Circulating Tissue Factor Levels and Risk of Stroke: Findings From the EPICOR Study.

BACKGROUND AND PURPOSE: Tissue factor (TF) expression is increased in inflammatory atherosclerotic plaques and has been related to their thrombogenicity. Blood-borne TF has been also demonstrated to contribute to thrombogenesis. However, few studies have evaluated the association of circulating levels of TF with stroke. We investigated the association of baseline circulating levels of TF with stroke events occurred in the European Prospective Investigation into Cancer and Nutrition-Italy cohort. METHODS: Using a nested case-cohort design, a center-stratified random sample of 839 subjects (66% women; age range, 35-71 years) was selected as subcohort and compared with 292 strokes in a mean follow-up of 9 years. Blood samples were collected at baseline in citrate, plasma was stored in liquid nitrogen and TF was measured by ELISA (IMUBIND, TF ELISA, Instrumentation Laboratory, Milan, Italy). The odd ratios and 95% confidence intervals, adjusted by relevant confounders (covariates of TF) and stratified by center, were estimated by a Cox regression model using Prentice method. RESULTS: Individuals in the highest compared with the lowest quartile of TF plasma levels had significantly increased risk of stroke (odds ratioIVvsI quartile, 2.01; 95% confidence interval, 1.25-3.23). The association was independent from several potential confounders (odds ratioIVvsI quartile, 1.91; 95% confidence interval, 1.15-3.19). No differences were observed between men and women. The increase in risk was restricted to ischemic strokes (odds ratioIVvsI quartile, 2.13; 95% confidence interval, 1.10-4.12; fully adjusted model), whereas high levels of TF were not associated with the risk of hemorrhagic stroke (odds ratioIVvsI quartile, 1.12; 95% confidence interval, 0.49-2.55; fully adjusted model). CONCLUSIONS: Our data provide evidence that elevated levels of circulating TF are potential risk factors for ischemic strokes.

Human peripheral blood endothelial progenitor cells synthesize and express functionally active tissue factor.

INTRODUCTION: Endothelial progenitor cells are circulating cells able to home to sites of vascular damage and to contribute to the revascularization of ischemic areas. We evaluated whether endothelial progenitor cells synthesize tissue factor, a procoagulant protein also involved in angiogenesis. MATERIALS AND METHODS: Endothelial progenitor cells were obtained from the peripheral blood mononuclear fraction of normal donors and cultured in endothelial medium supplemented with specific growth factors. The procoagulant activity expressed by cells disrupted by freeze-thaw cycles was assessed by a one stage clotting assay. Tissue factor mRNA expression was evaluated by RT-PCR. RESULTS: Endothelial progenitor cells do not express procoagulant activity in baseline conditions. However, lipopolysaccharide induces the expression of procoagulant activity. The effect is dose-dependent and reaches statistical significance at 100 ng/mL lipopolysaccharide. Inhibition with an anti-tissue factor antibody and amplification of cDNA with primers based on the tissue factor sequence confirm the identity of this activity with tissue factor. The kinetics of tissue factor expression by endothelial progenitor cells is identical to that of human umbilical vein endothelial cells showing maximal activity within 4 hours, and then decreasing; in contrast, tissue factor expression by mononuclear cells lasts for longer times. Both 5,6-dichloro-beta D-ribofuranosyl-benzimidazole and cycloheximide prevented the expression of procoagulant activity. Stimulation of endothelial progenitor cells with tumor necrosis factor-alpha did not elicit any detectable procoagulant activity. CONCLUSIONS: Endothelial progenitor cells can be stimulated by lipopolysaccharide to synthesize tissue factor. This protein might be involved in thrombotic phenomena and might contribute to endothelial progenitor cells related neovascularization.

Interleukin 1 gene cluster, myocardial infarction at young age and inflammatory response of human mononuclear cells.

