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1.
Prev Med ; 122: 148-154, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31078168

RESUMO

With a strong focus on end user, or knowledge user, engagement throughout the study, an integrated knowledge translation approach (iKT) is expected to enhance the quality, relevance and reach of research findings. From its initiation, the Canadian Population Attributable Risk of Cancer (ComPARe) study combined the expertise of the knowledge producers (cancer prevention researchers) and select knowledge users in an iKT approach. We describe in detail our iKT approach, including governance, outputs and early reflections. In our model, knowledge users were integrated as members of the research team or members of a KT Advisory Committee. The integrated knowledge users took a lead role on the KT activities for ComPARe, including developing the KT Blueprint, a four phase systematic approach to guide the planning and implementation of KT activities. This approach included planning, knowledge product development, dissemination and evaluation, with advisory committee engagement built in throughout. Our early reflections identified enablers and challenges of an iKT approach for this study. Enablers included co-investigators' commitment and attitude towards iKT, support for iKT from the funding agency, an established partnership early on, understanding of and experience in each other's area of expertise, dedicated funding, clearly delineated roles, advisory committee buy-in and existing tools. Challenges included anticipating all costs, continuity of involvement, competing priorities, relationship management and geographic distance. A future evaluation will determine the effectiveness and impact of the iKT approach and KT Blueprint. In the interim, the approach we describe here can be modeled by others interested in collaborative, action-oriented research.


Assuntos
Comportamento Cooperativo , Prestação Integrada de Cuidados de Saúde , Neoplasias/epidemiologia , Pesquisadores , Pesquisa Translacional Biomédica , Canadá , Humanos , Neoplasias/etiologia , Neoplasias/prevenção & controle
2.
Matrix Biol ; 27(2): 128-38, 2008 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-18029162

RESUMO

The human matrix metalloproteinase (MMP) gene family includes 24 genes whose regulated expression, together with that of four tissue inhibitors of metalloproteinases (TIMPs), is essential in tissue remodelling and cell signalling. Quantitative real-time-PCR (qPCR) analysis was used to evaluate the shared and unique patterns of control of these two gene families in human MRC-5 and WI-38 fibroblasts in response to the protein kinase C (PKC) activator phorbol-12-myristate-13-acetate (PMA). The requirement for ongoing translation was analysed using three protein synthesis inhibitors, anisomycin, cycloheximide and emetine. PMA induced MMP1, 3, 8, 9, 10, 12, 13, 14 and TIMP1 and TIMP3 RNAs after 4-8 h, and induction of all except MMP9 and TIMP3 was blocked by all protein synthesis inhibitors. However, even though all inhibitors effectively blocked translation, PMA-induction of MMP9 and TIMP3 was blocked by emetine but was insensitive to cycloheximide and anisomycin. Anisomycin alone induced MMP9 and TIMP3, along with MMP25 and MMP19. The extracellular signal-regulated kinases (ERKs)-1/2 were strongly activated by PMA, while anisomycin activated the c-Jun N-terminal kinase (JNK) and p38 pathways, and cycloheximide activated p38, but emetine had no effect on the stress-activated mitogen-activated protein kinase (MAPK) pathways. The involvement of the p38 and JNK pathways in the selective effects of anisomycin and cycloheximide on MMP/TIMP expression was supported by use of pharmacological inhibitors. These data confirm that most inducible MMPs and TIMP1 behave as "late" activated, protein synthesis-dependent genes in fibroblasts. However, the requirement of protein synthesis for PMA-induction of MMPs and TIMPs is not universal, since it is abrogated for MMP9 and TIMP3 by stimulation of the stress-activated MAPK pathways. The definition of clusters of co-regulated genes among the two gene families will aid in bioinformatic dissection of control mechanisms.


Assuntos
Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Metaloproteinases da Matriz/genética , Acetato de Tetradecanoilforbol/farmacologia , Inibidores Teciduais de Metaloproteinases/genética , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Western Blotting , Linhagem Celular , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Proteínas Ligadas por GPI , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/antagonistas & inibidores , Metaloproteinase 1 da Matriz/genética , Metaloproteinase 10 da Matriz/genética , Metaloproteinases da Matriz Associadas à Membrana/genética , Metaloproteinases da Matriz Secretadas/genética , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Modelos Biológicos , Biossíntese de Proteínas/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Inibidores da Síntese de Proteínas/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Inibidor Tecidual de Metaloproteinase-1/genética , Inibidor Tecidual de Metaloproteinase-3/genética , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores
3.
Cancer Res ; 66(24): 11771-80, 2006 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-17178873

RESUMO

The capacity of glioma cells to invade extensively within the central nervous system is a major cause of the high morbidity rate of primary malignant brain tumors. Glioma cell invasion involves the attachment of tumor cells to extracellular matrix (ECM), degradation of ECM components, and subsequent penetration into adjacent brain structures. These processes are accomplished in part by matrix metalloproteinases (MMP) within a three-dimensional milieu of the brain parenchyma. As the majority of studies have used a two-dimensional monolayer culture system, we have used a three-dimensional matrix of collagen type I gel to address glioma-secreted proteases, ECM, and invasiveness of glioma cells. We show that in a three-dimensional collagen type I matrix, the presence of tenascin-C, commonly elevated in high-grade gliomas, increased the invasiveness of glioma cells. The tenascin-C-mediated invasiveness was blocked by metalloproteinase inhibitors, but this did not involve the gelatinases (MMP-2 and MMP-9) commonly implicated in two-dimensional glioma growth. A thorough analysis of 21 MMPs and six members of a disintegrin and metalloproteinase domain showed that MMP-12 was increased in gliomas by tenascin-C in three-dimensional matrix. Furthermore, examinations of resected specimens revealed high MMP-12 levels in the high-grade glioblastoma multiforme tumors. Finally, a function-blocking antibody as well as small interfering RNA to MMP-12 attenuated the tenascin-C-stimulated glioma invasion. These results identify a new factor, MMP-12, in regulating glioma invasiveness through interaction with tenascin-C.


