RESUMEN
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.
Asunto(s)
Amidinas/farmacología , Anticuerpos Monoclonales/farmacología , Antineoplásicos/farmacología , Bencilaminas/farmacología , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/radioterapia , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/uso terapéutico , Macrófagos/efectos de los fármacos , Metaloproteinasa 14 de la Matriz/efectos de los fármacos , Metaloproteinasa 14 de la Matriz/metabolismo , Óxido Nítrico Sintasa de Tipo II/efectos de los fármacos , Animales , Anticuerpos Monoclonales/uso terapéutico , Antineoplásicos/uso terapéutico , Neoplasias de la Mama/irrigación sanguínea , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/inmunología , Línea Celular Tumoral , Fraccionamiento de la Dosis de Radiación , Femenino , Regulación Enzimológica de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Inmunoglobulina G/sangre , Macrófagos/enzimología , Neoplasias Mamarias Experimentales , Ratones , Neovascularización Patológica , Óxido Nítrico Sintasa de Tipo II/antagonistas & inhibidores , Óxido Nítrico Sintasa de Tipo II/metabolismo , Fenotipo , Transducción de Señal/efectos de los fármacos , Proteína Smad2/metabolismo , Proteína smad3/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Regulación hacia ArribaRESUMEN
MMP intervention strategies have met with limited clinical success due to severe toxicities. In particular, treatment with broad-spectrum MMP-inhibitors (MMPIs) caused musculoskeletal pain and inflammation. Selectivity may be essential for realizing the clinical potential of MMPIs. Here we review discoveries pinpointing membrane-bound MMPs as mediators of mechanisms underlying cancer and inflammation and as possible therapeutic targets for prevention/treatment of these diseases. We discuss strategies to target these therapeutic proteases using highly selective inhibitory agents (i.e., human blocking antibodies) against individual membrane-bound MMPs.
RESUMEN
Inhibition of specific matrix metalloproteinases (MMP) is an attractive noncytotoxic approach to cancer therapy. MMP-14, a membrane-bound zinc endopeptidase, has been proposed to play a central role in tumor growth, invasion, and neovascularization. Besides cleaving matrix proteins, MMP-14 activates proMMP-2 leading to an amplification of pericellular proteolytic activity. To examine the contribution of MMP-14 to tumor growth and angiogenesis, we used DX-2400, a highly selective fully human MMP-14 inhibitory antibody discovered using phage display technology. DX-2400 blocked proMMP-2 processing on tumor and endothelial cells, inhibited angiogenesis, and slowed tumor progression and formation of metastatic lesions. The combination of potency, selectivity, and robust in vivo activity shows the potential of a selective MMP-14 inhibitor for the treatment of solid tumors.
Asunto(s)
Anticuerpos Monoclonales , Antineoplásicos/uso terapéutico , División Celular/efectos de los fármacos , Inhibidores Enzimáticos/uso terapéutico , Inhibidores de la Metaloproteinasa de la Matriz , Neovascularización Patológica/prevención & control , Animales , Anticuerpos Monoclonales Humanizados , Neoplasias de la Mama/patología , Línea Celular Tumoral , Endotelio Vascular/citología , Endotelio Vascular/efectos de los fármacos , Femenino , Genes Reporteros , Humanos , Inmunohistoquímica , Ratones , Invasividad Neoplásica/patología , Transfección , Trasplante Heterólogo , Venas Umbilicales/citología , Venas Umbilicales/efectos de los fármacosRESUMEN
Novel inhibitors of the urokinase-mediated plasminogen (plg) activation system are potentially of great clinical benefit as anticancer treatments. Using phage display, we identified DX-1000 a tissue factor pathway inhibitor-derived Kunitz domain protein which is a specific high-affinity inhibitor of plasmin (pln) (K(i) = 99 pM). When tested in vitro, DX-1000 blocks plasmin-mediated pro-matrix metalloproteinase-9 (proMMP-9) activation on cells and dose-dependently inhibits tube formation, while not significantly affecting hemostasis and coagulation. However, this low-molecular weight protein inhibitor ( approximately 7 kDa) exhibits rapid plasma clearance in mice and rabbits, limiting its potential clinical use in chronic diseases. After site-specific PEGylation, DX-1000 retains its activity and exhibits a decreased plasma clearance. This PEGylated derivative is effective in vitro, as well as potent in inhibiting tumor growth of green fluorescent protein (GFP)-labeled MDA-MB-231 cells. 4PEG-DX-1000 treatment causes a significant reduction of urokinase-type plasminogen activator (uPA) and plasminogen expressions, a reduction of tumor proliferation, and vascularization. 4PEG-DX-1000 treatment significantly decreases the level of active mitogen-activated protein kinase (MAPK) in the primary tumors and reduces metastasis incidence. Together, our results demonstrate the potential value of plasmin inhibitors as therapeutic agents for blocking breast cancer growth and metastasis.
