disease (CAD). The exposure of vessel wall-derived TF at the site of vascular injury would play its main role in the initiation phase, whereas the blood-borne TF carried by platelets would be involved in the propagation phase of thrombus formation. More recent data indicate that megakaryocytes are committed to release into the bloodstream a well-defined number of TF-carrying platelets, which represents only a fraction of the whole platelet population. These findings are in line with the evidence that platelets are heterogeneous in their functions and only a subset of them is involved in the hemostatic process. In this review we summarize the existing knowledge on platelet associated TF and speculate on its relevance to physiology and to atherothrombosis and CAD.

Topics: Animals; Atherosclerosis; Blood Platelets; Coronary Artery Disease; Humans; Megakaryocytes; Thromboplastin; Thrombosis

Low-density lipoprotein receptor-related protein-1 (LRP1) is a large endocytic and signaling receptor that is widely expressed. In the liver, LRP1 plays an important role in regulating the plasma levels of blood coagulation factor VIII (fVIII) by mediating its uptake and subsequent degradation. fVIII is a key plasma protein that is deficient in hemophilia A and circulates in complex with von Willebrand factor. Because von Willebrand factor blocks binding of fVIII to LRP1, questions remain on the molecular mechanisms by which LRP1 removes fVIII from the circulation. LRP1 also regulates cell surface levels of tissue factor, a component of the extrinsic blood coagulation pathway. This occurs when tissue factor pathway inhibitor bridges the fVII/tissue factor complex to LRP1, resulting in rapid LRP1-mediated internalization and downregulation of coagulant activity. In the vasculature LRP1 also plays protective role from the development of aneurysms. Mice in which the lrp1 gene is selectively deleted in vascular smooth muscle cells develop a phenotype similar to the progression of aneurysm formation in human patient, revealing that these mice are ideal for investigating molecular mechanisms associated with aneurysm formation. Studies suggest that LRP1 protects against elastin fiber fragmentation by reducing excess protease activity in the vessel wall. These proteases include high-temperature requirement factor A1, matrix metalloproteinase 2, matrix metalloproteinase-9, and membrane associated type 1-matrix metalloproteinase. In addition, LRP1 regulates matrix deposition, in part, by modulating levels of connective tissue growth factor. Defining pathways modulated by LRP1 that lead to aneurysm formation and defining its role in thrombosis may allow for more effective intervention in patients.

Topics: Aneurysm; Animals; Atherosclerosis; Blood Coagulation; Elastin; Endocytosis; Extracellular Matrix; Factor VIII; Humans; Lipoproteins, LDL; Liver; Low Density Lipoprotein Receptor-Related Protein-1; Macrophages; Mice; Mice, Knockout; Models, Animal; Models, Molecular; Muscle, Smooth, Vascular; Organ Specificity; Peptide Hydrolases; Platelet-Derived Growth Factor; Protein Conformation; Receptors, LDL; Signal Transduction; Thromboplastin; Transforming Growth Factor beta; Tumor Suppressor Proteins; von Willebrand Factor

Tissue factor pathway inhibitor (TFPI) is the main inhibitor of tissue factor (TF)-mediated coagulation. In atherosclerotic plaques TFPI co-localizes with TF, where it is believed to play an important role in attenuating TF activity. Findings in animal models such as TFPI knockout models and gene transfer models are consistent on the role of TFPI in arterial thrombosis as they reveal an active role for TFPI in attenuating arterial thrombus formation. In addition, ample experimental evidence exists indicating that TFPI has inhibitory effects on both smooth muscle cell migration and proliferation, both which are recognized as important pathological features in atherosclerosis development. Nonetheless, the clinical relevance of these antithrombotic and atheroprotective effects remains unclear. Paradoxically, the majority of clinical studies find increased instead of decreased TFPI antigen and activity levels in atherothrombotic disease, particularly in atherosclerosis and coronary artery disease (CAD). Increased TFPI levels in cardiovascular disease might result from complex interactions with established cardiovascular risk factors, such as hypercholesterolemia, diabetes and smoking. Moreover, it is postulated that increased TFPI levels reflect either the amount of endothelial perturbation and platelet activation, or a compensatory mechanism for the increased procoagulant state observed in cardiovascular disease. In all, the prognostic value of plasma TFPI in cardiovascular disease remains to be established. The current review focuses on TFPI in clinical studies of asymptomatic and symptomatic atherosclerosis, coronary artery disease and ischemic stroke, and discusses potential atheroprotective actions of TFPI.

Topics: Animals; Anticoagulants; Atherosclerosis; Humans; Signal Transduction; Thromboplastin; Thrombosis

The prevalence of obesity has dramatically increased during the past two decades. Epidemiological studies suggest that obesity is an independent, modifiable risk factor for coronary heart disease, possibly due, at least in part, to the development of a pro-inflammatory and a pro-thrombotic state in obese subjects. In addition, numerous cohort studies have shown a link between obesity and different types of cancer. Accordingly, the regulation of body weight is becoming a serious concern for public health experts and scientists. Although the mechanisms responsible for these associations are still to be fully elucidated, a key role has been assigned to adipokines, a family of hormones which act as modulators of metabolism or inflammation, secreted by adipocytes. Tissue factor, the major physiological trigger of the blood coagulation cascade in vivo, which plays a central role in atherothrombosis and tumor biology, has also been proposed as one of the key molecules responsible for these associations.

Topics: Adipokines; Adipose Tissue; Animals; Atherosclerosis; Blood Coagulation; Humans; Inflammation Mediators; Neoplasms; Obesity; Risk Factors; Signal Transduction; Thromboplastin; Thrombosis

In the last ten years the contribution of both vessel wall-derived tissue factor (TF) and platelets to atherosclerosis has been revisited. At the beginning of the 2000 a circulating blood-borne TF has been proposed to sustain coagulation activation and propagation on the edge of a growing thrombus. Concomitantly with the observation that platelets not only contribute to thrombus formation, but also take part to the onset of the atherosclerotic lesion, evidences have been provided that they express functionally active TF, making them able to trigger the coagulation cascade.

Topics: Animals; Atherosclerosis; Blood Coagulation; Blood Platelets; Blood Vessels; Humans; Signal Transduction; Thromboplastin; Thrombosis

The transmembrane receptor tissue factor is a prominent protein expressed at macrophages and smooth muscle cells within human atherosclerotic lesions. While many coagulation proteins are detectable in atherosclerosis, a locally active thrombin and fibrin generating molecular machinery may be instrumental in manipulating cellular functions involved in atherogenesis. These include inflammation, angiogenesis and cell proliferation. Indeed, many experimental studies in mice show a correlation between hypercoagulability and increased atherosclerosis. In mice, the amount of atherosclerosis and/or the plaque phenotype, appear to be modifiable by specific anticoagulant interventions. While attempts to vary tissue factor level in the vasculature does not directly reduce plaque burden, the overexpression of tissue factor pathway inhibitor attenuates thrombogenicity and neo intima formation in mice. Moreover, inhibition of factor Xa or thrombin with novel selective agents, including rivaroxaban and dabigatran, inhibits inflammation associated with atherosclerosis in apoE(-/-) mice. The potential to modify a complex chronic disease like atherosclerosis with novel selective anticoagulants merits further clinical study.

Topics: Animals; Atherosclerosis; Humans; Inflammation; Mice; Thrombin; Thromboplastin

The term "vulnerable plaque" identifies atherosclerotic lesions prone to rupture. Plaque disruption facilitates the interaction of the inner components of the lesion, tissue factor (TF) among them, with the flowing blood. This results in activation of the coagulation cascade, ultimately leading to thrombus formation, and abrupt vascular occlusion. Despite the central role of vulnerable plaques in the onset of acute coronary syndromes (ACS), there are certain conditions (e.g., eroded plaques) where a hyperactive, "vulnerable" blood, may play a predominant pathophysiological role. Recently, two distinct pools of circulating TF have been identified. One, associated with cell-derived microparticles probably originating from apoptotic cells, such as macrophages, smooth muscle cells, and endothelium. The most recent, blood-borne TF, circulates in an "inactive" form (encryption) and has to be activated (decryption) to exert its thrombogenic activity. Certain pathological conditions associated with an increased rate of thrombotic complications have been associated with high levels of circulating TF. It is thought that the blood-borne TF perpetuates the initial thrombogenic stimulus, leading to the formation of larger or more stable thrombus, and thus, more severe ACS. Thus, the concept of vulnerable blood could represent a new link between the vulnerable lesion and the high-risk patient. Therefore, the assessment of selected biomarkers associated with "vulnerable or hyperreactive blood", e.g., blood-borne tissue factor, may represent a useful tool to identify patients with a high-risk profile of developing major cardiovascular events.

Topics: Acute Coronary Syndrome; Atherosclerosis; Biomarkers; Cell-Derived Microparticles; Coronary Artery Disease; Humans; Thromboplastin

SIRT1 is a NAD (+) -dependent class III histone deacetylase (HDAC) that mediates the effects of caloric restriction on lifespan and metabolic pathways in various organisms. It deacetylates both histone and non-histone proteins, and targets proteins with diverse cellular and tissue functions. In the vasculature of rodent models SIRT1 mediates vasodilatation via eNOS-derived nitric oxide (NO) and scavenging reactive oxygen species (ROS). Recent studies demonstrated further protective roles of SIRT1 in vascular biology and atherosclerosis. In endothelial cells and macrophages SIRT1 has anti-inflammatory functions by downregulating the expression of various pro-inflammatory cytokines by interfering with the NF-kB signaling pathway. Deacetylation of RelA/p65-NF-kB by SIRT1 in macrophages also suppresses the expression of Lox-1, a scavenger receptor for oxidized low-density lipoproteins (oxLDL), thereby preventing macrophage foam cell formation. Moreover, SIRT1 has been shown to regulate the activity of Liver X-receptor (LXR), thereby promoting ABCA1-driven reverse cholesterol transport in plaque macrophages. Finally, SIRT1 suppresses the expression of endothelial tissue factor (coagulation factor III) and hence exerts anti-thrombotic properties. These findings indicate atheroprotective effects of SIRT1 in atherogenesis and highlight the need for translational research from bench-to-bedside. Indeed, SIRT1 activators are available for experimental research and undergo clinical testing. Taken together, these studies suggest SIRT1 activation as a promising therapeutic approach in atherosclerosis. Further studies are necessary to better understand the exact role of SIRT1 in the protagonist cells orchestrating atherogenesis and to identify the specificity, target effects and putative off-target effects of these promising SIRT1 activators.

Topics: Animals; Anti-Inflammatory Agents; Atherosclerosis; Blood Vessels; Cholesterol; Cytokines; Endothelial Cells; Humans; Lipoproteins, LDL; Liver X Receptors; Macrophages; NF-kappa B; Orphan Nuclear Receptors; Reactive Oxygen Species; Sirtuin 1; Thromboplastin

Atherosclerosis is an inflammatory disease that involves the arterial wall and is characterised by the progressive accumulation of lipids in the vessel wall. The first step is the internalisation of lipids (LDL) in the intima with endothelial activation which enhances the permeability of the endothelial layer and the expression of cytokines/chemokines and adhesion molecules. These events increase LDL particles accumulation in the extracellular matrix where they aggregate/fuse, are retained by proteoglycans and become targets for oxidative and enzymatic modifications. In turn, retained pro-atherogenic LDLs enhance selective leukocyte recruitment and attachment to the endothelial layer inducing their transmigration across the endothelium into the intima. While smooth muscle cell numbers decline with the severity of plaque progression, monocytes differentiate into macrophages, a process associated with the upregulation of pattern recognition receptors including scavenger receptors and Toll-like receptors leading to foam cell formation. Foam cells release growth factors, cytokines, metalloproteinases and reactive oxygen species all of which perpetuate and amplify the vascular remodelling process. In addition, macrophages release tissue factor that, upon plaque rupture, contributes to thrombus formation. Smooth muscle cells exposed in eroded lesions are also able to internalise LDL through LRP-1 receptors acquiring a pro-thrombotic phenotype and releasing tissue factor. Platelets recognise ligands in the ruptured or eroded atherosclerotic plaque, initiate platelet activation and aggregation leading to thrombosis and to the clinical manifestation of the atherothrombotic disease. Additionally, platelets contribute to the local inflammatory response and may also participate in progenitor cell recruitment.

Topics: Animals; Atherosclerosis; Cell Communication; Endothelium, Vascular; Humans; Inflammation; Lipid Metabolism; Platelet Activation; Thromboplastin; Thrombosis

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.

Topics: Atherosclerosis; Humans; Inflammation; Thromboplastin; Thrombosis

The coagulation cascade represents a system of proteases responsible to maintain vascular integrity and to induce rapid clot formation after vessel injury. Tissue factor (TF), the key initiator of the coagulation cascade, binds to factor VIIa and thereby activates factor IX and factor X, resulting in thrombus formation. Different stimuli enhance TF gene expression in endothelial and vascular smooth muscle cells. In addition to these vascular cells, TF has recently been detected in the bloodstream in circulating cells such as leukocytes and platelets, as a component of microparticles, and as a soluble, alternatively spliced form of TF. Various cardiovascular risk factors like hypertension, diabetes, and dyslipidemia, increase levels of TF. In line with this observation, enhanced vascular TF expression occurs during atherogenesis, particularly in patients with acute coronary syndromes. (Circ J 2010; 74: 3 - 12).

Topics: Atherosclerosis; Blood Coagulation; Cardiovascular Diseases; Endothelium, Vascular; Hemostasis; Humans; Risk Factors; Thromboplastin

Coagulation, innate immunity, angiogenesis, and lipid metabolism represent fundamental and interdependent biological systems. While tissue factor pathway inhibitor (TFPI) is the major physiological inhibitor of TF, its unique structure and endothelial expression allow multi-modal interactions with constituent molecules in each of these systems. We review emerging data describing roles for TFPI beyond simply opposing the action of TF, particularly with regard to the highly basic c-terminus of TFPI, and highlight potentially exciting new areas for future research.

Topics: Algorithms; Atherosclerosis; Blood Coagulation; Humans; Immunity, Innate; Inflammation; Lipids; Lipoproteins; Models, Biological; Neovascularization, Pathologic; Protein Structure, Tertiary; Thromboplastin

Systemic lupus erythematosus (SLE) is a potentially fatal multiorgan inflammatory disease that primarily affects females. Due to the heterogeneity of clinical manifestations and lack of laboratory tests that are both specific and sensitive for the disease, diagnosis of SLE can often be difficult. Although the precise etiology remains to be fully elucidated, it is probable that various environmental, genetic, and hormonal factors contribute to the development of the disease. Patients with SLE have an increased risk for premature thrombosis and/or atherosclerosis, with up to half experiencing a thrombotic event. Furthermore, antiphospholipid antibodies probably play a key role in the development of thrombosis by affecting various hemostatic protein interactions with phospholipids and cell surfaces as well as platelet function. Despite recent advances in knowledge related to the factors that contribute to the pathophysiology of SLE, numerous challenges related to earlier diagnosis as well as the prediction and prevention of thrombotic events remain to be fully addressed.

Topics: Antibodies, Antinuclear; Antibodies, Antiphospholipid; Atherosclerosis; Blood Platelets; Female; Humans; Lupus Erythematosus, Systemic; Protein C; Risk Factors; Thromboplastin; Thrombosis

Tissue factor (TF) is the major initiator of blood coagulation, and a mediator of inflammation, angiogenesis, and carcinogenesis. According to recent evidence, preformed TF and inducible expression of the protein is observed in several blood components. TF is apparently constitutively expressed on circulating microparticles, and can be exposed within minutes on the cell membrane of activated eosinophils and platelets, and, potentially, on neutrophils. Expression in monocytes and neutrophils largely requires transcriptional activation of the TF gene. Eosinophils appear to harbour the highest concentration of preformed TF among all blood components under resting conditions. TF expression in eosinophil progenitor cells is substantially higher than in precursors of other granulocyte fractions. Eosinophil TF promotes transendothelial migration, which documents that presynthesized TF in blood supports functions apart from coagulation. It is still an open question how the intravascular TF is activated to trigger initiation of coagulation. TF activation in different blood components is likely to be differentially regulated according to the (patho)physiologic context.

Topics: Atherosclerosis; Blood Proteins; Endothelium, Vascular; Eosinophils; Humans; Monocytes; Thromboplastin; Thrombosis

Inflammation and thrombosis are key events in the long-lasting sequence of atherosclerotic plaque initiation, plaque growth, and eventual onset of complications leading to clinically manifest disease. Recent cellular and molecular studies have indicated that inside the plaque tissue complex, proinflammatory and prothrombotic mechanisms are intimately associated, and tissue factor (TF) is one of the main proteins that may link both processes. It is therefore not surprising that TF expression appeared to be a prominent feature in various types of vulnerable atherosclerotic plaques (i.e., lesions that specifically predispose to the onset of symptomatic atherosclerotic disease).

Topics: Animals; Atherosclerosis; Gene Expression Regulation; Humans; Inflammation; Thromboplastin; Thrombosis

Plaque disruption and subsequent thrombus formation play a critical role in the clinical manifestations of atherothrombosis. Vulnerable lesions are characterized by the existence of core rich in lipid, macrophages and tissue factor (TF). Plaque disruption facilitates the interaction between flowing blood with the inner components (TF) of disrupted atherosclerotic lesions triggering the coagulation cascade. TF, thrombin, platelets, fibrin and inflammatory cells are involved in this process of acute thrombus formation. This pathologic process is significantly accelerated by several "cardiovascular risk factors" such as diabetes, smoking, dyslipemia, etc. We will review on the role of TF, plaque cell apoptosis and blood thrombogenicity acting as a thread of inflammatory and prothrombotic mediators. We will also review the role of activated platelets as source for pro-inflammatory cytokines and enunciation of thrombotic process. Overall, we will try to emphasize the most recent understanding of the concepts involved in the interaction between inflammation and coagulation within the setting of atherothrombotic disease.

Topics: Animals; Apoptosis; Atherosclerosis; Humans; Inflammation; Thrombin; Thromboplastin; Thrombosis

The Liver X Receptor (LXR) alpha and beta isoforms are members of the type II nuclear receptor family which function as obligate heterodimers with the Retinoid X Receptor (RXR). Upon agonist binding, the DNA Binding Domain (DBD) of LXR interacts with LXR response elements on target genes to initiate transcription. A number of genes have been shown to be modulated by LXR function, including the ATP-binding cassette transporter A1 (ABCA1). ABCA1 is involved in the process of reverse cholesterol transport (RCT) from macrophages in atherosclerotic plaques to high-density lipoproteins (HDL) in the plasma. Both homozygous and heterozygous mutations in ABCA1 result in conditions characterised by decreased levels of HDL and an earlier onset of atherosclerosis. A number of other genes are upregulated by LXR activation which would be expected to have either pro- or anti-atherogenic effects. One such target gene is sterol regulatory element binding protein-1c (SREBP-1c), which is involved in the process of lipogenesis leading to increased levels of triglycerides which are pro-atherogenic. The complexity of LXR responses, however, makes it difficult to extrapolate the 'positive' or 'negative' effects of each target gene in isolation to a conclusion as to the outcome in humans when all target genes are being modulated in concert. This review will cover the structural features and associated biological data of non-steroidal LXR modulators claimed for the treatment of cardiovascular disease, as well as highlighting preferred compounds where this information can be discerned. In addition to this patent information a précis of literature data relevant to the utility of specific compounds in the treatment of cardiovascular disease will be given where available.

