Human Fetal Retinal Pigment Epithelial Research Articles

Divergent Metabolomic Signatures of TGFβ2 and TNFα in the Induction of Retinal Epithelial-Mesenchymal Transition.

Epithelial-mesenchymal transition (EMT) is a dedifferentiation program in which polarized, differentiated epithelial cells lose their cell-cell adhesions and transform into matrix-producing mesenchymal cells. EMT of retinal pigment epithelial (RPE) cells plays a crucial role in many retinal diseases, including age-related macular degeneration, proliferative vitreoretinopathy, and diabetic retinopathy. This dynamic process requires complex metabolic reprogramming to accommodate the demands of this dramatic cellular transformation. Both transforming growth factor-beta 2 (TGFβ2) and tumor necrosis factor-alpha (TNFα) have the capacity to induce EMT in RPE cells; however, little is known about their impact on the RPE metabolome. Untargeted metabolomics using high-resolution mass spectrometry was performed to reveal the metabolomic signatures of cellular and secreted metabolites of primary human fetal RPE cells treated with either TGFβ2 or TNFα for 5 days. A total of 638 metabolites were detected in both samples; 188 were annotated as primary metabolites. Metabolomics profiling showed distinct metabolomic signatures associated with TGFβ2 and TNFα treatment. Enrichment pathway network analysis revealed alterations in the pentose phosphate pathway, galactose metabolism, nucleotide and pyrimidine metabolism, purine metabolism, and arginine and proline metabolism in TNFα-treated cells compared to untreated control cells, whereas TGFβ2 treatment induced perturbations in fatty acid biosynthesis metabolism, the linoleic acid pathway, and the Notch signaling pathway. These results provide a broad metabolic understanding of the bioenergetic rewiring processes governing TGFβ2- and TNFα-dependent induction of EMT. Elucidating the contributions of TGFβ2 and TNFα and their mechanistic differences in promoting EMT of RPE will enable the identification of novel biomarkers for diagnosis, management, and tailored drug development for retinal fibrotic diseases.

Key Role for CRB2 in the Maintenance of Apicobasal Polarity in Retinal Pigment Epithelial Cells.

Apicobasal polarity is essential for epithelial cell function, yet the roles of different proteins in its completion is not fully understood. Here, we have studied the role of the polarity protein, CRB2, in human retinal pigment epithelial (RPE) cells during polarization in vitro, and in mature murine RPE cells in vivo. After establishing a simplified protocol for the culture of human fetal RPE cells, we studied the temporal sequence of the expression and localization of polarity and cell junction proteins during polarization in these epithelial cells. We found that CRB2 plays a key role in tight junction maintenance as well as in cell cycle arrest. In addition, our studies in vivo show that the knockdown of CRB2 in the RPE affects to the distribution of different apical polarity proteins and results in perturbed retinal homeostasis, manifested by the invasion of activated microglial cells into the subretinal space. Together our results demonstrate that CRB2 is a key protein for the development and maintenance of a polarized epithelium.

Vitamin C- and Valproic Acid-Induced Fetal RPE Stem-like Cells Recover Retinal Degeneration via Regulating SOX2

Retinal pigment epithelial (RPE) cell replacement therapy has provided promising outcomes in the treatment of retinal degenerative diseases (RDDs), but the resulting limited visual improvement has raised questions about graft survival and differentiation. Through combined treatment with vitamin C and valproic acid (together, VV), we activated human fetal RPE (fRPE) cells to become highly proliferative fetal RPE stem-like cells (fRPESCs). In this study, we report that SOX2 (SRY-box 2) activation contributed to mesenchymal-epithelial transition and elevated the retinal progenitor and mesenchymal stromal markers expressions of fRPESCs. These fRPESCs could differentiate into RPE cells, rod photoreceptors, and mesenchymal lineage progenies under defined conditions. Finally, fRPESCs were transplanted into the subretinal space of an RDD mouse model, and a photoreceptor rescue benefit was demonstrated. The RPE and rod photoreceptor differentiation of transplanted fRPESCs may account for the neural retinal recovery. This study establishes fRPESCs as a highly proliferative, multi-lineage differentiation potential (including RPE, rod photoreceptor, and mesenchymal lineage differentiation), mesenchymal-to-epithelial-transitioned retinal stem-like cell source for cell-based therapy of RDDs.

