Retinoid metabolism in cultured human retinal pigment epithelium.
Ontology highlight
ABSTRACT:
SUBMITTER: Das SR
PROVIDER: S-EPMC1148879 | biostudies-other | 1988 Mar
REPOSITORIES: biostudies-other
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Project description:In vertebrate eyes, the retinal pigment epithelium (RPE) provides structural and functional homeostasis to the retina. The RPE takes up retinol (ROL) to be dehydrogenated and isomerized to 11-cis-retinaldehyde (11-cis-RAL), which is a functional photopigment in mammalian photoreceptors. As excessive ROL is toxic, the RPE must also establish mechanisms to protect against ROL toxicity. Here, we found that the levels of retinol dehydrogenases (RDHs) are commonly decreased in phosphatase tensin homolog (Pten)-deficient mouse RPE, which degenerates due to elevated ROL and that can be rescued by feeding a ROL-free diet. We also identified that RDH gene expression is regulated by forkhead box O (FOXO) transcription factors, which are inactivated by hyperactive Akt in the Pten-deficient mouse RPE. Together, our findings suggest that a homeostatic pathway comprising PTEN, FOXO, and RDH can protect the RPE from ROL toxicity.
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Project description:The first event in light perception is absorption of a photon by an opsin pigment, which induces isomerization of its 11-cis-retinaldehyde chromophore. Restoration of light sensitivity to the bleached opsin requires chemical regeneration of 11-cis-retinaldehyde through an enzymatic pathway called the visual cycle. The isomerase, which converts an all-trans-retinyl ester to 11-cis-retinol, has never been identified. Here, we performed an unbiased cDNA expression screen to identify this isomerase. We discovered that the isomerase is a previously characterized protein called Rpe65. We confirmed our identification of the isomerase by demonstrating catalytic activity in mammalian and insect cells that express Rpe65. Mutations in the human RPE65 gene cause a blinding disease of infancy called Leber congenital amaurosis. Rpe65 with the Leber-associated C330Y and Y368H substitutions had no isomerase activity. Identification of Rpe65 as the isomerase explains the phenotypes in rpe65-/- knockout mice and in humans with Leber congenital amaurosis.
| S-EPMC2748856 | biostudies-literature
Project description:I. Exp Design 1. Type of experiment: Comparison of native versus cultured RPE cells 2. Experimental factors: Native RPE versus ARPE-19 cells grown on different matrices 3. How many hybridizations in exp: 21 4. If a common reference used for all the hybs: no 5. Quality control steps: three independent arrays for each condition 6. Description: The expression profile of ARPE-19 cells grown on different matrices were compared to morphologically normal native macular RPE cells that were laser capture microdissected from 3 donors. II. Samples used, extract prep, and labeling 1. Biosource: Human donor globes from NDRI (63, 71, 74 years old) and ARPE-19 cells. 2. Manipulations: Human donor globes were cryopreserved, and morphologically normal RPE cells from the macula were laser capture micodissected. ARPE-19 cells were grown on different matrices (plastic, Matrigel, collagen I, collagen IV, laminin, and fibronectin). 3. Extract preparation: Total RNA from cells were extracted with the RNeasy kit (Qiagen) using the manufacturerâs instructions. 4. Labeling protocol: Total RNA from cells was reverse transcribed with 33P-dCTP and 33P-dATP, and second strand cDNA was labeled with 33P-dCTP and 33P-dATP. 5. No external controls were added. III. Hybridization procedures and parameters 1. Sample, array type, batch and serial # used 2. Hybridization protocol: Hybridization was carried out using the manufacturerâs recommendations. Arrays were prehybridized with Microhyb solution containing denatured Cot-1 DNA and poly dA at 42oC for two hours. Hybridization was carried out at 42oC overnight using a hybridization oven set at 8-10 rpm. Arrays were washed twice at 50oC for 20 minutes using 2x SSC, 1%SDS and once at room temperature for 15 minutes using 0.5x SSC, 1%SDS. IV. Measurement data and specifications of data processing 1,2. Arrays were exposed to a phosphorimaging screen for 3 days and scanned at 50 mm resolution with a BioRad FX Pro-Plus phosphorimager. TIFF images from the phosphorimager were exported into ResGen Pathways 3 software for analysis. 3. Data processing: A gene was expressed if its background subtracted intensity was greater than 1.4 fold background. The data were normalized using a simple global scaling procedure, and Cluster/Treeview and Statistical Analysis of Microarrays (SAM version 1.12) programs were used for analysis.
