RESUMEN
The fgf-2 gene encodes low molecular weight (LMW, 18 kDa) and high molecular weight (HMW, 22-24 kDa) forms that originate from alternative translation of a single mRNA and exhibit diverse biological functions. HMW fibroblast growth factor-2 (FGF-2) inhibits cell migration and induces cell transformation or growth arrest in a cell type- and dose-dependent fashion. Conversely, LMW FGF-2 upregulates both cell proliferation and migration in most cell types. Although transcriptional and translational regulation of HMW and LMW FGF-2 has been extensively investigated, little is known about post-translational control of their relative expression. Here we report that thrombin, a key coagulation factor and inflammatory mediator, cleaves HMW FGF-2 into an LMW FGF-2-like form that stimulates endothelial cell migration and proliferation. The effect of thrombin on these cell functions requires HMW FGF-2 cleavage. This post-translational control mechanism adds a novel level of complexity to the regulation of FGF-2, and links the activities of thrombin and FGF-2 in patho-physiological processes in which both molecules are expressed.
Asunto(s)
Movimiento Celular/fisiología , Proliferación Celular , Células Endoteliales/metabolismo , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Mediadores de Inflamación/metabolismo , Procesamiento Proteico-Postraduccional/fisiología , Trombina/metabolismo , Animales , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Células Endoteliales/citología , Factor 2 de Crecimiento de Fibroblastos/farmacología , Humanos , Mediadores de Inflamación/farmacología , Ratones , Peso Molecular , Trombina/farmacología , Transcripción Genética/fisiologíaRESUMEN
Gelatinase A (MMP-2), a matrix metalloproteinase (MMP) involved in tumor invasion and angiogenesis, is secreted as an inactive zymogen (proMMP-2) and activated by proteolytic cleavage. Here we report that polymorphonuclear neutrophil (PMN)-derived elastase, cathepsin G, and proteinase-3 activate proMMP-2 through a mechanism that requires membrane-type 1 matrix metalloproteinase (MT1-MMP) expression. Immunoprecipitation of human PMN-conditioned medium with a mixture of antibodies to elastase, cathepsin G, and proteinase-3 abolished proMMP-2 activation, whereas individual antibodies were ineffective. Incubation of HT1080 cells with either purified PMN elastase or cathepsin G or proteinase-3 resulted in dose-and time-dependent proMMP-2 activation. Addition of PMN-conditioned medium to MT1-MMP expressing cells resulted in increased proMMP-2 activation and in vitro invasion of extracellular matrix (ECM), but had no effect with cells that express no MT1-MMP. MMP-2 activation by PMN-conditioned medium or purified elastase was blocked by the elastase inhibitor alpha(1)-antitrypsin but not by Batimastat, an MMP inhibitor, showing that elastase activation of MMP-2 is not mediated by MMP activities. The PMN-conditioned medium-induced increase in cell invasion was blocked by Batimastat as well as by alpha(1)-antitrypsin, showing that PMN serine proteinases trigger a proteinase cascade that entails proMMP-2 activation: this gelatinase is the downstream effector of the proinvasive activity of PMN proteinases. These findings indicate a novel role for PMN-mediated inflammation in a variety of tissue remodeling processes including tumor invasion and angiogenesis.
Asunto(s)
Precursores Enzimáticos/metabolismo , Gelatinasas/metabolismo , Metaloendopeptidasas/metabolismo , Invasividad Neoplásica , Neovascularización Patológica , Neutrófilos/enzimología , Neutrófilos/fisiología , Serina Endopeptidasas/farmacología , Catepsina G , Catepsinas/farmacología , Células Cultivadas , Medios de Cultivo Condicionados/farmacología , Activación Enzimática , Humanos , Elastasa de Leucocito/farmacología , Metaloproteinasas de la Matriz Asociadas a la Membrana , Metaloendopeptidasas/fisiología , Modelos Biológicos , Mieloblastina , Células Tumorales CultivadasRESUMEN
BACKGROUND: Matrix metalloproteinase-2 degrades a variety of basement membrane components and is essential for tumor invasion. We have previously reported that membrane type-1 matrix metalloproteinase (MT1-MMP) cooperates with neutrophil-derived serine proteinases (NDPs; elastase, cathepsin G, protease-3) to activate matrix metalloproteinase-2. We therefore hypothesized that NDPs enhance tumor-cell invasion. METHODS: Clones of human HT1080 fibrosarcoma cells transfected with MT1-MMP sense (HT-SE) or antisense CDNA (HT-AS) were used. These cells express either high (HT-SE) or extremely low levels (HT-AS) of MT1-MMP relative to nontransfected HT1080 cells (HT-WT). The cells were incubated in the presence or absence of purified NDP, with or without alpha 1-antitrypsin or the MMP inhibitor batimastat. Cell invasion was measured with the use of Boyden chambers with polycarbonate membranes coated with a reconstituted extracellular matrix. RESULTS: Under control conditions HT-WT and HT-SE cells were 4-fold more invasive than HT-AS cells. The addition of NDP increased HT-WT and HT-SE cell invasion 60% to 100% but had no effect on HT-AS cells. alpha 1-antitrypsin or batimastat did not decrease the baseline invasiveness of HT-WT and HT-SE cells; however, they abrogated the stimulatory effect of NDP. CONCLUSIONS: HT1080 cell invasion depends on MT1-MMP expression. MT1-MMP overexpression does not increase invasiveness by itself. NDPs increase invasion by MT1-MMP expressing cells by activating matrix metalloproteinase-2.
