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1.
Res Pract Thromb Haemost ; 3(2): 173-183, 2019 Apr.
Article in English | MEDLINE | ID: mdl-31011701

ABSTRACT

ABSTRACT: Fibrinogen, involved in coagulation, is a soluble protein composed of two sets of disulfide-bridged Aα, Bß, and γ-chains. In this review, we present the clinical implications of the αC domain of the molecule in Alzheimer's disease, hereditary renal amyloidosis and a number of thrombotic and hemorrhagic disorders. In Alzheimer's disease, amyloid beta peptide (Aß) is increased and binds to the αC domain of normal fibrinogen, triggering increased fibrin(ogen) deposition in patients' brain parenchyma. In hereditary renal amyloidosis, fibrinogen is abnormal, with mutations located in the fibrinogen αC domain. The mutant αC domain derived from fibrinogen degradation folds incorrectly so that, in time, aggregates form, leading to amyloid deposits in the kidneys. In these patients, no thrombotic tendency has been observed. Abnormal fibrinogens with either a point mutation in the αC domain or a frameshift mutation resulting in absence of a part of the αC domain are often associated with either thrombotic events or bleeding. Mutation of an amino acid into cysteine (as in fibrinogens Dusart and Caracas V) or a frameshift mutation yielding an unpaired cysteine in the αC domain is often responsible for thrombotic events. Covalent binding of albumin to the unpaired cysteine via a disulphide bridge leads to decreased accessibility to the fibrinolytic enzymes, hence formation of poorly degradable fibrin clots, which explains the high incidence of thrombosis. In contrast, anomalies due to a frameshift mutation in the αC connector of the molecule, provoking deletion of a great part of the αC domain, are associated with bleeding.

3.
PLoS One ; 9(3): e92379, 2014.
Article in English | MEDLINE | ID: mdl-24664182

ABSTRACT

AIM: To establish a new and reliable assay for quantification of the soluble fibrin (SF) in combination with that of D-dimer for early diagnosis of venous thromboembolism. METHODS AND SAMPLES: The SF assay is based on D-dimer generated after incubation of plasma with tissue-type plasminogen activator (t-PA). SF and standard D-dimer assays, run in blind, were used to test 119 untreated outpatients with clinically suspected deep-vein thrombosis (DVT, 49 patients) or pulmonary embolism (PE, 70 patients) consulting at the emergency unit of the hospital. Thromboses were confirmed by current imaging methods such as ultrasonography, scintigraphy, computed tomographic pulmonary angiography (CTPA) and ventilation/perfusion scan. RESULTS: SF assay was validated in 270 healthy volunteers [51.8% males; mean age years ± SD: 41±13; age range 19 to 65]. Among these normal plasmas, SF levels were ≤200 ng/mL in 97.8% of them, and 200-250 ng/mL in the remainder [26-46 years old; 50% males]. ROC curves were used to determine the SF cut-off value for plasma SF positivity, which was found to be 300 ng/mL. In patients with suspected venous thromboembolism, SF sensitivities for DVT and PE (92% and 94%, respectively) were comparable to those of D-dimer (96% and 94%), whereas SF specificities (86% and 95%) were higher than those of D-dimer (50% and 54%). Positive-predictive values for SF (89% and 94%) were again higher than those of D-dimer (70% and 65%) in DVT and PE. The amount of circulating SF normalized rapidly after anticoagulant therapy. CONCLUSION: Results from this small group of patients suggest that the evaluation of plasma SF, in combination with that of D-dimer, represents a potentially useful tool for the early diagnosis of venous thromboembolism, provided that the patients have not been treated previously by anticoagulants.


Subject(s)
Blood Chemical Analysis/methods , Fibrin Fibrinogen Degradation Products/chemistry , Fibrin Fibrinogen Degradation Products/metabolism , Venous Thromboembolism/blood , Venous Thromboembolism/diagnosis , Adult , Aged , Anticoagulants/therapeutic use , Blood Chemical Analysis/standards , Early Diagnosis , Female , Humans , Male , Middle Aged , Pulmonary Embolism/blood , Pulmonary Embolism/diagnosis , Reference Values , Reproducibility of Results , Solubility , Tissue Plasminogen Activator/pharmacology , Venous Thromboembolism/drug therapy , Young Adult
4.
Exp Hematol Oncol ; 3(1): 30, 2014.
Article in English | MEDLINE | ID: mdl-25601900

ABSTRACT

OBJECTIVES: Tissue factor (TF) exposed on activated monocytes and macrophages is involved in thrombosis through activation of factor X and cytokine release, responsible for inflammation and thrombosis. We investigated the effect of two anti-factor Xa drugs: rivaroxaban, a direct anti-Xa inhibitor, and fondaparinux, an antithrombin dependent anti-Xa inhibitor, on monocyte/macrophage procoagulant activity and cytokine release. METHODS: Rivaroxaban and fondaparinux were tested at pharmacological concentrations on LPS-activated monocytes and on THP-1 cells, a human monocytic cell line, to assess 1) TF expression by flow cytometry 2) prothrombinase activity by its coagulant activity and 3) cytokine release in cell supernatants by antibody based cytokine array and ELISA for IL-8 and TNFα. RESULTS AND CONCLUSION: Rivaroxaban and fondaparinux did not modify TF expression level on activated cells. In contrast procoagulant activity associated to monocytes and macrophages was dose dependently inhibited by rivaroxaban, but not significantly by fondaparinux. These results could explain why patients undergoing major orthopedic surgery with rivaroxaban prophylaxis were able to achieve significant reductions in venous thromboembolism, compared with drugs commonly used, i.e. fondaparinux and low molecular weight heparin. In addition, rivaroxaban and fondaparinux suppressed some chemokine secretion produced by activated macrophages. This may also contribute to their antithrombotic effect in clinic.