2010-06-05 | E-GEOD-741 | biostudies-arrayexpress
Project description:I. Exp Design 1. Type of experiment: Comparison of native versus cultured RPE cells 2. Experimental factors: Native RPE versus ARPE-19 cells grown on different matrices 3. How many hybridizations in exp: 21 4. If a common reference used for all the hybs: no 5. Quality control steps: three independent arrays for each condition 6. Description: The expression profile of ARPE-19 cells grown on different matrices were compared to morphologically normal native macular RPE cells that were laser capture microdissected from 3 donors. II. Samples used, extract prep, and labeling 1. Biosource: Human donor globes from NDRI (63, 71, 74 years old) and ARPE-19 cells. 2. Manipulations: Human donor globes were cryopreserved, and morphologically normal RPE cells from the macula were laser capture micodissected. ARPE-19 cells were grown on different matrices (plastic, Matrigel, collagen I, collagen IV, laminin, and fibronectin). 3. Extract preparation: Total RNA from cells were extracted with the RNeasy kit (Qiagen) using the manufacturer’s instructions. 4. Labeling protocol: Total RNA from cells was reverse transcribed with 33P-dCTP and 33P-dATP, and second strand cDNA was labeled with 33P-dCTP and 33P-dATP. 5. No external controls were added. III. Hybridization procedures and parameters 1. Sample, array type, batch and serial # used 2. Hybridization protocol: Hybridization was carried out using the manufacturer’s recommendations. Arrays were prehybridized with Microhyb solution containing denatured Cot-1 DNA and poly dA at 42oC for two hours. Hybridization was carried out at 42oC overnight using a hybridization oven set at 8-10 rpm. Arrays were washed twice at 50oC for 20 minutes using 2x SSC, 1%SDS and once at room temperature for 15 minutes using 0.5x SSC, 1%SDS. IV. Measurement data and specifications of data processing 1,2. Arrays were exposed to a phosphorimaging screen for 3 days and scanned at 50 mm resolution with a BioRad FX Pro-Plus phosphorimager. TIFF images from the phosphorimager were exported into ResGen Pathways 3 software for analysis. 3. Data processing: A gene was expressed if its background subtracted intensity was greater than 1.4 fold background. The data were normalized using a simple global scaling procedure, and Cluster/Treeview and Statistical Analysis of Microarrays (SAM version 1.12) programs were used for analysis. Keywords: other
2003-10-15 | GSE741 | GEO
Project description:The therapeutic potential of pluripotent stem cells is great as they promise to usher in a new era of medicine where cells or organs may be prescribed to replace dysfunctional tissue. At the forefront are efforts in the eye to develop this technology as it lends itself to in vivo monitoring and sophisticated non-invasive imaging modalities. In the retina, retinal pigment epithelium (RPE) is the most promising replacement cell as it has a single layer, is relatively simple to transplant, and is associated with several eye diseases. However, after transplantation, the cells may transform and cause complications. This transformation may be partially due to incomplete maturation. With the goal of learning how to mature RPE, we compared induced pluripotent stem cell-derived RPE (iPSC-RPE) cells with adult human primary RPE (ahRPE) cells and the immortalized human ARPE-19 line. We cultured ARPE-19, iPSC-RPE, and ahRPE cells for one month, and evaluated morphology, RPE marker staining, and transepithelial electrical resistance (TEER) as quality control indicators. We then isolated RNA for bulk RNA-sequencing and DNA for genotyping. We genotyped ahRPE lines for the top age-related macular degeneration (AMD) and proliferative vitreoretinopathy (PVR) risk allele polymorphisms. Transcriptome data verified that both adult and iPSC-RPE exhibit similar RPE gene expression signatures, significantly higher than ARPE-19. In addition, in iPSC-RPE, genes relating to stem cell maintenance, retina development, and muscle contraction were significantly upregulated compared to ahRPE. We compared ahRPE to iPSC-RPE in a model of epithelial-mesenchymal transition (EMT) and observed an increased sensitivity of iPSC-RPE to producing contractile aggregates in vitro which resembles incident reports upon transplantation. P38 inhibition was capable of inhibiting iPSC-RPE-derived aggregates. In summary, we find that the transcriptomic signature of iPSC-RPE conveys an immature RPE state which may be ameliorated by targeting "immature" gene regulatory networks.
