RESUMEN
Neuritin 1 (Nrn1) is an extracellular glycophosphatidylinositol-linked protein that stimulates axonal plasticity, dendritic arborization and synapse maturation in the central nervous system (CNS). The purpose of this study was to evaluate the neuroprotective and axogenic properties of Nrn1 on axotomized retinal ganglion cells (RGCs) in vitro and on the in vivo optic nerve crush (ONC) mouse model. Axotomized cultured RGCs treated with recombinant hNRN1 significantly increased survival of RGCs by 21% (n=6-7, P<0.01) and neurite outgrowth in RGCs by 141% compared to controls (n=15, P<0.05). RGC transduction with AAV2-CAG-hNRN1 prior to ONC promoted RGC survival (450%, n=3-7, P<0.05) and significantly preserved RGC function by 70% until 28 days post crush (dpc) (n=6, P<0.05) compared with the control AAV2-CAG-green fluorescent protein transduction group. Significantly elevated levels of RGC marker, RNA binding protein with multiple splicing (Rbpms; 73%, n=5-8, P<0.001) and growth cone marker, growth-associated protein 43 (Gap43; 36%, n=3, P<0.01) were observed 28 dpc in the retinas of the treatment group compared with the control group. Significant increase in Gap43 (100%, n=5-6, P<0.05) expression was observed within the optic nerves of the AAV2-hNRN1 group compared to controls. In conclusion, Nrn1 exhibited neuroprotective, regenerative effects and preserved RGC function on axotomized RGCs in vitro and after axonal injury in vivo. Nrn1 is a potential therapeutic target for CNS neurodegenerative diseases.
Asunto(s)
Axones/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Traumatismos del Nervio Óptico/metabolismo , Células Ganglionares de la Retina/citología , Animales , Western Blotting , Células Cultivadas , Proteínas Ligadas a GPI/genética , Proteínas Ligadas a GPI/metabolismo , Inmunohistoquímica , Hibridación in Situ , Ratones , Ratones Endogámicos BALB C , Compresión Nerviosa , Proteínas del Tejido Nervioso/genética , Neuritas/metabolismoRESUMEN
PURPOSE: To determine whether cells and tissue from the human lamina cribrosa (LC) express neurotrophin and tyrosine kinase (trk) receptor mRNA and protein and whether these cells secrete neurotrophins. METHODS: Synthesis of cDNA and the reverse transcription-polymerase chain reaction (RT-PCR) were conducted using total RNA obtained from well-characterized cell lines from the human LC and human optic nerve head (ONH) tissue. Immunofluorescent localization and Western blot analysis were used to evaluate neurotrophin and trk protein expression in cells and tissue from the human LC. Immunoassay systems (ELISAs) were used to detect the secretion of neurotrophins. RESULTS: Two morphologically distinct cell types (LC cells and ONH astrocytes) were isolated and characterized from the human LC. Messenger RNA for each of the neurotrophins, three full-length trk receptors and two truncated trk receptors were detected in both cell types and in human ONH tissue. Protein for the neurotrophins and trk receptors were detected in LC cells, ONH astrocytes, and ONH tissue. Neither cell type expressed mRNA or protein for the low-affinity neurotrophin receptor p75. The secretion of neurotrophins was observed in both cell types. CONCLUSIONS: Cells from the human LC express mRNA and protein of neurotrophins and trk receptors. In addition, cells from the LC secrete neurotrophins, which suggests that there is paracrine and/or autocrine signaling within the LC. Neurotrophin signaling within this region of the ONH may play important roles in the maintenance of the normal LC and in such diseases as glaucoma.
Asunto(s)
Factores de Crecimiento Nervioso/genética , Disco Óptico/metabolismo , Receptores de Factor de Crecimiento Nervioso/genética , Southern Blotting , Western Blotting , Factor Neurotrófico Derivado del Encéfalo/biosíntesis , Factor Neurotrófico Derivado del Encéfalo/genética , Células Cultivadas , Cartilla de ADN/química , Ensayo de Inmunoadsorción Enzimática , Técnica del Anticuerpo Fluorescente Indirecta , Proteína Ácida Fibrilar de la Glía/biosíntesis , Proteína Ácida Fibrilar de la Glía/genética , Humanos , Factores de Crecimiento Nervioso/biosíntesis , Disco Óptico/citología , ARN/aislamiento & purificación , ARN Mensajero/biosíntesis , Receptores de Factor de Crecimiento Nervioso/biosíntesis , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
The purpose of the present study was to establish a rat retinal ganglion cell line by transformation of rat retinal cells. For this investigation, retinal cells were isolated from postnatal day 1 (PN1) rats and transformed with the psi2 E1A virus. In order to isolate retinal ganglion cells (RGC), single cell clones were chosen at random from the transformed cells. Expression of Thy-1 (a marker for RGC), glial fibrillary acidic protein (GFAP, a positive marker for Muller cells), HPC-1/syntaxin (a marker for amacrine cells), 8A1 (a marker for horizontal and ganglion cells) and neurotrophins was studied using reverse transcriptase-polymerase chain reaction (RT-PCR), immunoblotting and immunocytochemistry. One of the retinal cell clones, designated RGC-5, was positive for Thy-1, Brn-3C, Neuritin, NMDA receptor, GABA-B receptor, and synaptophysin expression and negative for GFAP, HPC-1, and 8A1, suggesting that it represented a putative RGC clone. The results of RT-PCR analysis were confirmed by immunocytochemistry for Thy-1 and GFAP. Upon further characterization by immunoblotting, the RGC-5 clone was positive for Thy-1, negative for GFAP, 8A1 and syntaxin. RGC 5 cells were also positive for the expression of neurotrophins and their cognate receptors. To establish the physiological relevance of RGC-5, the effects of serum/trophic factor deprivation and glutamate toxicity were analyzed to determine if these cells would undergo apoptosis. The protective effects of neurotrophins on RGC-5 after serum deprivation was also investigated. Apoptosis was studied by terminal deoxynucleotidyl transferase-mediated fluoresceinated dUTP nick end labeling (TUNEL). Serum deprivation resulted in apoptosis and supplementation with both BDNF and NT-4 in the growth media, protected the RGC-5 cells from undergoing apoptosis. On differentiation with succinyl concanavalin A (sConA), RGC-5 cells became sensitive to glutamate toxicity, which could be reversed by inclusion of ciplizone (MK801). In conclusion, a transformed rat retinal cell line, RGC-5, has certain characteristics of retinal ganglion cells based on Thy-1 and Brn-3C expression and its sensitivity to glutamate excitotoxicity and neurotrophin withdrawal. These cells may be valuable in understanding of retinal ganglion cell biology and physiology including in vitro manipulations in experimental models of glaucoma.