5.
J Hematol Oncol ; 6: 49, 2013 Jul 09.
Article in English | MEDLINE | ID: mdl-23837843

ABSTRACT

BACKGROUND: Multidrug resistance (MDR) is one of the major problems in the treatment of cancer. Overcoming it is therefore expected to improve clinical outcomes for cancer patients. MDR is usually characterized by overexpression of ABC (ATP-binding cassette) protein transporters such as P-gp, MRP1, and ABCG2. Though the importance of ABC transporters for cancer cells is recognized, few studies have looked at its implications for the endothelial cells that are essential to tumor angiogenesis. This study investigated the expression and functions of these ABC transporters in endothelial cells in vitro and their potential contribution to cancer growth in mice. METHODS: Human micro vessel endothelial cells (HMEC-1) and human umbilical vein endothelial cells (HUVEC) were exposed to increasing doses of Doxorubicin (Dox) to induce ABC gene expression. Cell viability was then quantified by (3)H-thymidine and MTS assay. Flow cytometry, qPCR, and western blot were used to detect mRNA and the protein expression of P-gp, MRP1, and ABCG2. The intracellular accumulation of Rhodamine 123 (Rho) was used to evaluate drug efflux function and the inhibitors for P-gp, ABCG2, and MRP1 were used to verify their respective roles in vitro. In an attempt to evaluate drug resistance in endothelial cells in vivo, athymic mice were treated with Dox for 15 days before a MDA-MB-435 tumor graft to observe subsequent changes in the inhibition curves of tumor growth in response to Dox treatment. Furthermore, endothelial cells from multiple sites in these mice were also isolated to estimate their P-gp expression by flow cytometry. RESULTS: Drug resistance in HMEC-1 and HUVEC was successfully induced by the addition of Dox to the culture media. Two stabilized subcell lines of HMEC1 (HMECd1 and HMECd2) showed 15- and 24-fold increases in resistance. Tests also showed that these induced endothelial cells were cross-resistant to the structurally unrelated drugs Daunorubicin, Vinblastine, and Etoposide. P-gp protein levels increased four and six fold in HMECd1 and HMECd2 as revealed by western blot. The qPCR demonstrated 3.4- and 7.2-fold increases in P-gp, and a slight increase in ABCG2, gene expression. The Rho accumulation within these cells was inversely correlated with the expression levels of P-gp. The inhibitors of P-gp, but not of ABCG2 or MRP1, were able to block the induced endothelial cell resistance to Dox. Furthermore, we also showed that injecting Dox into healthy mice induced an increase in P-gp expression in endothelial cells. Using these pretreated mice in a tumor growth experiment, we observed a dramatic diminution in the therapeutic efficiency of Dox treatment, suggesting implications for drug resistance in mice endothelial cells supporting tumor growth. CONCLUSIONS: ABC transporter expression can be induced in endothelial cells in vitro. This study also indicates that P-gp plays an important role in the acquisition of resistance to Dox in endothelial cells and that this reduces the efficiency of chemotherapy.


Subject(s)
Endothelial Cells/drug effects , Endothelial Cells/pathology , Neoplasms/drug therapy , Neoplasms/pathology , Animals , Cell Line, Tumor , Cell Proliferation/physiology , Doxorubicin/pharmacology , Drug Resistance, Multiple , Drug Resistance, Neoplasm , Female , Heterografts , Human Umbilical Vein Endothelial Cells , Humans , Mice , Mice, Nude
6.
BMC Cancer ; 13: 63, 2013 Feb 06.
Article in English | MEDLINE | ID: mdl-23388133

