RESUMO
Fibroproliferative disorders such as systemic sclerosis (SSc) have no effective therapies and result in significant morbidity and mortality. We recently demonstrated that the C-terminal domain of endostatin, known as E4, prevented and reversed both dermal and pulmonary fibrosis. Our goal was to identify the mechanism by which E4 abrogates fibrosis and its cell surface binding partner(s). Our findings show that E4 activated the urokinase pathway and increased the urokinase plasminogen activator (uPA) to type 1 plasminogen activator inhibitor (PAI-1) ratio. In addition, E4 substantially increased MMP-1 and MMP-3 expression and activity. In vivo, E4 reversed bleomycin induction of PAI-1 and increased uPA activity. In patients with SSc, the uPA/PAI-1 ratio was decreased in both lung tissues and pulmonary fibroblasts compared with normal donors. Proteins bound to biotinylated-E4 were identified as enolase-1 (ENO) and uPA receptor (uPAR). The antifibrotic effects of E4 required uPAR. Further, ENO mediated the fibrotic effects of TGF-ß1 and exerted TGF-ß1-independent fibrotic effects. Our findings suggest that the antifibrotic effect of E4 is mediated, in part, by regulation of the urokinase pathway and induction of MMP-1 and MMP-3 levels and activity in a uPAR-dependent manner, thus promoting extracellular matrix degradation. Further, our findings identify a moonlighting function for the glycolytic enzyme ENO in fibrosis.
Assuntos
Proteínas E4 de Adenovirus/imunologia , Biomarcadores Tumorais/imunologia , Proteínas de Ligação a DNA/imunologia , Fibrose/imunologia , Fosfopiruvato Hidratase/imunologia , Proteínas Supressoras de Tumor/imunologia , Ativador de Plasminogênio Tipo Uroquinase/imunologia , Humanos , Transdução de Sinais , TransfecçãoAssuntos
Autoanticorpos/sangue , Dermatomiosite/diagnóstico , Adulto , Idoso , Antígenos Ly/imunologia , Autoanticorpos/imunologia , Autoanticorpos/isolamento & purificação , Biomarcadores/sangue , Dermatomiosite/sangue , Dermatomiosite/imunologia , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Helicase IFIH1 Induzida por Interferon/imunologia , Japão , Masculino , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase/imunologia , Pessoa de Meia-Idade , Estudos Retrospectivos , Índice de Gravidade de Doença , Fatores de Transcrição/imunologia , Ativador de Plasminogênio Tipo Uroquinase/imunologiaRESUMO
To develop a machine learning (ML) model that predicts disease groups or autoantibodies in patients with idiopathic inflammatory myopathies (IIMs) using muscle MRI radiomics features. Twenty-two patients with dermatomyositis (DM), 14 with amyopathic dermatomyositis (ADM), 19 with polymyositis (PM) and 19 with non-IIM were enrolled. Using 2D manual segmentation, 93 original features as well as 93 local binary pattern (LBP) features were extracted from MRI (short-tau inversion recovery [STIR] imaging) of proximal limb muscles. To construct and compare ML models that predict disease groups using each set of features, dimensional reductions were performed using a reproducibility analysis by inter-reader and intra-reader correlation coefficients, collinearity analysis, and the sequential feature selection (SFS) algorithm. Models were created using the linear discriminant analysis (LDA), quadratic discriminant analysis (QDA), support vector machine (SVM), k-nearest neighbors (k-NN), random forest (RF) and multi-layer perceptron (MLP) classifiers, and validated using tenfold cross-validation repeated 100 times. We also investigated whether it was possible to construct models predicting autoantibody status. Our ML-based MRI radiomics models showed the potential to distinguish between PM, DM, and ADM. Models using LBP features provided better results, with macro-average AUC values of 0.767 and 0.714, accuracy of 61.2 and 61.4%, and macro-average recall of 61.9 and 59.8%, in the LDA and k-NN classifiers, respectively. In contrast, the accuracies of radiomics models distinguishing between non-IIM and IIM disease groups were low. A subgroup analysis showed that classification models for anti-Jo-1 and anti-ARS antibodies provided AUC values of 0.646-0.853 and 0.692-0.792, with accuracy of 71.5-81.0 and 65.8-78.3%, respectively. ML-based TA of muscle MRI may be used to predict disease groups or the autoantibody status in patients with IIM and is useful in non-invasive assessments of disease mechanisms.
Assuntos
Dermatomiosite/diagnóstico , Interpretação de Imagem Assistida por Computador/métodos , Aprendizado de Máquina , Músculos/diagnóstico por imagem , Polimiosite/diagnóstico , Adulto , Idoso , Anticorpos Antinucleares/análise , Anticorpos Antinucleares/imunologia , Antígenos Ly/imunologia , Biópsia , Dermatomiosite/imunologia , Dermatomiosite/patologia , Diagnóstico Diferencial , Feminino , Humanos , Imageamento por Ressonância Magnética/métodos , Masculino , Pessoa de Meia-Idade , Músculos/imunologia , Músculos/patologia , Polimiosite/imunologia , Polimiosite/patologia , Curva ROC , Reprodutibilidade dos Testes , Estudos Retrospectivos , Ativador de Plasminogênio Tipo Uroquinase/imunologiaRESUMO
Plasminogen activator inhibitor-1 (PAI-1) is the main physiological inhibitor of plasminogen activators (PAs) and is therefore an important inhibitor of the plasminogen/plasmin system. Being the fast-acting inhibitor of tissue-type PA (tPA), PAI-1 primarily attenuates fibrinolysis. Through inhibition of urokinase-type PA (uPA) and interaction with biological ligands such as vitronectin and cell-surface receptors, the function of PAI-1 extends to pericellular proteolysis, tissue remodeling and other processes including cell migration. This review aims at providing a general overview of the properties of PAI-1 and the role it plays in many biological processes and touches upon the possible use of PAI-1 inhibitors as therapeutics.