Asunto(s)
Línea Celular Transformada/citología , Células Ganglionares de la Retina/citología , Animales , Animales Recién Nacidos , Anticuerpos Monoclonales , Antígenos de Superficie/análisis , Antígenos de Superficie/inmunología , Apoptosis/efectos de los fármacos , Apoptosis/fisiología , Biomarcadores , Factor Neurotrófico Derivado del Encéfalo/genética , Factor Neurotrófico Derivado del Encéfalo/farmacología , Técnicas de Cultivo de Célula/métodos , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/fisiología , Línea Celular Transformada/química , Medio de Cultivo Libre de Suero/farmacología , Proteínas de Unión al ADN/análisis , Proteínas de Unión al ADN/inmunología , Proteínas Ligadas a GPI , Glaucoma/patología , Proteína Ácida Fibrilar de la Glía/análisis , Proteína Ácida Fibrilar de la Glía/genética , Proteína Ácida Fibrilar de la Glía/inmunología , Ácido Glutámico/toxicidad , Etiquetado Corte-Fin in Situ , Factores de Crecimiento Nervioso/genética , Factores de Crecimiento Nervioso/farmacología , Proteínas del Tejido Nervioso/análisis , Proteínas del Tejido Nervioso/inmunología , Neuropéptidos/genética , Fármacos Neuroprotectores/metabolismo , Fármacos Neuroprotectores/farmacología , Ratas , Receptores de GABA-B/genética , Células Ganglionares de la Retina/química , Sinaptofisina/genética , Sintaxina 1 , Antígenos Thy-1/análisis , Antígenos Thy-1/genética , Antígenos Thy-1/inmunología , Factor de Transcripción Brn-3 , Factor de Transcripción Brn-3C , Factores de Transcripción/análisis , Factores de Transcripción/inmunologíaRESUMEN
PURPOSE: The purpose of this study was to compare the mRNA expression of neurotrophins (NTs) and NT receptors (Trk) in cultured human trabecular meshwork (HTM) cells and ex vivo HTM tissues, to immunolocalize both NT and Trk receptors in cultured HTM cells, and to demonstrate secretion of NTs by HTM cells. METHODS: Reverse transcription-polymerase chain reaction (RT-PCR) was used to detect the expression of NT and Trk receptor mRNAs in early-passaged, cultured HTM cells from donors of several ages. RT-PCR was used on ex vivo HTM tissues from donors to compare and contrast mRNA expression with cell culture results. In addition, immunohistochemistry was used to localize the translated NT and low- (p75) and high- (Trk) affinity NT receptor proteins within cultured HTM cells and trabecular meshwork tissues. Last, enzyme-linked immunoassay (ELISA) was used to demonstrate secretion of NTs by HTM cells. RESULTS: Amplification products of the expected size for NTs were detected in both cultured HTM cells and ex vivo HTM tissues. Specifically identified were amplification products for the following NTs: NGF, BDNF, NT-3, and NT-4. Amplification products for the full-length Trk A and Trk C high-affinity receptor were observed, as well as truncated isoforms for Trk B and Trk C. No amplification products were produced for the full-length Trk B receptor nor for the low-affinity p75 receptor. Immunohistochemistry indicated that proteins for the various NTs and full-length and truncated Trk receptors were translated by cultured HTM cells and tissues. Immunoassays (ELISA) detected BDNF, NT-4, NGF, and NT-3 in the culture media from HTM cells. CONCLUSIONS: The results demonstrate, for the first time, mRNA expression for NT and Trk receptors by both cultured HTM cells and ex vivo HTM tissues. NTs were immunolocalized in HTM tissues and cultured HTM cells are capable of secreting NTs. Specific NTs acting through high-affinity Trk receptors within the HTM may be involved in maintaining the normal function of this complex tissue.