Quantitative proteomic analysis of aqueous humor from patients with drusen and reticular pseudodrusen in age-related macular degeneration

BackgroundTo identify novel biomarkers related to the pathogenesis of dry age-related macular degeneration (AMD), we adopted a human retinal pigment epithelial (RPE) cell culture model that mimics some features of dry AMD including the accumulation of intra- and sub-RPE deposits. Then, we investigated the aqueous humor (AH) proteome using a data-independent acquisition method (sequential window acquisition of all theoretical fragment ion mass spectrometry) for dry AMD patients and controls.MethodsAfter uniformly pigmented polarized monolayers of human fetal primary RPE (hfRPE) cells were established, the cells were exposed to 4-hydroxy-2-nonenal (4-HNE), followed by Western blotting, immunofluorescence analysis and ELISA of cells or conditioned media for several proteins of interest. Data-dependent acquisition for identification of the AH proteome and SWATH-based mass spectrometry were performed for 11 dry AMD patients according to their phenotypes (including soft drusen and reticular pseudodrusen [RPD]) and 2 controls (3 groups).ResultsIncreased intra- and sub-RPE deposits were observed in 4-HNE-treated hfRPE cells compared with control cultures based on APOA1, cathepsin D, and clusterin immunoreactivity. Additionally, the differential abundance of proteins in apical and basal chambers with or without 4-HNE treatment confirmed the polarized secretion of proteins from hfRPE cells. A total of 119 proteins were quantified in dry AMD patients and controls by SWATH-MS. Sixty-five proteins exhibited significantly altered abundance among the three groups. A two-dimensional principal component analysis plot was generated to identify typical proteins related to the pathogenesis of dry AMD. Among the identified proteins, eight proteins, including APOA1, CFHR2, and CLUS, were previously considered major components or regulators of drusen. Three proteins (SERPINA4, LUM, and KERA proteins) have not been previously described as components of drusen or as being related to dry AMD. Interestingly, the LUM and KERA proteins, which are related to extracellular matrix organization, were upregulated in both RPD and soft drusen.ConclusionsDifferential protein expression in the AH between patients with drusen and RPD was quantified using SWATH-MS in the present study. Detailed proteomic analyses of dry AMD patients might provide insights into the in vivo biology of drusen and RPD.

Ascorbate Suppresses VEGF Expression in Retinal Pigment Epithelial Cells

PurposeTo investigate the impact of ascorbate, via DNA hydroxymethylation, on VEGF expression in retinal pigment epithelial (RPE) cells.MethodsDot-blot and hydroxymethylated DNA immunoprecipitation sequencing were applied to evaluate the impact of ascorbate on DNA hydroxymethylation in ARPE-19 cells. RNA sequencing (RNA-seq) was carried out to analyze the transcriptome. Quantitative RT-PCR and ELISA were conducted to examine the transcription and secretion of VEGF from cultured cells. Primary human fetal RPE cells and RPE-J cells were used to verify the effect of ascorbate on VEGF expression. ELISA was used to measure VEGF in the vitreous humor of Gulo−/− mice, which, like humans, cannot synthesize ascorbate de novo.ResultsTreatment with ascorbate (50 μM) promoted 5-hydroxymethycytosine (5hmC) generation and changed the genome-wide profiles of 5hmC in ARPE-19 cells. Ascorbate also caused a dramatic shift in the transcriptome—3186 differential transcripts, of which 69.3% are correlated with altered 5hmC in promoters or gene bodies. One of the most downregulated genes was VEGFA, which encodes the VEGF protein. The suppression of VEGF by ascorbate is independent of hypoxia-inducible factor 1-alpha (HIF-1α) but correlates with increased 5hmC in the gene body. The decreased transcription and secretion of VEGF by ascorbate were verified in primary human fetal RPE cells. Furthermore, adding ascorbate in the diet for Gulo−/− mice resulted in decreased levels of VEGF in the RPE/choroid and vitreous humor.ConclusionsAscorbate inhibits VEGF expression in RPE cells likely via DNA hydroxymethylation. Thus, ascorbate could be implicated in the prevention or treatment of diseases such as age-related macular degeneration (AMD).

