Ex-vivo models of the Retinal Pigment Epithelium (RPE) in long-term culture faithfully recapitulate key structural and physiological features of native RPE.
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ABSTRACT:
SUBMITTER: Lynn SA
PROVIDER: S-EPMC5545183 | biostudies-literature | 2017 Aug
REPOSITORIES: biostudies-literature
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Ex-vivo models of the Retinal Pigment Epithelium (RPE) in long-term culture faithfully recapitulate key structural and physiological features of native RPE.
Lynn Savannah A SA Ward Gareth G Keeling Eloise E Scott Jenny A JA Cree Angela J AJ Johnston David A DA Page Anton A Cuan-Urquizo Enrique E Bhaskar Atul A Grossel Martin C MC Tumbarello David A DA Newman Tracey A TA Lotery Andrew J AJ Ratnayaka J Arjuna JA
Tissue & cell 20170619 4
The Retinal Pigment Epithelium (RPE) forms the primary site of pathology in several blinding retinopathies. RPE cultures are being continuously refined so that dynamic disease processes in this important monolayer can be faithfully studied outside the eye over longer periods. The RPE substrate, which mimics the supportive Bruch's membrane (BrM), plays a key role in determining how well in-vitro cultures recapitulate native RPE cells. Here, we evaluate how two different types of BrM substrates; ( ...[more]
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Project description:BackgroundAge-related changes in the retina are often accompanied by visual impairment but their mechanistic details remain poorly understood.MethodologyProteomic studies were pursued toward a better molecular understanding of retinal pigment epithelium (RPE) aging mechanisms. RPE cells were isolated from young adults (3-4 month-old) and old (24-25 month-old) F344BN rats, and separated into subcellular fractions containing apical microvilli (MV) and RPE cell bodies (CB) lacking their apical microvilli. Proteins were extracted in detergent, separated by SDS-PAGE, digested in situ with trypsin and analyzed by LC MS/MS. Select proteins detected in young and old rat RPE were further studied using immunofluorescence and Western blot analysis.Principal findingsA total of 356 proteins were identified in RPE MV from young and 378 in RPE MV from old rats, 48% of which were common to each age group. A total of 897 proteins were identified in RPE CB from young rats and 675 in old CB, 56% of which were common to each age group. Several of the identified proteins, including proteins involved in response to oxidative stress, displayed both quantitative and qualitative changes in overall abundance during RPE aging. Numerous proteins were identified for the first time in the RPE. One such protein, collectrin, was localized to the apical membrane of apical brush border of proximal tubules where it likely regulates several amino acid transporters. Elsewhere, collectrin is involved in pancreatic β cell proliferation and insulin secretion. In the RPE, collectrin expression was significantly modulated during RPE aging. Another age-regulated, newly described protein was DJ-1, a protein extensively studied in brain where oxidative stress-related functions have been described.Conclusions/significanceThe data presented here reveals specific changes in the RPE during aging, providing the first protein database of RPE aging, which will facilitate future studies of age-related retinal diseases.
| S-EPMC3372495 | biostudies-literature
Project description:PurposeWe tested what native features have been preserved with a new culture protocol for adult human RPE.MethodsWe cultured RPE from adult human eyes. Standard protocols for immunohistochemistry, electron microscopy, electrophysiology, fluid transport, and ELISA were used.ResultsConfluent monolayers of adult human RPE cultures exhibit characteristics of native RPE. Immunohistochemistry demonstrated polarized expression of RPE markers. Electron microscopy illustrated characteristics of native RPE. The mean transepithelial potential (TEP) was 1.19 ± 0.24 mV (mean ± SEM, n = 31), apical positive, and the mean transepithelial resistance (RT) was 178.7 ± 9.9 Ω·cm2 (mean ± SEM, n = 31). Application of 100 μM adenosine triphosphate (ATP) apically increased net fluid absorption (Jv) by 6.11 ± 0.53 μL·cm2·h-1 (mean ± SEM, n = 6) and TEP by 0.33 ± 0.048 mV (mean ± SEM, n = 25). Gene expression of cultured RPE was comparable to native adult RPE (n = 5); however, native RPE RNA was harvested between 24 and 40 hours after death and, therefore, may not accurately reflect healthy native RPE. Vascular endothelial growth factor secreted preferentially basally 2582 ± 146 pg/mL/d, compared to an apical secretion of 1548 ± 162 pg/mL/d (n = 14, P < 0.01), while PEDF preferentially secreted apically 1487 ± 280 ng/mL/d compared to a basolateral secretion of 864 ± 132 ng/mL/d (n = 14, P < 0.01).ConclusionsThe new culture model preserves native RPE morphology, electrophysiology, and gene and protein expression patterns, and may be a useful model to study RPE physiology, disease, and transplantation.