Assuntos
Neoplasias Encefálicas/patologia , Glioma/patologia , Metaloproteinase 12 da Matriz/metabolismo , Tenascina/farmacologia , Neoplasias Encefálicas/cirurgia , Linhagem Celular Tumoral , Colágeno , Meios de Cultivo Condicionados , Primers do DNA , Glioma/cirurgia , Humanos , Cinética , Invasividade Neoplásica/patologia , Reação em Cadeia da Polimerase , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Transfecção
4.
Int J Oncol ; 30(5): 1263-71, 2007 May.
Artigo em Inglês | MEDLINE | ID: mdl-17390030

RESUMO

Selenium is considered to be one of the most promising micronutrients for cancer prevention and therapy, based on evidence from epidemiological studies, laboratory-based research and clinical trial intervention. There are ample reports of selenium methionine and sodium selenite's ability to induce apoptosis in various cancers in vitro. There are a few reports in the literature on the effects of selenium on established glioma cell lines but none on biopsy-derived short-term brain tumour cultures. In this in vitro study the effects of a range of concentrations (2-10 microg/ml) of sodium selenite were investigated in one low-passage culture of biopsy-derived glioma cells (IPSB-18, an anaplastic astrocytoma, P 18-22) and a normal human brain cell culture (CC2565, P11). Results from 2 viability assays, 3[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide (MTT) and sulphorodamine B (SRB) consistently showed that the IC50 for selenium in the astrocytoma was approximately 5 microg/ml whilst the normal brain cells were unaffected by selenium in the range of concentrations studied. Time-lapse video microscopy revealed that, while at 4 microg/ml selenium, the time taken to achieve 100% cell death was 17 h, with increasing concentrations of selenium from 6 to 8 microg/ml and finally at 10 microg/ml the IPSB-18 cells rounded up and died much more quickly. The time taken to achieve 100% cell death was 7 h, 7 h and 6 h, respectively, suggesting that the effect was similar at higher concentrations. Flow cytometry indicated that cell death was by apoptosis. RT-PCR results showed downregulation of the gene expression of 6 matrix metalloproteases (MMP2, 9, 14, 15, 16, 24), their inhibitors, TIMPs and epidermal growth factor receptor, in IPSB-18 cells treated with 2, 4 and 8 microg/ml of selenium. Collectively, the data in this study suggests that selenium, not only induces tumour cell-specific apoptosis but also has anti-invasive potential.


Assuntos
Apoptose , Astrocitoma/tratamento farmacológico , Neoplasias Encefálicas/tratamento farmacológico , Selênio/farmacologia , Adolescente , Antineoplásicos/farmacologia , Astrocitoma/patologia , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Sobrevivência Celular , Relação Dose-Resposta a Droga , Humanos , Técnicas In Vitro , Concentração Inibidora 50 , Masculino , Invasividade Neoplásica , RNA/metabolismo , Sais de Tetrazólio/farmacologia , Tiazóis/farmacologia
5.
Circ Res ; 97(4): 380-90, 2005 Aug 19.
Artigo em Inglês | MEDLINE | ID: mdl-16037568

RESUMO

Cytokine and extracellular matrix (ECM) homeostasis are distinct systems that are each dysregulated in heart failure. Here we show that tissue inhibitor of metalloproteinase (TIMP)-3 is a critical regulator of both systems in a mouse model of left ventricular (LV) dilation and dysfunction. Timp-3(-/-) mice develop precipitous LV dilation and dysfunction reminiscent of dilated cardiomyopathy (DCM), culminating in early onset of heart failure by 6 weeks, compared with wild-type aortic-banding (AB). Timp-3 deficiency resulted in increased TNFalpha converting enzyme (TACE) activity within 6 hours after AB leading to enhanced tumor necrosis factor-alpha (TNFalpha) processing. In addition, TNFalpha production increased in timp-3(-/-)-AB myocardium. A significant elevation in gelatinase and collagenase activities was observed 1 week after AB, with localized ECM degradation in timp-3(-/-)-AB myocardium. Timp-3(-/-)/tnfalpha(-/-) mice were generated and subjected to AB for comparative analyses with timp-3(-/-)-AB mice. This revealed the critical role of TNFalpha in the early phase of LV remodeling, de novo expression of Matrix metalloproteinases (MMP)-8 in the absence of TNFalpha, and highlighted the importance of interstitial collagenases (MMP-2, MMP-13, and MT1-MMP) for cardiac ECM degradation. Ablation of TNFalpha, or limiting MMP activity with a synthetic MMP inhibitor (PD166793), each partially attenuated LV dilation and cardiac dysfunction in timp-3(-/-)-AB mice. Notably, combining TNFalpha ablation with MMP inhibition completely rescued heart disease in timp-3(-/-)-AB mice. This study provides a basis for anti-TNFalpha and MMP inhibitor combination therapy in heart disease.


