RESUMEN
PURPOSE: Quantifying phenotypic variation at the level of protein expression (variegation) within populations of retinal pigment epithelium (RPE) cells may be important in the study of pathologies associated with this variation. The lack of quantitative methods for examining single cells, however, and the variable presence of pigment and/or lipofuscin complicate this experimental goal. We have applied the technique of laser scanning cytometry (LSC) to paraffin sections of mouse and human eyes to evaluate the utility of LSC for these measurements. METHODS: Mouse eyes were perfusion fixed in 4% paraformaldehyde and embedded in paraffin. Postmortem human eyes were fixed and dissected to obtain a 9-mm punch, which was then embedded in paraffin. A laser scanning cytometer equipped with violet, argon, and helium-neon lasers and the detectors for blue, green, and long red were used to record the fluorescence of each individual cell at all three wavelengths. Raw data were recorded and processed using the WinCyte software. Individual nuclei were identified by the fluorescence of the 4',6-diamidino-2-phenylindole (DAPI) nuclear counterstain. Next, RPE cells were uniquely identified in the green channel using an anti-retinal pigment epithelium-specific protein 65 kDa (anti-RPE65) monoclonal antibody with an Alexa Fluor 488-labeled secondary antibody. Mn-superoxide dismutase (MnSOD) was quantified in the long-red channel using an anti-MnSOD antibody and an Alexa Fluor 647-labeled secondary antibody. MnSOD(+) and RPE65(+) cells exhibited peaks in the plot of fluorescence intensity versus cell number, which could be characterized by the mean fluorescence intensity (MFI), the coefficient of variation (CV), and the percentage of total RPE cells that were also labeled for MnSOD. RESULTS: RPE cells can be uniquely identified in human and mouse paraffin sections by immunolabeling with anti-RPE65 antibody. A second antigen, such as MnSOD, can then be probed only within this set of RPE. Results are plotted primarily with the population frequency diagram, which can be subdivided into multiple regions. The data collected for each region include the MFI, the CV, and the number of cells that are immunolabeled in that region. Background interference from pigment or autofluorescent material can be successfully overcome by elevating the concentrations of fluorescent secondary antibodies. In the human and mouse eyes, age-related changes in MFI, CV, and percent RPE cells immunolabeled for MnSOD were observed. CONCLUSIONS: The extent of the variability of gene expression in RPE cells at the protein level can be quantified by LSC. Relative changes in the MFI, the CV, and/or percentage of RPE cells double labeled for a second antigen quantify the changes observed. The analysis of these data also suggest whether the effects observed are related to local changes in transcription (alterations of CV) or major changes of protein expression (MFI), which are likely to be due to changes in the chromatin structure. The changes of these variables with age suggest that the observed age-related variegation is primarily due to changes in the chromatin structure in individual cells.
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Proteínas del Ojo/metabolismo , Citometría de Barrido por Láser , Fenotipo , Epitelio Pigmentado de la Retina/citología , Epitelio Pigmentado de la Retina/metabolismo , Envejecimiento/metabolismo , Animales , Anticuerpos/análisis , Proteínas Portadoras/inmunología , Proteínas Portadoras/metabolismo , Proteínas del Ojo/inmunología , Femenino , Fluorescencia , Humanos , Inmunohistoquímica , Técnicas Inmunológicas , Técnicas In Vitro , Citometría de Barrido por Láser/normas , Masculino , Ratones , Superóxido Dismutasa/metabolismo , cis-trans-IsomerasasRESUMEN
AIM: To determine the transcriptional proximity of retinal pigment epithelium (RPE) cells grown under different culture conditions and native RPE. METHODS: ARPE-19 cells were grown under five conditions in 10% CO(2): "subconfluent" in DMEM/F12+10% FBS, "confluent" in serum and serum withdrawn, and "differentiated" for 2.5 months in serum and serum withdrawn medium. Native RPE was laser microdissected. Total RNA was extracted, reverse transcribed, and radiolabelled probes were hybridised to an array containing 5,353 genes. Arrays were evaluated by hierarchical cluster analysis and significance analysis of microarrays. RESULTS: 78% of genes were expressed by native RPE while 45.3--47.7% were expressed by ARPE-19 cells, depending on culture condition. While the most abundant genes were expressed by native and cultured cells, significant differences in low abundance genes were seen. Hierarchical cluster analysis showed that confluent and differentiated, serum withdrawn cultures clustered closest to native RPE, and that serum segregated cultured cells from native RPE. The number of differentially expressed genes and their function, and profile of expressed and unexpressed genes, demonstrate differences between native and cultured cells. CONCLUSIONS: While ARPE-19 cells have significant value for studying RPE behaviour, investigators must be aware of how culture conditions can influence the mRNA phenotype of the cell.