The objective was to investigate whether genotypes, haplotypes and haplotype-pairs of interleukin (IL) gene cluster are associated with risk of Myocardial Infarction (MI) at young age and with the release of IL-1B and expression of tissue factor pro-coagulant activity (TFPCA), after stimulation in vitro with lipopolysaccharide (LPS) of human peripheral blood mononuclear cells (PBMCs). Patients with MI at young age, frequency-matched for age, sex and recruitment centre, with healthy population-based controls and PBMCs from healthy volunteers were studied. Five single nucleotide polymorphisms (SNPs), identifying two haplotype-blocks, in IL-1B gene and one SNP in IL-1A and IL-RA genes were genotyped. In multivariate analyses, haplotype A2 (122) and A4 (112) were associated with decreased risk of MI [OR = 0.62 (95% CI = 0.40-0.95), p = 0.01; OR = 0.69 (95% CI = 0.51-0.92), p = 0.03, respectively]. Haplotype-pair A2/A2 showed significant reduction in the risk of MI [OR = 0.38 (95% CI = 0.18-0.81); p = 0.01]. Haplotype A2 and A4 were associated with lower levels of IL-1B (respectively p = 0.01; p = 0.04, multivariate analysis) and haplotype-pair A2/A4 showed decreased levels of IL-1beta (p = 0.02). No association was found between block "B" IL-1B haplotypes or IL-1A and IL-RA polymorphisms and risk of MI. IL-1B haplotypes influence the inflammatory response of human mononuclear cells to LPS and affect the risk of MI at young age.

Prevention of cardiovascular disease in type-2 diabetes: how to improve the clinical efficacy of aspirin.

Atherosclerotic cardiovascular disease and its thrombotic complications are the principal causes of morbidity and mortality in patients with type-2 diabetes. Aspirin reduces the risk of thrombotic events in a broad range of patients with vascular disease and, in selected individuals, is beneficial for primary prevention. Although recommended by existing guidelines, in secondary and in primary prevention trials the clinical efficacy of low-dose aspirin in patients with diabetes appears to be substantially lower than in individuals without diabetes. In this review, we discuss possible mechanisms that may contribute to reduce the antithrombotic activity of aspirin in diabetes. We also discuss adjuvant therapies used in diabetic patients that may potentially improve the antithrombotic efficacy of aspirin.

Long pentraxin PTX3 upregulates tissue factor expression in human endothelial cells: a novel link between vascular inflammation and clotting activation.

Inflammation is a major contributing factor to atherosclerotic plaque development and ischemic heart disease. PTX3 is a long pentraxin that was recently found to be increased in patients with acute myocardial infarction. Because tissue factor (TF), the in vivo trigger of blood coagulation, plays a dominant role in thrombus formation after plaque rupture, we tested the possibility that PTX3 could modulate TF expression. Human umbilical vein endothelial cells, incubated with endotoxin (lipopolysaccharide) or the inflammatory cytokines interleukin-1beta and tumor necrosis factor-alpha, expressed TF. The presence of PTX3 increased TF activity and antigen severalfold in a dose-dependent fashion. PTX3 exerted its effect at the transcription level, inasmuch as the increased levels of TF mRNA, mediated by the stimuli, were enhanced in its presence. The increase in mRNA determined by PTX3 originated from an enhanced nuclear binding activity of the transacting factor c-Rel/p65, which was mediated by the agonists and measured by electrophoretic mobility shift assay. The mechanism underlying the increased c-Rel/p65 activity resided in an enhanced degradation of the c-Rel/p65 inhibitory protein IkappaBalpha. In the area of vascular injury, during the inflammatory response, cell-mediated fibrin deposition takes place. Our results suggest that PTX3, by increasing TF expression, potentially plays a role in thrombogenesis and ischemic vascular disease.

The long pentraxin PTX3 up-regulates tissue factor in activated monocytes: another link between inflammation and clotting activation.

