RESUMO
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.
Assuntos
Neoplasias da Mama/enzimologia , Proteínas rac de Ligação ao GTP/metabolismo , Apoptose , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Adesão Celular , Movimento Celular , Forma Celular , Sobrevivência Celular , Colágeno/metabolismo , Citocinas/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Humanos , Células MCF-7 , Metaloproteinase 9 da Matriz/metabolismo , NF-kappa B/metabolismo , Invasividade Neoplásica , Interferência de RNA , Transdução de Sinais , Fatores de Tempo , Transfecção , Fator de Necrose Tumoral alfa/metabolismo , Proteínas rac de Ligação ao GTP/genética , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
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.
Assuntos
Inibidores da Angiogênese/farmacologia , Neoplasias da Mama/irrigação sanguínea , Neoplasias da Mama/patologia , Macrófagos/patologia , Molibdênio/farmacologia , Neovascularização Patológica/prevenção & controle , Animais , Apoptose/efeitos dos fármacos , Neoplasias da Mama/tratamento farmacológico , Células Cultivadas , Quimiocinas/metabolismo , Embrião de Galinha , Técnicas de Cocultura , Meios de Cultivo Condicionados/farmacologia , Citocinas/metabolismo , Ensaio de Imunoadsorção Enzimática , Feminino , Imunofluorescência , Humanos , Interferon gama/farmacologia , Lipopolissacarídeos/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Proteínas RecombinantesRESUMO
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.
Assuntos
Proteínas Sanguíneas/genética , Proteínas Sanguíneas/metabolismo , Infarto do Miocárdio/epidemiologia , Infarto do Miocárdio/genética , Adulto , Idade de Início , Fator Xa/metabolismo , Feminino , Predisposição Genética para Doença/epidemiologia , Genótipo , Humanos , Masculino , Infarto do Miocárdio/metabolismo , Polimorfismo Genético , Fatores de RiscoRESUMO
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.
RESUMO
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.
Assuntos
Coagulação Sanguínea , Fibrinólise/efeitos dos fármacos , Polissacarídeos/farmacologia , Antitrombina III , Fibrina/química , Fibrina/efeitos dos fármacos , Fibrinolíticos/farmacologia , Fondaparinux , Cinética , Conformação Proteica/efeitos dos fármacos , Trombina/biossíntese , Ativador de Plasminogênio Tecidual/farmacologiaRESUMO
Metargidin, a transmembrane protein of the adamalysin family, and integrins, e.g., alpha5beta1 and alphav, are preferentially expressed on endothelial cells on angiogenesis. Furthermore, metargidin interacts with these integrins via its disintegrin domain. In this study, recombinant human disintegrin domain (RDD) was produced in Escherichia coli by subcloning its cDNA into the pGEX-2T vector, and the effect of purified RDD on different steps of angiogenesis was evaluated. At concentrations of 2-10 micro g/ml, RDD exhibited inhibitory activities in a variety of in vitro functional assays, including endothelial cell proliferation and adhesion on the integrin substrates fibronectin, vitronectin, and fibrinogen. RDD (10 micro g/ml) totally abrogated endothelial cell migration and blocked most capillary formation in a three-dimensional fibrin gel. To test RDD efficacy in vivo, the RDD gene inserted into pBi vector containing a tetracycline-inducible promoter was electrotransferred into nude mouse muscle. RDD was successfully synthesized by muscle cells in vivo as shown by immunolabeling and Western blotting. In addition, 78% less MDA-MB-231 tumor growth, associated with strong inhibition of tumor angiogenesis, was observed in athymic mice bearing electrotransferred RDD. Moreover, in the presence of RDD, 74% fewer B16F10 melanoma lung metastases were found in C57BL/6 mice. Taken together, these results identified this RDD as a potent intrinsic inhibitor of angiogenesis, tumor growth, and metastasis, making it a promising tool for use in anticancer treatment.
Assuntos
Antineoplásicos/uso terapêutico , Desintegrinas/uso terapêutico , Neoplasias Pulmonares/prevenção & controle , Melanoma Experimental/prevenção & controle , Proteínas de Membrana/uso terapêutico , Metaloendopeptidases/uso terapêutico , Neovascularização Patológica/prevenção & controle , Proteínas ADAM , Animais , Apoptose/efeitos dos fármacos , Adesão Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/patologia , Escherichia coli/genética , Feminino , Neoplasias Pulmonares/irrigação sanguínea , Neoplasias Pulmonares/secundário , Melanoma Experimental/irrigação sanguínea , Melanoma Experimental/secundário , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Nus , Músculo Esquelético/patologia , Proteínas Recombinantes/uso terapêutico , Células Tumorais CultivadasRESUMO
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.
