Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 7 de 7
Filtrar
Más filtros












Base de datos
Intervalo de año de publicación
1.
Cell Microbiol ; 11(2): 323-36, 2009 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-19046337

RESUMEN

Previous experimental studies in a standard Transwell culture system have shown Streptococcus suis ability to compromise barrier function of porcine choroid plexus epithelial cells (PCPEC). The development of an 'inverted' Transwell filter system of PCPEC enables us now for the first time to investigate bacterial invasion and translocation from the physiologically relevant basolateral (blood) to the apical (cerebrospinal fluid) side. Most importantly, we observed specific invasion and translocation of S. suis across the PCPEC exclusively from the basolateral side. During this process, bacterial viability and the presence of a capsule as well as cytoskeletal regulation of PCPEC seemed to play an important role. No loss of barrier function was observed. Bacterial translocation could be significantly inhibited by the phosphatidylinositol 3-kinase inhibitor LY294002, but not by its inactive analogue Ly303511 or dexamethasone. Apotome imaging as well as electron microscopy revealed intracellular bacteria often in cell vacuoles. Thus, possibly regulated by the presence of a capsule, S. suis induces signals that depend on the lipid kinase phosphatidylinositol 3-kinase pathway, which paves the way for cellular uptake during the bacterial transcellular translocation process. Taken together, our data underline the relevance of the blood-cerebrospinal fluid barrier as a gate for bacterial entry into the central nervous system.


Asunto(s)
Barrera Hematoencefálica/microbiología , Células Epiteliales/microbiología , Streptococcus suis/fisiología , Animales , Células Cultivadas , Plexo Coroideo/microbiología , Células Epiteliales/ultraestructura , Microscopía Electrónica de Transmisión , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3 , Porcinos , Vacuolas/microbiología , Vacuolas/ultraestructura
2.
Graefes Arch Clin Exp Ophthalmol ; 244(3): 376-81, 2006 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-16088412

RESUMEN

PURPOSE: To evaluate the effects of verapamil isomers on in vitro proliferation of bovine choroidal endothelial cells (CECs). MATERIALS AND METHODS: CECs were isolated from bovine eyes and cultured in endothelial growth medium (EGM). For the proliferation assays, CECs were exposed to verapamil isomers (0.1-100 microM) in EGM with 2% fetal bovine serum or basic fibroblast growth factor (bFGF) (10 ng/ml). After 72 h of incubation with the desired drug, the cellular proliferation was determined by an MTT assay and a BrdU assay. In addition, the drug toxicity on CECs stimulated with EGM was evaluated by cell counting with trypan blue. RESULTS: All verapamil isomers inhibited the bFGF- or medium-stimulated growth significantly in a concentration range of 10-40 microM without toxicity. No significant differences were seen between the inhibitory effects of the various isomers. Cell toxicity was detected at a concentration of 100 microM verapamil isomers on EGM-stimulated CECs. CONCLUSION: The results demonstrate the efficacy of all verapamil isomers in inhibiting CEC proliferation involved in the process of choroidal neovascularization. D: -(+)-Verapamil may be recommended for further in vivo evaluation in an animal model of exudative AMD; it has fewer systemic and local side effects because calcium channels are not blocked.


Asunto(s)
Bloqueadores de los Canales de Calcio/farmacología , Proliferación Celular/efectos de los fármacos , Coroides/irrigación sanguínea , Endotelio Vascular/citología , Verapamilo/farmacología , Animales , Bovinos , Supervivencia Celular , Células Cultivadas , Medios de Cultivo , Endotelio Vascular/efectos de los fármacos , Factor 2 de Crecimiento de Fibroblastos/farmacología , Isomerismo
3.
Curr Eye Res ; 30(6): 479-89, 2005 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-16020281