| S-EPMC4640474 | biostudies-literature
Project description:Patients with certain defects in the dehydrodolichyl diphosphate synthase (DHDDS) gene (RP59; OMIM #613861) exhibit classic symptoms of retinitis pigmentosa, as well as macular changes, suggestive of retinal pigment epithelium (RPE) involvement. The DHDDS enzyme is ubiquitously required for several pathways of protein glycosylation. We wish to understand the basis for selective ocular pathology associated with certain DHDDS mutations and the contribution of specific ocular cell types to the pathology of mutant Dhdds-mediated retinal degeneration. To circumvent embryonic lethality associated with Dhdds knockout, we generated a Cre-dependent knockout allele of murine Dhdds (Dhddsflx/flx). We used targeted Cre expression to study the importance of the enzyme in the RPE. Structural alterations of the RPE and retina including reduction in outer retinal thickness, cell layer disruption, and increased RPE hyper-reflectivity were apparent at one postnatal month. At three months, RPE and photoreceptor disruption was observed non-uniformly across the retina as well as RPE transmigration into the photoreceptor layer, external limiting membrane descent towards the RPE, and patchy loss of photoreceptors. Functional loss measured by electroretinography was consistent with structural loss showing scotopic a- and b-wave reductions of 83% and 77%, respectively, at three months. These results indicate that RPE dysfunction contributes to DHDDS mutation-mediated pathology and suggests a more complicated disease mechanism than simply disruption of glycosylation.
| S-EPMC7140717 | 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:Endophthalmitis is a severe infection accompanied by inflammation that affects the anterior and posterior parts of the eye. It is typically treated with a combination of antibiotics that cover various microorganisms. However, retinal pigment epithelium (RPE) cells are highly susceptible to damage from intravitreal injection therapy. This study aimed to investigate the impact of clinically relevant concentrations of meropenem (alone or in combination with vancomycin) on the viability and inflammation of RPE cells. In-vitro Study. RPE cells from passages 5-7 were treated with different concentrations of meropenem (1/4x, x, and 4x; [x = 16 mg/L]), vancomycin (30 mg/L), and meropenem (x) plus vancomycin for 24 h. The morphology assessment and MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay were performed to evaluate cytotoxicity due to drug treatment. Real-time PCR was used to measure the relative expression of apoptotic genes (BCL-2 and BAX) and inflammation biomarkers (IL-1b and IL-6). Meropenem (alone or in combination with vancomycin) did not have any significant effect on RPE cell morphology, density, and viability. Gene expression analysis confirmed these results, showing no significant changes in the BCL-2/BAX ratio in drug-treated RPE cells compared to controls. Treatment with meropenem significantly induced the expression of IL-1b at all studied concentrations. Additionally, at concentrations of x and 4x, it also significantly increased the expression of IL-6, which was dose-dependent. However, this effect was not observed with vancomycin alone or in combination with meropenem. The results of this study suggest that meropenem, either alone or in combination with vancomycin, does not induce RPE cytotoxicity. There was an upregulation of IL-1b and IL-6 in meropenem monotherapy, the clinical implication of which should be elucidated in future in-vivo or clinical studies.
