RESUMO
OBJECTIVES: The objective was to assess the effect of ultrasound (US)-guidance compared to the anatomical landmark (AL) approach in patients requiring femoral artery (FA) access for coronary angiography/percutaneous coronary interventions (PCI). BACKGROUND: US-guidance has been proposed as a strategy to optimize FA access, potentially leading to decreased vascular complications. METHODS: Patients requiring FA access for coronary angiography/PCI were randomized to the US-guided or AL approaches. The primary endpoint was a composite of immediate procedural vascular outcomes, and access-site outcomes at day one. Results were subsequently pooled in a study-level meta-analysis of randomized trials comparing US-guided FA access to another strategy. RESULTS: A total of 129 patients were randomized (64 US-guided group; 65 AL group). The primary endpoint occurred in 30 patients (47%) with US, and in 39 patients (62%) with AL (P = 0.09). Four additional studies met the inclusion criteria and were included in the meta-analysis (1553 patients). Following data pooling, bleeding events (OR = 0.41; 95%CI 0.20-0.83; P = 0.01), venipunctures (OR = 0.18; 95%CI: 0.11-0.29; P < 0.0001), and multiple puncture attempts (OR = 0.24; 95%CI: 0.19-0.31; P < 0.0001) were significantly improved with US-guidance, but not successful common FA cannulation (OR = 0.84; 95%CI: 0.60-1.17; P = 0.29). CONCLUSION: Our study did not show significant benefits for the use of US to guide arterial femoral access compared to the anatomical landmark approach, but pooled analysis of five randomized trials showed decreased rates of bleeding events and venipunctures, and improved first-pass success. The clinical impact of these findings is uncertain, and do not warrant a systematic use of US-guidance in this clinical setting.
Assuntos
Perda Sanguínea Cirúrgica/estatística & dados numéricos , Cateterismo Periférico , Angiografia Coronária , Artéria Femoral , Intervenção Coronária Percutânea , Ultrassonografia de Intervenção/métodos , Idoso , Cateterismo Periférico/efeitos adversos , Cateterismo Periférico/métodos , Angiografia Coronária/efeitos adversos , Angiografia Coronária/métodos , Feminino , Artéria Femoral/diagnóstico por imagem , Artéria Femoral/cirurgia , Humanos , Masculino , Pessoa de Meia-Idade , Intervenção Coronária Percutânea/efeitos adversos , Intervenção Coronária Percutânea/métodos , Resultado do TratamentoRESUMO
Arginine methylation of histones is a well-known regulator of gene expression. Protein arginine methyltransferase 6 (PRMT6) has been shown to function as a transcriptional repressor by methylating the histone H3 arginine 2 [H3R2(me2a)] repressive mark; however, few targets are known. To define the physiological role of PRMT6 and to identify its targets, we generated PRMT6(-/-) mouse embryo fibroblasts (MEFs). We observed that early passage PRMT6(-/-) MEFs had growth defects and exhibited the hallmarks of cellular senescence. PRMT6(-/-) MEFs displayed high transcriptional levels of p53 and its targets, p21 and PML. Generation of PRMT6(-/-); p53(-/-) MEFs prevented the premature senescence, suggesting that the induction of senescence is p53-dependent. Using chromatin immunoprecipitation assays, we observed an enrichment of PRMT6 and H3R2(me2a) within the upstream region of Trp53. The PRMT6 association and the H3R2(me2a) mark were lost in PRMT6(-/-) MEFs and an increase in the H3K4(me3) activator mark was observed. Our findings define a new regulator of p53 transcriptional regulation and define a role for PRMT6 and arginine methylation in cellular senescence.