Topics: Acetyl-CoA Carboxylase; Animals; Atherosclerosis; ATP Binding Cassette Transporter 1; ATP-Binding Cassette Transporters; DNA-Binding Proteins; Fatty Acid Synthases; Humans; Hydrocarbons, Fluorinated; Liver X Receptors; Orphan Nuclear Receptors; Osteopontin; Receptors, Cytoplasmic and Nuclear; Sterol Regulatory Element Binding Protein 1; Sulfonamides; Thromboplastin

The development of atherosclerotic disease results from the interaction between environment and genetic make up. A key factor in atherogenesis is the oxidative modification of lipids, which is involved in the recruitment of mononuclear leukocytes to the arterial intima--a process regulated by several groups of adhesion molecules and cytokines. Activated leukocytes, as well as endothelial mitochondria, can produce reactive oxygen species (ROS) that are associated with endothelial dysfunction, a cause of reduced nitric oxide (NO) bioactivity and further ROS production. Peroxisome proliferator-activated receptors (PPAR) and liver X receptors (LXR) are nuclear receptors significantly involved in the control of lipid metabolism, inflammation and insulin sensitivity. Also, an emerging role has been suggested for G protein coupled receptors and for the small Ras and Rho GTPases in the regulation of the expression of endothelial NO synthase (eNOS) and of tissue factor, which are involved in thrombus formation and modulation of vascular tone. Further, the interactions among eNOS, cholesterol, oxidated LDL and caveola membranes are probably involved in some molecular changes observed in vascular diseases. Despite the relevance of oxidative processes in atherogenesis, anti-oxidants have failed to significantly improve atherosclerosis (ATS) prevention, while statins have proved to be the most successful drugs.

Topics: Animals; Arteries; Atherosclerosis; Cell Adhesion; Cytokines; DNA-Binding Proteins; Humans; Inflammation; Insulin; Leukocytes, Mononuclear; Lipids; Liver X Receptors; Mitochondria; Nitric Oxide; Nitric Oxide Synthase Type III; Orphan Nuclear Receptors; Oxygen; Peroxisome Proliferator-Activated Receptors; Reactive Oxygen Species; Receptors, Cytoplasmic and Nuclear; Receptors, G-Protein-Coupled; Thromboplastin

The effects of statins on platelet activation markers, chemokines and adiponectin, were investigated in 135 patients with hyperlipidemia. Of the 135 hyperlipidemic patients, 63 were allocated to the simvastatin group, treated with simvastatin at the dose of 10 mg daily, and the remaining 72 were allocated to the pitavastatin group, treated with pitavastatin at the dose of 2 mg daily. Plasma levels of platelet-derived microparticles (PDMP), cell adhesion molecules (sCD40L and sP-selectin), chemokines [monocyte chemoattractant protein-1 (MCP-1) and regulated on activation normally T-cell expressed and secreted] and adiponectin were measured at the baseline and after 6 months of treatment in both the groups. In addition, we carried out a basic study to investigate the MCP-1-dependent induction of tissue factor expression on a histiocytic cell line (U937 cells). The plasma levels of PDMP, sCD40L, sP-selectin, regulated on activation normally T-cell expressed and secreted and MCP-1 were higher, whereas those of adiponectin were lower, in the hyperlipidemic patients than in the normolipidemic controls. Plasma PDMP and sCD40L were positively correlated, whereas plasma adiponectin was negatively correlated, with the plasma levels of MCP-1. No significant differences in the plasma levels of PDMP, sCD40L, sP-selectin, regulated on activation normally T-cell expressed and secreted and MCP-1 measured before and after treatment were observed in either the simvastatin or pitavastatin group. A significant increase of the plasma adiponectin levels was observed after 6 months of treatment with pitavastatin but not after an equal duration of treatment with simvastatin. When pitavastatin-treated patients were divided into two groups according to the adiponectin response to pitavastatin treatment, significant decreases of the plasma MCP-1, PDMP and sCD40L levels were observed after pitavastatin treatment in the responder group. In the aforementioned basic study, MCP-1 by itself did not induce the expression of tissue factor on the U937 cells. However, the recombinant sCD40L-induced expression of tissue factor on U937 was enhanced by the addition of MCP-1. These findings suggest that PDMP, sCD40L and MCP-1 may participate in the development of atherothrombosis in patients with hyperlipidemia and that pitavastatin may exert an adiponectin-dependent antiatherothrombotic effect in hyperlipidemic patients.

Topics: Adiponectin; Adult; Aged; Atherosclerosis; CD40 Ligand; Cell-Derived Microparticles; Chemokine CCL2; Female; Humans; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Hyperlipidemias; Male; Middle Aged; P-Selectin; Quinolines; T-Lymphocytes; Thrombophilia; Thromboplastin; U937 Cells

Tissue factor (TF) plays a pivotal role in the generation of thrombin in atherothrombotic disease. The oxidized phospholipid 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (oxPAPC), an active compound of minimally oxidized low-density lipoprotein (MM-LDL), induces TF in endothelial cells (EC). The dietary soybean isoflavonoid genistein has been claimed to reverse several processes leading to atherosclerosis and related cardiovascular events via binding to estrogen receptors, generating nitric oxide (NO) or inhibiting tyrosine kinase-dependent pathways.. The effects and mechanisms of genistein on activity, antigen expression and mRNA levels of oxPAPC-induced TF were studied in human umbilical vein endothelial cells (HUVEC) and human aortic endothelial cells (HAEC).. Genistein abrogated oxPAPC-induced TF activity in arterial and venous human EC to basal levels, as measured by functional clotting assay, and downregulated oxPAPC-induced antigen expression measured by flow cytometry and mRNA levels quantified by real-time PCR. Western blotting and inhibitor experiments with the estrogen-receptor inhibitor ICI 182,780 and the NO-synthase inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME) showed that the effect may be mediated via inhibition of phosphorylation of ERK, but not upstream MEK1/2. The effect is not mediated by the tyrosine kinase inhibitor activity of genistein, as another tyrosine kinase inhibitor (tyrphostin 25) had no effect. Binding to the estrogen receptor or generation of NO are not involved in the action of genistein on TF. In conclusion genistein reduces oxPAPC-induced TF expression and thereby the prothrombotic phenotype of EC, further substantiating and explaining the beneficial effects of dietary genistein in preventing atherosclerosis and related cardiovascular events.

Topics: Anticoagulants; Atherosclerosis; Cell Culture Techniques; Endothelial Cells; Genistein; Humans; Phosphatidylcholines; Thromboplastin

Chronic inflammation is a major contributing factor to atherosclerosis and various markers of inflammation, fibrinolysis and coagulation are upregulated in patients with established atherosclerotic disease. The aim of this study was to investigate the direct and short-term effects of inflammation on platelet and monocyte activation with an in vivo model of endotoxemia in healthy volunteers.. In this study, 13 healthy male subjects with a mean age of 29.5+/-5.4 years received intravenous administration of lipopolysaccharide (LPS; 20 IU/kg IV). The kinetics of CD40-ligand and CD62P expression on platelets, tissue-factor binding on monocytes and platelet-monocyte aggregates were measured by whole blood flow cytometry at baseline and at 1, 2, 4, 6 and 24 hours after LPS administration. Plasma levels of soluble CD40-ligand were measured with an ELISA over the same time course. Platelet-monocyte aggregates, tissue-factor binding on monocytes and surface expression of platelet CD40L significantly increased in experimental endotoxemia in vivo, reaching peak values 1 hour after LPS administration. All values returned to baseline after 24 hours. Surface expression of CD62P on platelets and plasma levels of sCD40L did not change significantly in response to LPS.. In vivo administration of endotoxin leads to an activation of platelets and monocytes with an upregulation of proatherogenic CD40L on platelets. These findings underpin the role of inflammation in early atherogenesis through platelet and monocyte activation in an in vivo model.

Topics: Adult; Atherosclerosis; Blood Platelets; CD40 Ligand; Endotoxemia; Humans; Lipopolysaccharides; Male; Monocytes; P-Selectin; Platelet Activation; Thromboplastin; Up-Regulation

Topics: Atherosclerosis; Blotting, Western; Humans; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Real-Time Polymerase Chain Reaction; rho-Associated Kinases; rhoA GTP-Binding Protein; Rosuvastatin Calcium; Signal Transduction; Thromboplastin

Tissue factor (TF) is the initiator of the coagulation cascade, constitutively expressed in subendothelial cells such as vascular smooth muscle cells and initiating rapid coagulation when the vascular vessel is damaged. TF has been shown to be involved in the development and progression of atherosclerosis. Arsenic, an environmental pollutant, is related to the progression of atherosclerosis, although the pathogenic mechanisms are not fully elucidated. In the present study, we investigated the effect of arsenite on the expression of TF in human aortic smooth muscle cells (HASMCs) and the underlying molecular mechanisms. We found that (1) arsenite stimulated TF synthesis and activated the nuclear factor erythroid 2-related factor 2 (Nrf2) pathway in HASMCs, (2) sulforaphane, an Nrf2 activator, also stimulated TF synthesis in HASMCs, and (3) arsenite-induced upregulation of TF synthesis was prevented by Nrf2 knockdown in HASMCs. These results suggest that arsenite promotes TF synthesis by activating the Nrf2 pathway in HASMCs and that the induction of TF expression by arsenite may be related to the progression of atherosclerosis.

Topics: Aorta; Arsenites; Atherosclerosis; Cells, Cultured; Gene Expression; Humans; Isothiocyanates; Myocytes, Smooth Muscle; NF-E2-Related Factor 2; Signal Transduction; Sulfoxides; Thromboplastin

Antiphospholipid syndrome (APS) is associated with greater atherothrombotic risk and endothelial dysfunction, suggesting that endothelial glycocalyx is impaired in this disease.. The aim was to investigate the endothelial glycocalyx and the relationship between glycocalyx markers, endothelial dysfunction parameters and atherosclerotic markers in APS.. A total of 15 primary arterial APS patients and healthy controls were included in the study. Glycocalyx was assessed in both groups by sublingual sidestream dark field imaging and syndecan-1 plasma level. Endothelial function was evaluated by brachial artery flow-mediated dilatation (FMD) and early atherosclerosis by carotid intima media thickness (IMT). Thrombotic profile was also performed by measuring the plasma level of the tissue factor (TF).. APS patients had significantly increased syndecan-1 plasma level 38.6 ± 5.0 pg/ml vs. 19.1 ± 3.5 pg/ml;. This preliminary study supports, for the first time, that in APS patients endothelial glycocalyx is impaired, which could lead to thrombosis, endothelial dysfunction and early atherosclerosis.

Topics: Adolescent; Adult; Aged; Antiphospholipid Syndrome; Atherosclerosis; Autoantibodies; Biomarkers; Brachial Artery; Carotid Arteries; Carotid Intima-Media Thickness; Case-Control Studies; Cross-Sectional Studies; Endothelium, Vascular; Female; Glycocalyx; Humans; Male; Middle Aged; Risk Factors; Syndecan-1; Thromboplastin; Thrombosis; Vasodilation; Young Adult

The association of platelet and monocyte activity markers with long-term mortality was assessed in hemodialysis (HD) patients.. In 41 HD patients (25 male, 16 female), surface expression of CD40L and CD62P on platelets, tissue factor (TF) binding on monocytes, and platelet-monocyte aggregates were measured by flow cytometry. Plasma levels of MCP-1, IL-6, TNFα, and soluble CD40L were analyzed by enzyme linked immunosorbent assay. Cox proportional hazard regression analyses and Kaplan-Meier curve were calculated. The predefined endpoint was all-cause mortality.. The study follow-up was 11.54 years. Thirty-one patients (75.6%) died within the study period. Mean patient survival after study inclusion was 5.45 +/- 4.24 years. TF on monocytes above the median of the study population was significantly and independently associated with total mortality (HR (95% CI) 3.45 (1.32 - 9.07); p = 0.01). Cumulative mortality in patients with TF on monocytes above median was significantly higher compared to pa-tients with TF on monocytes below median value (log rank p < 0.01). Platelet-monocyte aggregates, the expression of CD40L and CD62P on platelets, and plasma levels of sCD40L, IL-6, MCP-1, and TNFα were not significantly correlated with mortality.. The present study confirms a high mortality in ESRD patients. TF binding on monocytes was significantly correlated with increased mortality and may identify a subgroup of patients at higher risk.

Topics: Aged; Atherosclerosis; Blood Platelets; Female; Humans; Kidney Failure, Chronic; Male; Middle Aged; Monocytes; Platelet Activation; Renal Dialysis; Thromboplastin

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.

Topics: Atherosclerosis; Cell Adhesion Molecules; Endothelial Cells; Human Umbilical Vein Endothelial Cells; Humans; Lipoproteins, LDL; NF-kappa B; Oxidation-Reduction; Receptors, Calcitriol; Signal Transduction; Thromboplastin; Thrombosis; Vitamin D; Vitamins

Atherosclerosis is the pathological process in arteries due to the plaque formation that is responsible for several diseases like heart disease, stroke and peripheral arterial disease. In this study, we performed in vitro and in vivo assays to evaluate the potential anti-atherosclerosis activity of peach kernel oil. For the in vitro assay, we incubated human umbilical vein endothelial cells (HUVEC) with tumor necrosis factor-α (TNF-α) to induce tissue factors (TF, an essential mediator of hemostasis and trigger of thrombosis) elevation. We found that TNF-α-induced TF elevation was suppressed by peach kernel oil in a dose-dependent manner at both mRNA and protein levels. Peach kernel oil can significantly improve HUVEC viability, protect the endothelial cells, which achieved the goal of prevention of thrombotic diseases. For the in vivo assay, we investigated the effect and mechanism of peach kernel oil on preventing atherosclerotic lesion formation in ApoE knockout mice. Results show that peach kernel oil could reduce total cholesterol, triglyceride, low-density lipoprotein cholesterol levels, elevate the high-density lipoprotein cholesterol level in serum, and reduce the area of the aortic atherosclerotic lesions in high-fat diet fed ApoE knockout mice. Moreover, peach kernel oil treatment can significantly down regulate the expression of TF protein to inhibit the formation of atherosclerotic plaque. In conclusion, peach kernel oil may be a potential health food to prevent atherosclerosis in cardiovascular diseases.

Topics: Animals; Aorta; Atherosclerosis; Cell Line; Cell Survival; Disease Models, Animal; Fatty Acids; Gene Expression Regulation; Human Umbilical Vein Endothelial Cells; Humans; Mice; Mice, Knockout, ApoE; Models, Biological; Phytochemicals; Plant Oils; Plaque, Atherosclerotic; Prunus persica; RNA, Messenger; Thromboplastin; Tumor Necrosis Factor-alpha

Trimethylamine-N-oxide (TMAO), one of the products in choline metabolite, is recently reported to be associated with cardiovascular diseases (CVD) that mainly attribute to atherothrombosis. However, the mechanisms how TMAO functions in the pathogenesis of CVD and atherothrombosis remain elusive. Tissue factor (TF) has been implicated in the thrombogenicity of atherosclerotic plaques. In the present study, we demonstrated that TMAO promoted TF (but not TF pathway inhibitor) expression via activation of NF-κB signaling pathway in primary human coronary artery endothelial cells (HCAECs). TMAO strongly increased TF activity and thrombin production. Further, a small dose of TMAO significantly increased TF expression and nuclear translocation of NF-κB with the synergistic action of low-dose of pro-atherosclerotic factors, such as TNF-α and HMGB1. Importantly, plasma TMAO level was positively correlated with TF activity in patients with ST-elevation myocardial infarction (STEMI). Altogether, our data revealed that TMAO promoted thrombosis through increasing TF expression and activity. The understanding of the new link between TMAO and atherothrombosis may facilitate therapeutic strategy in the prevention and treatment of atherothrombosis.

Topics: Aged; Atherosclerosis; Cells, Cultured; Endothelial Cells; Female; Humans; Male; Methylamines; Middle Aged; Thrombin; Thromboplastin; Thrombosis

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

Topics: Aged; Antigens, CD; Antigens, Differentiation, Myelomonocytic; Atherosclerosis; Cell Shape; Cells, Cultured; Coronary Artery Disease; Female; GTP-Binding Proteins; Humans; Lectins, C-Type; Macrophages; Male; Mannose Receptor; Mannose-Binding Lectins; Middle Aged; Plaque, Atherosclerotic; Protein Glutamine gamma Glutamyltransferase 2; Receptors, Cell Surface; Thromboplastin; Tomography, Optical Coherence; Transglutaminases

HIV infection is associated with an increased risk of cardiovascular disease in connection with atherosclerosis and thromboembolic complications. The pathogenesis of atherosclerosis is still unclear in this group of patients. Studies on pathogenesis of atherosclerosis in the general population emphasize the role of the extrinsic pathway of blood coagulation, particularly the tissue factor (TF) and tissue factor pathway inhibitor (TFPI). The effect of persistent activation of the immune system on enhanced expression of TF on the surface of monocytes in subjects infected with HIV is known to be correlated with the level of HIV RNA in blood serum.. The aim of this study was to evaluate the concentration of TF and its inhibitor TFPI in blood plasma, the impact of traditional and non-traditional cardiovascular risk factors on their concentration and the impact of both markers of haemostasis on the severity of subclinical atherosclerosis as assessed by the intima-media measurement of the carotid artery in HIV infected patients.. The study included 121 HIV-infected people with known clinical, immunological and virological status. The control group consisted of 42 healthy individuals, selected in terms of age and sex.. Higher concentrations of TF occurred in HIV-infected patients with a low current plasma HIV RNA level, nadir CD4+ T-cell count and longer duration of cumulative antiretroviral treatment. In multivariate analysis, it was the length of cumulative NRTI treatment that impacted on the concentration of TF. The determinants of cardiovascular disease (CVD) risk factors and inflammatory markers did not show any effect on the concentrations of TF. The TFPI level in HIV-infected patients was significantly higher than in the control group and was negatively correlated with the current level of HIV RNA and nadir CD4+ T-cell count, being higher in patients subjected to antiretroviral treatment. It was shown that the higher the cardiovascular risk and the higher the levels of total cholesterol, low-density lipoprotein cholesterol (LDL) and non-high-density lipoprotein cholesterol (non-HDL), the higher the concentrations of TFPI observed. The levels of TF and TFPI were positively correlated with carotid intima media thickness (cIMT); in the multivariate analysis, TF, non-HDL cholesterol and lifetime smoking (pack-years) independently affected the growth of cIMT. A similar effect on cIMT was demonstrated by TFPI.