Asunto(s)
Metaloproteinasa 2 de la Matriz/fisiología , Invasividad Neoplásica/fisiopatología , Neutrófilos/enzimología , Serina Endopeptidasas/fisiología , Catepsina G , Catepsinas/fisiología , Medios de Cultivo Condicionados , Fibrosarcoma/fisiopatología , Humanos , Inhibidores de la Metaloproteinasa de la Matriz , Mieloblastina , Elastasa Pancreática/fisiología , Fenilalanina/análogos & derivados , Fenilalanina/farmacología , Inhibidores de Serina Proteinasa/farmacología , Tiofenos/farmacología , Células Tumorales Cultivadas/fisiología , alfa 1-Antitripsina/farmacologíaRESUMEN
von Willebrand factor (vWF), a glycoprotein produced uniquely by endothelial cells and megakaryocytes, is routinely used to identify vessels in tissue sections. Vessel density in tumor specimens, as determined by immuno-histochemical staining for vWF or other endothelial cell markers, is a negative prognostic factor for many solid tumors. vWF is heterogeneously distributed throughout the vasculature, transcriptional control in response to the tissue microenvironment being responsible for local variations in endothelial cell levels of vWF. Here, we report that fibroblast growth factor-2 and vascular endothelial growth factor, potent angiogenesis inducers expressed in a variety of tumors, up-regulate expression of vWF mRNA and protein in cultured endothelial cells with a synergistic effect. Our data support the measurement of vWF mRNA in tumors to detect activated endothelium or angiogenesis. For this purpose, we developed a semi-quantitative RT-PCR for vWF mRNA. Preliminary results obtained with specimens from colon carcinoma and the corresponding normal colonic mucosa showed higher vWF mRNA levels in most tumors than in their normal counterparts. The differences in vWF mRNA levels were much larger than the differences in vessel counts between a tumor and the corresponding normal mucosa, indicating that high vWF mRNA levels in tumors may indeed be an early sign of activation of the endothelium. The rapidity, objectivity, sensitivity and specificity of this technique make it suitable for routine clinical application to identify aggressive, highly angiogenic tumors.
Asunto(s)
Factores de Crecimiento Endotelial/fisiología , Endotelio Vascular/metabolismo , Factor 2 de Crecimiento de Fibroblastos/fisiología , Linfocinas/fisiología , Neovascularización Patológica/metabolismo , Factor de von Willebrand/biosíntesis , Secuencia de Bases , Bioensayo , Biomarcadores de Tumor/metabolismo , Células Cultivadas , Neoplasias del Colon/irrigación sanguínea , Neoplasias del Colon/metabolismo , Humanos , Datos de Secuencia Molecular , ARN Mensajero/análisis , ARN Mensajero/biosíntesis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de Ácido Nucleico , Células Tumorales Cultivadas , Regulación hacia Arriba , Factor A de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial Vascular , Factor de von Willebrand/análisis , Factor de von Willebrand/genéticaRESUMEN
Vascular endothelial-cadherin (VE-cad) is an endothelial cell-specific adhesion molecule that is crucial for proper assembly of vascular tubes. Here we show that a monoclonal antibody (BV13) directed to the extracellular region of VE-cad inhibits formation of adherens junctions and capillary-like structures by endothelial cells and blocks angiogenesis in the mouse cornea and in Matrigel plugs in vivo. Systemic administration of BV13 markedly decreases the growth of s.c. Lewis lung or human A431 epidermoid tumors and strongly suppresses the growth of Lewis lung metastases. These data demonstrate that VE-cad is essential for postnatal angiogenesis and thus validate VE-cad as a novel target for antiangiogenesis agents.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Cadherinas/inmunología , División Celular/efectos de los fármacos , Metástasis de la Neoplasia , Neovascularización Patológica , Neovascularización Fisiológica/efectos de los fármacos , Animales , Antígenos CD , Carcinoma Pulmonar de Lewis/tratamiento farmacológico , Adhesión Celular , Colágeno/metabolismo , Córnea/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Combinación de Medicamentos , Endotelio/metabolismo , Humanos , Inmunoglobulina G/metabolismo , Inmunohistoquímica , Etiquetado Corte-Fin in Situ , Laminina/metabolismo , Neoplasias Pulmonares/tratamiento farmacológico , Ratones , Ratones Desnudos , Trasplante de Neoplasias , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/metabolismo , Antígeno Nuclear de Célula en Proliferación/metabolismo , Proteoglicanos/metabolismo , Factores de Tiempo , Células Tumorales CultivadasAsunto(s)
