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1.
J Diabetes Complications ; 37(4): 108421, 2023 04.
Artigo em Inglês | MEDLINE | ID: mdl-36905721

RESUMO

AIMS: The aim of this study was to investigate the effects of pioglitazone on reactive oxygen species (ROS), expressions/activities of MMPs and TIMP-2, and VSMC proliferation and vascular reactivity in high glucose (HG)-induced human saphenous vein (HSV) grafts. METHODS: HSV grafts (n = 10) obtained from patients undergoing CABG were incubated with 30 mM glucose and/or 10 µM pioglitazone or 0.1 % DMSO for 24 h after endothelium removal. ROS levels were examined by chemiluminescence assay, MMP-2,-9,-14, TIMP-2, and α-SMA expression/activity was determined by gelatine zymography/immunohistochemistry. Vascular reactivity to potassium chloride, noradrenaline, serotonin, prostaglandin F2α and papaverine was assessed in HSVs. RESULTS: HG induced superoxide anion (SA) (123 %) and other ROS levels (159 %), up-regulated MMP-2 expression (180 %)/activity (79 %), MMP-14 expression (24 %) and MMP-9 activity while down-regulating TIMP-2 expression (27 %). HG elevated total MMP-2/TIMP-2 ratio (483 %) and MMP-14/TIMP-2 ratio (78 %). However, HG plus pioglitazone inhibited SA (30 %) and other ROS levels (29 %), down-regulated MMP-2 expression (76 %)/activity (83 %), MMP-14 expression (38 %) and MMP-9 activity, while reversing TIMP-2 expression (44 %). HG plus pioglitazone decreased total MMP-2/TIMP-2 ratio (91 %) and MMP-14/TIMP-2 ratio (59 %). HG impaired contractions to all agents but pioglitazone improved them. CONCLUSIONS: Pioglitazone may contribute to the prevention of restenosis and maintaining vascular function in HSV grafts of DM patients undergoing CABG.


Assuntos
Metaloproteinase 2 da Matriz , Metaloproteinase 9 da Matriz , Estresse Oxidativo , Pioglitazona , Veia Safena , Humanos , Glucose/farmacologia , Glucose/metabolismo , Metaloproteinase 14 da Matriz/efeitos dos fármacos , Metaloproteinase 14 da Matriz/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Pioglitazona/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Veia Safena/metabolismo , Inibidor Tecidual de Metaloproteinase-1/metabolismo , Inibidor Tecidual de Metaloproteinase-1/farmacologia , Inibidor Tecidual de Metaloproteinase-2/metabolismo , Inibidor Tecidual de Metaloproteinase-2/farmacologia , Inflamação/metabolismo
2.
Drug Deliv ; 29(1): 111-127, 2022 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34964414

RESUMO

Naringenin, a flavonoid, possesses antiangiogenic potential and inhibits corneal neovascularization (CNV); however, its therapeutic use is restricted due to poor solubility and limited bioavailability. In this study, we developed a naringenin microemulsion (NAR-ME) for inhibiting CNV. NAR-ME formulation was composed of triacetin (oil phase), Cremophor RH40 (CRH40), PEG400, and water, its droplet size was 13.22 ± 0.13 nm with a narrow size distribution (0.112 ± 0.0014). The results demonstrated that NAR-ME released higher and permeated more drug than NAR suspension (NAR-Susp) in in vitro drug release and ex vivo corneal permeation study. Human corneal epithelial cells (HCECs) toxicity study showed no toxicity with NAR-ME, which is consistent with the result of ocular irritation study. NAR-ME had high bioavailability 1.45-fold, 2.15-fold, and 1.35-fold higher than NAR-Susp in the cornea, conjunctiva, and aqueous humor, respectively. Moreover, NAR-ME (0.5% NAR) presented efficacy comparable to that of dexamethasone (0.025%) in the inhibition of CNV in mice CNV model induced by alkali burning, resulting from the attenuation of corneal vascular endothelial growth factor (VEGF) and matrix metalloproteinase (MMP-14) expression. In conclusion, the optimized NAR-ME formulation demonstrated excellent physicochemical properties and good tolerance, enhanced ocular bioavailability and corneal permeability. This formulation is promising, safe, and effective for the treatment of CNV.


Assuntos
Neovascularização da Córnea/patologia , Portadores de Fármacos/química , Emulsões/química , Flavanonas/farmacologia , Animais , Linhagem Celular , Sobrevivência Celular , Química Farmacêutica , Córnea/metabolismo , Modelos Animais de Doenças , Liberação Controlada de Fármacos , Estabilidade de Medicamentos , Flavanonas/administração & dosagem , Flavanonas/efeitos adversos , Humanos , Concentração de Íons de Hidrogênio , Masculino , Metaloproteinase 14 da Matriz/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C , Soluções Oftálmicas , Tamanho da Partícula , Coelhos , Propriedades de Superfície , Fator A de Crescimento do Endotélio Vascular/efeitos dos fármacos
3.
Endocr J ; 66(11): 1029-1037, 2019 Nov 28.
Artigo em Inglês | MEDLINE | ID: mdl-31366822

RESUMO

In the present study, we investigate the effect of reduced cystathionine-γ-lyase (CSE) expression in high glucose induced metalloproteinases14 (MMP14) expression in adipocytes and visceral adipose tissues. Diabetic mice were prepared by injections of STZ and the expression of CSE, MMP14 in visceral adipose tissues were determined. Adipocytes were differentiated from 3T3-L1 cells and treated with high glucose (HG), H2S slow-releasing compound GYY4137 or transfected with CSE siRNA. Then the expression of CSE, MMP14 were determined by western blotting. CSE knockout mice were generated by crossing CSE+/- heterozygous mice and given intraperitoneally (i.p.) injections of GYY4137, and then the expression of CSE and MMP14 in visceral adipose tissues were determined by quantitative real-time PCR and western blotting. The following results were obtained from the study. In adipose tissues of diabetic mice, the mRNA and protein expression of MMP14 increased while the mRNA and protein expression of CSE decreased. In 3T3-L1 adipocytes, both HG DMEM and CSE siRNA transfection increased the mRNA and protein of MMP14. The addition of GYY4137 inhibited HG-induced upregulation of MMP14 expression. In CSE knockout mice, the mRNA and protein expression of MMP14 in adipose tissues increased, which could be inhibited by i.p. injections of GYY4137. In conclusion, high glucose increased the expression of MMP14 in adipocytes and visceral adipose tissues through inhibiting the expression of CSE.