Topics: Adult; Atherosclerosis; Biomarkers; Carotid Intima-Media Thickness; Case-Control Studies; Female; HIV; HIV Infections; Humans; Inflammation Mediators; Linear Models; Lipoproteins; Male; Middle Aged; Multivariate Analysis; Risk Factors; RNA, Viral; Thromboplastin

Topics: Animals; Antibodies; Apolipoproteins E; Arteries; Atherosclerosis; beta 2-Glycoprotein I; Humans; Interleukin-1beta; Male; Mice; Mice, Knockout; Thromboplastin; Toll-Like Receptor 4; Tumor Necrosis Factor-alpha; von Willebrand Factor

The activation of toll-like receptor 2 (TLR2) stimulates foam cell formation, which is a key early event in the process of atherosclerosis. In the present study, the role of toll/interleukin-1 receptor-domain-containing adaptor-inducing interferon-β (TRIF) in TLR2-mediated foam cell formation was investigated, and the importance of monocyte chemoattractant protein‑1 (MCP‑1), tissue factor (TF) and lectin‑like oxidized low‑density lipoprotein receptor‑1 (Lox‑1) were examined. Treatment of Raw 264.7 cells with the TLR2 agonist. Pam3CSK4, increased the gene expression of TRIF in a time‑dependent manner (RT‑PCR). The induced gene expression of TRIF stimulated by TLR2 was not observed in TLR2‑knockout mice‑derived bone marrow‑derived macrophages (BMDMs). Pam3CSK4 increased the mRNA expression of TRIF in the wild‑type BMDMs, but not in the TLR2‑knockout BMDMs. Knockdown of the expression of TRIF using small interfering RNA decreased Pam3CSK4‑induced foam cell formation (combination of oil‑red O and hematoxylin staining), suggesting a role of TRIF. Stimulation of TLR2 increased the expression levels of various genes, which are known to control atherosclerosis, including MCP‑1, TF and Lox‑1. The knockdown of TRIF also attenuated the Pam3CSK4‑induced expression of these genes. In addition, a reduction in TRIF affected the Pam3CSK4‑induced protein expression of MCP‑1 (EIA). Taken together, the results of the present study suggested that TRIF regulated foam cell formation via regulation of the expression levels of MCP‑1, TF and Lox‑1.

Topics: Adaptor Proteins, Vesicular Transport; Animals; Atherosclerosis; Chemokine CCL2; Foam Cells; Macrophages; Male; Mice; Mice, Inbred C57BL; RAW 264.7 Cells; RNA Interference; RNA, Messenger; RNA, Small Interfering; Scavenger Receptors, Class E; Thromboplastin; Toll-Like Receptor 2; Up-Regulation

Atherosclerosis is characterized by endothelial dysfunction, mainly induced by inflammation and oxidative stress. Increased reactive oxygen species (ROS) production together with increased adhesion molecules and thrombogenic tissue factor (TF) expression on endothelial cells has a key role in proatherogenic mechanisms. Therefore downmodulation of these molecules could be useful for reducing the severity of inflammation and atherosclerosis progression. Dehydrozingerone (DHZ) is a nutraceutical compound with anti-inflammatory and antioxidant activities. In this study we evaluated the ability of DHZ and its symmetric dimer to modulate hydrogen peroxide- (H

Topics: Atherosclerosis; Cell Adhesion; Cell Survival; Dietary Supplements; Dimerization; Fluorometry; Gene Expression Regulation; Human Umbilical Vein Endothelial Cells; Humans; Inflammation; Intercellular Adhesion Molecule-1; Intracellular Space; NF-kappa B; Oxidative Stress; Reactive Oxygen Species; Solubility; Styrenes; Thromboplastin; Vascular Cell Adhesion Molecule-1

Bone morphogenetic protein (BMP)-7, a major regulator of bone metabolism, inhibits ectopic calcification in atherosclerotic plaques. We have recently reported that BMP-7 is also a potent inducer of tissue factor (TF) in human mononuclear cells (MNCs). While nuclear factor kappa beta (NF-kB) and activation protein-1 (AP-1) are the transcription factors essential for inducible expression of human TF gene (F3), the mechanisms responsible for TF induction by BMP-7 are not known.. To elucidate the molecular mechanisms governing BMP-7-triggered TF expression in human MNCs.. Human blood monocytes were stimulated with BMP-7 and western blotting, qRT-PCR, and flow cytometry studies were carried out to assess F3 expression; promoter studies were also performed using a panel of reporter constructs. Procoagulant TF activity was measured using a validated FXa generation assay. The significance of NF-kB transcriptional activity was verified via pharmacological inhibition.. BMP-7 increased TF protein levels, procoagulant activity, surface presentation, and TF mRNA expression. This increase was accompanied by activation of NF-kB as evidenced by reduced IkB-α levels and elevated transcriptional activity of an NF-kB-sensitive reporter in transfected MNCs. Although treatment with BMP-7 also led to a strong phosphorylation of c-Jun, activation of AP-1 alone was not sufficient to induce TF expression: JSH-23, a potent and specific NF-kB inhibitor, completely blocked BMP-7-induced TF expression.. We report that BMP-7-dependent activation of TF in human MNCs is mediated via increased activity of NF-kB, leading to enhanced F3 transcription in human MNCs.

Topics: Atherosclerosis; Bone Morphogenetic Protein 7; Humans; Monocytes; Thromboplastin; Transfection

Occlusive renovascular disease and hypertension may progress to CKD. Circulating levels of several biomarkers, including fibroblast growth factor (FGF)-23, Klotho, and soluble urokinase plasminogen activator receptor (suPAR), are altered in patients with CKD, but their role in essential hypertension (EH) and renovascular hypertension (RVH) remains unclear.. Levels of FGF-23, Klotho, suPAR, plasminogen activator inhibitor (PAI)-1, tissue factor, and tissue factor pathway inhibitor (TFI) were measured in the inferior vena cava and renal vein of hypertensive patients with atherosclerotic renal artery stenosis (n=12) or age-matched participants with EH (n=12) and relatively preserved renal function. Single-kidney blood flow was measured to calculate renal release of markers. For control, peripheral vein levels were measured in healthy volunteers (HVs; n=12).. FGF-23 levels did not differ among the groups, whereas Klotho levels were lower in participants with RVH and EH than in HVs, and suPAR levels were elevated in patients with RVH compared with HVs and patients with EH (6.1±1.5 versus 4.4±1.9 and 3.2±1.2 ng/ml, P<0.05). PAI-1 levels were higher in patients with RVH than in patients with EH, but tissue factor and TFI levels were not statistically significantly different. After adjustment for GFR, Klotho levels remained decreased in both RVH and EH, and suPAR and PAI-1 levels remained elevated in RVH. eGFR correlated inversely with systemic and renal vein suPAR levels, and directly with systemic Klotho levels.. Klotho levels are low in hypertensive patients, whereas suPAR and PAI-1 levels are specifically elevated in RVH, correlating with GFR. Klotho, PAI-1, and suPAR may be markers of kidney injury in hypertensive patients.

Topics: Aged; Atherosclerosis; Biomarkers; Cross-Sectional Studies; Essential Hypertension; Female; Fibroblast Growth Factor-23; Fibroblast Growth Factors; Glomerular Filtration Rate; Glucuronidase; Humans; Hypertension; Hypertension, Renovascular; Klotho Proteins; Lipoproteins; Male; Middle Aged; Plasminogen Activator Inhibitor 1; Prospective Studies; Receptors, Urokinase Plasminogen Activator; Renal Artery Obstruction; Renal Circulation; Renal Veins; Thromboplastin; Vena Cava, Inferior

Atherosclerotic lesions represent a hypoxic milieu. However, the significance of this milieu in atherothrombosis has not been established. We aimed to assess the hypothesis that vascular wall hypoxia promotes arterial thrombus formation. We examined the relation between vascular wall hypoxia and arterial thrombus formation using a rabbit model in which arterial thrombosis was induced by 0.5 %-cholesterol diet and repeated balloon injury of femoral arteries. Vascular wall hypoxia was immunohistochemically detected by pimonidazole hydrochloride, a hypoxia marker. Rabbit neointima and THP-1 macrophages were cultured to analyse prothrombotic factor expression under hypoxic conditions (1 % O2). Prothrombotic factor expression and nuclear localisation of hypoxia-inducible factor (HIF)-1α and nuclear factor-kappa B (NF-κB) p65 were immunohistochemically assessed using human coronary atherectomy plaques. Hypoxic areas were localised in the macrophage-rich deep portion of rabbit neointima and positively correlated with the number of nuclei immunopositive for HIF-1α and NF-κB p65, and tissue factor (TF) expression. Immunopositive areas for glycoprotein IIb/IIIa and fibrin in thrombi were significantly correlated with hypoxic areas in arteries. TF and plasminogen activator inhibitor-1 (PAI-1) expression was increased in neointimal tissues and/or macrophages cultured under hypoxia, and both were suppressed by inhibitors of either HIF-1 or NF-κB. In human coronary plaques, the number of HIF-1α-immunopositive nuclei was positively correlated with that of NF-κB-immunopositive nuclei and TF-immunopositive and PAI-1-immunopositive area, and it was significantly higher in thrombotic plaques. Vascular wall hypoxia augments the thrombogenic potential of atherosclerotic plaque and thrombus formation on plaques via prothrombotic factor upregulation.

Topics: Aged; Angioplasty, Balloon; Animals; Atherosclerosis; Cell Hypoxia; Cell Line; Cholesterol, Dietary; Coronary Artery Disease; Disease Models, Animal; Female; Femoral Artery; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Macrophages; Male; Middle Aged; Neointima; Plaque, Atherosclerotic; Plasminogen Activator Inhibitor 1; Rabbits; Risk Factors; Signal Transduction; Thromboplastin; Thrombosis; Tissue Culture Techniques; Transcription Factor RelA; Vascular System Injuries

Intake of large amounts of added sweeteners has been associated with the pathogenesis of cardiometabolic risk. Several studies have shown that fructose increases the cardiovascular risk by modulating endothelial dysfunction and promoting atherosclerosis. Recently, a potential role for fructose in cardiovascular thrombosis has been suggested but with controversial results. Tissue factor (TF) plays a pivotal role in the pathophysiology of cardiovascular thrombosis by triggering the formation of intracoronary thrombi following endothelial injury. This study investigates the effects of fructose, in a concentration range usually observed in the plasma of patients with increased cardiovascular risk, on TF in human umbilical endothelial cells (HUVECs). Cells were stimulated with increasing concentrations of fructose (0.25, 1 and 2.5 mM) and then processed to evaluate TF-mRNA levels by real-time PCR as well as TF expression/activity by FACS analysis and procoagulant activity. Finally, a potential molecular pathway involved in modulating this phenomenon was investigated. We demonstrate that fructose induces transcription of mRNA for TF. In addition, we show that this monosaccharide promotes surface expression of TF that is functionally active. Fructose effects on TF appear modulated by the oxygen free radicals through activation of the transcription factor NF-κB since superoxide dismutase and NF-κB inhibitors suppressed TF expression. Data of the present study, although in vitro, indicate that fructose, besides promoting atherosclerosis, induces a prothrombotic phenotype in HUVECs, thus indicating one the mechanism(s) by which this sweetener might increase cardiometabolic risk.

Topics: Atherosclerosis; Fructose; Gene Expression Regulation; Human Umbilical Vein Endothelial Cells; Humans; NF-kappa B; Sweetening Agents; Thromboplastin; Thrombosis; Transcription, Genetic

Systemic Lupus Erythematosus (SLE) and antiphospholipid syndrome (APS) are associated with a high prevalence of atherosclerosis. β2 glycoprotein I (β2GPI) represents a link between autoimmunity and endothelial dysfunction. Recently, β2GPI reactive T cells have been identified; however, their role in atherosclerosis is still under investigation. We evaluated early atherosclerosis in patients with SLE and APS and investigated T cell reactivity to β2GPI and its relationship with atherosclerotic process.. Fifty SLE, 18 patients with primary APS (PAPS), and 25 healthy controls were enrolled. Demographic and clinical data, including traditional cardiovascular risk factors, were recorded. Monocyte β2GPI and Tissue Factor (TF) expression and peripheral blood mononuclear cell response to β2GPI stimulation were evaluated. Doppler ultrasound was performed to investigate flow-mediated dilatation (FMD) and carotid intima-media thickness (IMT). We detected an increase in mean IMT and a decrease in FMD in patients with SLE versus controls (P<0.05 and P=0.0001, respectively) and a decrease in FMD in patients with PAPS versus controls (P<0.05). Monocyte β2GPI and TF expression was higher in patients with SLE and PAPS than in controls (P=0.006 and P=0.001, respectively); no correlation of monocyte β2GPI and TF with IMT or FMD was detected. β2GPI induced peripheral blood mononuclear cell proliferation in 32% of patients with SLE, 25% of patients with PAPS yet in none of the controls. Proliferative response to β2GPI correlated with a history of arterial thrombosis, thrombocytopenia, and IMT >0.9 mm.. A significant percentage of patients with SLE and PAPS show a β2GPI-specific T cell reactivity, which is associated with subclinical atherosclerosis.

Topics: Adult; Aged; Antibody Specificity; Antiphospholipid Syndrome; Atherosclerosis; Autoantibodies; Autoantigens; beta 2-Glycoprotein I; Blood Pressure; Cardiovascular Diseases; Carotid Intima-Media Thickness; Cell Division; Endothelium, Vascular; Female; Hemorheology; Humans; Interferon-gamma Release Tests; Lipoproteins, HDL; Lupus Erythematosus, Systemic; Male; Middle Aged; Monocytes; Risk Factors; T-Cell Antigen Receptor Specificity; Thromboplastin; Vasodilation; Young Adult

Inflammation and possibly hypoxia largely affect glucose utilization in atherosclerotic arteries, which could alter many metabolic systems. However, metabolic changes in atherosclerotic plaques remain unknown. The present study aims to identify changes in metabolic systems relative to glucose uptake and hypoxia in rabbit atherosclerotic arteries and cultured macrophages.. Macrophage-rich or smooth muscle cell (SMC)-rich neointima was created by balloon injury in the iliac-femoral arteries of rabbits fed with a 0.5% cholesterol diet or a conventional diet. THP-1 macrophages stimulated with lipopolysaccharides (LPS) and interferon-γ (INFγ) were cultured under normoxic and hypoxic conditions. We evaluated comprehensive arterial and macrophage metabolism by performing metabolomic analyses using capillary electrophoresis-time of flight mass spectrometry. We evaluated glucose uptake and its relationship to vascular hypoxia using (18)F-fluorodeoxyglucose ((18)F-FDG) and pimonidazole, a marker of hypoxia.. The levels of many metabolites increased in the iliac-femoral arteries with macrophage-rich neointima, compared with those that were not injured and those with SMC-rich neointima (glycolysis, 4 of 9; pentose phosphate pathway, 4 of 6; tricarboxylic acid cycle, 4 of 6; nucleotides, 10 of 20). The uptake of (18)F-FDG in arterial walls measured by autoradiography positively correlated with macrophage- and pimonidazole-immunopositive areas (r = 0.76, and r = 0.59 respectively; n = 69 for both; p<0.0001). Pimonidazole immunoreactivity was closely localized with the nuclear translocation of hypoxia inducible factor-1α and hexokinase II expression in macrophage-rich neointima. The levels of glycolytic (8 of 8) and pentose phosphate pathway (4 of 6) metabolites increased in LPS and INFγ stimulated macrophages under hypoxic but not normoxic condition. Plasminogen activator inhibitor-1 protein levels in the supernatant were closely associated with metabolic pathways in the macrophages.. Infiltrative macrophages in atherosclerotic arteries might affect metabolic systems, and hypoxia but not classical activation might augment glycolytic and pentose phosphate pathways in macrophages.

Topics: Active Transport, Cell Nucleus; Animals; Atherosclerosis; Cell Hypoxia; Cell Line; Femoral Artery; Fluorodeoxyglucose F18; Glycolysis; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Macrophages; Male; Metabolome; Pentose Phosphate Pathway; Rabbits; Radionuclide Imaging; Radiopharmaceuticals; Thromboplastin; Tissue Distribution

To explore the role of toll-like receptor 4 (TLR4) on oxidized low-density/β₂-glycoprotein I/β₂-glycoprotein I (ox-LDL/β₂GPI/anti-β₂GPI) antibodies complex induced macrophage foam cell formation.. The peritoneal macrophages were separated from TLR4 intact C3H/HeN mice and TLR4 defective C3H/HeJ mice. The cells were treated with ox-LDL, ox-LDL/β₂GPI, ox-LDL/anti-β₂GPI, anti-β₂GPI/β₂GPI, ox-LDL/β₂GPI/anti-β₂GPI, lipopolysaccharide (LPS) for 48 h and the foam cells were identified by Oil red O staining for intracellular lipids. The total cellular RNA and the protein lysates were collected. The levels of tissue factor (TF) mRNA in two groups were detected by real-time PCR (RT-PCR), and the expression of phosphorylated nuclear factor-κB (NF-κB) p65 was detected by Western blotting. Monocyte chemotactic protein-1 (MCP-1) secretion from peritoneal macrophages was determined by enzyme linked immunosorbent assay (ELISA) kits.. Compared with C3H/HeJ mice, lipid droplets in the cytoplasm of peritoneal macrophages from C3H/HeN mice were significantly increased and phosphorylation-NF-κB expression was significantly upregulated after stimulating by ox-LDL/β₂GPI/anti-β₂GPI complex (P < 0.01). TF mRNA and MCP-1 expression were also upregulated post ox-LDL/β₂GPI/anti-β₂GPI complex stimulation [TF mRNA: 0.041 ± 0.023 vs. 0.005 ± 0.003; MCP-1: (6 200.2 ± 6.4) pg/ml vs. (803.3 ± 5.5) pg/ml, P < 0.01].. TLR4 can enhance ox-LDL/β₂GPI/anti-β₂GPI complex induced peritoneal macrophage foam cell formation via upregulating phosphorylation-NF-κB, TF and MCP-1 expression.