RESUMEN

PURPOSE: To investigate the potential role of endostatin, an endogenous angiogenesis inhibitor, in the prevention of choroidal angiogenesis-related disorders. METHODS: Bovine choroidal endothelial cells (CEC) were cultured and treated with basic fibroblast growth factor (bFGF) alone or combined with endostatin at concentrations ranging from 0.1 to 10 microg/ml. The proliferation and migration of CECs were evaluated by using 3, (4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) colorimetric assay and modified Boyden chamber assay, respectively. For evaluating expression and secretion of matrix metalloproteinase-2 (MMP-2), CEC-conditioned media were subjected to zymography and/or Western blot analysis, and the cells were used for semiquantitative reverse transcription polymerase chain reaction (RT-PCR) analysis. RESULTS: Endostatin did not inhibit bFGF-induced or nonstimulated CEC proliferation (p > 0.05). The bFGF-induced migration was significantly inhibited by endostatin at concentrations of 1 and 10 microg/ml (p < 0.05). The bFGF-upregulated expression of mRNA in CECs and the secretion of MMP-2 protein of CECs were both suppressed by endostatin. CONCLUSIONS: Inhibitory effect of endostatin on expression and secretion of MMP-2 and cell migration, but not on proliferation of CECs, could respond to its therapeutic action for choroidal neovascularization-dependent disorders.


Asunto(s)
Inhibidores de la Angiogénesis/farmacología , Coroides/irrigación sanguínea , Endostatinas/farmacología , Células Endoteliales/efectos de los fármacos , Células Endoteliales/enzimología , Factor 2 de Crecimiento de Fibroblastos/farmacología , Metaloproteinasa 2 de la Matriz/metabolismo , Animales , Bovinos , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Regulación hacia Abajo , Células Endoteliales/citología , Metaloproteinasa 2 de la Matriz/genética , Inhibidores de la Metaloproteinasa de la Matriz , ARN Mensajero/metabolismo
4.
Curr Eye Res ; 28(5): 359-66, 2004 May.
Artículo en Inglés | MEDLINE | ID: mdl-15287373

RESUMEN

PURPOSE: Integrin alpha(v)beta3 is predominantly expressed on endothelial cells in choroidal neovascularization (CNV). We evaluated the efficacy of cyclic RGD (Arg-Gly-Asp) peptide, an alpha(v)-integrin antagonist, in a rat model of laser-induced CNV METHODS: We evaluated the effect of cyclic RGD on the adhesion and cell viability of bovine choroidal endothelial cells (BCECs) by cell counting and the trypan blue dye exclusion test. CNV was induced by laser photocoagulation in female Long Evans rats (day 0), followed by intravitreal administration of one dose of cyclic RGD of 200 (n = 9), 100 (n = 10), 50 (n = 4), or 0 microg (n = 9) on days 9 and 11. We assessed the area of CNV by fluorescein angiography and the thickness microscopically on histologic sections. Neovascular vessels were detected by an antibody against factor VIII. RESULTS: Cyclic RGD (0.02 to 200 microg/ml) inhibited adhesion of BCECs in a dose-dependent manner without changing the cell viability (p < 0.01). In eyes treated with two injections of 200 or 100 microg of cyclic RGD peptide, the development of CNV was significantly (p < 0.01) inhibited in the area of leakage on fluorescein angiography. Histologically, the CNV membrane was observed beneath the retina and the factor VIII-positive cells and red blood cells were involved. The thickness of the lesions was significantly (p < 0.01) reduced in eyes that received 200 or 100 microg of RGD. CONCLUSIONS: Cyclic RGD effectively inhibited CNV progression in a rat model of laser-induced CNV, suggesting that this alpha(v)-integrin antagonist may be beneficial in the treatment of CNV.