Assuntos
Insuficiência Cardíaca/prevenção & controle , Inibidores de Metaloproteinases de Matriz , Inibidores de Proteases/uso terapêutico , Inibidor Tecidual de Metaloproteinase-3/fisiologia , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Proteínas ADAM , Proteína ADAM17 , Animais , Apoptose , Cardiomegalia/etiologia , Insuficiência Cardíaca/etiologia , Insuficiência Cardíaca/mortalidade , Metaloproteinase 8 da Matriz/genética , Metaloproteinases da Matriz/fisiologia , Metaloendopeptidases/metabolismo , Camundongos , Camundongos Knockout , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/fisiologia , Disfunção Ventricular Esquerda/prevenção & controle
6.
FASEB J ; 19(12): 1668-70, 2005 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-16081501

RESUMO

Inflammation in multiple sclerosis and its animal model, experimental autoimmune encephalomyelitis (EAE), is manifested by changes in matrix metalloproteinase (MMP) expression and in the ratio of T helper (Th) 1 and 2 effector cytokines. Here, we provide a comprehensive documentation of MMPs in EAE and report that of all the MMPs that could be measured at peak disease in spinal cord tissue, MMP-12 was the most highly up-regulated. In contrast to previously published findings of MMPs in EAE, this increase in MMP-12 expression was associated with protection, as MMP-12 null mice had significantly worse maximum severity and EAE disease burden compared with wild-type (WT) controls. When spleen and lymph node cells were removed from EAE-afflicted WT and MMP-12 null mice at the same disease score before divergence of disease and restimulated in vitro, the MMP-12 null cells had significantly higher Th1 to Th2 cytokine ratio. Measurements of the transcriptional regulators of T cell polarization revealed that MMP-12 null cells had increased T-bet and reduced GATA-3 expression, a condition that favors a Th1 bias. These results emphasize that specific MMPs can have beneficial roles in inflammation, and they implicate MMPs in T effector polarization for the first time.


Assuntos
Regulação da Expressão Gênica , Metaloendopeptidases/biossíntese , Células Th1/citologia , Células Th2/citologia , Animais , Autoimunidade , Citocinas/metabolismo , Progressão da Doença , Encefalomielite Autoimune Experimental/enzimologia , Feminino , Inflamação , Linfonodos/patologia , Metaloproteinase 12 da Matriz , Camundongos , Modelos Biológicos , Proteínas da Mielina , Glicoproteína Associada a Mielina/química , Glicoproteína Mielina-Oligodendrócito , RNA/metabolismo , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Medula Espinal/metabolismo , Baço/citologia , Baço/metabolismo , Baço/patologia , Linfócitos T/metabolismo , Células Th1/metabolismo , Células Th2/metabolismo , Transcrição Gênica , Resultado do Tratamento , Regulação para Cima
7.
Sarcoidosis Vasc Diffuse Lung Dis ; 23(1): 13-21, 2006 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-16933466

RESUMO

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is characterized by fibroblast expansion and extracellular matrix accumulation. Some secreted matrix metalloproteinases (MMPs) as MMP2 are highly upregulated in IPF lungs. Membrane-type (MT)-MMPs participate in the activation of pro-MMP2. However, they have not been examined in IPF. METHODS: Type I transmembrane MT-MMPs, MT1, MT2, MT3, and MT5-MMP were analyzed by real-time PCR and immunohistochemistry in IPF and normal lungs. MMP-2 was also immunolocalized and evaluated by gelatin zymography in BAL fluids. Additionally, the MT-MMPs were examined by real time PCR in lung fibroblasts stimulated with TGF-beta1 and IFN-gamma. RESULTS: MT1-MMP, was the most highly expressed followed by MT2- and MT5-MMP, and by a moderate expression of MT3-MMP. Regarding their localization, MT1- and MT2-MMPs were found in alveolar epithelial cells, MT3-MMP in fibroblasts from fibroblastic foci and alveolar epithelial cells and MT5-MMP in basal bronchiolar epithelial cells and in areas of squamous metaplasia. MMP2 was localized in alveolar and basal bronchiolar epithelial cells and fibroblasts, and increased active enzyme was observed in BAL fluids. In lung fibroblasts, TGF-beta1 induced a strong upregulation of MT3-MMP, both at the gene and protein level. This effect was blocked by genistein, a protein tyrosin kinase inhibitor and partially repressed by SB203580 a p38 MAP kinase inhibitor. IFN-gamma had no effect. CONCLUSIONS: MT-MMPs are expressed in IPF, in the same cell types as MMP2. Mostly by different types of epithelial cells a pivotal component in the aberrant remodeling of the lung microenvironment. Interestingly MT3-MMP that was found in fibroblastic foci was upregulated in vitro by TGF-beta1 a potent profibrotic mediator.


Assuntos
Metaloproteinases da Matriz Associadas à Membrana/análise , Fibrose Pulmonar/enzimologia , Líquido da Lavagem Broncoalveolar/citologia , Estudos de Casos e Controles , Células Cultivadas , Células Epiteliais/metabolismo , Fibroblastos/efeitos dos fármacos , Humanos , Imuno-Histoquímica , Isoenzimas/genética , Isoenzimas/metabolismo , Metaloproteinases da Matriz Associadas à Membrana/genética , Metaloproteinases da Matriz Associadas à Membrana/metabolismo , Fibrose Pulmonar/metabolismo , Fibrose Pulmonar/patologia , Fibrose Pulmonar/fisiopatologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Crescimento Transformador beta1/farmacologia
8.
Cardiovasc Res ; 67(1): 39-49, 2005 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-15949468

