RESUMO
The blood-brain barrier in the neonatal brain expresses the monocarboxylate transporter (MCT)-1 rather than the glucose transporter (GLUT)-1, due to the special energy supply during the suckling period. The hyaloid vascular system, consisting of the vasa hyaloidea propria and tunica vasculosa lentis, is a temporary vasculature present only during the early development of mammalian eyes and later regresses. Although the ocular vasculature manifests such a unique developmental process, no information is available concerning the expression of endothelial nutrient transporters in the developing eye. The present immunohistochemical study using whole mount preparations of murine eyes found that the hyaloid vascular system predominantly expressed GLUT1 in the endothelium, in contrast to the brain endothelium. Characteristically, the endothelium in peripheral regions of the neonatal hyaloid vessels displayed a mosaic pattern of MCT1-immunoreactive cells scattered within the GLUT1-expressing endothelium. The proper retinal vessels first developed by sprouting angiogenesis endowed with filopodia, which were absolutely free from the immunoreactivities of GLUT1 and MCT1. The remodeling retinal capillary networks and veins in the surface layer of the retina mainly expressed MCT1 until the weaning period. Immunostaining of MCT1 in the retina revealed fine radicular processes projecting from the endothelium, differing from the MCT1-immunonegative filopodia. These findings suggest that the expression of nutrient transporters in the ocular blood vessels is differentially regulated at a cellular level and that the neonatal eyes provide an interesting model for research on nutrient transporters in the endothelium.
Assuntos
Olho/crescimento & desenvolvimento , Transportador de Glucose Tipo 1/biossíntese , Transportadores de Ácidos Monocarboxílicos/biossíntese , Epitélio Pigmentado Ocular/metabolismo , Prenhez , Simportadores/biossíntese , Animais , Animais Recém-Nascidos , Transporte Biológico , Feminino , Imuno-Histoquímica , Camundongos , Microscopia Eletrônica , Modelos Animais , Epitélio Pigmentado Ocular/crescimento & desenvolvimento , Epitélio Pigmentado Ocular/ultraestrutura , GravidezRESUMO
The structural and biochemical changes of cytoskeletal components of retinal pigmented epithelial cells were studied during the development of chicken eyes. When the cytoskeletal components of the pigmented epithelial cells from various stages of development were examined by SDS PAGE, actin contents in the cells markedly increased between the 15-d-old and hatching stages. Immunofluorescence microscopy showed that chicken pigmented epithelial cells have two types of actin bundles. One is the circumferential bundle associated with the zonula adherens region as previously reported (Owaribe, K., and H. Masuda, 1982, J. Cell Biol., 95:310-315). The other is the paracrystalline bundle forming the core of the apical projections. The increase in actin contents after the 15-d-old stage is accompanied by the formation and elongation of core filaments of apical projections in the cells. During this period the apical projections extend into extracellular space among outer and inner segments of photoreceptor cells. Accompanying this change is an elongation of the paracrystalline bundles of actin filaments in the core of the projection. By electron microscopy, the bundles decorated with muscle heavy meromyosin showed unidirectional polarity, and had transverse striations with approximately 12-nm intervals, as determined by optical diffraction of electron micrographs. Since the shape of these bundles was not altered in the presence or absence of Ca2+, they seemed not to have villin-like proteins. Unlike the circumferential bundles, the paracrystalline bundles did not contract when exposed to Mg-ATP. These observations indicate that the paracrystalline bundles are structurally and functionally different from the circumferential actin bundles.
Assuntos
Actinas/biossíntese , Citoesqueleto/fisiologia , Olho/crescimento & desenvolvimento , Epitélio Pigmentado Ocular/metabolismo , Actinas/fisiologia , Animais , Embrião de Galinha , Citoesqueleto/ultraestrutura , Imunofluorescência , Peso Molecular , Epitélio Pigmentado Ocular/crescimento & desenvolvimento , Epitélio Pigmentado Ocular/ultraestrutura , Difração de Raios XRESUMO
Retinal pigment epithelial (RPE) cells apically polarize proteins that are basolateral in other epithelia. This reversal may be generated by the association of RPE with photoreceptors and the interphotoreceptor matrix, postnatal expansion of the RPE apical surface, and/or changes in RPE sorting machinery. We compared two proteins exhibiting reversed, apical polarities in RPE cells, neural cell adhesion molecule (N-CAM; 140-kD isoform) and extracellular matrix metalloproteinase inducer (EMMPRIN), with the cognate apical marker, p75-neurotrophin receptor (p75-NTR). N-CAM and p75-NTR were apically localized from birth to adulthood, contrasting with a basolateral to apical switch of EMMPRIN in developing postnatal rat RPE. Morphometric analysis demonstrated that this switch cannot be attributed to expansion of the apical surface of maturing RPE because the basolateral membrane expanded proportionally, maintaining a 3:1 apical/basolateral ratio. Kinetic analysis of polarized surface delivery in MDCK and RPE-J cells showed that EMMPRIN has a basolateral signal in its cytoplasmic tail recognized by both cell lines. In contrast, the basolateral signal of N-CAM is recognized by MDCK cells but not RPE-J cells. Deletion of N-CAM's basolateral signal did not prevent its apical localization in vivo. The data demonstrate that the apical polarity of EMMPRIN and N-CAM in mature RPE results from suppressed decoding of specific basolateral signals resulting in randomized delivery to the cell surface.