Pentraxin-3 (PTX3), an acute-phase protein that belongs to the family of the PTXs, is found elevated in septic shock and increased in patients with acute myocardial infarction. As tissue factor (TF) plays a key role in thrombosis and inflammation associated with atherosclerosis and as we have recently reported that PTX3 increases TF synthesis in endothelial cells, we tested whether PTX3 could modulate TF expression in monocytes. Monocytes from peripheral blood of healthy donors were incubated with highly purified PTX3 with or without lipopolysaccharide (LPS). Cells were then disrupted, and procoagulant activity was assessed by a one-stage clotting time. PTX3 enhanced TF activity and antigen from LPS-stimulated monocytes in a dose-dependent way. The effect was specific, as other PTXs, such as C-reactive protein and serum amyloid P component, were ineffective. Moreover, the increase in activity was specific for LPS, as in the presence of other TF-inducing agents such as interleukin-1beta and tumor necrosis factor alpha, PTX3 was not effective. The increase in TF activity requires mRNA synthesis, as assessed by polymerase chain reaction. The mechanism by which PTX3 modulates TF synthesis resides in an enhanced IkappaB, alpha phosphorylation and degradation and increased migration of the transacting factor c-Rel/p65 into the nucleus, as determined by Western blot and electro-mobility shift assay. These results show that PTX3 is an enhancer of the expression of TF by mononuclear cells. In the area of vascular injury, during the inflammatory response, cell-mediated fibrin deposition takes place. PTX3 increases TF expression, thus potentially playing a role in thrombogenesis and wound healing.

Caffeic acid phenethyl ester blocks apoptosis induced by low potassium in cerebellar granule cells.

Primary cultures of cerebellar granule neurons (CGNs) were prepared from 8-day-old Wistar rats, and maintained in an appropriate medium containing a high (25 mM) concentration of KCl. To induce apoptosis, culture medium was replaced with serum-free medium (containing 5mM KCl) 8 days after plating. Apoptosis was measured by the terminal deoxynucleotidyl transferase-mediated dUTP-fluorescein nick end-labeling (TUNEL) method, and by flow cytometry. Since there is evidence that an increased formation of reactive oxygen species (ROS) is involved in the apoptosis induced by low K(+) (5mM) concentrations, the potential anti-apoptotic effect of caffeic acid phenethyl ester (CAPE), a potent flavonoid antioxidant, was tested in this experimental model. It was found that CAPE (10 microg/ml) promoted cell survival and was capable of blocking the apoptotic process as assayed by both TUNEL and flow cytometric methods. The same concentration of CAPE prevented the formation of ROS induced by low K(+). Since there is evidence that low K(+)-induced apoptosis in CGNs is associated with a drop in intracellular Ca(2+) concentration ([Ca(2+)](i)), activation of the cell death effector proteases caspase-3 and caspase-9, and of the transcription factor nuclear factor kappa B (NF-kappaB), the interference of CAPE with these purported mediators of apoptosis was also evaluated. It was found that CAPE did not interfere with the marked decrease in [Ca(2+)](i) induced by low K(+), whereas it completely blocked caspase-3, caspase-9, and NF-kappaB activation. It is concluded that CAPE could exert its anti-apoptotic effect in CGNs by blocking ROS formation and by inhibiting caspase activity.

Both red and blond orange juice intake decreases the procoagulant activity of whole blood in healthy volunteers.

AIM: Numerous epidemiological studies suggest that exposure to flavonoid-rich fruits has beneficial influence on risk factors for cardiovascular disease. We investigated whether intake of orange juice (OJ) could affect whole blood (WB) procoagulant activity. METHODS: 17 healthy subjects (aged 31 ± 1.5 SEM 10 males) were randomized to receive, according to a cross-over design, either red or blond OJ, enriched or free of anthocyanins, respectively. After one week run-in period on a controlled diet, the subjects were randomly allocated to receive either type of OJ for 4 weeks, with a 4-week wash-out period. Venous blood was collected on citrate before and at the end of each treatment period. WB was incubated with or without an inflammatory stimulus (tumor necrosis factor-α or bacterial endotoxin LPS). Procoagulant activity was evaluated by a one-stage clotting assay. Tissue factor (TF) and TF pathway inhibitor (TFPI) were measured in plasma by ELISA. RESULTS: Intake of either type of OJ caused a prolongation of unstimulated and stimulated WB clotting times, without any difference between the two treatments. Intake of OJ did not modify TF levels. On the contrary, an increase in circulating TFPI antigen was detected following either treatment. CONCLUSIONS: Orange juice intake by healthy volunteers decreases procoagulant activity, possibly through mechanisms independent of its anthocyanin content.

