RESUMO
There is currently no therapy to limit the development of cardiac fibrosis and consequent heart failure. We have recently shown that cardiac fibrosis post-myocardial infarction (MI) can be regulated by resident cardiac cells with a fibrogenic signature and identified by the expression of PW1 (Peg3). Here we identify αV-integrin (CD51) as an essential regulator of cardiac PW1+ cells fibrogenic behavior. We used transcriptomic and proteomic approaches to identify specific cell-surface markers for cardiac PW1+ cells and found that αV-integrin (CD51) was expressed in almost all cardiac PW1+ cells (93% ± 1%), predominantly as the αVß1 complex. αV-integrin is a subunit member of the integrin family of cell adhesion receptors and was found to activate complex of latent transforming growth factor beta (TGFß at the surface of cardiac PW1+ cells. Pharmacological inhibition of αV-integrin reduced the profibrotic action of cardiac PW1+CD51+ cells and was associated with improved cardiac function and animal survival following MI coupled with a reduced infarct size and fibrotic lesion. These data identify a targetable pathway that regulates cardiac fibrosis in response to an ischemic injury and demonstrate that pharmacological inhibition of αV-integrin could reduce pathological outcomes following cardiac ischemia.
Assuntos
Integrina alfaV/efeitos dos fármacos , Infarto do Miocárdio/tratamento farmacológico , Venenos de Serpentes/uso terapêutico , Células Estromais/efeitos dos fármacos , Animais , Células Cultivadas , Avaliação Pré-Clínica de Medicamentos , Fibrose , Integrina alfaV/fisiologia , Fatores de Transcrição Kruppel-Like/análise , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Infarto do Miocárdio/genética , Infarto do Miocárdio/patologia , Miocárdio/patologia , Miócitos Cardíacos/metabolismo , RNA Mensageiro/biossíntese , Análise de Célula Única , Venenos de Serpentes/farmacologia , Células Estromais/química , Fator de Crescimento Transformador beta1/farmacologiaRESUMO
OBJECTIVES: To investigate the effect of simulated physiological stretch on the expression of extracellular matrix (ECM) proteins and the role of integrin α4/αv, focal adhesion kinase (FAK), extracellular regulated protein kinases 1/2 (ERK1/2) in the stretch-induced ECM protein expression of human bladder smooth muscle cells (HBSMCs). METHODS: HBSMCs were seeded onto silicone membrane and subjected to simulated physiological stretch at the range of 5, 10, and 15% elongation. Expression of primary ECM proteins in HBSMCs was analyzed by real-time polymerase chain reaction and Western blot. Specificity of the FAK and ERK1/2 was determined by Western blot with FAK inhibitor and ERK1/2 inhibitor (PD98059). Specificity of integrin α4 and integrin αv was determined with small interfering ribonucleic acid (siRNA) transfection. RESULTS: The expression of collagen I (Col1), collagen III (Col3), and fibronectin (Fn) was increased significantly under the simulated physiological stretch of 10 and 15%. Integrin α4 and αv, FAK, ERK1/2 were activated by 10% simulated physiological stretch compared with the static condition. Pretreatment of ERK1/2 inhibitor, FAK inhibitor, integrin α4 siRNA, or integrin αv siRNA reduced the stretch-induced expression of ECM proteins. And FAK inhibitor decreased the stretch-induced ERK1/2 activity and ECM protein expression. Integrin α4 siRNA or integrin αv siRNA inhibited the stretch-induced activity of FAK. CONCLUSION: Simulated physiological stretch increases the expression of ECM proteins in HBSMCs, and integrin α4/αv-FAK-ERK1/2 signaling pathway partly modulates the mechano-transducing process.
