RESUMEN
Smith-Lemli-Opitz Syndrome (SLOS) results from mutations in the gene encoding the enzyme DHCR7, which catalyzes conversion of 7-dehydrocholesterol (7DHC) to cholesterol (CHOL). Rats treated with a DHCR7 inhibitor serve as a SLOS animal model, and exhibit progressive photoreceptor-specific cell death, with accumulation of 7DHC and oxidized sterols. To understand the basis of this cell type specificity, we performed transcriptomic analyses on a photoreceptor-derived cell line (661W), treating cells with two 7DHC-derived oxysterols, which accumulate in tissues and bodily fluids of SLOS patients and in the rat SLOS model, as well as with CHOL (negative control), and evaluated differentially expressed genes (DEGs) for each treatment. Gene enrichment analysis and compilation of DEG sets indicated that endoplasmic reticulum stress, oxidative stress, DNA damage and repair, and autophagy were all highly up-regulated pathways in oxysterol-treated cells. Detailed analysis indicated that the two oxysterols exert their effects via different molecular mechanisms. Changes in expression of key genes in highlighted pathways (Hmox1, Ddit3, Trib3, and Herpud1) were validated by immunofluorescence confocal microscopy. The results extend our understanding of the pathobiology of retinal degeneration and SLOS, identifying potential new druggable targets for therapeutic intervention into these and other related orphan diseases.
Asunto(s)
Células Fotorreceptoras de Vertebrados/patología , Síndrome de Smith-Lemli-Opitz/genética , Síndrome de Smith-Lemli-Opitz/patología , Transcriptoma , Animales , Línea Celular , Supervivencia Celular , Daño del ADN , Modelos Animales de Enfermedad , Ratones , Oxiesteroles , Células Fotorreceptoras de Vertebrados/metabolismo , Ratas , Síndrome de Smith-Lemli-Opitz/inducido químicamenteRESUMEN
Treatment of rats with the cholesterol pathway inhibitor AY9944 produces an animal model of Smith-Lemli-Opitz syndrome (SLOS), an autosomal recessive disease caused by defective cholesterol synthesis. This SLOS rat model undergoes progressive and irreversible degeneration of the neural retina, with associated pathological features of the retinal pigmented epithelium (RPE). Here, we provide further insights into the mechanism involved in the RPE pathology. In the SLOS rat model, markedly increased RPE apical autofluorescence is observed, compared to untreated animals, which correlates with increased levels of A2E and other bisretinoids. Utilizing cultured human induced pluripotent stem cell (iPSC)- derived SLOS RPE cells, we found significantly elevated steady-state levels of 7-dehydrocholesterol (7DHC) and decreased cholesterol levels (key biochemical hallmarks of SLOS). Western blot analysis revealed altered levels of the macroautophagy/autophagy markers MAP1LC3B-II and SQSTM1/p62, and build-up of ubiquitinated proteins. Accumulation of immature autophagosomes was accompanied by inefficient degradation of phagocytized, exogenously supplied retinal rod outer segments (as evidenced by persistence of the C-terminal 1D4 epitope of RHO [rhodopsin]) in SLOS RPE compared to iPSC-derived normal human control. SLOS RPE cells exhibited lysosomal pH levels and CTSD activity within normal physiological limits, thus discounting the involvement of perturbed lysosomal function. Furthermore, 1D4-positive phagosomes that accumulated in the RPE in both pharmacological and genetic rodent models of SLOS failed to fuse with lysosomes. Taken together, these observations suggest that defective phagosome maturation underlies the observed RPE pathology. The potential relevance of these findings to SLOS and the requirement of cholesterol for phagosome maturation are discussed.