Endopeptidasas/fisiología , Proteínas de la Matriz Extracelular/metabolismo , Animales , Sitios de Unión , Adhesión Celular , Movimiento Celular , Endopeptidasas/química , Endopeptidasas/clasificación , Regulación de la Expresión Génica , Humanos , Metaloproteinasas de la Matriz/química , Metaloproteinasas de la Matriz/clasificación , Metaloproteinasas de la Matriz/fisiología , Proteínas de la Membrana/metabolismo , Serina Endopeptidasas/química , Serina Endopeptidasas/clasificación , Serina Endopeptidasas/fisiologíaRESUMEN
BACKGROUND: Endothelial damage, such as that associated with balloon angioplasty or preparation of veins for bypass grafts, results in intimal hyperplasia. Growth factors and cytokines that modulate endothelial cell functions through various intracellular signaling pathways mediate rapid endothelial repair, which may prevent or reduce restenosis. Here we investigated the effect of mechanical injury of endothelial cells on the mitogen-activated kinase signaling pathways, extracellular-signal-regulated kinases (ERKs), C-Jun N-terminal kinase (JNK/SAPK), and p38. METHODS: Confluent human umbilical vein endothelial cells or bovine aortic endothelial cells were wounded with a razor blade; mitogen-activated kinase activation was monitored by immunoblotting with antibodies to active ERK, JNK/SAPK, or p38. RESULTS: Wounding of human umbilical vein endothelial cell or bovine aortic endothelial cell monolayers resulted in rapid (5-minute) activation of ERK-1 and -2, which was abolished by monoclonal antibody to basic fibroblast growth factor (FGF-2). This antibody or an inhibitor of ERK activation, PD98059, also blocked endothelial cell migration after the wounding. Thus FGF-2-induced ERK activation mediates the endothelial response to wounding. CONCLUSIONS: ERK-1 and -2 are activated by FGF-2 released from endothelial cells in response to injury. Therapeutic strategies aimed at reducing FGF-2-induced intimal hyperplasia should preserve ERK activation in endothelial cells while abolishing it in smooth muscle cells.
Asunto(s)
Proteínas Quinasas Dependientes de Calcio-Calmodulina/fisiología , Endotelio Vascular/fisiología , Factor 2 de Crecimiento de Fibroblastos/fisiología , Proteínas Quinasas JNK Activadas por Mitógenos , Proteína Quinasa 1 Activada por Mitógenos/fisiología , Quinasas de Proteína Quinasa Activadas por Mitógenos , Proteínas Quinasas Activadas por Mitógenos , Animales , Bovinos , Células Cultivadas , Activación Enzimática , Humanos , MAP Quinasa Quinasa 4 , Proteína Quinasa 3 Activada por Mitógenos , Proteínas Quinasas/fisiología , Proteínas Quinasas p38 Activadas por MitógenosAsunto(s)
Membrana Celular/metabolismo , Metaloendopeptidasas/metabolismo , ADN sin Sentido , Fibrosarcoma , Gelatinasas/metabolismo , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Metaloproteinasas de la Matriz Asociadas a la Membrana , Metaloendopeptidasas/genética , Receptores de Vitronectina/metabolismo , Proteínas Recombinantes/metabolismo , Inhibidor Tisular de Metaloproteinasa-2/genética , Inhibidor Tisular de Metaloproteinasa-2/metabolismo , Transfección , Células Tumorales CultivadasRESUMEN