RESUMO

OBJECTIVES: Vascular cell adhesion molecule-1 (VCAM-1) is a cell surface adhesion molecule involved in the recruitment of leukocytes to endothelial cells on arterial walls during the pathogenesis of atherosclerosis. The soluble ectodomain of VCAM-1 (sVCAM-1) is proteolytically released from the cell surface into the circulation, a process which is up-regulated in patients with cardiovascular or inflammatory disease. Here we investigate mechanisms involved in sVCAM-1 generation in response to cytokine stimulation. METHODS: VCAM-1 ectodomain release into the conditioned media of MCEC-1 murine endothelial cells and cells grown from primary aortic explants from timp3-/- mice and wild-type littermates was measured by sandwich ELISA and Western blot after stimulation with tumor necrosis factor alpha (TNFalpha), interleukin-1beta (IL-1beta), or the phorbol ester PMA. Protease expression was inhibited (knocked down) with siRNA and validated using real-time PCR. RESULTS: Proinflammatory cytokines IL-1beta and TNFalpha up-regulated VCAM-1 ectodomain release from the MCEC-1 cells, and this was dependant on p38 and mitogen-activated protein kinases (MAP kinases) and inhibited by the matrix metalloproteinase (MMP) inhibitor BB94 and tissue inhibitor of metalloproteinase (TIMP)-3, but not TIMP-1 or TIMP-2. Timp-3-/- cells exhibited greater VCAM-1 ectodomain release following cytokine stimulation than TIMP-3-expressing cells. Additionally, cytokine stimulation of MCEC-1 cells was shown to cause down-regulation of TIMP-3 expression. Knockdown of the metalloproteinase ADAM17, but not ADAM10 or ADAM12, gene expression reduced cytokine-stimulated VCAM-1 shedding. CONCLUSIONS: TIMP-3 regulates the release of sVCAM-1 from cytokine-stimulated endothelial cells, which is mediated by ADAM17.


Assuntos
Citocinas/farmacologia , Células Endoteliais/metabolismo , Inibidor Tecidual de Metaloproteinase-3/fisiologia , Molécula 1 de Adesão de Célula Vascular/fisiologia , Animais , Aorta , Northern Blotting , Western Blotting/métodos , Linhagem Celular , Células Cultivadas , Meios de Cultivo Condicionados , Células Endoteliais/efeitos dos fármacos , Ensaio de Imunoadsorção Enzimática/métodos , MAP Quinases Reguladas por Sinal Extracelular , Humanos , Interleucina-1 , Camundongos , Camundongos Knockout , PPAR alfa , Estimulação Química , Inibidor Tecidual de Metaloproteinase-3/genética , Fator de Necrose Tumoral alfa , Veias Umbilicais , Molécula 1 de Adesão de Célula Vascular/análise , Proteínas Quinases p38 Ativadas por Mitógeno
9.
J Neurosci ; 23(31): 10107-15, 2003 Nov 05.
Artigo em Inglês | MEDLINE | ID: mdl-14602826

RESUMO

We investigated the role of matrix metalloproteinases (MMPs) in acute spinal cord injury (SCI). Transcripts encoding 22 of the 23 known mammalian MMPs were measured in the mouse spinal cord at various time points after injury. Although there were significant changes in the expression levels of multiple MMPs, MMP-12 was increased 189-fold over normal levels, the highest of all MMPs examined. To evaluate the role of MMP-12 in SCI, spinal cord compression was performed in wild-type (WT) and MMP-12 null mice. Behavioral analyses were conducted for 4 weeks using the Basso-Beattie-Bresnahan (BBB) locomotor rating scale as well as the inclined plane test. The results show that MMP-12 null mice exhibited significantly improved functional recovery compared with WT controls. Twenty-eight days after injury, the BBB score in the MMP-12 group was 7, representing extensive movement of all three hindlimb joints, compared with 4 in the WT group, representing only slight movement of these joints. Furthermore, MMP-12 null mice showed recovery of hindlimb strength more rapidly than control mice, with significantly higher inclined plane scores on days 14 and 21 after SCI. Mechanistically, there was decreased permeability of the blood-spinal barrier and reduced microglial and macrophage density in MMP-12 null mice compared with WT controls. This is the first study to profile the expression patterns of a majority of the known MMPs after spinal cord compression. The data indicate that MMP-12 expression after spinal cord trauma is deleterious and contributes to the development of secondary injury in SCI.


Assuntos
Perfilação da Expressão Gênica , Metaloproteinases da Matriz/genética , Metaloendopeptidases/genética , Traumatismos da Medula Espinal/enzimologia , Animais , Permeabilidade Capilar/genética , Contagem de Células , Linhagem da Célula , Modelos Animais de Doenças , Progressão da Doença , Membro Posterior/fisiopatologia , Hibridização In Situ , Macrófagos/enzimologia , Macrófagos/patologia , Masculino , Metaloproteinase 12 da Matriz , Camundongos , Camundongos Knockout , Microglia/enzimologia , Microglia/patologia , RNA Mensageiro/metabolismo , Recuperação de Função Fisiológica , Traumatismos da Medula Espinal/patologia , Traumatismos da Medula Espinal/fisiopatologia , Regulação para Cima
10.
Mol Cancer Res ; 1(5): 333-45, 2003 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-12651907

RESUMO

Matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) regulate proteolysis of the extracellular matrix and other extracellular proteins, including growth factors and their receptors. The aberrant expression of these genes is common in most cancers. We profiled the RNA levels of every human MMP and TIMP in a variety of cell types (fibroblast, endothelial, hematopoietic, carcinoma, melanoma, and glioma) using quantitative PCR, with the aim of identifying novel expression patterns. Almost all members of the membrane-type (MT-) MMP and TIMP families were elevated in glioma lines compared to carcinomas. In clinical glioma specimens, there were positive correlations between glioma grade and RNA levels of MT-1, MT-2, and MT-6 MMP, TIMP-1 and TIMP-2, and for several growth factors and receptors. These findings suggest that advanced malignant gliomas have elevated levels of membrane-associated MMPs and TIMPs, which may potentially regulate vascularization and invasion. Concurrent elevation of signaling molecules suggests potential bidirectional relationships that enhance tumor aggressiveness.