Asunto(s)
Fagosomas/metabolismo , Epitelio Pigmentado de la Retina/patología , Síndrome de Smith-Lemli-Opitz/patología , Animales , Biomarcadores/metabolismo , Catepsina D/metabolismo , Bovinos , Técnicas de Cultivo de Célula , Deshidrocolesteroles/metabolismo , Modelos Animales de Enfermedad , Humanos , Lisosomas/metabolismo , Fusión de Membrana , Fagocitosis , Biosíntesis de Proteínas , Ratas , Epitelio Pigmentado de la Retina/metabolismo , Retinoides/metabolismo , Segmento Externo de la Célula en Bastón/metabolismo , Síndrome de Smith-Lemli-Opitz/genética , Transcripción Genética , Proteínas Ubiquitinadas/metabolismo , Diclorhidrato de trans-1,4-Bis(2-clorobenzaminometil)ciclohexanoRESUMEN
The transient receptor potential cation channel mucolipin 1 (TRPML1) channel is a conduit for lysosomal calcium efflux, and channel activity may be affected by lysosomal contents. The lysosomes of retinal pigmented epithelial (RPE) cells are particularly susceptible to build-up of lysosomal waste products because they must degrade the outer segments phagocytosed daily from adjacent photoreceptors; incomplete degradation leads to accumulation of lipid waste in lysosomes. This study asks whether stimulation of TRPML1 can release lysosomal calcium in RPE cells and whether such release is affected by lysosomal accumulations. The TRPML agonist ML-SA1 raised cytoplasmic calcium levels in mouse RPE cells, hesRPE cells, and ARPE-19 cells; this increase was rapid, robust, reversible, and reproducible. The increase was not altered by extracellular calcium removal or by thapsigargin but was eliminated by lysosomal rupture with glycyl-l-phenylalanine-ß-naphthylamide. Treatment with desipramine to inhibit acid sphingomyelinase or YM201636 to inhibit PIKfyve also reduced the cytoplasmic calcium increase triggered by ML-SA1, whereas RPE cells from TRPML1-/- mice showed no response to ML-SA1. Cotreatment with chloroquine and U18666A induced formation of neutral, autofluorescent lipid in RPE lysosomes and decreased lysosomal Ca2+ release. Lysosomal Ca2+ release was also impaired in RPE cells from the ATP-binding cassette, subfamily A, member 4-/- mouse model of Stargardt's retinal dystrophy. Neither TRPML1 mRNA nor total lysosomal calcium levels were altered in these models, suggesting a more direct effect on the channel. In summary, stimulation of TRPML1 elevates cytoplasmic calcium levels in RPE cells, but this response is reduced by lysosomal accumulation.-Gómez, N. M., Lu, W. Lim, J. C., Kiselyov, K., Campagno, K. E., Grishchuk, Y., Slaugenhaupt, S. A., Pfeffer, B., Fliesler, S. J., Mitchell, C. H. Robust lysosomal calcium signaling through channel TRPML1 is impaired by lysosomal lipid accumulation.
Asunto(s)
Señalización del Calcio , Metabolismo de los Lípidos , Lisosomas/metabolismo , Epitelio Pigmentado de la Retina/metabolismo , Canales de Potencial de Receptor Transitorio/metabolismo , Animales , Calcio/metabolismo , Línea Celular , Modelos Animales de Enfermedad , Humanos , Lisosomas/patología , Degeneración Macular/congénito , Degeneración Macular/genética , Degeneración Macular/metabolismo , Degeneración Macular/patología , Ratones , Ratones Noqueados , Ftalimidas/farmacología , Quinolinas/farmacología , Epitelio Pigmentado de la Retina/patología , Enfermedad de Stargardt , Canales de Potencial de Receptor Transitorio/agonistas , Canales de Potencial de Receptor Transitorio/genéticaRESUMEN
A duplex fluorescence assay to assess the viability of cells cultured in multi-well plates is described, which can be carried out in the original culture plate using a plate reader, without exchanges of culture or assay medium, or transfer of cells or cell supernatant. The method uses freshly prepared reagents and does not rely on a proprietary, commercially supplied kit. Following experimental treatment, calcein acetoxymethyl ester (CaAM) is added to each well of cultured cells; after 30 min, the fluorescence intensity (emission λmax â¼ 530 nm) is measured. The signal is due to formation of calcein, which is produced from CaAM by action of esterase activity found in intact live cells. Since live cells may express plasma membrane multidrug transport proteins, especially of the ABC transporter family, the CaAM incubation is carried out in the presence of an inhibitor of this efflux process, thereby improving the dynamic range of the assay. Next, SYTOX® Orange (SO) is added to the culture wells, and, after a 30-min incubation, fluorescence intensity (emission λmax â¼ 590 nm) is measured again. SO is excluded from cells that have an intact plasma membrane, but penetrates dead/dying cells and can diffuse into the nucleus, where it binds to and forms a fluorescent complex with DNA. The CaAM already added to the wells causes no interference with the latter fluorescent signal. At the conclusion of the duplex assay, both live and dead cells remain in the culture wells and can be documented by digital imaging to demonstrate correlation of cellular morphology with the assay output. Two examples of the application of this method are provided, using cytotoxic compounds having different mechanisms of action.