BACKGROUND: Angiogenesis requires degradation of the vessel's basal lamina and endothelial cell migration into the tissue stroma. Matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) play important roles in this process. MMP activity is tightly regulated during vessel growth. This work was designed to characterize the effect of TIMP-1 upregulation on endothelial cell invasion of the extracellular matrix. METHODS: We constructed replication-deficient recombinant adenoviruses that encode either TIMP-1 (Ad.TIMP-1) or Escherichia coli lac Z (Ad.beta gal) cDNA. Bovine aortic endothelial (BAE) cells were infected with 100 infectious particles/cell. Gene expression was assessed by Northern and Western blotting. TIMP-1 activity in cell-conditioned media was measured by a resorufin-labeled casein protease assay. BAE cell migration was measured by Boyden chamber assays with 0.2% gelatin-coated, 8. 0-mcm polycarbonate membranes. RESULTS: TIMP-1 was overexpressed by Ad.TIMP-1-infected BAE cells relative to control, Ad. beta gal-infected or uninfected cells. TIMP-1 activity in Ad.TIMP-1 cell-conditioned medium was 2.8-fold higher than in control cells. By Boyden chamber assays with gelatin-coated membranes, Ad. TIMP-1-infected BAE cells showed 89.97 +/-1.64% (mean +/- SEM) reduction in migration relative to Ad.beta gal-infected cells (P < 0. 02) and 90.53 +/- 1.12% relative to uninfected cells (P < 0.02). Without gelatin coating, migration was equivalent in all groups. CONCLUSION: The replication-deficient recombinant adenovirus we constructed affords rapid and efficient upregulation of functional TIMP-1 in endothelial cells. Infection results in a dramatic decrease in cell migration and invasion of extracellular matrix. Thus, such a recombinant vector may provide a useful tool for the gene therapy of vascular remodeling and inhibition of angiogenesis.
Asunto(s)
Endotelio Vascular/fisiología , Técnicas de Transferencia de Gen , Inhibidor Tisular de Metaloproteinasa-1/genética , Inhibidor Tisular de Metaloproteinasa-1/fisiología , Animales , Bovinos , Movimiento Celular/fisiología , Células Cultivadas , Endotelio Vascular/citología , Matriz Extracelular/fisiología , Humanos , Inhibidor Tisular de Metaloproteinasa-1/metabolismo , Regulación hacia ArribaRESUMEN
BACKGROUND: The activation of the zymogen plasminogen to the serine protease plasmin by urokinase-type (uPA) and tissue-type (tPA) plasminogen activators (PA) is an important event in a variety of physiologic and pathophysiologic processes in mammals. Enhanced PA activity occurs during angiogenesis and has been correlated in vitro and in vivo with increased tumor aggressiveness and is an indicator of poor prognosis in a variety of tumors in humans. Preliminary studies suggest that the antiulcer drug irsogladine maleate (IM) diminishes PA activity in vitro and may inhibit angiogenesis in vivo. To define the precise mechanism of angiogenesis inhibition by IM in vivo, we tested the ability of IM to blunt angiogenesis in a mouse cornea neovascularization model performed in wild-type and PA-knockout mice. METHODS: Three days prior to pellet implantation, groups of C57Bl/6 wild-type, uPA-deficient (upA-/-), and tPA-deficient (tPA-/-) mice received IM (300 mg/kg), IM (500 mg/kg), or vehicle (0.5% carboxymethylcellulose) via oral gavage. After 3 days of treatment, hydron polymer-coated pellets of sucrose aluminum sulfate containing 100 ng of basic fibroblast growth factor (bFGF) were inserted into surgically created pockets in the cornea of each mouse. On postoperative day 6, the neovascularization of each cornea was evaluated by a blinded observer using slit lamp microscopy and photographed. Angiogenesis was quantified by calculating vascular area (mm2) +/- SEM using a modified formula for a half ellipse that incorporates calibrated vessel measurements [Vessel length (mm) x Clock hours x pi x 0.2]. RESULTS: IM treatment (300 and 500 mg/kg/day) resulted in a dose-dependent reduction of angiogenesis in wild-type mice by 21 and 45.3% (P < 0.02, P < 0.001), in tPA-deficient mice by 42.6 and 46% (P < 0.001, P < 0.001), and in uPA-deficient mice by 27.2 and 46% (P < 0.05, p < 0.001), respectively. No quantitative differences in neovascularization were observed in either treatment group between transgenic mouse strains. No toxicity was noted in any group. CONCLUSION: IM inhibits bFGF-induced angiogenesis in wild-type, tPA-knockout, and uPA-knockout mice. The observation that IM significantly diminishes angiogenesis in both PA-deficient mice and wild-type mice suggests that the mechanism of action of IM may be independent of plasminogen activation.