Assuntos
Proteínas da Matriz Extracelular/genética , Quinase 1 de Adesão Focal/genética , Integrina alfa4/genética , Integrina alfaV/genética , Sistema de Sinalização das MAP Quinases/genética , Miócitos de Músculo Liso/metabolismo , Fenômenos Biomecânicos , Western Blotting , Colágeno Tipo I/efeitos dos fármacos , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Colágeno Tipo III/efeitos dos fármacos , Colágeno Tipo III/genética , Colágeno Tipo III/metabolismo , Proteínas da Matriz Extracelular/efeitos dos fármacos , Proteínas da Matriz Extracelular/metabolismo , Fibronectinas/efeitos dos fármacos , Fibronectinas/genética , Fibronectinas/metabolismo , Flavonoides/farmacologia , Quinase 1 de Adesão Focal/efeitos dos fármacos , Quinase 1 de Adesão Focal/metabolismo , Humanos , Integrina alfa4/efeitos dos fármacos , Integrina alfa4/metabolismo , Integrina alfaV/efeitos dos fármacos , Integrina alfaV/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Miócitos de Músculo Liso/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , RNA Interferente Pequeno/farmacologia , Reação em Cadeia da Polimerase em Tempo Real , Transdução de Sinais , Bexiga Urinária/citologiaRESUMO
Low survival rates of metastatic cancers emphasize the need for a drug that can prevent and/or treat metastatic cancer. αv integrins are involved in essential processes for tumor growth and metastasis and targeting of αv integrins has been shown to decrease angiogenesis, tumor growth and metastasis. In this study, the role of αv integrin and its potential as a drug target in bladder cancer was investigated. Treatment with an αv integrin antagonist as well as knockdown of αv integrin in the bladder carcinoma cell lines, resulted in reduced malignancy in vitro, as illustrated by decreased proliferative, migratory and clonogenic capacity. The CDH1/CDH2 ratio increased, indicating a shift towards a more epithelial phenotype. This shift appeared to be associated with downregulation of EMT-inducing transcription factors including SNAI2. The expression levels of the self-renewal genes NANOG and BMI1 decreased as well as the number of cells with high Aldehyde Dehydrogenase activity. In addition, self-renewal ability decreased as measured with the urosphere assay. In line with these observations, knockdown or treatment of αv integrins resulted in decreased metastatic growth in preclinical in vivo models as assessed by bioluminescence imaging. In conclusion, we show that αv integrins are involved in migration, EMT and maintenance of Aldehyde Dehydrogenase activity in bladder cancer cells. Targeting of αv integrins might be a promising approach for treatment and/or prevention of metastatic bladder cancer.
Assuntos
Carcinoma de Células de Transição/patologia , Vetores Genéticos/farmacologia , Integrina alfaV/efeitos dos fármacos , Terapia de Alvo Molecular , Proteínas de Neoplasias/antagonistas & inibidores , Interferência de RNA , RNA Interferente Pequeno/farmacologia , Receptores de Vitronectina/antagonistas & inibidores , Neoplasias da Bexiga Urinária/patologia , Aldeído Desidrogenase/biossíntese , Aldeído Desidrogenase/genética , Animais , Adesão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Autorrenovação Celular/efeitos dos fármacos , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Coração , Humanos , Integrina alfaV/fisiologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Invasividade Neoplásica , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , Transplante de Neoplasias/métodos , Papiloma/patologia , Receptores de Vitronectina/fisiologia , Tíbia , Fatores de Transcrição/biossíntese , Fatores de Transcrição/genética , Transdução Genética , Ensaio Tumoral de Célula-Tronco , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
INTRODUCTION: It has been proved that integrin αv activity is related to cell proliferation, differentiation, migration, and organ development. However, the biological functions of integrin αv in human dental pulp cells (hDPCs) cultured on silicate-based materials have not been explored. The aim of this study was to investigate the role of integrin αv in the proliferation and odontogenic differentiation of hDPCs cultured with the effect of calcium silicate (CS) cement and ß-tricalcium phosphate (TCP) cement. METHODS: In this study, hDPCs were cultured on CS and TCP materials, and we evaluated fibronectin (FN) secretion and integrin αv expression during the cell attachment stage. After small interfering RNA transfection targeting integrin αv, the proliferation and odontogenesis differentiation behavior of hDPCs were analyzed. RESULTS: The results indicate that CS releases Si ion-increased FN secretion and adsorption, which promote cell attachment more effectively than TCP. The CS cement facilitates FN and αv subintegrin expression. However, the FN adsorption and integrin expression of TCP are similar to that observed in the control dish. Integrin αv small interfering RNA inhibited odontogenic differentiation of hDPCs with the decreased formation of mineralized nodules on CS. It also down-regulated the protein expression of multiple markers of odontogenesis and the expression of dentin sialophosphoprotein protein. CONCLUSIONS: These results establish composition-dependent differences in integrin binding and its effectiveness as a mechanism regulating cellular responses to biomaterial surface.