Asunto(s)
Factores de Crecimiento Nervioso/genética , ARN Mensajero/biosíntesis , Receptores de Factor de Crecimiento Nervioso/genética , Malla Trabecular/metabolismo , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Células Cultivadas , Niño , Preescolar , Cartilla de ADN/química , Ensayo de Inmunoadsorción Enzimática , Técnica del Anticuerpo Fluorescente Indirecta , Humanos , Lactante , Recién Nacido , Persona de Mediana Edad , Factor de Crecimiento Nervioso/metabolismo , Factores de Crecimiento Nervioso/biosíntesis , Reacción en Cadena de la Polimerasa , Receptores de Factor de Crecimiento Nervioso/biosíntesis , Malla Trabecular/citologíaRESUMEN
PURPOSE: This study was undertaken to determine the presence of retina-derived fetuin (RDF) protein and its message in retinal tissues and retinal pigment epithelial (RPE) cells. The techniques utilized in this study included light micros-copy, immunochemistry, Western blot, reverse transcriptase-polymerase chain reaction (RT-PCR) and Southern blot. METHODS: Retinal tissues and sections from embryonic, early postnatal and adult normal rats and retinal pigment epithe-lial (RPE) cells from postnatal rats were immunostained for fetuin with a polyclonal fetuin antibody and a peroxidase conjugated-secondary antibody using immunocytochemistry and Western blot analyses. The cDNA generated from RNA isolated from early postnatal rat retinas and RPE was probed with primers for rat fetuin, amplified by PCR and the PCR products were analyzed by Southern blot. RESULTS: Fetuin (RDF) was immunolocalized to cells of the neuroepithelium in retinas of early postnatal rats and most prominently in the nuclei and perinuclear region of cultured neonatal rat RPE cells. In adult retinas, ganglion cells, inner segments of photoreceptor cells, some components of the outer plexiform layer, ganglion cells and optic nerve processes were immunoreactive for the fetuin protein. As shown by Western blot, fetuin (RDF) was higher in embryonic and early postnatal retinas than in late postnatal retinas, indicating that this protein may be developmentally regulated. Using RT-PCR, the message for rat fetuin was demonstrated in the retina and RPE of normal postnatal rats. Southern blot confirmed that the PCR product from the retina and RPE was generated from rat fetuin mRNA as well as from rat liver, the primary source of fetuin. CONCLUSIONS: Fetuin, termed retina-derived fetuin (RDF), is reported for the first time in retinal tissues. Fetuin is a cysteine protease inhibitor that may play a role in support of neuronal cell survival during early retinal development and the maintenance of neuronal activity. RDF may interact with other growth factors and cytokines in providing trophic support for neurons and possibly other cells of the developing retina.
Asunto(s)
Retina/metabolismo , alfa-Fetoproteínas/análisis , Animales , Southern Blotting , Western Blotting , Células Cultivadas , Cuerpo Ciliar/química , Cuerpo Ciliar/metabolismo , Córnea/química , Córnea/metabolismo , Inmunohistoquímica , Epitelio Pigmentado Ocular/química , Epitelio Pigmentado Ocular/citología , Epitelio Pigmentado Ocular/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Ratas Long-Evans , Retina/química , Retina/citología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Cuerpo Vítreo/química , Cuerpo Vítreo/metabolismo , alfa-Fetoproteínas/genéticaRESUMEN
Glucocorticoid effects on the human trabecular meshwork can be used as a model system in which to study glaucomatous damage to the trabecular meshwork. One of the most important risk factors for glaucoma is an elevated intraocular pressure. The administration of glucocorticoids also can cause elevated intraocular pressure in some individuals. In addition, there is suggestive evidence linking glucocorticoids with the development of glaucoma. Glucocorticoids cause multiple effects on the human trabecular meshwork including changes in extracellular matrix metabolism, organisation of the cytoskeleton, and changes in gene expression and cell function. New discoveries on the molecular mechanisms of glucocorticoid receptor action provide new opportunities to study the possible role of this receptor in the development of glaucoma. For example, alternate spliced forms of the glucocorticoid receptor, glucocorticoid receptor response element half-sites, numerous modulatory factors, and direct effects of nuclear transcription factors have been recently described. Other recent information has shown that the new glaucoma gene (GLC1A/myocilin) is induced in the human trabecular meshwork by glucocorticoids. Although the exact function of myocilin is currently unknown, it offers the opportunity to dissect the molecular pathways regulating aqueous humor outflow. Future challenges include determining (1) which glucocorticoid effects in the human trabecular meshwork are responsible for elevated intraocular pressure; and (2) the significance of these findings to the development of glaucoma.
