RESUMEN
Transforming growth factor-beta1 (TGF-beta1), a tumour suppressing as well as tumour-promoting cytokine, is stored as an extracellular matrix-bound latent complex. We examined TGF-beta1 activation and localisation of TGF-beta1 activity in gastric cancer. Gastric tumours showed increased stromal and epithelial total TGF-beta1 staining by immunohistochemistry. Active TGF-beta1 was present in malignant epithelial cells, but most strongly in smooth muscle actin expressing fibroblasts. Normal gastric mucosa from the same patient showed some staining for total, and little for active TGF-beta1. Active TGF-beta1 levels were determined by ELISA on tissue homogenates, confirming a strong increase in active TGF-beta1 in tumours compared to corresponding normal mucosa. Moreover, high tumour TGF-beta1 activity levels were significantly associated with clinical parameters, including worse survival of the patients. Total and active TGF-beta1 levels were not correlated, suggesting a specific activation process. Of the different proteases tested, active TGF-beta1 levels were only correlated with urokinase activity levels. The correlation with urokinase activity suggests a role for plasmin in TGF-beta1 activation in the tumour microenvironment, resulting in transformation of resident fibroblasts to tumour promoting myofibroblasts. In conclusion we have shown localisation and clinical relevance of TGF-beta1 activity levels in gastric cancer.
Asunto(s)
Neoplasias Gástricas/metabolismo , Análisis de Supervivencia , Factor de Crecimiento Transformador beta1/metabolismo , Anciano , Femenino , Humanos , Inmunohistoquímica , Masculino , Persona de Mediana Edad , Neoplasias Gástricas/fisiopatologíaRESUMEN
OBJECTIVE: In the present study, the effects of plasmin antagonist tranexamic acid (TEA) on urinary pyridinoline excretion rates were investigated in rheumatoid arthritis (RA) patients. METHODS: The study was set up as a double-blind placebo-controlled pilot study. Ten patients received tranexamic acid and 9 received placebo for 12 weeks. Urinary excretion rates of hydroxylysylpyridinoline (HP) and lysylpyridinoline (LP) were used as molecular markers of articular cartilage and bone degradation. In addition, clinical parameters of disease activity were assessed and CRP levels were measured. RESULTS: Treatment with TEA did not reduce pyridinoline excretion, nor was any effect observed on clinical parameters of disease activity or on CRP levels. CONCLUSION: The results of the present pilot study show no beneficial effect of TEA as adjuvant therapy in RA patients with respect to joint destruction or disease activity.
Asunto(s)
Artritis Reumatoide/tratamiento farmacológico , Fibrinolisina/antagonistas & inhibidores , Ácido Tranexámico/administración & dosificación , Aminoácidos/orina , Artritis Reumatoide/diagnóstico , Distribución de Chi-Cuadrado , Relación Dosis-Respuesta a Droga , Método Doble Ciego , Esquema de Medicación , Femenino , Estudios de Seguimiento , Humanos , Masculino , Dimensión del Dolor/efectos de los fármacos , Proyectos Piloto , Probabilidad , Rango del Movimiento Articular/fisiología , Valores de Referencia , Índice de Severidad de la Enfermedad , Estadísticas no Paramétricas , Resultado del TratamientoRESUMEN
Matrix metalloproteinases (MMPs) are believed to be pivotal enzymes in the invasion of articular cartilage by synovial tissue in rheumatoid arthritis (RA). Here, we investigated the effects of gene transfer of tissue inhibitors of metalloproteinases (TIMPs) on the invasiveness of RA synovial fibroblasts (RASF) in vitro and in vivo. Adenoviral vectors (Ad) were used for gene transfer. The effects of AdTIMP-1 and AdTIMP-3 gene transfer on matrix invasion were investigated in vitro in a transwell system. Cartilage invasion in vivo was studied in the SCID mouse co-implantation model for 60 days. In addition, the effects of AdTIMP-1 and AdTIMP-3 on cell proliferation were investigated. A significant reduction in invasiveness was demonstrated in vitro as well as in vivo in both the AdTIMP-1- and AdTIMP-3-transduced RASF compared with untransduced SF or SF that were transduced with control vectors. in vitro, the number of invading cells was reduced to 25% (P<0.001) in the AdTIMP-1-transduced cells and to 13% (P<0.0001) in the AdTIMP-3-transduced cells (% of untransduced cells). Cell proliferation was significantly inhibited by AdTIMP-3 and, less, by AdTIMP-1. In conclusion, overexpression of TIMP-1 and TIMP-3 by Ad gene transfer results in a marked reduction of the invasiveness of RASF in vitro and in the SCID mouse model. Apart from the inhibition of MMPs, a reduction in proliferation rate may contribute to this effect. These results suggest that overexpression of TIMPs, particularly TIMP-3 at the invasive front of pannus tissue, may provide a novel therapeutic strategy for inhibiting joint destruction in RA.