Assuntos
Compostos de Cálcio/farmacologia , Polpa Dentária/citologia , Integrina alfaV/fisiologia , Cimento de Silicato/farmacologia , Silicatos/farmacologia , Adsorção , Materiais Biocompatíveis/farmacologia , Calcificação Fisiológica/efeitos dos fármacos , Fosfatos de Cálcio/farmacologia , Adesão Celular/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Cimentos Dentários/farmacologia , Polpa Dentária/efeitos dos fármacos , Proteínas da Matriz Extracelular/efeitos dos fármacos , Fibronectinas/análise , Fibronectinas/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Humanos , Integrina alfaV/efeitos dos fármacos , Integrina alfaV/genética , Odontogênese/efeitos dos fármacos , Fosfoproteínas/efeitos dos fármacos , RNA Interferente Pequeno/genética , Sialoglicoproteínas/efeitos dos fármacos , TransfecçãoRESUMO
A non-steroidal anti-inflammatory drug, diclofenac, acts efficiently against inflammation; however, down-regulation of diclofenac on bone remodeling has raised concerns. The inhibitory mechanisms of diclofenac are poorly understood. We hypothesized that diclofenac down-regulates osteoclast differentiation and activation via inhibition of the translocation of phosphorylated nuclear factor kappa B (NFkappaB). When osteoclasts prepared from mouse hematopoietic stem cells were treated with diclofenac, tartrateresistant acid phosphatase-positive multinucleated cells decreased in a concentration-dependent manner. Pit formation assay revealed the abolition of osteoclastic bone resorption; levels of cathepsin K transcripts, an osteoclastic resorption marker, were down-regulated time-dependently. Diclofenac induced the accumulation of the inhibitor of kappa B in cytosol, which led to suppression of the nuclear translocation of NFkappaB and phosphorylated NFkappaB. These results suggest that the novel mechanism of diclofenac for bone remodeling is associated with phosphorylated NFkappaB reduction, which regulates osteoclast differentiation and activation.
Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Diclofenaco/farmacologia , Subunidade p50 de NF-kappa B/antagonistas & inibidores , Osteoclastos/efeitos dos fármacos , Transcrição Gênica/efeitos dos fármacos , Fosfatase Ácida/análise , Animais , Anti-Inflamatórios não Esteroides/administração & dosagem , Biomarcadores/análise , Reabsorção Óssea/fisiopatologia , Catepsina K , Catepsinas/análise , Contagem de Células , Diferenciação Celular/efeitos dos fármacos , Núcleo Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Cisteína Endopeptidases/análise , Citosol/efeitos dos fármacos , Diclofenaco/administração & dosagem , Relação Dose-Resposta a Droga , Regulação para Baixo , Células-Tronco Hematopoéticas/citologia , Integrina alfaV/efeitos dos fármacos , Integrina beta3/efeitos dos fármacos , Isoenzimas/análise , Masculino , Camundongos , Fosfatase Ácida Resistente a Tartarato , Fatores de Tempo , Fator de Transcrição RelA/antagonistas & inibidoresRESUMO
In order to determine whether integrin dynamics is associated with intracellular Ca(2+) concentration ([Ca(2+)](i)) mobilization in ECs in response to hemodynamic forces, changes in [Ca(2+)](i) in fluo-4-loaded cultured bovine aortic endothelial cells (BAECs) under fluid flow conditions were visualized employing laser scanning confocal microscopy. Following the onset of flow stimulus, transient increases in [Ca(2+)](i) occurred several times in individual BAECs during the 30-min observation period. The frequency of these [Ca(2+)](i) transients was clearly reduced by the application of an integrin antagonist (GRGDSP peptide). Furthermore, treatment of cells with an integrin activator (Mn(2+)) resulted in reduction of peak [Ca(2+)](i) levels and elevated frequency, which was markedly rescued upon GRGDSP administration. In contrast, an actin de-polymerizing agent (cytochalasin D) exerted no inhibitory effects; rather, cytochalasin D more likely facilitated [Ca(2+)](i) transients. Moreover, [Ca(2+)](i) transients, which were suppressed by short interference RNA-induced silencing of alphav integrin, exhibited greater frequently in cells cultured on vitronectin substratum in comparison with those cultured on fibronectin or collagen substratum. Either removal of extracellular Ca(2+), application of an inhibitor of endoplasmic reticulum Ca(2+)-ATPase (thapsigargin) or non-selective cation channel blocker (La(3+)) inhibited the [Ca(2+)](i) transients. Additionally, [Ca(2+)](i) transients were attenuated by extracellular signal-regulated kinase (ERK) kinase inhibitor (U0126); in contrast, [Ca(2+)](i) transients were unaffected by tyrosine kinase inhibitor (genistein) or phosphatidylinositol 3-kinase (PI3K) inhibitor (LY294002). Therefore, our findings revealed that alphav integrin dynamics modulates the frequency of flow-induced [Ca(2+)](i) transients in BAECs in an ERK-dependent fashion.