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Epitelio Pigmentado Ocular/citología , Epitelio Pigmentado Ocular/metabolismo , Anciano , Anciano de 80 o más Años , Técnicas de Cultivo de Célula , Línea Celular , Análisis por Conglomerados , Medios de Cultivo , Medio de Cultivo Libre de Suero , Expresión Génica , Perfilación de la Expresión Génica/métodos , Humanos , Microdisección/métodos , Persona de Mediana Edad , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodosRESUMEN
PURPOSE: To investigate how the differentiation of ARPE-19 cells affects the relative expression of the FGFR genes in response to oxidative stress. METHODS: After differentiation in vitro, APRE-19 cells were treated with t-butyl hydroperoxide (tBH) or hydrogen peroxide (H2O2) to induce oxidative stress. Viability and reactive oxygen intermediate (ROI) production were measured using standard assays. The mRNA expression of FGFR1, FGFR2, cellular retinaldehyde-binding protein (CRALBP), RPE65, and heme oxygenase-1 (HO-1) were measured by Northern blot analysis as a function of treatment with tBH and H2O2. RESULTS: ARPE-19 cells were viable at all tBH concentrations tested but showed progressive loss of viability at concentrations greater than 300 microM H2O2. Differentiated ARPE-19 cells treated with tBH or H2O2 resulted in upregulation of the HO-1 and FGFR1 transcripts. The expression of RPE-differentiated specific genes, including FGFR2, CRALBP, and RPE65 mRNAs, was downregulated with tBH or H2O2 treatment. CONCLUSIONS: Oxidative stress in differentiated ARPE-19 cells alters the expression of FGFR1, FGFR2, CRALBP, and RPE65 toward levels characteristic of the undifferentiated state. If similar changes take place in vivo, these events could alter the proliferative potential, viability, and even the function of the RPE.
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Proteínas del Ojo/genética , Expresión Génica , Estrés Oxidativo , Células Ganglionares de la Retina/metabolismo , Northern Blotting , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Diferenciación Celular , Línea Celular , Regulación hacia Abajo , Proteínas del Ojo/metabolismo , Hemo Oxigenasa (Desciclizante)/genética , Hemo Oxigenasa (Desciclizante)/metabolismo , Hemo-Oxigenasa 1 , Humanos , Peróxido de Hidrógeno/farmacología , Proteínas de la Membrana , Proteínas/genética , Proteínas/metabolismo , ARN Mensajero/metabolismo , Proteínas Tirosina Quinasas Receptoras/genética , Proteínas Tirosina Quinasas Receptoras/metabolismo , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos , Receptores de Factores de Crecimiento de Fibroblastos/genética , Receptores de Factores de Crecimiento de Fibroblastos/metabolismo , Células Ganglionares de la Retina/citología , Células Ganglionares de la Retina/efectos de los fármacos , Factores de Tiempo , cis-trans-Isomerasas , terc-Butilhidroperóxido/farmacologíaAsunto(s)
ADN Complementario/biosíntesis , Disección/métodos , Rayos Láser , Degeneración Macular/metabolismo , Reacción en Cadena de la Polimerasa/métodos , Gliceraldehído-3-Fosfato Deshidrogenasas/biosíntesis , Gliceraldehído-3-Fosfato Deshidrogenasas/genética , Humanos , Degeneración Macular/genética , Degeneración Macular/patología , Persona de Mediana Edad , Epitelio Pigmentado Ocular/metabolismo , Epitelio Pigmentado Ocular/patología , ARN Mensajero/biosíntesis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sensibilidad y EspecificidadRESUMEN
PURPOSE: To demonstrate that chronic hyperoxia induces single-stranded breaks in chromosomal telomeres as a measure of oxidative DNA damage in cultured RPE cells. METHODS: RPE340 cells were cultured in 40% and 20% (control) O(2). DNA damage was assessed by mean terminal restriction fragment (TRF) length, and the S1 nuclease assay was used to determine the frequency of single-strand breaks in telomeric DNA. The degree of oxidative stress in cells was estimated by flow cytometric analysis of reactive oxygen intermediate (ROI)-induced 2',7'-dichlorodihydrofluorescein diacetate fluorescence and Northern blot analysis of heme oxygenase-1 (HO-1) mRNA induction. RESULTS: The mean TRF length of cells grown in 40% O(2) shortened at a faster rate than those grown in 20% O(2). The S1 nuclease assay showed that the accelerated mean TRF length shortening was due to an increased accumulation of single-stranded breaks in telomeric DNA. The degree of ROI production and HO-1 mRNA induction was greater in cells treated with 40% than 20% O(2), an effect that was also larger in old than young passaged cells. CONCLUSIONS: RPE340 cells in vitro grown in chronic hyperoxia exhibited evidence of DNA damage with accelerated telomeric shortening via an increased accumulation of single-strand breaks in telomeric DNA. These changes could provide insight into aging of RPE cells by oxidative DNA damage.