ABSTRACT

BACKGROUND: Rho GTPases are involved in cellular functions relevant to cancer. The roles of RhoA and Rac1 have already been established. However, the role of Rac3 in cancer aggressiveness is less well understood. METHODS: This work was conducted to analyze the implication of Rac3 in the aggressiveness of two breast cancer cell lines, MDA-MB-231 and MCF-7: both express Rac3, but MDA-MB-231 expresses more activated RhoA. The effect of Rac3 in cancer cells was also compared with its effect on the non-tumorigenic mammary epithelial cells MCF-10A. We analyzed the consequences of Rac3 depletion by anti-Rac3 siRNA. RESULTS: Firstly, we analyzed the effects of Rac3 depletion on the breast cancer cells' aggressiveness. In the invasive MDA-MB-231 cells, Rac3 inhibition caused a marked reduction of both invasion (40%) and cell adhesion to collagen (84%), accompanied by an increase in TNF-induced apoptosis (72%). This indicates that Rac3 is involved in the cancer cells' aggressiveness. Secondly, we investigated the effects of Rac3 inhibition on the expression and activation of related signaling molecules, including NF-κB and ERK. Cytokine secretion profiles were also analyzed. In the non-invasive MCF-7 line; Rac3 did not influence any of the parameters of aggressiveness. CONCLUSIONS: This discrepancy between the effects of Rac3 knockdown in the two cell lines could be explained as follows: in the MDA-MB-231 line, the Rac3-dependent aggressiveness of the cancer cells is due to the Rac3/ERK-2/NF-κB signaling pathway, which is responsible for MMP-9, interleukin-6, -8 and GRO secretion, as well as the resistance to TNF-induced apoptosis, whereas in the MCF-7 line, this pathway is not functional because of the low expression of NF-κB subunits in these cells. Rac3 may be a potent target for inhibiting aggressive breast cancer.


Subject(s)
Breast Neoplasms/enzymology , rac GTP-Binding Proteins/metabolism , Apoptosis , Breast Neoplasms/genetics , Breast Neoplasms/pathology , Cell Adhesion , Cell Movement , Cell Shape , Cell Survival , Collagen/metabolism , Cytokines/metabolism , Extracellular Signal-Regulated MAP Kinases/metabolism , Female , Humans , MCF-7 Cells , Matrix Metalloproteinase 9/metabolism , NF-kappa B/metabolism , Neoplasm Invasiveness , RNA Interference , Signal Transduction , Time Factors , Transfection , Tumor Necrosis Factor-alpha/metabolism , rac GTP-Binding Proteins/genetics , rhoA GTP-Binding Protein/metabolism
7.
Thromb Res ; 131(3): e100-9, 2013 Mar.
Article in English | MEDLINE | ID: mdl-23313382

ABSTRACT

INTRODUCTION: Defective thrombolysis, a thrombotic risk factor, can be attributed to the formation of a compact clot poorly accessible to fibrinolytic enzymes. Venous thrombi, rich in red blood cells (RBCs), and arterial thrombi containing various amounts of RBCS, plasma and whole blood (WB) clot permeability and degradability were compared. The effect of rivaroxaban, a potent direct factor Xa inhibitor, was also evaluated. MATERIALS AND METHODS: Fibrin permeability was determined by flow measurement through the clot. Clot degradability was evaluated by the amount of D-dimer generated by clot perfusion with plasminogen and tissue plasminogen activator. Fibrin clot structure was assessed by confocal microscopy. RESULTS: WB clot permeability (KS) and degradability were 6.7- and 38-fold lower, respectively, compared with plasma clots. This is attributed to 1) occlusion of fibrin pores by RBCs and 2) a consistent increase in thrombin generation due to platelets and RBCs inducing formation of a tighter clot. Rivaroxaban added to plasma or WB before clotting, in reducing thrombin generation, led to the formation of a looser clot that is more degradable by fibrinolytic enzymes. Permeability and degradability of whole blood clots formed in the presence of rivaroxaban were very similar to those of plasma clots. CONCLUSION: The resistance to fibrinolysis of WB clots was reduced considerably when clots were formed with rivaroxaban. These results may have implications for the development of antithrombotic agents.


Subject(s)
Anticoagulants/therapeutic use , Blood/drug effects , Fibrin/chemistry , Morpholines/therapeutic use , Plasma/drug effects , Thiophenes/therapeutic use , Thrombolytic Therapy/methods , Thrombosis/drug therapy , Blood Coagulation , Blood Platelets/cytology , Erythrocytes/cytology , Factor XIII/chemistry , Fibrin Fibrinogen Degradation Products/chemistry , Fibrinolysis/drug effects , Humans , Permeability , Risk Factors , Rivaroxaban , Thrombin/metabolism , Thromboplastin/chemistry , Thrombosis/metabolism , Time Factors
8.
J Hematol Oncol ; 5: 16, 2012 Jul 11.
Article in English | MEDLINE | ID: mdl-22534171