Assuntos
Neoplasias Encefálicas , Glioma , Metaloproteinases da Matriz/genética , Inibidor Tecidual de Metaloproteinase-1/genética , Inibidor Tecidual de Metaloproteinase-2/genética , Carcinoma , Primers do DNA , Endotélio/citologia , Fibroblastos/citologia , Fibrossarcoma , Perfilação da Expressão Gênica , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Neoplasias Hematológicas , Humanos , Melanoma , Miócitos de Músculo Liso/citologia , Reação em Cadeia da Polimerase , RNA Neoplásico/análise , Células Tumorais Cultivadas
11.
FEBS Lett ; 563(1-3): 129-34, 2004 Apr 09.
Artigo em Inglês | MEDLINE | ID: mdl-15063736

RESUMO

Matrix metalloproteinases (MMPs) and adamalysins (ADAMs) cleave many extracellular proteins, including matrix, growth factors, and receptors. We profiled the RNA levels of every MMP, several ADAMs, and inhibitors of metalloproteinases (TIMPs and RECK) in numerous mouse tissues during development and in the uterus during pregnancy. Observations include: most secreted MMPs are expressed at low to undetectable levels in tissues, whereas membrane-bound MMPs, ADAMs and inhibitors are abundant; almost every proteinase and inhibitor is present in the uterus or placenta at some time during gestation; the mouse collagenases mColA and mColB are found exclusively in the uterus and testis; and each tissue has its unique signature of proteinase and inhibitor expression.


Assuntos
Expressão Gênica , Metaloproteinases da Matriz/metabolismo , Camundongos/embriologia , Camundongos/crescimento & desenvolvimento , Inibidores Teciduais de Metaloproteinases/metabolismo , Animais , Animais Recém-Nascidos , Feminino , Proteínas Ligadas por GPI , Perfilação da Expressão Gênica , Masculino , Metaloproteinases da Matriz/genética , Glicoproteínas de Membrana/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos , Placenta/metabolismo , Gravidez , RNA Mensageiro/análise , Distribuição Tecidual , Inibidores Teciduais de Metaloproteinases/genética , Útero/metabolismo
12.
J Clin Invest ; 121(5): 1827-33, 2011 May.
Artigo em Inglês | MEDLINE | ID: mdl-21519144

RESUMO

Mycobacterium tuberculosis can cause lung tissue damage to spread, but the mechanisms driving this immunopathology are poorly understood. The breakdown of lung matrix involves MMPs, which have a unique ability to degrade fibrillar collagens at neutral pH. To determine whether MMPs play a role in the immunopathology of tuberculosis (TB), we profiled MMPs and their inhibitors, the tissue inhibitor of metalloproteinases (TIMPs), in sputum and bronchoalveolar lavage fluid from patients with TB and symptomatic controls. MMP-1 concentrations were significantly increased in both HIV-negative and HIV-positive patients with TB, while TIMP concentrations were lower in HIV-negative TB patients. In primary human monocytes, M. tuberculosis infection selectively upregulated MMP1 gene expression and secretion, and Ro32-3555, a specific MMP inhibitor, suppressed M. tuberculosis-driven MMP-1 activity. Since the mouse MMP-1 ortholog is not expressed in the lung and mice infected with M. tuberculosis do not develop tissue destruction equivalent to humans, we infected transgenic mice expressing human MMP-1 with M. tuberculosis to investigate whether MMP-1 caused lung immunopathology. In the MMP-1 transgenic mice, M. tuberculosis infection increased MMP-1 expression, resulting in alveolar destruction in lung granulomas and significantly greater collagen breakdown. In summary, MMP-1 may drive tissue destruction in TB and represents a therapeutic target to limit immunopathology.


Assuntos
Regulação Enzimológica da Expressão Gênica , Metaloproteinase 1 da Matriz/metabolismo , Tuberculose/enzimologia , Animais , Líquido da Lavagem Broncoalveolar , Movimento Celular , Inibidores Enzimáticos/farmacologia , Infecções por HIV/enzimologia , Soropositividade para HIV , Humanos , Concentração de Íons de Hidrogênio , Imidazóis/farmacologia , Sistema Imunitário , Camundongos , Camundongos Transgênicos , Monócitos/citologia
13.
Glia ; 55(5): 516-26, 2007 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-17216595

RESUMO

Microglia are resident immune cells within the central nervous system (CNS). They become activated following neurological insults and increase their expression of cytokines. Also elevated in CNS injuries are proteases, including matrix metalloproteinases (MMPs) and A disintegrin and metalloproteinases (ADAMs). The spectrum of metalloproteinase members expressed by microglia and by the systemic leukocytes that infiltrate the injured CNS is unknown, as are their functions. We determined the levels of transcripts encoding all 24 MMPs, nine ADAMs, and their four physiological antagonists, tissue inhibitor of metalloproteinases (TIMPs), in human microglia, B and T cells, monocytes, and neutrophils. We found a distinct pattern for each immune subset and an enrichment of metalloproteinases in microglia compared with leukocytes. When microglia were activated, there was an upregulation of transcripts for nine metalloproteinases, and reduction of TIMP3. Activation of microglia also resulted in increased levels of tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta, and IL-10 protein in the conditioned media of cells. The amount of secreted TNF-alpha, but not IL-1beta or IL-10, was suppressed by BB94, a broad spectrum metalloproteinase inhibitor, and by TIMP3 but not TIMP1 or TIMP2. This inhibitory profile suggests the involvement of an ADAM member in TNF-alpha secretion. We conclude that microglia bear a metalloproteinase signature distinct from systemic cells, and that following activation, microglia upregulate TNF-alpha protein levels through a combination of elevated cytokine transcripts, increased metalloproteinase level and activity, and through the decrease of TIMP3. The results have implications for the regulation of neuroinflammation and its outcomes following CNS injuries.


