RESUMEN
We evaluated the antiangiogenic activity of shark cartilage, tumor necrosis factor-alpha (TNF-alpha), and a combination of the two using a human umbilical vein endothelial cell proliferation assay. Proliferation of endothelium is a hallmark of angiogenesis, and inhibition of endothelial cell proliferation indicates potential antiangiogenic activity. Shark cartilage produced a concentration-dependent decline in endothelial cell 3H-thymidine incorporation. This activity was heat stable and was found in molecular weight fractions of less than 10 kd. The antiproliferative effect of shark cartilage was specific for vascular endothelium and did not affect the proliferative rate of human astrocytoma cells or human skin fibroblasts. Shark cartilage at a concentration of 500 mu g/ml and TNF-alpha at a concentration of 10 ng/ml reduced endothelial cell proliferation by 32% and 29%, respectively. Treatment of endothelial cells with the combination of shark cartilage and TNF-alpha resulted in a 44% reduction in endothelial cell proliferation. The isolation and identification of the active components of shark cartilage is continuing.
Asunto(s)
Cartílago/química , Endotelio Vascular/efectos de los fármacos , Tiburones , Extractos de Tejidos/farmacología , Venas Umbilicales/efectos de los fármacos , Animales , Técnicas de Cultivo de Célula , División Celular/efectos de los fármacos , Endotelio Vascular/citología , Humanos , Timidina/biosíntesis , Factor de Necrosis Tumoral alfa/farmacología , Venas Umbilicales/citologíaRESUMEN
An essential component of any in vitro model for endothelial permeability is a confluent cell monolayer. The model reported here utilizes primary human umbilical vein endothelial cells (HUVEC) cultured on recently developed polyethylene terephthalate micropore membranes. Using a modification of the Wright-Giemsa stain, confluent HUVEC monolayers grown on micropore membranes were routinely assessed using light microscopy. Determination of confluence using this method was confirmed by scanning electron microscopy. Transendothelial electrical resistance of HUVEC monolayers averaged 27.9 +/- 11.4 omega.cm2, 10 to 21% higher than literature values. Studies characterizing the permeability of the endothelial cell monolayer to 3H-inulin demonstrated a linear relationship between the luminal concentration of 3H-inulin and its flux across HUVEC monolayers. The slope of the flux versus concentration plot, which represents endothelial clearance of 3H-inulin, was 2.01 +/- 0.076 x 10(-4) ml/min (r2 = .9957). The permeability coefficient for the HUVEC monolayer-micropore membrane barrier was 3.17 +/- 0.427 x 10(-6) cm/s with a calculated permeability coefficient of the HUVEC monolayer alone of 4.07 +/- 0.617 x 10(-6) cm/s. The HUVEC monolayer reduced the permeability of the micropore membrane alone to 3H-inulin (1.43 +/- 0.445 x 10(-5) cm/s) by 78%. Evans blue dye-labeled bovine serum albumin could not be detected on the abluminal side without disruption of the HUVEC monolayer. These results demonstrate a model for endothelial permeability that can be extensively assessed for monolayer integrity by direct visualization, transendothelial electrical resistance, and the permeability of indicator macromolecules.