Asunto(s)
Antifibrinolíticos/farmacología , Antineoplásicos/farmacología , Polietilenglicoles/farmacología , Animales , Antifibrinolíticos/farmacocinética , Antineoplásicos/farmacocinética , Coagulación Sanguínea/efectos de los fármacos , Línea Celular , Relación Dosis-Respuesta a Droga , Precursores Enzimáticos/antagonistas & inhibidores , Femenino , Hemostasis/efectos de los fármacos , Humanos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Metaloproteinasa 9 de la Matriz , Inhibidores de la Metaloproteinasa de la Matriz , Ratones , Ratones Endogámicos BALB C , Conejos , Activador de Plasminógeno de Tipo Uroquinasa/antagonistas & inhibidores , Proteínas Quinasas p38 Activadas por Mitógenos/fisiologíaRESUMEN
The angiogenic mechanism and therapeutic potential of PDGF-CC, a recently discovered member of the VEGF/PDGF superfamily, remain incompletely characterized. Here we report that PDGF-CC mobilized endothelial progenitor cells in ischemic conditions; induced differentiation of bone marrow cells into ECs; and stimulated migration of ECs. Furthermore, PDGF-CC induced the differentiation of bone marrow cells into smooth muscle cells and stimulated their growth during vessel sprouting. Moreover, delivery of PDGF-CC enhanced postischemic revascularization of the heart and limb. Modulating the activity of PDGF-CC may provide novel opportunities for treating ischemic diseases.
Asunto(s)
Células Endoteliales/citología , Células Endoteliales/efectos de los fármacos , Isquemia/tratamiento farmacológico , Isquemia/patología , Neovascularización Fisiológica/efectos de los fármacos , Factor de Crecimiento Derivado de Plaquetas/farmacología , Células Madre/efectos de los fármacos , Animales , Diferenciación Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Células Cultivadas , Vasos Coronarios/citología , Vasos Coronarios/efectos de los fármacos , Miembro Posterior/irrigación sanguínea , Miembro Posterior/efectos de los fármacos , Humanos , Isquemia/inducido químicamente , Isquemia/metabolismo , Linfocinas , Ratones , Microcirculación/efectos de los fármacos , Miocardio/metabolismo , Miocardio/patología , Fosfotirosina/metabolismo , Receptores del Factor de Crecimiento Derivado de Plaquetas/metabolismo , Transducción de Señal , Células Madre/citologíaRESUMEN
Matrix metalloproteinases (MMPs) are known to play a role in cell growth, invasion, angiogenesis, metastasis, and bone degradation, all important events in the pathogenesis of cancer. Multiple myeloma is a B-cell cancer characterized by the proliferation of malignant plasma cells in the bone marrow, increased angiogenesis, and the development of osteolytic bone disease. The role of MMPs in the development of multiple myeloma is poorly understood. Using SC-964, a potent inhibitor of several MMPs (MMP-2, -3, -8, -9, and -13), we investigated the role of MMPs in the 5T2MM murine model. Reverse transcriptase-polymerase chain reaction demonstrated the presence of mRNA for MMP-2, -8, -9, and -13 in 5T2MM-diseased bone marrow. Mice bearing 5T2MM cells were given access to food containing SC-964. The concentration of SC-964 measured in the plasma of mice after 11 days of treatment was able to inhibit MMP-9 activity in gelatin zymography. Treatment of 5T2MM-bearing mice resulted in a significant reduction in tumor burden, a significant decrease in angiogenesis, and partially protective effect against the development of osteolytic bone disease. The direct role of MMPs in these different processes was confirmed by in vitro experiments. All these results support the multifunctional role of MMPs in the development of multiple myeloma.