Asunto(s)
Neovascularización Coroidal/tratamiento farmacológico , Integrina alfaVbeta3/antagonistas & inhibidores , Oligopéptidos/uso terapéutico , Animales , Bovinos , Adhesión Celular/efectos de los fármacos , Técnicas de Cultivo de Célula , Supervivencia Celular/efectos de los fármacos , Coroides/irrigación sanguínea , Neovascularización Coroidal/patología , Relación Dosis-Respuesta a Droga , Endotelio Vascular/efectos de los fármacos , Femenino , Angiografía con Fluoresceína , Modelos Animales , Ratas , Ratas Long-Evans
5.
Graefes Arch Clin Exp Ophthalmol ; 240(10): 860-6, 2002 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-12397436

RESUMEN

BACKGROUND: Nitric oxide (NO) is known as an important mediator of endothelial function. The aim of this investigation was to evaluate the influence of mediators of retinal pathology - vascular endothelial growth factor (VEGF) and advanced glycation end products (AGEs) - on NO release from choroidal endothelial cells (CEC) and retinal pigment epithelial (RPE) cells to elucidate the complex role of NO. METHODS: Bovine CEC were stimulated using VEGF (1, 10, and 100 ng/ml), and RPE cells were exposed to interferon-gamma (IFN-gamma 100 U/ml) and lipopolysaccharide (LPS 1 micro g/ml). NO release into the media was assessed by an amperometric NO sensor. The influence of AGEs (10 and 100 micro g/ml) on NO release from CEC and RPE cells was investigated. The competitive NO synthase inhibitor L(omega)-nitro-L-arginine methyl ester (L-NAME 2 nmol) was used to pretreat the cells 2 h before NO measurement. RESULTS: Unstimulated CEC produced low basal levels of NO in vitro (39.1+/-13.9 nmol), and unstimulated RPE cells produced minimal basal levels of NO (15.7+/-7.0 nmol). Exposure of CEC to VEGF for 30 min resulted in a dose-dependent rise of NO in the medium, which was significantly inhibited by L-NAME. Stimulation of RPE cells with IFN-gamma and LPS resulted in a rise of NO in the bath to 125.9+/-18.5% of basal values. Basal NO release from CEC, and stimulated NO release from RPE cells, was significantly reduced by AGE treatment and L-NAME. CONCLUSIONS: These data demonstrate that AGEs formed from the nonenzymatic glycation of proteins with reducing sugars quench NO activities in vitro. The results implicate AGEs as important modulators of NO activity and may be relevant to the impairment of endothelial functions observed in diabetes and aging.


Asunto(s)
Productos Finales de Glicación Avanzada/farmacología , Óxido Nítrico/antagonistas & inhibidores , Animales , Bovinos , Células Cultivadas , Coroides/citología , Coroides/metabolismo , Factores de Crecimiento Endotelial/farmacología , Endotelio/citología , Endotelio/metabolismo , Inhibidores Enzimáticos/farmacología , Péptidos y Proteínas de Señalización Intercelular/farmacología , Interferón gamma/farmacología , Lipopolisacáridos/farmacología , Linfocinas/farmacología , NG-Nitroarginina Metil Éster/farmacología , Óxido Nítrico/metabolismo , Epitelio Pigmentado Ocular/citología , Epitelio Pigmentado Ocular/metabolismo , Factor A de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial Vascular
6.
Invest Ophthalmol Vis Sci ; 43(8): 2767-73, 2002 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-12147614

RESUMEN

PURPOSE: The balance between matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs (TIMPs) is crucial for homeostasis of ocular extracellular matrices. To assess altered MMP activity as a determinant in the migration of human retinal pigment epithelial (RPE) cells, expression characteristics of several MMPs and TIMP-1 in RPE cell cultures were investigated. METHODS: Expression studies were performed with RT-PCR, ELISA, and immunofluorescence analysis. Secretion of MMP-2 was demonstrated by zymography. Migration of cytokine-stimulated RPE cells was evaluated with microporous membranes of permeable chambers. RESULTS: MMP-1, -2, -3, and -9; MT2-MMP; and TIMP-1 were expressed in cultured RPE cells. MMP-2 was detected on the cell surface and in secreted inactive and active forms. TGF-beta(2), IL-1beta, and TNF-alpha enhanced secretion of MMP-1, -2, and -3. TGF-beta(2) also stimulated MT2-MMP cell surface expression and release of TIMP-1. The mRNA levels of MMP-1, -2, and -3 and TIMP-1 were markedly increased by TNF-alpha and TGF-beta(2). MMP-2 mRNA levels were also upregulated by PDGF-BB. Migration of RPE cells stimulated by TGF-beta(2) or PDGF-BB was inhibited in presence of a synthetic MMP inhibitor. CONCLUSIONS: Proinflammatory cytokines and TGF-beta(2) play an important role in the upregulation of expression of MMP-1, -2, and -3 in RPE cells and account for a directional shift in the balance between MMPs and TIMPs. Facilitation of RPE cell migration stimulated by cytokines (i.e., TGF-beta(2) or PDGF-BB) in ocular diseases may be due to increased release of MMPs, in the presence of comparatively lower levels of their inhibitors.