Asunto(s)
Artritis Reumatoide/terapia , Cartílago Articular/patología , Terapia Genética/métodos , Metaloproteinasas de la Matriz/genética , Transducción Genética/métodos , Adenoviridae/genética , Animales , Artritis Reumatoide/patología , Cartílago Articular/enzimología , División Celular , Fibroblastos/patología , Expresión Génica , Vectores Genéticos/administración & dosificación , Humanos , Metaloproteinasas de la Matriz/metabolismo , Ratones , Ratones SCID , Membrana Sinovial/enzimología , Membrana Sinovial/patología , Inhibidor Tisular de Metaloproteinasa-1/genética , Inhibidor Tisular de Metaloproteinasa-3/genéticaRESUMEN
Although many epidemiological studies have shown an association between hyperfibrinogenemia and atherosclerosis, it is not established whether elevated fibrinogen has an etiological role in the pathogenesis or is only a reflection of the ongoing disease. We have studied the contribution of fibrinogen to the development of atherosclerosis in atherosclerosis-prone ApoE*3-Leiden mice that have been cross-bred with transgenic mice overexpressing fibrinogen. Genetic compound offspring were used to evaluate the progression of atherosclerotic lesions after being fed an atherogenic diet for 7 weeks. It was observed that the lesion area of the plaques as well as the severity of the lesions in the aortic valve was comparable in control single transgenic ApoE*3-Leiden mice and in double transgenic apoE*3-Leiden mice overexpressing fibrinogen. No thrombus or fibrin deposition was observed in atherosclerotic lesions in either group of mice. These results indicate that elevated plasma fibrinogen concentrations in ApoE*3-Leiden transgenic mice do not affect the progression of diet-induced atherosclerotic lesions.
Asunto(s)
Apolipoproteínas E , Arteriosclerosis/etiología , Dieta Aterogénica , Fibrinógeno/fisiología , Animales , Válvula Aórtica , Apolipoproteína E3 , Arteriosclerosis/patología , Progresión de la Enfermedad , Fibrinógeno/análisis , Humanos , Ratones , Ratones Transgénicos , Agregación PlaquetariaRESUMEN
Many epidemiological studies suggest that elevated plasma fibrinogen concentrations form one of the most important independent risk factors in blood for cardiovascular disease and particularly atherosclerosis in humans. To clarify the effect of genetic factors, diets and their interactions on plasma fibrinogen concentrations, we examined plasma fibrinogen levels in four strains of mice, which differ in their susceptibility to cholesterol-induced atherosclerosis. When maintained on basal diet, two strains 129/J and C3H/HeJ exhibited a significantly higher plasma fibrinogen concentration (2.1 and 1.9 mg/ml) than C57BL/6J and BALB/C strains (1.5 and 1.4 mg/ml). The strongest and most rapid (1 week) increase of plasma fibrinogen (by all semi-synthetic diets) is observed in C57BL/6J mice, which are known to be highly susceptible to diet-induced atherosclerosis. After a period of 8 weeks an increase in plasma fibrinogen of approximately 30-50% was observed in all strains on all semi-synthetic diets. Remarkably, no increase was observed in the fibrinogen Aalpha- Bbeta- and gamma-chain mRNA levels in the liver on the same diets. These mRNA levels were even decreased by approximately 20-50% in all strains on an extremely atherogenic diet. It was found that: genetic background determines the plasma fibrinogen levels on basal diet; plasma fibrinogen levels are altered by diet; the extent of these changes depends on the genetic background: surprisingly, this increase of fibrinogen in plasma is independent of transcription; the diet-induced increase of fibrinogen was very fast in the very highly atherosclerosis-susceptible strain C57BL/6J having a low basal fibrinogen level, and very slow in the atherosclerosis-resistant strain C3H/HeJ having a high basal fibrinogen level. It might be concluded that it is the kinetics of the response of fibrinogen to diet rather than the actual level, which relates to atherosclerosis susceptibility.