Assuntos
Adenoviridae , Terapia Genética , Vetores Genéticos , Neoplasias Experimentais/terapia , Receptores de Superfície Celular/metabolismo , Ativador de Plasminogênio Tipo Uroquinase , Animais , Linhagem Celular Tumoral , Feminino , Terapia Genética/métodos , Humanos , Camundongos , Camundongos Mutantes , Invasividade Neoplásica , Transplante de Neoplasias , Neoplasias Experimentais/metabolismo , Neoplasias Experimentais/patologia , Fragmentos de Peptídeos/administração & dosagem , Fragmentos de Peptídeos/genética , Receptores de Ativador de Plasminogênio Tipo Uroquinase , Ativador de Plasminogênio Tipo Uroquinase/administração & dosagem , Ativador de Plasminogênio Tipo Uroquinase/química , Ativador de Plasminogênio Tipo Uroquinase/genéticaRESUMO
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.
Assuntos
Testes de Química Clínica/métodos , Produtos de Degradação da Fibrina e do Fibrinogênio/biossíntese , Trombose Intracraniana/sangue , Trombose Intracraniana/diagnóstico , Trombose Venosa/sangue , Trombose Venosa/diagnóstico , Adulto , Feminino , Cefaleia/sangue , Cefaleia/diagnóstico , Humanos , Masculino , Pessoa de Meia-Idade , Modelos Estatísticos , Análise Multivariada , Fatores de Risco , Sensibilidade e Especificidade , Fatores de TempoRESUMO
Statins are currently used for the treatment of hypercholesterolemia. Recently, we demonstrated that cerivastatin also reduces the proliferation and invasion of aggressive breast cancer cells, MDA-MB-231. In this report, a molecular mechanism to explain its anti-cancer action is proposed by combining the study of cerivastatin effect on both gene expression (microarray) and signal transduction pathways. Firstly, the expression of 13 genes was modified by cerivastatin and confirmed at protein level. They could contribute to the inhibition of both cell proliferation (down-regulation of cyclin D1, PCNA, c-myc and up-regulation p21(Waf1), p19(INK4d), integrin beta8) and cell invasion, either directly (decrease in u-PA, MMP-9, u-PAR, PAI-1 and increase in anti-oncogenes Wnt-5a and H-cadherin) or indirectly by stimulating an anti-angiogenic gene (thrombospondin-2). The anti-angiogenic activity was confirmed by in vivo experiments. Secondly, we demonstrated that the biochemical mechanism of its anti-cancer action could be mainly explained by the inhibition of RhoA-dependent cell signalling. This hypothesis was supported by the fact that a RhoA inhibitor (C3 exoenzyme) or a dominant negative mutant RhoA (N19RhoA) induced similar effects to those of cerivastatin. In conclusion, cerivastatin, by preventing RhoA prenylation, inhibits (i) the RhoA/ROCK pathway, leading to defective actin stress fibres formation responsible for the loss of traction forces required for cell motility and (ii) the RhoA/FAK/AKT signalling pathway that could explain the majority of cancer-related gene modifications described above. Thus, the inhibition of RhoA cell signalling could be a good strategy in therapy of aggressive forms of breast cancer.