Assuntos
Doenças Cardiovasculares , Movimento Celular/imunologia , Fibrinólise/imunologia , Proteínas de Neoplasias , Neoplasias , Inibidor 1 de Ativador de Plasminogênio , Proteólise , Doenças Cardiovasculares/tratamento farmacológico , Doenças Cardiovasculares/imunologia , Doenças Cardiovasculares/metabolismo , Doenças Cardiovasculares/patologia , Fibrose , Humanos , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/imunologia , Proteínas de Neoplasias/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/imunologia , Neoplasias/patologia , Inibidor 1 de Ativador de Plasminogênio/imunologia , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Ativador de Plasminogênio Tipo Uroquinase/imunologia , Ativador de Plasminogênio Tipo Uroquinase/metabolismoRESUMO
Many studies have shown that urokinase plasminogen activator (uPA) is causally involved in promoting cancer invasion and metastasis. Thus, monitoring uPA levels could be very useful in cancer diagnosis, identification of initial metastasis, and guiding cancer treatment. Here, the authors developed a novel and scalable uPA sensor based on a graphene-gold nanoparticle platform that uses fluorescence of quantum dots to rapidly (<1 h) detect uPA up to 100 pM. Indeed, the authors' sensor is highly selective and showed an ability to sense up to 100 pM uPA even in the presence of complex biological milieu such as the fetal bovine serum.
Assuntos
Ouro/química , Grafite/química , Nanopartículas Metálicas/química , Papel , Ativador de Plasminogênio Tipo Uroquinase/análise , Anticorpos/química , Anticorpos/imunologia , Técnicas Biossensoriais/métodos , Teoria da Densidade Funcional , Humanos , Imunoensaio , Limite de Detecção , Neoplasias/diagnóstico , Proteínas Recombinantes/análise , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Ativador de Plasminogênio Tipo Uroquinase/imunologiaRESUMO
BACKGROUND: In early phase of acute pancreatitis (AP), systemic inflammatory response syndrome may lead to organ failure. The severe form of AP is associated with high mortality that may be prevented by timely diagnosis and treatment of the predicted severe cases. Serum interleukin 6 (IL-6) and urokinase-type plasminogen activator receptor (uPAR) have been proposed as accurate early markers of severe AP. The aim of the study was to assess whether widely available blood count indexes: neutrophil to lymphocyte (NLR), lymphocyte to monocyte (LMR) and platelet to lymphocyte ratios correlate with IL-6 and uPAR and may be utilized to predict organ complications at the early phase of AP. METHODS: The study included 95 adult patients with AP treated at the Surgical Ward Complex of Health Care Centers in Wadowice, Poland. Organ failure was diagnosed according to modi ed Marshall scoring system, as recommended by 2012 Atlanta classification. Blood samples for laboratory tests were collected on days 1, 2 and 3 following the onset of AP symptoms. RESULTS: Patients with organ failure presented significantly lower LMR on day 1 and significantly higher NLR on days 2 and 3. Strong positive correlations between NLR and IL-6 and moderate correlations between NLR and uPAR were observed throughout the study. Day 2 and 3 NLR values significantly predicted organ failure at the early phase of AP. CONCLUSIONS: Taking into account the wide availability of NLR, it may be considered as a surrogate of more expensive tests to help the early assessment of organ failure complicating AP.