Asunto(s)
Arteriosclerosis/sangre , Dieta , Fibrinógeno/metabolismo , alfa-Globulinas/metabolismo , Animales , Arteriosclerosis/genética , Northern Blotting , Dieta Aterogénica , Susceptibilidad a Enfermedades , Femenino , Haptoglobinas/metabolismo , Hígado/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Proteínas , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de RiesgoRESUMEN
Hyperfibrinogenemia is a risk predictor in several diseases, including cardiovascular disease. Nevertheless, it remains unknown whether elevated fibrinogen has an etiologic role in or is a reflection of disease pathogenesis, or both. To examine this question, we generated a mouse model of hyperfibrinogenemia. We isolated the mouse fibrinogen locus, containing the three fibrinogen genes, in a single P1 clone. This approximately 100 kb clone was injected into C57Bl/6J zygotes. Three transgenic lines were identified, two with elevated fibrinogen, 1.4- and 1.7-fold relative to normal. We characterized the line with the higher level. Northern blots of total RNA showed transgene expression was liver specific, and the message levels were 2- to 3-fold enhanced. Fibrinogen in transgenic mice was normal in both immunologic and clotting assays. Our data indicate that over-expression of all three fibrinogen genes is necessary to achieve hyperfibrinogenemia. We saw no increase in mortality or morbidity, no gross abnormalities in the organs, and no histologic differences in lung, liver, spleen or kidney, in transgenic mice relative to normal littermates. We conclude that elevated fibrinogen did not cause disease in mice. We anticipate that breeding these mice to other mouse models of disease will demonstrate whether hyperfibrinogenemia has a role in the initiation or progression of symptomatic disease.
Asunto(s)
Modelos Animales de Enfermedad , Fibrinógeno/metabolismo , Ratones Transgénicos , Animales , Clonación Molecular , Fibrinógeno/efectos adversos , Fibrinógeno/genética , Hígado/metabolismo , Ratones , Ratones Endogámicos C57BL , Especificidad de Órganos , ARN/metabolismo , Transgenes/genéticaAsunto(s)
Diabetes Mellitus Tipo 1/orina , Nefropatías Diabéticas/diagnóstico , Metaloproteinasa 2 de la Matriz/orina , Metaloproteinasa 9 de la Matriz/orina , Adulto , Albuminuria , Biomarcadores/sangre , Biomarcadores/orina , Creatinina/orina , Diabetes Mellitus Tipo 1/sangre , Diabetes Mellitus Tipo 1/enzimología , Nefropatías Diabéticas/sangre , Nefropatías Diabéticas/orina , Factores de Crecimiento Endotelial/sangre , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Linfocinas/sangre , Masculino , Metaloproteinasa 2 de la Matriz/sangre , Metaloproteinasa 9 de la Matriz/sangre , Valores de Referencia , Reproducibilidad de los Resultados , Factor A de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial VascularRESUMEN
The fibrinogen Aalpha, Bbeta, and gamma polypeptides are encoded by three separate genes, which are arranged in the order gamma-alpha-beta. In order to study the biosynthesis of fibrinogen in vivo we generated a line of transgenic mice carrying extra copies of the fibrinogen beta-gene. To clone the mouse fibrinogen Bbeta-chain gene, a mouse 129 Sv/Ev genomic cosmid library was screened, using the mouse fibrinogen Aalpha-, Bbeta-chain cDNA. A clone containing the complete fibrinogen Bbeta-chain gene including approximately 11-kb of the natural promoter region was identified and subsequently microinjected into mice. Southern blot analysis identified a founder that carried additional copies of the fibrinogen Bbeta-chain gene. Transgenic offspring of this founder were interbred and heterozygous and homozygous transgenic mice were obtained. Northern blot analysis demonstrated approximately a 3-fold increase in fibrinogen Bbeta mRNA in heterozygous mice as compared to wild-type, whereas homozygous transgenic mice showed approximately a 9-fold increase. The levels of the Aalpha and gamma mRNAs in transgenic homozygous mice were not changed as compared to those in wild-type mice. Fibrinogen levels in plasma were not significantly increased in transgenic mice as compared to wild-type mice. These results indicate that: additional copies of the fibrinogen Bbeta-chain gene lead to increased levels of the Bbeta-chain mRNA in the liver; the increased levels of Bbeta-chain mRNA in homozygous overexpression mice do not change the transcription levels of the two other fibrinogen mRNAs in vivo; the absence of an increased plasma fibrinogen level in the transgenic mice indicates that this level is not regulated solely by transcription of the Bbeta-chain gene.