Asunto(s)
Enfermedades Óseas/prevención & control , Modelos Animales de Enfermedad , Inhibidores Enzimáticos/farmacología , Metaloproteinasas de la Matriz/fisiología , Mieloma Múltiple/enzimología , Animales , Anexina A5/metabolismo , Apoptosis/efectos de los fármacos , Enfermedades Óseas/enzimología , División Celular/efectos de los fármacos , Dieta , Femenino , Ratones , Ratones Endogámicos C57BL , Mieloma Múltiple/irrigación sanguínea , Mieloma Múltiple/prevención & control , Neovascularización Patológica/enzimología , Neovascularización Patológica/prevención & control , Osteólisis/enzimología , Osteólisis/prevención & control , Células Plasmáticas/metabolismo , Células Plasmáticas/patología , Ratas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Timidina/metabolismoRESUMEN
PURPOSE: The implication of matrix metalloproteinases (MMPs) in the major stages of cancer progression has fueled interest in the design of synthetic MMP inhibitors (MMPIs) as a novel anticancer therapy. Thus far, drugs used in clinical trials are broad-spectrum MMPIs the therapeutic index of which proved disappointingly low. The development of selective MMPIs for tumor progression-associated MMPs is, thus, likely to offer improved therapeutic possibilities. EXPERIMENTAL DESIGN: The anti-invasive capacity of a series of pyrimidine-trione derivatives was tested in vitro in a chemoinvasion assay, and the most potent compound was further evaluated in vivo in different human tumor xenograft models. The activity of this novel selective MMPI was compared with BB-94, a broad-spectrum inhibitor. RESULTS: Ro-28-2653, an inhibitor with high selectivity for MMP-2, MMP-9, and membrane type 1 (MT1)-MMP, showed the highest anti-invasive activity in vitro. In vivo, Ro-28-2653 reduced the growth of tumors induced by the inoculation of different cell lines producing MMPs and inhibited the tumor-promoting effect of fibroblasts on breast adenocarcinoma cells. Furthermore, Ro-28-2653 reduced tumor vascularization and blocked angiogenesis in a rat aortic ring assay. In contrast, BB-94 up-regulated MMP-9 expression in tumor cells and promoted angiogenesis in the aortic ring assay. CONCLUSION: Ro-28-2653, a selective and orally bioavailable MMPI with inhibitory activity against MMPs expressed by tumor and/or stromal cells, is a potent antitumor and antiangiogenic agent. In contrast to broad-spectrum inhibitors, the administration of Ro-28-2653 was not associated with the occurrence of adverse side effects that might hamper the therapeutic potential of these drugs.