Assuntos
Proteínas de Drosophila/fisiologia , Regulação da Expressão Gênica , Proteína-Arginina N-Metiltransferases/fisiologia , Transcrição Gênica , Proteína Supressora de Tumor p53/genética , Animais , Células Cultivadas , Senescência Celular/genética , Proteínas de Drosophila/genética , Camundongos , Camundongos Knockout , Proteína-Arginina N-Metiltransferases/genética , Proteína Supressora de Tumor p53/metabolismo , Proteínas ras/genéticaRESUMO
Evidence has suggested that STAT3 functions as an oncogene in gliomagenesis. As a consequence, changes in the inflammatory microenvironment are thought to promote tumor development. Regardless of its origin, cancer-related inflammation has many tumor-promoting effects, such as the promotion of cell cycle progression, cell proliferation, cell migration and cell survival. Given that IL-6, a major cancer-related inflammatory cytokine, regulates STAT3 activation and is upregulated in glioblastoma, we sought to investigate the inhibitory effects of the chemopreventive flavonoid quercetin on glioblastoma cell proliferation and migration triggered by IL-6, and to determine the underlying mechanisms of action. In this study, we show that quercetin is a potent inhibitor of the IL-6-induced STAT3 signaling pathway in T98G and U87 glioblastoma cells. Exposure to quercetin resulted in the reduction of GP130, JAK1 and STAT3 activation by IL-6, as well as a marked decrease of the proliferative and migratory properties of glioblastoma cells induced by IL-6. Interestingly, quercetin also modulated the expression of two target genes regulated by STAT3, i.e. cyclin D1 and matrix metalloproteinase-2 (MMP-2). Moreover, quercetin reduced the recruitment of STAT3 at the cyclin D1 promoter and inhibited Rb phosphorylation in the presence of IL-6. Overall, these results provide new insight into the role of quercetin as a blocker of the STAT3 activation pathway stimulated by IL-6, with a potential role in the prevention and treatment of glioblastoma.
Assuntos
Glioblastoma/metabolismo , Interleucina-6/metabolismo , Quercetina/farmacologia , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Ciclina D1/biossíntese , Receptor gp130 de Citocina/biossíntese , Humanos , Janus Quinase 1/biossíntese , Metaloproteinase 2 da Matriz/biossíntese , Fosforilação/efeitos dos fármacos , Proteína do Retinoblastoma/metabolismo , Fator de Transcrição STAT3/antagonistas & inibidoresRESUMO
This work presents a fully flexible implantable neural probe fabricated with Polydimethylsiloxane (PDMS) and including a thermally-tunable stiffness microchannel filled with Polyester. The probe includes an optimized microfluidics mixer for drug delivery. Polyester, which is solid at room temperature and has a low melting point close to body temperature, is used to decrease the stiffness of the probe after insertion, after getting in contact with tissues. We designed a U-turn microchannel inside the PDMS neural probe and filled it up with melted polyester. The microchannel has a cross-section of 30 µm × 5 µm and a length of 14.7 mm. The following probe dimensions were chosen after extensive simulation: thickness = 20 µm, width = 300 µm, and length = 7 mm. These values yield a buckling force above 1 mN, which is sufficient for proper insertion into the brain tissues. Simulation results show that the microfluidics mixer with a cross-section of 90 µm × 5 µm and a length of 7 mm has optimum performance for the desired flow rate and quantity of drug to deliver. The pressure drop inside the microfluidic channel is less than 0.43 kPa, which is appropriate for PDMS-PDMS bonding, whereas the Reynolds number is near 1.91k in the laminar regime. No leakage or bubble occurred during the experimental validation, which suggests an appropriate pressure and a laminar flow in the channel.