Topics: Animals; Antigen-Antibody Complex; Atherosclerosis; beta 2-Glycoprotein I; Cells, Cultured; Foam Cells; Lipoproteins, LDL; Macrophages, Peritoneal; Male; Mice; Mice, Inbred C3H; NF-kappa B; Thromboplastin; Toll-Like Receptor 4

Atherogenesis, evolution of plaque, and outcomes following endovascular intervention depend heavily on the unique vascular architecture of each individual. Patient-specific, multiscale models able to correlate changes in microscopic cellular responses with relevant macroscopic flow, and structural conditions may help understand the progression of occlusive arterial disease, providing insights into how to mitigate adverse responses in specific settings and individuals.. Vascular architectures mimicking coronary and carotid bifurcations were derived from clinical imaging and used to generate conjoint computational meshes for in silico analysis and biocompatible scaffolds for in vitro models. In parallel with three-dimensional flow simulations, geometrically realistic scaffolds were seeded with human smooth muscle cells (SMC) or endothelial cells and exposed to relevant, physiological flows. In vitro surrogates of endothelial health, atherosclerotic progression, and thrombosis were locally quantified and correlated best with an quantified extent of flow recirculation occurring within the bifurcation models. Oxidized low-density lipoprotein uptake, monocyte adhesion, and tissue factor expression locally rose up to three-fold, and phosphorylated endothelial nitric oxide synthase and Krüppel-like factor 2 decreased up to two-fold in recirculation areas. Isolated testing in straight-tube idealized constructs subject to static, oscillatory, and pulsatile conditions, indicative of different recirculant conditions corroborated these flow-mediated dependencies.. Flow drives variations in vascular reactivity and vascular beds. Endothelial health was preserved by arterial flow but jeopardized in regions of flow recirculation in a quasi-linear manner. Similarly, SMC exposed to flow were more thrombogenic in large recirculating regions. Health, thrombosis, and atherosclerosis biomarkers correlate with the extent of recirculation in vascular cells lining certain vascular geometries.

Topics: Arteries; Atherosclerosis; Biomarkers; Biomechanical Phenomena; Carotid Arteries; Computer Simulation; Coronary Vessels; Disease Progression; Endothelial Cells; Humans; Kruppel-Like Transcription Factors; Lipoproteins, LDL; Models, Cardiovascular; Myocytes, Smooth Muscle; Nitric Oxide Synthase Type III; Regional Blood Flow; Thromboplastin; Thrombosis; Vascular Cell Adhesion Molecule-1

Atherothrombosis is the main cause of myocardial infarction and ischemic stroke. Although the extrinsic (tissue factor-factor VIIa [FVIIa]) pathway is considered as a major trigger of coagulation in atherothrombosis, the role of the intrinsic coagulation pathway via coagulation FXII herein is unknown. Here, we studied the roles of the extrinsic and intrinsic coagulation pathways in thrombus formation on atherosclerotic plaques both in vivo and ex vivo.. Plaque rupture after ultrasound treatment evoked immediate formation of subocclusive thrombi in the carotid arteries of Apoe(-/-) mice, which became unstable in the presence of structurally different FXIIa inhibitors. In contrast, inhibition of FVIIa reduced thrombus size at a more initial stage without affecting embolization. Genetic deficiency in FXII (human and mouse) or FXI (mouse) reduced ex vivo whole-blood thrombus and fibrin formation on immobilized plaque homogenates. Localization studies by confocal microscopy indicated that FXIIa bound to thrombi and fibrin particularly in luminal-exposed thrombus areas.. The FVIIa- and FXIIa-triggered coagulation pathways have distinct but complementary roles in atherothrombus formation. The tissue factor-FVIIa pathway contributes to initial thrombus buildup, whereas FXIIa bound to thrombi ensures thrombus stability.

Topics: Animals; Aorta, Thoracic; Aortic Diseases; Apolipoproteins E; Atherosclerosis; Blood Coagulation; Blood Platelets; Carotid Arteries; Carotid Artery Diseases; Cholesterol, Dietary; Disease Models, Animal; Factor VIIa; Factor XI; Factor XII; Factor XII Deficiency; Factor XIIa; Humans; Mice; Mice, Inbred C57BL; Mice, Knockout; Plaque, Atherosclerotic; Rupture, Spontaneous; Thromboplastin; Thrombosis; Time Factors

It has been reported that oxidized low-density lipoprotein (oxLDL) forms a stable and non-dissociable complex with β2-glycoprotein I (β2GPI) and that IgG anti-β2GPI autoantibodies are able to recognize this complex, thus facilitating macrophage-derived foam cell formation in patients with antiphospholipid syndrome (APS). However, the immunopathological mechanisms of oxLDL/β2GPI complexes in promoting foam cell formation are not fully understood. In this study, we examined the role of toll-like receptor 4 (TLR4) in the oxLDL/β2GPI/anti-β2GPI complex-induced transformation of mouse peritoneal macrophages to foam cells.. Oil red O staining and optical density (OD) measurements of intracellular stained oil red O solution were used to monitor the transformation of peritoneal macrophages to foam cells in TLR4-competent C3H/HeN and TLR4-mutant C3H/HeJ mice. During foam cell formation induced by the oxLDL/β2GPI/anti-β2GPI complex, the expression of TLR4 and activation of nuclear factor kappa B (NF-κB) were confirmed by analyzing the protein and mRNA levels of these compounds. Furthermore, the related active molecule expression during foam cell formation induced by the oxLDL/β2GPI/anti-β2GPI complex was examined in the presence or absence of TLR4.. The data showed that treatment with the oxLDL/β2GPI/anti-β2GPI complex markedly increased foam cell formation, the TLR4 expression, NF-κB activation, the tissue factor (TF) expression and tumor necrosis factor-α (TNF-α) and monocyte chemotactic protein-1 (MCP-1) secretion in the C3H/HeN mice. However, the transformation of macrophages to foam cells and the expression levels of phosphorylated NF-κB, TF, TNF-α and MCP-1 were significantly reduced in the C3H/HeJ mice treated with the oxLDL/β2GPI/anti-β2GPI complex. In addition, compared with that achieved by oxLDL alone, the oxLDL/β2GPI complex decreased foam cell formation and the related signaling molecule expression in the C3H/HeN mice.. Our results indicate that TLR4 plays an important role in the process of oxLDL/β2GPI/anti-β2GPI complex-induced transformation of macrophages to foam cells, which may accelerate the development of atherosclerosis in the setting of APS. However, β2GPI alone functions as an antiatherogenic protein by preventing the foam cell formation induced by oxLDL.

Topics: Animals; Atherosclerosis; beta 2-Glycoprotein I; Chemokine CCL2; Foam Cells; Lipoproteins, LDL; Macrophages, Peritoneal; Male; Mice; Mice, Inbred C3H; NF-kappa B; Oxidation-Reduction; Phosphorylation; Signal Transduction; Thromboplastin; Toll-Like Receptor 4; Tumor Necrosis Factor-alpha

Atherosclerosis is understood to be a blood vessel inflammation. High-mobility group box-1 (HMGB-1) plays a key role in the systemic inflammation. Tissue factor (TF) is known to lead to inflammation which promotes thrombus formation. Membrane type1 matrix metalloprotease (MT1-MMP) associates with advanced glycation endproducts (AGE) triggered-TF protein expression and phosphorylation of NF-κB. However, it is still unclear about the correlation of MT1-MMP and HMBG-1-mediated TF expression. In this study, we investigated the molecular mechanisms of TF expression in response to HMGB-1 stimulation and the involvement of MT1-MMP in endothelial cells.. Pull-down assays and Western blotting revealed that HMGB-1 induced RhoA/Rac1 activation and NF-kB phosphorylation in cultured human aortic endothelial cells. HMGB-1 increased the activity of MT1-MMP, and inhibition of RAGE or MT1-MMP by siRNA suppressed HMGB-1-induced TF upregulation as well as HMGB-1-triggered RhoA/Rac1 activation and NF-kB phosphorylation.. The present study showed that RAGE/MT1-MMP axis modified HMBG-1-mediated TF expression through RhoA and Rac1 activation and NF-κB phosphorylation in endothelial cells. These results suggested that MT1-MMP was involved in vascular inflammation and might be a good target for treating atherosclerosis.

Topics: Atherosclerosis; Endothelial Cells; Enzyme Activation; Gene Expression Regulation; Gene Silencing; HMGB1 Protein; Humans; Matrix Metalloproteinase 14; NF-kappa B; Phosphorylation; rac1 GTP-Binding Protein; Receptor for Advanced Glycation End Products; Receptors, Immunologic; rhoA GTP-Binding Protein; RNA, Small Interfering; Signal Transduction; Thromboplastin

Hyperlipidaemia is a causal factor in the ethiopathogenesis of atherosclerosis. Statins are the cornerstone drug therapy for LDL-cholesterol (LDL-c) lowering, that exert beneficial effects beyond lipid lowering. Circulating microparticles (cMPs), microvesicles released by activated cells into the bloodstream, are markers of vascular and inflammatory cell activation with tentative role in disease progression. However, the role of statins on cMPs seems controversial. We aimed at the evaluation of the effects of lipid-lowering treatment (LLT) on cMP generation in patients in primary prevention of atherosclerosis. A case-control study was conducted in hypercholesterolaemic patients receiving LLT with statins and normocholesterolaemic controls (LLT+ and LLT-, respectively, n=37/group), matched by age, gender and LDL-c levels. cMPs were characterised by flow cytometry using annexin-V and cell-specific antibodies. In LLT+-patients overall numbers of cMPs (p<0.005) were lower than in controls. Levels of cMPs carrying parental cell markers from vascular and circulating cell origin (platelet, endothelial cell, pan-leukocyte and specific-leukocyte subsets) were significantly lower in blood of LLT+ compared to LLT--patients. Moreover, MPs from LLT+-patients had reduced markers of activated platelets (αIIbβ3-integrin), activated inflammatory cells (αM-integrin) and tissue factor. The effect of LLT on cMP shedding was found to be accumulative in years. cMP shedding associated to cardiovascular risk in LLT+-patients. In summary, at similar plasma cholesterol levels patients on statin treatment had a significant lower number of cMPs carrying markers of activated cells. These findings indicate that statins protect against vascular cell activation.

Topics: Adult; Annexin A5; Atherosclerosis; Blood Platelets; Case-Control Studies; Cell-Derived Microparticles; Cholesterol, LDL; Endothelial Cells; Female; Humans; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Hyperlipidemias; Hypolipidemic Agents; Inflammation; Leukocytes; Male; Middle Aged; Primary Prevention; Thromboplastin

Imaging modalities to assess atherosclerotic plaque thrombogenicity have not been established, so in this study the relationship between [(18)F]-fluorodeoxyglucose ((18)F-FDG) uptake and thrombus formation was investigated in rabbit atherosclerotic arteries.. Atherosclerotic plaque was induced in the iliacofemoral artery by balloon injury and a 0.5% cholesterol diet. At 3 weeks after the first balloon injury, the arteries were visualized by (18)F-FDG positron emission tomography (PET) imaging 2h after an (18)F-FDG infusion, and then arterial thrombus was induced by a second balloon injury of both iliacofemoral arteries. Imaging with (18)F-FDG-PET revealed significantly more radioactivity along the injured (0.63 ± 0.12 SUVmax), than the contralateral non-injured artery (0.34 ± 0.08 SUV max, n=17, P<0.0001). Arterial radioactivity measured by autoradiography positively correlated with macrophage area, the number of nuclei that were immunopositive for nuclear factor κ B (NF-κB), and tissue factor (TF) expression. The immunopositive areas for glycoprotein IIb/IIIa and fibrin in thrombi were significantly larger in the atherosclerotic than in the contralateral arteries, and significantly correlated with radioactivity in PET (r=0.92, P<0.001, n=10) and autoradiography (r=0.73, P<0.0001, n=50) in the arteries. Inhibition of NF-κB significantly reduced TF expression in cultured atherosclerotic plaque.. Arterial (18)F-FDG uptake reflects the thrombogenicity of atherosclerotic plaque following balloon injury. 

Topics: Animals; Atherosclerosis; Catheters; Fluorodeoxyglucose F18; Gene Expression Regulation; Magnetic Resonance Angiography; Male; NF-kappa B; Plaque, Atherosclerotic; Positron-Emission Tomography; Rabbits; Radiography; Radiopharmaceuticals; Thromboplastin; Thrombosis

Topics: Animals; Atherosclerosis; Catheters; Fluorodeoxyglucose F18; Magnetic Resonance Angiography; Male; NF-kappa B; Positron-Emission Tomography; Radiopharmaceuticals; Thromboplastin; Thrombosis

Acyl-CoA:cholesterol acyltransferase (ACAT) converts cholesterol to cholesteryl esters in plaque foam cells. Complete deficiency of macrophage ACAT has been shown to increase atherosclerosis in hypercholesterolemic mice because of cytotoxicity from free cholesterol accumulation, whereas we previously showed that partial ACAT inhibition by Fujirebio compound F1394 decreased early atherosclerosis development. In this report, we tested F1394 effects on preestablished, advanced lesions of apolipoprotein-E-deficient mice.. Apolipoprotein-E-deficient mice on Western diet for 14 weeks developed advanced plaques, and were either euthanized (Baseline), or continued on Western diet with or without F1394 and euthanized after 14 more weeks. F1394 was not associated with systemic toxicity. Compared with the baseline group, lesion size progressed in both groups; however, F1394 significantly retarded plaque progression and reduced plaque macrophage, free and esterified cholesterol, and tissue factor contents compared with the untreated group. Apoptosis of plaque cells was not increased, consistent with the decrease in lesional free cholesterol. There was no increase in plaque necrosis and unimpaired efferocytosis (phagocytic clearance of apoptotic cells). The effects of F1394 were independent of changes in plasma cholesterol levels.. Partial ACAT inhibition by F1394 lowered plaque cholesterol content and had other antiatherogenic effects in advanced lesions in apolipoprotein-E-deficient mice without overt systemic or plaque toxicity, suggesting the continued potential of ACAT inhibition for the clinical treatment of atherosclerosis, in spite of recent trial data.

Topics: Acetyl-CoA C-Acyltransferase; Animals; Aorta; Aortic Diseases; Apolipoproteins E; Apoptosis; Atherosclerosis; Cholesterol; Cyclohexanes; Diet, Atherogenic; Dioxanes; Disease Models, Animal; Disease Progression; Enzyme Inhibitors; Foam Cells; Male; Mice; Mice, Knockout; Necrosis; Plaque, Atherosclerotic; Thromboplastin

Tissue factor (TF) is a core protein with an essential function in the coagulation cascade that maintains the homeostasis of the blood vessels. TF not only participates in neointima formation, but also causes the development of atherosclerosis. This study investigated the mechanism regulating TF expression in macrophages using Pam3 CSK4 , a TLR2 ligand. Pam3 CSK4 induced TF expression in two types of macrophages (Raw264.7 and BMDM), but not in TLR2 KO mice derived BMDM. Pam3 CSK4 induced TF expression was inhibited by pretreatment with pan-JAK inhibitor or JAK2 inhibitor AG490. JAK2 knock-down by siRNA inhibited Pam3 CSK4 induced TF expression. Pam3 CSK4 stimulated STAT3 phosphorylation (S727), while STAT3 knock-down by siRNA reduced Pam3 CSK4 induced TF expression. These results suggest that Pam3 CSK4 induced TF expression is regulated by the JAK2-STAT3 signaling pathway. Pam3 CSK4 , unlike increased TF expression, significantly decreased RGS2 expression, while RGS2 overexpression decreased Pam3 CSK4 induced TF expression. Inhibition of TF by RGS2 WT did not occur in mutants with flawed RGS domains. We also investigated the correlation between RGS2 and STAT3 phosphorylation. RGS2 knock-down elevated Pam3 CSK4 induced STAT3 phosphorylation, but RGS2 overexpression had the opposite effect on STAT3 phosphorylation. These results suggest that, while Pam3 CSK4 induced TF expression is regulated by JAK2-STAT3 signaling, RGS2 is a negative regulator targeted to STAT3.

Topics: Animals; Atherosclerosis; Cell Line; Janus Kinase 2; Lipopeptides; Macrophages; Mice; Mice, Knockout; Phosphorylation; Protein Processing, Post-Translational; RGS Proteins; Signal Transduction; STAT3 Transcription Factor; Thromboplastin; Toll-Like Receptor 2; Transcription, Genetic; Transcriptional Activation

Women with polycystic ovary syndrome (PCOS) have chronic low-grade inflammation that can increase the risk of atherothrombosis. We performed a cross-sectional study to examine the effect of glucose ingestion on markers of atherothrombotic inflammation in mononuclear cells (MNC) of 16 women with PCOS (8 lean, 8 obese) and 16 weight-matched controls. Activator protein-1 (AP-1) activation and the protein content of early growth response-1 (EGR-1), matrix matalloproteinases-2 (MMP2), and tissue factor (TF) were quantified from MNC obtained from blood drawn fasting and 2 h after glucose ingestion. Plasma MMP9 and C-reactive protein (CRP) were measured from fasting blood samples. Truncal fat was determined by DEXA. Lean women with PCOS exhibited greater AP-1 activation and MMP2 protein content after glucose ingestion and higher plasma MMP9 and CRP levels than lean controls. Obese women with PCOS exhibited greater EGR-1 and TF protein content after glucose ingestion, and plasma CRP levels were even higher compared with lean subjects regardless of PCOS status. Truncal fat correlated with MMP9 and CRP levels and glucose-stimulated increases in AP-1 activation and EGR-1 and TF protein content. Testosterone correlated with glucose-stimulated AP-1 activation, and androstenedione correlated with MMP9 and CRP levels and glucose-stimulated AP-1 activation. Thus, both PCOS and obesity contribute to an atherothrombotic state in which excess abdominal adiposity and hyperandrogenism may be specific risk factors for developing atherothrombosis.

Topics: Adult; Atherosclerosis; Biomarkers; Body Mass Index; C-Reactive Protein; Cross-Sectional Studies; Diet, Atherogenic; Early Growth Response Protein 1; Female; Gelatinases; Glucose; Humans; Hyperandrogenism; Leukocytes, Mononuclear; Obesity, Abdominal; Polycystic Ovary Syndrome; Thromboplastin; Thrombosis; Transcription Factor AP-1; Young Adult

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.. 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.. 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.