Assuntos
Proteínas ADAM/metabolismo , Citocinas/metabolismo , Leucócitos/enzimologia , Metaloproteases/metabolismo , Microglia/enzimologia , Proteínas ADAM/classificação , Proteínas ADAM/genética , Adulto , Células Cultivadas , Sistema Nervoso Central/lesões , Citocinas/imunologia , Humanos , Inflamação , Interleucina-10/imunologia , Interleucina-10/metabolismo , Interleucina-1beta/imunologia , Interleucina-1beta/metabolismo , Leucócitos/imunologia , Lipopolissacarídeos/imunologia , Metaloproteases/classificação , Metaloproteases/genética , Microglia/citologia , Microglia/imunologia , RNA/análise , RNA Mensageiro/análise , Fator de Necrose Tumoral alfa/imunologia , Fator de Necrose Tumoral alfa/metabolismo , Regulação para Cima/fisiologia
14.
J Immunol ; 178(2): 1199-207, 2007 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-17202385

RESUMO

CNS tuberculosis (CNS-TB) is the most deadly form of tuberculous disease accounting for 10% of clinical cases. CNS-TB is characterized by extensive tissue destruction, in which matrix metalloproteinases (MMPs) may play a critical role. We investigated the hypothesis that Mycobacterium tuberculosis activates monocyte-astrocyte networks increasing the activity of key MMPs. We examined the expression of all human MMPs and the tissue inhibitors of metalloproteinases (TIMPs) in human astrocytes stimulated by conditioned medium from M. tuberculosis-infected monocytes (CoMTB). Real-time RT-PCR showed that gene expression of MMP-1, -2, -3, -7, and -9 was increased (p < 0.05). MMP-9 secretion was significantly up-regulated at 24 h and increased over 120 h (p < 0.01). MMP-1, -3, and -7 secretion was not detected. Secretion of MMP-2 was constitutive and unaffected by CoMTB. Astrocyte gene expression and secretion of TIMP-1 was not affected by CoMTB although TIMP-2 secretion increased 3-fold at 120 h. Immunohistochemical analysis of human brain biopsies confirmed that astrocyte MMP-9 secretion is a predominant feature in CNS-TB in vivo. Dexamethasone inhibited astrocyte MMP-9, but not TIMP-1/2 secretion in response to CoMTB. CoMTB stimulated the nuclear translocation of NF-kappaB, inducing a 6-fold increase in nuclear p65 and a 2-fold increase in nuclear p50. This was associated with degradation of IkappaBalpha and beta within 30 min, persisting for 24 h. In summary, networks active between monocytes and astrocytes regulate MMP-9 activity in tuberculosis and astrocytes are a major source of MMP-9 in CNS-TB. Astrocytes may contribute to a matrix degrading environment within the CNS and subsequent morbidity and mortality.


Assuntos
Astrócitos/enzimologia , Astrócitos/imunologia , Metaloproteinases da Matriz/genética , Monócitos/enzimologia , Monócitos/imunologia , Tuberculose do Sistema Nervoso Central/enzimologia , Tuberculose do Sistema Nervoso Central/imunologia , Astrócitos/metabolismo , Comunicação Celular , Células Cultivadas , Dexametasona/farmacologia , Expressão Gênica , Regulação Enzimológica da Expressão Gênica , Granuloma/enzimologia , Humanos , Metaloproteinases da Matriz/metabolismo , Monócitos/efeitos dos fármacos , NF-kappa B/metabolismo , Inibidores Teciduais de Metaloproteinases/metabolismo , Tuberculose do Sistema Nervoso Central/patologia , Regulação para Cima
15.
Exp Lung Res ; 32(5): 201-14, 2006 May.
Artigo em Inglês | MEDLINE | ID: mdl-16908447

RESUMO

Idiopathic pulmonary fibrosis (IPF) is characterized by fibroblast expansion and extracellular matrix accumulation. However, the mechanisms involved in matrix remodeling have not been elucidated. In this study, the authors aimed to evaluate the expression of the tissue inhibitors of matrix metalloproteinases (TIMPs) in human fibroblasts and whole tissues from IPF and normal lungs. They also determined the role of mitogen-activated protein kinase (MAPK) in TIMP3 expression. TIMP1, TIMP2, and TIMP3 were highly expressed in lung fibroblasts. Transforming growth factor (TGF)-beta1, a profibrotic mediator, induced strong up-regulation of TIMP3 at the mRNA and protein levels. The authors examined whether the MAPK pathway was involved in TGF-beta1-induced TIMP3 expression. TGF-beta1 induced the phosphorylation of p38 and extracellular signal-regulated kinase (ERK)1/2. Biochemical blockade of p38 by SB203580, but not of the ERK MAPK pathway, inhibited the effect of this factor. The effect was also blocked by the tyrosine kinase inhibitor genistein and by antagonizing TGF-beta1 receptor type I (activin-linked kinase [ALK5]). In IPF tissues TIMP3 gene expression was significantly increased and the protein was localized to fibroblastic foci and extracellular matrix. Our findings suggest that TGF-beta1-induced TIMP3 may be an important mediator in lung fibrogenesis.


Assuntos
Fibroblastos/metabolismo , Fibrose Pulmonar/metabolismo , Inibidor Tecidual de Metaloproteinase-3/metabolismo , Fator de Crescimento Transformador beta/fisiologia , Regulação para Cima/efeitos dos fármacos , Células Cultivadas , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/fisiologia , Inibidores Enzimáticos/farmacologia , Fibroblastos/patologia , Fibroblastos/fisiologia , Genisteína/farmacologia , Humanos , Imidazóis/farmacologia , Interferon gama/farmacologia , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Fibrose Pulmonar/patologia , Fibrose Pulmonar/fisiopatologia , Piridinas/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Inibidor Tecidual de Metaloproteinase-1/genética , Inibidor Tecidual de Metaloproteinase-1/metabolismo , Inibidor Tecidual de Metaloproteinase-3/genética , Fator de Crescimento Transformador beta1 , Regulação para Cima/fisiologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
16.
Am J Respir Crit Care Med ; 172(12): 1596-604, 2005 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-16141443