Assuntos
Microfluídica , Poliésteres , Microfluídica/métodos , Dimetilpolisiloxanos , Simulação por ComputadorRESUMO
BACKGROUND: The 3-minute chair rise test (3-minute CRT) and the Disability Related to COPD Tool (DIRECT) are two reproducible and valid short tests that can assess the benefit of pulmonary rehabilitation (PR) in terms of functional capacity and dyspnea in everyday activities. METHODS: We determined the minimal clinically important difference (MCID) of the DIRECT questionnaire and 3-minute CRT using distribution methods and anchor encroaches with a panel of eight standard tests in a cohort of 116 COPD patients who completed a PR program in real-life settings. RESULTS: The estimated MCID for the 3-minute CRT and DIRECT scores was five repetitions and two units, respectively, using separate and combined independent anchors. The all-patient (body mass index-obstruction-dyspnea-exercise [BODE] scores 0-7), BODE 0-2 (n=42), and BODE 3-4 (n=50) groups showed improvements greater than the MCID in most tests and questionnaires used. In contrast, the BODE 5-7 group (n=24) showed improvements greater than MCID in only the 3-minute CRT, 6-minute walk test, endurance exercise test, and DIRECT questionnaire. DISCUSSION AND CONCLUSION: This study demonstrates that the short and simple DIRECT questionnaire and 3-minute CRT are responsive to capture the beneficial effects of a PR program in COPD patients, including those with severe disease. TRIAL REGISTRATION NUMBER: NCT03286660.
Assuntos
Avaliação da Deficiência , Teste de Esforço , Pulmão/fisiopatologia , Diferença Mínima Clinicamente Importante , Doença Pulmonar Obstrutiva Crônica/diagnóstico , Doença Pulmonar Obstrutiva Crônica/reabilitação , Inquéritos e Questionários , Atividades Cotidianas , Idoso , Europa (Continente) , Tolerância ao Exercício , Feminino , Nível de Saúde , Humanos , Masculino , Pessoa de Meia-Idade , Valor Preditivo dos Testes , Estudos Prospectivos , Doença Pulmonar Obstrutiva Crônica/fisiopatologia , Quebeque , Recuperação de Função Fisiológica , Índice de Gravidade de Doença , Fatores de Tempo , Resultado do Tratamento , Teste de CaminhadaRESUMO
We have previously demonstrated that human recombinant soluble melanotransferrin (hr-sMTf) interacts with the single-chain zymogen pro urokinase-type plasminogen activator (scu-PA) and plasminogen. In the present work, the impact of exogenous hr-sMTf on endothelial cells (EC) migration and morphogenic differentiation into capillary-like structures (tubulogenesis) was assessed. hr-sMTF at 10 nM inhibited by 50% the migration and tubulogenesis of human microvessel EC (HMEC-1). In addition, in hr-sMTf-treated HMEC-1, the expression of both urokinase-type plasminogen activator receptor (u-PAR) and low-density lipoprotein receptor-related protein (LRP) are down-regulated. However, fluorescence-activated cell sorting analysis revealed a 25% increase in cell surface u-PAR in hr-sMTf-treated HMEC-1, whereas the binding of the urokinase-type plasminogen activator (u-PA)*plasminogen activator inhibitor-1 (PAI-1) complex is decreased. This reduced u-PA-PAI-1 binding is correlated with a strong inhibition of the HMEC-1 plasminolytic activity, indicating that exogenous hr-sMTf treatment alters the internalization and recycling processes of free and active u-PAR at the cellular surface. Overall, these results demonstrate that exogenous hr-sMTf affects plasminogen activation at the cell surface, thus leading to the inhibition of EC movement and tubulogenesis. These results are the first to consider the potential use of hr-sMTf as a possible therapeutic agent in angiogenesis-related pathologies.