Assuntos
Adipócitos/metabolismo , Cistationina gama-Liase/genética , Diabetes Mellitus Experimental/genética , Gordura Intra-Abdominal/metabolismo , Metaloproteinase 14 da Matriz/genética , Células 3T3-L1 , Adipócitos/efeitos dos fármacos , Animais , Western Blotting , Cistationina gama-Liase/efeitos dos fármacos , Cistationina gama-Liase/metabolismo , Diabetes Mellitus Experimental/metabolismo , Glucose/farmacologia , Metaloproteinase 14 da Matriz/efeitos dos fármacos , Metaloproteinase 14 da Matriz/metabolismo , Camundongos , Camundongos Knockout , Morfolinas/farmacologia , Compostos Organotiofosforados/farmacologia , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/metabolismo , RNA Interferente Pequeno , Reação em Cadeia da Polimerase em Tempo Real
4.
Bauru; s.n; 2017. 54 p. ilus, graf.
Tese em Inglês | BBO - odontologia (Brasil) | ID: biblio-880409

RESUMO

A espécie vegetal Qualea grandiflora (QG), popularmente conhecida como "pauferro", "pau-terra-da-folha-grande", "pau-terra" ou "pau-de-tucano", muito comum no Cerrado brasileiro, é bem conhecida devido às suas variadas propriedades terapêuticas. Suas indicações incluem ações preventivas no aparecimento de lesões de mucosa gástrica, efeitos analgésicos, antibacterianos, anti-inflamatórios e antifúngicos. Assim, os componentes da QG poderiam ter alguma ação sobre moléculas amplamente envolvidas em processos angiogênicos e de desenvolvimento/reparo, como a Metaloproteinase de matriz 14 (MMP-14) e o Fator Induzido por hipóxia 1α (HIF-1alfa). Dessa maneira, o objetivo deste estudo foi investigar os efeitos do extrato hidroalcoólico das folhas de QG na viabilidade celular e expressão de MMP-14 e HIF-1alpha em culturas de fibroblastos da linhagem NIH/3T3 e pré-osteoblastos da linhagem MC3T3-E1. Para o teste de viabilidade celular e expressão das moléculas, concentrações de 0.1; 1.0 e 10 µg/mL do extrato hidroalcoólico das folhas de QG foram administrados por períodos de 24, 48, 72 e 96h. Após cada período, a viabilidade celular foi avaliada pelo método de redução de MTT e a análise da expressão das moléculas foi feita por meio da técnica de imunofluorescência. Os resultados mostram que o extrato de QG não promove redução da viabilidade celular de fibroblastos e pré-osteoblastos em concentrações até 10 µg/mL, nos períodos iniciais (24 e 48h). Porém, uma redução significativa da viabilidade pode ser verificada nos períodos de 72h e 96h para os fibroblastos e 96h para os pré-osteoblastos, expostos a mais alta concentração do extrato (10 µg/mL). O ensaio de imunofluorescência indica que o extrato, nas concentrações de 0.1; 1.0 e 10 µg/mL foi capaz de aumentar a expressão de MMP-14 e HIF-1alpha, em ambos os tipos celulares. Em conclusão, nossos resultados indicam que o extrato de QG exerce um efeito capaz de aumentar a expressão das duas moléculas em estudo (MMP-14 e HIF-1alpha), tanto para os fibroblastos da linhagem NIH/3T3 como para os pré- osteoblastos da linhagem MC3T3-E1. Assim, os compostos de QG podem apresentar potencial para serem utilizados como agentes terapêuticos moduladores da angiogênese, por meio do aumento da expressão de MMP-14 e HIF-1alpha.(AU)


The vegetable specie Qualea grandiflora (QG), popularly known as "pau-ferro", "pauterra-da-folha-grande", "pau-terra" or "pau-de-tucano", very common in the Brazilian Cerrado, is well known due to its varied therapeutic properties. Its indications include preventive actions in the appearance of lesions of gastric mucosa, analgesic, antibacterial, anti-inflammatory and antifungal effects. Thus, QG components could have some action on molecules widely involved in angiogenic and developmental / repair processes, such as Matrix metalloproteinase 14 (MMP-14) and HypoxiaInducible Factor-1α (HIF-1alpha). Thus, the objective of our study was to investigate the effects of QG hydroalcoholic extract on cell viability and expression of MMP-14 and HIF-1alpha in NIH/3T3 fibroblasts and MC3T3-E1 pre-osteoblasts cell lines. For the cell viability assay and expression of the molecules, concentrations of 0.1; 1.0 and 10 µg / mL of the hydroalcoholic extract of leaves of QG, were administered for periods of 24, 48, 72 and 96h. After each period, the cell viability was evaluated by MTT assay and the expression of the molecules was analyzed using the immunofluorescence technique. The results show that the QG extract does not promote reduction of the cellular viability of fibroblasts and pre-osteoblasts in concentrations up to 10 µg/mL in the initial periods (24 and 48h). However, a significant reduction in viability can be observed in 72h and 96h for fibroblasts and 96h for pre-osteoblasts exposed to the highest extract concentration (10 µg/mL). The immunofluorescence assay indicates that the extract, at concentrations of 0.1; 1.0 and 10 µg/mL was able to increase the expression of MMP-14 and HIF-1alpha in both cell types. In conclusion, our results indicate that the QG extract exerts an effect capable of increasing the expression of the two molecules under study (MMP-14 and HIF-1alpha) both for the NIH/3T3 fibroblasts as well as for the MC3T3-E1 pre-osteoblasts cells. Thus, the QG compounds could have potential to be used as angiogenesis modulating therapeutic agents, by increasing the expression of MMP-14 and HIF-1alpha.(AU)


Assuntos
Animais , Sobrevivência Celular/efeitos dos fármacos , Subunidade alfa do Fator 1 Induzível por Hipóxia/efeitos dos fármacos , Magnoliopsida/química , Metaloproteinase 14 da Matriz/efeitos dos fármacos , Células NIH 3T3/efeitos dos fármacos , Osteoblastos/efeitos dos fármacos , Extratos Vegetais/farmacologia , Células Cultivadas , Imunofluorescência/métodos , Subunidade alfa do Fator 1 Induzível por Hipóxia/análise , Metaloproteinase 14 da Matriz/análise , Muridae , Reprodutibilidade dos Testes , Espectrofotometria/métodos , Fatores de Tempo
5.
Arthritis Rheumatol ; 68(2): 521-31, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26315469

RESUMO

OBJECTIVE: In rheumatoid arthritis (RA), destruction of articular cartilage by the inflamed synovium is considered to be driven by increased activities of proteolytic enzymes, including matrix metalloproteinases (MMPs). The purpose of this study was to investigate the therapeutic potential of selective inhibition of membrane type 1 MMP (MT1-MMP) and its combination with tumor necrosis factor (TNF) blockage in mice with collagen-induced arthritis (CIA). METHODS: CIA was induced in DBA/1 mice by immunization with bovine type II collagen. From the onset of clinical arthritis, mice were treated with MT1-MMP selective inhibitory antibody DX-2400 and/or TNFR-Fc fusion protein. Disease progression was monitored daily, and serum, lymph nodes, and affected paws were collected at the end of the study for cytokine and histologic analyses. For in vitro analysis, bone marrow-derived macrophages were stimulated with lipopolysaccharide for 24 hours in the presence of DX-2400 and/or TNFR-Fc to analyze cytokine production and phenotype. RESULTS: DX-2400 treatment significantly reduced cartilage degradation and disease progression in mice with CIA. Importantly, when combined with TNF blockade, DX-2400 acted synergistically, inducing long-term benefit. DX-2400 also inhibited the up-regulation of interleukin-12 (IL-12)/IL-23 p40 via polarization toward an M2 phenotype in bone marrow-derived macrophages. Increased production of IL-17 induced by anti-TNF, which correlated with an incomplete response to anti-TNF, was abrogated by combined treatment with DX-2400 in CIA. CONCLUSION: Targeting MT1-MMP provides a potential strategy for joint protection, and its combination with TNF blockade may be particularly beneficial in RA patients with an inadequate response to anti-TNF therapy.