Asunto(s)
Fibrinógeno/biosíntesis , Fibrinógeno/genética , Hígado/metabolismo , Regulación hacia Arriba/genética , Animales , Northern Blotting , Fibrinógeno/fisiología , Humanos , Masculino , Ratones , Ratones Transgénicos , Subunidades de Proteína , ARN Mensajero/biosíntesis , Transcripción GenéticaRESUMEN
BACKGROUND: Inhibition of matrix metalloproteinase (MMP) activity after balloon angioplasty by intraperitoneal injection of batimastat reduces late lumen loss by inhibition of constrictive remodeling. In the present study, we investigated whether the oral MMP inhibitor marimastat inhibits constrictive remodeling in favor of neutral or expansive remodeling. METHODS AND RESULTS: In 26 pigs, balloon dilation was performed in 101 peripheral arteries. Pigs were treated with marimastat or served as controls and were euthanized 42 days after intervention. Intravascular ultrasound was performed at all time points. Vessel area (VA) loss was assessed by calculating the change in VA at termination relative to after intervention. Arteries were divided in 3 categories: expansive remodeling (VA loss < -5%), neutral (-5%
Asunto(s)
Arterias/diagnóstico por imagen , Arterias/fisiopatología , Cateterismo/efectos adversos , Inhibidores Enzimáticos/administración & dosificación , Ácidos Hidroxámicos/administración & dosificación , Inhibidores de la Metaloproteinasa de la Matriz , Administración Oral , Animales , Arterias/efectos de los fármacos , Arterias/enzimología , Hiperplasia , Porcinos , Túnica Íntima/diagnóstico por imagen , Ultrasonografía IntervencionalRESUMEN
OBJECTIVE: To investigate the cartilage-degrading capacity of granzyme B and the presence of granzyme B-positive cells at sites of erosion in the rheumatoid synovium. METHODS: Granzyme B was added to [(3)H]proline/[(35)S]sulphate-labelled cartilage matrices and to cartilage explants. Proteoglycan degradation was assessed by the release of (35)S and glycosaminoglycans into the medium and collagen degradation was assessed by the release of (3)H and hydroxyproline and by measuring the fraction of denatured collagen. Granzyme B expression was studied at the invasive front of the synovium by immunohistochemistry. RESULTS: Granzyme B induced loss of both newly synthesized, radiolabelled proteoglycans in cartilage matrices and resident proteoglycans of the cartilage explants. No effect on collagen degradation was found. Granzyme B-positive cells were present throughout the synovium and at the invasive front. CONCLUSION: The presence of granzyme B-positive cells at the invasive front of the synovium together with its ability to degrade articular proteoglycans supports the view that granzyme B may contribute to joint destruction in rheumatoid arthritis.
Asunto(s)
Artritis Reumatoide/enzimología , Cartílago/enzimología , Proteoglicanos/metabolismo , Serina Endopeptidasas/metabolismo , Membrana Sinovial/enzimología , Animales , Artritis Reumatoide/metabolismo , Artritis Reumatoide/patología , Cartílago/metabolismo , Cartílago/patología , Bovinos , Células Cultivadas , Condrocitos/metabolismo , Colágeno/metabolismo , Matriz Extracelular/metabolismo , Granzimas , Humanos , Articulación Metacarpofalángica/enzimología , Membrana Sinovial/metabolismo , Membrana Sinovial/patologíaRESUMEN
BACKGROUND: Smooth muscle cell migration, in addition to proliferation, contributes to a large extent to the neointima formed in humans after balloon angioplasty or bypass surgery. Plasminogen activator/plasmin-mediated proteolysis is an important mediator of this smooth muscle cell migration. Here, we report the construction of a novel hybrid protein designed to inhibit the activity of cell surface-bound plasmin, which cannot be inhibited by its natural inhibitors, such as alpha(2)-antiplasmin. This hybrid protein, consisting of the receptor-binding amino-terminal fragment of uPA (ATF), linked to the potent protease inhibitor bovine pancreas trypsin inhibitor (BPTI), can inhibit plasmin activity at the cell surface. METHODS AND RESULTS: The effect of adenovirus-mediated ATF.BPTI expression on neointima formation was tested in human saphenous vein organ cultures. Infection of human saphenous vein segments with Ad.CMV.ATF.BPTI (5x10(9) pfu/mL) resulted in 87.5+/-3.8% (mean+/-SEM, n=10) inhibition of neointima formation after 5 weeks, whereas Ad.CMV.ATF or Ad.CMV.BPTI virus had only minimal or no effect on neointima formation. The efficacy of ATF.BPTI in vivo was demonstrated in a murine model for neointima formation. Neointima formation in the femoral artery of mice, induced by placement of a polyethylene cuff, was strongly inhibited (93.9+/-2%) after infection with Ad.CMV.mATF.BPTI, a variant of ATF.BPTI able to bind specifically to murine uPA receptor; Ad.CMV.mATF and Ad.CMV.BPTI had no significant effect. CONCLUSIONS: These data provide evidence that adenoviral transfer of a hybrid protein that binds selectively to the uPA receptor and inhibits plasmin activity directly on the cell surface is a powerful approach to inhibiting neointima formation and restenosis.