Asunto(s)
Antineoplásicos/farmacología , Inhibidores de la Metaloproteinasa de la Matriz , Fenilalanina/análogos & derivados , Piperazinas/farmacología , Pirimidinas/farmacología , Adenocarcinoma/tratamiento farmacológico , Administración Oral , Animales , Aorta/patología , Neoplasias de la Mama/tratamiento farmacológico , Línea Celular Tumoral , ADN Complementario/metabolismo , Progresión de la Enfermedad , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/farmacología , Fibroblastos/metabolismo , Humanos , Concentración 50 Inhibidora , Metaloproteinasa 2 de la Matriz/metabolismo , Microscopía Fluorescente , Modelos Químicos , Invasividad Neoplásica , Trasplante de Neoplasias , Neovascularización Patológica , Fenilalanina/farmacología , Inhibidores de Proteasas/farmacología , Ratas , Tiofenos/farmacología , Factores de Tiempo , Regulación hacia ArribaRESUMEN
To generate doxorubicin (Dox) specifically at the tumor site, the chemotherapeutic agent was incorporated into a prodrug by linkage to a peptide specifically recognized by plasmin, which is overproduced in many cancers. ST-9905, which contains an elongated self-elimination spacer, is activated more rapidly in vitro by plasmin than is ST-9802. Prodrug activation in vitro depended on the level of urokinase produced by tumor cells and was inhibited by aprotinin, a plasmin inhibitor. Comparison of equimolar concentrations of ST-9905, ST-9802, and Dox in EF43.fgf-4 and MCF7 models revealed that both prodrugs, in sharp contrast to Dox, displayed antiproliferative and antiangiogenic activities without discernible toxicity. Although MCF7 cells are poor urokinase producers in vitro, prodrug efficacy in this model may be explained by production of plasmin by tumor-infiltrating host cells. Mice treated with equitoxic concentrations (maximum tolerated doses) of prodrugs showed 100% survival and negligible body weight loss, in contrast to results after Dox treatment. ST-9905 was substantially more effective than ST-9802 and induced similar tumor growth inhibition as Dox but without apparent toxicity. This finding may be explained by the elongated spacer, which facilitates enzymatic prodrug activation. These data validate both the use of elongated spacers in vivo and the concept of targeting anticancer prodrugs to tumor-associated plasmin.
Asunto(s)
Adenocarcinoma/tratamiento farmacológico , Doxorrubicina/farmacología , Fibrinolisina/farmacología , Neoplasias Mamarias Experimentales/tratamiento farmacológico , Neovascularización Patológica/tratamiento farmacológico , Profármacos/farmacología , Adenocarcinoma/irrigación sanguínea , Adenocarcinoma/enzimología , Adenocarcinoma/patología , Animales , Biotransformación , Peso Corporal/efectos de los fármacos , Neoplasias de la Mama/irrigación sanguínea , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/enzimología , Neoplasias de la Mama/patología , Línea Celular Tumoral/trasplante , Doxorrubicina/análogos & derivados , Doxorrubicina/química , Doxorrubicina/metabolismo , Doxorrubicina/uso terapéutico , Doxorrubicina/toxicidad , Femenino , Humanos , Neoplasias Mamarias Experimentales/irrigación sanguínea , Neoplasias Mamarias Experimentales/enzimología , Neoplasias Mamarias Experimentales/patología , Dosis Máxima Tolerada , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Estructura Molecular , Proteínas de Neoplasias/metabolismo , Profármacos/química , Profármacos/metabolismo , Profármacos/uso terapéutico , Profármacos/toxicidad , Activador de Plasminógeno de Tipo Uroquinasa/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
PURPOSE: VEGF has been shown to be necessary, but not sufficient alone, for the development of subretinal pathologic angiogenesis. In the current study, the influence of placental growth factor (PlGF), a member of the VEGF family, in human and experimental choroidal neovascularization (CNV) was investigated. METHODS: The presence of VEGF family member mRNA was evaluated by RT-PCR in neovascular membranes extracted during surgery. The spatial and temporal pattern of VEGF isoforms and PlGF mRNA expression were explored by using the laser capture catapulting technique and RT-PCR in a murine laser-induced model and in vitro. PlGF expression was also studied in human donor eyes. The influence of endogenous PlGF was evaluated in deficient mice (PlGF(-/-)) and by antibody-mediated neutralization of the PlGF receptor. RESULTS: Human neovascular membranes consistently expressed VEGF-A, -B, and -C; PlGF; and VEGFR-1 and -2. The VEGF(120) isoform mRNA was primarily induced in early stages of angiogenesis in vivo and in vitro. PlGF mRNA expression was present in the intact choroid and significantly upregulated during the course of experimental CNV. Both deficient PlGF expression in PlGF(-/-) mice and PlGF receptor neutralization in wild-type mice prevented the development of choroidal neovascularization induced by laser. CONCLUSIONS: These observations demonstrate the participation of PlGF in experimental CNV. They identify therefore PlGF as an additional promising target for ocular antiangiogenic strategies.