Assuntos
Células Endoteliais/efeitos dos fármacos , Proteínas Relacionadas a Receptor de LDL/metabolismo , Proteínas de Neoplasias/farmacologia , Receptores de Superfície Celular/metabolismo , Antígenos de Neoplasias , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Regulação para Baixo , Células Endoteliais/fisiologia , Humanos , Antígenos Específicos de Melanoma , Microtúbulos/fisiologia , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Receptores de Ativador de Plasminogênio Tipo Uroquinase , Proteínas Recombinantes/farmacologia , Ativador de Plasminogênio Tipo Uroquinase/metabolismoRESUMO
The concept of cancer prevention by use of naturally occuring substances that could be included in the diet is under investigation as a practical approach towards reducing cancer incidence, and therefore the mortality and morbidity associated with this disease. Tea, which is the most popularly consumed beverage aside from water, has been particularly associated with decreased risk of various proliferative diseases such as cancer and atherosclerosis in humans. Various studies have provided evidence that polyphenols are the strongest biologically active agents in green tea. Green tea polyphenols (GTPs) mainly consist of catechins (3-flavanols), of which (-)-epigallocatechin gallate is the most abundant and the most extensively studied. Recent observations have raised the possibility that green tea catechins, in addition to their antioxidative properties, also affect the molecular mechanisms involved in angiogenesis, extracellular matrix degradation, regulation of cell death and multidrug resistance. This article will review the effects and the biological activities of green tea catechins in relation to these mechanisms, each of which plays a crucial role in the development of cancer in humans. The extraction of polyphenols from green tea, as well as their bioavailability, are also discussed since these two important parameters affect blood and tissue levels of the GTPs and consequently their biological activities. In addition, general perspectives on the application of dietary GTPs as novel antiangiogenic and antitumor compounds are also presented.
Assuntos
Inibidores da Angiogênese/química , Antineoplásicos/química , Catequina/farmacologia , Flavonoides , Chá/química , Inibidores da Angiogênese/farmacocinética , Inibidores da Angiogênese/farmacologia , Animais , Antineoplásicos/farmacocinética , Antineoplásicos/farmacologia , Catequina/química , Catequina/farmacocinética , Humanos , Fenóis/química , Fenóis/farmacocinética , Fenóis/farmacologia , Polímeros/química , Polímeros/farmacocinética , Polímeros/farmacologiaRESUMO
Protein arginine methyltransferase 6 (PRMT6) is known to catalyze the generation of asymmetric dimethylarginine in polypeptides. Although the cellular role of PRMT6 is not well understood, it has been implicated in human immunodeficiency virus pathogenesis, DNA repair, and transcriptional regulation. PRMT6 is known to methylate histone H3 Arg-2 (H3R2), and this negatively regulates the lysine methylation of H3K4 resulting in gene repression. To identify in a nonbiased manner genes regulated by PRMT6 expression, we performed a microarray analysis on U2OS osteosarcoma cells transfected with control and PRMT6 small interfering RNAs. We identified thrombospondin-1 (TSP-1), a potent natural inhibitor of angiogenesis, as a transcriptional repression target of PRMT6. Moreover, we show that PRMT6-deficient U2OS cells exhibited cell migration defects that were rescued by blocking the secreted TSP-1 with a neutralizing peptide or blocking alpha-TSP-1 antibody. PRMT6 associates with the TSP-1 promoter and regulates the balance of methylation of H3R2 and H3K4, such that in PRMT6-deficient cells H3R2 was hypomethylated and H3K4 was trimethylated at the TSP-1 promoter. Using a TSP-1 promoter reporter gene, we further show that PRMT6 directly regulates the TSP-1 promoter activity. These findings show that TSP-1 is a transcriptional repression target of PRMT6 and suggest that neutralizing the activity of PRMT6 could inhibit tumor progression and therefore may be of cancer therapeutic significance.
Assuntos
Regulação Neoplásica da Expressão Gênica , Proteínas Nucleares/metabolismo , Proteína-Arginina N-Metiltransferases/metabolismo , Trombospondina 1/genética , Trombospondina 1/fisiologia , Transcrição Gênica , Linhagem Celular Tumoral , Movimento Celular , Metilação de DNA , Reparo do DNA , Progressão da Doença , Epigênese Genética , Humanos , Modelos Biológicos , Peptídeos/química , Regiões Promotoras GenéticasRESUMO
Histone tail post-translational modification results in changes in cellular processes, either by generating or blocking docking sites for histone code readers or by altering the higher order chromatin structure. H3K4me3 is known to mark the promoter regions of active transcription. Proteins bind H3K4 in a methyl-dependent manner and aid in the recruitment of histone-remodeling enzymes and transcriptional cofactors. The H3K4me3 binders harbor methyl-specific chromatin binding domains, including plant homeodomain, Chromo, and tudor domains. Structural analysis of the plant homeodomains present in effector proteins, as well as the WD40 repeats of WDR5, reveals critical contacts between residues in these domains and H3R2. The intimate contact between H3R2 and these domain types leads to the hypothesis that methylation of this arginine residue antagonizes the binding of effector proteins to the N-terminal tail of H3. Here we show that H3 tail binding effector proteins are indeed sensitive to H3R2 methylation and that PRMT6, not CARM1/PRMT4, is the primary methyltransferase acting on this site. We have tested the expression of a select group of H3K4 effector-regulated genes in PRMT6 knockdown cells and found that their levels are altered. Thus, PRMT6 methylates H3R2 and is a negative regulator of N-terminal H3 tail binding.