Assuntos
Biomarcadores/sangue , Interleucina-6/imunologia , Linfócitos/imunologia , Neutrófilos/imunologia , Pancreatite/imunologia , Ativador de Plasminogênio Tipo Uroquinase/imunologia , Doença Aguda , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Interleucina-6/sangue , Masculino , Pessoa de Meia-Idade , Pancreatite/sangue , Pancreatite/fisiopatologia , Polônia , Prognóstico , Estudos Prospectivos , Ativador de Plasminogênio Tipo Uroquinase/sangueRESUMO
OBJECTIVE AND DESIGN: To determine the requirement of plasminogen activator inhibitor-1-knockout (PAI-1) for monocyte adhesion in animals and cells under diabetic conditions. METHODS AND SUBJECTS: Monocyte adhesion assay, enzyme-linked immunosorbent assay, and Western blotting were used in analyzing samples from PAI-1-knockout (PAI-1-KO) mice or cultured human umbilical vein endothelial cells (HUVEC). TREATMENTS: Diabetes in PAI-1-KO and wild-type mice was induced by intraperitoneal injection of streptozotocin (STZ). HUVEC was transfected with short interference RNA (siRNA) against PAI-1, tumor necrosis factor-α (TNFα), or toll-like receptor (TLR4), and then was treated with glycated low-density lipoproteins (glyLDL). RESULTS: The adhesion of monocytes to aortic intima was reduced in PAI-1-KO mice, which was associated with decreased levels of TNFα and monocyte chemotactic protein-1 (MCP-1) in plasma and cardiovascular tissue, and increased abundances of urokinase plasminogen activator (uPA) and uPA receptor (uPAR) in cardiovascular tissue compared to wild-type mice. Significant reductions in monocyte adhesion, inflammatory, and fibrinolytic regulators were detected in cardiovascular tissue or plasma in diabetic PAI-1-KO mice compared to wild-type diabetic mice. Transfection of PAI-1, TNFα or TLR4 siRNA to HUVEC inhibited glyLDL-induced monocyte adhesion to EC. PAI-1 siRNA inhibited the abundances of TLR4 and TNFα in EC. CONCLUSION: The findings suggest that PAI-1 is required for diabetes-induced monocyte adhesion via interactions with uPA/uPAR, and it also regulates TLR4 and TNFα expression in vascular EC. Inhibition of PAI-1 potentially reduces vascular inflammation under diabetic condition.
Assuntos
Diabetes Mellitus Experimental/imunologia , Monócitos/imunologia , Serpina E2/imunologia , Animais , Antígenos/sangue , Aorta/fisiologia , Adesão Celular , Quimiocina CCL2/sangue , Quimiocina CCL2/imunologia , Diabetes Mellitus Experimental/sangue , Diabetes Mellitus Experimental/fisiopatologia , Células Endoteliais da Veia Umbilical Humana/imunologia , Células Endoteliais da Veia Umbilical Humana/fisiologia , Humanos , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Monócitos/fisiologia , RNA Interferente Pequeno/genética , Receptores de Ativador de Plasminogênio Tipo Uroquinase/imunologia , Serpina E2/genética , Receptor 4 Toll-Like/genética , Fator de Necrose Tumoral alfa/sangue , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologia , Ativador de Plasminogênio Tipo Uroquinase/imunologiaRESUMO
Hepatocellular carcinoma (HCC) is the second leading cause of cancer-related death and its prognosis remains poor due to the high risk of tumor recurrence and metastasis. Berberine (BBR) is a natural compound derived from some medicinal plants, and accumulating evidence has shown its potent anti-tumor activity with diverse action on tumor cells, including inducing cancer cell death and blocking cell cycle and migration. Molecular targets of berberine involved in its inhibitory effect on the invasiveness remains not yet clear. In this study, we identified that berberine exhibits a potent inhibition on the invasion and migration of HCC cells. This was accompanied by a dose-dependent down-regulation of expression of Cyclooxygenase-2 (COX-2), nuclear factor kappa B (NF-κB), urokinase-type plasminogen activator (uPA) and matrix metalloproteinase (MMP)-9 in berberine-treated HCC cells. Furthermore, berberine inactivated p38 and Erk1/2 signaling pathway in HCC cells. Primarily, this may be attributed to the up-regulation of plasminogen activator inhibitor-1 (PAI-1), a tumor suppressor that can antagonize uPA receptor and down-regulation of uPA. Blockade of uPA receptor-associated pathways leads to reduced invasiveness and motility of berberine-treated HCC cells. In conclusion, our findings identified for the first time that inactivation of uPA receptor by up-regulation of PAI-1 and down-regulation of uPA is involved in the inhibitory effect of berberine on HCC cell invasion and migration.
Assuntos
Anti-Inflamatórios/farmacologia , Berberina/farmacologia , Carcinoma Hepatocelular/tratamento farmacológico , Neoplasias Hepáticas/tratamento farmacológico , Inibidor 1 de Ativador de Plasminogênio/imunologia , Ativador de Plasminogênio Tipo Uroquinase/imunologia , Carcinoma Hepatocelular/imunologia , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Humanos , Inflamação/tratamento farmacológico , Inflamação/imunologia , Inflamação/patologia , Fígado/efeitos dos fármacos , Fígado/imunologia , Fígado/patologia , Neoplasias Hepáticas/imunologia , Neoplasias Hepáticas/patologia , Invasividade Neoplásica/patologia , Invasividade Neoplásica/prevenção & controle , Inibidor 1 de Ativador de Plasminogênio/análise , Regulação para Cima/efeitos dos fármacos , Ativador de Plasminogênio Tipo Uroquinase/análiseRESUMO
Ischaemic stroke, accompanied by neuroinflammation, impairs blood-brain barrier integrity through a complex mechanism involving both protein kinase C (PKC) and urokinase. Using an in vitro model of human blood-brain barrier (BBB) composed of brain microvascular endothelial cells (HBMEC) and astrocytes, this study assessed the putative roles of these elements in BBB damage evoked by enhanced availability of pro-inflammatory cytokine, TNF-α. Treatment of HBMEC with TNF-α significantly increased the mRNA and protein expressions of all plasminogen-plasmin system (PPS) components, namely tissue plasminogen activator, urokinase, urokinase plasminogen activator receptor and plasminogen activator inhibitor-1 and also the activities of urokinase, total PKC and extracellular MMP-2. Inhibition of urokinase by amiloride abated the effects of TNF-α on BBB integrity and MMP-2 activity without affecting that of total PKC. Conversely, pharmacological inhibition of conventional PKC isoforms dramatically suppressed TNF-α-induced overactivation of urokinase. Knockdown of PKC-α gene via specific siRNA in HBMEC suppressed the stimulatory effects of TNF-α on protein expression of all PPS components, MMP-2 activity, DNA fragmentation rates and pro-apoptotic caspase-3/7 activities. Establishment of co-cultures with BMEC transfected with PKC-α siRNA attenuated the disruptive effects of TNF-α on BBB integrity and function. This was partly due to elevations observed in expression of a tight junction protein, claudin-5 and partly to prevention of stress fibre formation. In conclusion, specific inhibition of PKC-α in cerebral conditions associated with exaggerated release of pro-inflammatory cytokines, notably TNF-α may be of considerable therapeutic value and help maintain endothelial cell viability, appropriate cytoskeletal structure and basement membrane.