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Daño del ADN , ADN de Cadena Simple/metabolismo , Estrés Oxidativo , Epitelio Pigmentado Ocular/metabolismo , Telómero/metabolismo , Hipoxia de la Célula , Células Cultivadas , Senescencia Celular , Fibroblastos/citología , Fibroblastos/metabolismo , Citometría de Flujo , Fluoresceínas , Hemo Oxigenasa (Desciclizante)/genética , Hemo-Oxigenasa 1 , Humanos , Proteínas de la Membrana , Epitelio Pigmentado Ocular/citología , ARN Mensajero/biosíntesis , Especies Reactivas de Oxígeno/metabolismoRESUMEN
PURPOSE: To determine an extensive mRNA phenotype of the established RPE cell line ARPE-19 when grown on a matrix modified by advanced glycation end products (AGEs). METHODS: Growth Factor Reduced Matrigel (Collaborative Biomedical Products, Bedford, MA) was nonenzymatically glycated with glycolaldehyde. ARPE-19 cells were seeded on both AGE-Matrigel and Matrigel and grown to confluence, and serum was withdrawn for 3 days. RNA was extracted, and microarray analysis was performed to characterize the genes, which are altered by a matrix modified by AGEs. Gene expression changes were confirmed by RT-PCR/Southern and Northern blot analysis. Apoptosis was measured by annexin V/propidium iodide labeling. RESULTS: Clusters of genes with altered expression were found related to cell differentiation, growth factors that regulate the RPE cell and basement membrane, and apoptosis. RT-PCR/Southern and Northern blot analysis confirmed the expression patterns of selected genes, and flow cytometry showed increased annexin V/propidium iodide-labeled cells when grown on AGE-Matrigel. CONCLUSIONS: Microarray analysis identified clusters of genes that could promote an aging RPE phenotype in vitro induced by a matrix modified with AGEs.
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Envejecimiento/metabolismo , Proteínas del Ojo/genética , Productos Finales de Glicación Avanzada/farmacología , Epitelio Pigmentado Ocular/efectos de los fármacos , ARN Mensajero/biosíntesis , Anexina A5/metabolismo , Membrana Basal/fisiología , Northern Blotting , Southern Blotting , Diferenciación Celular/genética , Línea Celular , Cartilla de ADN/química , Proteínas del Ojo/biosíntesis , Citometría de Flujo , Perfilación de la Expresión Génica , Sustancias de Crecimiento/genética , Humanos , Análisis de Secuencia por Matrices de Oligonucleótidos , Fenotipo , Epitelio Pigmentado Ocular/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Cryopreservation can alter cellular function under certain conditions. In this report, we demonstrate the induction of cellular senescence after cells have been cryopreserved using a standard protocol. A retinal pigment epithelial cell line frozen at a specific freezing rate and subsequently thawed showed severely impaired proliferation compared to cells that were not cryopreserved. The induction of senescence was suggested by senescent associated beta-galactosidase activity and diminished bromo-deoxyuridine incorporation. A remarkable increase of single-strand DNA breaks in terminal restriction fragment (TRF) were found in cryopreserved cells immediately after thawing. The rate of mean TRF length shortening was accelerated after cryopreservation. Given this evidence, we hypothesize that cryopreservation may cause telomere shortening and cellular senescence under certain freezing conditions.
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División Celular , Senescencia Celular , Criopreservación , Telómero/metabolismo , Bromodesoxiuridina/metabolismo , Línea Celular , Daño del ADN , Humanos , Epitelio Pigmentado Ocular/citología , Epitelio Pigmentado Ocular/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Endonucleasas Específicas del ADN y ARN con un Solo Filamento , beta-Galactosidasa/metabolismoRESUMEN
PURPOSE: Previous studies have shown that insulin-like growth factor-binding protein (IGFBP)-2 is markedly upregulated in senescent RPE cells in vitro, and might therefore be a marker of senescent cells in vivo. This study was conducted to determine whether IGFBP-2 expression in human RPE cells from the macula and periphery varies with age in vivo. METHODS: Paraformaldehyde (4%)-fixed and optimal cutting temperature (OCT) compound-embedded human eyes from 17 patients were cryosectioned and subjected to high-sensitivity digoxigenin (DIG)-labeled cRNA in situ hybridization to determine the expression of IGFBP-2. Complementary immunohistochemistry experiments using a polyclonal anti-IGFBP-2 antibody were performed to confirm IGFBP-2 protein expression. Specimens were examined by light microscopy, and images were captured with a digital camera. The total numbers of RPE cells and IGFBP-2 mRNA expression-positive RPE cells were counted for each section, and the ratio of labeled RPE cells to total RPE cells counted was calculated for both macular and peripheral regions of each donor. RESULTS: IGFBP-2 mRNA expression was detected in the ganglion cell layer, inner and outer nuclear layers, and inner segments of photoreceptor cells in all 17 eyes. In 16 of 17 eyes, IGFBP-2 mRNA expression was detected in the RPE. In 11, the ratio of labeled cells to total RPE cells counted per section in the macula was 1.2 times greater than the ratio in the periphery (P = 0.008). The ratio of labeled RPE cells in the macula decreased with age (P = 0.0064). Immunohistochemistry studies for IGFBP-2 confirmed the expression pattern found by in situ hybridization. CONCLUSIONS: There is a topographical and age-related change in IGFBP-2 expression in RPE cells from human donor eyes. This distribution is likely not to represent senescent RPE cells in vivo.