Assuntos
Arginina/química , Regulação da Expressão Gênica , Histonas/química , Animais , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , Humanos , Metilação , Proteínas Nucleares/química , Peptídeos/química , Ligação Proteica , Processamento de Proteína Pós-Traducional , Proteína-Arginina N-Metiltransferases/química , RNA/metabolismo , Transcrição GênicaRESUMO
Melanotransferrin (MTf), the membrane-bound human melanoma antigen p97, binds to plasminogen and stimulates its activation, thus regulating a crucial step involved in angiogenesis. In our study, a truncated soluble form of MTf (sMTf) inhibits, in a dose-dependent manner, the in vitro tubulogenesis of human umbilical vessel endothelial cells (HUVEC) induced by media conditioned with MTf-expressing cells. Following these results, we used the in vivo Matrigel plug angiogenesis assay to investigate whether truncated sMTf could inhibit neovascularization in mice. We found that basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF) and MTf-expressing cells strongly stimulate in vivo Matrigel neovascularization. However, subcutaneous (s.c.) administration of truncated sMTf inhibits both bFGF- and VEGF- as well as human melanoma SK-Mel-28-induced angiogenesis. This inhibition was dependent on the truncated sMTf concentration and reached maximal inhibition at 40 mg/kg/week. Furthermore, we found that a single s.c. injection of truncated sMTf into nude mice at 5 mg/kg produced a maximal blood concentration of approximately 40 nM, which is comparable with the level required to inhibit in vitro HUVEC tubulogenesis. Overall, our results strongly suggest that s.c. administration of truncated sMTf may provide a novel therapeutic strategy for the treatment of angiogenesis-related disorders.
Assuntos
Inibidores da Angiogênese/farmacologia , Proteínas de Neoplasias/farmacologia , Neovascularização Patológica , Antígenos de Neoplasias , Células Cultivadas , Fibrinolisina/fisiologia , Fator 2 de Crescimento de Fibroblastos/farmacologia , Humanos , Antígenos Específicos de Melanoma , Proteínas Recombinantes/farmacologia , Solubilidade , Fator A de Crescimento do Endotélio Vascular/farmacologiaRESUMO
We investigated the effect of plasminogen (Plg) on the internalization of recombinant soluble melanotransferrin (sMTf) using U87 human glioblastoma cells and murine embryonic fibroblasts (MEF) deficient in the low-density lipoprotein receptor-related protein (LRP). Using biospecific interaction analysis, both Glu- and Lys-Plg were shown to interact with immobilized sMTf. The binding of sMTf at the cell surface increased in the presence of both forms of Plg in control and in LRP-deficient MEF cells, whereas the uptake was strongly stimulated only by Lys-Plg in control MEF and U87 cells. In addition, in the presence of Lys-Plg, the internalization of sMTf was a saturable process, sensitive to temperature and dependent on the integrity of lysine residues. The addition of the receptor-associated protein, lactoferrin and aprotinin, as well as a monoclonal antibody (mAb) directed against LRP, inhibited the Lys-Plg-dependent uptake of sMTf. These results suggest an important role for LRP in this process. In addition, using binding and uptake assays in the presence of anti-annexin II mAb, we showed that annexin II might be responsible for the initial binding of sMTf in the presence of Plg. Our results suggest a Plg-mediated internalization mechanism for the clearance of sMTf via annexin II and LRP.