Assuntos
Barreira Hematoencefálica/patologia , Fibrinolisina/imunologia , Inflamação/patologia , Metaloproteinase 2 da Matriz/imunologia , Plasminogênio/imunologia , Proteína Quinase C-alfa/imunologia , Fator de Necrose Tumoral alfa/imunologia , Apoptose , Barreira Hematoencefálica/imunologia , Linhagem Celular , Inativação Gênica , Humanos , Inflamação/imunologia , Proteína Quinase C-alfa/genética , Ativador de Plasminogênio Tipo Uroquinase/imunologiaRESUMO
Transplantation of the pancreatic islets of Langerhans (islets) is a promising cell therapy for treating insulin-dependent type 1 diabetes mellitus. Islet transplantation is a minimally-invasive technique involving relatively simple surgery. However, after intraportal transplantation, the transplanted islets are attacked by the recipient's immune system, because they activate a number of systems, including coagulation, complement response, inflammation, immune rejection, and recurrence of autoimmune disease. We have developed a surface modification and microencapsulation technique that protects cells and islets with biomaterials and bioactive substances, which may be useful in clinical settings. This approach employs amphiphilic polymers, which can interact with lipid bilayer membranes, without increasing cell volume. Molecules attached to these polymers can protect transplanted cells and islets from attack by the host immune system. We expect that this surface modification technique will improve graft survival in clinical islet transplantation.
Assuntos
Diabetes Mellitus Experimental/terapia , Rejeição de Enxerto/prevenção & controle , Transplante das Ilhotas Pancreáticas/métodos , Ilhotas Pancreáticas/química , Bicamadas Lipídicas/química , Polietilenoglicóis/química , Animais , Glicemia/metabolismo , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/imunologia , Diabetes Mellitus Experimental/patologia , Rejeição de Enxerto/imunologia , Rejeição de Enxerto/patologia , Sobrevivência de Enxerto , Proteínas Imobilizadas/química , Proteínas Imobilizadas/imunologia , Ilhotas Pancreáticas/citologia , Ilhotas Pancreáticas/imunologia , Transplante das Ilhotas Pancreáticas/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Transplante Isogênico , Ativador de Plasminogênio Tipo Uroquinase/química , Ativador de Plasminogênio Tipo Uroquinase/imunologiaRESUMO
Urokinase-type plasminogen activator (uPA) is a trypsin-like serine protease that plays a vital role in extracellular conversion of inactive plasminogen into catalytically active plasmin. Activated plasmin facilitates the release of several proteolytic enzymes, which control processes like pericellular proteolysis and remodeling of ECM. uPA and the receptor uPAR, are overexpressed in a number of malignant tumours and uPA/uPAR play major roles in adhesion, migration, invasion and metastasis of cancer cells. Elevated levels of uPA have been reported as a risk biomarker for disease relapse, increased cancer malignancy and poor survival prognosis. For these reasons uPA is considered an important target for anticancer drug therapy. In this study we isolated two camel single domain antibodies (nanobodies) from a naïve library by phage display. The nanobody sequences were sequence-optimized for Escherichia coli expression, cloned into the pET22-B(+) vector system, expressed in BL-21 cells and purified from the periplasmic fraction by IMAC. ELISA tests demonstrated that the purified nanobodies were specific for uPA when tested towards other trypsin-like serine proteases. The apparent affinities of the nanobodies were determined by competitive ELISA to 80 nM and 522 nM, respectively. The best binder did not inhibit uPA (nAb-C3), however the lowest affinity binder (nAb-C8) was able to inhibit the uPA-mediated cleavage of the substrate S-2444. The results validate the naïve library as a resource for retrieval of relevant lead molecules and the novel uPA-nanobodies can be useful pharmacological tools to study uPA structure-function relationships.