Assuntos
Anticorpos Monoclonais/farmacologia , Artrite Experimental/imunologia , Cartilagem Articular/efeitos dos fármacos , Etanercepte/farmacologia , Subunidade p40 da Interleucina-12/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Metaloproteinase 14 da Matriz/efeitos dos fármacos , Inibidores de Metaloproteinases de Matriz/farmacologia , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Animais , Anticorpos Monoclonais Humanizados , Artrite Experimental/patologia , Cartilagem Articular/patologia , Progressão da Doença , Técnicas In Vitro , Interferon gama/efeitos dos fármacos , Interferon gama/imunologia , Interleucina-10/imunologia , Subunidade p40 da Interleucina-12/imunologia , Interleucina-17/imunologia , Lipopolissacarídeos/farmacologia , Linfonodos/efeitos dos fármacos , Linfonodos/imunologia , Linfonodos/patologia , Macrófagos/imunologia , Metaloproteinase 14 da Matriz/imunologia , Camundongos , Camundongos Endogâmicos DBA
6.
J Natl Cancer Inst ; 107(4)2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25710962

RESUMO

BACKGROUND: Matrix metalloproteinase (MMP) 14 may mediate tumor progression through vascular and immune-modulatory effects. METHODS: Orthotopic murine breast tumors (4T1 and E0771 with high and low MMP14 expression, respectively; n = 5-10 per group) were treated with an anti-MMP14 inhibitory antibody (DX-2400), IgG control, fractionated radiation therapy, or their combination. We assessed primary tumor growth, transforming growth factor ß (TGFß) and inducible nitric oxide synthase (iNOS) expression, macrophage phenotype, and vascular parameters. A linear mixed model with repeated observations, with Mann-Whitney or analysis of variance with Bonferroni post hoc adjustment, was used to determine statistical significance. All statistical tests were two-sided. RESULTS: DX-2400 inhibited tumor growth compared with IgG control treatment, increased macrophage numbers, and shifted the macrophage phenotype towards antitumor M1-like. These effects were associated with a reduction in active TGFß and SMAD2/3 signaling. DX-2400 also transiently increased iNOS expression and tumor perfusion, reduced tissue hypoxia (median % area: control, 20.2%, interquartile range (IQR) = 6.4%-38.9%; DX-2400: 1.2%, IQR = 0.2%-3.2%, P = .044), and synergistically enhanced radiation therapy (days to grow to 800mm(3): control, 12 days, IQR = 9-13 days; DX-2400 plus radiation, 29 days, IQR = 26-30 days, P < .001) in the 4T1 model. The selective iNOS inhibitor, 1400W, abolished the effects of DX-2400 on vessel perfusion and radiotherapy. On the other hand, DX-2400 was not capable of inducing iNOS expression or synergizing with radiation in E0771 tumors. CONCLUSION: MMP14 blockade decreased immunosuppressive TGFß, polarized macrophages to an antitumor phenotype, increased iNOS, and improved tumor perfusion, resulting in reduced primary tumor growth and enhanced response to radiation therapy, especially in high MMP14-expressing tumors.


Assuntos
Amidinas/farmacologia , Anticorpos Monoclonais/farmacologia , Antineoplásicos/farmacologia , Benzilaminas/farmacologia , Neoplasias da Mama/metabolismo , Neoplasias da Mama/radioterapia , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/uso terapêutico , Macrófagos/efeitos dos fármacos , Metaloproteinase 14 da Matriz/efeitos dos fármacos , Metaloproteinase 14 da Matriz/metabolismo , Óxido Nítrico Sintase Tipo II/efeitos dos fármacos , Animais , Anticorpos Monoclonais/uso terapêutico , Antineoplásicos/uso terapêutico , Neoplasias da Mama/irrigação sanguínea , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/imunologia , Linhagem Celular Tumoral , Fracionamento da Dose de Radiação , Feminino , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Imunoglobulina G/sangue , Macrófagos/enzimologia , Neoplasias Mamárias Experimentais , Camundongos , Neovascularização Patológica , Óxido Nítrico Sintase Tipo II/antagonistas & inibidores , Óxido Nítrico Sintase Tipo II/metabolismo , Fenótipo , Transdução de Sinais/efeitos dos fármacos , Proteína Smad2/metabolismo , Proteína Smad3/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Regulação para Cima
7.
J Pediatr Gastroenterol Nutr ; 61(2): 194-9, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-25539192

RESUMO

OBJECTIVES: Eosinophilic esophagitis (EoE) is a chronic, antigen-mediated disease in children and adults associated with substantial esophageal remodeling and fibrosis. The expression of the remodeling-associated matrix metalloproteinases (MMPs) has not been previously detailed in EoE. METHODS: MMP-2 and -14 expression and cellular localization were assessed using real-time quantitative polymerase chain reaction and immunohistochemistry/immunofluorescence in EoE fibroblasts, active and inactive pediatric EoE biopsies, and nondiseased control biopsies. The effect of transforming growth factor (TGF)-ß1 treatment on MMP-2 expression in cultured esophageal epithelial (HET1A) cells was analyzed. RESULTS: MMP-2 and -14 mRNA were expressed in EoE fibroblasts and biopsies. Proliferating epithelial cells produced MMP-14 more abundantly in EoE than in controls (P < 0.001) and the degree of epithelial MMP-14 expression correlated positively with basal zone hyperplasia (r = 0.65, P = 0.002). EoE lamina propria had higher numbers of MMP-2- and -14-positive cells (906 ±â€Š167 and 701 ±â€Š93 cells/mm²) as compared with controls (258 ±â€Š93 cells/mm², P < 0.01 and 232 ±â€Š54 cells/mm², P < 0.01), and MMP-14 expression correlated with the severity of fibrosis. Following therapy with topical corticosteroids, MMP-14 and -2 were significantly diminished (P < 0.01). TGF-ß1 increased the expression and secretion of MMP-2 from esophageal epithelial HET1A cells. CONCLUSIONS: MMP-2 and -14 are elevated in pediatric patients with EoE and significantly decrease following topical corticosteroid therapy. TGF-ß1 increases MMP-2 in esophageal epithelial cells. This alludes to previously unappreciated role for MMPs in EoE-associated esophageal remodeling and a potential positive feedback loop via TGF-ß1.