The importance of the polarity proteins CRB in the differentiation process of the RPE cells

SummaryApicobasal polarity is essential for epithelial cell function, and it is determined by the expression of three polarity protein complexes: Scribble, Par and Crumbs. Both the Par and the Crumbs complex, promote apical membrane identity. Crumbs complex is composed by PALS1, PATJ and the Crumbs homologs (CRB) proteins. We have recently described the expression of one of the Crumbs proteins, CRB2, in the retinal pigment epithelial (RPE) cells. To better understand this process, we have studied the role of CRB2 in human RPE cells during differentiation in vitro and in the mouse retina in vivo. To achieve this goal, we analyzed the temporal sequence of the establishment of the polarity proteins during human fetal RPE cells polarization and knocking‐down CRB2, we analyzed the establishment of junctional complexes and features involved in the acquisition of polarization compared to the control RPE cultures. We demonstrate that, in differentiating RPE cells, CRB2 functions, as the sole CRB protein, in the formation of the junctional complex and apicobasal polarity. Additionally, the absence of the CRB2 protein from these cells results in a delay in the formation of cell‐cell junctions and in an increase in cell proliferation in RPE cells.

The Retinal Pigment Epithelium Utilizes Fatty Acids for Ketogenesis: IMPLICATIONS FOR METABOLIC COUPLING WITH THE OUTER RETINA

Every day, shortly after light onset, photoreceptor cells shed approximately a tenth of their outer segment. The adjacent retinal pigment epithelial (RPE) cells phagocytize and digest shed photoreceptor outer segment, which provides a rich source of fatty acids that could be utilized as an energy substrate. From a microarray analysis, we found that RPE cells express particularly high levels of the mitochondrial HMG-CoA synthase 2 (Hmgcs2) compared with all other tissues (except the liver and colon), leading to the hypothesis that RPE cells, like hepatocytes, can produce β-hydroxybutyrate (β-HB) from fatty acids. Using primary human fetal RPE (hfRPE) cells cultured on Transwell filters with separate apical and basal chambers, we demonstrate that hfRPE cells can metabolize palmitate, a saturated fatty acid that constitutes .15% of all lipids in the photoreceptor outer segment, to produce β-HB. Importantly, we found that hfRPE cells preferentially release β-HB into the apical chamber and that this process is mediated primarily by monocarboxylate transporter isoform 1 (MCT1). Using a GC-MS analysis of (13)C-labeled metabolites, we showed that retinal cells can take up and metabolize (13)C-labeled β-HB into various TCA cycle intermediates and amino acids. Collectively, our data support a novel mechanism of RPE-retina metabolic coupling in which RPE cells metabolize fatty acids to produce β-HB, which is transported to the retina for use as a metabolic substrate.

Enhancement of retinal pigment epithelial culture characteristics and subretinal space tolerance of scaffolds with 200 nm fiber topography

Tissue engineered retinal pigment epithelial (RPE) transplantation is a promising cell-based therapy for age-related macular degeneration. The aim of this work is to develop a supportive scaffold with a favorable topography to aid functional RPE monolayer maintenance while being tolerated underneath the retina. To this end, films and electrospun substrates with fiber diameters ranging from 200 to 1000 nm were made of polyethylene terephthalate or poly(l-lactide-co-ε-caprolactone), and then tested using human fetal RPE cells in vitro and transplanted subretinally in rabbits. The results indicated that RPE on both 200 nm fiber variants showed the highest cell densities, adherent monolayers achieved deeper pigmentation, and more uniform hexagonal tight junctions. Facile subretinal implantation of flat 200 nm fiber membranes was achieved by electrospinning them onto a porous rigid-elastic carrier. Spectral-domain optical coherence tomography showed a reattached, slightly thinned retina overlying the implants over 2 weeks observation. Histology demonstrated native RPE variably migrated onto the nanofibers, and a reactive gliosis with some photoreceptor degeneration. In conclusion, scaffolds with 200 nm fiber topography enhanced RPE culture, showed subretinal biocompatibility, and should thus be considered for future cell-based therapies in blinding retinal diseases.