Assuntos
Anexina A2/metabolismo , Proteínas Relacionadas a Receptor de LDL/metabolismo , Proteínas de Neoplasias/metabolismo , Plasminogênio/metabolismo , Animais , Antígenos de Neoplasias , Linhagem Celular , Humanos , Proteínas Relacionadas a Receptor de LDL/deficiência , Proteínas Relacionadas a Receptor de LDL/genética , Ligantes , Lisina/metabolismo , Antígenos Específicos de Melanoma , Camundongos , Camundongos Knockout , Proteínas de Neoplasias/genética , Ligação Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , SolubilidadeRESUMO
Melanotransferrin is a glycoprotein expressed at the cell membrane and secreted in the extracellular environment. Recombinant truncated form of membrane-bound melanotransferrin (sMTf) was reported to exert in vitro anti-angiogenic properties. Here we show that sMTf treatment leads to a 50% inhibition of neovascularization in Matrigel implants when stimulated by growth factors. Using a glioblastoma xenograft model, we demonstrate that sMTf delivery at 2.5 and 10 mg/kg/day by micro-osmotic pump inhibits tumor growth by 73% and 91%, respectively. In a lung carcinoma xenograft model, sMTf treatment at 2.5 and 10 mg/kg/day impeded tumor growth by 87% and 97%. Furthermore, subcutaneous glioblastoma and lung carcinoma tumors from mice treated with 10 mg/kg/day of sMTf present insignificant growth toward the study. In association with a reduction in endoglin mRNA expression, the hemoglobin content decreased by half in sMTf-treated glioblastoma tumors. In vitro experiments revealed that NCI-H460 cells treated with sMTf display an inhibition in their invasive capabilities with a concomitant reduction in the expression of the low-density lipoprotein receptor protein and urokinase plasminogen activator receptor. Altogether, our results demonstrate that sMTf exerts anti-cancer and anti-angiogenic activities, suggesting that its administration may provide novel therapeutic strategies for the treatment of cancer.
Assuntos
Inibidores da Angiogênese/uso terapêutico , Antineoplásicos/uso terapêutico , Proteínas de Neoplasias/uso terapêutico , Neoplasias/tratamento farmacológico , Neovascularização Patológica/tratamento farmacológico , Animais , Antígenos de Neoplasias , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Fatores de Crescimento de Fibroblastos/antagonistas & inibidores , Fatores de Crescimento de Fibroblastos/farmacologia , Humanos , Antígenos Específicos de Melanoma , Camundongos , Camundongos Nus , Proteínas de Neoplasias/análise , Proteínas de Neoplasias/metabolismo , Proteínas de Neoplasias/farmacologia , Neoplasias/irrigação sanguínea , Neoplasias/metabolismo , Distribuição Tecidual , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Fator A de Crescimento do Endotélio Vascular/farmacologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The expression of melanotransferrin (MTf), a membrane-bound glycoprotein highly expressed in melanomas, is correlated with tumor vascularization and progression, suggesting a proinvasive function associated with MTf in malignant tumors. To test this hypothesis, we silenced MTf in human melanoma SK-MEL-28 cells using small interfering RNA (siRNA) and examined the plasmin activity and invasiveness of MTf-silenced melanoma. In vitro, the siRNA-mediated MTf knockdown inhibited by 58% the cell surface activation of plasminogen into plasmin. In addition, decreased expression of MTf in melanoma cells reduced cell migration. In vivo, we used a nude mice invasion model in which tissue factor (TF) induces vascular [125I]-fibrin deposition following injection. Using this metastasis model, the invasive potential of MTf-silenced cells into the lungs was reduced by fivefold. Altogether, these findings strongly suggest that MTf overexpression in melanoma cells contributes to tumor progression by stimulating plasmin generation as well as cell migration and invasion.