Assuntos
Corticosteroides/uso terapêutico , Esofagite Eosinofílica/enzimologia , Metaloproteinase 14 da Matriz/análise , Metaloproteinase 2 da Matriz/análise , Biópsia , Criança , Esofagite Eosinofílica/tratamento farmacológico , Esofagite Eosinofílica/patologia , Células Epiteliais/enzimologia , Células Epiteliais/patologia , Feminino , Fibroblastos/enzimologia , Imunofluorescência , Expressão Gênica/efeitos dos fármacos , Humanos , Imuno-Histoquímica , Masculino , Metaloproteinase 14 da Matriz/efeitos dos fármacos , Metaloproteinase 14 da Matriz/genética , Metaloproteinase 2 da Matriz/efeitos dos fármacos , Metaloproteinase 2 da Matriz/genética , RNA Mensageiro/análise , Reação em Cadeia da Polimerase em Tempo Real , Fator de Crescimento Transformador beta1/farmacologia
8.
Circulation ; 124(24): 2725-34, 2011 Dec 13.
Artigo em Inglês | MEDLINE | ID: mdl-22082680

RESUMO

BACKGROUND: Outcomes for organ transplantation are constantly improving because of advances in organ preservation, surgical techniques, immune clinical monitoring, and immunosuppressive treatment preventing acute transplant rejection. However, chronic rejection including transplant vasculopathy still limits long-term patient survival. Transplant vasculopathy is characterized by progressive neointimal hyperplasia leading to arterial stenosis and ischemic failure of the allograft. This work sought to decipher the manner in which the humoral immune response, mimicked by W6/32 anti-HLA antibody, contributes to transplant vasculopathy. METHODS AND RESULTS: Studies were performed in vitro on cultured human smooth muscle cells, ex vivo on human arterial segments, and in vivo in a model consisting of human arterial segments grafted into severe combined immunodeficiency/beige mice injected weekly with anti-HLA antibodies. We report that anti-HLA antibodies are mitogenic for smooth muscle cells through a signaling mechanism implicating matrix metalloproteinases (MMPs) (membrane type 1 MMP and MMP2) and neutral sphingomyelinase-2. This mitogenic signaling and subsequent DNA synthesis are blocked in smooth muscle cells silenced for MMP2 or for neutral sphingomyelinase-2 by small interfering RNAs, in smooth muscle cells transfected with a vector coding for a dominant-negative form of membrane type 1 MMP, and after treatment by pharmacological inhibitors of MMPs (Ro28-2653) or neutral sphingomyelinase-2 (GW4869). In vivo, Ro28-2653 and GW4869 reduced the intimal thickening induced by anti-HLA antibodies in human mesenteric arteries grafted into severe combined immunodeficiency/beige mice. CONCLUSIONS: These data highlight a crucial role for MMP2 and neutral sphingomyelinase-2 in vasculopathy triggered by a humoral immune response and open new perspectives for preventing transplant vasculopathy with the use of MMP and neutral sphingomyelinase inhibitors, in addition to conventional immunosuppression.


Assuntos
Anticorpos Anti-Idiotípicos/farmacologia , Artérias/transplante , Antígenos HLA/imunologia , Metaloproteinase 14 da Matriz/metabolismo , Metaloproteinase 2 da Matriz/metabolismo , Esfingomielina Fosfodiesterase/metabolismo , Doenças Vasculares/fisiopatologia , Compostos de Anilina/farmacologia , Animais , Anticorpos Anti-Idiotípicos/efeitos adversos , Artérias/patologia , Artérias/fisiopatologia , Compostos de Benzilideno/farmacologia , Células Cultivadas , Constrição Patológica/etiologia , Constrição Patológica/fisiopatologia , Modelos Animais de Doenças , Humanos , Hiperplasia/etiologia , Hiperplasia/fisiopatologia , Técnicas In Vitro , Metaloproteinase 14 da Matriz/efeitos dos fármacos , Metaloproteinase 2 da Matriz/efeitos dos fármacos , Inibidores de Metaloproteinases de Matriz , Camundongos , Camundongos SCID , Modelos Animais , Músculo Liso Vascular/citologia , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/metabolismo , Neointima/patologia , Neointima/fisiopatologia , Piperazinas/farmacologia , Pirimidinas/farmacologia , RNA Interferente Pequeno/farmacologia , Esfingomielina Fosfodiesterase/antagonistas & inibidores , Esfingomielina Fosfodiesterase/efeitos dos fármacos , Doenças Vasculares/etiologia , Enxerto Vascular
9.
Oral Dis ; 17(3): 291-7, 2011 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-20860766

RESUMO

OBJECTIVE: Osteonecrosis of the jaw is a serious complication of bisphosphonate treatment for which the pathophysiology is unknown. The purpose of this study was to investigate whether in vivo zoledronic acid (ZA) induces alterations in cell proliferation, apoptosis, and matrix metalloproteinases (MMPs) expression in oral mucosal epithelial cells. METHODS: One-year-old dogs were either untreated (control group) or given high doses of intravenous ZA (ZA group) for 3 months. The doses of ZA were equivalent to those given to cancer patients, yet were administered two times more frequently (every 2 weeks). Mucosal tissues were assessed immunohistochemically for cell proliferation (proliferating cell nuclear antigen, PCNA), matrix metalloproteinase (MMP) expression, and apoptosis (caspase 3 and TUNEL). RESULTS: There were no significant differences between the groups with respect to PCNA, MMP-2, MMP-14, and TUNEL positive cells. However, the expression of MMP-9 was significantly higher in the control group than in the ZA group (P < 0.05), whereas the expression of caspase 3 was significantly lower in the control group than in the ZA group (P < 0.05). CONCLUSION: These results suggest that high doses of ZA resulted in higher levels of apoptosis and lower levels of MMP-9 in the oral epithelial cells supporting the idea of bisphosphonate treatment affects the oral mucosa.