Asunto(s)
Antineoplásicos/farmacología , Córnea/irrigación sanguínea , Neovascularización Patológica/tratamiento farmacológico , Activadores Plasminogénicos/genética , Triazinas/farmacología , Animales , Relación Dosis-Respuesta a Droga , Factor 2 de Crecimiento de Fibroblastos/farmacología , Ratones , Ratones Endogámicos C57BL , Ratones NoqueadosRESUMEN
OBJECTIVE: Polymorphonuclear leukocyte (PMN) infiltration and microvascular injury are hallmarks of the tissue remodeling associated with multiple organ failure. These processes require the concerted action of various proteolytic enzymes, including serine and matrix metalloproteinases (MMPs). Matrix metalloproteinase-2 (MMP-2) plays an important role in the turnover of various ECM components, including type IV collagen, fibronectin, and gelatins. Like all MMPs, MMP-2 is secreted as an inactive zymogen (proMMP-2) and activated extracellularly by limited proteolytic cleavage. The physiologic mechanism(s) of proMMP-2 activation remains unclear. This study was designed to characterize the effect of PMNs on the activation of proMMP-2 produced by endothelial cells. METHODS: PMNs and human umbilical vein endothelial cells (HUVECs) were grown either separately or together for 2-16 h. To evaluate the role of cell-cell contact, cocultures were also established in which the two cell types were separated by a semipermeable polycarbonate membrane. Alternatively, PMN-conditioned medium was added to HUVEC cultures with or without various proteinase inhibitors (aprotinin, 1,10-phenanthroline, Batimastat, E-64, eglin c peptide, or pepstatin A). After incubation, the culture supernatants were analyzed by gelatin zymography to characterize the gelatinases. RESULTS: HUVECs produce MMP-2 in its inactive (72 kDa) form. PMNs produce high levels of MMP-9 (gelatinase B, 92 kDa) but no MMP-2. Coculture of PMNs with or addition of PMN-conditioned medium to HUVECs results in the production of active (62 kDa) MMP-2. ProMMP-2 activation by PMN-conditioned medium is not blocked by inhibitors of plasmin, cysteine-, acid-, or metalloproteinases. CONCLUSION: PMNs release a soluble factor that activates endothelial cell MMP-2 through a novel mechanism independent of cell-cell contact and not attributable to the activities of plasmin, cysteine-, acid-, or metalloproteinases. These findings may provide insight into the tissue remodeling that accompanies PMN-mediated microvascular injury.
Asunto(s)
Endotelio Vascular/enzimología , Gelatinasas/metabolismo , Metaloendopeptidasas/metabolismo , Activación Neutrófila/fisiología , Neutrófilos/metabolismo , Aprotinina/farmacología , Células Cultivadas , Medios de Cultivo Condicionados , Inhibidores de Cisteína Proteinasa/farmacología , Endotelio Vascular/citología , Endotelio Vascular/inmunología , Activación Enzimática/inmunología , Gelatinasas/antagonistas & inhibidores , Gelatinasas/biosíntesis , Humanos , Leucina/análogos & derivados , Leucina/farmacología , Metaloproteinasa 2 de la Matriz , Metaloendopeptidasas/antagonistas & inhibidores , Metaloendopeptidasas/biosíntesis , Pepstatinas/farmacología , Fenantrolinas/farmacología , Fenilalanina/análogos & derivados , Fenilalanina/farmacología , Inhibidores de Proteasas/farmacología , Proteínas , Inhibidores de Serina Proteinasa/farmacología , Serpinas/farmacología , Solubilidad , Tiofenos/farmacología , Factores de Tiempo , Venas Umbilicales/citologíaRESUMEN
BACKGROUND: Neointima formation after human saphenous vein grafting (hSVG) involves several matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs). This study assessed the feasibility of modulating MMP activity in hSVGs by adenovirus-mediated gene transfer. METHODS: First, 1 x 10(9) plaque-forming units (pfu) of replication-deficient recombinant adenoviruses encoding either beta-galactosidase (ad beta gal), MMP-3 (AdMMP-3), or TIMP-1 (AdTIMP-1) were added into the lumen of hSVGs for 1 hour. After incubation at 37 degrees C for 24 hours, specimens were analyzed by immunohistochemistry, in situ zymography, and X-gal staining. RESULTS: By X-gal staining ad beta gal-infected hSVGs stained positively in the intima and occasionally in the media. Immunohistochemistry of AdMMP-3- and AdTIMP-1-infected hSVGs localized these proteins to the intima. In situ zymography showed increased MMP activity in the intima of AdMMP-3-infected hSVGs relative to AdTIMP-1- or Ad beta gal-infected vessels. CONCLUSIONS: MMP-3 and TIMP activity can be regulated in hSVGs by replication-deficient recombinant adenoviruses. We have previously demonstrated that MMP-3 or TIMP-1 transduction, or both, inhibit SMC migration in an in vitro reconstituted vessel wall. Modulation of MMP activity may thus afford high patency rates in genetically engineered hSVGs. However, adenovirus-mediated gene delivery is limited to the vessel's intima; strategies to infect medial smooth muscle cells need to be developed.