Asunto(s)
Glaucoma de Ángulo Abierto/inducido químicamente , Glucocorticoides/efectos adversos , Malla Trabecular/efectos de los fármacos , Animales , Proteínas del Citoesqueleto/genética , Proteínas del Citoesqueleto/metabolismo , Proteínas del Ojo/genética , Proteínas del Ojo/metabolismo , Expresión Génica , Glaucoma de Ángulo Abierto/genética , Glucocorticoides/farmacología , Glicoproteínas/genética , Glicoproteínas/metabolismo , Humanos , Presión Intraocular/efectos de los fármacos , Hipertensión Ocular/inducido químicamente , Hipertensión Ocular/genética , Malla Trabecular/metabolismo , Factores de Transcripción/metabolismoRESUMEN
A gradual loss of cells occurs within the human trabecular meshwork during normal aging and appears to be increased in patients with primary open-angle glaucoma. The exact mechanism by which cells are lost in either condition is not known, however phagocytosis, cell migration and cell death have been suggested. Apoptosis is one method by which cell death can occur. We have examined the modulators for apoptosis within the human trabecular meshwork using both cell lines and ex-vivo dissected trabecular meshwork tissues obtained from normal donors. Using RT-PCR it was shown that mRNA for several modulators of apoptosis (Fas, Bcl-2, Bcl-xl, Bax, and ICE) are expressed by both cell lines and ex-vivo tissues. Apoptosis was stimulated to occur by treating cell lines with a monoclonal antibody (IgM) to Fas. Apoptosis was verified via morphological changes to the cells, transferase-mediated dUTP nick-end labeling TUNEL Immunofluorescence, and DNA laddering. Control cells exposed to IgM did not undergo apoptosis. These results represent the first report of apoptosis modulators within the human trabecular meshwork and demonstrate that human trabecular meshwork cells can be stimulated to undergo apoptosis via the Fas/FasL pathway.
Asunto(s)
Envejecimiento/fisiología , Apoptosis , Glicoproteínas de Membrana/metabolismo , Malla Trabecular/fisiología , Proteínas Virales , Receptor fas/metabolismo , Anciano , Anciano de 80 o más Años , Anticuerpos Monoclonales/farmacología , Línea Celular , Preescolar , Inhibidores de Cisteína Proteinasa/genética , Fragmentación del ADN , Proteína Ligando Fas , Humanos , Etiquetado Corte-Fin in Situ , Lactante , Recién Nacido , Persona de Mediana Edad , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-bcl-2/genética , ARN Mensajero/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Serpinas/genética , Malla Trabecular/citología , Proteína X Asociada a bcl-2 , Proteína bcl-X , Receptor fas/genética , Receptor fas/inmunologíaRESUMEN
PURPOSE: Growth factors act through high-affinity cell surface receptors expressed by target cells and are critical modulators of cell function. Because aqueous humor is known to contain growth factors, these molecules may play a key role in maintaining the normal function of the human trabecular meshwork (HTM). Alternate mRNA splicing is an important mechanism used by cells to generate diverse isoforms of growth factor receptors. Although previous investigators have suggested that HTM cells may express alternative isoforms of several growth factor receptors, there have been no studies to verify these preliminary findings. The objective of this study was to determine whether cultured and ex vivo HTM cells express alternate isoforms of hepatocyte, keratinocyte, and transforming growth factor beta (TGFbeta)-II receptors and to characterize the isoform molecular sequences. METHODS: To determine whether cells within the HTM express mRNA for alternate isoforms of growth factor receptors, total RNA was isolated from several well-characterized HTM cell lines that were established from donors of various ages and from fresh ex vivo HTM tissues from healthy donors. After cDNA synthesis, polymerase chain reaction was initiated using specific primers for alternate forms of the following receptors: hepatocyte growth factor (HGFR), keratinocyte growth factor (KGFR), and transforming growth factor beta receptor II (TGFbetaR-II). Specificity and characterization of the polymerase chain reaction amplification products were determined by nucleic acid sequencing. RESULTS: Amplification products of the expected size for the growth factor isoforms were expressed in cell lines and in ex vivo tissues. Nucleic acid sequencing showed that cultured HTM cells and fresh ex vivo trabecular meshwork tissues expressed specific mRNA for alternatively spliced isoforms of HGFR, KGFR, and TGFbetaR-II The HGFR alternate isoform contained a 96-bp insert in the C-terminal coding region of the cytoplasmic tyrosine kinase domain. The KGFR alternate isoform is a soluble, truncated form, because it has no transmembrane or cytoplasmic domain as does the normal membrane-associated form. The TGFbetaR-II alternate isoform contained a 75-bp insert in the N-terminal coding region of the extracellular domain. CONCLUSIONS: In vitro and ex vivo HTM cells express mRNA for alternatively spliced isoforms of HGFR, KGFR, and TGFbetaR-II. These alternatively spliced receptor isoforms may be functional within the HTM and may play a critical role in maintaining the normal microenvironment of this important tissue.