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Envejecimiento/metabolismo , Proteína 2 de Unión a Factor de Crecimiento Similar a la Insulina/metabolismo , Epitelio Pigmentado Ocular/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Femenino , Humanos , Técnicas para Inmunoenzimas , Hibridación in Situ , Proteína 2 de Unión a Factor de Crecimiento Similar a la Insulina/genética , Masculino , Persona de Mediana Edad , ARN Mensajero/metabolismo , Donantes de Tejidos , Fijación del TejidoRESUMEN
PURPOSE: To establish a model of mild and chronic oxidative stress using hyperoxia for retinal pigment epithelial (RPE) cells in vitro. METHODS: RPE340 cells and WI38 lung fibroblasts were grown in normal oxygen (20% O2) and hyperoxia (40% O2 or 60% O2). After cell viability was examined, the levels of reactive oxygen intermediates (ROI) by flow cytometry and heme oxygenase-1 (HO-1) mRNA by northern analysis were measured as markers of oxidative stress in both cell types. Proliferative ability and gene expression pattern of growth factors were studied to demonstrate the phenotypic changes induced by mild oxidative stress upon these cells. RESULTS: While decreased by 60% O2, 40% O2 did not affect viability in both cell types, ROI production and HO-1 mRNA expression were elevated in hyperoxia compared to controls, but were inhibited with the antioxidant dehydro-ascorbic acid (DHA). The proliferation of cells by hyperoxia was inhibited in both cell types. The expression of growth factors induced by hyperoxia was cell type dependent. Fibroblast growth factor-2 mRNA was unchanged in RPE cells, but was increased in fibroblasts. Transforming growth factor-beta2 was decreased in RPE cells, but unchanged in fibroblasts. Vascular endothelial growth factor was downregulated in RPE cells, while upregulated in fibroblasts. Connective tissue growth factor was decreased in RPE cells, but was unchanged in fibroblasts. CONCLUSIONS: The results demonstrate that hyperoxia induces mild oxidative stress which alters the phenotype of cells in a cell type specific manner.
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Hiperoxia/metabolismo , Estrés Oxidativo , Epitelio Pigmentado Ocular/metabolismo , Northern Blotting , División Celular , Línea Celular , Supervivencia Celular , Enfermedad Crónica , Fibroblastos/citología , Fibroblastos/metabolismo , Citometría de Flujo , Expresión Génica , Hemo Oxigenasa (Desciclizante)/genética , Hemo-Oxigenasa 1 , Humanos , Lactante , Pulmón/citología , Pulmón/metabolismo , Proteínas de la Membrana , Fenotipo , Epitelio Pigmentado Ocular/citología , ARN Mensajero/metabolismo , Especies Reactivas de OxígenoRESUMEN
PURPOSE: The purpose of this study is to demonstrate the effect of culture density on the steady state mRNA levels of fibroblast growth factor-2 (FGF-2) when retinal pigment epithelial (RPE) cells are subjected to oxidative stress in vitro. METHODS: Subconfluent and confluent cultures of the established RPE cell line ARPE-19, were treated with increasing concentrations of tert-butyl hydroperoxide (tBH) or hydrogen peroxide (H(2)O(2)). Cell viability was measured using the WST-1 assay, and intracellular reactive oxygen intermediate (ROI) production was quantified by dichlorofluoroscein (DCF) fluorescence. Steady state changes in heme oxygenase-1 (HO-1) and FGF-2 mRNAs were measured by Northern blot analysis. RESULTS: Confluent cultures of ARPE-19 cells were less susceptible than subconfluent cultures to the toxic effects of the chemical oxidants. The intracellular reactive oxygen intermediate production was higher in subconfluent than confluent cultures with increasing tBH concentration. At nontoxic concentrations of tBH and H(2)O(2), a dose dependent increase in FGF-2 expression was seen as a function of culture density. FGF-2 mRNA expression was induced after tBH treatment in subconfluent, but not confluent cells. On the other hand, FGF-2 mRNA induction was observed after H(2)O( 2) treatment in confluent, but not subconfluent cultures. In contrast, no density dependent induction of HO-1 mRNA was seen after treatment with either tBH or H(2)O(2). CONCLUSIONS: These results suggest that care should be taken to control for cell density in similar types of in vitro experiments.