Thrombosis and cancer: 40 years of research.

We have taken here the task to go back to a brief history concerning the evolution of the concept of "cancer and thrombosis" according to the experience accumulated by our group in the last 40years. Since its first description by Armand Trousseau in 1865, the association between cancer and thrombosis only received attention again during the last decades of the XXth century: from scattered reports on experimental material (tumor extracts) or on animal models of tumor/metastasis growth, through the progress of cell biology and experimental pharmacology, the interest moved to clinical questions, such as: how to prevent and treat thrombosis, a frequent complication of both solid and hematologic malignancies? Has an occult cancer to be suspected in the majority of cases of idiopathic deep vein thrombosis? Do we need to prevent pharmacologically the occurrence of chemotherapy-associated thrombosis? Do anticoagulants have an impact on the natural history of some tumors? Why antiangiogenetic agents may be associated to a thrombotic risk? Presently, a continuous cross-talk between clinical results and experimental data is required to provide answers to these questions, taking advantage from a multidisciplinary approach to this still partially mysterious issue. If one would take a paradigm of the evolution of the subject during the past 40years, a symbol could be the knowledge accumulated on tissue factor, the "middleman" of the clotting cascade as well as of the interactions between thrombosis and cancer, as briefly reviewed at the end of this chapter.

Thrombosis and obesity: cellular bases.

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.

Inhibition of the renin-angiotensin system downregulates tissue factor and vascular endothelial growth factor in human breast carcinoma cells.

INTRODUCTION: The renin-angiotensin system (RAS) promotes angiogenesis and growth of neoplastic cells. Angiotensin-converting enzyme (ACE) inhibitors and angiotensin II receptor AT1 blockers may protect against cancer. Tissue factor (TF), for its involvement in tumor growth, angiogenesis, and metastasis is considered a hallmark of cancer progression. In this study we evaluated whether RAS blockade modulates TF constitutive expression by the metastatic breast carcinoma MDA-MB-231 cell line. MATERIALS AND METHODS: Cell TF activity was assessed by one stage clotting time, TF and VEGF antigens and mRNA levels by ELISA and RT-PCR, respectively. AT(1) was detected by flow-cytometry and angiotensin-II levels by EIA. RESULTS: Captopril reduced in a concentration-dependent way both the strong constitutive TF activity (983.2±55.2 vs. 686.7±135.1U/5×10(5) cells with 10µg/ml captopril) and antigen (32.3±5.9 vs. 13.2±6.6ng/ml) in MDA-MB-231. Similar results were observed with enalapril. AT1 was present on cell membrane and losartan, a competitive inhibitor of AT1, reduced TF expression to a degree similar as that exerted by ACE inhibitors. Moreover, captopril and losartan downregulated the constitutive mRNA TF expression by ~35%. Similar results were observed with anti-AT1 and angiotensin II antibodies. In addition, the constitutive VEGF antigen and mRNA levels were reduced in the presence of captopril or losartan, and an anti-VEGF antibody downregulated cell TF activity by ~40%. CONCLUSIONS: These results could, at least in part, contribute to the discussion about the possible effects of ACE inhibitors and AT1 receptor antagonists in malignancy, and offer new clues to support their use for tumor control.

Leptin upregulates tissue factor expression in human breast cancer MCF-7 cells.