Asunto(s)
Aprotinina/fisiología , Vasos Sanguíneos/fisiología , Túnica Íntima/crecimiento & desarrollo , Activador de Plasminógeno de Tipo Uroquinasa/fisiología , Adenoviridae/genética , Animales , Aprotinina/genética , Células CHO , Bovinos , Cricetinae , Arteria Femoral/crecimiento & desarrollo , Arteria Femoral/lesiones , Vena Femoral/citología , Vena Femoral/metabolismo , Fibrinolisina/metabolismo , Expresión Génica , Vectores Genéticos/genética , Humanos , Ratones , Ratones Endogámicos C57BL , Músculo Liso Vascular/citología , Músculo Liso Vascular/metabolismo , Técnicas de Cultivo de Órganos , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/fisiología , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/fisiología , Vena Safena/citología , Transfección , Activador de Plasminógeno de Tipo Uroquinasa/química , Activador de Plasminógeno de Tipo Uroquinasa/genéticaRESUMEN
OBJECTIVE: To determine the role of chondrocytes and factors released from chondrocytes in cartilage destruction by fibroblast-like synoviocytes (FLS) derived from patients with rheumatoid arthritis (RA). METHODS: RA FLS from 2 patients were implanted into SCID mice, together with fresh articular cartilage or with cartilage that had been stored for 24 hours at 4 degrees C or at 37 degrees C. The invasion of the same RA FLS into the fresh and stored cartilage was compared histologically using a semiquantitative scoring system. In addition, we investigated whether protein synthesis in chondrocytes affects the invasion of RA FLS in vitro. A 3-dimensional cartilage-like matrix formed by cultured chondrocytes was labeled with 35S. After formation of the cartilage-like matrix, protein synthesis was blocked with cycloheximide. The invasion of RA FLS from 6 patients into cycloheximide-treated and untreated matrix was assessed by measuring the released radioactivity in coculture with and without interleukin-1beta (IL-1beta) and tumor necrosis factor alpha (TNFalpha). RESULTS: The SCID mouse experiments showed a significant invasion of RA FLS into the cartilage (overall mean score 3.2) but revealed significant differences when the invasion of the same RA FLS into fresh and stored cartilage was compared. RA FLS that were implanted with fresh articular cartilage showed a significantly higher invasiveness than those implanted with pieces of cartilage that had been stored for 24 hours (overall mean score 2.3). Storage at 37 degrees C and 4 degrees C resulted in the same reduction of invasion (35% and 37%, respectively). In the in vitro experiments, RA FLS rapidly destroyed the cartilage-like matrix. Blocking of chondrocyte protein biosynthesis significantly decreased the invasion of RA FLS, as shown by a decreased release of radioactivity. Addition of IL-1beta, but not TNFalpha, to the cocultures partially restored the invasiveness of RA FLS. CONCLUSION: These data underline the value of the SCID mouse in vivo model of rheumatoid cartilage destruction and demonstrate that chondrocytes contribute significantly to the degradation of cartilage by releasing factors that stimulate RA FLS. Among those, IL-1beta-mediated mechanisms might be of particular importance.
Asunto(s)
Artritis Reumatoide/patología , Cartílago Articular/metabolismo , Animales , Cartílago Articular/citología , Movimiento Celular/efectos de los fármacos , Condrocitos/citología , Condrocitos/metabolismo , Citocinas/farmacología , Modelos Animales de Enfermedad , Femenino , Humanos , Ratones , Ratones SCID , Biosíntesis de Proteínas , Proteínas/efectos de los fármacos , Membrana Sinovial/citologíaRESUMEN
Smooth muscle cell migration plays a role in the development of intimal hyperplasia. Given the established role of the plasminogen activation system in cell migration, an approach to therapy is to overexpress an inhibitor of plasmin. Therefore, an adenoviral vector was constructed encoding the hybrid protein ATF.BPTI, which contains the active domain of bovine pancreas trypsin inhibitor (BPTI), fused to ATF, the amino terminal fragment or receptor-binding domain of u-PA. Adenoviral vectors expressing ATF and BPTI individually were also constructed, and a fourth vector was constructed encoding ATF.BPTI linked by an internal ribosomal entry site to Green Fluorescent Protein (ABIG). Both the expression and functionality of the recombinant proteins were established in human vascular smooth muscle cells. Adenoviral gene transfer of ATF.BPTI inhibited SMC migration more efficiently than the expression of ATF or BPTI individually. Expression of ABIG resulted in the co-expression of ATF.BPTI and Green Fluorescent Protein, thereby providing a tool to monitor transfection efficiency and the behavior of the transfected cells.