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Recuento de Células , Factor 2 de Crecimiento de Fibroblastos/biosíntesis , Estrés Oxidativo/fisiología , Epitelio Pigmentado Ocular/citología , Northern Blotting , Supervivencia Celular , Células Cultivadas , Relación Dosis-Respuesta a Droga , Factor 2 de Crecimiento de Fibroblastos/genética , Hemo Oxigenasa (Desciclizante)/genética , Hemo Oxigenasa (Desciclizante)/metabolismo , Hemo-Oxigenasa 1 , Humanos , Peróxido de Hidrógeno/farmacología , Proteínas de la Membrana , Epitelio Pigmentado Ocular/efectos de los fármacos , Epitelio Pigmentado Ocular/metabolismo , ARN Mensajero/metabolismo , Especies Reactivas de Oxígeno , terc-Butilhidroperóxido/farmacologíaRESUMEN
PURPOSE: Our goals were to produce a functional recombinant RPE retinal G protein-coupled receptor (RGR) opsin for biochemical studies and to test the efficiency of a lentiviral vector for transgene expression of human RGR. METHODS: A human RGR cDNA was cloned into a replication-defective lentiviral vector, and recombinant hRGR-Lentivirus was prepared for transduction of the ARPE-19, a human retinal pigment epithelium (RPE) cell line, and COS-7 cells. Recombinant RGR expression was detected by Western blot analysis, and functionality of the protein was tested by a [3H]all-trans-retinal binding assay. RESULTS: RGR protein was detected in each cell type after transduction with recombinant virus and was not observed in untreated cells. RGR expression in ARPE-19 cells increased steadily for up to 10 days after transduction and was stable for at least 6 months. The transduced ARPE-19 cells produced approximately 100-fold higher amounts of RGR protein than the transduced COS-7 cells. When cell membranes from the ARPE-19 cells were incubated with [3H]all-trans-retinal, the chromophore bound specifically to the expressed protein. Uptake of [3H]all-trans-retinol into the ARPE-19 cells was followed by specific binding of radiolabeled retinoid to RGR. CONCLUSIONS: Using a Lentivirus-derived gene delivery system, we were able to express high amounts of human RGR protein in the ARPE-19 human RPE cell line. The transduced ARPE-19 cells remain able to process all-trans-retinol, and the expressed protein is capable of binding to the all-trans-retinal chromophore. The Lentivirus-based expression of functional RGR can be used to study RGR in cultured cells and to test in vivo transduction of quiescent RPE cells.
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Proteínas del Ojo/biosíntesis , VIH/genética , Epitelio Pigmentado Ocular/metabolismo , Receptores de Superficie Celular/biosíntesis , Receptores Acoplados a Proteínas G , Opsinas de Bastones/biosíntesis , Transducción Genética , Animales , Autorradiografía , Western Blotting , Células COS/metabolismo , Bovinos , Células Cultivadas , Virus Defectuosos , Electroforesis en Gel de Poliacrilamida , Proteínas del Ojo/genética , Vectores Genéticos , Técnicas para Inmunoenzimas , Ratones , Ratones Endogámicos C57BL , Microsomas/metabolismo , Receptores de Superficie Celular/genética , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Retinaldehído/metabolismo , Opsinas de Bastones/genética , Vitamina A/metabolismoRESUMEN
PURPOSE: The expression and alternative splicing of the four FGF receptor (FGFR) mRNAs are regulated in a developmental- and tissue-specific fashion. Capability of differentiation in vitro of the retinal pigment epithelial cell line ARPE-19 has been previously demonstrated. In this study, the hypothesis that FGF receptor gene expression and the alternative splicing of the FGFR1 mRNA is regulated as a function of ARPE-19 differentiation in vitro was tested. METHODS: ARPE-19 cells were plated at sparse or confluent densities and maintained in culture up to 14 months. The expression of FGF receptors and the ratio of the FGFR1beta to FGFR1alpha splice variants of the FGFR1 transcript were quantified by a published PCR technique. Two in vivo samples of human RPE served as controls. RESULTS: Sparse cultures of ARPE-19 cells predominantly express FGFR1. When these cultures are allowed to differentiate, FGFR2 is also expressed. Samples of mRNA from RPE cells in vivo exhibit FGFR1 and FGFR2 expression as well as FGFR3 expression, a form that is minimally apparent in vitro. The ratio of the FGFR1beta to FGFR1alpha splice variant decreases as a function of cell differentiation in vitro and approaches the ratio observed in human RPE cells in vivo. Stimulation of cultures in vitro with FGF2 as a prototypical differentiation agent does not regulate the ratio of the FGFR1beta to FGFR1alpha splice variant. CONCLUSIONS: Differentiation of the ARPE-19 cell line in vitro recapitulates many but not all the in vivo patterns of FGFR expression and splicing. This in vitro system may be useful for selected studies on how cellular differentiation regulates FGF receptor gene expression and splicing.