Assuntos
Antígenos CD , Antígenos de Neoplasias , Polaridade Celular , Glicoproteínas de Membrana/metabolismo , Moléculas de Adesão de Célula Nervosa/metabolismo , Epitélio Pigmentado Ocular/metabolismo , Sinais Direcionadores de Proteínas/metabolismo , Animais , Basigina , Transporte Biológico , Linhagem Celular , Linhagem Celular Transformada , Cães , Feminino , Técnicas de Transferência de Genes , Humanos , Masculino , Glicoproteínas de Membrana/genética , Moléculas de Adesão de Célula Nervosa/genética , Epitélio Pigmentado Ocular/crescimento & desenvolvimento , Ratos , Receptor de Fator de Crescimento Neural , Receptores de Fator de Crescimento Neural/metabolismoRESUMO
PURPOSE: Visually guided ocular growth is facilitated by scleral extracellular matrix remodeling at the posterior pole of the eye. Coincident with scleral remodeling, significant changes in choroidal morphology, blood flow, and protein synthesis have been shown to occur in eyes undergoing ocular growth changes. The current study is designed to identify gene expression changes that may occur in the choroid/retinal pigment epithelium (RPE) of marmoset eyes during their compensation for hyperopic defocus as compared to eyes compensating for myopic defocus. METHODS: Total RNA was isolated from choroid/RPE from four common marmosets (Callithrix jacchus) undergoing binocular lens treatment using extended wear soft contact lenses of equal magnitude but opposite sign (+/-5 diopter [D]). After reverse transcription, cDNA was labeled and hybridized to a human oligonucleotide microarray and gene transcript expression profiles were determined. Real-time polymerase chain reaction (PCR) and western blot analysis were used to confirm genes and proteins of interest, respectively. RESULTS: Microarray analyses in choroid/RPE indicated 204 genes were significantly changed in minus lens-treated as compared with plus lens-treated eyes (p<0.05, Student's t-test). Differential choroid/RPE expression of protein tyrosine phosphatase, receptor type, B (PTPRB), transforming growth factor beta-induced (TGFBI), and basic fibroblast growth factor 2 (FGF-2) were confirmed by real-time PCR. TGFBIp was confirmed at the protein level by western blot analysis in marmoset and human cornea, choroid/RPE, and sclera. CONCLUSIONS: The present study demonstrated that significant gene expression changes occur in the marmoset choroid/RPE during visually guided ocular growth. The identification of novel candidate genes in choroid/RPE of marmoset eyes actively accelerating or decelerating their rates of ocular elongation may elucidate the choroidal response during the regulation of postnatal ocular growth and may lead to the identification of choroid/RPE signaling molecules that participate in scleral remodeling.