Assuntos
Cálcio/metabolismo , Células Endoteliais/metabolismo , Integrina alfaV/metabolismo , Actinas/metabolismo , Animais , Butadienos/farmacologia , Cálcio/farmacologia , Bovinos , Adesão Celular/efeitos dos fármacos , Células Cultivadas , Colágeno Tipo IV/farmacologia , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/metabolismo , Citoesqueleto/fisiologia , Relação Dose-Resposta a Droga , Células Endoteliais/citologia , Inibidores Enzimáticos/farmacologia , MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Fibronectinas/farmacologia , Integrina alfaV/efeitos dos fármacos , Integrina alfaV/genética , Manganês/farmacologia , Microscopia Confocal , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Modelos Biológicos , Nitrilas/farmacologia , Oligopeptídeos/farmacologia , Interferência de RNA , Reologia/métodos , Estresse Mecânico , Vitronectina/farmacologiaRESUMO
Integrin-mediated cell adhesion and signaling is essential to vascular development and inflammatory processes. Elevated expression of integrin alpha(v)beta(3) has been detected in ischemia-reperfusion injury and rejecting heart allografts. We thus hypothesized that the inhibition of alpha(v)-associated integrins may have potent anti-inflammatory effects in acute kidney allograft rejection. We studied the effects of a peptidomimetic antagonist of alpha(v) integrins in two rat models of renal allotransplantation, differing in degree of major histocompatibility complex mismatch. Integrin alpha(v)beta(3) was up-regulated in rejecting renal allografts. Integrin antagonist reduced the histological signs of acute rejection, the intensity of the mononuclear cell infiltration, and cell proliferation in the grafted kidneys. This could be correlated to a reduced leukocyte-endothelial interaction and an improved peritubular microcirculation observed by intravital microscopy. In vitro under laminar flow conditions, the arrest of monocytes to interleukin-1beta-activated endothelium was decreased. Furthermore, in co-culture models the proliferation and transmigration of monocytes/macrophages, endothelium, and fibroblasts induced by renal tubular epithelia was efficiently inhibited by alpha(v) integrin antagonism. These data reveal an important role of this integrin subclass in leukocyte recruitment and development and maintenance of acute rejection; blockade of alpha(v) integrins may provide a new therapeutic strategy to attenuate acute allograft rejection.
Assuntos
Rejeição de Enxerto/prevenção & controle , Integrina alfaVbeta3/antagonistas & inibidores , Transplante de Rim , Compostos Orgânicos/uso terapêutico , Animais , Adesão Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Técnicas de Cocultura , Fibroblastos/efeitos dos fármacos , Rejeição de Enxerto/patologia , Humanos , Inflamação/tratamento farmacológico , Integrina alfaV/efeitos dos fármacos , Migração e Rolagem de Leucócitos/efeitos dos fármacos , Leucócitos Mononucleares/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Masculino , Compostos Orgânicos/farmacologia , Ratos , Ratos Endogâmicos , Transplante HomólogoRESUMO
Angiogenesis is now understood to play a major role in the pathology of chronic inflammatory diseases and is indicated to exacerbate disease pathology. Recent evidence shows that angiogenesis is crucial during inflammatory bowel disease (IBD) and in experimental models of colitis. Examination of the relationship between angiogenesis and inflammation in experimental colitis shows that initiating factors for these responses simultaneously increase as disease progresses and correlate in magnitude. Recent studies show that inhibition of the inflammatory response attenuates angiogenesis to a similar degree and, importantly, that inhibition of angiogenesis does the same to inflammation. Recent data provide evidence that differential regulation of the angiogenic mediators involved in IBD-associated chronic inflammation is the root of this pathological angiogenesis. Many factors are involved in this phenomenon, including growth factors/cytokines, chemokines, adhesion molecules, integrins, matrix-associated molecules, and signaling targets. These factors are produced by various vascular, inflammatory, and immune cell types that are involved in IBD pathology. Moreover, recent studies provide evidence that antiangiogenic therapy is a novel and effective approach for IBD treatment. Here we review the role of pathological angiogenesis during IBD and experimental colitis and discuss the therapeutic avenues this recent knowledge has revealed.