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Empalme Alternativo/genética , Diferenciación Celular/fisiología , Epitelio Pigmentado Ocular/citología , ARN Mensajero/biosíntesis , Receptores de Factores de Crecimiento de Fibroblastos/genética , Northern Blotting , Línea Celular , Expresión Génica , Sustancias de Crecimiento/farmacología , Semivida , Humanos , Epitelio Pigmentado Ocular/efectos de los fármacos , Epitelio Pigmentado Ocular/metabolismo , Receptores de Factores de Crecimiento de Fibroblastos/biosíntesis , Proteínas Recombinantes/farmacología , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Senescence of human cells has largely been studied as an in vitro phenomenon resulting from replicative exhaustion. The literature contains many studies of retinal pigment epithelium (RPE) cells which document replicative senescence. Several studies by Burke and others illustrate the relationship between donor age and replicative lifespan, the relationship between geographical location of RPE in the posterior pole and replicative lifespan, and the phenomena of altered cellular morphology and decreased culture saturation density for senescent RPE cells. Other studies have focused on the alterations of the expression of specific genes or the alteration of enzymatic activities during the senescence of RPE cells in vitro. Recently, a technique utilizing a histochemical staining procedure for beta galactosidase has been developed which identifies senescent cells. Normal beta galactosidase histochemistry which identifies the lysosomal form of the enzyme is performed at pH 4.0, while senescence-associated beta galactosidase activity is observed at pH 6.0 and is observed in the cytoplasm. We have studied the replicative senescence of human RPE cells in vitro using this procedure and have also measured the length of chromosomal telomeres to identify the aging of cultures in vitro. Our results show that RPE cultures accumulate beta galactosidase positive cells as a function of the number of population doublings and that these data correlate with the shortening of chromosomal telomeres to a functional limit observed for many human cell types at senescence. We have also recently extended this work to the development of a senescence-associated beta galactosidase procedure for observing senescent RPE cells in vivo. Basically, the same histochemical procedure is used with a post-staining bleaching step to clearly visualize staining in the RPE. Our first studies were performed on globes from Rhesus monkeys at a variety of ages from 1 year to 29 years of age. The results show the accumulation of beta galactosidase positive cells in the older monkey eyes. We have also examined several human eyes in an attempt to observe whether any relationship exists between beta galactosidase staining and age, pathology (diabetes, basal laminar deposits), and geographical location (macula vrs. periphery). These studies represent a first effort to determine if senescent RPE are present in vivo. It will be important to extend these studies so that these data might be expressed on a quantitative bases.
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Senescencia Celular , Proteínas del Ojo , Factores de Crecimiento Nervioso , Epitelio Pigmentado Ocular/citología , Envejecimiento , Animales , Regulación hacia Abajo , Histocitoquímica , Lipofuscina/biosíntesis , Lisosomas/fisiología , Macaca mulatta , Degeneración Macular/etiología , Degeneración Macular/patología , Epitelio Pigmentado Ocular/metabolismo , Epitelio Pigmentado Ocular/ultraestructura , Proteínas/metabolismo , ARN Mensajero/metabolismo , Serpinas/metabolismoRESUMEN
PURPOSE: To develop an antibody that recognizes a variety of advanced glycation endproduct (AGE) epitopes. METHODS: Glycolaldehyde was used to modify bovine serum albumin and HPLC analysis was used to measure pentosidine formation as an indicator of AGE formation. A polyclonal anti-AGE antibody was synthesized by injecting glycolaldehyde-incubated keyhole limpet hemocyanin into rabbits, affinity purified using AGE modified bovine serum albumin coupled to an affinity resin column, and characterized by immunoblot analysis. RESULTS: HPLC analysis of glycolaldehyde treated bovine serum albumin detected high levels of pentosidine formation, suggesting that glycolaldehyde is a potent precursor for pentosidine. By immunoblot analysis, our antibody recognized carboxymethyllysine and pentosidine, two well-characterized AGEs, as well as other AGE epitopes. Immunohistochemical evaluation showed evidence of AGEs in Bruch's membrane (including basal laminar deposits and drusen), choroidal extracellular matrix, and vessel walls in an 82 year old nondiabetic globe. A similar staining pattern was observed in an age-matched diabetic control. In contrast, no staining was seen with the antibody in a 20 month old nondiabetic globe. CONCLUSIONS: A unique anti-AGE antibody was synthesized that recognizes a variety of AGE epitopes including carboxymethyllysine and pentosidine. Its best use might be in broad surveys of the age-dependent accumulation of a large number of AGE epitopes that might not be revealed by antibodies to pentosidine or CML.