Assuntos
Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Melanoma/metabolismo , Melanoma/patologia , Invasividade Neoplásica/patologia , Proteínas de Neoplasias/metabolismo , Animais , Antígenos de Neoplasias , Linhagem Celular Tumoral , Movimento Celular , Humanos , Neoplasias Pulmonares/secundário , Masculino , Melanoma/secundário , Antígenos Específicos de Melanoma , CamundongosRESUMO
Integrins play an essential role in endothelial cell motility processes during angiogenesis and thus present interesting targets for the development of new anti-angiogenic agents. Snake venoms naturally contain a variety of proteins that can affect integrin-ligand interactions. Recently, the C-type lectin proteins (CLPs) have been characterized as efficient modulators of integrin functions. In this study, we investigated the anti-angiogenic activity of lebectin, a newly discovered CLP from Macrovipera lebetina venom. Human brain microvascular endothelial cells (HBMEC), used as an in vitro model, express alphavbeta3, alphavbeta5, and alpha5beta1 integrins, as well as the alpha2, alpha3, alpha6, and beta4 subunits. Our data show that lebectin acts as a very potent inhibitor (IC(50) approximately 0.5 nM) of HBMEC adhesion and migration on fibronectin by blocking the adhesive functions of both the alpha5beta1 and alphaV integrins. In addition, lebectin strongly inhibits both HBMEC in vitro tubulogenesis on Matrigel trade mark (IC(50) = 0.4 nM) and proliferation. Finally, using both a chicken CAM assay and a Matrigel trade mark Plug assay in nude mice, our results show that lebectin displays potent anti-angiogenic activity in vivo. Lebectin thus represents a new C-type lectin with anti-angiogenic properties with great potential for the treatment of angiogenesis-related diseases.
Assuntos
Inibidores da Angiogênese/farmacologia , Células Endoteliais/efeitos dos fármacos , Lectinas Tipo C/fisiologia , Neovascularização Patológica/prevenção & controle , Neovascularização Fisiológica/efeitos dos fármacos , Viperidae , Inibidores da Angiogênese/isolamento & purificação , Inibidores da Angiogênese/uso terapêutico , Animais , Encéfalo/irrigação sanguínea , Capilares/citologia , Capilares/efeitos dos fármacos , Adesão Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Embrião de Galinha , Membrana Corioalantoide/irrigação sanguínea , Membrana Corioalantoide/efeitos dos fármacos , Colágeno , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Combinação de Medicamentos , Técnicas de Cultura Embrionária , Células Endoteliais/metabolismo , Fibronectinas/farmacologia , Humanos , Integrinas/antagonistas & inibidores , Integrinas/metabolismo , Laminina , Lectinas Tipo C/isolamento & purificação , Lectinas Tipo C/uso terapêutico , Camundongos , Camundongos Nus , Neovascularização Patológica/induzido quimicamente , Proteoglicanas , Tela Subcutânea/irrigação sanguínea , Fatores de Tempo , Venenos de Víboras/isolamento & purificação , Venenos de Víboras/farmacologia , Venenos de Víboras/uso terapêuticoRESUMO
Epidemiological studies have shown that a diet rich in fruits and vegetables has a beneficial preventive effect on cardiovascular diseases and cancer by mechanisms that have not yet been elucidated. In this work, we investigated the antiangiogenic activities of anthocyanidins, a class of polyphenols present at high levels in fruits. Among the tested anthocyanidins (cyanidin, delphinidin, malvidin, pelargonidin, peonidin and petunidin), delphinidin was the most potent angiogenic inhibitor. In vitro, low concentrations of delphinidin inhibited vascular endothelial growth factor (VEGF)-induced tyrosine phosphorylation of VEGF receptor (VEGFR)-2, leading to the inhibition of downstream signaling triggered by VEGFR-2. Inhibition of VEGFR-2 by delphinidin inhibited the VEGF-induced activation of ERK-1/2 signaling and the chemotactic motility of human EC as well as their differentiation into capillary-like tubular structures in Matrigel and within fibrin gels. In vivo, delphinidin was able to suppress basic fibroblast growth factor-induced vessel formation in the mouse Matrigel plug assay. The identification of delphinidin as a naturally occurring inhibitor of VEGF receptors suggests that this molecule possesses important antiangiogenic properties that may be helpful for the prevention and treatment of cancer.