Asunto(s)
Células Fotorreceptoras/citología , Transportadoras de Casetes de Unión a ATP/metabolismo , Derivados del Benceno/farmacología , Bioensayo , Muerte Celular/fisiología , Línea Celular , Supervivencia Celular/fisiología , Células Cultivadas , Dimetilsulfóxido/farmacología , Fluoresceínas/química , Fluorescencia , Colorantes Fluorescentes/química , Humanos , Microscopía Fluorescente , Compuestos Orgánicos/química , Células Fotorreceptoras/efectos de los fármacos , Células Fotorreceptoras/metabolismo , Saponinas/farmacología , Estaurosporina/farmacologíaRESUMEN
PURPOSE: The retinal pigment epithelium (RPE) tight junctions play a pivotal role in maintaining the homeostatic environment of the neural retina. Herein, we investigated the role of X-box binding protein 1 (XBP1), an endoplasmic reticulum (ER) stress-responsive transcription factor, in regulation of RPE tight junctions. METHODS: Human RPE cell line (ARPE-19) and primary primate RPE cells were used for in vitro experiments and RPE-specific XBP1 knockout (KO) mice were used for in vivo study. Endoplasmic reticulum stress was induced by a sublethal dose of thapsigargin or tunicamycin. XBP1 activation was manipulated by IRE inhibitor 4µ8C, which suppresses XBP1 mRNA splicing. The integrity of tight junctions and the involvement of calcium-dependent RhoA/Rho kinase pathway were examined. RESULTS: Induction of ER stress by thapsigargin, but not tunicamycin, disrupted RPE tight junctions in ARPE-19 cells. Inhibition of XBP1 activation by 4µ8C resulted in a remarkable downregulation of tight junction proteins (ZO-1 and occludin) and defects in tight junction formation in the presence or absence of ER stress inducers. Overexpression of active XBP1 partially reversed 4µ8C-induced anomalies in tight junctions. Mechanistically, XBP1 inhibition resulted in increased intracellular Ca2+ concentration, upregulation of RhoA expression, redistribution of F-actin, and tight junction damage, which was attenuated by Rho kinase inhibitor Y27632. In vivo, deletion of XBP1 in the RPE resulted in defective RPE tight junctions accompanied by increased VEGF expression. CONCLUSIONS: Taken together, these results suggest a protective role of XBP1 in maintaining RPE tight junctions possibly through regulation of calcium-dependent RhoA/Rho kinase signaling and actin cytoskeletal reorganization.
Asunto(s)
Regulación de la Expresión Génica , ARN Mensajero/genética , Degeneración Retiniana/genética , Epitelio Pigmentado de la Retina/metabolismo , Proteína 1 de Unión a la X-Box/genética , Animales , Western Blotting , Supervivencia Celular , Células Cultivadas , Modelos Animales de Enfermedad , Estrés del Retículo Endoplásmico , Humanos , Macaca mulatta , Ratones , Ratones Noqueados , Reacción en Cadena en Tiempo Real de la Polimerasa , Degeneración Retiniana/metabolismo , Degeneración Retiniana/patología , Epitelio Pigmentado de la Retina/patología , Uniones Estrechas/genética , Uniones Estrechas/metabolismo , Proteína 1 de Unión a la X-Box/biosíntesisRESUMEN
Modification of proteins by 4-hydroxy-2-nonenal (HNE), a reactive by-product of ω6 polyunsaturated fatty acid oxidation, on specific amino acid residues is considered a biomarker for oxidative stress, as occurs in many metabolic, hereditary, and age-related diseases. HNE modification of amino acids can occur either via Michael addition or by formation of Schiff-base adducts. These modifications typically occur on cysteine (Cys), histidine (His), and/or lysine (Lys) residues, resulting in an increase of 156 Da (Michael addition) or 138 Da (Schiff-base adducts), respectively, in the mass of the residue. Here, we employed biochemical and mass spectrometry (MS) approaches to determine the MS "signatures" of HNE-modified amino acids, using lysozyme and BSA as model proteins. Using direct infusion of unmodified and HNE-modified lysozyme into an electrospray quadrupole time-of-flight mass spectrometer, we were able to detect up to seven HNE modifications per molecule of lysozyme. Using nanoLC-MS/MS, we found that, in addition to N-terminal amino acids, Cys, His, and Lys residues, HNE modification of arginine (Arg), threonine (Thr), tryptophan (Trp), and histidine (His) residues can also occur. These sensitive and specific methods can be applied to the study of oxidative stress to evaluate HNE modification of proteins in complex mixtures from cells and tissues under diseased versus normal conditions.