ABSTRACT

BACKGROUND: Tissue factor (TF), an initiator of blood coagulation, participates in cancer progression and metastasis. We recently found that inhibition of MAPK/ERK upregulated both full length TF (flTF) and soluble isoform TF (asTF) gene expression and cell-associated TF activity in breast cancer MDA-MB-231 cells. We explored the possible mechanisms, especially the possible interaction with EGFR and PI3K/Akt pathways. METHODS: A plasmid containing TF promoter -2174 ~ +128 plus luciferase reporter gene was introduced into MDA-MB-231 cells to evaluate TF promoter activity. In order to study the interaction of these pathways, ERK inhibitor (PD98059), PI3K inhibitors (LY294002, wortmannin), Akt inhibitor (A6730), and EGFR inhibitor (erlotinib) as well as the corresponding siRNAs were used to treat MDA-MB-231 cells, and ovarian cancer OVCAR-3 and SKOV-3 cells. Quantitative PCR and western blot were used to determine TF expression. One stage clotting assays were used to measure pro-coagulation activity of the MDA-MB-231 cells. RESULTS: We show that PI3K inhibitors LY294002, wortmannin and A6730 significantly inhibited TF promoter activity, and reduced TF mRNA and protein levels due to the inhibition of Akt phosphorylation. In contrast, ERK inhibitor PD98059 and ERK siRNA enhanced TF promoter activity by 2.5 fold and induced an increase in TF mRNA and protein levels in a dose dependent manner in these cells. The PI3K/Akt pathway was shown to be involved in PD98059-induced TF expression because the induction was inhibited by PI3K/Akt inhibitors. Most interestingly, the EGFR inhibitor erlotinib and EGFR siRNA also significantly suppressed PD98059- or ERK siRNA-induced TF promoter activity and TF protein expression. Similar results were found with ovarian cancer cells SKOV-3 and OVCAR-3. Furthermore, in MDA-MB-231, mRNA levels of asTF were regulated in a similar way to that of TF in response to the cell treatment. CONCLUSIONS: This study showed a regulatory mechanism in which MAPK/ERK signals inhibit EGFR/PI3K/Akt-mediated TF expression in breast cancer MDA-MB-231 cells. The same regulation was observed in ovarian cancer OVCAR-3 and SKOV-3 cells. Interestingly, we observed that both flTF and asTF could be regulated in a parallel manner in MDA-MB-231. As the PI3K/Akt pathway and EGFR regulate TF expression in cancer cells, targeting these signaling components is expected to potentially inhibit TF expression-associated tumor progression.


Subject(s)
Breast Neoplasms/metabolism , Cell Movement , MAP Kinase Signaling System , Mitogen-Activated Protein Kinases/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Thromboplastin/metabolism , Blood Coagulation/drug effects , Blotting, Western , Breast Neoplasms/drug therapy , Breast Neoplasms/pathology , Cell Proliferation , Collagen/metabolism , Drug Combinations , Enzyme Inhibitors , ErbB Receptors/genetics , ErbB Receptors/metabolism , Female , Gene Expression Regulation, Neoplastic , Humans , Laminin/metabolism , Mitogen-Activated Protein Kinases/antagonists & inhibitors , Mitogen-Activated Protein Kinases/genetics , Neoplasm Invasiveness , Ovarian Neoplasms/drug therapy , Ovarian Neoplasms/metabolism , Ovarian Neoplasms/pathology , Phosphatidylinositol 3-Kinases/genetics , Phosphoinositide-3 Kinase Inhibitors , Phosphorylation , Promoter Regions, Genetic/genetics , Proteoglycans/metabolism , Proto-Oncogene Proteins c-akt/antagonists & inhibitors , Proto-Oncogene Proteins c-akt/genetics , RNA, Messenger/genetics , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction , Signal Transduction/drug effects , Thromboplastin/genetics , Transcription, Genetic , Tumor Cells, Cultured
9.
Cancer Lett ; 317(2): 207-17, 2012 Apr 28.
Article in English | MEDLINE | ID: mdl-22120672

ABSTRACT

Seeking to improve ovarian cancer therapy, we compared biological characteristics of the moderately-aggressive OVCAR-3 cell line with two highly aggressive ovarian cancer cell populations: the SK-OV-3 cell line, and HASCJ primary cells isolated from the ascitic fluid of a patient with FIGO stage IV ovarian cancer. Secretion of angiogenic factors was not discriminative, whereas cell invasion through Matrigel and vasculogenic mimicry were much greater in the more aggressive cells. Among 10 agents tested for their ability to decrease cancer cell aggressivity using these two models, inhibitors of Stat3, IGF-IR and Rho GTPase were found to be the most promising.


Subject(s)
Ovarian Neoplasms/metabolism , Receptor, IGF Type 1/metabolism , STAT3 Transcription Factor/metabolism , rhoA GTP-Binding Protein/metabolism , Angiogenesis Inhibitors/pharmacology , Angiogenesis Inhibitors/therapeutic use , Antineoplastic Agents/pharmacology , Antineoplastic Agents/therapeutic use , Blotting, Western , Cell Line , Cell Line, Tumor , Drug Screening Assays, Antitumor/methods , Female , Humans , Models, Biological , Ovarian Neoplasms/drug therapy , Ovarian Neoplasms/pathology , Signal Transduction/drug effects , Tumor Cells, Cultured
10.
BMC Cancer ; 10: 375, 2010 Jul 17.
Article in English | MEDLINE | ID: mdl-20637124