Asunto(s)
Empalme Alternativo , Proteínas Proto-Oncogénicas c-met/genética , ARN Mensajero/metabolismo , Receptores de Factores de Crecimiento de Fibroblastos , Receptores de Factores de Crecimiento/genética , Receptores de Factores de Crecimiento Transformadores beta/genética , Malla Trabecular/metabolismo , Anciano , Anciano de 80 o más Años , Células Cultivadas , Cartilla de ADN/química , Electroforesis en Gel de Agar , Humanos , Reacción en Cadena de la Polimerasa , Proteínas Serina-Treonina Quinasas , Proteínas Proto-Oncogénicas c-met/metabolismo , ARN/aislamiento & purificación , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos , Receptor Tipo II de Factor de Crecimiento Transformador beta , Receptores de Factores de Crecimiento/metabolismo , Receptores de Factores de Crecimiento Transformadores beta/metabolismoRESUMEN
PURPOSE: To compare the mRNA expression of growth factor receptors in cultured human trabecular meshwork (HTM) cells with ex vivo HTM tissues and to determine whether HTM cells generate a physiologic response after exposure to exogenous growth factors. METHODS: The reverse transcription-polymerase chain reaction (RT-PCR) method was used to detect the expression of various growth factor receptor mRNAs using early passaged, cultured HTM cells from donors of several ages. RT-PCR on ex vivo HTM tissues from healthy donors and donors with glaucoma were also used to compare and contrast mRNA expression with cell culture results. After the exogenous administration of growth factors, cell proliferation and extracellular acidification rate studies were used to measure the functional responses of HTM cells to growth factors. RESULTS: Amplification products of the expected size for 15 growth factor receptors were detected in cultured HTM cells and in ex vivo HTM tissues. The administration of exogenous growth factors showed that (a) hepatocyte growth factor (HGF), epidermal growth factor (EGF), insulinlike growth factor (IGF)-1, tumor necrosis factor (TNF) alpha, platelet-derived growth factor (PDGF)-AA, PDGF-BB, PDGF-AB, and basic fibroblast growth factor (FGF-2) stimulated cell proliferation, whereas FGF-1 (acidic), transforming growth factor (TGF) alpha, interleukin (IL)-1alpha, nerve growth factor (NGF), and FGF-7 (keratinocyte growth factor [KGF]) had no significant influence on cell proliferation; (b) TGF-beta isoforms significantly inhibited EGF-stimulated trabecular meshwork cell proliferation; and (c) FGF-1 (acidic), TGF-alpha, EGF, IL-1alpha, IL-1beta, HGF, TNF-alpha, PDGF-AA, and IGF-1 significantly stimulated extracellular acidification, whereas FGF-2 (basic), FGF-7 (KGF), TGF-beta1-beta3 and NGF had no significant influence on extracellular acidification. CONCLUSIONS: These studies show that mRNA for numerous growth factor receptors can be detected in cultured HTM cells and in ex vivo HTM tissues. They also show that many of the receptors are functional, because exogenous growth factor administration elicits a physiologic response. In vivo, these receptors may be activated by growth factors present within the aqueous humor (aquecrine/paracrine) or by growth factors synthesized and released locally by trabecular meshwork cells themselves (autocrine). Specific growth factors acting through high-affinity receptors may be involved in maintaining the normal microenvironment of the HTM and also may be involved in the pathogenesis of primary open-angle glaucoma.
Asunto(s)
Receptores de Factores de Crecimiento/metabolismo , Malla Trabecular/metabolismo , Adolescente , Anciano , Anciano de 80 o más Años , División Celular/efectos de los fármacos , Células Cultivadas , Preescolar , Cartilla de ADN/química , Glaucoma/metabolismo , Glaucoma/patología , Sustancias de Crecimiento/farmacología , Humanos , Lactante , Recién Nacido , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa , ARN Mensajero/metabolismo , Receptores de Factores de Crecimiento/genética , Malla Trabecular/citología , Malla Trabecular/efectos de los fármacosRESUMEN
The introduction of viral transforming genes into mammalian cells has been used in establishing cultures of unlimited lifespan. Although Müller cells, the predominant glial cells in the mammalian retina, have been isolated using a variety of techniques, most of these cultures have limited capacity for cell division and are often contaminated by other cell types especially astrocytes, endothelial cells and microglial cells. We have established pure cultures of retinal cells which express Müller cell characteristics and exhibit unlimited growth in vitro. We now report the techniques involved in the propagation and characterization of these cultures. Mixed retinal cultures isolated from dystrophic rat retinas were infected with defective retroviruses coding for human papillomavirus (HPV) type 16 E6 and E7 proteins. The disabled viral constructs also contained the neomycin gene allowing selection of the cultures using Geneticin, a neomycin analogue. Pure cultures were then obtained from Geneticin-selected populations by limiting end-dilution techniques. The expression of the HPV-16 E6/E7 genes in the transfected cell line was established using an HPV-16 E6/E7 PCR product to probe Northern blots. Cloned cells were found to be highly reactive for Müller cell markers including S-100, carbonic anhydrase-C, cellular retinaldehyde binding protein, and glial fibrillary acidic protein but not for glutamine synthetase. Ultrastructural studies showed stacks of cells with long elaborate processes, short microvilli, coated pits, cytoplasmic filaments, abundant perinuclear rough endoplasmic reticulum, and smooth endoplasmic reticulum extending to the cell processes. Growth patterns of late passage cells (> 50 passages) showed a lag phase of 48 hr followed by exponential growth extending past visual confluence at day 5. Since the cultures have undergone more than 240 population doublings, they can be characterized as a continuous cell line with unlimited lifespan. The HPV-16 E6/E7 transfected Müller cell line may prove useful in studies requiring abundant and pure cultures of Müller cells.