Topics: Animals; Anticholesteremic Agents; Aorta, Abdominal; Aortic Diseases; Atherosclerosis; Chemokine CCL2; Cyclooxygenase 2; Disease Progression; Drug Combinations; Drug Synergism; Indazoles; Liver X Receptors; Magnetic Resonance Angiography; Orphan Nuclear Receptors; Plaque, Atherosclerotic; Rabbits; Random Allocation; Simvastatin; Thromboplastin; Up-Regulation

Phospholipases are produced from bacterial pathogens causing very different diseases. One of the most intriguing aspects of phospholipases is their potential to interfere with cellular signaling cascades and to modulate the host-immune response. Here, we investigated the role of the innate and acquired immune responses elicited by Chlamydophila pneumoniae phospholipase D (CpPLD) in the pathogenesis of atherosclerosis. We evaluated the cytokine and chemokine production induced by CpPLD in healthy donors' monocytes and in vivo activated T cells specific for CpPLD that infiltrate atherosclerotic lesions of patients with C. pneumoniae antibodies. We also examined the helper function of CpPLD-specific T cells for monocyte matrix metalloproteinase (MMP)-9 and tissue factor (TF) production as well as the CpPLD-induced chemokine expression by human venular endothelial cells (HUVECs). We report here that CpPLD is a TLR4 agonist able to induce the expression of IL-23, IL-6, IL-1β, TGF-β, and CCL-20 in monocytes, as well as CXCL-9, CCL-20, CCL-4, CCL-2, ICAM-1, and VCAM-1 in HUVECs. Plaque-derived T cells produce IL-17 in response to CpPLD. Moreover, CpPLD-specific CD4(+) T lymphocytes display helper function for monocyte MMP-9 and TF production. CpPLD promotes Th17 cell migration through the induction of chemokine secretion and adhesion molecule expression on endothelial cells. These findings indicate that CpPLD is able to drive the expression of IL-23, IL-6, IL-1β, TGF-β, and CCL-20 by monocytes and to elicit a Th17 immune response that plays a key role in the genesis of atherosclerosis.

Topics: Aged; Atherosclerosis; Cell Line; Chemokines; Chlamydophila pneumoniae; Cytokines; Enzyme-Linked Immunosorbent Assay; Female; Gene Expression Regulation; Human Umbilical Vein Endothelial Cells; Humans; Male; Matrix Metalloproteinase 9; Middle Aged; Monocytes; Phospholipase D; Real-Time Polymerase Chain Reaction; Th17 Cells; Thromboplastin; Toll-Like Receptor 4

Infection and inflammation are risk factors in the initiation and progression of atherosclerosis. Periodontitis is one of the most prevalent chronic inflammations of the oral cavity, and has been reported to be associated with systemic disease. In this study, we evaluated whether the heat-shock protein GroEL of Fusobacterium nucleatum, one of the most prevalent bacteria in periodontitis, induces factors that predispose to atherosclerosis in human microvascular endothelial cells (HMEC-1) and apolipoprotein E-deficient (ApoE(-/-)) mice. GroEL induced the expression of chemokines such as monocyte chemoattractant protein-1 and interleukin-8 as well as cell adhesion molecules, such as intercellular adhesion molecule 1, vascular cell adhesion molecule 1, and E-selectin. GroEL induced the activity of tissue factor and reduced the activity of the tissue factor pathway inhibitor. Foam cell formation was induced by GroEL. GroEL-injected ApoE(-/-) mice showed significant atherosclerotic lesion progression compared with control mice. Serum levels of risk factors for atherosclerosis such as interleukin-6, C-reactive protein, and low-density lipoprotein were increased in GroEL-injected ApoE(-/-) mice compared with control mice, whereas serum levels of high-density lipoprotein were decreased. We could detect significantly higher levels of anti-F. nucleatum GroEL antibody in serum and F. nucleatum DNA in gingival crevicular fluid from patients with periodontitis than in that from healthy subjects. Our results indicate that the host response to the GroEL of periodontal pathogens like F. nucleatum may be a mechanism involved in atherosclerosis, supporting the association of periodontitis and systemic infection.

Topics: Animals; Antibodies, Bacterial; Apolipoproteins E; Atherosclerosis; Cell Adhesion Molecules; Cells, Cultured; Chaperonin 60; Chemokines; Chronic Periodontitis; Cloning, Molecular; Dental Plaque; Endothelium, Vascular; Foam Cells; Fusobacterium nucleatum; Humans; Lipoproteins; Male; Mice; Mice, Knockout; Monocytes; Recombinant Proteins; Risk Factors; Thromboplastin

Accelerated atherosclerosis represents a major problem in both systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA) patients, and endothelial damage is a key feature of atherogenesis. We aimed to assess early endothelial changes in SLE and RA female patients (127 SLE and 107 RA) without previous CV events. Biomarkers of endothelial cell activation (intercellular adhesion molecule-1 (sICAM-1), vascular cell adhesion molecule-1 (sVCAM-1), thrombomodulin (TM), and tissue factor (TF)) were measured and endothelial function was assessed using peripheral artery tonometry. Reactive hyperemia index (RHI), an indicator of microvascular reactivity, and augmentation index (AIx), a measure of arterial stiffness, were obtained. In addition, traditional CV risk factors, disease activity and medication were determined. Women with SLE displayed higher sICAM-1 and TM and lower TF levels than women with RA (p = 0.001, p<0.001 and p<0.001, respectively). These differences remained significant after controlling for CV risk factors and medication. Serum levels of vascular biomarkers were increased in active disease and a moderate correlation was observed between sVCAM-1 levels and lupus disease activity (rho = 0.246) and between TF levels and RA disease activity (rho = 0.301). Although RHI was similar across the groups, AIx was higher in lupus as compared to RA (p = 0.04). Also in active SLE, a trend towards poorer vasodilation was observed (p = 0.06). In conclusion, women with SLE and RA present with distinct patterns of endothelial cell activation biomarkers not explained by differences in traditional CV risk factors. Early vascular alterations are more pronounced in SLE which is in line with the higher CV risk of these patients.

Topics: Adult; Arthritis, Rheumatoid; Atherosclerosis; Biomarkers; Endothelium, Vascular; Female; Humans; Intercellular Adhesion Molecule-1; Lupus Erythematosus, Systemic; Manometry; Middle Aged; Risk Factors; Thrombomodulin; Thromboplastin; Vascular Cell Adhesion Molecule-1; Vascular Stiffness

Atherosclerotic coronary arteries are more calcified in patients with than without chronic kidney disease (CKD). We addressed the potential for coronary microvascular obstruction in patients with and without CKD during stenting for saphenous vein aorto-coronary graft (SVG) stenosis under protection with a distal occlusion/aspiration device. In patients with and without CKD (n = 20/20), SVG plaque composition was analyzed from virtual histology using intravascular ultrasound analysis before stent implantation. There was more dense calcium and more necrotic core in patients with than without CKD (14 ± 3 vs. 3 ± 1 % and 21 ± 3 vs. 12 ± 2 % of plaque volume, respectively). Coronary aspirate was retrieved during stent implantation and divided into particulate debris and plasma. Patients with CKD had more particulate debris and calcium release than patients without CKD. In contrast, the release of serotonin was less in patients with than without CKD (0.4 ± 0.1 vs. 1.2 ± 0.3 μmol/L), whereas that of catecholamines, endothelin, tissue factor, thromboxane, tumor necrosis factor α, and C reactive protein was not significantly different. Confirming the biochemical results, aspirate plasma from patients with CKD induced less vasoconstriction of rat mesenteric arteries than that from patients without CKD (with endothelium (+E), 26 ± 7 %; without endothelium (-E): 28 ± 7 % vs. +E, 68 ± 12 %; -E: 95 ± 16 % of maximum KCl-induced vasoconstriction). Graft atherosclerosis of patients with CKD is more degenerated and releases more particulate debris and calcium, but the aspirate has surprisingly less serotonin and vasoconstrictor potential.

Topics: Adult; Aged; Aged, 80 and over; alpha-2-HS-Glycoprotein; Animals; Atherosclerosis; Blood Vessel Prosthesis Implantation; C-Reactive Protein; Calcium; Coronary Artery Disease; Graft Occlusion, Vascular; Humans; Male; Middle Aged; Necrosis; Plaque, Atherosclerotic; Rats; Renal Insufficiency, Chronic; Saphenous Vein; Stents; Thromboplastin; Tumor Necrosis Factor-alpha; Ultrasonography, Interventional; Vascular Calcification; Vasoconstriction

Tissue factor (TF) is expressed in atherosclerotic lesions. Since mechanical forces influence endothelial cell (EC) function and are thought to account for the unique distribution of atherosclerosis in areas exposed to disturbed flow, we hypothesized that disturbed to-fro flow (TFF) and unidirectional pulsatile forward flow (PFF) would have different effects on TF expression in EC. TF RNA expression in HUVEC exposed to mechanical stress in the presence or absence of chemical stimulation with thrombin was determined. TFF induced a significantly higher TF expression than PFF that was sustained for 8 h. Combination of mechanical and chemical stimuli induced significantly higher TF expression than only mechanical stresses, and this effect was synergistic in both TFF and PFF. The MAPK p38 inhibitor SB-203580 significantly inhibited TF expression induced by mechanical and chemical stimulations, but the MEK inhibitor PD-98059 did not inhibit TF induced by TFF. Immunoblotting revealed that ERK1/2 phosphorylation induced by TFF was sustained for 120 min, whereas that induced by PFF was not. We conclude that disturbed flow induced greater and sustained amplification of TF expression, and this synergistic effect may be regulated by p38 MAPK and ERK1/2. These results provide added insight into the mechanism of atherosclerosis in areas of disturbed flow.

Topics: Atherosclerosis; Cells, Cultured; Endothelial Cells; Enzyme Inhibitors; Flavonoids; Humans; Imidazoles; MAP Kinase Kinase Kinases; p38 Mitogen-Activated Protein Kinases; Pulsatile Flow; Pyridines; Stress, Mechanical; Thrombin; Thromboplastin

Oxidized low-density lipoprotein (oxLDL) interacts with macrophages and is implicated in atherogenesis. Macrophages are also the major source within the atherosclerotic plaque of tissue factor (TF), the membrane-bound glycoprotein receptor that triggers the coagulation cascade in vivo and contributes to plaque thrombogenicity. In this study we tested the hypothesis that oxLDL modulates TF expression in human monocyte-derived macrophages (MDMs).. Mononuclear cells were isolated from human blood, allowed to differentiate into MDMs during 8 days in cell culture, and then exposed to varying concentrations of oxLDL in the presence or absence of lipopolysaccharide (LPS). TF procoagulant activity (TF-PCA) of MDMs was measured by one-stage recalcification clotting assay using human recombinant TF as standard. TF protein was evaluated by Western blotting, and TF mRNA was determined by Northern blot analysis.. OxLDL at 5-10 µg/mL increased TF-PCA, TF protein, and mRNA in MDMs, whereas 20-100 µg/mL oxLDL inhibited TF-PCA, protein expression, and mRNA expression in these cells even in the face of LPS stimulation.. Low concentrations of oxLDL enhance TF expression in MDMs, whereas higher concentrations attenuate TF expression both at baseline as well as following LPS stimulation. Both TF-PCA and TF protein follow this dose-response pattern that is preceded by concordant mRNA changes. Thus, we have demonstrated modulation by oxLDL of TF protein and bioactivity in MDMs.

Topics: Atherosclerosis; Blotting, Northern; Blotting, Western; Cell Differentiation; Coagulants; Flow Cytometry; Humans; Lipopolysaccharides; Lipoproteins, LDL; Macrophages; Recombinant Proteins; RNA, Messenger; Thromboplastin

Atherosclerotic lesions predominantly localize in areas exposed to distinct hemodynamic conditions. In such lesions, tissue factor (TF) is over-expressed. Therefore, we hypothesized that varying types of mechanical forces may induce different effects on TF expression in endothelial cell, and may also influence the effects of chemical stimuli.. TF RNA expression in human umbilical vein endothelial cells (HUVEC) exposed to mechanical stress in the presence or absence of chemical stimulation with thrombin (Th) was determined. The forces examined were: steady unidirectional laminar flow (LF), pulsatile unidirectional laminar flow (PF), constant oscillatory flow (OF), pulsatile to-fro flow (TFF), and cyclic strain (CS).. Mechanical stimulation of HUVEC with LF for 2 h induced an 8.7 ± 0.7-fold increase in TF RNA expression, while PF induced 4.7 ± 0.9 and TFF induced 8.6 ± 1.7-fold, respectively. These responses were significantly higher than static controls. Exposure to OF or CS did not result in any significant increase, whereas chemical stimulation with Th led to significant TF expression (4.9 ± 0.3-fold). The combination of mechanical-chemical stimuli induced significantly higher TF expression than mechanical stresses alone, and this effect was synergistic. Combination of LF+Th for 2 h induced significantly increased TF expression (16.6 ± 1.7-fold), as did PF+Th (14.8 ± 2.4) and TFF+Th (17.4 ± 1.0). Furthermore, after 6 h exposure, only TFF demonstrated significantly higher TF expression both with and without Th.. While uniform laminar flow resulted in transient TF expression, disturbed flow induced sustained amplification of TF expression. Further investigation is needed to elucidate the mechanism of localized atherosclerosis in areas exposed to disturbed flow.

Topics: Atherosclerosis; Cells, Cultured; Endothelial Cells; Hemodynamics; Humans; Pulsatile Flow; RNA, Messenger; Stress, Mechanical; Thrombin; Thromboplastin

The acute-phase protein serum amyloid A (SAA) is elevated during inflammation and may be deposited in atheroma where it promotes atherosclerosis. We investigated the proatherogenic effects of SAA on the vascular endothelium and their regulation by high-density lipoprotein (HDL). Exposure of human aortic endothelial cells (HAEC) to SAA (0.25-25μg/ml) decreased nitric oxide ((•)NO) synthesis/bioavailability, although the endothelial NO synthase monomer-to-dimer ratio was unaffected. SAA (10μg/ml) stimulated a Ca(2+) influx linked to apocynin-sensitive superoxide radical anion (O(2)(•-)) production. Gene expression for arginase-1, nuclear factor κB (NF-κB), interleukin-8, and tissue factor (TF) increased within 4h of SAA stimulation. Enzymatically active Arg-1/2 was detected in HAEC cultured with SAA for 24h. Therefore, in addition to modulating (•)NO bioavailability by stimulating O(2)(•-) production in the endothelium, SAA modulated vascular l-Arg bioavailability. SAA also diminished relaxation of preconstricted aortic rings induced by acetylcholine, and added superoxide dismutase restored the vascular response. Preincubation of HAEC with HDL (100 or 200, but not 50, μg/ml) before (not after) SAA treatment ameliorated the Ca(2+) influx and O(2)(•-) production; decreased TF, NF-κB, and Arg-1 gene expression; and preserved overall vascular function. Thus, SAA may promote endothelial dysfunction by modulating (•)NO and l-Arg bioavailability, and HDL pretreatment may be protective. The relative HDL to SAA concentrations may regulate the proatherogenic properties of SAA on the vascular endothelium.

Topics: Animals; Aorta; Arginase; Atherosclerosis; Blotting, Western; Calcium; Endothelial Cells; Endothelium, Vascular; Gene Expression; Humans; Inflammation; Lipoproteins, HDL; Male; NF-kappa B; Nitric Oxide; Rats; Rats, Wistar; Serum Amyloid A Protein; Signal Transduction; Superoxide Dismutase; Superoxides; Thromboplastin

Early growth response gene-1 (Egr-1) regulates the expression of genes important to cardiovascular disease. Within atherosclerotic lesions, Egr-1 is expressed in smooth muscle cells, endothelial cells, and macrophages. Since macrophages play a pivotal role in atherosclerotic lesion initiation and progression, this study investigated the effects of Egr-1 deficiency within bone marrow-derived cells on the development of atherosclerosis in a hyperlipidaemic mouse model.. Bone marrow from Egr-1-deficient mice and wild-type controls was transplanted into lethally irradiated LDL receptor null mice. After 26 weeks on a high fat diet, atherosclerotic lesion size within the aortic sinus of recipients was evaluated. Mice receiving Egr-1-deficient bone marrow had significantly decreased lesion size compared with controls. Lesions of these mice contained fewer macrophages and had reduced expression of vascular cell adhesion molecule-1 (VCAM-1), tissue factor, as well as transforming growth factor receptor type II, which are target genes of Egr-1. These results were validated by in vitro analysis of Egr-1-deficient peritoneal macrophages which, after lipopolysaccharide stimulation, had decreased VCAM-1 and tissue factor mRNA expression compared with wild-type controls.. This study demonstrates that bone marrow-derived Egr-1 promotes macrophage accumulation, atherosclerotic lesion development, and lesion complexity.

Topics: Animals; Aorta, Thoracic; Atherosclerosis; Bone Marrow Cells; Bone Marrow Transplantation; Disease Models, Animal; Early Growth Response Protein 1; Female; Gene Expression Regulation; Hyperlipidemias; Lipids; Macrophages; Mice; Mice, Inbred C57BL; Mice, Knockout; Protein Serine-Threonine Kinases; Radiation Chimera; Receptor, Transforming Growth Factor-beta Type II; Receptors, LDL; Receptors, Transforming Growth Factor beta; RNA, Messenger; Thromboplastin; Time Factors; Vascular Cell Adhesion Molecule-1; Whole-Body Irradiation

Adipocytes are nowadays recognised as cells able to produce and secrete a large variety of active substances termed adipokines, which exert direct effects on vascular cells. Among these adipokines, leptin has been proposed to play a role in the pathophysiology of acute coronary syndromes, as well as in increasing cardiovascular risk. At the moment, however, the mechanisms linking leptin to cardiovascular disease are not completely understood. This study investigates the effects of leptin, in a concentration range usually observed in the plasma of patients with increased cardiovascular risk or measurable in patients with acute coronary syndromes, on tissue factor (TF) and cellular adhesion molecules (CAMs) expression in human coronary endothelial cells (HCAECs). We demonstrate that leptin induces transcription of mRNA for TF and CAMs by real-time PCR. In addition, we show that this adipokine promotes surface expression of TF and CAMs that are functionally active since we observed increased procoagulant activity and leukocyte adhesion on cell surface. Leptin effects appear modulated by eNOS-production of oxygen free radicals through the activation of the transcription factor, nuclear factor(NF)-kappaB, since L-NAME, Superoxide Dismutase and NF-kappaB inhibitors suppressed CAMs and TF expression. Data of the present study, although in vitro , indicate that leptin may exert direct effects on human coronary endothelial cells by promoting CAMs and TF expression and support the hypothesis that this adipokines, besides being involved in the pathophysiology of obesity, might play a relevant role as an active mediator linking obesity to cardiovascular disease.

Topics: Acute Coronary Syndrome; Atherosclerosis; Cell Adhesion Molecules; Cells, Cultured; Coronary Thrombosis; Coronary Vessels; Endothelial Cells; Enzyme Inhibitors; Humans; Leptin; NF-kappa B; NG-Nitroarginine Methyl Ester; Nitric Oxide Synthase Type III; Reactive Oxygen Species; Thromboplastin

Acadesine, an adenosine-regulating agent and activator of AMP-activated protein kinase, has been shown to possess antiinflammatory activity. This study investigated whether and how acadesine inhibits tissue factor (TF) expression and thrombus formation.. Human umbilical vein endothelial cells and human peripheral blood monocytes were stimulated with lipopolysaccharide to induce TF expression. Pretreatment with acadesine dramatically suppressed the clotting activity and expression of TF (protein and mRNA). These inhibitory effects of acadesine were unchanged for endothelial cells treated with ZM241385 (a specific adenosine A(2A) receptor antagonist) or AMP-activated protein kinase inhibitor compound C, and in macrophages lacking adenosine A(2A) receptor or alpha1-AMP-activated protein kinase. In endothelial cells and macrophages, acadesine activated the phosphoinositide 3-kinase/Akt signaling pathway, reduced the activity of mitogen-activated protein kinases, and consequently suppressed TF expression by inhibiting the activator protein-1 and NF-kappaB pathways. In mice, acadesine suppressed lipopolysaccharide-mediated increases in blood coagulation, decreased TF expression in atherosclerotic lesions, and reduced deep vein thrombus formation.. Acadesine inhibits TF expression and thrombus formation by activating the phosphoinositide 3-kinase/Akt pathway. This novel finding implicates acadesine as a potentially useful treatment for many disorders associated with thrombotic pathology, such as angina pain, deep vein thrombosis, and sepsis.