Comparison of growth of human fetal RPE cells on electrospun nanofibers and etched pore polyester membranes

To investigate and compare the growth of human fetal retinal pigment epithelial (RPE) cells seeded onto electrospun polyamide nanofibers (EPN) or etched pore polyester (EPP), and, further, to explore their possible use as prosthetic Bruch's membrane. Human fetal RPE cells were planted onto the EPN, EPP and plastic (control) substrates in Transwells. The cultures were assessed with respect to cell attachment at 2, 4, 8 hours and proliferation at 1, 4, 8 days after seeding. Growth and morphology of the cells were monitored under the phase contrast microscope, and the phenotype was identified by immunofluorescence staining with antibodies against tight junction protein ZO-1. Strips of single EPP coated with nothing or EPP coated with EPN was differently implanted into the subretinal space of two P21 RCS rats for two weeks and the histologic slides of the retina were assessed. Cultured human fetal RPE cells were attached to either EPN or EPP substrates (with seeding on plastic substrate as control). After 8 h, the numbers of adherent cells in the EPN, EPP and control groups were 1.23*10(5)/cm(2), 1.70*10(5)/cm(2), and 1.64*10(5)/cm(2), respectively. The number of RPE cells attached to EPN was obviously less than that to both EPP and control (P<0.05). On the first day, the proliferation of cells on EPN was less than that of EPP and control (P<0.05); but by the 8th day in culture, the proliferation of cells on EPN had increased and was higher than proliferation on both EPP and control (P<0.05). All of the RPE cells cultured on EPN and EPP substrates were in monolayer, and the EPN-attached cells resembled the inner collagenous layer of Bruch's membrane. Immunofluorescence staining showed that the RPE cells cultured on EPN and EPP substrates adopted a higher expression of ZO-1 than that on the plastic control substrate. Subretinal implantation of either EPP alone or EPP as a carrier for free EPN for 2 weeks in P21RCS rats resulted in an expected encapsulation and loss of photoreceptor layer. No toxicity or other adverse reaction was observed in the vicinity of the transplant. EPN and EPP could maintain human fetal RPE cell attachment and proliferation. Both EPN and EPP appeared to be grossly tolerance and biocompatible with subretinal implantation. EPN represents an intriguing prospect for prosthetic Bruch's membrane replacement because of its similarity in structure to native Bruch's membrane.

Mechanism of the aging phenomenon with passage of human retinal pigment epithelial cell