Assuntos
Conservadores da Densidade Óssea/farmacologia , Difosfonatos/farmacologia , Imidazóis/farmacologia , Mucosa Bucal/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Conservadores da Densidade Óssea/administração & dosagem , Caspase 3/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Difosfonatos/administração & dosagem , Cães , Células Epiteliais/efeitos dos fármacos , Feminino , Imidazóis/administração & dosagem , Imuno-Histoquímica , Marcação In Situ das Extremidades Cortadas , Injeções Intravenosas , Metaloproteinase 14 da Matriz/efeitos dos fármacos , Metaloproteinase 2 da Matriz/efeitos dos fármacos , Metaloproteinase 9 da Matriz/efeitos dos fármacos , Modelos Animais , Mucosa Bucal/citologia , Antígeno Nuclear de Célula em Proliferação/efeitos dos fármacos , Ácido Zoledrônico
10.
J Periodontol ; 82(7): 1071-9, 2011 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-21142980

RESUMO

BACKGROUND: Members of the matrix metalloproteinase (MMP) family have been shown to be involved in periodontal disease. Risk factors for periodontal disease include tobacco smoking. Cigarette smoke condensate (CSC) is comprised of thousands of chemicals. Nicotine is one of the active components in tobacco. This study compares the effects of CSC and nicotine at the level in CSC on the collagen-degrading ability of human gingival fibroblasts (HGFs) and the expression of selected MMPs and tissue inhibitors of metalloproteinases (TIMPs). METHODS: HGFs were seeded in six-well collagen-coated plates, exposed to 100 µg/mL (2.4 µg/mL nicotine) of CSC or 2.4 µg/mL nicotine for 3 days, and then collagen degradation was analyzed. After 3 days exposure to CSC or nicotine, the conditioned media from HGFs was collected and the membrane proteins were extracted for gelatin zymography and Western blot analyses. The mRNA levels of MMP-2, MMP-14, and TIMP-2 were measured by reverse transcription-polymerase chain reaction. RESULTS: The CSC increased collagen degradation, and increased the levels of TIMP-2, MMP-14, and the active MMP-2 in the membrane extracts, and their mRNA levels. CSC also increased the level of active MMP-2 in the conditioned media. Nicotine at the level in CSC (2.4 µg/mL) had little influence on collagen degradation, as well as on the protein and mRNA levels of MMP-2, MMP-14, and TIMP-2. CONCLUSIONS: CSC may increase HGF-mediated collagen degradation by affecting membrane-associated MMPs and TIMPs.


Assuntos
Colágeno Tipo I/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Gengiva/efeitos dos fármacos , Nicotiana , Nicotina/farmacologia , Fumaça , Western Blotting , Células Cultivadas , Colágeno Tipo I/análise , Misturas Complexas/farmacologia , Meios de Cultivo Condicionados , Meios de Cultura Livres de Soro , Ciclofilinas/análise , Ciclofilinas/efeitos dos fármacos , Precursores Enzimáticos/análise , Precursores Enzimáticos/efeitos dos fármacos , Fibroblastos/metabolismo , Gelatinases/análise , Gelatinases/efeitos dos fármacos , Gengiva/citologia , Humanos , Metaloproteinase 14 da Matriz/análise , Metaloproteinase 14 da Matriz/efeitos dos fármacos , Metaloproteinase 2 da Matriz/análise , Metaloproteinase 2 da Matriz/efeitos dos fármacos , Metaloproteinases da Matriz Associadas à Membrana/análise , Metaloproteinases da Matriz Associadas à Membrana/efeitos dos fármacos , Inibidores de Proteases/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Inibidor Tecidual de Metaloproteinase-2/análise , Inibidor Tecidual de Metaloproteinase-2/efeitos dos fármacos , Inibidores Teciduais de Metaloproteinases/análise , Inibidores Teciduais de Metaloproteinases/efeitos dos fármacos
11.
Bioorg Med Chem Lett ; 20(12): 3557-60, 2010 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-20529684

RESUMO

A series of phenyl piperidine alpha-sulfone hydroxamate derivatives has been prepared utilizing a combination of solution-phase and resin-bound library technologies to afford compounds that are potent and highly selective for MMP-13, are dual-sparing of MMP-1 and MMP-14 (MT1-MMP) and exhibit oral bioavailability in rats.


Assuntos
Inibidores de Metaloproteinases de Matriz , Administração Oral , Animais , Disponibilidade Biológica , Ácidos Hidroxâmicos/administração & dosagem , Metaloproteinase 1 da Matriz/efeitos dos fármacos , Metaloproteinase 14 da Matriz/efeitos dos fármacos , Piperidinas , Ratos , Bibliotecas de Moléculas Pequenas , Solubilidade , Especificidade por Substrato , Sulfonas
12.
Int J Cancer ; 126(5): 1055-66, 2010 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-19551865

RESUMO

We previously demonstrated that the CNYYSNS peptide derived from tumstatin inhibited in vivo tumor progression. The YSNS motif formed a beta-turn crucial for biological activity. More recently, a YSNSG cyclopeptide with a constrained beta-turn on the YSNS residues was designed. Intraperitoneal administration of the YSNSG cyclopeptide inhibited in vivo melanoma progression more efficiently than the native linear peptide. In the present article, we showed that the YSNSG cyclopeptide also triggered an inhibition of in vivo tumor neovascularization and we further analyzed its in vitroantiangiogenic effect. The YSNSG cyclopeptide did not alter endothelial cell proliferation but inhibited cell migration by 83% in an in vitro wound healing model. The inhibition was mediated by a decrease in active MT1-MMP at the migration front as well as a decrease in u-PA and u-PAR expression. The cyclopeptide also altered beta1-integrin distribution in endothelial cell lamellipodia, induced a strong decrease in the phosphorylated focal adhesion kinase (p125(FAK)), disorganized F-actin stress fibers and decreased the number of lamellipodia, resulting in a non migratory phenotype. Our results confirm the YSNSG cyclopeptide as a potent antitumor agent, through both the inhibition of invasive properties of tumor cells and the antiangiogenic activity.


Assuntos
Antineoplásicos/farmacologia , Movimento Celular/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , Neovascularização Patológica/tratamento farmacológico , Peptídeos Cíclicos/farmacologia , Animais , Autoantígenos/química , Western Blotting , Proliferação de Células/efeitos dos fármacos , Colágeno Tipo IV/química , Regulação para Baixo , Imunofluorescência , Humanos , Imuno-Histoquímica , Metaloproteinase 14 da Matriz/efeitos dos fármacos , Metaloproteinase 14 da Matriz/metabolismo , Melanoma Experimental/tratamento farmacológico , Melanoma Experimental/patologia , Camundongos , Camundongos Endogâmicos C57BL , Receptores de Ativador de Plasminogênio Tipo Uroquinase/efeitos dos fármacos , Receptores de Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Ativador de Plasminogênio Tipo Uroquinase/efeitos dos fármacos , Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
13.
J Periodontal Res ; 44(1): 73-80, 2009 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-19515020

