RESUMEN
PURPOSE: To determine the in vivo effect of doxycycline on choroidal angiogenesis and pterygium growth by using a choroidal neovascular (CNV) murine model, a directed in vivo angiogenesis assay (DIVAA) and a pterygium murine model. DESIGN: Experimental study. PARTICIPANTS: Three murine models were investigated with 4 mice minimum per group and 22 maximum per group. METHODS: Mice received water with or without doxycycline. For the CNV, the neovascular lesion volume was determined in choroid-retinal pigment epithelial flat mounts using confocal microscopy 7 days after laser induction. For DIVAA, silicone capsules containing 10,000 human pterygium epithelial cells were implanted in the flanks of mice subcutaneously. After 11 days, neovascularization (NV) was quantified using spectrofluorometry after murine tail-vein injection of fluorescein isothiocyanate-labeled dextran. A pterygium epithelial cell model was developed by injecting 10,000 human pterygium epithelial cells in the nasal subconjunctival space in athymic nude mice. Doxycycline was started on day 6 at 50 mg/kg per day; corneal lesions that resulted from the injections were compared at days 6 and 15. MAIN OUTCOME MEASURES: The Student t-test was used to evaluate the data for the CNV and DIVAA models and histologic preparations were used to evaluate pterygia lesions. RESULTS: There was significantly less NV and lesion volume with doxycycline taken in drinking water versus plain water. With doxycycline treatment, the laser-induced CNV showed a maximal 66% decrease in choroidal blood vessel volume (P< or =0.008) and the DIVAA showed a 30% reduction of blood vessel growth and migration (P<0.004). Histologic preparations demonstrated that pterygium cell lesions regressed when mice were administered doxycycline for 9 days. CONCLUSIONS: Doxycycline significantly inhibited angiogenesis in 3 murine models. The most dramatic effect was found in the CNV model followed by the pterygia epithelial cell DIVAA model. The anterior segment pterygium model also showed regression histologically. This suggests that doxycycline may be successful as an adjunctive treatment for CNV and pterygia in humans; clinical trials would be necessary to determine if there is a benefit.
Asunto(s)
Antibacterianos/farmacología , Neovascularización Coroidal/prevención & control , Neovascularización de la Córnea/prevención & control , Modelos Animales de Enfermedad , Doxiciclina/farmacología , Administración Oral , Animales , Antibacterianos/administración & dosificación , Movimiento Celular/efectos de los fármacos , Trasplante de Células , Células Cultivadas , Doxiciclina/administración & dosificación , Endotelio Vascular/efectos de los fármacos , Células Epiteliales/efectos de los fármacos , Células Epiteliales/patología , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Desnudos , Microscopía Confocal , Pterigion/tratamiento farmacológico , Pterigion/patología , Espectrometría de FluorescenciaRESUMEN
BACKGROUND: Human cripto-1 (CR-1) promotes cell transformation and increases migration and invasion of various mouse and human epithelial cell lines. We investigated whether CR-1 also stimulates angiogenesis. METHODS: We used human umbilical vein endothelial cells (HUVECs) to measure in vitro migration with fibronectin-coated Boyden chambers, invasion with Matrigel-coated Boyden chambers, proliferation with a tetrazolium salt, and differentiation with an in vitro Matrigel assay. We investigated new blood vessel formation in vivo by use of Matrigel-filled silicone cylinders implanted under the skin of nude mice and by use of a breast cancer xenograft model with CR-1-transfected or control Neo-transfected MCF-7 human breast cancer cells. We also used a blocking anti-CR-1 monoclonal antibody to investigate the role of CR-1 in angiogenesis in vivo and in vitro. All statistical tests were two-sided. RESULTS: CR-1 stimulated HUVEC proliferation, migration, and invasion and induced HUVEC differentiation into vascular-like structures on Matrigel. In vivo, recombinant CR-1 protein induced microvessel formation in Matrigel-filled silicone cylinders, and microvessel formation was statistically significantly inhibited with a blocking anti-CR-1 monoclonal antibody (CR-1 and antibody = 127% of microvessel formation compared with that in untreated control cylinders and CR-1 alone = 259%; difference = 132%, 95% confidence interval [CI] = 123% to 140%; P<.001). Tumors formed by CR-1-transfected MCF-7 cells in the cleared mammary fat pad of nude mice had higher microvessel density than tumors formed by control Neo-transfected MCF-7 cells (CR-1-transfected cells = 4.66 vessels per field and Neo-transfected cells = 2.33 vessels per field; difference = 2.33 vessels per field, 95% CI = 1.2 to 2.8; P = .004). CONCLUSION: CR-1 appears to have an important role in the multistep process of angiogenesis.