Asunto(s)
Inductores de la Angiogénesis/genética , Neovascularización Coroidal/metabolismo , Proteínas Gestacionales/genética , Inductores de la Angiogénesis/metabolismo , Animales , Animales Modificados Genéticamente , Neovascularización Coroidal/patología , Modelos Animales de Enfermedad , Factores de Crecimiento Endotelial/genética , Factores de Crecimiento Endotelial/metabolismo , Femenino , Técnica del Anticuerpo Fluorescente Indirecta , Humanos , Péptidos y Proteínas de Señalización Intercelular/genética , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Linfocinas/genética , Linfocinas/metabolismo , Masculino , Ratones , Factor de Crecimiento Placentario , Proteínas Gestacionales/metabolismo , Isoformas de Proteínas , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor A de Crecimiento Endotelial Vascular , Receptor 1 de Factores de Crecimiento Endotelial Vascular/genética , Receptor 1 de Factores de Crecimiento Endotelial Vascular/metabolismo , Receptor 2 de Factores de Crecimiento Endotelial Vascular/genética , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismo , Factores de Crecimiento Endotelial VascularRESUMEN
Endostatin and angiostatin are known as tumor-derived angiogenesis inhibitors, but their mechanisms of action are not yet completely defined. We report here that endostatin and angiostatin, delivered by adenoviral vectors, reduced in vitro the neovessel formation in the mouse aortic ring assay by 85 and 40%, respectively. We also demonstrated in vivo that both endostatin and angiostatin inhibited local invasion and tumor vascularization of transplanted murine malignant keratinocytes, and reduced by 50 and 90% the development of highly vascularized murine mammary tumors. This inhibition of tumor growth was associated with a reduction of tumor vascularization. Expression analysis of vascular endothelial growth factor (VEGF) carried out in the mouse aortic ring model revealed a 3- to 10-fold down-regulation of VEGF mRNA expression in endostatin-treated rings. A similar down-regulation of VEGF expression at both mRNA and protein levels was also observed in the two in vivo cancer models after treatment with each angiogenesis inhibitor. This suggests that endostatin and angiostatin effects may be mediated, at least in part, by their ability to down-regulate VEGF expression within the tumor. This work provides evidence that endostatin and angiostatin act on tumor cells themselves.
Asunto(s)
Colágeno/fisiología , Factores de Crecimiento Endotelial/genética , Péptidos y Proteínas de Señalización Intercelular/genética , Linfocinas/genética , Fragmentos de Péptidos/fisiología , Plasminógeno/fisiología , Adenoviridae/genética , Angiostatinas , Animales , Aorta Torácica/metabolismo , Vasos Sanguíneos/crecimiento & desarrollo , Western Blotting , Colágeno/genética , Técnicas de Cultivo , Regulación hacia Abajo , Endostatinas , Factores de Crecimiento Endotelial/metabolismo , Regulación Neoplásica de la Expresión Génica , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Linfocinas/metabolismo , Neoplasias Mamarias Experimentales/irrigación sanguínea , Neoplasias Mamarias Experimentales/genética , Neoplasias Mamarias Experimentales/patología , Ratones , Ratones Endogámicos BALB C , Neovascularización Patológica/genética , Neovascularización Patológica/fisiopatología , Fragmentos de Péptidos/genética , Plasminógeno/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Células Tumorales Cultivadas , Factor A de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial VascularRESUMEN
Bisphosphonate drugs inhibit osteoclastic bone resorption and are widely used to treat skeletal complications in patients with tumor-induced osteolysis. We now show that zoledronic acid, a new generation bisphosphonate with a heterocyclic imidazole substituent, is also a potent inhibitor of angiogenesis. In vitro, zoledronic acid inhibits proliferation of human endothelial cells stimulated with fetal calf serum, basic fibroblast growth factor (bFGF), and vascular endothelial growth factor (IC(50) values 4.1, 4.2, and 6.9 microM, respectively), and modulates endothelial cell adhesion and migration. In cultured aortic rings and in the chicken egg chorioallantoic membrane assay, zoledronic acid reduces vessel sprouting. When administered systemically to mice, zoledronic acid potently inhibits the angiogenesis induced by subcutaneous implants impregnated with bFGF [ED(50), 3 microg/kg (7.5 nmol/kg) s.c.]. These findings indicate that zoledronic acid has marked antiangiogenic properties that could augment its efficacy in the treatment of malignant bone disease and extend its potential clinical use to other diseases with an angiogenic component.