INTRODUCTION: Obesity is a risk factor for both cardiovascular disease and cancer development. Leptin, a cytokine produced by adipose tissue, controls different processes in peripheral tissues, including cancer development and thrombotic disorders in patients with a variety of clinical disorders. Tissue factor (TF), the trigger of blood clotting, is abundant in the adipose tissue. Since TF, often expressed by cancer cells, is considered a hallmark of cancer progression, we investigated whether leptin could modulate TF in the human metastatic breast carcinoma cell line MCF-7. MATERIALS AND METHODS: MCF-7 cells were incubated with or without the different reagents at 37 °C. At the end of incubation, cells were tested for procoagulant activity by a one-stage clotting assay, TF and TNF-α antigen levels and mRNA by ELISA and real-time RT-PCR, respectively. Leptin receptor was studied by FACS. RESULTS: Both TF activity and antigen constitutively expressed by MCF-7 were significantly increased by leptin in a dose-dependent fashion. TF mRNA levels were also enhanced indicating that leptin exerts its effect at the transcription level. The effect of leptin was specific and required binding to its receptor (Ob-R), which was found on the surface of the cells, since antibodies against leptin and Ob-R completely prevented TF expression upregulation. In addition, leptin enhanced both TNF-α mRNA synthesis and secretion from MCF7. An anti-TNF-α MoAb completely abolished the leptin-induced TF expression. CONCLUSIONS: These data support the hypothesis that leptin, by its upregulation of TF, possibly mediated by TNF-α synthesis, may contribute to processes underlying both cancer and vascular cell disorders.

Platelet - leukocyte interactions: multiple links between inflammation, blood coagulation and vascular risk.

The aim of this review is to summarize the contribution of platelets and leukocytes and their interactions in inflammation and blood coagulation and its possible relevance in the pathogenesis of thrombosis. There is some evidence of an association between infection/inflammation and thrombosis. This is likely a bidirectional relationship. The presence of a thrombus may serve as a nidus of infection. Vascular injury indeed promotes platelet and leukocyte activation and thrombus formation and the thrombus and its components facilitate adherence of bacteria to the vessel wall. Alternatively, an infection and the associated inflammation can trigger platelet and leukocyte activation and thrombus formation. In either case platelets and leukocytes co-localize and interact in the area of vascular injury, at sites of inflammation and/or at sites of thrombosis. Following vascular injury, the subendothelial tissue, a thrombogenic surface, becomes available for interaction with these blood cells. Tissue factor, found not only in media and adventitia of the vascular wall, but also on activated platelets and leukocytes, triggers blood coagulation. Vascular-blood cell interactions, mediated by the release of preformed components of the endothelium, is modulated by both cell adhesion and production of soluble stimulatory or inhibitory molecules that alter cell function: adhesion molecules regulate cell-cell contact and facilitate the modulation of biochemical pathways relevant to inflammatory and/or thrombotic processes.

Paclitaxel downregulates tissue factor in cancer and host tumour-associated cells.

Paclitaxel, a microtubule-stabilising compound with potent anti-tumour activity, has been clinically used in a wide variety of malignancies. Tissue factor (TF) is often expressed by tumour-associated endothelial and inflammatory cells, as well as by cancer cells themselves, and it is considered a hallmark of cancer progression. We investigated whether paclitaxel could modulate TF in human mononuclear (MN) cells, human umbilical vein endothelial cells (HUVEC) and the metastatic breast carcinoma cell line MDA-MB-231. Cells were incubated with or without paclitaxel at 37 degrees C. At the end of incubation, cells were disrupted and tested for procoagulant activity by a one-stage clotting assay, for TF antigen levels by ELISA and TF mRNA by real-time RT-PCR. IL-6 and IL-1beta were tested by ELISA in conditioned medium. Both the strong TF activity and antigen constitutively expressed by MDA-MB-231 and the TF induced by LPS, TNF-alpha and IL-1beta in MN cells and HUVEC were significantly reduced by paclitaxel. In the presence of paclitaxel, lower TF mRNA levels were also detected. Since paclitaxel has been shown to induce the expression of inflammatory genes in monocytes and tumour cells, we tested whether paclitaxel could influence IL-6 and IL-1beta release from the cells used in this paper. Neither the constitutive expression of IL-6 and IL-1beta by MDA-MB-231 nor the basal and LPS-induced release from MN cells and HUVEC was affected. Our data support the hypothesis that the anti-tumour effects of paclitaxel may, at least in part, be mediated by the capacity of this drug to modulate the procoagulant potential of cancer and host cells.