Assuntos
Callithrix/genética , Corioide/crescimento & desenvolvimento , Perfilação da Expressão Gênica , Análise de Sequência com Séries de Oligonucleotídeos , Epitélio Pigmentado Ocular/crescimento & desenvolvimento , Refração Ocular/genética , Animais , Adesão Celular/efeitos dos fármacos , Células Cultivadas , Corioide/efeitos dos fármacos , Corioide/metabolismo , Eletroforese , Proteínas do Olho/genética , Proteínas do Olho/metabolismo , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Cristalino/efeitos dos fármacos , Cristalino/metabolismo , Epitélio Pigmentado Ocular/efeitos dos fármacos , Epitélio Pigmentado Ocular/metabolismo , RNA/isolamento & purificação , Refração Ocular/efeitos dos fármacos , Reprodutibilidade dos Testes , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Esclera/citologia , Fator de Crescimento Transformador beta/farmacologia , Visão Binocular/efeitos dos fármacos , Corpo Vítreo/efeitos dos fármacos , Corpo Vítreo/metabolismoRESUMO
The iris plays a key role in visual function. It regulates the amount of light entering the eye and falling on the retina and also operates in focal adjustment of closer objects. The iris is involved in circulation of the aqueous humor and hence functions in regulation of intraocular pressure. Intriguingly, iris pigmented cells possess the ability to transdifferentiate into different ocular cell types of retinal pigmented epithelium, photoreceptors and lens cells. Thus, the iris is considered a potential source for cell-replacement therapies. During embryogenesis, the iris arises from both the optic cup and the periocular mesenchyme. Its interesting mode of development includes specification of the peripheral optic cup to a non-neuronal fate, migration of cells from the surrounding periocular mesenchyme and an atypical formation of smooth muscles from the neuroectoderm. This manner of development raises some interesting general topics concerning the early patterning of the neuroectoderm, the specification and differentiation of diverse cell types and the interactions between intrinsic and extrinsic factors in the process of organogenesis. In this review, we discuss iris anatomy and development, describe major pathologies of the iris and their molecular etiology and finally summarize the recent findings on genes and signaling pathways that are involved in iris development.
Assuntos
Padronização Corporal/genética , Diferenciação Celular/genética , Linhagem da Célula/genética , Regulação Enzimológica da Expressão Gênica/genética , Iris/embriologia , Iris/crescimento & desenvolvimento , Animais , Movimento Celular/genética , Ectoderma/citologia , Ectoderma/metabolismo , Humanos , Iris/citologia , Mesoderma/citologia , Mesoderma/metabolismo , Epitélio Pigmentado Ocular/citologia , Epitélio Pigmentado Ocular/embriologia , Epitélio Pigmentado Ocular/crescimento & desenvolvimento , Vertebrados/embriologia , Vertebrados/crescimento & desenvolvimentoRESUMO
p21/WAF1/CIP1/MDA6 is a key cell cycle regulator. Cell cycle regulation is an important part of development, differentiation, DNA repair and apoptosis. Following DNA damage, p53 dependent expression of p21 results in a rapid cell cycle arrest. p21 also appears to be important for the development of melanocytes, promoting their differentiation and melanogenesis. Here, we examine the effect of p21 deficiency on the development of another pigmented tissue, the retinal pigment epithelium. The murine mutation pink-eyed unstable (p(un)) spontaneously reverts to a wild-type allele by homologous recombination. In a retinal pigment epithelium cell this results in pigmentation, which can be observed in the adult eye. The clonal expansion of such cells during development has provided insight into the pattern of retinal pigment epithelium development. In contrast to previous results with Atm, p53 and Gadd45, p(un) reversion events in p21 deficient mice did not show any significant change. These results suggest that p21 does not play any role in maintaining overall genomic stability by regulating homologous recombination frequencies during development. However, the absence of p21 caused a distinct change in the positions of the reversion events within the retinal pigment epithelium. Those events that would normally arrest to produce single cell events continued to proliferate uncovering a cell cycle dysregulation phenotype. It is likely that p21 is involved in controlling the developmental pattern of the retinal pigment. We also found a C57BL/6J specific p21 dependent ocular defect in retinal folding, similar to those reported in the absence of p53.
Assuntos
Padronização Corporal/fisiologia , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Epitélio Pigmentado Ocular/embriologia , Recombinação Genética , Animais , Proliferação de Células , Inibidor de Quinase Dependente de Ciclina p21/genética , Dano ao DNA/fisiologia , Olho/citologia , Olho/crescimento & desenvolvimento , Anormalidades do Olho/genética , Regulação da Expressão Gênica no Desenvolvimento , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Epitélio Pigmentado Ocular/citologia , Epitélio Pigmentado Ocular/crescimento & desenvolvimento , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
Sonic hedgehog (Shh) signaling plays a major role in vertebrate development, from regulation of proliferation to the patterning of various organs. In amniotes, Shh affects dorsoventral patterning in the inner ear but affects anteroposterior patterning in teleost ears. It remains unknown how altered function of Shh relates to morphogenetic changes that coincide with the evolution of limbs and novel auditory organs in the ear. In this study, we used the tetrapod, Xenopus laevis, to test how increasing concentrations of the Shh signal pathway antagonist, Vismodegib, affects ear development. Vismodegib treatment dose dependently alters the development of the ear, hypaxial muscle, and indirectly the Mauthner cell through its interaction with the inner ear afferents. Together, these phenotypes have an effect on escape response. The altered Mauthner cell likely contributes to the increased time to respond to a stimulus. In addition, the increased hypaxial muscle in the trunk likely contributes to the subtle change in animal C-start flexion angle. In the ear, Vismodegib treatment results in decreasing segregation between the gravistatic sensory epithelia as the concentration of Vismodegib increases. Furthermore, at higher doses, there is a loss of the horizontal canal but no enantiomorphic transformation, as in bony fish lacking Shh. Like in amniotes, Shh signaling in frogs affects dorsoventral patterning in the ear, suggesting that auditory sensory evolution in sarcopterygians/tetrapods evolved with a shift of Shh function in axis specification. © 2017 Wiley Periodicals, Inc. Develop Neurobiol 77: 1385-1400, 2017.