Asunto(s)
Inhibidores de la Angiogénesis/farmacología , Difosfonatos/farmacología , Imidazoles/farmacología , Apoptosis/efectos de los fármacos , Adhesión Celular/efectos de los fármacos , Ciclo Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Células Cultivadas , Quimiotaxis/efectos de los fármacos , Corion/química , Factores de Crecimiento Endotelial/farmacología , Endotelio Vascular/citología , Endotelio Vascular/efectos de los fármacos , Factor 2 de Crecimiento de Fibroblastos/farmacología , Sustancias de Crecimiento/administración & dosificación , Sustancias de Crecimiento/farmacología , Humanos , Citometría de Imagen , Linfocinas/farmacología , Músculo Liso Vascular/citología , Músculo Liso Vascular/efectos de los fármacos , Pamidronato , Factor A de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial Vascular , Ácido ZoledrónicoRESUMEN
Angiogenesis is a complex biological process involving the coordinated modulation of many genes. Histone deacetylases (HDAC) are a growing family of enzymes that mediate the availability of chromatin to the transcriptional machinery. Trichostatin-A (TSA) and suberoylanilide hydroxamic acid (SAHA), two HDAC inhibitors known to relieve gene silencing, were evaluated as potential antiangiogenic agents. TSA and SAHA were shown to prevent vascular endothelial growth factor (VEGF)-stimulated human umbilical cord endothelial cells (HUVEC) from invading a type I collagen gel and forming capillary-like structures. SAHA and TSA inhibited the VEGF-induced formation of a CD31-positive capillary-like network in embryoid bodies and inhibited the VEGF-induced angiogenesis in the CAM assay. TSA also prevented, in a dose-response relationship, the sprouting of capillaries from rat aortic rings. TSA inhibited in a dose-dependent and reversible fashion the VEGF-induced expression of VEGF receptors, VEGFR1, VEGFR2, and neuropilin-1. TSA and SAHA upregulated the expression by HUVEC of semaphorin III, a recently described VEGF competitor, at both mRNA and protein levels. This effect was specific to endothelial cells and was not observed in human fibroblasts neither in vascular smooth muscle cells. These observations provide a conspicuous demonstration that HDAC inhibitors are potent anti-angiogenic factors altering VEGF signaling.
Asunto(s)
Inhibidores de la Angiogénesis/farmacología , Factores de Crecimiento Endotelial/farmacología , Inhibidores de Histona Desacetilasas , Linfocinas/farmacología , Semaforina-3A , Transducción de Señal/efectos de los fármacos , Animales , Aorta/efectos de los fármacos , Western Blotting , Proteínas Portadoras/genética , Células Cultivadas , Embrión de Pollo , Corion/irrigación sanguínea , Corion/efectos de los fármacos , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/metabolismo , Histona Desacetilasas/metabolismo , Humanos , Ácidos Hidroxámicos/farmacología , Microscopía Fluorescente , Microscopía de Contraste de Fase , Proteínas del Tejido Nervioso/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Ribosómico 28S/genética , Ratas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Venas Umbilicales/citología , Venas Umbilicales/efectos de los fármacos , Venas Umbilicales/metabolismo , Regulación hacia Arriba/efectos de los fármacos , Factor A de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial Vascular , VorinostatRESUMEN
In myeloid leukemia, immature leukemic cells are able to egress into peripheral blood to infiltrate extra-medullary organs. We therefore analyzed the migrating and invasive potential of human HL-60 and NB4 cell lines, representative of acute myelogenous leukemia, their ability to express matrix metalloproteases (MMPs), tissue inhibitors of metalloproteases (TIMPs) and urokinase plasminogen activator (uPA) in response to differentiating agents. Granulocytic differentiation by all-trans-retinoic acid (ATRA) and aclacinomycin (ACLA) strongly increased HL-60 and NB4 cell migration and invasion. At mRNA and protein levels, these cell lines produced significant amounts of MMP-9 (HL-60Asunto(s)