Asunto(s)
Aldehídos/análisis , Muramidasa/análisis , Estrés Oxidativo , Proteómica/métodos , Albúmina Sérica Bovina/análisis , Espectrometría de Masas en Tándem/métodos , Aldehídos/química , Aldehídos/metabolismo , Aminoácidos/análisis , Aminoácidos/química , Aminoácidos/metabolismo , Animales , Bovinos , Cromatografía Liquida/métodos , Modelos Químicos , Muramidasa/química , Muramidasa/metabolismo , Nanotecnología/métodos , Sensibilidad y Especificidad , Albúmina Sérica Bovina/química , Albúmina Sérica Bovina/metabolismoRESUMEN
Tissue accumulation of 7-dehydrocholesterol (7DHC) is a hallmark of Smith-Lemli-Opitz Syndrome (SLOS), a human inborn error of the cholesterol (CHOL) synthesis pathway. Retinal 7DHC-derived oxysterol formation occurs in the AY9944-induced rat model of SLOS, which exhibits a retinal degeneration characterized by selective loss of photoreceptors and associated functional deficits, Müller cell hypertrophy, and engorgement of the retinal pigment epithelium (RPE) with phagocytic inclusions. We evaluated the relative effects of four 7DHC-derived oxysterols on three retina-derived cell types in culture, with respect to changes in cellular morphology and viability. 661W (photoreceptor-derived) cells, rMC-1 (Müller glia-derived) cells, and normal diploid monkey RPE (mRPE) cells were incubated for 24 h with dose ranges of either 7-ketocholesterol (7kCHOL), 5,9-endoperoxy-cholest-7-en-3ß,6α-diol (EPCD), 3ß,5α-dihydroxycholest-7-en-6-one (DHCEO), or 4ß-hydroxy-7-dehydrocholesterol (4HDHC); CHOL served as a negative control (same dose range), along with appropriate vehicle controls, while staurosporine (Stsp) was used as a positive cytotoxic control. For 661W cells, the rank order of oxysterol potency was: EPCD > 7kCHOL >> DHCEO > 4HDHC ≈ CHOL. EC50 values were higher for confluent vs. subconfluent cultures. 661W cells exhibited much higher sensitivity to EPCD and 7kCHOL than either rMC-1 or mRPE cells, with the latter being the most robust when challenged, either at confluence or in sub-confluent cultures. When tested on rMC-1 and mRPE cells, EPCD was again an order of magnitude more potent than 7kCHOL in compromising cellular viability. Hence, 7DHC-derived oxysterols elicit differential cytotoxicity that is dose-, cell type-, and cell density-dependent. These results are consistent with the observed progressive, photoreceptor-specific retinal degeneration in the rat SLOS model, and support the hypothesis that 7DHC-derived oxysterols are causally linked to that retinal degeneration as well as to SLOS.
Asunto(s)
Deshidrocolesteroles/farmacología , Células Ependimogliales/efectos de los fármacos , Células Epiteliales/efectos de los fármacos , Oxiesteroles/farmacología , Células Fotorreceptoras de Vertebrados/efectos de los fármacos , Retina/citología , Animales , Recuento de Células , Muerte Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Deshidrocolesteroles/química , Deshidrocolesteroles/metabolismo , Macaca mulatta , Modelos Animales , Ratas , Retina/metabolismo , Epitelio Pigmentado de la Retina/citologíaRESUMEN
This series of review articles highlights how in vitro models of RPE can be effectively used to understand essential functions of the RPE that are not only fundamental to epithelial biology, but also have direct relevance to the visual system. The issue contains reviews from experts in the field covering aspects of basic cell and epithelial biology, namely: the barrier properties of the RPE (Rizzolo, 2014), epithelial polarity (Lehmann et al., 2014), cytoskeleton (Bonilha, 2014), and lysosomes (Guha et al., 2014), as well as properties more unique to the RPE, e.g., vitamin A metabolism (Hu and Bok, 2014), bioenergetics (Adijanto and Philp, 2014), phagocytosis (Mazzoni et al., 2014), ion transport (Reichhart and Strauß, 2014), and melanin/lipofuscin (Boulton, 2014).