Asunto(s)
Antifibrinolíticos/metabolismo , Técnicas de Transferencia de Gen , Proteínas Luminiscentes/genética , Inhibidores de Tripsina/genética , Activador de Plasminógeno de Tipo Uroquinasa/genética , Adenoviridae/genética , Animales , Bovinos , División Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Virus de la Encefalomiocarditis/genética , Fibrinolisina/antagonistas & inhibidores , Fibrinolisina/metabolismo , Fibrinolíticos/metabolismo , Vectores Genéticos , Proteínas Fluorescentes Verdes , Humanos , Indicadores y Reactivos , Proteínas Luminiscentes/metabolismo , Proteínas de la Membrana/metabolismo , Músculo Liso Vascular/citología , Músculo Liso Vascular/metabolismo , Páncreas , Activadores Plasminogénicos/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Vena Safena/citología , Vena Safena/metabolismo , Inhibidores de Tripsina/metabolismo , Inhibidores de Tripsina/farmacología , Activador de Plasminógeno de Tipo Uroquinasa/metabolismoRESUMEN
OBJECTIVE: Joint destruction in rheumatoid arthritis (RA) is a result of degradation and invasion of the articular cartilage by the pannus tissue. The present study was undertaken to examine the role of the plasminogen activation system in cartilage degradation and invasion by synovial fibroblasts and investigate a novel gene therapeutic approach using a cell surface-targeted plasmin inhibitor (ATF.BPTI). METHODS: Adenoviral vectors were used for gene transfer. The effects of ATF.BPTI gene transfer on RA synovial fibroblast-dependent cartilage degradation were studied in vitro, and cartilage invasion was studied in vivo in the SCID mouse coimplantation model. RESULTS: The results indicate that cartilage matrix degradation by rheumatoid synovial fibroblasts is plasmin mediated and depends on urokinase-type plasminogen activator for activation. Targeting plasmin inhibition to the cell surface of the fibroblasts by gene transfer of a cell surface-binding plasmin inhibitor resulted in a significant reduction of cartilage matrix degradation in vitro and of cartilage invasion in vivo. Compared with uninfected rheumatoid synovial fibroblasts, the mean +/-SEM cartilage degradation in vitro was reduced to 87.9+/-0.9% after LacZ gene transfer versus a reduction to 24.0+/-1.6% after ATF.BPTI gene transfer (P<0.0001). The mean +/- SEM in vivo cartilage invasion score was 3.1+/-0.4 in the control-transduced fibroblasts and 1.8+/-0.4 in the ATF.BPTI-transduced fibroblasts (P<0.05). CONCLUSION: These results indicate a role of the plasminogen activation system in synovial fibroblast-dependent cartilage degradation and invasion in RA, and demonstrate an effective way to inhibit this by gene transfer of a cell surface-targeted plasmin inhibitor.
Asunto(s)
Antifibrinolíticos/metabolismo , Artritis Reumatoide/patología , Cartílago Articular/metabolismo , Fibrinolisina/farmacología , Factores de Transcripción Activadores , Infecciones por Adenoviridae , Animales , Proteínas Sanguíneas/genética , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Fibroblastos/virología , Expresión Génica , Técnicas de Transferencia de Gen , Humanos , Ratones , Ratones SCID , Membrana Sinovial/patología , Factores de Transcripción/genética , Transfección , Activador de Plasminógeno de Tipo Uroquinasa/farmacologíaRESUMEN
BACKGROUND: Arterial remodeling after balloon angioplasty has been recognized as a major determinant of restenosis. Perturbation of collagen metabolism might be important. After balloon injury, matrix metalloproteinase (MMP) expression is upregulated. We investigated the effect of Batimastat, a nonspecific MMP inhibitor, on late lumen loss, arterial remodeling, and neointima formation after balloon dilation. METHODS AND RESULTS: In atherosclerotic iliac arteries of 12 Yucatan micropigs, balloon dilation was performed, with intravascular ultrasound and quantitative angiography used before and after balloon dilation and at 42-day follow-up. The animals were randomly divided into 2 groups, the Batimastat group (n=6) and the vehicle group (n=6). All animals were intraperitoneally injected with either Batimastat or a vehicle immediately after balloon dilation and at 2 weeks and 4 weeks after balloon dilation. Angiographic and echographic late lumen loss in the Batimastat group versus the vehicle group was 0.3+/-0.1 versus 0.8+/-0.1 mm (P=0.01) and 2.2+/-0.5 versus 4.9+/-0.7 mm(2) (P=0.004), respectively. Late media-bounded area loss was used as a measure of remodeling after balloon dilation and was 0.9+/-0.6 mm(2) in the Batimastat group compared with 3.8+/-0.8 mm(2) in the vehicle group (P=0.003, mixed model analysis P=0.01). Neointima formation was 1.3+/-0.3 mm(2) in the Batimastat group and 1.0+/-0.2 mm(2) in the vehicle group (P=0. 542). CONCLUSIONS: Metalloproteinase inhibition by Batimastat significantly reduced late lumen loss after balloon angioplasty by inhibition of constrictive arterial remodeling, whereas neointima formation was not inhibited by MMP inhibition.