Assuntos
Especificidade de Anticorpos/genética , Expressão Gênica , Oligopeptídeos/química , Anticorpos de Domínio Único , Ativador de Plasminogênio Tipo Uroquinase , Animais , Camelus , Linhagem Celular , Biblioteca Gênica , Humanos , Anticorpos de Domínio Único/química , Anticorpos de Domínio Único/genética , Anticorpos de Domínio Único/imunologia , Ativador de Plasminogênio Tipo Uroquinase/química , Ativador de Plasminogênio Tipo Uroquinase/imunologiaRESUMO
BACKGROUND: Members of the urokinase-type plasminogen activator (uPA) system including uPA, its receptor uPAR and the plasminogen activator inhibitor 1 (PAI-1) play an important role in tumour invasion and progression in a variety of tumour types. Since the majority of clear cell renal cell carcinoma (ccRCC) shows distant metastasis at time of diagnosis or later, the interplay of uPA, uPAR and PAI-1 might be of importance in this process determining the patients' outcome. METHODS: Corresponding pairs of malignant and non-malignant renal tissue specimens were obtained from 112 ccRCC patients without distant metastasis who underwent tumour nephrectomy. Tissue extracts prepared from fresh-frozen tissue samples by detergent extraction were used for the determination of antigen levels of uPA, uPAR and PAI-1 by ELISA. Antigen levels were normalised to protein concentrations and expressed as ng per mg of total protein. RESULTS: Antigen levels of uPA, uPAR, and PAI-1 correlated with each other in the malignant tissue specimens (rs=0.51-0.65; all P<0.001). Antigen levels of uPA system components were significantly higher in tissue extracts of non-organ confined tumours (pT3+4) compared to organ-confined tumours (pT1+2; all P<0.05). Significantly elevated levels of uPAR and PAI-1 were also observed in high grade ccRCC. When using median antigen levels as cut-off points, all three uPA system factors were significant predictors for disease-specific survival (DSS) in univariate Cox's regression analyses. High levels of uPA and uPAR remained independent predictors for DSS with HR=2.86 (95% CI 1.07-7.67, P=0.037) and HR=4.70 (95% CI 1.51-14.6, P=0.008), respectively, in multivariate Cox's regression analyses. A combination of high antigen levels of uPA and/or uPAR further improved the prediction of DSS in multivariate analysis (HR=14.5, 95% CI 1.88-111.1, P=0.010). Moreover, high uPA and/or uPAR levels defined a patient subgroup of high risk for tumour-related death in ccRCC patients with organ-confined disease (pT1+2) (HR=9.83, 95% CI 1.21-79.6, P=0.032). CONCLUSIONS: High levels of uPA and uPAR in tumour tissue extracts are associated with a significantly shorter DSS of ccRCC patients without distant metastases.
Assuntos
Carcinoma de Células Renais/química , Neoplasias Renais/química , Inibidor 1 de Ativador de Plasminogênio/análise , Receptores de Ativador de Plasminogênio Tipo Uroquinase/análise , Ativador de Plasminogênio Tipo Uroquinase/análise , Idoso , Antígenos/análise , Carcinoma de Células Renais/mortalidade , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Rim/química , Neoplasias Renais/mortalidade , Masculino , Pessoa de Meia-Idade , Inibidor 1 de Ativador de Plasminogênio/imunologia , Valor Preditivo dos Testes , Prognóstico , Receptores de Ativador de Plasminogênio Tipo Uroquinase/imunologia , Taxa de Sobrevida , Ativador de Plasminogênio Tipo Uroquinase/imunologiaRESUMO
Urokinase-type plasminogen activator (uPA) and uPA receptor (uPAR) are expressed in many tumors and have been reported to be correlated to protein expression and poor prognosis in malignant tumors. In a previous study, we reported on the selection of human single-chain variable fragment (scFv) A8 specific to uPA from phage-displayed human naïve scFv library. In this study, scFv A8 was converted to minibody form and evaluated for its functional ability on the uPA system involved in cellular signaling and cancer cell metastasis. A8 minibody increased enzyme activity of uPA and enhanced the migration and invasion of HT1080 colon cancer cells in a dose-dependent manner. A8 increased ERK phosphorylation, and enhanced migration was blocked by U0126, but not by LY0294002, SB2203580, and SP600125. A8 minibody also enhanced migration of MDA-MB231 by mediated expressing surface uPA, but not that of MCF-7 non-expressing surface uPA. Taken together, the A8 anti-uPA antibody is a uPA agonistic antibody, enhancing migration and invasion of cancer cells that express uPA via activation of ERK pathway.
Assuntos
Anticorpos Monoclonais/imunologia , Sistema de Sinalização das MAP Quinases/imunologia , Metástase Neoplásica/imunologia , Transdução de Sinais/imunologia , Anticorpos de Cadeia Única/imunologia , Ativador de Plasminogênio Tipo Uroquinase/imunologia , Movimento Celular/imunologia , Células HEK293 , Humanos , Células MCF-7 , Fosforilação/imunologia , Receptores de Superfície Celular/imunologia , Células Tumorais CultivadasRESUMO
The urokinase plasminogen activator receptor (uPAR) plays a role in tumor progression and has been proposed as a target for the treatment of cancer. We recently described the development of a novel humanized monoclonal antibody that targets uPAR and has anti-tumor activity in multiple xenograft animal tumor models. This antibody, ATN-658, does not inhibit ligand binding (i.e. uPA and vitronectin) to uPAR and its mechanism of action remains unclear. As a first step in understanding the anti-tumor activity of ATN-658, we set out to identify the epitope on uPAR to which ATN-658 binds. Guided by comparisons between primate and human uPAR, epitope mapping studies were performed using several orthogonal techniques. Systematic site directed and alanine scanning mutagenesis identified the region of aa 268-275 of uPAR as the epitope for ATN-658. No known function has previously been attributed to this epitope Structural insights into epitope recognition were obtained from structural studies of the Fab fragment of ATN-658 bound to uPAR. The structure shows that the ATN-658 binds to the DIII domain of uPAR, close to the C-terminus of the receptor, corroborating the epitope mapping results. Intriguingly, when bound to uPAR, the complementarity determining region (CDR) regions of ATN-658 closely mimic the binding regions of the integrin CD11b (αM), a previously identified uPAR ligand thought to be involved in leukocyte rolling, migration and complement fixation with no known role in tumor progression of solid tumors. These studies reveal a new functional epitope on uPAR involved in tumor progression and demonstrate a previously unrecognized strategy for the therapeutic targeting of uPAR.