Assuntos
Colite/fisiopatologia , Doenças Inflamatórias Intestinais/patologia , Doenças Inflamatórias Intestinais/terapia , Neovascularização Patológica , Inibidores da Angiogênese/uso terapêutico , Animais , Antígenos CD/fisiologia , Caderinas/fisiologia , Moléculas de Adesão Celular , Colo/química , Modelos Animais de Doenças , Humanos , Imunoglobulinas/fisiologia , Mediadores da Inflamação/fisiologia , Integrina alfaV/efeitos dos fármacos , Integrina alfaV/fisiologia , Molécula 1 de Adesão Intercelular/fisiologia , Peptídeos e Proteínas de Sinalização Intercelular/fisiologia , Metaloproteinases da Matriz/fisiologia , Camundongos , Microcirculação , Mucoproteínas/fisiologia , Neovascularização Patológica/prevenção & controle , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Molécula 1 de Adesão de Célula Vascular/fisiologia , Fator A de Crescimento do Endotélio Vascular/fisiologiaRESUMO
Anti-angiogenesis is a promising strategy for cancer therapy currently evaluated in clinical trials. The aim of the study was to investigate the effects of an antiangiogenic combination therapy inhibiting alpha(v)-integrins by a c(yclic)RGD-peptide (EMD270179) and blocking VEGFR-2 by SU5416 on tumor angiogenesis and progression in vivo. Experiments were performed in dorsal skinfold chamber preparations of Syrian golden hamsters (60 +/- 5 g) bearing A-Mel-3 tumors. From day 3-10 after tumor-cell implantation, animals (n = 6 per group) were treated by monotherapies using the cRGD-peptide (114 mg/kg/day; i.p.), the VEGFR-2 antagonist (6 mg/kg/day; i.p.) or by the combination of both monotherapies. A control group received only the solvent DMSO. Using intravital microscopy parameters of intratumoral microcirculation were analyzed on day 5, 7 and 10. In separate experiments subcutaneous tumor growth and metastasis formation was evaluated starting therapy on day 0. Functional vessel density was significantly reduced by the combination therapy compared to that by all other groups on day 10. Although intratumoral red blood cell velocity and vessel diameters were less affected, blood flow in vessel segments and the microcirculatory perfusion index were lower after combined therapy compared to controls. In addition, we observed a significantly stronger inhibition of subcutaneous tumor growth and metastasis formation using the combination therapy. These data clearly support the concept of antiangiogenic combination therapy and demonstrate that it may especially be effective when scheduled as an early or prophylactic treatment regimen, thus avoiding angiogenesis-dependent tumor and metastasis initiation or tumor recurrence.
Assuntos
Inibidores da Angiogênese/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Indóis/farmacologia , Integrina alfaV/efeitos dos fármacos , Neovascularização Patológica/prevenção & controle , Pirróis/farmacologia , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Animais , Cricetinae , Progressão da Doença , Masculino , Mesocricetus , Microcirculação/efeitos dos fármacos , Metástase Neoplásica/prevenção & controle , Recidiva Local de Neoplasia/prevenção & controleRESUMO
The intricate intracellular communication between stromal and epithelial cells, which involves cell-cell-, cell-insoluble extracellular matrix- (ECM), and cell-soluble factor-mediated signaling processes, is an attractive target for therapeutic intervention in hormone-refractory and bone-metastatic prostate cancer. In the present study we demonstrated that androgen-independent PC3 prostate cancer cells adhered to and migrated on vitronectin (VN), a major noncollagenous ECM in mature bone, through the expression of alphav-containing integrin receptors alphavbeta1 and alphavbeta5 on the cell surface, as determined by antibody function blocking assay and flow cytometry analysis. Small interfering RNAs (siRNAs) targeting human integrin alphav markedly reduced their respective mRNA and protein expression in cells, resulting in nearly complete reduction in VN-mediated cancer progression in vitro. In vivo quantitative bioluminescence analysis of human prostate cancer bone xenografts demonstrated for the first time that intratumoral administration of liposome-encapsulated human alphav-siRNAs significantly inhibits the growth of luciferase-tagged PC3 tumors in skeleton, which was associated with decreased integrin alphav expression and increased apoptosis in tumor cells. This integrin-based gene therapy is particularly suitable for the treatment of prostate cancer bone metastasis.