Assuntos
Anilidas/farmacologia , Padronização Corporal/efeitos dos fármacos , Orelha Interna/crescimento & desenvolvimento , Orelha Interna/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Proteínas Hedgehog/antagonistas & inibidores , Proteínas Hedgehog/metabolismo , Piridinas/farmacologia , Animais , Padronização Corporal/fisiologia , Dextranos/metabolismo , Relação Dose-Resposta a Droga , Reação de Fuga/efeitos dos fármacos , Feminino , Imageamento Tridimensional , Larva , Locomoção/efeitos dos fármacos , Locomoção/fisiologia , Miosina Tipo IV/metabolismo , Mucosa Olfatória/efeitos dos fármacos , Mucosa Olfatória/crescimento & desenvolvimento , Epitélio Pigmentado Ocular/efeitos dos fármacos , Epitélio Pigmentado Ocular/crescimento & desenvolvimento , Pigmentação/efeitos dos fármacos , Natação , Xenopus laevisRESUMO
The retinal pigment epithelium (RPE) lies between the retina and the choroid of the eye and plays a vital role in ocular metabolism. The RPE develops from the same sheet of neuroepithelium as the retina and the two derivatives become distinguished by different expression patterns of a number of transcription factors during embryonic development. As the RPE layer differentiates it expresses a set of unique molecules, many of which are restricted to certain regions of the cell. PRE cells undergo both a loss of polarity and a loss of expression of many of these cell type-specific molecules when placed in monolayer culture. The RPE of many species, including mammals, can be induced to transdifferentiate by growth factors such as basic fibroblast growth factor. Under the influence of such factors the RPE is triggered to alter expression of a wide array of molecules and to take on a retinal epithelium fate, from which differentiated retinal cell types including rod photoreceptors can be produced.
Assuntos
Epitélio Pigmentado Ocular/citologia , Animais , Técnicas de Cultura de Células , Diferenciação Celular , Humanos , Epitélio Pigmentado Ocular/crescimento & desenvolvimento , Epitélio Pigmentado Ocular/transplanteRESUMO
Development of the eye can be subdivided into three phases. The first phase is the formation of the major structures of the eye by the processes of induction and regional specification. The second is the maturation of these structures to form the functional eye, and the third phase is the formation of neuronal connections between retina and the optic tectum. These processes are tightly regulated by signalling cascades that direct axonal outgrowth, cellular proliferation and differentiation. Some members of these signalling cascades have been identified in recent studies. These include secreted factors which transmit signals extracellularly, and receptors and transcription factors which are members of intracellular signalling pathways that respond to extracellular signals. This review summarizes the recent research that has implicated these factors in playing a role in eye development on the basis of functional or expression criteria.