Assuntos
Anticorpos Monoclonais/química , Antígeno CD11b/química , Epitopos/química , Receptores de Ativador de Plasminogênio Tipo Uroquinase/química , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/farmacologia , Antígeno CD11b/imunologia , Linhagem Celular Tumoral , Chlorocebus aethiops , Cães , Drosophila melanogaster , Mapeamento de Epitopos , Epitopos/imunologia , Humanos , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Ligação Proteica/efeitos dos fármacos , Receptores de Ativador de Plasminogênio Tipo Uroquinase/antagonistas & inibidores , Receptores de Ativador de Plasminogênio Tipo Uroquinase/imunologia , Proteínas Recombinantes/química , Proteínas Recombinantes/imunologia , Homologia de Sequência de Aminoácidos , Ativador de Plasminogênio Tipo Uroquinase/química , Ativador de Plasminogênio Tipo Uroquinase/imunologia , Vitronectina/química , Vitronectina/imunologiaRESUMO
Assessments of disease activity and organ damage in systemic lupus erythematosus (SLE) remain challenging because of the lack of reliable biomarkers and disease heterogeneity. Ongoing inflammation can be difficult to distinguish from permanent organ damage caused by previous flare-ups or medication side effects. Circulating soluble urokinase plasminogen activator receptor (suPAR) has emerged as a potential marker of inflammation and disease severity, and an outcome predictor in several disparate conditions. This study was done to evaluate suPAR as a marker of disease activity and organ damage in SLE. Sera from 100 healthy donors and 198 patients with SLE fulfilling the 1982 American College of Rheumatology classification criteria and/or the Fries criteria were analyzed for suPAR by enzyme immunoassay. Eighteen patients with varying degree of disease activity were monitored longitudinally. Disease activity was assessed by the SLE disease activity index 2000 and the physician's global assessment. Organ damage was evaluated by the Systemic Lupus International Collaborating Clinics/American College of Rheumatology damage index (SDI). Compared with healthy control subjects, serum suPAR levels were elevated significantly in patients with SLE. No association was recorded regarding suPAR levels and SLE disease activity in cross-sectional or consecutive samples. However, a strong association was observed between suPAR and SDI (P < 0.0005). Considering distinct SDI domains, renal, neuropsychiatric, ocular, skin, and peripheral vascular damage had a significant effect on suPAR levels. This study is the first to demonstrate an association between serum suPAR and irreversible organ damage in SLE. Further studies are warranted to evaluate suPAR and other biomarkers as predictors of evolving organ damage.
Assuntos
Biomarcadores/sangue , Lúpus Eritematoso Sistêmico/diagnóstico , Receptores de Ativador de Plasminogênio Tipo Uroquinase/sangue , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores/química , Feminino , Seguimentos , Humanos , Inflamação , Lúpus Eritematoso Sistêmico/imunologia , Masculino , Pessoa de Meia-Idade , Receptores de Ativador de Plasminogênio Tipo Uroquinase/imunologia , Solubilidade , Ativador de Plasminogênio Tipo Uroquinase/imunologiaRESUMO
OBJECTIVE: Cardiac neonatal lupus (cardiac-NL), initiated by surface binding of anti-Ro60 autoantibodies to apoptotic cardiocytes during development, activates the urokinase plasminogen activator/urokinase plasminogen activator receptor (uPA/uPAR) system. Subsequent accumulation of apoptotic cells and plasmin generation facilitates increased binding of anti-Ro60 by disrupting and cleaving circulating ß2-glycoprotein I (ß2GPI) thereby eliminating its protective effect. The association of soluble levels of components of the uPA/uPAR system with cardiac-NL was examined. METHODS: Levels of the uPA/uPAR system were assessed by ELISA in cord blood and immunohistological evaluation of autopsies. RESULTS: uPA, uPAR and plasminogen levels were each significantly higher in cord blood from cardiac-NL (n = 35) compared with non-cardiac-NL (n = 26) anti-Ro-exposed neonates: 3.3 ± 0.1 vs 1.9 ± 0.05 ng/ml (P < 0.0001), 6.6 ± 0.3 vs 2.1 ± 0.2 ng/ml (P < 0.0001) and 435 ± 34 vs 220 ± 19 ng/ml (P < 0.0001), respectively. In three twin pairs discordant for cardiac-NL, the twin with cardiac-NL had higher levels of uPA, uPAR and plasminogen than the unaffected twin (3.1 ± 0.1 vs 1.9 ± 0.05 ng/ml; P = 0.0086, 6.2 ± 1.4 vs 2.2 ± 0.7 ng/ml; P = 0.147 and 412 ± 61 vs 260 ± 27 ng/ml; P = 0.152, respectively). Immunohistological evaluation of three hearts from fetuses dying with cardiac-NL revealed macrophages and giant cells expressing uPA and plasminogen in the septal region. CONCLUSION: Increased soluble uPA, uPAR and plasminogen in cord blood and expression in affected tissue of fetuses with cardiac-NL supports the hypothesis that fetal cardiac injury is in part mediated by plasmin generation initiated by anti-Ro binding to the apoptotic cardiocyte.