ABSTRACT

BACKGROUND: Infiltration by macrophages (Mphi) indicates a poor prognosis in breast cancers, in particular by inducing angiogenesis. Our study aimed 1) to investigate the mechanism by which cooperation between Mphi and aggressive breast cancer cells (MDA-MB-231) induces angiogenesis; 2) to examine the effect of tetrathiomolybdate (TM) on this angiogenic activity. METHODS: Mphi coincubated with MDA-MB-231 were used as a model to mimic the inflammatory microenvironment. Angiogenesis induced by the culture media was tested in the chick chorioallantoic membrane (CAM). Mphi phenotype was evaluated by 1) expression of the M1 marker CD80, and secretion of interleukin 10 (IL-10), an M2 marker; 2) capacity to secrete Tumour Necrosis Factor alpha (TNFalpha) when stimulated by lipopolysaccharide/interferon gamma (LPS/IFNgamma); 3) ability to induce MDA-MB-231 apoptosis. To explore the molecular mechanisms involved, cytokine profiles of conditioned media from MDA-MB-231, Mphi and the coculture were characterised by an antibody cytokine array. All experiments were carried out both in presence and in absence of TM. RESULTS: Incubation of Mphi with MDA-MB-231 induced a pro-angiogenic effect in the CAM. It emerged that the angiogenic activity of the coculture is due to the capacity of Mphi to switch from M1 Mphi towards M2, probably due to an increase in Macrophage Colony Stimulating Factor. This M1-M2 switch was shown by a decreased expression of CD80 upon LPS/IFNgamma stimulation, an increased secretion of IL-10, a decreased secretion of TNFalpha in response to LPS/IFNgamma and an inability to potentiate apoptosis. At the molecular level, the angiogenic activity of the coculture medium can be explained by the secretion of CXC chemokines/ELR+ and CC chemokines. Although TM did not modify either the M2 phenotype in the coculture or the profile of the secreted chemokines, it did decrease the angiogenic activity of the coculture medium, suggesting that TM inhibited angiogenic activity by interfering with the endothelial cell signalling induced by these chemokines. CONCLUSIONS: Cooperation between Mphi and MDA-MB-231 transformed M1 Mphi to an angiogenic, M2 phenotype, attested by secretion of CXC chemokines/ELR+ and CC chemokines. TM inhibited this coculture-induced increase in angiogenic activity, without affecting either Mphi phenotype or cytokine secretion profiles.


Subject(s)
Angiogenesis Inhibitors/pharmacology , Breast Neoplasms/blood supply , Breast Neoplasms/pathology , Macrophages/pathology , Molybdenum/pharmacology , Neovascularization, Pathologic/prevention & control , Animals , Apoptosis/drug effects , Breast Neoplasms/drug therapy , Cells, Cultured , Chemokines/metabolism , Chick Embryo , Coculture Techniques , Culture Media, Conditioned/pharmacology , Cytokines/metabolism , Enzyme-Linked Immunosorbent Assay , Female , Fluorescent Antibody Technique , Humans , Interferon-gamma/pharmacology , Lipopolysaccharides/pharmacology , Macrophages/drug effects , Macrophages/metabolism , Recombinant Proteins
11.
Gastroenterol Res Pract ; 2010: 640797, 2010.
Article in English | MEDLINE | ID: mdl-21528105

ABSTRACT

Background and Aims. An arterial blood supply and phenotypic changes of the sinusoids characterise the liver vasculature in human hepatocellular carcinoma (HCC). We investigated the effects of rosuvastatin on liver vessel anomalies, tumour growth and survival in HCC. Methods. We treated transgenic mice developing HCC, characterized by vessel anomalies similar to those of human HCC, with rosuvastatin. Results. In the rosuvastatin group, the survival time was longer (P < .001), and liver weight (P < .01) and nodule surface (P < .01) were reduced. Rosuvastatin decreased the number of smooth muscle actin-positive arteries (P < .05) and prevented the sinusoid anomalies, with decreased laminin expression (P < .001), activated hepatic stellate cells (P < .001), and active Notch4 expression. Furthermore, rosuvastatin inhibited endothelial cell but not tumour hepatocyte functions. Conclusions. Rosuvastatin reduced the vessel anomalies and tumour growth and prolonged survival in HCC. These results represent new mechanisms of the effects of statin on tumour angiogenesis and a potential target therapy in HCC.

12.
Thromb Haemost ; 102(1): 131-6, 2009 Jul.
Article in English | MEDLINE | ID: mdl-19572077

ABSTRACT

Protein Z (PZ) is the cofactor of PZ dependent inhibitor (ZPI) that inhibits activated coagulation factor X. PZ was expected to play a role in coronary artery disease (CAD) but with inconsistent clinical findings. We therefore evaluated whether PZ plasma level and/or three genetic variants encoding for low PZ plasma level were associated with premature CAD in stable young post-myocardial infarction (MI) patients. PZ plasma level and three polymorphisms A-13G, G-103A and G79A were determined in 176 young stable post-MI patients and in 176 sex- and age-matched controls (FITE-NAT population). Moreover the genotypes, resulting from the combination of the three polymorphisms (A-13G/G-103A/G79A), were studied. PZ plasma level and the number of patients disclosing a PZ deficiency did not differ between post-MI patients and controls. The presence of the mutated allele for each polymorphism was associated with a significantly reduced level of PZ. The A-13G polymorphism was associated with premature CAD only in univariate analysis. Whereas, the presence of rare genotypes of PZ gene was an independent risk factor for premature CAD. In conclusion, PZ plasma level is not a key player in the pathophysiology of premature coronary artery disease. But, rare genotypes of PZ gene were found to be associated with premature CAD.