Asunto(s)
Citocinas/farmacología , Metaloproteinasas de la Matriz/biosíntesis , Epitelio Pigmentado Ocular/efectos de los fármacos , Inhibidor Tisular de Metaloproteinasa-1/biosíntesis , Movimiento Celular/fisiología , Células Cultivadas , Ensayo de Inmunoadsorción Enzimática , Matriz Extracelular/metabolismo , Técnica del Anticuerpo Fluorescente Indirecta , Gelatinasas/metabolismo , Humanos , Metaloproteinasas de la Matriz/genética , Epitelio Pigmentado Ocular/citología , Epitelio Pigmentado Ocular/metabolismo , ARN Mensajero/biosíntesis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Inhibidor Tisular de Metaloproteinasa-1/genética , Regulación hacia Arriba
7.
Graefes Arch Clin Exp Ophthalmol ; 240(1): 42-8, 2002 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-11954780

RESUMEN

BACKGROUND: Angiostatic drugs might provide desirable modulation of choroidal angiogenesis-related diseases, including histoplasmosis and the exudative form of age-related macular degeneration. However, the precise effects of this class of compounds in the choroidal neovascularization are still unclear. In the present study, we investigated the effects of triamcinolone acetonide (TA), an angiostatic steroid, on choroidal angiogenesis in vitro. METHODS: Bovine choroidal endothelial cells (CEC), which are the critical cellular component of choroidal angiogenesis in vivo, were isolated with Lycopersicon esculentum agglutinin-coated Dynabeads and cultured in EGM medium. CEC were treated with basic fibroblast growth factor (bFGF) and TA at various concentrations ranging from 50 to 300 mg/l. The capacities for CEC migration and tube formation were evaluated with the modified Boyden chamber and the Vitrogen collagen assay, respectively. The activities of matrix metalloproteinases (MMP)-2 and -9 were examined using gelatin zymography. RESULTS: The stimulation of CEC with 50 ng/ml bFGF resulted in an increase of about 100% in migration activity (P<0.01). Preincubation of CEC with TA at the indicated concentrations for 20 min inhibited the bFGF-stimulated migration in a dose-dependent manner (P<0.01). After 5 days, the bFGF-stimulated tube formation in CEC was inhibited by TA at the concentrations 100, 150 and 300 mg/l (P<0.01). Gelatin zymography of the culture media of CEC showed that the bFGF-induced activation of MMP-2 was attenuated by 300 mg/l TA (P<0.05). CONCLUSION: Downregulation of the activation of MMPs in CEC could be one of the mechanisms by which angiostatic steroids inhibit choroidal angiogenesis.


Asunto(s)
Coroides/irrigación sanguínea , Neovascularización Coroidal/prevención & control , Endotelio Vascular/efectos de los fármacos , Factor 2 de Crecimiento de Fibroblastos/farmacología , Inmunosupresores/farmacología , Triamcinolona Acetonida/farmacología , Animales , Bovinos , Movimiento Celular/efectos de los fármacos , Células Cultivadas , Neovascularización Coroidal/inducido químicamente , Neovascularización Coroidal/enzimología , Relación Dosis-Respuesta a Droga , Regulación hacia Abajo , Endotelio Vascular/enzimología , Endotelio Vascular/patología , Metaloproteinasa 2 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo
SELECCIÓN DE REFERENCIAS
DETALLE DE LA BÚSQUEDA
...