Assuntos
Olho/embriologia , Olho/crescimento & desenvolvimento , Regulação da Expressão Gênica no Desenvolvimento , Vertebrados/crescimento & desenvolvimento , Animais , Indução Embrionária , Cristalino/embriologia , Cristalino/crescimento & desenvolvimento , Disco Óptico/embriologia , Disco Óptico/crescimento & desenvolvimento , Epitélio Pigmentado Ocular/embriologia , Epitélio Pigmentado Ocular/crescimento & desenvolvimento , Retina/embriologia , Retina/crescimento & desenvolvimento , Células Ganglionares da Retina/fisiologiaRESUMO
Neurothelin is a cell surface protein of chicken endothelial cells at the blood-brain barrier and also of pigment epithelial cells forming the blood-eye barrier. Peptide sequencing of affinity-purified neurothelin revealed that it is likely to be identical with the protein called HT7. It belongs to the immunoglobulin superfamily having two extracellular C2-type domains, a membrane spanning region and a cytoplasmic tail. During development neurothelin cell surface localization changed on pigment epithelial cells. In early embryogenesis neurothelin was found preferentially at lateral sites of neighboring epithelial cells, but after hatching, predominantly on basal cell surfaces and on apical microvilli of epithelial cells that contact retinal photoreceptors. Disruption of cell-cell contacts induced a rapid change of neurothelin distribution on the cell surface in vitro as could be shown by confocal laser microscopy. Disintegration of microfilaments by cytochalasin D hampered this specific cell surface rearrangement of neurothelin, whereas depolymerization of microtubules by demecolcine had no effect. In double-labeling experiments neurothelin and F-actin were colocalized. The data suggest that the polarized cell surface distribution of neurothelin is influenced intracellularly by F-actin and extracellularly by cell-cell interactions.
Assuntos
Actinas/metabolismo , Antígenos CD , Antígenos de Neoplasias , Antígenos de Superfície , Proteínas Aviárias , Proteínas Sanguíneas , Galinhas/metabolismo , Proteínas do Olho/química , Glicoproteínas de Membrana/química , Epitélio Pigmentado Ocular/metabolismo , Citoesqueleto de Actina/efeitos dos fármacos , Sequência de Aminoácidos , Animais , Basigina , Barreira Hematoencefálica , Barreira Hematorretiniana , Comunicação Celular , Embrião de Galinha , Galinhas/crescimento & desenvolvimento , Citocalasina D/farmacologia , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/metabolismo , Citoesqueleto/ultraestrutura , Endotélio Vascular/citologia , Endotélio Vascular/metabolismo , Proteínas do Olho/metabolismo , Glicoproteínas de Membrana/metabolismo , Dados de Sequência Molecular , Epitélio Pigmentado Ocular/embriologia , Epitélio Pigmentado Ocular/crescimento & desenvolvimentoRESUMO
Cell generation and the early stages of maturation of the retinal pigment epithelium (RPE) and photoreceptors were examined in a marsupial, the quokka, Setonix brachyurus. Results are presented for animals aged up to postnatal day (P)250. RPE cell generation was studied by analysis of cell number from wholemounted retinae and by tritiated thymidine (3HThy) autoradiography in sectioned material. For 3HThy autoradiography, quokkas aged P1-P200 were injected with 3HThy and killed either 6-20 hours later (pulse-kill) or at P100 or P250 (pulse-leave). The extent of pigmentation of the RPE sheet was examined from sections of embryonic and early postnatal stages. Retinae from animals aged P5 to P160 were also examined at the electron microscope. By P100, RPE cell number is within the range found in adults. New RPE cells are generated in a peripheral band which moves outwards as cells leave the cell cycle in more central locations. RPE cells thus complete their last cell division in a centre-to-periphery wave centred about the optic nerve head. At any given retinal location, RPE cells complete their last cell division earlier than the overlying layers of the neural retina. Cells of the RPE rapidly develop a mature morphology. For example, melanin granules are observed at P5 and Verhoeff's membrane (the terminal bar complex) is evident by P25. By contrast, photoreceptor development in this species is protracted; cone inner segments are observed by P40, whilst the first rod inner segments are observed at P60. Despite being generated earlier, morphological maturation of the cones appears retarded and prolonged compared with that of the rods. The last stages of RPE cell maturation occur late in development, in synchrony with the generation of rods.
Assuntos
Macropodidae/crescimento & desenvolvimento , Células Fotorreceptoras/fisiologia , Epitélio Pigmentado Ocular/crescimento & desenvolvimento , Retina/crescimento & desenvolvimento , Animais , Autorradiografia , Contagem de Células , Ciclo Celular/fisiologia , Macropodidae/anatomia & histologia , Microscopia Eletrônica , Epitélio Pigmentado Ocular/ultraestrutura , Retina/ultraestruturaRESUMO
Cell cultures of adult bovine retinal pigment epithelium (RPE) were propagated from posterior and equatorial regions of the same eyes to study topographical differences in cell growth. To generate the cultures, the retina was removed, eyecups were incubated in collagenase followed by trephining three zones from the posterior area centralis and three zones from the equatorial region of the tapetal retina. Outgrowth in primary cultures and proliferation in passaged cultures were evaluated in paired cultures derived from the same eyes. Relative to cells from the more peripheral regions, RPE cells from the area centralis showed lower growth potential which was manifested as reduced cell numbers in primary outgrowth and a smaller fraction of serum-responsive cells when cultures were serially passaged. Cells from older donors have been shown to exhibit similar reductions in in vitro growth potential, making it appear as if, with regards to proliferation, RPE from the area centralis is more "aged" than RPE from more peripheral regions of the same eyes.