Topics: Adenosine A2 Receptor Antagonists; Aminoimidazole Carboxamide; AMP-Activated Protein Kinases; Animals; Apolipoproteins E; Atherosclerosis; Blood Coagulation; Cells, Cultured; Disease Models, Animal; Dose-Response Relationship, Drug; Endothelial Cells; Enzyme Activation; Fibrinolytic Agents; Humans; Lipopolysaccharides; Macrophages; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Monocytes; NF-kappa B; Phosphatidylinositol 3-Kinases; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Pyrazoles; Pyrimidines; Receptor, Adenosine A2A; Ribonucleosides; RNA, Messenger; Sepsis; Signal Transduction; Thromboplastin; Transcription Factor AP-1; Triazines; Triazoles; Up-Regulation; Venous Thrombosis

The aim of this study was to understand the initial mechanism of arterial thrombus formation induced by vulnerable human atherosclerotic plaques to re-assess and improve current antithrombotic strategies.. Rupture of atherosclerotic plaques causes arterial thrombus formation that might lead to myocardial infarction and ischemic stroke. Atherothrombosis is considered as an inseparable tangle of platelet activation and coagulation processes, involving plaque components such as tissue factor (TF) and collagen as well as blood-borne TF and coagulation factor XIIa (FXIIa). A combination of anticoagulants and antiplatelet agents is the present treatment.. Human atheromatous plaque material was exposed to blood or blood components at physiological calcium/magnesium concentration. Platelet aggregation and coagulation were measured under static and arterial flow conditions by state-of-the-art microscopic and physiological techniques. Plaque TF, plaque collagen, FXIIa, and platelet glycoprotein VI (GPVI) were specifically inhibited.. Plaques induced thrombus formation by 2 discrete steps. The rapid first phase of GPVI-mediated platelet adhesion and aggregation onto plaque collagen occurred within 1 min. The second phase of coagulation started after a delay of >3 min with the formation of thrombin and fibrin, and was driven entirely by plaque TF. Coagulation occurred only in flow niches provided by platelet aggregates, with no evidence for a role of blood-borne TF and FXIIa. Inhibition of GPVI but not plaque TF inhibited plaque-induced thrombus formation.. The major thrombogenic plaque components--collagen and TF--induce platelet activation and coagulation, respectively, in 2 consecutive steps. Targeting specifically the first step is crucial and might be sufficient to inhibit atherothrombus formation.

Topics: Atherosclerosis; Collagen; Female; Fibrin; Humans; Male; Platelet Activation; Platelet Membrane Glycoproteins; Thrombin; Thromboplastin; Thrombosis

Although recent studies have suggested a role for the receptor activator of nuclear factor κB ligand (RANKL) in the late stages of atherosclerosis (eg, plaque destabilization and rupture), the underlying mechanisms and subsequent events are unclear.. Because blood clotting is common after plaque rupture, we hypothesized that RANKL influenced tissue factor (TF) expression and activity to initiate the coagulation cascade.. RANKL increased the TF mRNA level and procoagulant activity in macrophages, as determined by semiquantitative reverse transcription polymerase chain reaction (semiquantitative RT-PCR) and a chromogenic assay. TF promoter analysis revealed that AP-1 and Egr-1 are responsible for RANKL-induced TF transcription. In addition, RANKL increased phosphorylation of c-Jun NH(2)-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK)1/2. RANKL-induced TF expression was attenuated by JNK- and MEK1-specific inhibitors and by small interfering RNA knockdown of c-Jun and Egr-1.. Our results indicate that RANKL induces TF in macrophages mainly through the cooperative action of AP-1 and Egr-1 via JNK and ERK1/2 pathways. These findings provide strong mechanistic support for the role of RANKL in the thrombogenicity of atherosclerotic plaques.

Topics: Animals; Atherosclerosis; Cell Line; DNA, Single-Stranded; Extracellular Signal-Regulated MAP Kinases; Humans; JNK Mitogen-Activated Protein Kinases; Macrophages, Peritoneal; MAP Kinase Signaling System; Mice; Phosphorylation; RANK Ligand; Risk Factors; RNA, Messenger; RNA, Small Interfering; Thromboplastin; Thrombosis; Transcription, Genetic; Up-Regulation

Thrombin generation in vivo may be important in regulating atherosclerotic progression. In the present study, we examined for the first time the activity and presence of relevant coagulation proteins in relation to the progression of atherosclerosis.. Both early and stable advanced atherosclerotic lesions were collected pairwise from each individual (n=27) during autopsy. Tissue homogenates were prepared from both total plaques and isolated plaque layers, in which the activity of factors (F) II, X, and XII and tissue factor was determined. Microarray analysis was implemented to elucidate local messenger RNA synthesis of coagulation proteins. Part of each specimen was paraffin embedded, and histological sections were immunohistochemically stained for multiple coagulation markers with the use of commercial antibodies. Data are expressed as median (interquartile range [IQR]). Tissue factor, FII, FX, and FXII activities were significantly higher in early atherosclerotic lesions than in stable advanced atherosclerotic lesions. Endogenous thrombin potential and thrombin-antithrombin complex values consolidated a procoagulant profile of early atherosclerotic lesions (endogenous thrombin potential, 1240 nmol/L x min [IQR, 1173 to 1311]; thrombin-antithrombin complex, 1045 ng/mg [IQR, 842.6 to 1376]) versus stable advanced atherosclerotic lesions (endogenous thrombin potential, 782 nmol/L x min [IQR, 0 to 1151]; thrombin-antithrombin complex, 718.4 ng/mg [IQR, 508.6 to 1151]). Tissue factor, FVII, and FX colocalized with macrophages and smooth muscle cells. In addition, multiple procoagulant and anticoagulant proteases were immunohistochemically mapped to various locations throughout the atherosclerotic vessel wall in both early and advanced atherosclerotic stages.. This study shows an enhanced procoagulant state of early-stage atherosclerotic plaques compared with advanced-stage plaques, which may provide novel insights into the role of coagulation during atherosclerotic plaque progression.

Topics: Aged; Aged, 80 and over; Atherosclerosis; Blood Coagulation; Blood Coagulation Factors; Factor VII; Factor X; Factor XII; Female; Humans; Immunohistochemistry; Lipoproteins; Male; Middle Aged; Thrombin; Thromboplastin

We recently demonstrated in an experimental model the expression of tissue factor (TF) in aortic valves. Thrombin, generated at the end of the TF-initiated coagulation cascade, has been shown to cleave the anti-calcific osteopontin (OSP) liberating the pro-inflammatory OSP N-half.. We hypothesized that TF might play an important role in calcific aortic valve stenosis (AS) through thrombin generation and hence evaluated the valvular expression of TF and its inhibitor (TF pathway inhibitor), α-thrombin, OSP and its thrombin-cleaved form (OSP N-half).. Calcified aortic valves were obtained from patients undergoing valve replacement. Protein expression was evaluated by immunostaining and measured using ELISA kits. Transcripts were analyzed by RT-PCR.. We included 52 patients (31 men; age 70 ± 10 years; aortic valve area index 0.35 ± 0.13 cm(2)/m(2)). Immunohistochemistry revealed that TF, OSP and α-thrombin expressions were associated with calcifications at the aortic side of the leaflets. There was an overexpression in calcified regions as compared to non-calcified zones of TF (733.3 ± 70.5 pg/mg vs. 429.4 ± 73.2 pg/mg; p<0.0001), OSP (88.9 ± 12.7 ng/mg vs. 15.0 ± 3.3 ng/mg; p<0.0001) and OSP N-half (0.41 ± 0.06 pmol/mg vs. 0.056 ± 0.011 pmol/mg; p<0.0001). Additionally, both TF and α-thrombin expressions were associated with OSP N-half (r=0.52; p<0.0001 and r=0.33; p=0.019, respectively).. Aortic leaflet TF and α-thrombin expressions and their association with the thrombin-cleaved form of OSP, are a new and important feature of AS. We hypothesize that TF may be involved in the mineralization process of aortic valves by enhancing the generation of the pro-inflammatory OSP N-half through thrombin induction. This pathway deserves further studies to address its implication in the aortic valve calcification process.

Topics: Aged; Aged, 80 and over; Aortic Valve; Aortic Valve Stenosis; Atherosclerosis; Calcinosis; Female; Gene Expression Profiling; Humans; Male; Middle Aged; Osteopontin; Thrombin; Thromboplastin

Tissue factor (TF), a transmembrane glycoprotein that acts as an essential cofactor to factor VII/VIIa, initiates the exogenous blood coagulation cascade leading to thrombin generation and subsequent thrombus formation in vivo. TF expression is closely related to plaque vulnerability, and high TF expression is shown in macrophage-rich atheromatous lesions, making TF a potential target for detecting atheromatous lesions in vivo. Thus, we prepared (99m)Tc-labeled anti-TF-monoclonal antibody (TF-mAb) IgG as a molecular probe and evaluated its usefulness to achieve TF-specific imaging using myocardial infarction-prone Watanabe heritable hyperlipidemic (WHHLMI) rabbits.. Anti-TF-mAb was created using a standard hybridoma technique and was labeled by (99m)Tc with 6-hydrazinonicotinic acid (HYNIC) as a chelating agent to obtain (99m)Tc-TF-mAb. The immunoreactivity of HYNIC-TF-mAb was estimated by flow cytometry. WHHLMI and control rabbits were injected intravenously with (99m)Tc-TF-mAb. Twenty-four hours after the injection, the aorta was removed and radioactivity was measured. Autoradiography and histologic studies were performed using serial aorta sections. Subclass matched antibody (IgG(1)) was used as a negative control.. HYNIC-TF-mAb showed 93% immunoreactivity of the anti-TF-mAb. The radioactivity accumulation in WHHLMI aortas was 6.1-fold higher than that of control rabbits. Autoradiograms showed a heterogeneous distribution of radioactivity in the intima of WHHLMI aortas. Regional radioactivity accumulation was positively correlated with TF expression density (R = 0.64, P < 0.0001). The highest radioactivity accumulation in percentage injected dose × body weight/mm(2) × 10(2) was found in atheromatous lesions (5.2 ± 1.9) followed by fibroatheromatous (2.1 ± 0.7), collagen-rich (1.8 ± 0.7), and neointimal lesions (1.8 ± 0.6). In contrast, (99m)Tc-IgG(1) showed low radioactivity accumulation in WHHLMI aortas that was independent of the histologic grade of lesions.. The TF-detecting ability and preferential accumulation in atheromatous lesions of (99m)Tc-TF-mAb were demonstrated, indicating its potential for selective imaging of macrophage-rich atheromatous lesions in vivo.

Topics: Animals; Antibodies, Monoclonal; Atherosclerosis; Autoradiography; Flow Cytometry; Hydrazines; Hyperlipidemias; Immunoglobulin G; Male; Molecular Probes; Nicotinic Acids; Rabbits; Radionuclide Imaging; Radiopharmaceuticals; Regression Analysis; Technetium Compounds; Thromboplastin; Tissue Distribution

Serum C-reactive protein (CRP) is a strong risk predictor of cardiovascular events, and tissue factor (TF) plays a central role in thrombus formation of advanced atherosclerotic plaques. Aim of the present study was to quantify in situ CRP and TF in coronary atherectomy specimens associated with acute coronary syndromes (ACS) or stable angina (SA). In addition, the effect of statin treatment on both intimal determinants was analyzed.. Serial sections from atherectomy probes retrieved from coronary primary target lesions of 42 ACS and 70 SA patients were examined for CRP and TF expression using immunostaining. CRP and TF intimal expression was consistently higher in ACS lesions and a positive correlation between both determinants was detected. In both subgroups intimal staining intensity of CRP but not TF was strongly associated with serum CRP levels. Using angioscopy, complex plaques revealed a higher intimal CRP and TF expression than white/yellow plaques. Both CRP and TF were consistently lower expressed in target lesions of patients with pre-existing statin treatment.. CRP and TF expression is markedly increased in plaques derived from patients with ACS as compared to SA patients. Statin treatment appears to reduce vascular expression of CRP and TF.

Topics: Acute Coronary Syndrome; Aged; Angina Pectoris; Angioscopy; Atherectomy, Coronary; Atherosclerosis; C-Reactive Protein; Female; Gene Expression Regulation; Humans; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Male; Middle Aged; Thromboplastin; Thrombosis

In the ApoE(-/-) mouse model of atherosclerosis (AS) stable plaque, the expression and location of intracellular tissue factor (TF) in the cellular components of AS stable plaque were investigated in order to explore the cellular mechanism of AS thrombosis. Pathological changes of the stable plaque were observed under a microscope. The expression of TF protein was examined in aortic stable plaque of mice by using immunohistochemistry. Color image planimetric system was used to analyze the histological components of the stable plaque and the TF distribution. Under the confocal microscope, the intracellular TF location in the stable plaque of mice was observed. The results showed the cellular area was the major part of stable plaque (67.36%+/-6.52%, P<0.01). The percentage of total area occupied by cellular area was significantly larger than atheromatous gruel and acellular area (P<0.01). Macrophages and smooth muscle cells (SMC) were major cells in the cellular area. The percentage of total area occupied by SMC was significantly larger than by macrophages (P<0.01). Multiple linear regression analysis showed there was a positive correlation between TF area and SMC area (r=0.616, P=0.008), and no correlation was found between TF area and macrophage area (r=0.437, P=0.08). Pictures of color image planimetric analysis of TF and SMC were merged to highlight areas with co-localization (yellow), it was concluded that the process could be a cell-mediated TF expression in the stable plaque. SMC may be the major source of TF in AS without plaque rupture.

Topics: Animals; Aorta; Apolipoproteins E; Atherosclerosis; Gene Knockdown Techniques; Linear Models; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Muscle, Smooth; Thromboplastin

Activated blood monocytes are a major source of tissue factor (TF), the principal initiator of blood coagulation. TF can be shed from the monocyte surface in association with microparticles (MPs) and increased numbers of circulating MPs are indicative of poor clinical outcome in a number of inflammatory disorders, including atherosclerosis. The mechanisms coupling inflammation with aberrant TF production/activity remain obscure but the protease-activated receptor (PAR) family has been implicated. We have previously shown (i) that freshly isolated human monocytes express low levels of cell surface PAR-2, (ii) that cell surface PAR-2 is rapidly upregulated from intracellular stores following mechanical stimulation, and (iii) that PAR-2 stimulation results in elevation of intracellular calcium and cytokine release. Here, we have investigated the expression of PAR-2 on monocytes exposed to TNF-activated endothelial cells both under static conditions and in our newly-established model of arterial flow, using diluted whole blood. Monocyte surface PAR-2 expression was upregulated following static exposure to activated EC and with laminar (atheroprotective) arterial flow there was a further increase in monocyte PAR-2 expression. We have also shown under arterial flow conditions that exposure to TNF-stimulated EC resulted in a significant increase in expression of TF on monocytes compared to that on cells exposed to quiescent EC. These data strongly suggest that direct or indirect interactions with inflamed EC can modulate expression of PAR-2 and TF on the monocyte cell surface.

Topics: Adult; Atherosclerosis; Blood Flow Velocity; Calcium; Cells, Cultured; Endothelium, Vascular; Female; Humans; Male; Models, Cardiovascular; Monocytes; Receptor, PAR-2; Thromboplastin; Tumor Necrosis Factor-alpha; Up-Regulation

Plaque rupture and subsequent embolism as well as thrombosis are major causes of acute myocardial infarction and stroke secondary to atherosclerosis. Pai-1, t-PA, TF and ET-1 are thrombosis- and thrombolysis-related factors which play important roles in thrombosis formation and plaque rupture. Since acute myocardial infarction and stroke are more likely to occur between 6 a.m. and 12 p.m. than at another time of the day, we studied the relationship between circadian rhythm and Pai-1, t-PA, TF and ET-1 in normal and atherosclerotic mice. Atherosclerosis was developed in apoE-/- mice fed a normal diet or a high cholesterol diet. The expression of Pai-1, t-PA, TF and ET-1 in the hearts of control C57BL/6J mice and atherosclerotic mice was measured by real-time RT-PCR at different Zeitgeber times (ZT) including ZT0, ZT4, ZT8, ZT10, ZT12, ZT14, ZT16 and ZT20. The expression of Pai-1, t-PA, TF and ET-1 peaked between ZT14 and ZT16 and bottomed at ZT10 in C57BL/6J mice. Their expression in apoE-/- mice fed a normal diet lost circadian rhythm. Their expression in apoE-/- mice fed a high cholesterol diet peaked at ZT4, indicating a reverse circadian rhythm. Our result indicates that circadian changes in the expression of Pai-1, t-PA, TF and ET-1 may be involved in the onset of myocardial infarction and stroke.

Topics: Animals; Apolipoproteins E; Atherosclerosis; Cholesterol, Dietary; Chronobiology Disorders; Circadian Rhythm; Endothelin-1; Gene Expression; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Myocardium; Plasminogen Activator Inhibitor 1; Thromboplastin; Thrombosis; Tissue Plasminogen Activator

Despite increasing in vitro evidence that lectin-like oxidized low density lipoprotein (LDL) receptor-1 (LOX-1), a cell-surface receptor for oxidized LDL, is implicated in the atherogenesis and thrombus formation, its in vivo participation to the atherosclerotic plaque destabilization, rupture and thrombus formation remains unclear. Here, we compared the in vivo expression of LOX-1, with tissue factor (TF) expression and cell apoptosis, in atherosclerotic lesions of myocardial infarction-prone Watanabe heritable hyperlipidemic (WHHLMI) rabbits.. We prepared sixty series of cross sections in the aortic arch and the thoracic aorta from four WHHLMI rabbits. LOX-1 and TF expression, as well as apoptotic events were determined by immunohistochemical staining and TUNEL methods, respectively. LOX-1 expression was mainly observed in the macrophage-rich lipid areas of vulnerable plaque-like atheromatous lesions where TF expression and apoptotic events were prominent. LOX-1 expression was positively correlated with TF expression (r=0.53, p<0.0001), apoptotic events (r=0.52, p<0.0001) and morphological vulnerability (r=0.63, p<0.0001).. LOX-1 expression appears to be closely associated with TF expression, apoptotic events and the morphological vulnerability, suggesting the in vivo involvement of LOX-1 in the destabilization and rupture of atherosclerotic lesions and the subsequent thrombus formation. The present findings in hypercholesterolemic rabbits should help advance our understanding of the pathophysiology of atherosclerosis.