| S-EPMC9461284 | biostudies-literature
Project description:Previous reports have described a decrease in retinal temperature and clinical improvement of wet age-related macular degeneration (AMD) after vitrectomy. We hypothesized that the retinal temperature decrease after vitrectomy plays a part in the suppression of wet AMD development. To test this hypothesis, we evaluated the temperature dependence of the expression of vascular endothelial growth factor-A (VEGF-A) and in vitro angiogenesis in retinal pigment epithelium (RPE).We cultured ARPE-19 cells at 37, 35, 33 and 31 °C and measured the expression of VEGF-A, VEGF-A splicing variants, and pigment epithelium-derived factor (PEDF). We performed an in vitro tube formation assay. The dehydrogenase activity was also evaluated at each temperature. Expression of VEGF-A significantly decreased with decreased temperature while PEDF expression did not. VEGF165 expression and in vitro angiogenesis also were temperature dependent. The dehydrogenase activity significantly decreased as the culture temperature decreased.RPE cultured under hypothermia that decreased cellular metabolism also had decreased VEGF-A and sustained PEDF expression, creating an anti-angiogenic environment. This mechanism may be associated with a beneficial effect after vitrectomy in patients with wet AMD.
| S-EPMC4518530 | biostudies-literature
Project description:BACKGROUND: To determine level, variability and functional annotation of gene expression of the human retinal pigment epithelium (RPE), the key tissue involved in retinal diseases like age-related macular degeneration and retinitis pigmentosa. Macular RPE cells from six selected healthy human donor eyes (aged 63-78 years) were laser dissected and used for 22k microarray studies (Agilent technologies). Data were analyzed with Rosetta Resolver, the web tool DAVID and Ingenuity software. RESULTS: In total, we identified 19,746 array entries with significant expression in the RPE. Gene expression was analyzed according to expression levels, interindividual variability and functionality. A group of highly (n = 2,194) expressed RPE genes showed an overrepresentation of genes of the oxidative phosphorylation, ATP synthesis and ribosome pathways. In the group of moderately expressed genes (n = 8,776) genes of the phosphatidylinositol signaling system and aminosugars metabolism were overrepresented. As expected, the top 10 percent (n = 2,194) of genes with the highest interindividual differences in expression showed functional overrepresentation of the complement cascade, essential in inflammation in age-related macular degeneration, and other signaling pathways. Surprisingly, this same category also includes the genes involved in Bruch's membrane (BM) composition. Among the top 10 percent of genes with low interindividual differences, there was an overrepresentation of genes involved in local glycosaminoglycan turnover. CONCLUSION: Our study expands current knowledge of the RPE transcriptome by assigning new genes, and adding data about expression level and interindividual variation. Functional annotation suggests that the RPE has high levels of protein synthesis, strong energy demands, and is exposed to high levels of oxidative stress and a variable degree of inflammation. Our data sheds new light on the molecular composition of BM, adjacent to the RPE, and is useful for candidate retinal disease gene identification or gene dose-dependent therapeutic studies.