Background Retinal pigment epithelial (RPE) cell senescence damage the metabolism of photoreceptor,leading to retinal dysfunction and loss of vision.To understand RPE cell senescence mechanism will contribute to the study of age-related macular degeneration ( AMD). Objective The present study was to prepare the ageing RPE cell model with passage and explore its potential mechanisms. Methods This study was approved by the Ethic Committee of Qingdao University Medical College,and the informed consent was obtained from each gravida.Six human eyeballs were obtained from artificial labor fetusl with the gestational age 16-28 weeks.RPE cells were isolated,cultured and passaged in vitro to establish the cell replicative aging model.The third to twelfth cells were collected to be used to this experiment.Human keratin was used to identify the cells by immunochemistry,and MTT method was utilized to assess the proliferation and viability of different generations of cells as the A490 value.The cellular cycles and transmembrane potential (△ψm) of mitochondrion (△ψm) with passage were detected and compared using Flow Cytometry. Results Cultured and passaged cells showed the hexagon in shape with the melanin in 1-2 generations of cells and presented with the brown staining in cytoplasm for human keratin.The melanin was absent in the third generation cells.Vibrant growth statues were seen from the 3-6 generations cells and thereafter the proliferation ability reduced.The cells of G0/G1 phase were gradually elevated with the passage from 3 - 12 generations with a percentage of 68.40％ in the third generation of cells to 87.33％ in the twelfth generation of cells,showing a significant difference among various generations ( F =180.43,P =0.00),and that of the sixth,ninth and twelfth generation of cells was significant higher than the third,sixth and ninth generation respectively (t =4.002,P＜0.05 ; t=12.885,P＜0.01 ;t=16.387,P＜0.01 ).MTT assay showed that of RPE cells were significantly declined with the passage ( F =38.77,P =0.00),and the A490 value of the ninth,twelfth generations of cells was considerably lower in comparison with sixth and ninth generation respectively ( t =5.991,11.983,P＜0.01 ).From 3 through 12 generations of cells,the staining intensity of rhodamine 123 was gradually decreased ( F =121.68,P =0.00 ),and the staining intensity in the sixth,ninth and twelfth generation of cells was significant lower than that the third,sixth and ninth generation respectively(t=6.918,7.620,11.207,P＜0.01 ). Conclusions A replicative aging model can be successfully created by the passage in vitro using human fetal RPE cells.The reduce of transmembrane potential and damage of mitochondria might be one of mechanisms of senescence of RPE cells. Key words: Retina/pigment epithelium; Cell ageing; Cell cycle; Mitochondria; Transmembrane potential

CNTF Mediates Neurotrophic Factor Secretion and Fluid Absorption in Human Retinal Pigment Epithelium

Ciliary neurotrophic factor (CNTF) protects photoreceptors and regulates their phototransduction machinery, but little is known about CNTF's effects on retinal pigment epithelial (RPE) physiology. Therefore, we determined the expression and localization of CNTF receptors and the physiological consequence of their activation in primary cultures of human fetal RPE (hfRPE). Cultured hfRPE express CNTF, CT1, and OsM and their receptors, including CNTFRα, LIFRβ, gp130, and OsMRβ, all localized mainly at the apical membrane. Exogenous CNTF, CT1, or OsM induces STAT3 phosphorylation, and OsM also induces the phosphorylation of ERK1/2 (p44/42 MAP kinase). CNTF increases RPE survivability, but not rates of phagocytosis. CNTF increases secretion of NT3 to the apical bath and decreases that of VEGF, IL8, and TGFβ2. It also significantly increases fluid absorption (J V) across intact monolayers of hfRPE by activating CFTR chloride channels at the basolateral membrane. CNTF induces profound changes in RPE cell biology, biochemistry, and physiology, including the increase in cell survival, polarized secretion of cytokines/neurotrophic factors, and the increase in steady-state fluid absorption mediated by JAK/STAT3 signaling. In vivo, these changes, taken together, could serve to regulate the microenvironment around the distal retinal/RPE/Bruch's membrane complex and provide protection against neurodegenerative disease.

Interactive Expressions of HtrA1 and VEGF in Human Vitreous Humors and Fetal RPE Cells

High-temperature requirement factor A1 (HtrA1) is associated with exudative age-related macular degeneration, an angiogenic retinal disease related to vascular endothelial growth factor (VEGF). This study investigates the interactive relationship between the expressions of HtrA1 and VEGF. METHODS; The vitreous humor levels of HtrA1, VEGF, and pigment epithelium-derived factor were determined in 55 unrelated Han Chinese patients who underwent ocular surgeries. Expressions of HTRA1 and VEGFA were studied interactively and under stress conditions in primary human fetal retinal pigment epithelial (RPE) cells to evaluate their regulations. Vitreous levels of HtrA1 were significantly associated with that of VEGF in vitreous samples from all patients (Pearson's correlation coefficient test, r = 0.650, P = 7.91 × 10(-8)) and from patients with retinal detachment (r = 0.835, P = 2.14 × 10(-7)). On stress induction, HTRA1 and VEGFA were upregulated in human fetal RPE cells treated by tunicamycin and dithiothreitol, but reduced after treatment by MG132. However, HtrA1 and VEGF did not regulate each other in their expressions. This study revealed an association between HtrA1 and VEGF in human vitreous humors and RPE cells. They are both related to stress and inflammatory conditions.