Assuntos
Epitélio Pigmentado Ocular/crescimento & desenvolvimento , Animais , Bovinos , Divisão Celular , Sobrevivência Celular , Células Cultivadas , Epitélio Pigmentado Ocular/citologiaRESUMO
The structure and development of junctional complexes during redifferentiation of chick pigmented epithelial cells in clonal culture have been studied with TEM and colloidal lanthanum. The mature junctional complex consists of a zonula adherens which is usually surmounted by one or more macular gap junctions of varying length. Tight junctions (zonulae occludentes) appear to surround the gap junctions and extend into the zonula adherens. Punctate intermediate junctions appear first. As differentiation progresses, these extend to form fasciae and zonulae adherentes. Focal membrane fustions are found both within and above the developing adherens junctions; gap junctions appear to form adjacent to the latter structures. Colloidal lanthanum passes through the junctional regions between cells in the outer zone of the colony but is stopped by those of the differentiated cells, suggesting that these cells are sealed by fasciae or zonulae occludentes. During redifferentiation, groups of cells undergo slow, coordinated contractions which appear to be involved in developing the differentiated cell shape. These begin shortly after the formation of the junctional complexes and are most active during junctional complex maturation. Once cellular junctional complex differentiation is well established, the contractions cease. The possible roles of the different junctions in the development of cellular shape are discussed.
Assuntos
Epitélio Pigmentado Ocular/citologia , Animais , Células Cultivadas , Galinhas , Lantânio , Epitélio Pigmentado Ocular/crescimento & desenvolvimento , Epitélio Pigmentado Ocular/ultraestruturaRESUMO
The in vitro growth characteristics and morphology of human retinal pigment epithelium (RPE) cells from adult donor eyes (15 to 100 years of age) have been studied. Although RPE cells are viable in culture for several months, only a fraction of the cells actually divide. Rapid cell proliferation and confluency of a culture occur from 10 to 30 days after seeding. The time for confluency is both age- and media-dependent; the number of cells that are potential dividers and contribute to confluency decreases with increasing donor age. Since melanolipofuscin granules do not form in vitro and are diluted by cell division, stationary (nondividing) cells can be distinguished from the dividing cells by the presence of dense clusters of melanolipofuscin granules in the stationary cells. Confluent cultures contain a monolayer of relatively clear polygonal cells with densely pigmented stationary cells scattered throughout. Stationary cells can often represent as much as 95% of the original RPE cell population. Ultrastructurally all cells appear epithelioid, with apical-basal polarity, junctional complexes, and cytoplasmic organization characteristic of RPE cells in vivo. Stationary cells are extremely large, with melanolipofuscin granules clustered around the nucleus. Melanolipofuscin is a unique marker for nondividing cells and may facilitate studies of the age-dependent loss of replicability of human RPE.
Assuntos
Epitélio Pigmentado Ocular/crescimento & desenvolvimento , Adolescente , Adulto , Fatores Etários , Idoso , Divisão Celular , Humanos , Técnicas In Vitro , Microscopia Eletrônica , Microscopia de Contraste de Fase , Pessoa de Meia-Idade , Epitélio Pigmentado Ocular/citologia , Epitélio Pigmentado Ocular/ultraestrutura , Pigmentos BiológicosRESUMO
Retinal pigment epithelium (RPE) was isolated from human donors with no known eye disease and normal-appearing fundi and was grown in culture. Population doubling times were measured for fifth passage RPE cells; the mean was 2.25 +/- 0.75 days and showed a correlation with the age of the donor. Metabolic studies were performed by perfusing the cells with 1-[13C]-D-glucose and monitoring the perfusate with magnetic resonance spectroscopy. Glucose uptake was 10.5 +/- 3.3 nmoles/min/mg protein. Lactate production from glucose was 20.0 +/- 5.00 nmoles/min/mg protein. The percentage of glucose through the hexose monophosphate shunt was 21 +/- 2%. Oxygen (O) consumption was measured at 20.1 +/- 4.1 nmoles O/min/mg endogenous and 14.5 +/- 4.8 nmoles O/min/mg with 10 mM glucose. These cells exhibit high rates of lactate production concomitantly with high oxygen consumptions and high flux through the hexose monophosphate shunt.