RESUMO

BACKGROUND AND OBJECTIVES: Membrane type 1-matrix metalloproteinase (MT1-MMP) is a collagenolytic enzyme involved in connective tissue remodeling. In periodontal tissues, either cytokines or growth factors regulate the production of proteolytic enzymes. Mice deficient in epidermal growth factor receptor (EGFR) show a reduced expression of MT1-MMP, suggesting that this receptor may play an important role in MT1-MMP production. The present study evaluated the role of the inflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) and EGFR in the production of MT1-MMP in gingival fibroblasts. MATERIAL AND METHODS: Primary cultures of human gingival fibroblasts were cultured over plastic or a type I collagen matrix and stimulated with TNF-alpha and EGF. A selective EGFR inhibitor (AG1478) was used to interfere with this signaling pathway. Production of MT1-MMP and activation of proMMP-2 were studied using Western blot and gelatin zymography, respectively. Activation of EGFR signaling was assessed through immunoprecipitation and Western blot. Expression of EGFR ligands was determined through reverse transcriptase-polymerase chain reaction. RESULTS: Treatment of gingival fibroblasts cultured over a collagen matrix with TNF-alpha stimulated proMMP-2 activation and MT1-MMP production. However, after using AG1478, both responses were inhibited. Tumor necrosis factor-alpha induced EGFR transactivation and stimulated the expression of the mRNA for the EGFR ligands heparin binding-epidermal growth factor (HB-EGF) and transforming growth factor-alpha (TGF-alpha). CONCLUSIONS: The present study shows that TNF-alpha may stimulate MT1-MMP production through transactivation of EGFR. Tumor necrosis factor-alpha may also modulate the expression of the EGFR ligands TGF-alpha and HB-EGF. Production of MT1-MMP by TNF-alpha requires interaction with EGFR, suggesting that tissue remodeling is controlled by cross-communication between diverse signaling pathways in gingival fibroblasts.


Assuntos
Receptores ErbB/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Gengiva/efeitos dos fármacos , Metaloproteinase 14 da Matriz/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Fator de Necrose Tumoral alfa/farmacologia , Células Cultivadas , Colágeno Tipo I/farmacologia , Meios de Cultura , Inibidores Enzimáticos/farmacologia , Precursores Enzimáticos/efeitos dos fármacos , Fator de Crescimento Epidérmico/farmacologia , Receptores ErbB/antagonistas & inibidores , Fibroblastos/enzimologia , Gelatinases/efeitos dos fármacos , Gengiva/citologia , Gengiva/enzimologia , Heparina/análise , Fator de Crescimento Semelhante a EGF de Ligação à Heparina , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/análise , Ligantes , Fosforilação , Proteínas Tirosina Fosfatases/antagonistas & inibidores , Quinazolinas , Receptores de Superfície Celular/análise , Ativação Transcricional/efeitos dos fármacos , Fator de Crescimento Transformador alfa/efeitos dos fármacos , Tirfostinas/farmacologia
14.
J Clin Invest ; 119(3): 492-503, 2009 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-19197139

RESUMO

The mechanisms governing hematopoietic progenitor cell mobilization are not fully understood. We report higher membrane type 1-MMP (MT1-MMP) and lower expression of the MT1-MMP inhibitor, reversion-inducing cysteine-rich protein with Kazal motifs (RECK), on isolated circulating human CD34+ progenitor cells compared with immature BM cells. The expression of MT1-MMP correlated with clinical mobilization of CD34+ cells in healthy donors and patients with lymphoid malignancies. Treatment with G-CSF further increased MT1-MMP and decreased RECK expression in human and murine hematopoietic cells in a PI3K/Akt-dependent manner, resulting in elevated MT1-MMP activity. Blocking MT1-MMP function by Abs or siRNAs impaired chemotaxis and homing of G-CSF-mobilized human CD34+ progenitors. The mobilization of immature and maturing human progenitors in chimeric NOD/SCID mice by G-CSF was inhibited by anti-MT1-MMP treatment, while RECK neutralization promoted motility and egress of BM CD34+ cells. BM c-kit+ cells from MT1-MMP-deficient mice also exhibited inferior chemotaxis, reduced homing and engraftment capacities, and impaired G-CSF-induced mobilization in murine chimeras. Membranal CD44 cleavage by MT1-MMP was enhanced following G-CSF treatment, reducing CD34+ cell adhesion. Accordingly, CD44-deficient mice had a higher frequency of circulating progenitors. Our results reveal that the motility, adhesion, homing, and mobilization of human hematopoietic progenitor cells are regulated in a cell-autonomous manner by dynamic and opposite changes in MT1-MMP and RECK expression.


Assuntos
Antígenos CD34/análise , Fator Estimulador de Colônias de Granulócitos/farmacologia , Células-Tronco Hematopoéticas/fisiologia , Metaloproteinase 14 da Matriz/genética , Glicoproteínas de Membrana/genética , Animais , Antígenos CD/análise , Células da Medula Óssea/fisiologia , Movimento Celular/fisiologia , Quimiotaxia , Quimera/genética , Proteínas Ligadas por GPI , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Mobilização de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/imunologia , Humanos , Metaloproteinase 14 da Matriz/deficiência , Metaloproteinase 14 da Matriz/efeitos dos fármacos , Metaloproteinase 14 da Matriz/metabolismo , Glicoproteínas de Membrana/efeitos dos fármacos , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , RNA Mensageiro/genética , RNA Interferente Pequeno/genética
15.
Eur J Pharmacol ; 599(1-3): 24-31, 2008 Dec 03.
Artigo em Inglês | MEDLINE | ID: mdl-18851960

RESUMO

Human adipose tissue-derived stromal cells (hADSCs) demonstrate promising potential in various clinical applications, including the transplantation to regenerate injured or degenerative tissues. The migration of engrafted hADSCs to the correct site of injure is essential for the curative effect of stem cell therapy. We found that protocatechuic acid (PCA) from Alpinia (A.) oxyphylla could promote the migration capacity of hADSCs through transwell coated with gelatin in vitro. PCA enhanced the cell migration rate in a dose-dependent and time-dependent manner. Meanwhile, RT-PCR and quantitative RT-PCR analysis revealed the elevation of membrane-type matrix metalloproteinase-1 (MT1-MMP) mRNA expression in 1.5 mM PCA-treated hADSCs. In the supernatants of these cells, the active matrix metalloproteinase-2 (MMP-2) increased compared with control cells with zymography. Moreover, the promotion of cell migration by PCA could be effectively and obviously inhibited by anti-MT1-MMP or anti-MMP-2 antibodies. Furthermore, flow cytometric analysis of the cell surface antigens, osteogenic induction, adipogenic induction and cardiomyocyte-like cell induction demonstrated that hADSCs retained their functional characteristics of multipotential mesenchymal progenitors after PCA treatment. These results suggest that PCA from A. oxyphylla promote the migration of hADSCs in vitro, which is partially due to the increased expression of MT1-MMP and the promotion of MMP-2 zymogen activation.