Asunto(s)
Adenoviridae , Técnicas de Transferencia de Gen , Metaloproteinasa 3 de la Matriz/genética , Vena Safena/trasplante , Inhibidor Tisular de Metaloproteinasa-1/genética , Animales , Aorta/citología , Northern Blotting , Bovinos , Células Cultivadas , Endotelio Vascular/citología , Endotelio Vascular/enzimología , Regulación Enzimológica de la Expresión Génica/genética , Genes Reporteros , Humanos , Técnicas para Inmunoenzimas , Operón Lac , Metaloproteinasa 3 de la Matriz/análisis , Metaloproteinasa 3 de la Matriz/metabolismo , ARN Mensajero/análisis , Inhibidor Tisular de Metaloproteinasa-1/análisis , Inhibidor Tisular de Metaloproteinasa-1/metabolismo , Túnica Íntima/citología , Túnica Íntima/enzimología , beta-Galactosidasa/análisis , beta-Galactosidasa/genética , beta-Galactosidasa/metabolismoRESUMEN
BACKGROUND: Matrix metalloproteinase-2 (MMP-2), an enzyme involved in tumor invasion, is secreted as an inactive proenzyme and requires interaction with membrane-type 1 MMP (MT1-MMP) for activation. We have previously demonstrated that polymorphonuclear neutrophils (PMNs) release a soluble factor(s) that activates pro-MMP-2. Therefore, we tested the hypothesis that PMN-derived proteinases act in concert with MT1-MMP to activate pro-MMP-2. METHODS: Human HT-1080 cells transfected with MT1-MMP cDNA (HT-SE) or the corresponding antisense cDNA (HT-AS) or an empty vector (HT-V), which expressed differing levels of MT1-MMP, were incubated with serum-free, human PMN-conditioned medium with or without proteinase inhibitors. The culture supernatants were analyzed by gelatin zymography. RESULTS: Ht-1080 cells expressing basal (HT-V) or low levels (HT-AS) of MT1-MMP secreted MMP-2 in proenzyme from (72 kd). Ht-1080 cells with high levels of MT1-MMP (HT-SE) secreted pro MMP-2 and a 68 kd intermediate activation product. Addition of PMN-conditioned medium to either HT-SE or HT-V clones resulted in dose-dependent generation of active, 62 kd MMP-2. In contrast, when PMN-conditioned medium was added to HT-AS clones, no MMP-2 activation occurred. CONCLUSIONS: PMN-derived serine proteinases act in concert with MT1-MMP to activate proMMP-2. This finding indicates a potential role for inflammatory cells in promoting extracellular matrix breakdown during tumor invasion.