Subject(s)
Blood Proteins/genetics , Blood Proteins/metabolism , Myocardial Infarction/epidemiology , Myocardial Infarction/genetics , Adult , Age of Onset , Factor Xa/metabolism , Female , Genetic Predisposition to Disease/epidemiology , Genotype , Humans , Male , Myocardial Infarction/metabolism , Polymorphism, Genetic , Risk Factors
14.
Thromb Haemost ; 97(1): 27-31, 2007 Jan.
Article in English | MEDLINE | ID: mdl-17200767

ABSTRACT

Fondaparinux is a synthetic pentasaccharide consisting of the minimal sequence of heparin which interacts with antithrombin (AT). It represents a new class of selective factor Xa inhibitors without any antithrombin activity. It has been shown to exhibit potent antithrombotic properties in clinical studies. However, the mechanism of its antithrombotic action has not yet been fully established. In the present study it was shown that fondaparinux, used at pharmacological concentration (500 ng/ml), rendered the clot more susceptible to fibrinolysis induced by t-PA: plasma fibrin clots formed in the presence of fondaparinux and perfused with t-PA were degraded at a faster rate than those formed in the absence of fondaparinux. This fibrinolytic activity of fondaparinux is mainly due to a modification of clot structure characterized by a loose fibrin conformation with less branched fibers and the presence of large pores in comparison to control clots which present a tighter conformation. The difference in fibrin structure was responsible for an increase in clot porosity leading to a better availability of t-PA to the fibrin network. It is related to the decrease in thrombin generation, in an AT-dependent pathway. It was also demonstrated that in the presence of exogenous thrombomodulin, the inhibition of TAFI activation by fondaparinux could contribute, to a lesser extent, to the increased thrombus lysis. The increase in t-PA induced thrombus lysis could contribute to the antithrombotic activity of fondaparinux.


Subject(s)
Blood Coagulation , Fibrinolysis/drug effects , Polysaccharides/pharmacology , Antithrombin III , Fibrin/chemistry , Fibrin/drug effects , Fibrinolytic Agents/pharmacology , Fondaparinux , Kinetics , Protein Conformation/drug effects , Thrombin/biosynthesis , Tissue Plasminogen Activator/pharmacology
15.
Br J Haematol ; 131(3): 356-65, 2005 Nov.
Article in English | MEDLINE | ID: mdl-16225656

ABSTRACT

Monocytes can be induced to express both tissue factor (TF) and its inhibitor, TF pathway inhibitor-1 (TFPI-1). A short incubation (<6 h) with interleukin (IL)-4 and IL-10, two potent deactivators of monocyte functions, has been shown to modulate the synthesis and expression of TF by monocytes activated by lipopolysaccharide, but the consequences of longer incubations (up to 96 h) on both TF and TFPI-1 are unknown. The results of this study showed that adherent monocytes in culture spontaneously expressed TF and TFPI and that prolonged incubation with IL-10 induced a time- and dose-dependent decrease of monocyte TF synthesis, and an accumulation of TF/TFPI-1 complexes at the moncyte surface, suggesting a decreased clearance of these complexes. In contrast, IL-4 induced a time- and dose-dependent increase in TF synthesis, which remained intracytoplasmic, as shown by confocal microscopy. Surprisingly, TF:antigen (Ag) was decreased at the monocyte surface, but the procoagulant activity (PCA) of IL-4-treated monocytes was increased, as a result of more pronounced decrease of TFPI-1:Ag expression than that of TF. In conclusion, prolonged incubation with IL-4 and IL-10 oppositely modified PCA of cultured monocytes, and altered TF and TFPI trafficking and clearance. These data explain the respective deleterious or benefit effects of IL-4 or IL-10 in atherothrombosis.


Subject(s)
Blood Coagulation Factors/metabolism , Interleukin-10/pharmacology , Interleukin-4/pharmacology , Monocytes/drug effects , Thromboplastin/metabolism , Antibodies, Monoclonal/immunology , Cells, Cultured , Cytoplasm/metabolism , Dose-Response Relationship, Drug , Enzyme-Linked Immunosorbent Assay/methods , Gene Expression , Humans , Lipoproteins/antagonists & inhibitors , Lipoproteins/genetics , Lipoproteins/metabolism , Microscopy, Confocal , Monocytes/metabolism , RNA, Messenger/genetics , Reverse Transcriptase Polymerase Chain Reaction/methods , Thromboplastin/genetics , Time Factors
16.
Hum Gene Ther ; 16(10): 1157-67, 2005 Oct.
Article in English | MEDLINE | ID: mdl-16218777