| S-EPMC2679759 | biostudies-literature
Project description:Diabetic retinopathy is the most common complication of diabetes and a leading cause of blindness among working-age adults. Anatomical and functional changes occur in the retina and retinal pigment epithelium (RPE) prior to clinical symptoms of the disease. However, the molecular mechanisms responsible for these early changes, particularly in the RPE, remain unclear. To begin defining the molecular changes associated with pre-retinopathic diabetes, we conducted a comparative proteomics study of human donor RPE.The RPE was dissected from diabetic human donor eyes with no clinically apparent diabetic retinopathy (n=6) and from eyes of age-matched control donors (n=17). Soluble proteins were separated based upon their mass and charge using two-dimensional (2-D) gel electrophoresis. Protein spots were visualised with a fluorescent dye and spot densities were compared between diabetic and control gels. Proteins from spots with significant disease-related changes in density were identified using mass spectrometry.Analysis of 325 spots on 2-D gels identified 31 spots that were either up- or downregulated relative to those from age-matched control donors. The protein identity of 18 spots was determined by mass spectrometry. A majority of altered proteins belonged to two major functional groups, metabolism and chaperones, while other affected categories included protein degradation, synthesis and transport, oxidoreductases, cytoskeletal structure and retinoid metabolism.Changes identified in the RPE proteome of pre-retinopathic diabetic donor eyes compared with age-matched controls suggest specific cellular alterations that may contribute to diabetic retinopathy. Defining the pre-retinopathic changes affecting the RPE could provide important insight into the molecular events that lead to this disease.
| S-EPMC4397501 | biostudies-literature
Project description:PurposeLittle is known about the susceptibility of posterior segment tissues, particularly the human retinal pigment epithelium (hRPE), to Chlamydia trachomatis. The purpose of the study was to investigate the possibility of infecting the hRPE with Chlamydia trachomatis, and to examine the infectivity of different Chlamydia trachomatis clinical isolates for hRPE cells and the hRPE cell response to the infection.MethodsCultured hRPE and McCoy cells were inoculated with eight Chlamydia trachomatis (serovar E) clinical isolates at multiplicity of infection (MOI) of 2.0 or 0.3. To detect Chlamydia trachomatis, samples were stained immunohistochemically with anti-major outer membrane protein antibodies at 24h, 48h, and 72h postinoculation (PI). The changes in the expression of signaling molecules and proteins of cytoskeleton and extracellular matrix in hRPE cells were examined immunohistochemically.ResultsAll eight clinical isolates demonstrated ability to infect hRPE cells. At equal MOI of 0.3, the infectivity of Chlamydia trachomatis clinical isolates for RPE culture was found to be at least as high as that for McCoy cell culture. At 24h PI, the percentage of inclusion-containing cells varied from 1.5 ± 0.52 to 14.6 ± 3.3% in hRPE cell culture infected at MOI of 2.0 against 0.37 ± 0.34 to 8.9 ± 0.2% in McCoy cell culture infected at MOI of 0.3. Collagen type I, collagen type IV, basic fibroblast growth factor, transforming growth factor-beta and interleukin-8 expression at 48h PI were maximally increased, by 2.1-, 1.3-, 1.5-, 1.5- and 1.6-fold, respectively, in the Chlamydia trachomatis-infected compared with control hRPE cell culture specimens (P < 0.05).ConclusionsThis study, for the first time, proved the possibility of infecting hRPE cultured cells with Chlamydia trachomatis, which leads to proproliferative and proinflammatory changes in the expression of signaling molecules and extracellular matrix components.
| S-EPMC4633144 | biostudies-literature
Project description:Antibody-mediated rejection is characterized by donor-specific antibody produced by B cells. However, to our knowledge, B cell invasion and antibody in the inflamed retina after transplantation of retinal pigment epithelial (RPE) cells has not been reported. To determine if RPE transplantation could be performed using allografts, we established in vivo immune rejection models with induced pluripotent stem cell (iPSC)-RPE allografts and determined whether RPE-specific antibody could be detected in these models. We detected alloantibodies in the serum from recipient monkeys that had immune attacks in the retina in an immunofluorescent assay using the transplanted iPSC-RPE cells as the antigen. In addition to T cell and antigen-presenting cell immunity, peripheral blood cells and lymph nodes in animal models with allogeneic iPSC-RPE cells also had activated B cells, which were probably secreting alloantibodies. Using serum and transplanted cells, alloreactive antibody can be detected for the diagnosis of immune rejection after transplantation.
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