Asunto(s)
Colagenasas/metabolismo , Gelatinasas/metabolismo , Metaloendopeptidasas/metabolismo , Neutrófilos/enzimología , Aprotinina/farmacología , Inhibidores de Cisteína Proteinasa/farmacología , Endopeptidasas/metabolismo , Activación Enzimática/inmunología , Precursores Enzimáticos/metabolismo , Fibrosarcoma , Gelatinasas/antagonistas & inhibidores , Humanos , Leucina/análogos & derivados , Leucina/farmacología , Metaloproteinasa 1 de la Matriz , Metaloproteinasa 2 de la Matriz , Metaloproteinasa 9 de la Matriz , Inhibidores de la Metaloproteinasa de la Matriz , Metaloendopeptidasas/antagonistas & inhibidores , Invasividad Neoplásica , Pepstatinas/farmacología , Fenilalanina/análogos & derivados , Fenilalanina/farmacología , Inhibidores de Proteasas/farmacología , Inhibidores de Serina Proteinasa/farmacología , Tiofenos/farmacología , Transfección , Células Tumorales Cultivadas/efectos de los fármacos , Células Tumorales Cultivadas/enzimología , alfa 1-Antitripsina/farmacologíaRESUMEN
FGF-2 and VEGF are potent angiogenesis inducers in vivo and in vitro. Here we show that FGF-2 induces VEGF expression in vascular endothelial cells through autocrine and paracrine mechanisms. Addition of recombinant FGF-2 to cultured endothelial cells or upregulation of endogenous FGF-2 results in increased VEGF expression. Neutralizing monoclonal antibody to VEGF inhibits FGF-2-induced endothelial cell proliferation. Endogenous 18-kD FGF-2 production upregulates VEGF expression through extracellular interaction with cell membrane receptors; high-Mr FGF-2 (22-24-kD) acts via intracellular mechanism(s). During angiogenesis induced by FGF-2 in the mouse cornea, the endothelial cells of forming capillaries express VEGF mRNA and protein. Systemic administration of neutralizing VEGF antibody dramatically reduces FGF-2-induced angiogenesis. Because occasional fibroblasts or other cell types present in the corneal stroma show no significant expression of VEGF mRNA, these findings demonstrate that endothelial cell-derived VEGF is an important autocrine mediator of FGF-2-induced angiogenesis. Thus, angiogenesis in vivo can be modulated by a novel mechanism that involves the autocrine action of vascular endothelial cell-derived FGF-2 and VEGF.
Asunto(s)
Comunicación Autocrina/fisiología , Factores de Crecimiento Endotelial/metabolismo , Endotelio Vascular/fisiología , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Linfocinas/metabolismo , Neovascularización Fisiológica/fisiología , Células 3T3 , Animales , Capilares/fisiología , Bovinos , División Celular , Células Cultivadas , Endotelio Vascular/citología , Factor 2 de Crecimiento de Fibroblastos/farmacología , Humanos , Ratones , Comunicación Paracrina/fisiología , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacología , Factor A de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial VascularRESUMEN
Membrane vesicles are shed by tumor cells both in vivo and in vitro. Although their functions are not well understood, it has been proposed that they may play multiple roles in tumor progression. We characterized membrane vesicles from human HT1080 fibrosarcoma cell cultures for the presence of proteinases involved in tumor invasion. By gelatin zymography and Western blotting, these vesicles showed major bands corresponding to the zymogen and active forms of gelatinase B (MMP-9) and gelatinase A (MMP-2) and to the MMP-9. tissue inhibitor of metalloproteinase 1 complex. Both gelatinases appeared to be associated with the vesicle membrane. HT1080 cell vesicles also showed a strong, plasminogen-dependent fibrinolytic activity in 125I fibrin assays; this activity was associated with urokinase plasminogen activator, as shown by casein zymography and Western blotting. Urokinase was bound to its high affinity receptor on the vesicle membrane. Addition of plasminogen resulted in activation of the progelatinases associated with the vesicles, indicating a role of the urokinase-plasmin system in MMP-2 and MMP-9 activation. We propose that vesicles shed by tumor cells may provide a large membrane surface for the activation of membrane-associated proteinases involved in extracellular matrix degradation and tissue invasion.
Asunto(s)
Matriz Extracelular/enzimología , Fibrosarcoma/enzimología , Gelatinasas/metabolismo , Activador de Plasminógeno de Tipo Uroquinasa/metabolismo , Sangre , Western Blotting , Colagenasas/metabolismo , Precursores Enzimáticos/metabolismo , Fibrinolisina/metabolismo , Glicoproteínas/metabolismo , Humanos , Sustancias Macromoleculares , Metaloproteinasa 2 de la Matriz , Metaloproteinasa 9 de la Matriz , Metaloendopeptidasas/metabolismo , Inhibidores Tisulares de Metaloproteinasas , Células Tumorales CultivadasRESUMEN
The urokinase-type plasminogen activator (uPA) and the matrix-degrading metalloproteinases MMP-2 and MMP-9 (type IV collagenases/gelatinases) have been implicated in a variety of invasive processes, including tumor invasion, metastasis and angiogenesis. MMP-2 and MMP-9 are secreted in the form of inactive zymogens that are activated extracellularly, a fundamental process for the control of their activity. The physiological mechanism(s) of gelatinase activation are still poorly understood; their comprehension may provide tools to control cell invasion. The data reported in this paper show multiple roles of the uPA-plasmin system in the control of gelatinase activity: (i) both gelatinases are associated with the cell surface; binding of uPA and plasmin(ogen) to the cell surface results in gelatinase activation without the action of other metallo- or acid proteinases; (ii) inhibition of uPA or plasminogen binding to the cell surface blocks gelatinase activation; (iii) in soluble phase plasmin degrades both gelatinases; and (iv) gelatinase activation and degradation occur in a dose- and time-dependent manner in the presence of physiological plasminogen and uPA concentrations. Thus, the uPA-plasmin system may represent a physiological mechanism for the control of gelatinase activity.