Asunto(s)
Neoplasias de la Mama/irrigación sanguínea , Células Endoteliales/metabolismo , Factor de Crecimiento Epidérmico/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Neovascularización Patológica/metabolismo , Animales , Anticuerpos Monoclonales , Biomarcadores de Tumor/metabolismo , Diferenciación Celular , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Colágeno , Combinación de Medicamentos , Factor de Crecimiento Epidérmico/inmunología , Factor de Crecimiento Epidérmico/farmacología , Femenino , Factor 2 de Crecimiento de Fibroblastos/análisis , Proteínas Ligadas a GPI , Sustancias de Crecimiento/metabolismo , Humanos , Inmunoensayo , Péptidos y Proteínas de Señalización Intercelular , Laminina , Glicoproteínas de Membrana/inmunología , Glicoproteínas de Membrana/farmacología , Ratones , Ratones Desnudos , Proteínas de Neoplasias/inmunología , Proteínas de Neoplasias/farmacología , Proteoglicanos , Proteínas Recombinantes/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal , Trasplante Heterólogo , Venas Umbilicales , Factor A de Crecimiento Endotelial Vascular/análisisRESUMEN
Tissue inhibitors of metalloproteinases (TIMPs) suppress matrix metalloproteinase (MMP) activity critical for extracellular matrix turnover associated with both physiologic and pathologic tissue remodeling. We demonstrate here that TIMP-2 abrogates angiogenic factor-induced endothelial cell proliferation in vitro and angiogenesis in vivo independent of MMP inhibition. These effects require alpha 3 beta 1 integrin-mediated binding of TIMP-2 to endothelial cells. Further, TIMP-2 induces a decrease in total protein tyrosine phosphatase (PTP) activity associated with beta1 integrin subunits as well as dissociation of the phosphatase SHP-1 from beta1. TIMP-2 treatment also results in a concomitant increase in PTP activity associated with tyrosine kinase receptors FGFR-1 and KDR. Our findings establish an unexpected, MMP-independent mechanism for TIMP-2 inhibition of endothelial cell proliferation in vitro and reveal an important component of the antiangiogenic effect of TIMP2 in vivo.