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Envejecimiento/metabolismo , Anticuerpos/inmunología , Lámina Basal de la Coroides/metabolismo , Productos Finales de Glicación Avanzada/inmunología , Productos Finales de Glicación Avanzada/metabolismo , Acetaldehído/análogos & derivados , Acetaldehído/inmunología , Acetaldehído/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Animales , Anticuerpos/metabolismo , Especificidad de Anticuerpos , Arginina/análogos & derivados , Arginina/inmunología , Arginina/metabolismo , Bovinos , Plexo Coroideo/inmunología , Plexo Coroideo/metabolismo , Proteínas de la Matriz Extracelular/inmunología , Proteínas de la Matriz Extracelular/metabolismo , Humanos , Inmunohistoquímica , Lactante , Lisina/análogos & derivados , Lisina/inmunología , Lisina/metabolismo , Persona de Mediana EdadRESUMEN
BACKGROUND/AIMS: Preretinal neovascular formations called sea fans develop at the border of non-perfused peripheral retina in sickle cell retinopathy. Angiogenic factors which could contribute to their development, however, have not been examined previously. The objective of this study was to determine immunohistochemically if vascular endothelial growth factor (VEGF) or basic fibroblast growth factor (bFGF) were associated with sea fan formations. METHODS: Immunohistochemistry on cryosections was used to localise bFGF, VEGF, heparan sulphate proteoglycan, human serum albumin, collagens IV and II, and von Willebrand factor in tissue from five sickle cell and one control subject. RESULTS: The greatest immunoreactivity for VEGF and bFGF was in the feeder and preretinal vessels of sea fans (p<0.01). The most prominent reaction product was localised to vascular endothelial cells. In retinal vessels, VEGF and bFGF immunoreactivities were greater in sickle cell subjects (both proliferative and non-proliferative) than in the control subject (p<0.01 and p<0.02 respectively). In the sickle cell retina, no angiogenic factor immunoreactivity was detected in non-perfused periphery and there was no significant difference in bFGF or VEGF immunoreactivity between perfused retina and the border of perfused and non-perfused areas. CONCLUSION: Our results demonstrate for the first time that VEGF and bFGF are associated with sea fan formations in sickle cell retinopathy. Both factors may function in an autocrine manner because immunoreactivity for these factors was greater within the neovascularisation than in adjacent retina.
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Anemia de Células Falciformes/metabolismo , Factores de Crecimiento Endotelial/metabolismo , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Linfocinas/metabolismo , Neovascularización Retiniana/metabolismo , Adolescente , Adulto , Anciano , Anemia de Células Falciformes/patología , Biomarcadores , Femenino , Humanos , Inmunohistoquímica , Masculino , Neovascularización Retiniana/patología , Factor A de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial VascularRESUMEN
PURPOSE: To develop a senescence-associated beta-galactosidase histochemistry and bleaching protocol for the primate posterior pole. METHODS: Rhesus monkey eyes of different ages were enucleated after death, fixed in 4% paraformaldehyde for up to 16 hours, and cryoprotected using a graded sucrose infiltration technique. Ten-micrometer tissue sections were treated with beta-galactosidase, pH 4 (lysosomal) or pH 6 (senescence-associated) activity, for various times. Bleaching of retinal pigment epithelial (RPE) cell and choroidal melanocyte pigment was performed after beta-galactosidase histochemistry using 0.1% to 1% potassium permanganate incubation for 1 minute to 2 hours followed by 0.5% oxalic acid immersion. RESULTS: A 6-hour incubation with beta-galactosidase, pH 4 or 6, demonstrated optimal staining of the RPE. Uniform staining of the RPE for pH 4 beta-galactosidase was seen in both young and old eyes. In contrast, senescence-associated beta-galactosidase (pH 6) staining was seen in the RPE of 16 and 29-year-old, but not 1- and 2-year-old eyes. Senescence-associated beta-galactosidase staining was evident in RPE cells adjacent to cuticular drusen. Optimal bleaching without loss of beta-galactosidase staining was obtained using a 25-minute incubation with 0.05% permanganate. CONCLUSIONS: The senescence-associated beta-galactosidase histochemistry assay, adapted for use in the primate posterior pole, showed staining of RPE cells in older eyes. Visualization of beta-galactosidase activity in the RPE was enhanced by permanganate bleaching of melanin pigment. This technique could be valuable for identifying senescent RPE cells in human eyes.