Assuntos
Neoplasias Ósseas/secundário , Inativação Gênica , Terapia Genética/métodos , Integrina alfaV/metabolismo , Neoplasias da Próstata/terapia , RNA Interferente Pequeno/uso terapêutico , Vitronectina/metabolismo , Animais , Neoplasias Ósseas/genética , Neoplasias Ósseas/terapia , Linhagem Celular Tumoral , Movimento Celular , Modelos Animais de Doenças , Inativação Gênica/efeitos dos fármacos , Humanos , Integrina alfaV/efeitos dos fármacos , Lipossomos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/farmacologia , Transplante HeterólogoRESUMO
This work reports on the role of the synergy peptide PHSRN in mediating the adhesion of cells. The attachment of baby hamster kidney cells and 3T3 Swiss fibroblasts to model substrates presenting either GRGDS or PHSRN was evaluated using self-assembled monolayers of alkanethiolates on gold presenting the peptide ligands mixed with tri(ethylene glycol) groups. These substrates permit rigorous control over the structures and densities of peptide ligands and at the same time prevent nonspecific interactions with adherent cells. Both cell types attached efficiently to monolayers presenting either the RGD or the PHSRN peptide but not to monolayers presenting scrambled peptide GRDGS or HRPSN. Cell attachment was comparable on substrates presenting either peptide ligand but less efficient than on substrates presenting the protein fibronectin. The degree of cell spreading, however, was substantially higher on substrates presenting RGD relative to PHSRN. Staining of 3T3 fibroblasts with anti-vinculin and phalloidin revealed clear cytoskeletal filaments and focal adhesions for cells attached by way of either RGD or PHSRN. Inhibition experiments showed that the attachment of 3T3 fibroblasts to monolayers presenting RGD could be inhibited completely by a soluble RGD peptide and partially by a soluble PHSRN peptide. IMR 90 fibroblast attachment to monolayers presenting PHSRN could be inhibited with anti-integrin alpha(5) or anti-integrin beta(1) antibody. This work demonstrates unambiguously that PHSRN alone can support the attachment of cells and that the RGD and PHSRN bind competitively to the integrin receptors.
Assuntos
Adesão Celular , Fibronectinas/farmacologia , Integrinas/efeitos dos fármacos , Oligopeptídeos/farmacologia , Fragmentos de Peptídeos/farmacologia , Citoesqueleto de Actina/química , Citoesqueleto de Actina/metabolismo , Animais , Anticorpos/imunologia , Ligação Competitiva , Cricetinae , Fibroblastos/química , Fibroblastos/efeitos dos fármacos , Fibronectinas/química , Integrina alfaV/efeitos dos fármacos , Integrina alfaV/imunologia , Integrina alfaV/fisiologia , Integrina beta1/efeitos dos fármacos , Integrina beta1/imunologia , Integrina beta1/fisiologia , Integrinas/imunologia , Integrinas/fisiologia , Camundongos , Oligopeptídeos/química , Fragmentos de Peptídeos/química , Células Swiss 3T3Assuntos
Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Ensaios Clínicos como Assunto , Integrina alfaV/efeitos dos fármacos , Venenos de Serpentes/farmacologia , Venenos de Serpentes/uso terapêutico , Animais , Antineoplásicos/química , Humanos , Neoplasias/tratamento farmacológico , Venenos de Serpentes/químicaRESUMO