Topics: Animals; Aorta, Thoracic; Apoptosis; Atherosclerosis; Female; Hypercholesterolemia; Immunohistochemistry; In Situ Nick-End Labeling; Macrophages; Rabbits; Receptors, LDL; Thromboplastin

Thrombus formation plays a critical role in pathogenesis of cardiovascular complications in atherosclerotic peripheral arterial occlusive disease (PAOD). Tissue factor (TF) initiates the clotting cascade and is considered an important regulator of hemostasis and thrombosis. TF activity is regulated by TF pathway inhibitor (TFPI). The aim of our study was to evaluate plasma levels of the TF, TFPI and their relation to coagulation system and various other risk factors of atherosclerosis in patients with chronic limbs ischemia.. Plasma TF, total TFPI, truncated TFPI, full-length TFPI were assessed by ELISA using commercially available kits (IMUBIND Tissue Factor; Total TFPI; Truncated TFPI ELISA Kit; American Diagnostica Inc. Stamford) in 62 claudicant patients with PAOD and 20 healthy controls.. We observed statistically higher levels of TF (94+/-52 pg/mL), total TFPI (43+/-8 ng/mL), and truncated TFPI (22+/-7 ng/mL) in patients with PAOD compared to healthy individuals (TF: 66+/-15 pg/mL; total TFPI: 36+/-4 ng/mL; truncated TFPI: 14+/-5 ng/mL). Full-length TFPI (20+/-4 ng/mL) is lower in patients with PAOD than in controls (23+/-5 ng/mL). The study indicated a positive correlation between TF and truncated TFPI (r=0.34), total TFPI and full TFPI (r=0.5), total TFPI and truncated TFPI (r=0.83) in patients with PAOD, and negative correlation between full TFPI and truncated TFPI (r=-0.65) in the control.. Elevated levels of TF, disorders of balance between full-length TFPI and truncated TFPI as well as significantly increased truncated TFPI level in patients with PAOD can be independent risk factors of atherosclerotic complications.

Topics: Arterial Occlusive Diseases; Atherosclerosis; Biomarkers; Blood Coagulation; Case-Control Studies; Female; Humans; Ischemia; Lipoproteins; Lower Extremity; Male; Middle Aged; Peripheral Vascular Diseases; Pilot Projects; Risk Assessment; Risk Factors; Thromboplastin; Up-Regulation

Decreased heart rate variability (HRV) has been associated with an increased risk of atherosclerosis. We hypothesized that a decrease in frequency domains of resting HRV would be associated with elevated plasma levels of interleukin (IL)-6 and soluble tissue factor (sTF) both previously shown to prospectively predict atherothrombotic events in healthy subjects. Subjects were 102 healthy and unmedicated black and white middle-aged men and women. We determined IL-6 and sTF antigen in plasma and HRV measures from surface electrocardiogram data using spectral analysis. All statistical analyses controlled for age, gender, ethnicity, smoking status, blood pressure, and body mass index. Low amounts of low frequency (LF) power (beta=-0.31, p=0.007) and high frequency (HF) power (beta=-0.36, p=0.002) were associated with increased amounts of IL-6, explaining 7% and 9% of the variance, respectively. Interactions between LF power and IL-6 (p=0.002) and between HF power and IL-6 (p=0.012) explained 8% and 5%, respectively, of the variance in sTF. Post hoc analyses showed associations between IL-6 and sTF when LF power (beta=0.51, p<0.001) and HF power (beta=0.48, p<0.001) were low but not when LF power and high HF power were high. The findings suggest that systemic low-grade inflammatory activity is associated with a decrease in HRV. Furthermore, there was a positive relationship between plasma levels of IL-6 and sTF antigen when HRV was low. Inflammation and related hypercoagulability might particularly contribute to atherothrombotic events in a setting of decreased HRV.

Topics: Adult; Atherosclerosis; Autonomic Nervous System; Blood Pressure; Female; Heart Rate; Humans; Interleukin-6; Male; Middle Aged; Predictive Value of Tests; Regression, Psychology; Risk Factors; Smoking; Solubility; Thromboplastin; Vagus Nerve

Recent studies have suggested a link between inhaled particulate matter (PM) exposure and atherogenesis. We investigated tissue factor (TF) expression with ambient fine particulate matter (diameter < 2.5 microm, PM(2.5)) exposure and in response to in vitro exposure to fine and ultrafine PM in cultured human bronchial epithelial cells, vascular smooth muscle cells (hSMCs), and monocytes. ApoE-/- mice, fed with normal chow (NC) or high-fat chow (HFC), were exposed to concentrated PM(2.5) or filtered air (FA) for 6 mo (6 h/day, 5 day/wk, n = 28). Following in vivo ultrasound bio-microscopy (UBM) assessment of plaque area, macrophage infiltration (CD68) and TF expression in the aorta were quantified. Cultured cells were incubated with size-fractionated PM from cascade impactors, or with standard reference PM material (SRM, number 1649a) and assayed for TF protein, mRNA, and activity. UBM-derived plaque areas were 7 +/- 1% larger in the PM(2.5)-HFC than the FA-HFC group (p = .04), but not significantly different between the PM(2.5)-NC and FA-NC groups (p = .07). Immunohistochemistry revealed increased TF (15 +/- 3% vs. 8 +/- 2%, p < .01) and macrophage infiltration (19 +/- 2% vs. 14 +/- 3%, p < .01) in the plaques of PM(2.5)-HFC compared with FA-HFC groups. Impactor-collected PM(2.5) and ultrafine particles consistently increased TF protein in bronchial epithelial cells, monocytes, and hSMCs. TF mRNA expression increased rapidly (within 1 h) in response to SRM PM. We conclude that in vivo and in vitro exposure to ambient air PM(2.5) induces TF expression.

Topics: Air Pollution; Animals; Apolipoproteins E; Atherosclerosis; Bronchi; Cells, Cultured; Disease Models, Animal; Dose-Response Relationship, Drug; Environmental Exposure; Epithelial Cells; Gene Expression Regulation; Gene Silencing; Humans; Image Processing, Computer-Assisted; Inhalation Exposure; Male; Mice; Mice, Knockout; Monocytes; Muscle, Smooth, Vascular; Particulate Matter; RNA, Messenger; Thromboplastin; Ultrasonography

Posttraumatic stress disorder (PTSD) confers an increased cardiovascular risk. In 14 otherwise healthy patients with PTSD and in 14 age- and gender-matched non-PTSD controls, we investigated whether the categorical diagnosis of PTSD and severity of PTSD symptom clusters (i.e. re-experiencing, avoidance, arousal, and overall score) would be associated with plasma concentrations of three markers of endothelial dysfunction [soluble tissue factor (sTF), von Willebrand factor (VWF), and soluble intercellular adhesion molecule (sICAM)-1]. Compared with controls, patients had significantly higher sTF; this difference became nonsignificant when controlling for psychological distress. VWF and sICAM-1 levels were not significantly different between patients and controls. In the entire sample virtually all PTSD symptom clusters correlated significantly and positively with sTF and VWF but not with sICAM-1. The correlation between symptoms of re-experiencing and sTF was significantly different between patients and controls. Controlling for symptoms of anxiety and depression (i.e. psychological distress) rendered most associations between PTSD symptom clusters and sTF nonsignificant, whereas controlling for age retained significance of associations with VWF. Posttraumatic stress showed a continuous relationship with sTF and VWF, with the former relationship being partly affected by psychological distress. This suggests one mechanism by which posttraumatic stress could contribute to atherosclerosis.

Topics: Adult; Age Factors; Atherosclerosis; Biomarkers; Cluster Analysis; Control Groups; Endothelium, Vascular; Female; Humans; Intercellular Adhesion Molecule-1; Life Change Events; Life Style; Male; Psychiatric Status Rating Scales; Risk Factors; Severity of Illness Index; Stress Disorders, Post-Traumatic; Stress, Psychological; Thromboplastin; von Willebrand Factor

The resistance of internal mammary artery (IMA) toward thrombotic occlusion and accelerated atherosclerosis is not well understood. This study analyzed gene expression profiles of vascular smooth muscle cells (VSMCs) from IMA versus saphenous vein (SV).. 54'675 probe sets were examined by Affymetrix microarrays. Thirty-one genes belonged to the coagulation system; 2 were differentially expressed, namely tissue factor (TF) and tissue-type plasminogen activator (tPA). TF was 3.1-fold lower in IMA than SV (P=0.006), whereas tPA was 9.0-fold higher (P<0.001). TF mRNA expression was lower in IMA than SV (P<0.05); tPA was higher (P<0.001). TF protein expression was 4.2+/-0.5-fold lower in IMA than SV (P<0.001); tPA was 2.6+/-0.4-fold higher (P<0.01). In IMA VSMC supernatant, TF protein and activity was lower (P<0.05), TFPI and tPA protein higher (P<0.05 and P<0.005), and clotting time of human plasma prolonged (P<0.05) as compared to SV. Migration to TF/FVIIa (10(-9) mol/L) was 3-fold lower in IMA than SV (P=0.01); PAR-2 protein expression was similar (P=NS), PAR-2 blockade without effect (P=NS).. Among the genes of the coagulation system, TF and tPA are differentially expressed in VSMCs from IMA versus SV. This is consistent with protection of IMA from thrombus formation and vascular remodeling.

Topics: Atherosclerosis; Blood Coagulation; Coronary Artery Bypass; Gene Expression Profiling; Graft Occlusion, Vascular; Humans; Lipoproteins; Mammary Arteries; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Oligonucleotide Array Sequence Analysis; Plasminogen Activator Inhibitor 1; RNA, Messenger; Saphenous Vein; Thromboplastin; Thrombosis; Tissue Distribution; Tissue Plasminogen Activator; Transplantation, Autologous

Patients with obstructive sleep apnea (OSA) are at increased risk of atherothrombosis independent of the Framingham risk factors. Studies on hemostasis factors in OSA are scarce and inconsistent. We sought to understand the variation in atherothrombotic propensity as a function of apoptotic circulating endothelial cells (CECs) in OSA by investigating the relationship between CEC apoptosis and plasma levels of hemostatic factors tissue factor (TF) and von Willebrand Factor (vWF) in apneic subjects. Apoptotic CECs were detected by flow cytometry in 35 male subjects free of cardiovascular diseases (AHI range 8-43) and 12 healthy male controls (AHI range 2-5) before and after 8 weeks of nasal continuous positive airway pressure (nCPAP). Quantitative determination of TF and vWF was performed using an enzyme-linked immunosorbent assay (ELISA) kit. The mean levels of TF (66.78 +/- 41.59 pg/ml) and vWF (189.70 +/- 69.24 IU/dl) were significantly higher in OSA patients compared with those in healthy subjects (42.83 +/- 14.18 pg/ml; and 124.48 +/- 31.43 IU/dl). Apoptotic CECs were elevated in patients with OSA and correlated strongly with TF and vWF levels (p = 0.02 and p < 0.001; respectively). There were no correlations between TF, vWF and apnea hypopnea index, or arousal index. Only the percentage of time spent <90% oxygen saturation was inversely associated with TF (r = 0.38; p = 0.02). Following nCPAP therapy, there was significant decrease in TF levels that correlated with decrease in apoptotic CECs. In patients with OSA, increased prothrombotic factors are strongly determined by apoptotic CECs. Treatment with nCPAP may alleviate the coagulation propensity.

Topics: Adult; Apoptosis; Atherosclerosis; Continuous Positive Airway Pressure; Endothelium, Vascular; Enzyme-Linked Immunosorbent Assay; Hemostasis; Humans; Male; Oxygen; Polysomnography; Reference Values; Risk Factors; Sleep Apnea, Obstructive; Thrombophilia; Thromboplastin; Thrombosis; von Willebrand Factor

We have recently demonstrated that tissue factor (TF) expression increases in adipose tissues/adipocytes of cholesterol-fed rabbit, which is associated with a hypercoagulable state that contributes to thrombosis. In this study, we evaluated the ability of atorvastatin to modulate TF expression in cholesterol-fed rabbit and the regulatory mechanism.. Male rabbits were randomly fed with normal diet and high-cholesterol diet for 8 weeks, following 4 weeks, those fed high-cholesterol diet were randomly assigned to atorvastatin or starch. At the end of 12 weeks, subcutaneous adipose was collected, and culture adipocyte. TF mRNA was detected by reverse transcription-polymerase chain reaction (RT-PCR). TF concentrations were determined with ELISA. The in vitro effect of atorvastatin and mevalonate (MVA) on TF production in adipocytes was observed.. Atorvastatin reduced serum TC and LDL-C levels (P<0.05), and decreased plasma TF concentration and expression in adipose tissues/adipocytes from cholesterol-fed rabbits. In vitro, atorvastatin dose-dependently suppressed TF expression and protein secretion in adipocytes. MVA reversed the inhibitory effect of atorvastatin on TF expression in concentration-dependent manner.. Results provide further support for the antithrombotic effect of atorvastatin. It also indicated that mevalonate pathway may play an important role in TF expression in adipocyte.

Topics: Animals; Atherosclerosis; Atorvastatin; Heptanoic Acids; Male; Pyrroles; Rabbits; Subcutaneous Fat; Thromboplastin

Biologically significant amounts of two procoagulant molecules, phosphatidylserine (PS) and tissue factor (TF), are transported by monocyte/macrophage-derived microvesicles (MVs). Because cellular cholesterol accumulation is an important feature of atherosclerotic vascular disease, we now examined effects of cholesterol enrichment on MV release from human monocytes and macrophages.. Cholesterol enrichment of human THP-1 monocytes, alone or in combination with lipopolysaccharide (LPS), tripled their total MV generation, as quantified by flow cytometry based on particle size and PS exposure. The subset of these MVs that were also TF-positive was likewise increased by cellular cholesterol enrichment, and these TF-positive MVs exhibited a striking 10-fold increase in procoagulant activity. Moreover, cholesterol enrichment of primary human monocyte-derived macrophages also increased their total as well as TF-positive MV release, and these TF-positive MVs exhibited a similar 10-fold increase in procoagulant activity. To explore the mechanisms of enhanced MV release, we found that cholesterol enrichment of monocytes caused PS exposure on the cell surface by as early as 2 hours and genomic DNA fragmentation in a minority of cells by 20 hours. Addition of a caspase inhibitor at the beginning of these incubations blunted both cholesterol-induced apoptosis and MV release.. Cholesterol enrichment of human monocyte/macrophages induces the generation of highly biologically active, PS-positive MVs, at least in part through induction of apoptosis. Cholesterol-induced monocyte/macrophage MVs, both TF-positive and TF-negative, may be novel contributors to atherothrombosis.

Topics: Apoptosis; Atherosclerosis; Blood Coagulation; Cell Membrane; Cells, Cultured; Cholesterol; Cytoplasmic Vesicles; Flow Cytometry; Humans; Lipopolysaccharides; Macrophages; Monocytes; Phosphatidylserines; Thromboplastin

Receptors for extracellular nucleotides are the focus of increasing attention for their ability to cause release of plasma membrane vesicles (microparticles, MPs). Here, we show that monocyte-derived human dendritic cells (DCs) stimulated with a P2X7 receptor (P2X7R) agonist undergo a large release of MPs endowed with procoagulant activity. Functional and Western blot studies revealed that MPs contain the membrane-bound form of tissue factor (TF), a glycoprotein acting as essential cofactor of activated factor VII and triggering blood coagulation. Quiescent DCs express the membrane-bound (full length), as well as truncated alternatively spliced TF forms. DC reactivity to anti-TF Abs disappeared almost completely on stimulation with ATP or benzoyl ATP (BzATP), as shown by immunoblot and confocal microscopy analysis. Concurrently, TF reactivity and activity appeared in the vesicular fraction, indicating that MPs are important carriers for the dissemination of full-length TF form. Activity of MP-bound TF, comparable to that of relipidated recombinant TF, was dose dependently inhibited by the addition of a specific anti-human TF antibody. We infer that a large fraction of this protein, and its procoagulant potential, are "deliverable" after physiological or pathological stimuli. These findings might have implications for triggering and propagating coagulation in healthy and atherosclerotic vessels.

Topics: Atherosclerosis; Blood Coagulation; Cell Membrane; Dendritic Cells; Humans; Particle Size; Receptors, Purinergic P2; Receptors, Purinergic P2X7; Thromboplastin

Inflammation contributes importantly to all stages of atherosclerosis, including the onset of acute thrombotic complications. In clinical trials, statins are beneficial in the primary and secondary prevention of coronary heart disease. Moreover, statins have been shown to possess several pleiotropic properties independent of cholesterol lowering in experimental settings. Based on these premises, we investigated the anti-inflammatory and anti-atherothrombotic properties of rosuvastatin in vivo, testing its effect on cholesterol and monocyte accumulation, and on adhesion molecules and tissue factor (TF) expression. ApoE-deficient female mice were fed a cholesterol-rich diet containing rosuvastatin (0, 1, 2 or 10 mg kg(-1)d(-1)) for 12 weeks. Treatment with rosuvastatin did not significantly affect either body weight gain or plasma total cholesterol (C) and triglyceride levels. However, rosuvastatin treatment dose-dependently reduced ICAM-1 expression in the aortic valves (V) (up to 40% inhibition, p<0.05) and in the proximal segment of the ascending aorta (AA) (-50%, p<0.001). Similarly, rosuvastatin inhibited VCAM-1 expression in the V (-40%) and in the AA (-35%, p<0.05). Moreover, there was a reduced accumulation of macrophages in the V in a dose-dependent and statistically significant manner (-45%, p<0.01). These anti-inflammatory effects were reflected in a reduction of cholesterol deposition in the entire aorta, both in the free and in the esterified form. Finally, the expression of tissue factor, the most potent pro-thrombogenic agent, was consistently reduced in AA by rosuvastatin treatment (-71%, p<0.001). Altogether, these data demonstrate that rosuvastatin has anti-inflammatory and anti-atherothrombotic activities in apoE-deficient mice that could translate in a beneficial effect on atherogenesis.