| S-EPMC11292269 | biostudies-literature
Project description:Age-related macular degeneration (AMD) is a leading cause of vision loss and blindness among the elderly population in the industrialized world. One of the typical features of this pathology is the gradual death of retinal pigment epithelial (RPE) cells, which are essential for maintaining photoreceptor functions and survival. The etiology is multifactorial, and oxidative stress is clearly one of the key factors involved in disease pathogenesis (Plafker, Adv. Exp. Med. Biol. 664 (2010) 447-56; Qin, Drug Dev. Res. 68 (2007) 213-225). Recent work has revealed the presence of phosphorylated signaling proteins in the vitreous humour of patients affected by AMD or other retinal diseases. While the location of these signaling proteins is typically the cell membrane or intracellular compartments, vitreous samples were proven to be cell-free (Davuluri et al., Arch. Ophthalmol. 127 (2009) 613-21). To gain a better understanding of how these proteins can be shed into the vitreous, we used reverse phase protein arrays (RPMA) to analyze the protein and phosphoprotein content of exosomes shed by cultured ARPE-19 cells under oxidative stress conditions. Seventy two proteins were shown to be released by ARPE-19 cells and compartmentalized within exosomes. Forty one of them were selectively detected in their post-translationally modified form (i.e., phosphorylated or cleaved) for the first time in exosomes. Sets of these proteins were linked together reflecting activation of pathway units within exosomes. A subset of (phospho)proteins were altered in exosomes secreted by ARPE-19 cells subjected to oxidative stress, compared to that secreted by control/non stressed cells. Stress-altered exosome proteins were found to be involved in pathways regulating apoptosis/survival (i.e, Bak, Smac/Diablo, PDK1 (S241), Akt (T308), Src (Y416), Elk1 (S383), ERK 1/2 (T202/Y204)) and cell metabolism (i.e., AMPKα1 (S485), acetyl-CoA carboxylase (S79), LDHA). Exosomes may thus represent the conduit through which membrane and intracellular signaling proteins are released into the vitreous. Changes in their (phospho)protein content upon stress conditions suggest their possible role in mediating cell-cell signaling during physio-pathological events; furthermore, exosomes may represent a potential source of biomarkers.
| S-EPMC3727419 | 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 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:Age-related macular degeneration (AMD) is the leading cause of vision loss in the western world. Recent evidence suggests that RPE and photoreceptors have an interconnected metabolism and that mitochondrial damage in RPE is a trigger for degeneration in both RPE and photoreceptors in AMD. To test this hypothesis, this study was designed to induce mitochondrial damage in RPE in mice to determine whether this is sufficient to cause RPE and photoreceptor damage characteristic of AMD. In this study, we conditionally deleted the gene encoding the mitochondrial antioxidant enzyme, manganese superoxide dismutase (MnSOD encoded by Sod2) in the retinal pigment epithelium (RPE) of albino BALB/cJ mice. VMD2-Cre;Sod2flox/flox BALB/cJ mice were housed in either 12-h dark, 12-h 200 lux white lighting (normal light), or 12-h dark, 12-h <10 lux red lighting (dim light). Electroretinography (ERG) and spectral-domain optical coherence tomography (SD-OCT) were performed to assess retinal function and morphology. Immunofluorescence was used to examine protein expression; quantitative RT-PCR was used to measure gene expression. Sod2 knockout (KO) mice had reduced RPE function with age and increased oxidative stress compared to wild type (WT) controls as expected by the cell-specific deletion of Sod2. This was associated with alterations in RPE morphology and the structure and function of RPE mitochondria. In addition, data show a compensatory increase in RPE glycolytic metabolism. The metabolic shift in RPE correlated with severe disruption of photoreceptor mitochondria including a reduction in TOMM20 expression, mitochondrial fragmentation, and reduced COXIII/β-actin levels. These findings demonstrate that mitochondrial oxidative stress can lead to RPE dysfunction and metabolic reprogramming of RPE. Secondary to these changes, photoreceptors also undergo metabolic stress with increased mitochondrial damage. These data are consistent with the hypothesis of a linked metabolism between RPE and photoreceptors and suggest a mechanism of retinal degeneration in dry AMD.