Assuntos
Antineoplásicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Piridinas/farmacologia , ADP Ribose Transferases/farmacologia , Animais , Toxinas Botulínicas/farmacologia , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Divisão Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Citosol/metabolismo , Humanos , Camundongos , Camundongos Nus , Invasividade Neoplásica , Neovascularização Patológica/tratamento farmacológico , Análise de Sequência com Séries de Oligonucleotídeos , Prenilação de Proteína/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Células Tumorais Cultivadas/citologia , Células Tumorais Cultivadas/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
Cerivastatin is an inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase. It inhibits the biosynthesis of cholesterol and its precursors: farnesyl pyrophosphate and geranylgeranyl pyrophosphate (GGPP), which are involved in Ras and RhoA cell signaling, respectively. Statins induce greater protection against vascular risk than that expected by cholesterol reduction. Therefore, cerivastatin could protect plaque against rupture, an important cause of ischemic events. In this study, the effect of cerivastatin was tested on angiogenesis because it participates in plaque progression and plaque destabilization. Cerivastatin inhibits in vitro the microvascular endothelial cell proliferation induced by growth factors, whereas it has no effect on unstimulated cells. This growth arrest occurs at the G(1)/S phase and is related to the increase of the cyclin-dependent kinase inhibitor p21(Waf1/Cip1). These effects are reversed by GGPP, suggesting that the inhibitory effect of cerivastatin is related to RhoA inactivation. This mechanism was confirmed by RhoA delocalization from cell membrane to cytoplasm and actin fiber depolymerization, which are also prevented by GGPP. It was also shown that RhoA-dependent inhibition of cell proliferation is mediated by the inhibition of focal adhesion kinase and Akt activations. Moreover, cerivastatin inhibits in vivo angiogenesis in matrigel and chick chorioallantoic membrane models. These results demonstrate the antiangiogenic activity of statins and suggest that it may contribute to their therapeutic benefits in the progression and acute manifestations of atherosclerosis.
Assuntos
Endotélio Vascular/efeitos dos fármacos , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Piridinas/farmacologia , Arteriosclerose/patologia , Arteriosclerose/prevenção & controle , Divisão Celular/efeitos dos fármacos , Inibidor de Quinase Dependente de Ciclina p21 , Ciclinas/metabolismo , Endotélio Vascular/citologia , Fase G1/efeitos dos fármacos , Humanos , Microcirculação , Neovascularização Patológica/prevenção & controle , Fosfatos de Poli-Isoprenil/metabolismo , Fosfatos de Poli-Isoprenil/farmacologia , Sesquiterpenos , Transdução de Sinais/efeitos dos fármacos , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
Endostatin has been considered a highly specific inhibitor of endothelial cell proliferation and/or migration. To explore the use of endostatin in antiangiogenic gene therapy, we generated a recombinant adenovirus, AdEndo, carrying the gene for mouse endostatin. Injection of 10(9) PFU of AdEndo resulted in a low but significant suppression (25%) of preestablished tumor growth in murine models involving murine Lewis lung carcinoma (LLC) and human breast cancer MDA-MB-231 tumors. Greater anticancer activity was observed when the same dose of AdEndo was injected into two other preestablished murine models involving C51 murine colon cancer and HT29 human colon cancer (55 and 47% tumor growth reduction, respectively). In vitro, endostatin derived from AdEndo-infected MRC-5 fibroblasts inhibited the growth of C51 and HT29 cell lines (72 and 61%, respectively). The extent of this inhibition was comparable to that observed in endothelial cells: 75% for microcapillary endothelial cell line HMEC-1, 52% for human dermal microvascular endothelial cells, 46% for human umbilical vein endothelial cells, and 67% for calf pulmonary arterial endothelial cells. Both endothelial and colon cancer cells showed a clear increase in cell apoptosis (4- to 5-fold for endothelial cells and 5- to 10-fold for colon cancer cells) and an accumulation in the G(1) phase of the cell cycle. This antiproliferative activity was not observed in other tumor cell lines: LLC, MDA-MB-231, murine colon adenocarcinoma MC38, human prostate cancer cell line DU145, and human breast cancer cell line CAL51. Taken together, these results provide evidence that, in addition to its antiangiogenic activity, endostatin exerts a direct anticancer action that appears to be restricted to some tumor cell lines. Thus, endostatin could be used in some colon cancer treatments and its clinical efficacy would depend on the response of tumor cells themselves.