We sought to assess how one tablet of non-enteric coated aspirin (325 mg) affects human platelets in subjects with risk factors for coronary artery disease. Data from 63 individuals with multiple cardiac risk factors were analyzed. Platelets were assessed twice at baseline (pre-aspirin), and after 3-4 h (post-aspirin). We employed 5 microM epinephrine-induced conventional aggregometry, closure time with epinephrine/collagen cartridge by PFA-100(R) (Dade-Behring), and aspirin response units (ARU) stimulated by propyl gallat with Ultegra (Accumetrics, San Diego, CA, USA) for measuring platelet function. In addition, the expression of platelet receptors was determined by using the following monoclonal antibodies: anti-CD31, CD41, CD42b, CD51/CD61, CD62p, CD63, CD107a, and CD151. Platelet-leukocyte formation was detected utilizing dual antibodies for a pan-platelet marker CD151, and CD14, a monocyte/macrophage marker. PAC-1 was used to measure fibrinogen-platelet binding. One pill of aspirin significantly decreased platelet-rich plasma (PRP) aggregation (74.18+/-16.75% vs. 24.92+/-8.64%; p<0.0001) and resulted in reduction of the aspirin response units (ARU) (662.24+/-65.65 vs. 451.05+/-69.31; p<0.0001). There was also prolongation of the closure time (194.4+/-25.3 vs. 258.63+/-55.61 s; p<0.0001). High correlation (r(2)=0.73-0.86) between platelet analyzer readings and aggregation was observed. One tablet of aspirin moderately inhibited expression of most surface platelet receptors measured, and such inhibition reached significance (p<0.05) for PAC-1, CD31, CD41, CD42, CD62p, and CD151. We conclude that a single dose of aspirin affects major platelet receptors, presumably directly or indirectly through the inhibition of prostanoids via platelet cyclooxygenase-1 blockade. The Ultegra Analyzer with a novel cartridge seems to be reliable in reflecting aspirins' effects on platelets and could be used in the future in clinical practice for monitoring aspirin therapy.
Assuntos
Aspirina/farmacologia , Doença da Artéria Coronariana/sangue , Inibidores da Agregação Plaquetária/farmacologia , Adulto , Antígenos CD/sangue , Antígenos CD/efeitos dos fármacos , Doença da Artéria Coronariana/prevenção & controle , Feminino , Citometria de Fluxo/instrumentação , Citometria de Fluxo/métodos , Humanos , Integrina alfaV/sangue , Integrina alfaV/efeitos dos fármacos , Receptores de Lipopolissacarídeos/sangue , Receptores de Lipopolissacarídeos/efeitos dos fármacos , Proteína 1 de Membrana Associada ao Lisossomo , Proteínas de Membrana Lisossomal , Masculino , Pessoa de Meia-Idade , Selectina-P/sangue , Selectina-P/efeitos dos fármacos , Agregação Plaquetária/efeitos dos fármacos , Molécula-1 de Adesão Celular Endotelial a Plaquetas/sangue , Molécula-1 de Adesão Celular Endotelial a Plaquetas/efeitos dos fármacos , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/efeitos dos fármacos , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Glicoproteínas da Membrana de Plaquetas/efeitos dos fármacos , Fatores de Risco , Tetraspanina 24 , Tetraspanina 30 , Resultado do TratamentoRESUMO
We have investigated the inhibitory effect of salmosin on integrin-mediated human tumour cell proliferation. SK-Mel-2 human melanoma cell adhesion to denatured collagen or vitronectin was found to be significantly and statistically inhibited by salmosin in a dose-dependent manner (P<0.05). Moreover, the binding of SK-Mel-2 cells to salmosin-coated plates was specifically disrupted by anti-integrin alphav monoclonal antibody at 8 microg mL(-1), but not by anti-integrin monoclonal antibody. These findings indicated that salmosin inhibited the adhesion of SK-Mel-2 cells to denatured collagen by specifically blocking integrin alphav. The proliferation of SK-Mel-2 cells on a denatured collagen-coated plate was statistically and significantly inhibited by salmosin induced apoptosis in a dose-dependent manner (P<0.05). Anti-integrin alphav monoclonal antibody, anti-integrin alphavbeta3 monoclonal antibody, and synthetic RGD peptide also suppressed SK-Mel-2 cell proliferation. Several lines of experimental evidence strongly suggested that the inhibition of SK-Mel-2 cell proliferation by salmosin was due to the induction of apoptosis via the blocking of integrin alphav-mediated cell survival.