ABSTRACT

The urokinase plasminogen activator (uPA) is implicated in both cancer cell invasion and angiogenesis. It can interact with a specific receptor (uPAR) via the epidermal growth factor (EGF)-like domain in the urokinase amino-terminal fragment (ATF) in a species-specific manner. Our previous studies showed that adenovirusmediated delivery of murine ATF (AdmATF) suppressed human tumor growth in mouse models, by inhibiting murine angiogenesis. However, we cannot exclude its putative inhibitory action on human cancer cell invasion through a uPAR-independent pathway. To further investigate the mechanisms of ATF, we constructed another adenovirus, AdhmATF, expressing humanized murine ATF (hmATF). hmATF binds to human uPAR but not to murine uPAR. We compared the antagonist effect of both AdmATF and AdhmATF on human and murine cancer cells. In vitro, the supernatant from AdhmATF-infected cells repressed 79% of membrane-associated uPA activity on human MDA-MB-231 cells, whereas that from AdmATF-infected cells repressed 35% of membrane-associated uPA activity. On murine LLC cells, the supernatant from AdhmATF-infected cells inhibited 29% of cell surface uPA activity, whereas that from AdmATF-infected cells inhibited 74% of cell surface uPA activity. Similar results were obtained in a cell invasion assay. In vivo, intratumoral injection of the adenoviruses into LLC tumors on day 24 postinjection induced lower but significant tumor growth suppression by AdhmATF (tumor volume was 1185 +/- 128 mm3), whereas suppression by AdmATF was greater (407 +/- 147 mm3). In the MDA-MB-231 tumor model, on day 52 postinjection, tumor size was 187 +/- 47 mm3 in the AdhmATF-treated group and 468 +/- 65 mm3 in the AdmATF-treated group. The LLC and MDA-MB- 231 cell lines transfected by mATF or hmATF genes showed growth inhibition In vivo equivalent to the results obtained by adenovirus treatment. These results demonstrate the strong anticancer activity of ATF even when its uPAR-binding affinity has been suppressed, and indicate that ATF exerts an antitumor effect via dual mechanisms: essentially through targeting the uPA-uPAR system via the EGF-like domain and partially through targeting a uPAR-independent interaction via the kringle domain.


Subject(s)
Adenoviridae , Genetic Therapy , Genetic Vectors , Neoplasms, Experimental/therapy , Receptors, Cell Surface/metabolism , Urokinase-Type Plasminogen Activator , Animals , Cell Line, Tumor , Female , Genetic Therapy/methods , Humans , Mice , Mice, Mutant Strains , Neoplasm Invasiveness , Neoplasm Transplantation , Neoplasms, Experimental/metabolism , Neoplasms, Experimental/pathology , Peptide Fragments/administration & dosage , Peptide Fragments/genetics , Receptors, Urokinase Plasminogen Activator , Urokinase-Type Plasminogen Activator/administration & dosage , Urokinase-Type Plasminogen Activator/chemistry , Urokinase-Type Plasminogen Activator/genetics
17.
Stroke ; 36(8): 1716-9, 2005 Aug.
Article in English | MEDLINE | ID: mdl-16020765

ABSTRACT

BACKGROUND AND PURPOSE: Cerebral venous thrombosis (CVT) is an infrequent variety of cerebrovascular disease with a wide spectrum of clinical presentations and a notoriously difficult diagnosis. Previous reports have emphasized the potential clinical utility of D-dimer assay in CVT diagnosis. METHODS: A rapid sensitive D-dimer assay was performed at entry in 73 patients with CVT <30 days duration, examined in our institution between 1999 and 2003. RESULTS: The mean value of D-dimer levels was 1521 ng/mL; 7 patients (10% of all patients and 26% of those presenting with isolated headache) had values <500 ng/mL. In a multivariate analysis, isolated headache was the only variable associated with a negative D-dimer assay. CONCLUSIONS: A negative D-Dimer assay does not confidently rule out CVT, particularly in the setting of recent isolated headache.


Subject(s)
Clinical Chemistry Tests/methods , Fibrin Fibrinogen Degradation Products/biosynthesis , Intracranial Thrombosis/blood , Intracranial Thrombosis/diagnosis , Venous Thrombosis/blood , Venous Thrombosis/diagnosis , Adult , Female , Headache/blood , Headache/diagnosis , Humans , Male , Middle Aged , Models, Statistical , Multivariate Analysis , Risk Factors , Sensitivity and Specificity , Time Factors
18.
J Cell Sci ; 118(Pt 2): 343-56, 2005 Jan 15.
Article in English | MEDLINE | ID: mdl-15632106

ABSTRACT

Elastin-derived peptides display a wide range of biological activities in a number of normal and transformed cells but their involvement in angiogenesis has not been reported. In the present study, we show that kappa-elastin and VGVAPG hexapeptide elastin motif accelerated angiogenesis in the chick chorio-allantoic membrane in an in vivo model. They also stimulated pseudotube formation from human vascular and microvascular endothelial cells in the matrigel and collagen models as well as cell migration in an in vitro wound healing assay. Confocal and scanning electron microscopy analyses revealed the main reorganization of actin filaments mediated by elastin-derived peptides and changes in cell shape that correlated with a decrease of the cell form factor determined by computerized image analysis. Such elastin-derived peptide effects were attributed to upregulation of proMT1-MMP and proMMP-2 expression and activation at both the mRNA and protein levels. Batimastat, an inhibitor of furin convertase and TIMP-2, but not TIMP-1, totally abolished the influence of elastin-derived peptides (EDPs) on cell migration and tubulogenesis, thus favoring the involvement of MT1-MMP in such processes. To assess its contribution to EDP-mediated angiogenesis further, we used a small interfering RNA (siRNA) approach for specifically silencing MT1-MMP in human microvascular endothelial cells. Four sets of 21 bp siRNA duplexes targeting MT1-MMP mRNA were synthesized by in vitro transcription. Two of them proved to inhibit MT1-MMP expression efficiently but did not affect MT2-, MT3- and MT5-MMP expression. Seventy-two hours after transfection with 25 nM siRNAs EDP-induced MT1-MMP expression at the mRNA and protein levels was decreased fourfold. In parallel, proMMP-2 activation was inhibited. A scrambled siRNA, used as a negative control, had no effect. Finally, the effect of elastin peptides on pseudotube formation in MT1-MMP-siRNA transfected cells was totally abolished. These data emphasise the crucial role of MT1-MMP in the elastin-induced angiogenic phenotype of endothelial cells.