Topics: Animals; Anti-Inflammatory Agents; Aorta; Aortic Valve; Apolipoproteins E; Atherosclerosis; Cardiovascular Agents; Cholesterol; Cholesterol, Dietary; Disease Models, Animal; Dose-Response Relationship, Drug; Female; Fluorobenzenes; Intercellular Adhesion Molecule-1; Macrophages; Mice; Mice, Knockout; Pyrimidines; Rosuvastatin Calcium; Sulfonamides; Thromboplastin; Time Factors; Vascular Cell Adhesion Molecule-1

Hypericin and pseudohypericin are polycyclic-phenolic structurally related compounds found in Hypericum perforatum L. (St John's wort). As hypericin has been found to bind to LDL one may assume that it can act as antioxidant of LDL lipid oxidation, a property which is of prophylactic/therapeutic interest regarding atherogenesis as LDL oxidation may play a pivotal role in the onset of atherosclerosis. Therefore, in the present paper hypericin, pseudohypericin and hyperforin, an other structurally unrelated constituent in St John's wort were tested in their ability to inhibit LDL oxidation. LDL was isolated by ultracentrifugation and oxidation was initiated either by transition metal ions (copper), tyrosyl radical (myeloperoxidase/hydrogen peroxide/tyrosine) or by endothelial cells (HUVEC). LDL modification was monitored by conjugated diene and malondialdehyde formation. The data show that all compounds (hypericin, pseudohypericin and hyperforin) at doses as low as 2.5 micromol/l are potent antioxidants in the LDL oxidation systems used. The results indicate that the derivatives found in Hypericum perforatum have possible antiatherogenic potential.

Topics: Anthracenes; Antidepressive Agents; Antioxidants; Atherosclerosis; Bridged Bicyclo Compounds; Cells, Cultured; Chromatography, High Pressure Liquid; Circular Dichroism; Depression; Drug Evaluation, Preclinical; Endothelial Cells; Humans; Hypericum; Lipoproteins, LDL; Malondialdehyde; Mass Spectrometry; Molecular Structure; Nonprescription Drugs; Oxidation-Reduction; Peroxidase; Perylene; Phloroglucinol; Phytotherapy; Protein Binding; Structure-Activity Relationship; Terpenes; Thiobarbituric Acid Reactive Substances; Thromboplastin; Tyrosine; Umbilical Veins

Advanced atherogenesis is characterized by the presence of markers of enhanced prothrombotic capacity, attenuated fibrinolysis, and by clinical conditions associated with defective coagulation. Diabetes may be associated with enhanced lesion instability and atherosclerotic plaque rupture. Plaques obtained from 206 patients undergoing carotid endarterectomy were divided into diabetic (type 2) and nondiabetic and analyzed by Western blotting and immunohistochemistry to detect tissue factor (TF), metalloproteinases (MMP)-2, -8, -9, and fibrin/fibrinogen related antigens, and in situ zymography to detect MMP activity. Plasma samples were quantified for TF procoagulant activity, C-reactive protein, fibrinogen and D-dimer. Diabetic and symptomatic patients with hypoechogenic plaques had increased plasma TF activity and D-dimer, compared with those with hyperechogenic plaques (p = 0.03, p = 0.007, respectively). Diabetic, symptomatic patients had higher plasma D-dimer levels than asymptomatic patients (p = 0.03). There was a significant correlation between intramural TF levels and D-dimer in diabetic patients with symptomatic disease (p = 0.001, r2 = 0.4). In diabetic patients, plasma fibrinogen levels were higher in patients with hypoechogenic plaques (p = 0.007). Diabetic patients with ulcerated plaques had higher plasma D-dimer and MMP-8 levels than those with fibrous plaques (p = 0.02, p = 0.01, respectively). This data suggests that currently available circulating markers may be clinically useful to select diabetic patients at higher risk of atherothrombosis. Increased procoagulant activity in diabetic patients may be linked to increased mural remodeling.

Topics: Aged; Atherosclerosis; Biomarkers; Carotid Artery Diseases; Diabetes Complications; Diabetes Mellitus; Endarterectomy, Carotid; Female; Fibrin Fibrinogen Degradation Products; Humans; Male; Matrix Metalloproteinase 2; Matrix Metalloproteinase 8; Matrix Metalloproteinase 9; Thromboplastin

Tissue factor (TF) is a transmembrane glycoprotein that binds its zymogen cofactor, Factor VIIa (FVIIa) on the cell surface. Together (TF/FVIIa) they activate Factor X (FX) and Factor IX (FIX) and start the extrinsic pathway of blood coagulation. As such, the TF/FVIIa complex plays an important role in normal physiology as well as in thrombotic diseases such as unstable angina (UA), disseminated intravascular coagulation (DIC), and deep vein thrombosis (DVT). In addition to its function as an initiator of coagulation, TF/FVIIa plays an important role in inflammation. Expression of TF on the cell surface and its appearance as a soluble molecule are characteristic features of acute and chronic inflammation in conditions such as sepsis and atherosclerosis. Here we demonstrate that BCX-3607, a small molecule potent inhibitor of TF/FVIIa, reduces thrombus weight in an animal model of DVT. BCX-3607 also decreases the level of interleukin-6 (IL-6) in a LPS-stimulated mouse model of endotoxemia. Additionally, in vitro studies indicate that BCX-3607 blocks the generation of TF/FVIIa-induced IL-8 mRNA in human keratinocytes and reduces the TF/FVIIa-mediated generation of IL-6 and IL-8 in human umbilical vein endothelial cells (HUVEC). Therefore, BCX-3607 might block the TF/FVIIa-mediated coagulation and inflammation associated with pathological conditions.

Topics: Animals; Anti-Inflammatory Agents; Atherosclerosis; Blotting, Northern; Cells, Cultured; Disease Models, Animal; Dose-Response Relationship, Drug; Endothelium, Vascular; Endotoxemia; Factor VIIa; Fibrinolytic Agents; Humans; Inflammation; Interleukin-6; Interleukin-8; Keratinocytes; Lipopolysaccharides; Male; Mice; Models, Biological; Models, Chemical; Prothrombin Time; Pyridines; Rats; Rats, Sprague-Dawley; RNA, Messenger; Sepsis; Thromboplastin; Time Factors

To determine whether tissue factor (TF) contributes to the progression of atherosclerotic lesions in mice.. We determined the effect of a 50% reduction of TF levels in all cells on atherosclerosis in apolipoprotein E-deficient (apoE(-/-)) mice. No differences were observed in the extent of atherosclerosis in apoE(-/-)/TF(+/+) and apoE(-/-)/TF(+/-) mice fed regular chow for 34 weeks. Atherosclerosis could not be analyzed in apoE(-/-) mice expressing low levels of TF because of premature death of these mice. Macrophages are a major source of TF in atherosclerotic plaques. Therefore, in a second series of experiments, we investigated the effect on atherosclerosis of selectively reducing hematopoietic cell-derived TF by transplanting bone marrow from mice expressing low levels of TF into low-density lipoprotein receptor deficient (LDLR(-/-)) mice. Atherosclerosis within the arterial tree and aortic root were similar in LDLR(-/-) mice with low-TF bone marrow compared with control bone marrow (TF(+/+) or TF(+/-)) after 4 and 16 weeks on an atherogenic diet. Furthermore, the cellular composition of the aortic root lesions was similar between the 2 groups.. Our data indicate that either a 50% reduction of TF in all cells or a selective reduction in hematopoietic cell-derived TF does not affect the development of atherosclerotic lesions in mice.

Topics: Animals; Aorta; Apolipoproteins E; Atherosclerosis; Bone Marrow Transplantation; Gene Expression; Hematopoietic Stem Cells; Mice; Mice, Inbred C57BL; Mice, Mutant Strains; Receptors, LDL; Thromboplastin

Obstructive sleep apnoea hypopnoea syndrome (OSAHS) is associated with increased morbidity and mortality due to cardiovascular disease. In order to examine the association between OSAHS and cardiovascular disease, this study measured hypoxia-inducible and atherosclerosis-associated molecules in the peripheral blood.. In this study peripheral blood was obtained early in the morning from 60 consecutive male patients with OSAHS (AHI > or =10 events/h) and 30 male control subjects without OSAHS (AHI <5 events/h). Serum levels of heat shock protein-70 (Hsp-70), tissue factor (TF), monocyte chemotactic protein-1 (MCP-1), highly sensitive C-reactive protein (hs-CRP), hepatocyte growth factor and plasma vascular endothelial growth factor were measured and their relationship with severity and hypoxaemia in OSAHS examined.. Serum hs-CRP, TF, MCP-1 and Hsp-70 levels were significantly higher in OSAHS compared with control subjects. Categorization of the patients into mild (10 < or = AHI < 30 events/h), moderate (30 < or = AHI < 60 events/h) and severe (AHI > or = 60 events/h) OSAHS subgroups showed that serum levels of hs-CRP, TF and Hsp-70 increased with severity. The hs-CRP, TF, MCP-1 and Hsp-70 levels in the non-obese OSAHS group were also significantly higher than those in the control group whereas there was no difference in BMI between the two groups. Repetitive hypoxaemia significantly correlated with hs-CRP, TF and Hsp-70 levels and appeared to be a significant determinant for these molecules.. These findings suggest that CRP, TF and Hsp-70 may be upregulated by repetitive hypoxaemia in OSAHS and may be involved in the development of the atherogenic process in OSAHS.

Topics: Analysis of Variance; Atherosclerosis; C-Reactive Protein; Case-Control Studies; HSP70 Heat-Shock Proteins; Humans; Hypoxia; Intercellular Signaling Peptides and Proteins; Linear Models; Male; Middle Aged; Polysomnography; Risk Factors; Severity of Illness Index; Sleep Apnea, Obstructive; Thromboplastin

Topics: Animals; Atherosclerosis; Humans; Neoplasms; Sepsis; Thromboplastin; Wounds and Injuries

Cocaine consumption can lead to myocardial infarction. Tissue factor (TF) has been implicated in acute coronary syndromes, and the balance of TF and tissue factor pathway inhibitor (TFPI) determines initiation of thrombus formation. This study was designed to investigate the effect of cocaine on endothelial TF and TFPI expression. Cocaine (10(-8)-10(-5) mol/l) increased thrombin-induced TF expression by 24% at 10(-7) mol/l (P < 0.001) without affecting basal TF expression. In contrast, cocaine reduced endothelial TFPI expression by 47% at 10(-7) mol/l (P < 0.01). Moreover, thrombin impaired endothelial TFPI expression, and cocaine (10(-8) mol/l) further reduced TFPI expression by 33% as compared to thrombin (P < 0.02). These effects occur at cocaine concentrations usually present in plasma of consumers. Given the importance of TF in the pathogenesis of acute coronary syndromes, TF induction in conjunction with TFPI suppression may be relevant for the increased frequency of myocardial infarction observed in cocaine consumers.

Topics: Acute Disease; Aorta; Atherosclerosis; Cells, Cultured; Cocaine; Endothelial Cells; Endothelium, Vascular; Gene Expression Regulation; Heart Diseases; Humans; Lipoproteins; Myocardial Infarction; Thrombin; Thromboplastin; Vasoconstrictor Agents

Systemic hypertension is one of the main risk factors for atherothrombosis. Tissue factor (TF) is found in the adventitia of blood vessels and in the lipid core of atherosclerotic plaques, and is specifically expressed on monocyte or macrophage cell membrane surfaces. TF plays a pivotal role in blood clotting physiology and is involved in pro-inflammatory action and atherosclerotic plaque destabilization.. In this study we investigated whether there is any relationship between TF messenger RNA expression and activity in blood monocytes isolated from hypertensive patients with clinical signs of atherosclerosis, uncomplicated hypertensive individuals and normotensive control subjects.. Eighty subjects (41 men and 39 women, mean age 41 +/- 12 years) with untreated essential hypertension and 41 control subjects matched for sex and age were enrolled in the study. Patients were classified according to whether they had a normal ( 1 mm, 39 patients) intima-media thickness (IMT).. TF mRNA expression and activity in hypertensive individuals with no carotid atherosclerosis were no different from control subjects in unstimulated and stimulated monocytes. Abnormal IMT patients showed a higher TF mRNA expression compared with normal IMT hypertensive subjects (P < 0.001).. We demonstrated that TF mRNA and activity levels in monocytes obtained from uncomplicated hypertensive individuals are comparable with those of normotensive subjects, whereas atherosclerotic hypertensive patients showed increased levels of these parameters.

Topics: Adult; Atherosclerosis; Blood Pressure; Case-Control Studies; Female; Gene Expression; Humans; Hypertension; Lipopolysaccharides; Male; Middle Aged; Monocytes; Multivariate Analysis; Regression Analysis; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Thromboplastin

In endothelial cells (EC), celecoxib inhibits expression of tissue factor (TF), a key protein for initiation and propagation of thrombus formation. The current study was designed to examine the effect of celecoxib on TF expression and activity in VSMC. In contrast to EC, celecoxib increased TNF-alpha-induced TF expression and surface activity in VSMC by 33% and 20%, respectively, as compared to TNF-alpha alone, while rofecoxib or NS-398 had no effect. Celecoxib increased p38 MAP kinase (p38), p44/42 MAP kinase (ERK), and p70S6 kinase (p70S6K) phosphorylation while leaving JNK activation unaffected. Simultaneous inhibition of p38 and ERK reduced TNF-alpha-induced TF expression by 59%, while inhibition of JNK with SP600125 did not affect TF expression. Thus, in contrast to endothelial cells, celecoxib does not inhibit TF expression in VSMC, but instead enhances it. As neither rofecoxib nor NS-398 affected TF expression, this effect does not seem to be related to COX-2 inhibition but rather appears to be mediated by an increase in p38, ERK, and p70S6K activation. The observation that the inhibiting effect of celecoxib on endothelial TF expression does not extend to VSMC may have important implications for patients with cardiovascular disease.

Topics: Anti-Inflammatory Agents, Non-Steroidal; Atherosclerosis; Celecoxib; Cell Line; Endothelium, Vascular; Gene Expression Regulation; Heart Diseases; Humans; Lactones; Muscle, Smooth, Vascular; Pyrazoles; Sulfonamides; Sulfones; Thromboplastin; Thrombosis; Transcription, Genetic; Tumor Necrosis Factor-alpha

To test the hypothesis that Grp78 negatively regulates cell surface tissue factor (TF) procoagulant activity and whether this is mediated by physical interaction.. Biopanning with phage-displayed peptidyl libraries has identified peptide probes that bind selectively in vivo to the surface of atherosclerotic plaque endothelium. The highest affinity peptide, EKO130, binds 78-kDa glucose regulated protein (Grp78). Grp78 participates in numerous pathological processes, including the regulation of the coagulation cascade, but the mechanism of Grp78 regulation of coagulation is unknown. To characterize this function, we analyzed the effect of Grp78 on TF-mediated procoagulant activity on murine brain endothelial cells (bEND.3) and macrophage-like (RAW) cells, which are relevant in mediation of atherothrombosis. We show that Grp78 is present on the surface of endothelium and monocyte/macrophage-like cells in atherosclerotic lesions. Inhibition of Grp78 resulted in increased procoagulant activity. We demonstrate that Grp78 negatively regulates procoagulant activity by interacting physically with the TF extracellular domain on the cell surface.. The evidence indicates that Grp78 negatively regulates TF functional activity via direct binding to and functional inhibition of TF. Identification of the mechanism by which Grp78 regulates TF function may advance insight into the pathobiology of atherosclerosis and associated arterial thrombosis.

Topics: Animals; Atherosclerosis; Blood Coagulation; Cells, Cultured; Cerebrovascular Circulation; Endoplasmic Reticulum Chaperone BiP; Endothelium, Vascular; Factor Xa; Foam Cells; Heat-Shock Proteins; Macrophages; Membrane Proteins; Mice; Molecular Chaperones; Thromboplastin; Thrombosis

Topics: Atherosclerosis; Endoplasmic Reticulum Chaperone BiP; Heat-Shock Proteins; Humans; Molecular Chaperones; Thromboplastin; Thrombosis

The oxidative modification of LDL may play an important role in the early events of atherogenesis. Thus the identification of antioxidative compounds may be of therapeutic and prophylactic importance regarding cardiovascular disease. Copper-chlorophyllin (Cu-CHL), a Cu(2+)-protoporphyrin IX complex, has been reported to inhibit lipid oxidation in biological membranes and liposomes. Hemin (Fe(3+)-protoporphyrin IX) has been shown to bind to LDL thereby inducing lipid peroxidation. As Cu-CHL has a similar structure as hemin, one may assume that Cu-CHL may compete with the hemin action on LDL. Therefore, in the present study Cu-CHL and the related compound magnesium-chlorophyllin (Mg-CHL) were examined in their ability to inhibit LDL oxidation initiated by hemin and other LDL oxidizing systems. LDL oxidation by hemin in presence of H(2)O(2) was strongly inhibited by both CHLs. Both chlorophyllins were also capable of effectively inhibiting LDL oxidation initiated by transition metal ions (Cu(2+)), human umbilical vein endothelial cells (HUVEC) and tyrosyl radicals generated by myeloperoxidase (MPO) in presence of H(2)O(2) and tyrosine. Cu- and Mg-CHL showed radical scavenging ability as demonstrated by the diphenylpicrylhydracylradical (DPPH)-radical assay and estimation of phenoxyl radical generated diphenyl (dityrosine) formation. As assessed by ultracentrifugation the chlorophyllins were found to bind to LDL (and HDL) in serum. The present study shows that copper chlorophyllin (Cu-CHL) and its magnesium analog could act as potent antagonists of atherogenic LDL modification induced by various oxidative stimuli. As inhibitory effects of the CHLs were found at concentrations as low as 1 mumol/l, which can be achieved in humans, the results may be physiologically/therapeutically relevant.

Topics: Atherosclerosis; Biphenyl Compounds; Cardiovascular Diseases; Catalysis; Cells, Cultured; Chlorophyll; Chlorophyllides; Copper; Dose-Response Relationship, Drug; Endothelium, Vascular; Free Radical Scavengers; Free Radicals; Hemin; Humans; Hydrazines; Hydrogen Peroxide; Ions; Iron; Lipid Peroxidation; Lipids; Lipoproteins; Lipoproteins, LDL; Magnesium; Malondialdehyde; Models, Chemical; Octanols; Oxygen; Picrates; Protoporphyrins; Pyrazoles; Pyrimidines; Thiobarbituric Acid Reactive Substances; Thromboplastin; Time Factors; Tyrosine; Umbilical Veins; Water

Topics: Administration, Intravenous; Atherosclerosis; Chlorides; Chlorpromazine; Dicumarol; Diet; Diet, Atherogenic; Pharmacology; Potassium; Prothrombin Time; Rats; Research; Thromboplastin; Thrombosis; Toxicology

Topics: Animals; Aorta; Arteriosclerosis; Atherosclerosis; Blood Coagulation; Cholesterol; Fibrinolysin; Heparin; Heparinoids; Plasminogen; Rabbits; Thrombolytic Therapy; Thromboplastin

Topics: Anticoagulants; Arteriosclerosis; Atherosclerosis; Blood Platelets; Fibrinolysis; Humans; Thromboplastin