RESUMO

RATIONALE: Pulmonary cavitation is fundamental to the global success of Mycobacterium tuberculosis. However, the mechanisms of this lung destruction are poorly understood. The biochemistry of lung matrix predicts matrix metalloproteinase (MMP) involvement in immunopathology. METHODS: We investigated gene expression of all MMPs, proteins with a disintegrin and metalloproteinase domain, and tissue inhibitors of metalloproteinases in M. tuberculosis-infected human macrophages by real-time polymerase chain reaction. MMP secretion was measured by zymography and Western analysis, and expression in patients with pulmonary tuberculosis was localized by immunohistochemistry. RESULTS: MMP-1 and MMP-7 gene expression and secretion are potently upregulated by M. tuberculosis, and no increase in tissue inhibitor of metalloproteinase expression occurs to oppose their activity. Dexamethasone completely suppresses MMP-1 but not MMP-7 gene expression and secretion. In patients with active tuberculosis, macrophages express MMP-1 and MMP-7 adjacent to areas of tissue destruction. MMP-1 but not MMP-7 expression and secretion are relatively M. tuberculosis specific, are not upregulated by tuberculosis-associated cytokines, and are prostaglandin dependent. In contrast, the vaccine M. bovis bacillus Calmette-Guérin (BCG) does not stimulate MMP-1 secretion from human macrophages, although M. tuberculosis and BCG do upregulate MMP-7 equally. BCG-infected macrophages secrete reduced prostaglandin E2 concentrations compared with M. tuberculosis-infected macrophages, and prostaglandin pathway supplementation augments MMP-1 secretion from BCG-infected cells. CONCLUSIONS: M. tuberculosis specifically upregulates MMP-1 in a cellular model of human infection and in patients with tuberculosis. In contrast, vaccine BCG, which does not cause lung cavitation, does not upregulate prostaglandin E2-dependent MMP-1 secretion.


Assuntos
Proteínas ADAM/metabolismo , Metaloproteinases da Matriz/metabolismo , Monócitos/metabolismo , Mycobacterium bovis/fisiologia , Mycobacterium tuberculosis/fisiologia , Tuberculose Pulmonar/metabolismo , Proteínas ADAM/genética , Estudos de Casos e Controles , Técnicas de Cultura de Células , Humanos , Metaloproteinases da Matriz/genética , RNA Mensageiro/metabolismo , Inibidores Teciduais de Metaloproteinases/genética , Inibidores Teciduais de Metaloproteinases/metabolismo
17.
J Immunol ; 173(8): 5209-18, 2004 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-15470066

RESUMO

Metalloproteinases (MPs) include matrix metalloproteinases (MMPs) and metalloproteinase-disintegrins (ADAMs). Their physiological inhibitors are tissue inhibitor of metalloproteinases (TIMPs). MPs are thought to be mediators of cellular infiltration in the pathogenesis of multiple sclerosis and its animal model, experimental autoimmune encephalomyelitis (EAE). We used real-time RT-PCR to profile the expression of all 22 known mouse MMPs, seven ADAMs, and all four known TIMPs in spinal cord from SJL/J mice and mice with adoptively transferred myelin basic protein (MBP)-specific EAE. A significant and >3-fold alteration in expression was observed for MMP-8, MMP-10, MMP-12, ADAM-12, and TIMP-1, which were up-regulated, and for MMP-15, which was down-regulated. Expression levels correlated with disease course, with all but ADAM-12 returning toward control levels in remission. To examine potential cellular sources of these strongly affected proteins in the inflamed CNS, we isolated macrophages, granulocytes, microglia, and T cells by cell sorting from the CNS of mice with EAE and analyzed their expression by real-time RT-PCR. This identified macrophages as a major source of MMP-12 and TIMP-1. Granulocytes were a major source of MMP-8. ADAM-12 was expressed primarily by T cells. Cellular localization of MMP-10, TIMP-1, and ADAM-12 in perivascular infiltrates was confirmed by immunostaining or in situ hybridization. Microglia from control mice expressed strong signal for MMP-15. Strikingly, the expression of MMP-15 by microglia was significantly down-regulated in EAE, which was confirmed by immunostaining. Our study identifies the cellular sources of key MPs in CNS inflammation.


Assuntos
Encefalomielite Autoimune Experimental/enzimologia , Metaloproteases/genética , Proteínas ADAM , Proteína ADAM12 , Animais , Feminino , Imuno-Histoquímica , Hibridização In Situ , Metaloproteinase 10 da Matriz , Metaloproteinase 8 da Matriz/genética , Metaloproteinases da Matriz Associadas à Membrana , Proteínas de Membrana/genética , Metaloendopeptidases/genética , Metaloproteases/análise , Camundongos , Células Mieloides/enzimologia , Medula Espinal/citologia , Medula Espinal/enzimologia , Inibidor Tecidual de Metaloproteinase-1/genética
18.
Biochem J ; 364(Pt 1): 89-99, 2002 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-11988080

RESUMO

We have used real-time quantitative reverse transcriptase PCR (TaqMan) to quantify the expression of the four tissue inhibitor of metalloproteinases (Timp) genes in mouse tissues during development and in the adult. Among the four Timp genes, Timp-4 shows the most restricted pattern of expression, with highest RNA levels in brain, heart and testes. These data indicate that in the brain, Timp-4 transcripts are temporally regulated during development, becoming more abundant than those of the other Timps after birth. Cloning of the Timp-4 gene confirmed a five-exon organization resembling that of Timp-2 and Timp-3, and like all Timps, Timp-4 is located within an intron of a synapsin gene. Ribonuclease protection analysis and 5'-rapid amplification of cDNA ends PCR identified multiple transcription starts for Timp-4 from brain and heart mRNA. The promoter region of Timp-4 was functional in transient transfection analysis in mouse C3H10T1/2 fibroblasts, where it directed basal expression that was non-inducible by serum. The TATA-less promoter contains consensus motifs for Sp1 and an inverted CCAAT box upstream of an initiator-like element that is in close proximity to a transcription start site. Mutation of the CCAAT box caused a 2-fold increase in reporter expression. More significantly, mutation of the Sp1 motif or initiator-like element almost completely abolished reporter expression. This first functional characterization of the Timp-4 promoter shows it to be distinct from other members of the Timp family and provides insights into potential mechanisms controlling the tight spatio-temporal expression pattern of the gene.