Expression and regulation of enzymes in the ceramide metabolic pathway in human retinal pigment epithelial cells and their relevance to retinal degeneration

Ceramide and its metabolic derivatives are important modulators of cellular apoptosis and proliferation. Dysregulation or imbalance of their metabolic pathways may promote the development of retinal degeneration. The aim of this study was to identify the expression and regulation of key enzymes of the ceramide pathway in retinal pigment epithelial (RPE) cells. RT-PCR was used to screen the enzymes involved in ceramide metabolism that are expressed in RPE. Over-expression of neutral sphingomyelinase-2 (SMPD3) or sphingosine kinase 1 (Sphk1) in ARPE-19 cells was achieved by transient transfection of SMPD3 or Sphk1 cDNA subcloned into an expression vector. The number of apoptotic or proliferating cells was determined using TUNEL and BrdU assays, respectively. Neutral sphingomyelinase-1, neutral sphingomyelinase-2, acidic ceramidase, ceramide kinase, SphK1 and Sphk2 were expressed in both ARPE-19 and early passage human fetal RPE (fRPE) cells, while alkaline ceramidase 2 was only expressed in fRPE cells. Over-expression of SMPD3 decreased RPE cell proliferation and increased cell apoptosis. The percentage of apoptotic cells increased proportionally with the amount of transfected SMPD3 DNA. Over-expression of SphK1 promoted cell proliferation and protected ARPE-19 cells from ceramide-induced apoptosis. The effect of C 2 ceramide on induction of apoptosis was evaluated in polarized vs. non-polarized RPE cultures; polarization of RPE was associated with much reduced apoptosis in response to ceramide. In conclusion, RPE cells possess the synthetic machinery for the production of ceramide, sphingosine, ceramide-1-phosphate (C1P), and sphingosine-1-phosphate (S1P). Over-expression of SMPD3 may increase cellular ceramide levels, leading to enhanced cell death and arrested cell proliferation. The selective induction of apoptosis in non-polarized RPE cultures by C 2 ceramide suggests that increased ceramide levels will preferentially affect non-polarized RPE, as are found in late age-related macular degeneration lesions, and may spare the normal RPE monolayer. SphK1 over-expression increased cellular S1P, which promoted cell proliferation and protected RPE from ceramide-induced apoptosis. Understanding the relationship between the metabolism of sphingolipids and their effects in RPE cell survival/death may help us to develop effective and efficient therapies for retinal degeneration.

Retinal Pigment Epithelium-Retinal G Protein Receptor-Opsin Mediates Light-dependent Translocation of All-trans-retinyl Esters for Synthesis of Visual Chromophore in Retinal Pigment Epithelial Cells

Visual perception begins with the absorption of a photon by an opsin pigment, inducing isomerization of its 11-cis-retinaldehyde chromophore. After a brief period of activation, the resulting all-trans-retinaldehyde dissociates from the opsin apoprotein rendering it insensitive to light. Restoring light sensitivity to apo-opsin requires thermal re-isomerization of all-trans-retinaldehyde to 11-cis-retinaldehyde via an enzyme pathway called the visual cycle in retinal pigment epithelial (RPE) cells. Vertebrates can see over a 10(8)-fold range of background illumination. This implies that the visual cycle can regenerate a visual chromophore over a similarly broad range. However, nothing is known about how the visual cycle is regulated. Here we show that RPE cells, functionally or physically separated from photoreceptors, respond to light by mobilizing all-trans-retinyl esters. These retinyl esters are substrates for the retinoid isomerase and hence critical for regenerating visual chromophore. We show in knock-out mice and by RNA interference in human RPE cells that this mobilization is mediated by a protein called "RPE-retinal G protein receptor" (RGR) opsin. These data establish that RPE cells are intrinsically sensitive to light. Finally, we show that in the dark, RGR-opsin inhibits lecithin:retinol acyltransferase and all-trans-retinyl ester hydrolase in vitro and that this inhibition is released upon exposure to light. The results of this study suggest that RGR-opsin mediates light-dependent translocation of all-trans-retinyl esters from a storage pool in lipid droplets to an "isomerase pool" in membranes of the endoplasmic reticulum. This translocation permits insoluble all-trans-retinyl esters to be utilized as substrate for the synthesis of a new visual chromophore.