| S-EPMC6488819 | biostudies-literature
Project description:Age-related macular degeneration (AMD) is characterized by the loss or dysfunction of retinal pigment epithelium (RPE) and is the most common cause of vision loss among the elderly. Stem-cell-based strategies, using human embryonic stem cells (hESCs) or human-induced pluripotent stem cells (hiPSCs), may provide an abundant donor source for generating RPE cells in cell replacement therapies. Despite a significant amount of research on deriving functional RPE cells from various stem cell sources, it is still unclear whether stem-cell-derived RPE cells fully mimic primary RPE cells. In this report, we demonstrate that functional RPE cells can be derived from multiple lines of hESCs and hiPSCs with varying efficiencies. Stem-cell-derived RPE cells exhibit cobblestone-like morphology, transcripts, proteins and phagocytic function similar to human fetal RPE (fRPE) cells. In addition, we performed global gene expression profiling of stem-cell-derived RPE cells, native and cultured fRPE cells, undifferentiated hESCs and fibroblasts to determine the differentiation state of stem-cell-derived RPE cells. Our data indicate that hESC-derived RPE cells closely resemble human fRPE cells, whereas hiPSC-derived RPE cells are in a unique differentiation state. Furthermore, we identified a set of 87 signature genes that are unique to human fRPE and a majority of these signature genes are shared by stem-cell-derived RPE cells. These results establish a panel of molecular markers for evaluating the fidelity of human pluripotent stem cell to RPE conversion. This study contributes to our understanding of the utility of hESC/hiPSC-derived RPE in AMD therapy.
| S-EPMC3115666 | biostudies-literature
Project description:BackgroundDJ-1 is found in many tissues, including the brain, where it has been extensively studied due to its association with Parkinson's disease. DJ-1 functions as a redox-sensitive molecular chaperone and transcription regulator that robustly protects cells from oxidative stress.MethodologyRetinal pigment epithelial (RPE) cultures were treated with H2O2 for various times followed by biochemical and immunohistological analysis. Cells were transfected with adenoviruses carrying the full-length human DJ-1 cDNA and a mutant construct, which has the cysteine residues at amino acid 46, 53 and 106 mutated to serine (C to S) prior to stress experiments. DJ-1 localization, levels of expression and reactive oxygen species (ROS) generation were also analyzed in cells expressing exogenous DJ-1 under baseline and oxidative stress conditions. The presence of DJ-1 and oxidized DJ-1 was evaluated in human RPE total lysates. The distribution of DJ-1 was assessed in AMD and non-AMD cryosectionss and in isolated human Bruch's membrane (BM)/choroid from AMD eyes.Principal findingsDJ-1 in RPE cells under baseline conditions, displays a diffuse cytoplasmic and nuclear staining. After oxidative challenge, more DJ-1 was associated with mitochondria. Increasing concentrations of H2O2 resulted in a dose-dependent increase in DJ-1. Overexpression of DJ-1 but not the C to S mutant prior to exposure to oxidative stress led to significant decrease in the generation of ROS. DJ-1 and oxDJ-1 intensity of immunoreactivity was significantly higher in the RPE lysates from AMD eyes. More DJ-1 was localized to RPE cells from AMD donors with geographic atrophy and DJ-1 was also present in isolated human BM/choroid from AMD eyes.Conclusions/significanceDJ-1 regulates RPE responses to oxidative stress. Most importantly, increased DJ-1 expression prior to oxidative stress leads to decreased generation of ROS, which will be relevant for future studies of AMD since oxidative stress is a known factor affecting this disease.
| S-EPMC3699467 | biostudies-literature