Asunto(s)
Angioplastia de Balón/efectos adversos , Arteriosclerosis/etiología , Arteriosclerosis/terapia , Arteria Ilíaca/fisiopatología , Metaloendopeptidasas/antagonistas & inhibidores , Fenilalanina/análogos & derivados , Inhibidores de Proteasas/uso terapéutico , Tiofenos/uso terapéutico , Angiografía , Animales , Arteriosclerosis/diagnóstico , Arteriosclerosis/metabolismo , Arteria Ilíaca/diagnóstico por imagen , Arteria Ilíaca/patología , Inmunohistoquímica , Macrófagos/patología , Metaloendopeptidasas/metabolismo , Fenilalanina/sangre , Fenilalanina/uso terapéutico , Periodo Posoperatorio , Porcinos , Porcinos Enanos , Tiofenos/sangre , Túnica Íntima/diagnóstico por imagen , Túnica Íntima/patología , Ultrasonografía IntervencionalRESUMEN
Angiostatin is a tumor-derived angiogenesis inhibitor consisting of an internal fragment of plasminogen. Little is known about the production of angiostatin by human tumors. In this study, we examined the in vitro angiostatin-generating capacities of a panel of human tumor cell lines (total n = 75) and the proteolytic molecule(s) involved. Angiostatin formation was determined by assessing the level of plasminogen digestion in conditioned medium by Western-blot analysis. We found that the capacity to produce angiostatin is a common feature of many cell lines, depending on the tumor type. All 6 bladder-carcinoma and 6 out of 7 prostate-carcinoma cell lines showed intermediate to potent angiostatin-generating activity. In contrast, only 2 out of 7 colon-carcinoma and 2 out of 9 renal-cell carcinoma cell lines were able to generate angiostatin at intermediate levels. Out of 25 melanoma cell lines, only one line failed to generate angiostatin. In the other cell-line groups (cervix, breast and ovary), angiostatin formation varied. Remarkably, angiostatin bands were not of equal size in all plasminogen digests. Since reported data have indicated that plasminogen activators (uPA and tPA) were able to excise the angiostatin fragment from the plasminogen parent molecule via plasmin generation, we determined levels of uPA and tPA and PAI-1 antigen in the conditioned media, and correlated the results with angiostatin-generating capacity. Whereas prostate- and bladder-carcinoma lines capable of generating high levels of angiostatin showed high uPA levels, angiostatin generation in melanoma cell lines was correlated with tPA levels. Generally, angiostatin non-producers did not express uPA or tPA. In 6 out of 75 cell lines, however, we found angiostatin generation combined with low or absent levels of plasminogen activator, suggesting the involvement of alternative proteolytic pathways in the generation of angiostatin.
Asunto(s)
Neoplasias/metabolismo , Fragmentos de Péptidos/biosíntesis , Plasminógeno/biosíntesis , Activador de Tejido Plasminógeno/fisiología , Activador de Plasminógeno de Tipo Uroquinasa/fisiología , Angiostatinas , Medios de Cultivo Condicionados , Endopeptidasas/fisiología , Femenino , Humanos , Masculino , Neoplasias/patología , Activador de Tejido Plasminógeno/análisis , Células Tumorales Cultivadas , Activador de Plasminógeno de Tipo Uroquinasa/análisisRESUMEN
Matrix metalloproteinases (MMPs) are involved in tumor growth and metastasis, promoting the migration and invasion of cells. In this study, the amount of MMP-2 and MMP-9 activity was measured in urine from superficial bladder carcinoma patients (pTa, pT1) to evaluate their possible diagnostic value. The active and total amount of MMP-2 and MMP-9, respectively, in urine from tumor patients were compared with the levels in urine from age- and gender-matched healthy volunteers. Both MMP-2 and MMP-9 activity levels were significantly enhanced in urine from patients with high invasive cancers (pT2, PT3), whereas in urine from healthy controls no or very low MMP activities were found. More importantly, a substantial number of urine samples from patients with superficial tumors contained elevated MMP-2 and MMP-9 activities, suggesting that enhanced urinary MMP activity levels, indeed, might be indicative for early-stage bladder cancer. Overall, urinary MMP-2 and MMP-9 activity levels were significantly correlated to each other, with some individual exceptions. A comparison between urinary MMP-9 activity and a recently proposed urinary marker for bladder cancer, NMP-22, showed slightly lower numbers of patients with elevated levels for MMP-9. But because MMP-9 and NMP-22 levels were not correlated, enhanced urinary MMP activity might be useful as a marker for superficial bladder carcinoma like, or especially in combination with, other markers.