Asunto(s)
Neuroglía/metabolismo , Retina/metabolismo , Transfección , Animales , Northern Blotting , Anhidrasas Carbónicas/metabolismo , Proteínas Portadoras/metabolismo , División Celular , Línea Celular , Genes Virales/genética , Proteína Ácida Fibrilar de la Glía/metabolismo , Glutamato-Amoníaco Ligasa/metabolismo , Inmunohistoquímica , Oncogenes/genética , Papillomaviridae/genética , Papillomaviridae/metabolismo , ARN Viral/análisis , Ratas , Retina/citología , Proteínas S100/metabolismoRESUMEN
PURPOSE: A newly-derived transformed neonatal rat retinal pigment epithelial (tnrRPE) cell line was investigated: for secreted proteins by electrophoresis, and for basic and acidic fibroblast growth factor (FGF) by immunocytochemistry, Northern blot and reverse transcriptase-polymerase chain reaction (RT-PCR). The FGFR-1 (flg) receptor, which is recognized by aFGF and bFGF, was studied by RT-PCR. METHODS: Retinal pigment epithelial (RPE) cells were isolated from 6-day-old pigmented normal Long Evans rats, and became spontaneously transformed after the second passage. RESULTS: RPE cells at the 5th through 28th passages expressed the epithelial cell marker cytokeratin and cellular retinaldehyde binding protein (CRALBP), an RPE cell marker, but were negative for glial fibrillary acidic protein (GFAP), as shown by immunofluorescence. Secreted proteins of late passage tnrRPE cells were in a narrow molecular weight range of 60-80kDa, while early passage cells exhibited multiple proteins from 20-200kDa. These tnrRPE cells increased by 17-30 fold over a 4-day culture period. At 5th and 28th passage, immunostaining for bFGF and aFGF was dense within nuclei, but light and diffuse within the cytoplasm of transformed RPE cells. As shown by Northern blot, similar levels of message for bFGF were detected in 5th and 30th passage RPE cells. As shown by Northern blot, similar levels of message for bFGF were detected in 5th and 30th passage RPE cells. Furthermore, as shown by RT-PCR, bFGF mRNA was found in freshly isolated and transformed neonatal rat RPE cells. However, the message for FGFR-1(flg) receptor was detected only in the transformed RPE cells. CONCLUSIONS: This study demonstrated a neonatal rat RPE cell line that proliferated rapidly in vitro, expressed high levels of message for hFGF and FGFR-1(flg) receptor, and continued to express RPE-cell characteristics. Importantly, mRNA levels of confluent cultures of these cells were sufficient for bFGF mRNA blot analysis, which eliminates the necessity for PCR and for using excessive numbers of animals for such studies.
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Proteínas del Ojo/metabolismo , Factores de Crecimiento de Fibroblastos/metabolismo , Epitelio Pigmentado Ocular/citología , Receptores de Factores de Crecimiento de Fibroblastos/metabolismo , Animales , Animales Recién Nacidos , Northern Blotting , División Celular , Línea Celular Transformada , Electroforesis en Gel de Poliacrilamida , Inmunohistoquímica , Epitelio Pigmentado Ocular/metabolismo , Reacción en Cadena de la Polimerasa , Ratas , Ratas EndogámicasRESUMEN
In the rds mutant mouse the photoreceptor cells differentiate normally for the first few postnatal days, with the inner segments projecting an extended cilium. However, outer segments fail to form and only rudimentary disks and opsin-laden vesicles assemble at the tip of the cilium. These are shed into the interphotoreceptor space where they are phagocytosed by the retinal pigment epithelial cells. In this animal model, the photoreceptors undergo a slow degeneration via apoptosis leading to eventual loss of the entire photoreceptor population. Since increased expression of clusterin has been implicated in apoptosis, we studied the expression of clusterin in the rds mutant mouse retina and compared it to normal BALB/ c retinas. Small intestinal microvillus epithelium was used as a positive control tissue for apoptosis. Immunocytochemistry revealed the presence of clusterin in the ganglion cell, inner nuclear and outer plexiform layers and in the retinal pigment epithelium of both the rds and the BALB/c retinas. Interestingly, scattered clusterin-positive cells were observed in the outer nuclear layer (onl) of dystrophic retinas. Since the increased presence of clusterin protein in the onl of dystrophic retina may indicate dying photoreceptor cells due to apoptosis, we utilized a co-localization procedure for apoptotic nuclei and clusterin. For apoptosis we utilized an in situ 3' end labeling of fragmented DNA (TUNEL) and immunohistochemistry for clusterin using brown and red colored substrates respectively. Small intestine tissue sections were also included as positive controls for apoptosis. Our results show that clusterin is co-localized with apoptotic nuclei both in the onl of rds mutant retinas as well as in the small intestine epithelial cells undergoing cell turnover and exfoliation. These results are of interest since overexpression of clusterin is also observed in other neuro-degenerative diseases such as Alzheimer's and Pick's disease.