Acrolein, a Toxicant in Cigarette Smoke, Causes Oxidative Damage and Mitochondrial Dysfunction in RPE Cells: Protection by (R)-α-Lipoic Acid

To understand better the cell and molecular basis for the epidemiologic association between cigarette smoke, oxidant injury, and age-associated macular degeneration, the authors examined the effects of acrolein, a major toxicant in cigarette smoke, on oxidative mitochondrial damage in retinal pigment epithelial (RPE) cells and the reduction of this damage by lipoic acid. Cultured human ARPE19 cells and primary cultures of human fetal (hf)RPE were treated with acrolein. The toxicity of acrolein and the protective effects of R-alpha-lipoic acid were examined with a variety of previously described techniques. Acute acrolein exposure exceeding 50 microM (24 hours) in ARPR19 cells caused toxicity, including decreases in cell viability, mitochondrial potential, GSH, antioxidant capacity, Nrf2 expression, enzyme activity (mitochondrial complexes I, II, III; superoxide dismutase; and glutathione peroxidase). Acute exposure also increased oxidant levels, protein carbonyls, and calcium. Continuous acrolein exposure over 8 or 32 days caused similar toxicity but from 10- to 100-fold lower doses (0.1-5 microM). Pretreatment with R-alpha-lipoic acid effectively protected ARPE-19 cells from acrolein toxicity. Primary hfRPE cells were comparable to the ARPE-19 cells in sensitivity to acrolein toxicity and lipoic acid protection. These results show that acrolein is a mitochondrial toxicant in RPE cells and that acrolein-induced oxidative mitochondrial dysfunction is reduced by lipoic acid. The similar sensitivity of the ARPE-19 and hfRPE cells suggests that both models are useful for studying RPE toxicity and protection. These experiments indicate that mitochondria-targeted antioxidants such as lipoic acid may be an effective strategy for reducing or preventing chronic oxidant-induced RPE degeneration in vivo from a variety of sources, including cigarette smoke.

The effect of type I and II interferons on human fetal retinal pigment epithelium-induced apoptosis in Jurkat T cells.

To examine the regulatory effects of interferon (IFN)-alpha, IFN-gamma, transforming growth factor (TGF)-beta, and tumor necrosis factor (TNF)-alpha on human fetal retinal pigment epithelial (HFRPE) cell-induced apoptosis of human Jurkat T (Jkt) cells. Pure cultures of HFRPE cells were isolated. The cells were precultured with medium alone or with addition of IFN-alpha, IFN-gamma, TNF-alpha, or TGF-beta for 72 hours. Thereafter, HFRPE cells were extensively washed before they were cocultured jointly with Jkt cells (standard) or cultured alone for another 48 hours to accumulate conditioned medium that is collected and added as cell-free conditioned medium to Jkt cell cultures (supernatant). Jkt cells were cocultured under the two culture conditions for 48, 72, and 96 hours. The rate of apoptosis in Jkt T cells was determined with annexin V staining and flow cytometry. Both IFN-alpha and -gamma upregulated HFRPE-induced apoptosis in Jkt T cells. However, the apoptosis induced by IFN-alpha-activated HFRPE cells was significant only in the absence of cell-cell contact (supernatant). The supernatant induced a higher rate of apoptosis in Jkt T cells when compared to the direct coculture of the cells. TGF-beta and TNF-alpha did not upregulate HFRPE-induced apoptosis in Jkt T cells. These results indicate that type I and type II IFNs can upregulated HFRPE-induced apoptosis in Jkt T cells, IFN-gamma being the more effective cytokine. Neither, TGF-beta nor TNF-alpha upregulated the HFRPE-induced apoptosis in Jkt T cells. Although HFRPE-induced apoptosis was mediated in a cell-cell-contact-independent pathway, HFRPE cells may also express membrane-bound antiapoptotic molecules. These findings may help us to understand better the modulatory effects of pro- and anti-inflammatory cytokines on immune suppressive characteristics of RPE cells.