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Inhibidores de la Angiogénesis/farmacología , Endotelio Vascular/efectos de los fármacos , Metaloproteinasas de la Matriz/metabolismo , Neovascularización Fisiológica/fisiología , Inhibidor Tisular de Metaloproteinasa-2/farmacología , Inhibidores de la Angiogénesis/genética , Animales , Unión Competitiva , División Celular/efectos de los fármacos , Células Cultivadas , Relación Dosis-Respuesta a Droga , Embrión de Mamíferos/citología , Factores de Crecimiento Endotelial/metabolismo , Endotelio Vascular/citología , Endotelio Vascular/metabolismo , Inhibidores Enzimáticos/farmacología , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Fibroblastos/metabolismo , Humanos , Integrina alfa3beta1/metabolismo , Péptidos y Proteínas de Señalización Intracelular , Ratones , Neovascularización Fisiológica/efectos de los fármacos , Proteína Tirosina Fosfatasa no Receptora Tipo 1 , Proteína Tirosina Fosfatasa no Receptora Tipo 6 , Proteínas Tirosina Fosfatasas/efectos de los fármacos , Proteínas Tirosina Fosfatasas/metabolismo , Proteínas Tirosina Quinasas Receptoras/farmacología , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos , Receptores de Factores de Crecimiento de Fibroblastos , Proteínas Recombinantes/farmacología , Inhibidor Tisular de Metaloproteinasa-2/genética , Vanadatos/farmacología , Factor A de Crecimiento Endotelial Vascular , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismoRESUMEN
One of the major problems in angiogenesis research remains the lack of suitable methods for quantifying the angiogenic response in vivo. We describe the development and application of the directed in vivo angiogenesis assay (DIVAA) and demonstrated that it is reproducible and quantitative. This assay consists of subcutaneous implantation of semiclosed silicone cylinders (angioreactors) into nude mice. Angioreactors are filled with only 18 micro l of extracellular matrix premixed with or without angiogenic factors. Vascularization within angioreactors is quantified by the intravenous injection of fluorescein isothiocyanate (FITC)-dextran before their recovery, followed by spectrofluorimetry. Angioreactors examined by immunofluorescence show cells and invading angiogenic vessels at different developmental stages. The minimally detectable angiogenic response requires 9 days after implantation and >/=50 ng/ml (P < 0.01) of either fibroblast growth factor-2 or vascular endothelial growth factor. Characterization of this assay system demonstrates that the FITC-labeled dextran quantitation is highly reproducible and that levels of FITC-dextran are not significantly influenced by vascular permeability. DIVAA allows accurate dose-response analysis and identification of effective doses of angiogenesis-modulating factors in vivo. TNP-470 potently inhibits angiogenesis (EC(50) = 88 pmol/L) induced by 500 ng/ml of fibroblast growth factor-2. This inhibition correlates with decreased endothelial cell invasion. DIVAA efficiently detects differences in anti-angiogenic potencies of thrombospondin-1 peptides (25 micro mol/L) and demonstrates a partial inhibition of angiogenesis ( approximately 40%) in a matrix metalloprotease (MMP)-2-deficient mouse compared with that in wild-type animals. Zymography of angioreactors from MMP-deficient and control animals reveals quantitative changes in MMP expression. These results support DIVAA as an assay to compare potencies of angiogenic factors or inhibitors, and for profiling molecular markers of angiogenesis in vivo.
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Endotelio Vascular/patología , Neovascularización Patológica/fisiopatología , Animales , Bioensayo , Permeabilidad Capilar/fisiología , Factores de Crecimiento Endotelial/farmacología , Gelatinasas/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intercelular/farmacología , Linfocinas/farmacología , Ratones , Neovascularización Patológica/patología , Proteínas Recombinantes/farmacología , Reproducibilidad de los Resultados , Trombospondina 1/fisiología , Factor A de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial VascularRESUMEN
We studied 86 specimens from 24 patients with hairy cell leukemia (HCL) to determine the sensitivity of routine flow cytometry (FC) and consensus primer PCR (cpPCR) in this disease. FC was more sensitive, detecting HCL in 48 (56%) of 86 specimens, while clonal B-cell populations were detected by cpPCR in only 23 (27%) of 86 specimens. FC and cpPCR were both more sensitive than morphologic examination. A positive cpPCR result is associated with higher tumor cell numbers than a negative cpPCR result, as determined by FC (P = .0017). We determined cutoff values for number of tumor cells at which cpPCR is consistently positive. At 6.8 tumor cells per microliter, cpPCR would be expected to be positive in at least 90% of the samples. FC was adequate in 86 cases (100%), while cpPCR was adequate in 74 cases (86%). FC is superior to cpPCR for detecting minimal residual HCL. It is more sensitive and more specific and permits quantitation of tumor cell number.