Assuntos
Sangue Fetal/imunologia , Fibrinolisina/imunologia , Cardiopatias/imunologia , Lúpus Eritematoso Sistêmico/congênito , Receptores de Ativador de Plasminogênio Tipo Uroquinase/imunologia , Ribonucleoproteínas/imunologia , Anticorpos Antinucleares/imunologia , Anticorpos Antinucleares/metabolismo , Biomarcadores , Estudos de Casos e Controles , Ensaio de Imunoadsorção Enzimática , Feminino , Fibrinolisina/metabolismo , Cardiopatias/mortalidade , Cardiopatias/patologia , Humanos , Imuno-Histoquímica , Recém-Nascido , Lúpus Eritematoso Sistêmico/imunologia , Lúpus Eritematoso Sistêmico/mortalidade , Lúpus Eritematoso Sistêmico/patologia , Macrófagos/imunologia , Miócitos Cardíacos/imunologia , Miócitos Cardíacos/metabolismo , Gravidez , Receptores de Ativador de Plasminogênio Tipo Uroquinase/sangue , Valores de Referência , Ribonucleoproteínas/metabolismo , Taxa de Sobrevida , Ativador de Plasminogênio Tipo Uroquinase/sangue , Ativador de Plasminogênio Tipo Uroquinase/imunologiaRESUMO
Streptococcus pneumoniae infections remain a major cause of morbidity and mortality worldwide. Therefore a detailed understanding and characterization of the mechanism of host cell colonization and dissemination is critical to gain control over this versatile pathogen. Here we identified a novel 72-kDa pneumococcal protein endopeptidase O (PepO), as a plasminogen- and fibronectin-binding protein. Using a collection of clinical isolates, representing different serotypes, we found PepO to be ubiquitously present both at the gene and protein level. In addition, PepO protein was secreted in a growth phase-dependent manner to the culture supernatants of the pneumococcal isolates. Recombinant PepO bound human plasminogen and fibronectin in a dose-dependent manner and plasminogen did not compete with fibronectin for binding PepO. PepO bound plasminogen via lysine residues and the interaction was influenced by ionic strength. Moreover, upon activation of PepO-bound plasminogen by urokinase-type plasminogen activator, generated plasmin cleaved complement protein C3b thus assisting in complement control. Furthermore, direct binding assays demonstrated the interaction of PepO with epithelial and endothelial cells that in turn blocked pneumococcal adherence. Moreover, a pepO-mutant strain showed impaired adherence to and invasion of host cells compared with their isogenic wild-type strains. Taken together, the results demonstrated that PepO is a ubiquitously expressed plasminogen- and fibronectin-binding protein, which plays role in pneumococcal invasion of host cells and aids in immune evasion.
Assuntos
Proteínas de Bactérias/imunologia , Endopeptidases/imunologia , Fibronectinas/imunologia , Evasão da Resposta Imune/imunologia , Imunidade Inata/imunologia , Plasminogênio/imunologia , Aderência Bacteriana/genética , Aderência Bacteriana/imunologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Western Blotting , Linhagem Celular Tumoral , Células Cultivadas , Complemento C3b/imunologia , Complemento C3b/metabolismo , Endopeptidases/genética , Endopeptidases/metabolismo , Células Epiteliais/imunologia , Células Epiteliais/metabolismo , Células Epiteliais/microbiologia , Fibrinolisina/imunologia , Fibrinolisina/metabolismo , Fibronectinas/metabolismo , Células Endoteliais da Veia Umbilical Humana/imunologia , Células Endoteliais da Veia Umbilical Humana/metabolismo , Células Endoteliais da Veia Umbilical Humana/microbiologia , Humanos , Microscopia Confocal , Mutação , Plasminogênio/metabolismo , Ligação Proteica , Streptococcus pneumoniae/genética , Streptococcus pneumoniae/imunologia , Streptococcus pneumoniae/metabolismo , Ativador de Plasminogênio Tipo Uroquinase/imunologia , Ativador de Plasminogênio Tipo Uroquinase/metabolismoRESUMO
Cystic fibrosis (CF) transmembrane conductance regulator (CFTR) is expressed in the epithelial cells of a wide range of organs/tissues from which most cancers are derived. Although accumulating reports have indicated the association of cancer incidence with genetic variations in CFTR gene, the exact role of CFTR in cancer development and the possible underlying mechanism have not been elucidated. Here, we report that CFTR expression is significantly decreased in both prostate cancer cell lines and human prostate cancer tissue samples. Overexpression of CFTR in prostate cancer cell lines suppresses tumor progression (cell growth, adhesion and migration), whereas knockdown of CFTR leads to enhanced malignancies both in vitro and in vivo. In addition, we demonstrate that CFTR knockdown-enhanced cell proliferation, cell invasion and migration are significantly reversed by antibodies against either urokinase plasminogen activator (uPA) or uPA receptor (uPAR), which are known to be involved in various malignant traits of cancer development. More interestingly, overexpression of CFTR suppresses uPA by upregulating the recently described tumor suppressor microRNA-193b (miR-193b), and overexpression of pre-miR-193b significantly reverses CFTR knockdown-enhanced malignant phenotype and abrogates elevated uPA activity in prostate cancer cell line. Finally, we show that CFTR gene transfer results in significant tumor repression in prostate cancer xenografts in vivo. Taken together, the present study has demonstrated a previously undefined tumor-suppressing role of CFTR and its involvement in regulation of miR-193b in prostate cancer development.