Assuntos
Venenos de Crotalídeos/farmacologia , Integrina alfaV/efeitos dos fármacos , Melanoma/tratamento farmacológico , Apoptose/efeitos dos fármacos , Adesão Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Humanos , Melanoma/metabolismo , Células Tumorais CultivadasRESUMO
Anchorage of cells to the extracellular matrix and integrin-mediated signals play crucial roles in cell survival. We have previously shown that during growth factor deprivation-induced apoptosis in human umbilical vein endothelial cells (HUVECs), key molecules in focal adhesions and adherens junctions are cleaved by caspases. In this study we provide evidence for a selective upregulation of cell-associated matrix metalloproteinases (MMPs). We observe a physical association of MMP2 with beta1 and alphav integrins, which increased three- to fourfold during apoptosis and is dependent upon integrin beta1 levels and activation state. Both enforced activation of beta1 integrin by a specific antibody and inhibition of MMPs protect HUVECs from apoptosis. We hypothesize that, prior to the commitment to apoptosis, 'inside-out' signals initiated by the apoptotic stimulus alter cell shape together with the activation states and/or the availability of integrins, which promote matrix-degrading activity around dying cells. This 'auxiliary' apoptotic pathway may interrupt ECM-mediated survival signaling, and thus accelerate the efficient execution of the cell death program.
Assuntos
Apoptose/fisiologia , Adesão Celular/fisiologia , Sobrevivência Celular/fisiologia , Endotélio Vascular/enzimologia , Matriz Extracelular/enzimologia , Integrinas/metabolismo , Metaloendopeptidases/metabolismo , Apoptose/efeitos dos fármacos , Adesão Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Proteínas do Citoesqueleto/metabolismo , Endotélio Vascular/citologia , Endotélio Vascular/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Humanos , Integrina alfaV/efeitos dos fármacos , Integrina alfaV/metabolismo , Integrina beta1/efeitos dos fármacos , Integrina beta1/metabolismo , Integrinas/efeitos dos fármacos , Metaloproteinase 2 da Matriz/efeitos dos fármacos , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinases da Matriz Associadas à Membrana , Metaloendopeptidases/antagonistas & inibidores , Paxilina , Fosfoproteínas/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologiaRESUMO
A novel series of potent and specific alpha(v) integrin antagonists has been obtained by aminoalkyl substitutions on benzocyloheptene acetic acids as a rigid GD bioisostere. The preferred compounds 1-2, 1-3 and 1-8, showed nano- to subnanomolar IC(50) values on alpha(v)beta(3) and alpha(v)beta(5) integrins, with favorable pharmacokinetics.
Assuntos
Benzocicloeptenos/síntese química , Benzocicloeptenos/farmacologia , Integrina alfaV/efeitos dos fármacos , Animais , Disponibilidade Biológica , Adesão Celular/efeitos dos fármacos , Permeabilidade da Membrana Celular , Humanos , Concentração Inibidora 50 , Integrina alfaVbeta3/antagonistas & inibidores , Integrinas/antagonistas & inibidores , Microssomos Hepáticos/metabolismo , Conformação Molecular , Oligopeptídeos , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/antagonistas & inibidores , Ratos , Receptores de Vitronectina/antagonistas & inibidores , Relação Estrutura-Atividade , Células Tumorais CultivadasRESUMO
It has recently been suggested by several investigators that the epithelial-mesenchymal transition-inducing capacity of TGFbetas contributes to invasive transition of tumors at later stages of carcinogenesis. In the present study, we examined the possibility of TGFbeta1-stimulated epithelial-mesenchymal transition in SiHa cell line, detailed molecular events in the process, and its possible contribution to the invasive transition of tumors. TGFbeta1-induced epithelial-mesenchymal transition of SiHa cells was based on morphological and biochemical criteria; actin stress fiber formation, focal translocalization of integrin alphav, talin, and vinculin, fibronectin-based matrix assembly at the cell periphery, and translocalization and down-regulation of E-cadherin. TGFbeta1 also stimulated surface expression of integrin alphavbeta3 and FAK activation. Focal translocalization of integrin alphav preceded actin reorganization and fibronectin matrix assembly, and functional blocking of the integrin suppressed actin stress fiber formation. Furthermore, induction of actin reorganization and fibronectin matrix assembly by TGFbeta1 were shown to be mutually independent events. These changes were irreversible because 5 minutes pulse exposure to TGFbeta1 was sufficient to stimulate progress of actin reorganization and fibronectin matrix assembly. In further studies with raft culture, TGFbeta1 was found to stimulate invasion of SiHa cells into a type I collagen gel matrix. In conclusion, TGFbeta1 stimulated epithelial-mesenchymal transition of SiHa cells, indicating a positive role in the invasive transition of tumors.