Asunto(s)
Modelos Biológicos , Epitelio Pigmentado de la Retina , Células Cultivadas/fisiología , Humanos , Epitelio Pigmentado de la Retina/citología , Epitelio Pigmentado de la Retina/fisiologíaRESUMEN
Smith-Lemli-Opitz syndrome (SLOS) is one of the most common recessive human disorders and is characterized by multiple congenital malformations as well as neurosensory and cognitive abnormalities. A rat model of SLOS has been developed that exhibits progressive retinal degeneration and visual dysfunction; however, the molecular events underlying the degeneration and dysfunction remain poorly understood. Here, we employed a well-controlled, ion-current-based approach to compare retinas from the SLOS rat model to retinas from age- and sex-matched control rats (n = 5/group). Retinas were subjected to detergent extraction and subsequent precipitation and on-pellet-digestion procedures and then were analyzed on a long, heated column (75 cm, with small particles) with a 7-h gradient. The high analytical reproducibility of the overall proteomics procedure enabled reliable expression profiling. In total, 1,259 unique protein groups, ~40% of which were membrane proteins, were quantified under highly stringent criteria, including a peptide false discovery rate of 0.4%, with high quality ion-current data (e.g. signal-to-noise ratio ≥ 10) obtained independently from at least two unique peptides for each protein. The ion-current-based strategy showed greater quantitative accuracy and reproducibility over a parallel spectral counting analysis. Statistically significant alterations of 101 proteins were observed; these proteins are implicated in a variety of biological processes, including lipid metabolism, oxidative stress, cell death, proteolysis, visual transduction, and vesicular/membrane transport, consistent with the features of the associated retinal degeneration in the SLOS model. Selected targets were further validated by Western blot analysis and correlative immunohistochemistry. Importantly, although photoreceptor cell death was validated by TUNEL analysis, Western blot and immunohistochemical analyses suggested a caspase-3-independent pathway. In total, these results provide compelling new evidence implicating molecular changes beyond the initial defect in cholesterol biosynthesis in this retinal degeneration model, and they might have broader implications with respect to the pathobiological mechanism underlying SLOS.
Asunto(s)
Proteínas del Ojo/aislamiento & purificación , Proteínas de la Membrana/aislamiento & purificación , Proteoma/aislamiento & purificación , Retina/metabolismo , Degeneración Retiniana/genética , Síndrome de Smith-Lemli-Opitz/genética , Animales , Muerte Celular , Cromatografía de Fase Inversa , Modelos Animales de Enfermedad , Proteínas del Ojo/genética , Proteínas del Ojo/metabolismo , Femenino , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Humanos , Inmunohistoquímica , Masculino , Espectrometría de Masas/instrumentación , Espectrometría de Masas/métodos , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Anotación de Secuencia Molecular , Proteoma/genética , Proteoma/metabolismo , Ratas , Reproducibilidad de los Resultados , Retina/química , Retina/patología , Degeneración Retiniana/metabolismo , Degeneración Retiniana/patología , Síndrome de Smith-Lemli-Opitz/metabolismo , Síndrome de Smith-Lemli-Opitz/patología , Visión Ocular/fisiologíaRESUMEN
PURPOSE: To assess in vitro myocilin (MYOC) expression in trabecular meshwork (TM) cells exposed to BOL-303242-X, a selective glucocorticoid receptor (GR) agonist (SEGRA), in comparison with dexamethasone (DEX), and prednisolone acetate (PA). METHODS: After drug treatment of monkey TM cultures, MYOC protein in conditioned media (CM) was measured by Western blot and densitometry. MYOC mRNA levels were analyzed by qRT-PCR. RU-486 was tested for antagonism of MYOC protein expression induced by DEX and BOL-303242-X. RESULTS: Baseline MYOC protein released into CM and MYOC mRNA were detected. DEX or PA elicited dose-dependent increases in MYOC in CM and also in MYOC mRNA. BOL-303242-X effects typified partial agonism, with significantly reduced MYOC protein and mRNA, compared with DEX. Maximum efficacy for BOL-303242-X was 53% of that for DEX. Mean EC(50) across all strains tested was lower, but not significantly different, for BOL-303242-X versus DEX. Compared with DEX, MYOC mRNA levels were significantly lower in BOL-303242-X-treated TM cells at the highest doses tested. EC(50)s for PA were higher than DEX, for both myocilin protein and mRNA. RU-486 displayed a dose-dependent antagonism to drug-induced increases in myocilin levels. CONCLUSIONS: In vitro quantitative assays of myocilin expression in TM cells can be used for characterizing anti-inflammatory drugs that are GR ligands. The results suggest that, compared with traditional ocular steroids, therapeutic doses of BOL-303242-X elicit a reduced myocilin expression profile in TM cells by virtue of the partial agonist properties of this compound.