Subject(s)
Cell Movement/drug effects , Elastin/pharmacology , Endothelium, Vascular/physiology , Metalloendopeptidases/metabolism , Neovascularization, Physiologic/physiology , Peptide Fragments/pharmacology , Animals , Cell Movement/physiology , Chick Embryo , Dose-Response Relationship, Drug , Elastin/chemistry , Endothelium, Vascular/cytology , Endothelium, Vascular/drug effects , Humans , Matrix Metalloproteinases, Membrane-Associated , Metalloendopeptidases/drug effects , Neovascularization, Physiologic/drug effects , Phenotype , RNA, Small Interfering/pharmacology , Time Factors , Up-Regulation
19.
Eur J Pharmacol ; 502(1-2): 21-30, 2004 Oct 11.
Article in English | MEDLINE | ID: mdl-15464086

ABSTRACT

Although dehydroepiandrosterone (DHEA) is widely used in the elderly to prevent some adverse effects of ageing, possible deleterious side effects have not been fully assessed. We evaluated the direct actions of DHEA and DHEA sulphate on angiogenesis, a critical event in pathologies that are common in the elderly (cancer, atherosclerosis, inflammation... etc.). At physiological concentrations found in human plasma following DHEA therapy (1-50 nM), DHEA had no action on angiogenesis in vitro. In contrast, higher concentrations of DHEA (10-100 microM), which can be found in tissues after local administration or storage, inhibited in vitro endothelial cell proliferation (blockage in G2/M), migration and capillary tube formation and in vivo angiogenesis in the Matrigel plug assay. This inhibition might be due to a decreased glucose-6-phosphate dehydrogenase activity and to a modification of the tubulin network involved in cell proliferation and migration. The sulphate ester form of DHEA had no effect on angiogenesis.


Subject(s)
Angiogenesis Inhibitors/pharmacology , Dehydroepiandrosterone Sulfate/pharmacology , Dehydroepiandrosterone/pharmacology , Endothelial Cells/drug effects , Cells, Cultured , Dose-Response Relationship, Drug , Endothelial Cells/cytology , Humans
20.
Clin Chim Acta ; 344(1-2): 163-71, 2004 Jun.
Article in English | MEDLINE | ID: mdl-15149885

ABSTRACT

BACKGROUND: We investigated whether the increase of urokinase-type plasminogen activator (uPA) monocyte expression and chemokine releases induced by oxidised low density lipoproteins (LDL), which participate to vascular tissue remodeling and to atherosclerotic plaque rupture, involved proinflammatory phospholipid products having platelet-activating factor (PAF)-like activity via the PAF-receptor pathway. METHODS: uPA monocyte expression was stimulated by either copper ions-oxidised or O2*-/HO* free radical-oxidised LDL. The effects of PAF and oxidised LDL on the production of monocyte chemoattractant protein-1 and interleukin-8 were also examined. RESULTS: Synthetic PAF significantly enhanced chemokine releases (P<0.001) without modifying uPA expression. Copper-oxidised LDL, which exhibit a higher content in lysophosphatidylcholines than free radical-oxidised LDL, induced a significantly higher enhancement in uPA expression (P<0.05). By contrast, free radical-oxidised LDL were more efficient than copper-oxidised LDL to increase chemokine releases (P<0.01). Oxidised LDL-enhanced uPA expressions were not altered by the PAF-receptor antagonist SR27417, whereas increases in chemokine releases induced by oxidised LDL and by PAF were abolished. PAF-acetylhydrolase activity was rapidly and largely inhibited in free radical-oxidised LDL when compared to copper-oxidised LDL, suggesting that free radical-oxidised LDL would contain a higher content in PAF-like products than copper-oxidised LDL. CONCLUSION: Our results indicated that PAF-like oxidation products are responsible for the monocyte chemokine releases, but did not contribute to the enhanced monocyte uPA expression by oxidised LDL.


Subject(s)
Chemokines/metabolism , Lipoproteins, LDL/metabolism , Monocytes/metabolism , Platelet Membrane Glycoproteins/metabolism , Receptors, G-Protein-Coupled/metabolism , Urokinase-Type Plasminogen Activator/genetics , Cells, Cultured , Chemokine CCL4 , Copper/metabolism , Free Radicals/metabolism , Humans , Inflammation/metabolism , Interleukin-8/biosynthesis , Lipoproteins, LDL/physiology , Macrophage Inflammatory Proteins/biosynthesis , Phospholipids/metabolism
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