Assuntos
Inibidores Teciduais de Metaloproteinases/biossíntese , Inibidores Teciduais de Metaloproteinases/genética , Regiões 5' não Traduzidas , Animais , Sequência de Bases , Linhagem Celular , Núcleo Celular/metabolismo , DNA Complementar/metabolismo , Feminino , Fibroblastos/metabolismo , Genes Reporter , Íntrons , Luciferases/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C3H , Modelos Genéticos , Dados de Sequência Molecular , Mutação , Plasmídeos/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , Estrutura Terciária de Proteína , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Transcrição Sp1/metabolismo , Fatores de Tempo , Distribuição Tecidual , Transcrição Gênica , Transfecção , Inibidor Tecidual 4 de Metaloproteinase
19.
Biochem Biophys Res Commun ; 322(3): 759-65, 2004 Sep 24.
Artigo em Inglês | MEDLINE | ID: mdl-15336529

RESUMO

P19CL6 are a clonal derivative of P19 embryonal carcinoma cells, a euploid, multipotent mouse cell line, that differentiate efficiently into cardiac myocytes, with spontaneous beating evident within 10 days, following DMSO treatment. Using real-time quantitative RT-PCR we have profiled the expression of the complete matrix metalloproteinase and tissue inhibitor of metalloproteinase gene families during P19CL6 differentiation to cardiac myocytes. The genes subdivide into eight groups based upon their expression profile. Their expression was both qualitatively and quantitatively highly homologous to that seen during mouse heart development.


Assuntos
Dimetil Sulfóxido/farmacologia , Metaloproteinases da Matriz/metabolismo , Células Musculares/citologia , Miocárdio/citologia , Inibidores Teciduais de Metaloproteinases/metabolismo , Animais , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Linhagem Celular Tumoral , Cinética , Metaloproteinases da Matriz/efeitos dos fármacos , Camundongos , Células Musculares/efeitos dos fármacos , Teratoma , Inibidores Teciduais de Metaloproteinases/efeitos dos fármacos
20.
J Immunol ; 170(9): 4497-505, 2003 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-12707326

RESUMO

Circulating B cells enter the CNS as part of normal immune surveillance and in pathologic states, including the common and disabling illness multiple sclerosis. However, little is known about the molecular mechanisms that mediate human B cell interaction with the specialized brain endothelial cells comprising the blood-brain barrier (BBB). We studied the molecular mechanisms that regulate the migration of normal human B cells purified ex vivo, across human adult brain-derived endothelial cells (HBECs). We found that B cells migrated across HBECs more efficiently than T cells from the same individuals. B cell migration was significantly inhibited by blocking Abs to the adhesion molecules ICAM-1 and VLA-4, but not VCAM-1, similar to the results previously reported for T cells. Blockade of the chemokines monocyte chemoattractant protein-1 and IL-8, but not RANTES or IFN-gamma-inducible protein-10, significantly inhibited B cell migration, and these results were correlated with the chemokine receptor expression of B cells measured by flow cytometry and by RNase protection assay. Tissue inhibitor of metalloproteinase-1, a natural inhibitor of matrix metalloproteinases, significantly decreased B cell migration across the HBECs. A comprehensive RT-PCR comparative analysis of all known matrix metalloproteinases and tissue inhibitors of metalloproteinases in human B and T cells revealed distinct profiles of expression of these molecules in the different cell subsets. Our results provide insights into the molecular mechanisms that underlie human B cell migration across the BBB. Furthermore, they identify potential common, and unique, therapeutic targets for limiting CNS B cell infiltration and predict how therapies currently developed to target T cell migration, such as anti-VLA-4 Abs, may impact on B cell trafficking.


Assuntos
Linfócitos B/citologia , Barreira Hematoencefálica/imunologia , Movimento Celular/imunologia , Endotélio Vascular/citologia , Endotélio Vascular/imunologia , Adulto , Linfócitos B/enzimologia , Linfócitos B/imunologia , Linfócitos B/metabolismo , Inibição de Migração Celular , Movimento Celular/efeitos dos fármacos , Separação Celular , Quimiocina CCL2/biossíntese , Quimiocina CCL2/genética , Quimiocina CCL2/metabolismo , Cultura em Câmaras de Difusão , Endotélio Vascular/enzimologia , Fibronectinas/metabolismo , Humanos , Integrina alfa4beta1/metabolismo , Integrina alfa4beta1/fisiologia , Interleucina-8/biossíntese , Interleucina-8/genética , Interleucina-8/metabolismo , Inibidores de Metaloproteinases de Matriz , Metaloproteinases da Matriz/biossíntese , Receptores CCR2 , Receptores de Quimiocinas/biossíntese , Receptores de Quimiocinas/genética , Receptores de Quimiocinas/metabolismo , Receptores de Interleucina-8A/biossíntese , Receptores de Interleucina-8A/genética , Receptores de Interleucina-8A/metabolismo , Receptores de Interleucina-8B/biossíntese , Receptores de Interleucina-8B/genética , Receptores de Interleucina-8B/metabolismo , Linfócitos T/citologia , Inibidor Tecidual de Metaloproteinase-1/farmacologia , Molécula 1 de Adesão de Célula Vascular/metabolismo
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