Asunto(s)
Benzofuranos/farmacología , Proteínas del Citoesqueleto/genética , Proteínas del Citoesqueleto/metabolismo , Proteínas del Ojo/genética , Proteínas del Ojo/metabolismo , Glucocorticoides/farmacología , Glicoproteínas/genética , Glicoproteínas/metabolismo , Pentanoles/farmacología , Quinolinas/farmacología , Receptores de Glucocorticoides/agonistas , Malla Trabecular/efectos de los fármacos , Animales , Western Blotting , Células Cultivadas , Dexametasona/farmacología , Relación Dosis-Respuesta a Droga , Expresión Génica , Antagonistas de Hormonas/farmacología , Macaca mulatta , Mifepristona/farmacología , Prednisolona/análogos & derivados , Prednisolona/farmacología , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Malla Trabecular/metabolismoRESUMEN
BACKGROUND: The vitreous body is implicated in the etiology and pathology of a variety of retinal conditions. Many such conditions are treated surgically to remove the posterior vitreous from the inner limiting lamina (ILL) of the retina, and there is current interest in the adjunct use of enzymes for this purpose. To evaluate the efficacy of these agents in future preclinical studies, improved preservation and assessment methods were developed to establish a baseline histological profile of the vitreous and retina of the rabbit, to identify and distinguish artifactual vitreoretinal separation from authentic posterior vitreous detachment, and to preserve structural integrity while maintaining antigenicity for immunohistochemical analysis. METHODS: Two pigmented rabbits each underwent perfusion with one of three fixatives, either: (1) 10% neutral buffered formalin + cetylpyridinium chloride (NBF/CPC), (2) acid-ethanolic formalin + CPC (AEF/CPC), or (3) formaldehyde-glutaraldehyde + CPC (FG/CPC). An eye fixed in NBF/CPC by immersion, from an additional rabbit, was also included for comparison. Eyes were processed whole through paraffin infiltration. Treatments were assessed by immunohistochemical labeling for retinal and cortical vitreous (CV) markers. RESULTS: In contrast to immersion fixation, perfusion with either NBF/CPC or AEF/CPC maintained vitreous adherence to the ILL during histological processing. NBF/CPC proved best for highlighting intralaminar structure and for labeling type II collagen in the CV, particularly with antigen retrieval. AEF/CPC caused condensation of fibrillar elements in the CV. Collagen XVIII in the ILL was observed with AEF/CPC exclusively. Only retinal vessels near the optic nerve head were labeled for type IV collagen. The labeling of glia was useful for distinguishing between cellular and extracellular elements. GF/CPC hindered detection of collagen II and disrupted posterior segment structure. Expression of type II collagen extended from the ONH directly to CV affiliated with the central canal of Cloquet, a feature characteristic of rabbit eyes. DISCUSSION: Careful tissue preservation and processing techniques minimize artifactual separation of the CV from the ILL. By optimizing the tissue architecture and antigenicity of the vitreoretinal complex, CV may be distinguished from the ILL immunohistochemically. The techniques described may be used to evaluate more effectively the utility of pharmacologic vitreolysis, using experimental animal models.