Assuntos
Glucose/metabolismo , Consumo de Oxigênio , Via de Pentose Fosfato/fisiologia , Epitélio Pigmentado Ocular/metabolismo , Divisão Celular/fisiologia , Células Cultivadas , Metabolismo Energético , Glicólise , Humanos , Lactatos/biossíntese , Ácido Láctico , Espectroscopia de Ressonância Magnética , Perfusão , Epitélio Pigmentado Ocular/crescimento & desenvolvimentoRESUMO
PURPOSE: To measure the total amount of rhodopsin in human eyes across the life span and to test the hypothesis that the rhodopsin content of infants' and the elderly's eyes is lower than at other ages. METHODS: Rhodopsin was extracted from retinal and pigment epithelial fractions of 196 eyes of 102 donors, ages 27 weeks' gestation through 94 years, using quantitative procedures. To recover photopigment bleached by unavoidable light exposure, the fractions from 78 eyes were incubated with 9-cis retinal. The total photopigment (retinal plus pigment epithelial fractions) per eye was examined for significant changes with age, using the higher value from pairs of eyes. RESULTS: The median rhodopsin content of the higher eye of adults is 6.45 nmoles (range, 3.33-10.84 nmoles) with 8 nmoles or more recovered from 28% of all adult eyes. The rhodopsin content of infants' eyes (< 12 months post-term) is significantly lower than that of older individuals and increases with age. After infancy, no change with age is found. For both infants and adults, 9-cis retinal significantly increases the amount of photopigment recovered without reducing the variance in the amount of photopigment recovered. The rhodopsin content is estimated to be 50% of the median adult amount early in infancy, approximately 5 weeks postterm (95% confidence interval, 0-10 weeks postterm). CONCLUSIONS: A developmental increase in rhodopsin content occurs during infancy. Thereafter rhodopsin content remains constant. The amount of rhodopsin recovered from human eyes is quite variable. Bleaching alone cannot explain the variability.
Assuntos
Envelhecimento , Epitélio Pigmentado Ocular/química , Retina/química , Rodopsina/análise , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Envelhecimento/fisiologia , Criança , Pré-Escolar , Humanos , Lactente , Recém-Nascido , Pessoa de Meia-Idade , Epitélio Pigmentado Ocular/embriologia , Epitélio Pigmentado Ocular/crescimento & desenvolvimento , Retina/embriologia , Retina/crescimento & desenvolvimentoRESUMO
PURPOSE: This study sought To learn more about the mechanisms that determine and maintain the differentiated state of the retinal pigment epithelium (RPE). METHODS: Monoclonal antibodies were raised against human RPE and used in conjunction with other antibodies. Immunocytochemical and biochemical analyses were performed on tissue sections and cells in culture. RESULTS: An RPE-specific epitope, RET-PE10, has been detected as a 61 kD cytoplasmic polypeptide in a variety of mammalian, amphibian, and avian species. In the rat, RPE-PE10 was expressed late in eye development, with a faint initial labelling of the RPE in central regions at postnatal day 9 (PN9) that increased to adult levels and extent of staining by PN14. RET-PE10 expression initially was present in overnight cultures of dissociated rat RPE cells but was lost rapidly from these cultures during the first week. Comparison of the staining patterns of RET-PE10 with those of various cytoskeletal elements suggests that RET-PE10 may be associated with part of the intermediate filament network. Culture of whole eyecups also resulted in a loss of RET-PE10 expression. RET-PE10 expression was normal in eyes of adult rd/rd mutant mice. CONCLUSIONS: RET-PE10 is a late-appearing marker of RPE differentiation. The results also suggest that the maintained expression of RET-PE10 depends upon extrinsic factors but that these do not include maintained contact with Bruch's membrane or light-induced retinal activity.