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Apoptosis , Glicoproteínas/biosíntesis , Chaperonas Moleculares , Células Fotorreceptoras/metabolismo , Degeneración Retiniana/genética , Animales , Clusterina , Glicoproteínas/análisis , Técnicas para Inmunoenzimas , Inmunohistoquímica/métodos , Ratones , Ratones Endogámicos BALB C , Ratones Mutantes , Proteínas del Tejido Nervioso/análisis , Proteínas del Tejido Nervioso/biosíntesis , Células Fotorreceptoras/patología , Epitelio Pigmentado Ocular/metabolismo , Epitelio Pigmentado Ocular/patología , Valores de Referencia , Degeneración Retiniana/metabolismo , Degeneración Retiniana/patología , Células Ganglionares de la Retina/metabolismo , Células Ganglionares de la Retina/patologíaRESUMEN
PURPOSE: Previous studies have suggested that the disappearance of anterior keratocytes after injury to the overlying epithelium is mediated by apoptosis. The authors examined the expression of the apoptosis-related modulators, Fas (receptor), Fas ligand, Bax, Bcl-2, Bcl-XL, and interleukin-1 beta converting enzyme (ICE) in corneal cells as candidate mediators of this response and tested the effect of Fas receptor-stimulating antibody on corneal stromal fibroblast cells in vitro. METHODS: Reverse-transcription-polymerase chain reaction was used to detect FAS, FAS ligand, Bax, Bcl-2, Bcl-XL, and ICE mRNA expression in primary cultures of human corneal epithelial, stromal fibroblast, and endothelial cells. Immunohistochemistry was applied to detect Fas and Fas ligand proteins in fresh-frozen sections of normal human cornea. The effect of FAS-stimulating monoclonal antibody on first-passage stromal fibroblasts was studied using a DNA fragmentation assay, the live-dead assay with fluorescent microscopy, toluidene blue staining with light microscopy, and electron microscopy. RESULTS: FAS, Fas ligand, Bax, Bcl-2, Bcl-XL, and ICE mRNAs are expressed in all three major cell types of the cornea. Fas protein is expressed in corneal epithelial, keratocyte, and endothelial cells in fresh-frozen human cornea. Fas ligand protein, however, was detected in corneal epithelial and endothelial, but not keratocyte, cells. Fas-stimulating antibody induced first-passage stromal fibroblast cell death with morphologic changes and DNA fragmentation consistent with apoptosis. CONCLUSIONS: The Fas system (Fas and Fas ligand) modulators and final common pathway mediators of apoptosis are expressed in corneal cells. The distribution of Fas (epithelial, keratocyte, and endothelial cells) and Fas ligand (epithelial and endothelial cells) protein expression in fresh-frozen corneal tissue suggests that Fas ligand expressed in corneal epithelial and endothelial cells modulates functions in keratocyte cells and, possibly, autocrine-juxtacrine functions in epithelium and endothelium. The Fas-Fas ligand system is expressed in the cornea and could have important functions in normal corneal physiology and in the pathophysiology of corneal disease, including modulation of keratocyte apoptosis after epithelial injury.
Asunto(s)
Apoptosis , Córnea/metabolismo , Cisteína Endopeptidasas/biosíntesis , Proteínas Proto-Oncogénicas/biosíntesis , Receptor fas/biosíntesis , Secuencia de Aminoácidos , Secuencia de Bases , Caspasa 1 , Muerte Celular , Células Cultivadas , Córnea/citología , Sustancia Propia/citología , Sustancia Propia/metabolismo , Cisteína Endopeptidasas/genética , Daño del ADN , Cartilla de ADN/química , Endotelio Corneal/citología , Endotelio Corneal/metabolismo , Epitelio/metabolismo , Fibroblastos/metabolismo , Fibroblastos/ultraestructura , Humanos , Técnicas para Inmunoenzimas , Ligandos , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-bcl-2 , ARN Mensajero/biosíntesis , Transcripción Genética , Proteína X Asociada a bcl-2 , Proteína bcl-X , Receptor fas/genéticaRESUMEN
Mammalian embryo implantation involves a series of complex interactions between maternal and embryonic cells. Uterine polypeptide growth factors may play critical roles in these cell interactions. Basic fibroblast growth factor (basic FGF) is a member of a family of growth factors. This growth factor may be potentially important for the process of embryo implantation because it (a) is stored within the extracellular matrix and is thus easily available during embryo invasion, (b) is a potent modulator of cell proliferation and differentiation and (c) stimulates angiogenesis. The immunolocalization of basic FGF in the uterus during the peri-implantation period of pregnancy is presented in this study. Uterine tissue samples were obtained on days 6-9 of pregnancy with day 1 of pregnancy being the day of a vaginal copulatory plug. Uterine samples were fixed in Bouin's fluid for no longer than 18 h. Following fixation and paraffin embedding, sections were exposed to primary antisera made in rabbits against either (a) human recombinant basic FGF or (b) 1-24 synthetic fragment of bovine basic FGF. The primary antibody was followed by biotinylated goat anti-rabbit IgG and a biotin-avidin-peroxidase complex. There were no differences in the immunolocalization of basic FGF using either source of primary antibody. Our results demonstrated both temporal and spatial changes in the localization of immunoreactive basic FGF within the implantation chamber during days 6-9 of pregnancy. Inter-implantation sites resembled the non-pregnant uterus with basic FGF present in extracellular matrices including basal laminae. On day 6 of pregnancy, decidual cells within the primary decidual zone lacked both intracellular and pericellular basic FGF while non-decidualized uterine stroma resembled inter-implantation sites. By days 7-8 of pregnancy, the secondary decidual zone had formed and was characterized by the distinct pericellular localization of basic FGF around individual decidual cells. By day 9 of pregnancy, the mesometrial region was forming and contained cords of decidual cells and a labyrinth of maternal blood vessels. The decidual cells contained diffuse intracellular basic FGF. Trophoblast cells were devoid of basic FGF at all times examined. These results indicate that basic FGF is present within the implantation chamber on days 6-9 of pregnancy and may be involved in the decidual cell response, trophoblast cell invasion and angiogenesis.