Asunto(s)
Inmunohistoquímica/métodos , Retina/citología , Fijación del Tejido/métodos , Vitrectomía/métodos , Cuerpo Vítreo/citología , Aldehídos/farmacología , Animales , Antígenos/metabolismo , Artefactos , Cetilpiridinio/farmacología , Colágeno Tipo II/metabolismo , Detergentes/farmacología , Enzimas/farmacología , Lectinas , Masculino , Modelos Animales , Nervio Óptico/citología , Nervio Óptico/metabolismo , Nervio Óptico/cirugía , Conejos , Retina/metabolismo , Retina/cirugía , Cuerpo Vítreo/metabolismo , Cuerpo Vítreo/cirugía , Desprendimiento del VítreoRESUMEN
PURPOSE: To document the expression of mRNA for transthyretin (TTR) and retinol binding protein (RBP) in native and cultured Rhesus monkey retinal pigmented epithelium (RPE); to compare mRNA transcripts for these two proteins expressed in RPE with those found in whole monkey liver and brain; to demonstrate the secretion of TTR by RPE during short-term maintenance in a protein-free, defined medium, as a manifestation of the differentiated state of these cells in vitro. METHODS: Total RNA was isolated from cultured RPE in first passage, after incubation for eight days in defined, protein-free medium. Conditioned medium was collected for western analysis at this time. Total RNA was also extracted from RPE/choroid freshly dissected from monkey eyes. Using cDNA probes for human TTR and RBP, northern analysis was performed on the total RNA from fresh and cultured RPE samples, together with poly(A+) mRNA purified from monkey liver and brain. RESULTS: Conditioned medium from RPE yielded TTR protein of the expected monomer subunit molecular size. The TTR secreted de novo from the cultured cells was detectable in the absence of biosynthetic labeling. With the exception of some extremely low abundance transcripts expressed in cultured RPE, all samples contained a single 900 bp transcript for TTR. Based on relative amounts of actual message, RPE ranks higher than liver in abundance of TTR mRNA. In contrast, both native monkey RPE and cultured RPE cells expressed comparatively low levels of mRNA for RBP. All samples displayed a single RBP mRNA transcript at 1100 bp. CONCLUSIONS: Our results indicate that TTR is a significant gene product of the RPE, and may be considered as a marker for a differentiated phenotype for these cells in culture. There is increased recognition of various forms of ocular pathology associated with mutations or other malfunctions involving TTR and RBP, warranting a greater understanding of mechanisms of transcriptional and translational control for these two proteins.
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Proteínas Portadoras/genética , Regulación de la Expresión Génica/fisiología , Epitelio Pigmentado Ocular/metabolismo , Prealbúmina/genética , ARN Mensajero/metabolismo , Animales , Northern Blotting , Western Blotting , Encéfalo/metabolismo , Proteínas Portadoras/metabolismo , Células Cultivadas , Electroforesis en Gel de Poliacrilamida , Marcadores Genéticos , Hígado/metabolismo , Macaca mulatta , Prealbúmina/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Pigment epithelium-derived factor (PEDF) is an extracellular protein derived from the retinal pigment epithelium (RPE), a tissue formed by polarized cells that release growth and trophic factors in a directional fashion. We have investigated the distribution and directional release of PEDF protein by the monkey RPE. We established primary cultures of monkey RPE cells that expressed the PEDF gene, and that synthesized and secreted the PEDF protein. Northern analysis of RPE cultures and monkey ocular tissues showed that PEDF transcripts were highly expressed in RPE as compared with several other monkey ocular tissues, being even more abundant in cultured cells than they were in the native RPE. The differentiated RPE cells in culture secreted protein that shared the immunological, biochemical and biological characteristics of PEDF. The overall PEDF levels in the RPE conditioned media reached 6.5 mg ml- after 8 days in culture (i.e. 1.1 pg of PEDF per RPE cell). RPE cells were cultivated on permeable supports as monolayers forming a barrier between apical and basal compartments. Apical and basal culture media were sampled at three or four-day intervals for 18 cycles, and the PEDF content was quantified. Most of the PEDF protein was significantly higher in the apical than in the basal medium (>4 times) at the initial recovery intervals, to be detected only in the apical medium at the latter intervals. In the native monkey eye, the concentration of soluble PEDF in the interphotoreceptor matrix (144 nM) was 7-fold and 25-fold greater than in vitreous and aqueous, respectively. PEDF was abundant in the interphotoreceptor matrix surrounding rod and cone outer segments, and was detectable at lower levels in the RPE as visualized by confocal microscopy. We concluded that PEDF synthesized by the RPE is secreted preferentially from the apical surface and is distributed apically to the RPE bordering the outer segments of photoreceptors. PEDF can be a useful marker for RPE polarization and differentiation. The polarization of RPE may be an important mechanism to control PEDF secretion and our results offer interesting possibilities on regulation of PEDF.