Asunto(s)
Endotelio Vascular/citología , Modelos Biológicos , Albúmina Sérica Bovina/farmacocinética , Venas Umbilicales/citología , Permeabilidad de la Membrana Celular , Células Cultivadas , Electrofisiología , Humanos , Membranas ArtificialesRESUMEN
The synergistic effects of combining fish oil (FO) diet, which reduces thromboxane A production, with the free radical scavenger, dimethylthiourea (DMTU), were evaluated in acute adriamycin nephrosis, because proteinuria in adriamycin nephrosis is mediated by increased renal thromboxane A and free radical production. The effects of combined evening primrose oil (EPO) and DMTU were compared with the DMTU + FO combination because EPO increases prostaglandin E but not thromboxane A. After 7, 14, and 21 days, proteinuria was significantly (p < 0.05) reduced in rats receiving either DMTU + corn oil (CO) or DMTU + FO compared with untreated control rats. However, after 21 days, rats receiving DMTU + FO had significantly reduced urine protein excretion compared with those receiving DMTU + CO (103.9 +/- 20 mg daily vs 351.8 +/- 29.8 mg daily; P < 0.05). In contrast to FO, rats receiving EPO + DMTU had similar urine protein excretion to rats receiving DMTU + CO after 21 days (170.2 +/- 20.34 mg daily vs 179.45 +/- 26.38 mg daily). The mean serum cholesterol concentration was significantly (P < 0.01) reduced in rats receiving DMTU + FO (195.2 +/- 23.8 mg/dL) compared with DMTU + CO (377.9 +/- 28.5 mg/dL). Serum triglyceride levels also were significantly (P < 0.01) reduced in rats receiving DMTU + FO (52.5 +/- 26.4 mg/dL) compared with DMTU + CO (100.5 +/- 36.9 mg/dL). No significant differences in serum cholesterol concentrations or triglycerides occurred between rats receiving DMTU + CO and DMTU + EPO. Renal glutathione content was significantly (P < 0.05) increased by 23% in normal rats receiving FO diet and by 34% in rats receiving combined DMTU + FO compared with CO alone.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Grasas de la Dieta/uso terapéutico , Doxorrubicina/toxicidad , Aceites de Pescado/uso terapéutico , Riñón/efectos de los fármacos , Nefrosis/terapia , Tiourea/análogos & derivados , Animales , Colesterol/sangre , Aceite de Maíz , Quimioterapia Combinada , Ácidos Grasos Esenciales/farmacología , Glutatión/metabolismo , Hipolipemiantes/farmacología , Riñón/metabolismo , Riñón/patología , Ácidos Linoleicos , Nefrosis/inducido químicamente , Oenothera biennis , Aceites de Plantas , Proteinuria , Distribución Aleatoria , Ratas , Ratas Sprague-Dawley , Valores de Referencia , Tiourea/uso terapéutico , Factores de Tiempo , Triglicéridos/sangre , Ácido gammalinolénicoAsunto(s)
Factores de Crecimiento de Fibroblastos , Factor de Crecimiento Derivado de Plaquetas , Compuestos de Tosilo , Animales , Línea Celular , Cloraminas , Replicación del ADN/efectos de los fármacos , Factores de Crecimiento de Fibroblastos/metabolismo , Factores de Crecimiento de Fibroblastos/farmacología , Humanos , Indicadores y Reactivos , Radioisótopos de Yodo , Marcaje Isotópico/métodos , Lactoperoxidasa , Factor de Crecimiento Derivado de Plaquetas/metabolismo , Factor de Crecimiento Derivado de Plaquetas/farmacología , Receptores de Superficie Celular/metabolismo , Receptores de Factores de Crecimiento de Fibroblastos , Receptores del Factor de Crecimiento Derivado de Plaquetas , Succinimidas , PorcinosRESUMEN
Previous studies have shown that cell density can regulate the binding of several growth factors. To determine whether cell density exerts a uniform effect on the expression of epidermal growth factor (EGF) receptors, seven cell lines were examined in detail. For each cell line, EGF binding was found to decrease as cell density increases. Scatchard analysis of the binding data reveals that decreases in EGF binding are due to reductions in the number of cell surface EGF receptors. The only apparent exception is the effect of cell density on the binding of EGF to A-431 cells. For these cells, increases in cell density lead to two effects: decreases in the number of high affinity EGF receptors and increases in the total number of EGF receptors. In addition to the effects of cell density on EGF receptors, it was determined that increases in cell density can coordinately down-regulate receptors for as many as four different growth factors. Overall, the findings described in this report for EGF and those previously described for transforming growth factor type-beta (TGF-beta) and fibroblast growth factor (FGF) demonstrate the existence of a common mechanism for down-regulating growth factor receptors.