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Implantación del Embrión/fisiología , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Útero/metabolismo , Animales , Membrana Basal/metabolismo , Matriz Extracelular/metabolismo , Femenino , Factor 2 de Crecimiento de Fibroblastos/inmunología , Técnicas para Inmunoenzimas , Ratones , Embarazo , Trofoblastos/metabolismo , Útero/ultraestructuraRESUMEN
Basic fibroblast growth factor is a biologically active peptide with a strong affinity for heparin. This growth factor has been previously shown to be mitogenic for a variety of mesoderm and neuroectoderm-derived cells. The immunohistochemical localization of basic FGF within mouse growing and atretic ovarian follicles is presented in the study. Ovarian tissue samples were obtained either (a) randomly from mice housed in a controlled light environment or (b) following the administration of exogenous gonadotropins to stimulate follicle development. Ovarian samples were fixed in Bouin's fluid for no longer than 18 h. Following fixation and paraffin embedding, sections were exposed to a primary antibody made in rabbits against either (a) human recombinant basic FGF or (b) the 1-24 synthetic fragment of bovine basic FGF. The primary antibody was followed by biotinylated goat anti-rabbit IgG and a biotin-avidin-peroxidase complex. There were no differences in the immunolocalization of basic FGF using either source of primary antibody or between randomly obtained ovarian samples and those obtained from mice given exogenous gonadotropins. Basic FGF was immunolocalized in follicle basal laminae and was also closely associated with individual follicle cells during all stages of ovarian follicle development. Basic FGF was absent in the theca interna, oocyte cytoplasm, zona pellucida and follicle fluid of normal growing follicles. Individual corpora luteal cells were surrounded by basic FGF but lacked cytoplasmic staining. Atretic follicles exhibited staining patterns similar to their respective stage of follicle development. However, when present, follicle fluid within atretic follicles was strongly positive for basic FGF. These results indicate that basic FGF may be an important factor involved in intraovarian control mechanisms.
Asunto(s)
Factor 2 de Crecimiento de Fibroblastos/análisis , Atresia Folicular/metabolismo , Folículo Ovárico/química , Animales , Cuerpo Lúteo/química , Femenino , Gonadotropinas/farmacología , Técnicas para Inmunoenzimas , Ratones , Folículo Ovárico/efectos de los fármacos , Folículo Ovárico/fisiologíaRESUMEN
Uterine samples were either rapidly frozen in liquid nitrogen or placed in Bouin's fixative. A commercial primary polyclonal antibody made in rabbits against human recombinant basic fibroblast growth factor (bFGF) was used. Western blot analysis indicated that the antibody was specific for bFGF and did not react with acidic FGF. The primary antibody was followed by either goat anti-rabbit immunoglobulin G (IgG) conjugated to the fluorescent phycobiliprotein tracer phycoerythrin or biotinylated goat anti-rabbit IgG and a biotin-avidin-peroxidase complex. Specificity controls using adjacent sections were carried out by (i) substituting normal rabbit sera for the primary antisera, (ii) omitting the primary antisera or (iii) extracting sections with NaCl (2 mol l-1) prior to the immunochemical procedures. No binding of the antibody was observed with any of the specificity control sections. The connective tissue stroma and the basal lamina associated with uterine glandular and surface epithelial layers were positive for bFGF. Localization was not observed within surface or glandular epithelial cells. The basal lamina and endothelial cells associated with blood vessels within the uterus and the smooth muscle cells of the myometrium were positive for bFGF. There were no differences in uterine localization patterns or intensity during the oestrous cycle or after ovariectomy and steroid hormone supplementation. These studies demonstrate the specific localization of bFGF within the mouse uterus.
Asunto(s)
Factor 2 de Crecimiento de Fibroblastos/análisis , Útero/química , Animales , Membrana Basal/química , Western Blotting , Endometrio/química , Estradiol/farmacología , Estro/metabolismo , Femenino , Técnicas para Inmunoenzimas , Inmunohistoquímica , Ratones , Miometrio/química , Ovariectomía , Progesterona/farmacología , Células del Estroma/química , Útero/efectos de los fármacosRESUMEN
Guinea-pig (intrusive) and mouse (displacement) blastocysts display different cellular mechanisms of implantation. Blastocysts were placed in CMRL-1066 supplemented with either 10 or 20% fetal calf serum, 0.1M L-glutamine and antibiotics and then transferred to dishes previously coated with either Matrigel or type I collagen. After culture for 48 or 72 h, the dishes were processed for transmission electron microscopy. Blastocysts had attached to both extracellular matrices by 48 h. Matrigel elicited minimal trophoblast cell activity. Trophoblast cell projections were oriented parallel to the Matrigel and displayed little invasive activity, but trophoblast cells displayed active interaction with type I collagen. By 72 h, trophoblast cells exhibited slender, anastomosing projections which extended into the collagen matrix. Bundles of microfilaments running parallel with the long axis of the projections were observed. The morphology of type I collagen was altered in the immediate vicinity of the trophoblast projections. The projections interdigitated and desmosomes developed between processes. Projections appeared to meet, fuse and entrap matrix. These results suggest that trophoblast cells do not significantly interact with Matrigel, but penetrate into type I collagen.