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Envejecimiento/fisiología , Coroides/enzimología , Epitelio Pigmentado Ocular/enzimología , beta-Galactosidasa/metabolismo , Animales , Senescencia Celular/fisiología , Histocitoquímica , Concentración de Iones de Hidrógeno , Macaca mulatta , Microtomía , Adhesión del TejidoRESUMEN
PURPOSE: To determine whether there is an age-related increase of pentosidine in human Bruch's membranes and to localize pentosidine and carboxymethyllysine (CML), two well-characterized, advanced glycation end products (AGEs) in aged human Bruch's membranes and choroid in vivo. METHODS: Human Bruch's membrane samples were isolated from the retinal pigment epithelium (RPE) and choroid and subjected to reversed-phase high-performance liquid chromatography to determine pentosidine content. A polyclonal anti-pentosidine antibody and a monoclonal antibody specific for carboxymethyllysine were used to localize AGEs in 20-month-old nondiabetic, 82-year-old nondiabetic, and 82-year-old diabetic globes. RESULTS: Human Bruch's membranes (n = 20) showed a linear age-dependent increase in pentosidine that reached approximately 0.17 millimoles pentosidine per mole hydroxyproline in late life (r = 0.896; P < 0.001). Immunohistochemical evaluation showed evidence of pentosidine in Bruch's membrane, choroidal extracellular matrix, and vessel walls in the 82-year-old nondiabetic and diabetic globes. A similar staining pattern was found with the anti-CML antibody. Basal laminar deposits and drusen stained with both antibodies in the elderly nondiabetic eye. In contrast, neither antibody stained the 20-month-old tissue. CONCLUSIONS: We provide biochemical and immunohistochemical evidence for the formation of pentosidine and CML structures in human Bruch's membrane and choroid with age. These changes could promote aging of the RPE-Bruch's membrane-choroid complex.
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Envejecimiento/metabolismo , Arginina/análogos & derivados , Lámina Basal de la Coroides/metabolismo , Productos Finales de Glicación Avanzada/metabolismo , Lisina/análogos & derivados , Adulto , Anciano , Anciano de 80 o más Años , Arginina/metabolismo , Coroides/metabolismo , Cromatografía Líquida de Alta Presión , Retinopatía Diabética/metabolismo , Humanos , Técnicas para Inmunoenzimas , Lactante , Lisina/metabolismo , Persona de Mediana Edad , Epitelio Pigmentado Ocular/metabolismo , Drusas Retinianas/metabolismoRESUMEN
PURPOSE: To investigate the relation of senescence-related beta-galactosidase activity and telomere shortening to replicative senescence in cultured human retinal pigment epithelial (RPE) cells. METHODS: A human RPE cell line was serially passaged until 80% of cells were nondividing in a 72-hour 5-bromo-2'-deoxyuridine (BrdU) labeling study. Early- and late-passage cells were double-stained for BrdU and senescence-related beta-galactosidase activity (pH 6). The average chromosomal telomere length at several population doublings was estimated by Southern blot analysis after double digestion of DNA with RsaI and HinfI and using a telomere-specific probe. RESULTS: BrdU-beta-galactosidase double-staining revealed an inverse correlation between the number of BrdU-labeled nuclei and beta-galactosidase-labeled cells as a function of population doubling level (PDL). At PDL 58, only 20% of all cells labeled for BrdU, whereas 57% stained for beta-galactosidase. The mean terminal restriction fragment length (TRF) was reduced from 10 kb in early (PDL 12) cultures to 4 kb in late (PDL 57) cultures. CONCLUSIONS: Senescence-related beta-galactosidase activity and mean TRF length may prove useful in studying the senescence of RPE cells in vitro. These techniques may be valuable in determining senescence of the retinal pigment epithelium in vivo, where senescent RPE cells could be involved in the development of age-related maculopathy and age-related macular degeneration.
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Senescencia Celular , Epitelio Pigmentado Ocular/enzimología , Telómero/metabolismo , beta-Galactosidasa/metabolismo , Southern Blotting , Bromodesoxiuridina/metabolismo , División Celular , Línea Celular , Células Cultivadas , Senescencia Celular/fisiología , ADN/análisis , Replicación del ADN , Histocitoquímica , Humanos , Lactante , Epitelio Pigmentado Ocular/citologíaRESUMEN
PURPOSE: To explore the changes in expression of a set of genes in a single retinal pigment epithelial (RPE) cell line and two fibroblast cell lines as controls under culture conditions previously used for the analysis of senescent gene expression. METHODS: A single human RPE cell line, which had previously been characterized using known markers of senescence, and two fibroblast cell lines were grown to replicative exhaustion. The mRNA phenotype of genes known to be altered by senescence were studied by quantitative Northern analysis. RESULTS: The mRNA phenotype of cells changes at replicative senescence yielding a synthetic phenotype which is similar to cells found in repairing wounds. Of the genes studied, urokinase-type plasminogen activator and plasminogen activator inhibitor-1 were regulated in RPE cells similar to fibroblasts at senescence. The largest changes noted for any single gene were the upregulation of insulin growth factor binding protein 2, and the downregulation of collagen I alpha 2, basic fibroblast growth factor, and fibroblast growth factor-5. CONCLUSIONS: This study demonstrates an altered mRNA phenotype of a human RPE cell line grown to replicative exhaustion. This analysis of a single cell line emphasizes the variability of results based on a single cell line or tissue specimen and indicates the need for additional study.