Assuntos
Antocianinas/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Inibidores da Angiogênese/farmacologia , Caspase 3 , Caspases/metabolismo , Movimento Celular , Sobrevivência Celular , Células Cultivadas , Colágeno/metabolismo , Relação Dose-Resposta a Droga , Combinação de Medicamentos , Humanos , Laminina/metabolismo , Modelos Químicos , Neoplasias/metabolismo , Fosforilação , Proteoglicanas/metabolismo , Veias Umbilicais/citologiaRESUMO
The activation of plasminogen at the cell surface is a crucial step in cell migration and invasion. In the present study, the effect of membrane-bound melanotransferrin (mMTf), also known as human melanoma antigen p97, on cell surface plasminogen binding and activation was investigated by using Chinese Hamster Ovary (CHO) cells transfected with full-length melanotransferrin (MTf) cDNA and SK-MeL-28 melanoma cells. The expression of mMTf in CHO increased cell surface plasminogen binding by about 2-fold. In addition, application of the monoclonal antibody L235 against MTf as well as truncated, soluble MTf (sMTf) abolished plasminogen binding to MTf-transfected and SK-MeL-28 cells, indicating that mMTf is a potential cell surface plasminogen receptor. Moreover, mMTf expression in CHO cells stimulates plasminogen activation at the cell surface by about 2.5-fold. In addition to the induced binding and activation of plasminogen, cell motility, migration and invasion were about 3-fold higher in CHO cells expressing mMTf. Both monoclonal antibody L235 and truncated sMTf inhibited mMTf-stimulated CHO cell motility, migration and invasion. Overall, our results indicate a key role for mMTf in cell surface plasminogen binding and in activation processes involved during cell migration and invasion.
Assuntos
Membrana Celular/metabolismo , Invasividade Neoplásica/fisiopatologia , Proteínas de Neoplasias/metabolismo , Plasminogênio/metabolismo , Receptores de Superfície Celular/metabolismo , Animais , Anticorpos Monoclonais/farmacologia , Antígenos de Neoplasias , Células CHO , Movimento Celular/fisiologia , Cricetinae , DNA Complementar/genética , Humanos , Antígenos Específicos de Melanoma , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/genética , Ativadores de Plasminogênio/metabolismo , Ligação Proteica/fisiologia , Receptores de Superfície Celular/genética , Receptores de Ativador de Plasminogênio Tipo Uroquinase , Transfecção , Regulação para Cima/fisiologiaRESUMO
We recently reported that human recombinant melanotransferrin (p97) presents a high transport rate across the blood-brain barrier that might involve the low-density lipoprotein receptor-related protein (LRP). We now report new interactions between p97 and another LRP ligand, the urokinase plasminogen activator (uPA) complex. By using biospecific interaction analysis, both pro-uPA and plasminogen are shown to interact with immobilized p97. Moreover, the activation of plasminogen by pro-uPA is increased by soluble p97. Because the uPA system plays a crucial role in cell migration, both in cancer and in angiogenesis, we also measured the impact of both endogenous membrane-bound and exogenous p97 on cell migration. The monoclonal antibody L235 (which recognizes a conformational epitope on p97) inhibited the migration of human microvascular endothelial cells (HMECs-1) and of human melanoma SK-MEL-28 cells, indicating that endogenous membrane-bound p97 could be associated with this process. In addition, low concentrations of exogenous p97 (10 and 100 nM) inhibited HMEC-1 and SK-MEL28 cell migration by more than 50%. These results indicate that membrane-bound and soluble p97 affect the migration capacity of endothelial and melanoma cells and suggest that p97 could be involved in the regulation of plasminogen activation by interacting with pro-uPA and plasminogen.