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/genética , MicroRNAs/genética , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Animais , Sequência de Bases , Linhagem Celular Tumoral , Movimento Celular/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Regulação para Baixo , Feminino , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Masculino , Camundongos , Camundongos Nus , Dados de Sequência Molecular , Neoplasias da Próstata/metabolismo , Receptores de Ativador de Plasminogênio Tipo Uroquinase/genética , Receptores de Ativador de Plasminogênio Tipo Uroquinase/imunologia , Receptores de Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Ativador de Plasminogênio Tipo Uroquinase/genética , Ativador de Plasminogênio Tipo Uroquinase/imunologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
We have designed bispecific antibodies that bind one target (anti-Her3) in a bivalent IgG-like manner and contain one additional binding entity (anti-cMet) composed of one V(H) and one V(L) domain connected by a disulfide bond. The molecules are assembled by fusing a V(H,Cys44) domain via flexible connector peptides to the C-terminus of one H-chain (heavy chain), and a V(L,Cys100) to another H-chain. To ensure heterodimerization during expression in mammalian cells, we introduced complementary knobs-into-holes mutations into the different H-chains. The IgG-shaped trivalent molecules carry as third binding entity one disulfide-stabilized Fv (dsFv) without a linker between V(H) and V(L). Tethering the V(H) and V(L) domains at the C-terminus of the C(H)3 domain decreases the on-rates of the dsFv to target antigens without affecting off-rates. Steric hindrance resolves upon removal of one side of the double connection by proteolysis: this improves flexibility and accessibility of the dsFv and fully restores antigen access and affinity. This technology has multiple applications: (i) in cases where single-chain linkers are not desired, dsFvs without linkers can be generated by addition of furin site(s) in the connector that are processed during expression within mammalian cells; (ii) highly active (toxic) entities which affect expression can be produced as inactive dsFvs and subsequently be activated (e.g. via PreScission cleavage) during purification; (iii) entities can be generated which are targeted by the unrestricted binding entity and can be activated by proteases in target tissues. For example, Her3-binding molecules containing linkers with recognition sequences for matrix metalloproteases or urokinase, whose inactivated cMet binding site is activated by proteolytic processing.
Assuntos
Anticorpos Biespecíficos/química , Anticorpos Biespecíficos/metabolismo , Anticorpos de Cadeia Única/química , Anticorpos de Cadeia Única/metabolismo , Anticorpos Biespecíficos/genética , Anticorpos Biespecíficos/imunologia , Sítios de Ligação de Anticorpos , Linhagem Celular , Dissulfetos/química , Expressão Gênica , Humanos , Metaloproteinase 2 da Matriz/imunologia , Metaloproteinase 9 da Matriz/imunologia , Peptídeo Hidrolases/metabolismo , Engenharia de Proteínas , Proteólise , Receptor ErbB-3/imunologia , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Anticorpos de Cadeia Única/genética , Anticorpos de Cadeia Única/imunologia , Ativador de Plasminogênio Tipo Uroquinase/imunologiaRESUMO
Synthetic compounds for controlling or creating human immunity have the potential to revolutionize disease treatment. Motivated by challenges in this arena, we report herein a strategy to target metastatic cancer cells for immune-mediated destruction by targeting the urokinase-type plasminogen activator receptor (uPAR). Urokinase-type plasminogen activator (uPA) and uPAR are overexpressed on the surfaces of a wide range of invasive cancer cells and are believed to contribute substantially to the migratory propensities of these cells. The key component of our approach is an antibody-recruiting molecule that targets the urokinase receptor (ARM-U). This bifunctional construct is formed by selectively, covalently attaching an antibody-binding small molecule to the active site of the urokinase enzyme. We demonstrate that ARM-U is capable of directing antibodies to the surfaces of target cancer cells and mediating both antibody-dependent cellular phagocytosis (ADCP) and antibody-dependent cellular cytotoxicity (ADCC) against multiple human cancer cell lines. We believe that the reported strategy has the potential to inform novel treatment options for a variety of deadly, invasive cancers.