Assuntos
Epitopos/metabolismo , Proteínas de Filamentos Intermediários/metabolismo , Peptídeos/metabolismo , Epitélio Pigmentado Ocular/metabolismo , Animais , Anticorpos Monoclonais , Anuros , Diferenciação Celular , Células Cultivadas , Galinhas , Citoplasma/metabolismo , Imunofluorescência , Humanos , Camundongos , Peso Molecular , Epitélio Pigmentado Ocular/crescimento & desenvolvimento , Coelhos , RatosRESUMO
PURPOSE: To investigate the expression of alpha B-crystallin in the developing rat retina by immunohistochemical techniques. METHODS: Rat eyes were enucleated on embryonic day 18 and on postnatal days 1, 4, 9, 15, 19, and 90. The avidin-biotin-peroxidase technique was applied to show alpha B-crystallin immunostaining. After the cornea and lens were removed, the developing rat eyeballs were solubilized by 1% sodium dodecyl sulfate. Western blot analysis and enzyme-linked immunosorbent assay (ELISA) were performed to detect alpha B-crystallin in the extracts. RESULTS: From postnatal day 4 to postnatal day 9, alpha B-crystallin was present in retinal pigment epithelium, but it disappeared after postnatal day 15. alpha A-crystallin immunoreactivity was negative in retina and retinal pigment epithelium throughout development. The results of Western blot analysis and ELISA coincided with those of the immunostaining. CONCLUSIONS: These results show that retinal pigment epithelium in mammalian rat retina demonstrates alpha B-crystallin expression in the early developmental stages but loses it after 15 days of age.
Assuntos
Cristalinas/biossíntese , Epitélio Pigmentado Ocular/crescimento & desenvolvimento , Epitélio Pigmentado Ocular/metabolismo , Animais , Western Blotting , Cristalinas/imunologia , Ensaio de Imunoadsorção Enzimática , Técnicas Imunoenzimáticas , Imunoglobulina G/imunologia , Dados de Sequência Molecular , Oligopeptídeos/química , Oligopeptídeos/imunologia , Epitélio Pigmentado Ocular/embriologia , Coelhos , RatosRESUMO
Human retinal pigment epithelium (RPE) contains two genetically distinct forms of superoxide dismutase (SOD) enzymes that scavenge harmful superoxide anions. Biochemical and immunochemical techniques were used to compare levels of copper-zinc- and manganese-containing forms of SOD (CuZn-SOD and Mn-SOD) in human adult and fetal RPE cells. It was found that Mn-SOD activity was higher in adult than fetal RPE cells, both in vivo and in vitro. Immunolocalization of Mn-SOD in cultured RPE cells showed a greater reactivity in the mitochondria of the adult cells. Primary cultures of adult RPE contained cells with various patterns of mitochondria as shown by immunolabeling for Mn-SOD. Adult RPE cells were more resistant to the effects of a superoxide generator, paraquat, which appeared to disrupt mitochondrial integrity as judged by staining with rhodamine 123. These results suggest that high levels of Mn-SOD protect mitochondria from oxidative damage that probably occurs with aging in the RPE.
Assuntos
Mitocôndrias/enzimologia , Epitélio Pigmentado Ocular/embriologia , Superóxido Dismutase/análise , Idoso , Idoso de 80 Anos ou mais , Linhagem Celular , Células Cultivadas , Eletroforese em Gel de Poliacrilamida , Feto/enzimologia , Imunofluorescência , Humanos , Pessoa de Meia-Idade , Mitocôndrias/efeitos dos fármacos , Epitélio Pigmentado Ocular/enzimologia , Epitélio Pigmentado Ocular/crescimento & desenvolvimentoRESUMO
Transthyretin (TTR) is a 55-kilodalton tetrameric protein that plays an important role in the plasma transport of thyroxine and retinol. Plasma TTR is synthesized in the liver, but major sites of synthesis also have been described in the choroid plexus (CP) epithelium, the visceral yolk sac, and the eye. Recently, the retinal pigment epithelium (RPE) was identified as the specific site of TTR synthesis in the rat eye, and it was suggested that this established a functional homology between the RPE and the CP epithelium. In this study, the temporal pattern of TTR mRNA expression was investigated in the rat eye and brain during development (embryonic day 10 [e10]-postnatal day 7 [P7]) by in situ hybridization and quantitative densitometry. The TTR mRNA was present in abundance in the primordial CP before organogenesis (e10-12), but in the eye, TTR mRNA first was detected at considerably lower levels after organogenesis (e16) and only in a subset of RPE cells in the equatorial region. The relative abundance of TTR mRNA in RPE rose gradually until e21, but on the first day of life a surge was seen, followed by stabilization at adult levels by P7. These findings suggest that the requirement for TTR in CP and RPE, and possibly its function, may differ during development. The postnatal surge in RPE TTR message levels raises the possibility that transcription of the TTR gene in the newborn animal may be responsive to newly encountered environmental stimuli in the perinatal period, such as incident light.