Assuntos
Alpinia/química , Movimento Celular/efeitos dos fármacos , Hidroxibenzoatos/farmacologia , Metaloproteinase 14 da Matriz/efeitos dos fármacos , Tecido Adiposo/citologia , Tecido Adiposo/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Citometria de Fluxo , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Humanos , Hidroxibenzoatos/administração & dosagem , Hidroxibenzoatos/isolamento & purificação , Metaloproteinase 14 da Matriz/metabolismo , Metaloproteinase 2 da Matriz/efeitos dos fármacos , Metaloproteinase 2 da Matriz/metabolismo , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Estromais/efeitos dos fármacos , Células Estromais/metabolismo , Fatores de Tempo
16.
J Periodontol ; 79(10): 1934-41, 2008 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-18834249

RESUMO

BACKGROUND: Matrix metalloproteinases (MMPs) play important roles in tissue-destruction mechanisms-associated periodontitis. MMP-8 and -13 are the predominant collagenases that are important in the extracellular matrix degradation in periodontal tissues. MMP-14 is a membrane-type MMP, whereas laminin-5 indicates basal membrane modification and epithelial induction. The purpose of the present study was to evaluate the effects of celecoxib and omega-3 fatty acid administration on the gingival tissue expression of MMP-8, -13, and -14, tissue inhibitor of MMP (TIMP)-1, and laminin (Ln)-5gamma2-chain in rat experimental periodontitis induced by Escherichia coli endotoxin (lipopolysaccharide [LPS]). METHODS: Experimental periodontitis was induced in rats by repeated LPS injection. Fifty-one adult male Sprague-Dawley rats were divided into six study groups: saline control, LPS, LPS + celecoxib, LPS + therapeutic omega-3 (TO3), prophylactic omega-3 + LPS + omega-3 (P+TO3), and LPS + celecoxib + omega-3 fatty acid. Celecoxib and omega-3 fatty acid were given as a single agent or as combination therapy for 14 days. On day 15, all rats were sacrificed, and gingival tissues were analyzed immunohistochemically for the expression of MMP-8, -13, and -14, TIMP-1, and Ln-5gamma2-chain. Alveolar bone loss was evaluated morphometrically under a stereomicroscope. Data were tested statistically by Kruskal-Wallis and Mann-Whitney tests and Spearman correlation analysis. RESULTS: Alveolar bone loss was significantly higher in all study groups compared to the saline control group (all P <0.01). MMP-8 expression was significantly higher in the LPS group than in the saline group (P = 0.001). Very low expression of MMP-8 was found in the celecoxib, P+TO3, and combination groups. TO3 increased TIMP-1 expression significantly compared to the LPS group (P <0.05). Individual celecoxib and P+TO3 administration increased MMP-14 significantly compared to saline control and LPS groups (P <0.05). No significant differences were found among the study groups with regard to Ln-5gamma2-chain and MMP-13 expressions (P >0.05). CONCLUSIONS: Selective cyclooxygenase-2 inhibitor, prophylactic omega-3 fatty acid, and a combination of these two agents can inhibit gingival tissue MMP-8 expression. Moreover, the individual administration of therapeutic omega-3 may increase gingival TIMP-1 expression in contrast to no effect on MMP-8, -13, and -14 expressions in experimental periodontitis. These experimental findings in a rat model of LPS-induced periodontitis need to be verified by clinical human studies.


Assuntos
Inibidores de Ciclo-Oxigenase 2/uso terapêutico , Ácidos Graxos Ômega-3/uso terapêutico , Laminina/efeitos dos fármacos , Metaloproteinases da Matriz/efeitos dos fármacos , Periodontite/tratamento farmacológico , Pirazóis/uso terapêutico , Sulfonamidas/uso terapêutico , Inibidor Tecidual de Metaloproteinase-1/efeitos dos fármacos , Perda do Osso Alveolar/tratamento farmacológico , Perda do Osso Alveolar/enzimologia , Perda do Osso Alveolar/patologia , Animais , Celecoxib , Modelos Animais de Doenças , Combinação de Medicamentos , Escherichia coli , Gengiva/efeitos dos fármacos , Gengiva/enzimologia , Gengivite/tratamento farmacológico , Gengivite/enzimologia , Gengivite/patologia , Laminina/análise , Lipopolissacarídeos , Masculino , Metaloproteinase 13 da Matriz/análise , Metaloproteinase 13 da Matriz/efeitos dos fármacos , Metaloproteinase 14 da Matriz/análise , Metaloproteinase 14 da Matriz/efeitos dos fármacos , Metaloproteinase 8 da Matriz/análise , Metaloproteinase 8 da Matriz/efeitos dos fármacos , Metaloproteinases da Matriz/análise , Periodontite/enzimologia , Periodontite/patologia , Ratos , Ratos Sprague-Dawley , Inibidor Tecidual de Metaloproteinase-1/análise
17.
J Toxicol Environ Health A ; 71(16): 1053-5, 2008.
Artigo em Inglês | MEDLINE | ID: mdl-18569616

RESUMO

This study focused on the effects of arsenic (As) on fibroblast-derived matrix metalloproteinase (MMP)-2 and -14 levels, as these proteins were reported to be associated with tumor progression. Arsenic was found to promote production of the fibroblast-derived active form of MMP-2. Further, As at 100 or 1000 microM increased MMP-14 expression levels in fibroblasts. In addition, 1000 microM mercury (Hg) but not As increased pro-MMP-2 protein, which is involved in the conversion of the proenzyme into its active form. Since MMP-14 is an activator of pro-MMP-2, data suggest that As promotes production of fibroblast-derived active form of MMP-2 through increased expression of MMP-14. Evidence indicates that As appeared to be less effective than Hg in the conversion of pro-MMP-2 into its active form.


Assuntos
Arsênio/farmacologia , Fibroblastos/metabolismo , Metaloproteinase 14 da Matriz/efeitos dos fármacos , Metaloproteinase 2 da Matriz/efeitos dos fármacos , Células Cultivadas , Ativação Enzimática/efeitos dos fármacos , Fibroblastos/enzimologia , Metaloproteinase 14 da Matriz/metabolismo , Metaloproteinase 2 da Matriz/metabolismo
18.
Cardiovasc Res ; 79(1): 34-41, 2008 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-18310679

RESUMO

AIMS: Expression and activation of matrix metalloproteinase (MMP)-2 play pivotal roles in the migration and invasion of human aortic vascular smooth muscle cells (VSMC) originating from normal human tissue, which is strongly linked to atherosclerosis. The present study investigated the possible inhibitory effects of cocoa procyanidin on thrombin-induced expression and activation of MMP-2 in VSMC. METHODS AND RESULTS: Cocoa procyanidin fraction (CPF) and procyanidin B2, one of major procyanidins in cocoa (3 microg/mL and 5 microM, respectively), strongly inhibited thrombin-induced activation and expression of pro-MMP-2 in VSMC, as determined by zymography. The thrombin-induced invasion and migration of VSMC were inhibited by CPF or procyanidin B2 (P < 0.05), as assessed by a modified Boyden chamber and wound healing assays, respectively. An enzymatic assay data demonstrated that CPF and procyanidin B2 directly inhibited membrane type-1 (MT1)-MMP activity (P < 0.05), and this inhibition of CPF was greater than those of red wine polyphenols. Western blot data showed that CPF and procyanidin B2 inhibited thrombin-induced phosphorylation of extracellular signal-regulated protein kinase but not mitogen-activated protein kinase kinase (MEK) in VSMC. Kinase and pull-down data revealed that CPF and procyanidin B2 inhibited MEK1 activity and directly bound with glutathione-S-transferase-MEK1. In addition, the thrombin-induced invasion and migration and the activation and expression of pro-MMP-2 in VSMC were attenuated by U0126 (a well-known inhibitor of MEK1). CONCLUSION: Cocoa procyanidins are potent inhibitors of MEK and MT1-MMP, and subsequently inhibit the expression and activation of pro-MMP-2, and also the invasion and migration of VSMC, which may in part explain the molecular action of antiatherosclerotic effects of cocoa.