Assuntos
Adenoviridae/genética , Inibidores da Angiogênese , Antineoplásicos , Colágeno/genética , Neoplasias do Colo/terapia , Fragmentos de Peptídeos/genética , Animais , Apoptose , Ciclo Celular , Divisão Celular , Neoplasias do Colo/irrigação sanguínea , Neoplasias do Colo/patologia , Endostatinas , Endotélio Vascular/citologia , Feminino , Citometria de Fluxo , Vetores Genéticos , Humanos , Integrina alfa5beta1/genética , Integrina alfa5beta1/metabolismo , Integrina alfaV/genética , Integrina alfaV/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Microscopia Confocal , Neovascularização Patológica , Células Tumorais CultivadasRESUMO
BACKGROUND: Sneddon's syndrome is characterized by the association of ischemic cerebrovascular events and widespread livedo racemosa. The pathophysiology of Sneddon's syndrome remains elusive, but various prothrombotic abnormalities have been previously reported in this setting. Low levels of protein Z, a downregulator of coagulation, have been recently linked to an increased risk of arterial thrombosis. The purpose of this study was to investigate the levels of protein Z in a series of Sneddon's syndrome patients without circulating antiphospholipid antibodies in comparison with an age- and sex-matched control population. METHODS: Twenty-six patients and 78 healthy controls had determination of their protein Z blood levels by an enzyme-linked immunoassay test. Patients' thrombotic and vascular risk factors, including tobacco smoking, arterial hypertension, oral contraceptive agents, dyslipidemia, factor V Leiden, and factor II mutation were recorded. RESULTS: Protein Z plasma levels were significantly lower in patients (mean 1.47 mg/L) than in controls (mean 1.93 mg/L) (P=0.02). Prevalence of protein Z deficiency (level <1 mg/L) was significantly higher (P=0.001) among patients (31%) than among controls (3.8%). Factor V Leiden and heavy smoking were observed in 4 and 7 patients, respectively. CONCLUSIONS: Sneddon's syndrome could be viewed as the peculiar clinical expression of various and sometimes associated coagulation abnormalities. Low levels of protein Z may account, at least partly, for the thrombotic events observed in Sneddon's syndrome and shed a new light on its pathophysiology. Clinical implications for protein Z deficiency in this setting deserve further investigations.
Assuntos
Proteínas Sanguíneas/análise , Síndrome de Sneddon/sangue , Adulto , Anticorpos Antifosfolipídeos/sangue , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Síndrome de Sneddon/diagnósticoRESUMO
3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins) reduce the risk of coronary event by cholesterollowering dependent and independent mechanisms. We have already described that the inhibitory effect of cerivastatin on angiogenesis contribute to the cholesterol-independent beneficial effect and was due to the inhibition of the cell signaling cascade RhoA/FAK/Akt. In this study, new insights in the molecular mechanism of action were provided. It indicates an inhibition of exposure of alpha V beta 3 integrin on cell membrane and a modification of gene expression. The inhibition of angiogenesis could be related to 1) an increase in genes involved in the inhibition of cell proliferation (p19(INK4), p21(Waf/Cip1),Wnt-5a), the inhibition of cell migration (Rho-GDI 1, alpha E-catenin) and 2) a downregulation of genes involved in angiogenesis (PAI-1, Vitronectin, HoxD3, Notch4) or in cell invasion (Semaphorin E). In addition, DNA repair protein genes (MLH1, XRCC1) were increased. This study may indicate new biological interest of genes involved in angiogenesis control.
Assuntos
Inibidores da Angiogênese/farmacologia , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Neovascularização Patológica/tratamento farmacológico , Piridinas/farmacologia , Linhagem Celular , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/metabolismo , Expressão Gênica/efeitos dos fármacos , Humanos , Integrina alfaVbeta3/genética , Integrinas/genética , Neovascularização Patológica/genética , Neovascularização Patológica/fisiopatologia , Receptores de Vitronectina/genética , Transdução de Sinais/efeitos dos fármacos , Proteína rhoA de Ligação ao GTP/antagonistas & inibidoresRESUMO
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.
Assuntos
Inibidores da Angiogênese/farmacologia , Sulfato de Desidroepiandrosterona/farmacologia , Desidroepiandrosterona/farmacologia , Células Endoteliais/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Células Endoteliais/citologia , HumanosRESUMO
UNLABELLED: Recombinant human erythropoietin (rHuEPO) is considered to be a mitogenic and chemotactic agent in cultured endothelial cells (ECs). The effect of recombinant granulocyte-macrophage (rGM-CSF) and granulocyte (rG-CSF) colony-stimulating factors on the proliferation of human umbilical vein endothelial cells (HUVECs) has been controversial. METHODS: HUVEC proliferation and the release of endothelial markers in HUVEC culture stimulated by rHuEPO, rGM-CSF and G-CSF were measured. RESULTS: We found the dose-dependent stimulating effect of rHuEPO and rGM-CSF on HUVEC proliferation, but we did not achieve this with rG-CSF. rHuEPO like rG-CSF was an effective agent in stimulating the plasminogen activator inhibitor (PAI)-1 release from HUVECs to a cultured medium, while rGM-CSF failed. CONCLUSION: We suggest that rHuEPO showed prothrombotic changes in HUVEC culture. Our results support the idea of suspected rHuEPO direct prothrombotic role in haemodialysed patients treated with rHuEPO.