Aclarubicina/análogos & derivados
, Aclarubicina/farmacología
, Diferenciación Celular/efectos de los fármacos
, Leucemia/enzimología
, Fenilalanina/análogos & derivados
, Serina Endopeptidasas/biosíntesis
, Tretinoina/farmacología
, Antineoplásicos/farmacología
, Diferenciación Celular/fisiología
, Movimiento Celular/efectos de los fármacos
, Células HL-60
, Humanos
, Leucemia/metabolismo
, Leucemia/patología
, Metaloproteinasa 9 de la Matriz/biosíntesis
, Invasividad Neoplásica
, Fenilalanina/farmacología
, Tiofenos/farmacología
, Inhibidores Tisulares de Metaloproteinasas/biosíntesis
, Células Tumorales Cultivadas
RESUMEN
Plasminogen activator inhibitor 1 (PAI-1) is believed to control proteolytic activity and cell migration during angiogenesis. We previously demonstrated in vivo that this inhibitor is necessary for optimal tumor invasion and vascularization. We also showed that PAI-1 angiogenic activity is associated with its control of plasminogen activation but not with the regulation of cell-matrix interaction. To dissect the role of the various components of the plasminogen activation system during angiogenesis, we have adapted the aortic ring assay to use vessels from gene-inactivated mice. The single deficiency of tPA, uPA, or uPAR, as well as combined deficiencies of uPA and tPA, did not dramatically affect microvessel formation. Deficiency of plasminogen delayed microvessel outgrowth. Lack of PAI-1 completely abolished angiogenesis, demonstrating its importance in the control of plasmin-mediated proteolysis. Microvessel outgrowth from PAI-1-/- aortic rings could be restored by adding exogenous PAI-1 (wild-type serum or purified recombinant PAI-1). Addition of recombinant PAI-1 led to a bell-shaped angiogenic response clearly showing that PAI-1 is proangiogenic at physiological concentrations and antiangiogenic at higher levels. Using specific PAI-1 mutants, we could demonstrate that PAI-1 promotes angiogenesis at physiological (nanomolar) concentrations through its antiproteolytic activity rather than by interacting with vitronectin.
Asunto(s)
Neovascularización Fisiológica/efectos de los fármacos , Inhibidor 1 de Activador Plasminogénico/farmacología , Animales , Aorta/efectos de los fármacos , Aorta/crecimiento & desarrollo , Vasos Sanguíneos/efectos de los fármacos , Vasos Sanguíneos/crecimiento & desarrollo , Técnicas de Cultivo , Relación Dosis-Respuesta a Droga , Femenino , Genotipo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos , Ratones Noqueados , Plasminógeno/genética , Inhibidor 1 de Activador Plasminogénico/genética , Inhibidor 1 de Activador Plasminogénico/fisiología , Receptores de Superficie Celular/genética , Receptores del Activador de Plasminógeno Tipo Uroquinasa , Activador de Tejido Plasminógeno/genética , Activador de Plasminógeno de Tipo Uroquinasa/genéticaRESUMEN
Angiogenesis, a key step in many physiological and pathological processes, involves proteolysis of the extracellular matrix. To study the role of two enzymatic families, serine-proteases and matrix metalloproteases in angiogenesis, we have adapted to the mouse, the aortic ring assay initially developed in the rat. The use of deficient mice allowed us to demonstrate that PAI-1 is essential for angiogenesis while the absence of an MMP, MMP-11, did not affect vessel sprouting. We report here that this model is attractive to elucidate the cellular and molecular mechanisms of angiogenesis, to identify, characterise or screen "pro- or anti-angiogenic agents that could be used for the treatment of angiogenesis-dependent diseases. Approaches include using recombinant proteins, synthetic molecules and adenovirus-mediated gene transfer.