Assuntos
Cacau , MAP Quinase Quinase 1/metabolismo , Metaloproteinase 14 da Matriz/metabolismo , Metaloproteinase 2 da Matriz/metabolismo , Músculo Liso Vascular/metabolismo , Preparações de Plantas/farmacologia , Proantocianidinas/farmacologia , Aorta/citologia , Aorta/efeitos dos fármacos , Aorta/metabolismo , Biflavonoides/farmacologia , Catequina/farmacologia , Movimento Celular/efeitos dos fármacos , Células Cultivadas , MAP Quinases Reguladas por Sinal Extracelular/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Humanos , MAP Quinase Quinase 1/efeitos dos fármacos , Metaloproteinase 14 da Matriz/efeitos dos fármacos , Músculo Liso Vascular/citologia , Músculo Liso Vascular/efeitos dos fármacos , Trombina/farmacologia
19.
Int J Cancer ; 121(12): 2808-14, 2007 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-17721919

RESUMO

Tumor progress depends on the proliferation of cancer cells, their interactions with stroma and the proteolytic action of enzymes. Colon cancer is c-kit positive and responsive to the specific tyrosine kinase inhibitor imatinib. We investigated the effect of imatinib on the proliferation of a panel of epithelial colon cancer cell lines in presence and absence of the antimetabolite 5-FU, and the effect of conditioned media (CM) derived from colon stromal fibroblasts with and without previous exposure to imatinib. The effects of imatinib on gene expression of MMPs and TIMPs were also studied. Imatinib effectively inhibited the proliferation of all cell lines, showing IC(50) from 0.3 to 3 microM. Its combination with 5-FU significantly enhances the growth inhibition of the highly tumourigenic HT-29 cells. CM derived from stromal fibroblasts induced the proliferation of the HT-29 cells; this stimulatory effect was abolished upon treatment with CM obtained after exposure of fibroblasts to imatinib. Gene expression of MT1-, MT2-MMP and MMP-7 was also inhibited depending on the cell line, whereas that of TIMP-2 was not affected. CM stimulated MT1-MMP protein expression by HT-29; this stimulatory effect was suppressed in the presence of imatinib. Activation of pro-MMP2 to MMP2 in culture medium of HT-29 treated with CM was increased and this activity was inhibited in presence of imatinib. The obtained data showed that imatinib is a powerful inhibitor of human colon cancer cell growth and effectively suppresses the stromal-induced stimulation of cancer cell growth and activation of proMMP2. Further studies are warranted to evaluate the in vivo effects.


Assuntos
Adenocarcinoma/tratamento farmacológico , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Neoplasias do Colo/tratamento farmacológico , Precursores Enzimáticos/metabolismo , Fluoruracila/farmacologia , Gelatinases/metabolismo , Metaloproteinase 14 da Matriz/metabolismo , Metaloendopeptidases/metabolismo , Piperazinas/farmacologia , Pirimidinas/farmacologia , Antimetabólitos Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Benzamidas , Western Blotting , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Precursores Enzimáticos/efeitos dos fármacos , Gelatinases/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Mesilato de Imatinib , Metaloproteinase 14 da Matriz/efeitos dos fármacos , Metaloendopeptidases/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-kit/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Inibidores Teciduais de Metaloproteinases/metabolismo
20.
Stem Cells ; 24(8): 1892-903, 2006 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-16690780

RESUMO

Human mesenchymal stem cells (hMSCs) exhibit the potential to contribute to a wide variety of endogenous organ tissue repair. However, the signals governing hMSC mobilization out of the bone marrow, release into the bloodstream, and migration/invasion into the target tissue are largely unknown. Since canonical Wnt signaling regulates not only tumor but also various stem cell attributes, we hypothesized that this signal transduction pathway might also be involved in governing the transmigration of hMSCs through human extracellular matrix (ECM). Stimulation of hMSCs with recombinant Wnt3a or LiCl resulted in the accumulation of the transcriptional activator beta-catenin, its translocation into the nucleus, and the upregulation of typical Wnt target genes such as cyclin D1 and membrane-type matrix metalloproteinase-1 (MT1-MMP). Moreover, both stimuli significantly enhanced hMSC proliferation up to 40%. In addition, an increase of more than twofold in the ability of hMSCs to transmigrate through Transwell filters coated with human ECM was observed. In a reverse approach, Wnt signaling in hMSCs was inhibited by knocking down the expression of either beta-catenin or low-density lipoprotein receptor-related protein 5 using RNA interference technology. These inhibition strategies resulted in downregulation of the Wnt target genes cyclin D1 and MT1-MMP, in a reduced proliferation rate, and in a strikingly diminished invasion capacity (64% and 52%). Taken together, this study provides for the first time decisive evidence that canonical Wnt signaling is critically involved in the regulation of the proliferation, as well as of the migration/invasion capacity of hMSCs, representing essential stem cell features indispensable during tissue regeneration processes.


Assuntos
Movimento Celular/fisiologia , Células-Tronco Mesenquimais/fisiologia , Transdução de Sinais/fisiologia , Proteínas Wnt/metabolismo , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Ciclina D , Ciclinas/efeitos dos fármacos , Ciclinas/genética , Ciclinas/metabolismo , Humanos , Proteínas Relacionadas a Receptor de LDL/efeitos dos fármacos , Proteínas Relacionadas a Receptor de LDL/genética , Proteínas Relacionadas a Receptor de LDL/metabolismo , Cloreto de Lítio/química , Cloreto de Lítio/metabolismo , Proteína-5 Relacionada a Receptor de Lipoproteína de Baixa Densidade , Metaloproteinase 14 da Matriz/efeitos dos fármacos , Metaloproteinase 14 da Matriz/genética , Metaloproteinase 14 da Matriz/metabolismo , Células-Tronco Mesenquimais/citologia , Dados de Sequência Molecular , Interferência de RNA , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/genética , Proteínas Wnt/antagonistas & inibidores , Proteínas Wnt/genética , Proteína Wnt3 , Proteína Wnt3A , beta Catenina/efeitos dos fármacos , beta Catenina/genética , beta Catenina/metabolismo
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