Asunto(s)
Recuento de Células , Regulación hacia Abajo , Receptores ErbB/metabolismo , Animales , Línea Celular , Supervivencia Celular , Factor de Crecimiento Epidérmico/metabolismo , Factores de Crecimiento de Fibroblastos , Humanos , Factor de Crecimiento Derivado de Plaquetas/metabolismo , Factores de Crecimiento Transformadores/metabolismo , Células Tumorales CultivadasRESUMEN
Previous studies have shown that the binding of fibroblast growth factor to several different non-transformed cell lines decreases as cell density increases. However, it was not determined whether this is due to a reduction in receptor number or to a decrease in receptor affinity. In this study, we demonstrate that the reduction in fibroblast growth factor binding is due to a reduction in receptor number. In addition, flow cytometric analysis indicated little or no difference in the cell cycle distribution of the cells at low and high cell densities, yet the binding of fibroblast growth factor was reduced substantially at high cell densities. Thus, the reduction in growth factor binding observed in this study does not appear to result from cell cycle-dependent regulation of growth factor receptors.
Asunto(s)
Recuento de Células , Factores de Crecimiento de Fibroblastos/metabolismo , Fibroblastos/metabolismo , Receptores de Superficie Celular/análisis , Animales , Ciclo Celular , Línea Celular , Fibroblastos/citología , Citometría de Flujo , Ratas , Receptores de Factores de Crecimiento de FibroblastosRESUMEN
The work described in this paper demonstrates that the cellular binding of transforming growth factor beta, epidermal growth factor, platelet-derived growth factor, and fibroblast growth factor is reduced as cell density is increased. The reduction in transforming growth factor beta binding was observed in five different cell lines. Examination of several of the cell lines, under conditions where transforming growth factor beta binding is reduced, revealed that epidermal growth factor binding, platelet-derived growth factor binding, and fibroblast growth factor binding are also reduced. In the case of NRK-49F cells, the reduction in transforming growth factor beta binding results from a decrease in the number of high-affinity receptors and not from a change in receptor affinity. Similarly, it was determined that the reduction in epidermal growth factor binding is due to a selective reduction in the high-affinity receptors for epidermal growth factor. Overall, the data suggest that the effect of cell density on growth factor binding, which we refer to as density-induced down regulation of growth factor receptors, differs both from down regulation induced by a specific growth factor and from receptor transmodulation.
Asunto(s)
Recuento de Células , Factor de Crecimiento Epidérmico/metabolismo , Factores de Crecimiento de Fibroblastos/metabolismo , Péptidos/metabolismo , Factor de Crecimiento Derivado de Plaquetas/metabolismo , Animales , Línea Celular , Riñón/metabolismo , Cinética , Ratones , Ratas , Temperatura , Factores de Crecimiento TransformadoresRESUMEN
A-431 cells, which exhibit large numbers of epidermal growth factor (EGF) receptors and respond to EGF by growth inhibition, are widely used to study EGF receptors and the effects of EGF. In this report, we describe the isolation and characterization of variant A-431 cells that respond to EGF by growth stimulation. One variant, which is referred to as A-431R-1, has been characterized in detail. EGF stimulates both monolayer and soft agar growth of A-431R-1 cells cultured in serum-free medium. In contrast to the original A-431 cells, growth of A-431R-1 cells is not inhibited by EGF, even at high concentrations. Scatchard analysis of EGF binding to A-431R-1 cells and A-431 cells indicates that both cell populations exhibit approximately 1.8 x 10(6) EGF receptors per cell. Thus, unlike other variants of A-431 cells that are not inhibited by EGF, A-431R-1 cells exhibit as many EGF receptors as the parental A-431 cells. It was also determined that the phorbol ester 12-O-tetradecanoylphorbol-13-acetate reduces EGF binding to the high affinity receptors of A-431R-1 cells; whereas, transforming growth factor type beta did not significantly affect EGF binding. Our results suggest that A-431R-1 cells should be useful for studying the biochemical effects of EGF and for examining why some cells are inhibited by EGF, whereas others are stimulated by EGF.