Asunto(s)
Carcinoma/diagnóstico , Carcinoma/orina , Metaloproteinasa 2 de la Matriz/orina , Metaloproteinasa 9 de la Matriz/orina , Neoplasias de la Vejiga Urinaria/diagnóstico , Neoplasias de la Vejiga Urinaria/orina , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores de Tumor/orina , Estudios de Casos y Controles , Catepsina B/orina , Creatinina/metabolismo , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Masculino , Persona de Mediana Edad , Invasividad Neoplásica , Proteínas Nucleares/orina , Activador de Plasminógeno de Tipo Uroquinasa/orinaRESUMEN
Matrix metalloproteinases (MMP) types 2 and 9 (also known as gelatinase A and B) are thought to be causally involved in cancer invasion and metastasis. In normal as well as in malignant tissue, both these MMPs occur in multiple forms such as inactive precursors, active enzymes and enzyme-inhibitor complexes. Using newly developed quantitative activity assays, the levels of active MMP-2, total (active and activatable) MMP-2 and total MMP-9 were found to be significantly higher in breast carcinomas than in fibroadenomas. In addition, active MMP-2 and MMP-9 were detected more frequently in malignant than in benign breast carcinoma. These new quantitative activity assays are likely to be of use in studying the mechanism of action of both MMP-2 and -9, assessing their potential prognostic value in different cancers and in the design of MMP inhibitors for preventing cancer metastasis.
Asunto(s)
Neoplasias de la Mama/enzimología , Fibroadenoma/enzimología , Metaloproteinasa 2 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Catálisis , Ensayo de Inmunoadsorción Enzimática , Humanos , Metaloproteinasa 2 de la Matriz/análisis , Metaloproteinasa 9 de la Matriz/análisis , PronósticoRESUMEN
Recent reports suggest that elevated levels of plasminogen activator inhibitor-1 (PAI-1) may contribute to tumour progression. The studies reported here were designed to help elucidate PAI-1's contribution to the invasive and migratory phenotype. Antibodies to PA-1 dose-dependently, and significantly, inhibited the invasive and migratory potential of human HT1080 fibrosarcoma cells, as did an antibody to uPA and the plasmin inhibitor aprotinin. Invasion of the human melanoma cell line, BLM, was also attenuated by the anti-PAI-1 monoclonal antibody MAI-12. The non-invasive human melanoma cell line, IF6, which does not express uPA, provided further confirmation of PAI-1 and uPA's role as, upon transfection with uPA, this cell line attained an invasive phenotype, which was again attenuated by MAI-12. Although antibodies to PAI-1 did not affect the adhesion of HT1080 cells to vitronectin, the antibody to uPA reduced their attachment. Addition of exogenous PAI-1, however, prevented HT1080 cell adhesion (IC50 180 nM) and promoted cell detachment from vitronectin. Furthermore melanoma cells transfected with a uPA variant, which had an impaired interaction with PAI-1, were not invasive and had impaired binding to vitronectin. These data highlight the importance of a balanced proteolysis and suggest an additional role for PAI-1 distinct from its role in proteolysis. These data also suggest that uPA and PAI-1 may co-operate in the migratory process by respectively facilitating the attachment to, and subsequent detachment from, vitronectin in the extracellular matrix. These results support the clinical findings and indicate that modulation of PAI-1 activity may be of therapeutic benefit for the treatment of cancer.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Invasividad Neoplásica/inmunología , Metástasis de la Neoplasia/inmunología , Inhibidor 1 de Activador Plasminogénico/inmunología , Animales , Células CHO , Adhesión Celular/fisiología , Cricetinae , Fibrosarcoma/patología , Humanos , Melanoma/patología , Inhibidor 1 de Activador Plasminogénico/fisiología , Transfección , Células Tumorales Cultivadas , Activador de Plasminógeno de Tipo Uroquinasa/genética , Activador de Plasminógeno de Tipo Uroquinasa/fisiología , Vitronectina/metabolismoRESUMEN
New prodrugs of daunorubicin and doxorubicin designed for selective activation by the serine protease plasmin are described. The low toxic prodrugs 3, 4, and 5 are converted to the corresponding cytotoxic drugs upon proteolysis by the tumor-associated protease plasmin. Application of a self-eliminating spacer was essential for enzyme activation. A prodrug containing a chloro-substituted spacer was synthesized with the aim of enhancing the rate of conversion by plasmin. All prodrugs were highly stable in buffer solution and in serum and on the average 15-fold less cytotoxic than the parent drugs in seven human tumor cell lines. A marked in vitro selectivity was demonstrated by incubation of the doxorubicin prodrugs with a plasmin generating MCF-7 breast cancer cell line transfected with urokinase-type plasminogen activator (u-PA) in comparison with the nontransfected nonplasmin generating cell line. Prodrugs 4 and 5 showed the same cytotoxic effect as the free parent drug doxorubicin in the u-PA transfected cells, indicating complete conversion of the prodrug by plasmin. Addition of the plasmin inhibitor Trasylol drastically increased the ID(50) values in the u-PA transfected MCF-7 cells for both prodrugs 4 and 5.