Asunto(s)
Colagenasas/metabolismo , Fibrinolisina/metabolismo , Gelatinasas/metabolismo , Metaloendopeptidasas/metabolismo , Activador de Plasminógeno de Tipo Uroquinasa/metabolismo , Activación Enzimática , Precursores Enzimáticos/metabolismo , Humanos , Metaloproteinasa 2 de la Matriz , Metaloproteinasa 9 de la Matriz , Peso Molecular , Activadores Plasminogénicos/metabolismo , Receptores de Superficie Celular/metabolismo , Receptores del Activador de Plasminógeno Tipo Uroquinasa , Propiedades de Superficie , Células Tumorales CultivadasRESUMEN
We have previously reported that culture medium conditioned by human SK-Hep1 hepatoma cells or mouse S180 sarcoma cells induces in vitro angiogenesis and stimulates production of urokinase plasminogen activator (uPA) in vascular endothelial cells. These activities are mediated by a 3.5-10 kDa, heparin-binding peptide that upregulates endothelial cell expression of basic fibroblast growth factor (bFGF; Peverali et al., 1994, J. Cell. Physiol. 161:1-14.) We now report that SK-Hep 1 or S180 cell-conditioned medium rapidly induces a 4- to 5-fold increase in cell-bound uPA activity and in the high-affinity binding of 125I-prouPA to vascular endothelial cells. Ligand blotting and purification experiments show an equivalent increase in the synthesis of a cell surface protein corresponding to the endothelial cell uPA receptor (uPAR) on the basis of M, (45-50 kDa) and sensitivity to phosphatidylinositol-specific phospholipase C (PI-PLC). The tumor cell-conditioned media also upregulate uPAR mRNA levels in endothelial cells. Thus, the increase in uPA binding capacity of endothelial cells is mediated by an increased expression of uPAR. The uPAR-inducing activity of SK-Hep 1 or S180 cell-conditioned medium is not neutralized by antibodies to bFGF, and is associated with a peptide that has a M, higher than 10 kDa and no affinity for heparin. Therefore, it appears to be distinct from the bFGF/uPA-inducing factor secreted by the same cells, and from other heparin-binding cytokines that upregulate uPAR expression in endothelial cells.
Asunto(s)
Medios de Cultivo Condicionados/farmacología , Endotelio Vascular/metabolismo , Receptores de Superficie Celular/metabolismo , Animales , Anticuerpos/inmunología , Anticuerpos/farmacología , Northern Blotting , Bovinos , Cromatografía en Agarosa , Medios de Cultivo Condicionados/química , Electroforesis en Gel de Poliacrilamida , Factores de Crecimiento de Fibroblastos/inmunología , Humanos , Ratones , Unión Proteica , Receptores del Activador de Plasminógeno Tipo Uroquinasa , Células Tumorales Cultivadas , Activador de Plasminógeno de Tipo Uroquinasa/metabolismoRESUMEN
In the initial stages of capillary formation (angiogenesis) microvascular endothelial cells of preexisting blood vessels locally degrade the underlying basal lamina and invade into the stroma of the tissue to be vascularized. A consistent body of experimental evidence has shown that this process requires a wide array of dedradative enzymes. Components of the plasminogen activator (PA)-plasmin system and of the matrix metalloproteinase (MMP) family play important roles. PAs trigger a proteinase cascade that results is the generation of high local concentrations of plasmin and active MMPs. This increase in proteolytic activity has three major consequences: it permits endothelial cell degradation and invasion of the vessel basal lamina, generates extracellular matrix (ECM) degradation products that are chemotactic for endothelial cells, and activates and mobilizes growth factors localized in the ECM. In addition, urokinase-type PA modulates some endothelial cell functions, including proliferation and migration, with a mechanism independent of proteolytic activity. PA and MMP activities are modulated in endothelial cells by complex mechanisms, including transcriptional regulation by a variety of growth factors and cytokines with angiogenic activity, extracellular control of the proteolytic activities by tissue inhibitors, and interaction with binding sites on the cell membrane and ECM.