Assuntos
Endotélio Vascular/citologia , Eritropoetina/farmacologia , Fator Estimulador de Colônias de Granulócitos/farmacologia , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Biomarcadores/análise , Divisão Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Endotélio Vascular/metabolismo , Humanos , Inibidor 1 de Ativador de Plasminogênio/análise , Proteínas Recombinantes/farmacologia , Trombose/induzido quimicamente , Veias Umbilicais/citologiaRESUMO
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.
Assuntos
Quimiocinas/metabolismo , Lipoproteínas LDL/metabolismo , Monócitos/metabolismo , Glicoproteínas da Membrana de Plaquetas/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Ativador de Plasminogênio Tipo Uroquinase/genética , Células Cultivadas , Quimiocina CCL4 , Cobre/metabolismo , Radicais Livres/metabolismo , Humanos , Inflamação/metabolismo , Interleucina-8/biossíntese , Lipoproteínas LDL/fisiologia , Proteínas Inflamatórias de Macrófagos/biossíntese , Fosfolipídeos/metabolismoRESUMO
Fucans are sulphated polysaccharides extracted from brown seaweed, which display a wide scale of activities including inhibition of tumour cell invasion. Like several sulphated polysaccharides, they have been shown to be potent inhibitors of experimental metastasis. However, their mechanism of action is not fully understood Using standard adhesion and chemoinvasion assays, we demonstrated that fucans can inhibit MDA-MB231 cell invasion through matrigel. This effect is correlated with a direct interaction of the fucans with laminin that leads to an inhibition of cell adhesion. It depends upon the sulphate content and the molecular weight of the fucans. Moreover, chromogenic assays allowed us to bring to the fore an increase of u-PA activity in the MDA-MB231 culture medium when tests were performed in the presence of fucans. Since tumour cell adhesion is a prerequisite step in the invasion process, our results suggest that the inhibitory effect of fucans on MDA-MB231 cell invasion is caused, at least in part, by the blockage of tumour cell adhesion to the extracellular matrix and by the increase of the proteolysis of the extracellular membrane.
Assuntos
Neoplasias da Mama/patologia , Adesão Celular/efeitos dos fármacos , Fucose/farmacologia , Fitoterapia , Polissacarídeos/farmacologia , Adenocarcinoma/patologia , Biotinilação , Relação Dose-Resposta a Droga , Feminino , Humanos , Cinética , Invasividade Neoplásica , Alga Marinha , Células Tumorais CultivadasRESUMO
OBJECTIVE: To investigate the potential effects of angiogenic process by secretory phospholipase A2 (sPLA2) inhibitor-HyPE (linking N-derivatized phosphatidyl-ethanolamine to hyaluronic acid) on human bone marrow endothelial cell line (HBME-1). METHODS: In order to examine the suppressing effects of HyPE on HBME-1 proliferation, migration, and capillary-like tube formation, HBME-1 were activated hy angiogenic factor, specifically by basic fibroblast growth factor (b-FGF), vascular endothelial growth factor (VEGF), and oncostatin M (OSM) (at a final concentration of 25, 20, and 2.5 ng/mL, respectively), then HBME-1 proliferation, migration, and tube formation were studied in the absence or presence of HyPE. HBME-1 tube formation was specially analyzed in fibrin gel. RESULTS: HyPE effectively inhibited HBME-1 proliferation and migration as a dose-dependent manner, whatever HBME-1 were grown in the control culture medium or stimulated with b-FGF, VEGF, or OSM. In fibrin, the formations of HBME-1 derived tube-like structures were enhanced by all angiogenic factors, but these were strongly suppressed by HyPE. CONCLUSIONS: The results support the involvement of sPLA2 in angiogenesis. It is proposed that sPLA2 inhibitor introduces a novel approach in the control of cancer development.
Assuntos
Inibidores Enzimáticos/farmacologia , Neovascularização Patológica/patologia , Fosfolipases A/antagonistas & inibidores , Células da Medula Óssea/citologia , Capilares/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Células Endoteliais/citologia , Fator 2 de Crescimento de Fibroblastos/antagonistas & inibidores , Humanos , Ácido Hialurônico/farmacologia , Oncostatina M , Peptídeos/antagonistas & inibidores , Fosfatidiletanolaminas/farmacologia , Fosfolipases A2 , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidoresRESUMO
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.