Aquaporin-1 channels in human retinal pigment epithelium: role in transepithelial water movement.

Aquaporin (AQP) is a hexahelical integral membrane protein that functions as a constitutive channel for water and regulated channel for cations in fluid transporting tissues, including many in the eye. Although AQP1 has been cloned from a cDNA library prepared from cultures of retinal pigment epithelial (RPE) cells isolated from human fetal tissue, three separate studies failed with various immunochemical techniques to detect AQP1 protein in adult human or rat RPE preparations. The purpose of this study was to examine specifically the expression and distribution of AQP1 in adult human RPE in situ by using alternative methodologies and model systems and to determine the contribution of AQP1 to water movement across cultured RPE cells isolated from human cadaveric and fetal eyes. AQP1 in human RPE in situ was determined after biotinylation of proteins on cell surfaces and streptavidin chromatography, followed by immunoblot analyses. AQP1 distribution in a polarized in vitro RPE model was determined with indirect immunofluorescence confocal microscopy. The role of channel-mediated transport of water across RPE cell monolayers on filters was assessed by osmotic challenge assay. Expression levels of AQP1 were controlled with an adenovirus expression system and monitored by immunoblot analyses. AQP1 protein was detected in human RPE in situ and in cultures of human adult and fetal RPE cells. In functional assays, AQP1 facilitated water movement across RPE monolayers in an expression-dependent manner in two complementary model systems. The expression of AQP1 by RPE in vivo probably contributes to the efficient transepithelial water transport across RPE, maintains retinal attachment, and prevents subretinal edema.

Human fetal retinal pigment epithelium induces apoptosis in human T-cell line Jurkat which is independent from its expression of TRAIL

Purpose. To evaluate whether human fetal retinal pigment epithelial (HFRPE) cells express TRAIL (tumor necrosis factor related apoptosis inducing ligand). The role of TRAIL in HFRPE induced apoptosis was evaluated. Methods. Pure cultures of HFRPE cells were isolated. The expression of TRAIL protein and mRNA in non-activated and IFN-? activated HFRPE cells was evaluated with RT-PCR. The role of TRAIL in HFRPE induced apoptosis was assessed by incubating HFRPE cells with human T-cell leukemia line Jurkat (Jkt) in the presence or absence of neutralizing TRAIL antibodies. Cultures were pulsed with [ 3 H]-thymidine to measure Jkt cell proliferation. The role of TRAIL was further examined by western blott evaluating the cleavage of caspases 8 and 10 in Jkt cells after their incubation with HFRPE cells. Results. HFRPE cells expressed TRAIL mRNA. The expression of TRAIL mRNA and protein was up-regulated by IFN-? activation. However, anti-TRAIL antibodies were not able to prevent the HFRPE induced suppression of Jkt cell proliferation. The caspases 8 and 10 were also not cleaved in Jkt cells after their incubation with IFN-? activated HFRPE cells. Conclusions. Although HFRPE cells express TRAIL and its expression is upregulated by IFN-? activation, TRAIL is not involved in HFRPE induced apoptosis in Jkt cells. Currently the role of TRAIL in HFRPE cells is under investigation.

Superantigen Presentation by Human Retinal Pigment Epithelial Cells to T Cells is Dependent on CD2–CD58 and CD18–CD54 Molecule Interactions

Human retinal pigment epithelial (RPE) cells are capable of presenting bacterial superantigens (SAg) to T cells in vitro by ligation of MHC class II molecules on RPE cells with the T cell receptor. The purpose of this study was to evaluate the involvement of adhesion molecules in presentation of SAg. Cultured human fetal and adult RPE cells were treated with interferon-γ (IFN-γ, 500Uml−1 for 72hr) and afterwards pulsed with the SAg staphylococcal enterotoxin A (SEA, 500ngml−1 for 2hr) followed by coculture with freshly obtained T cells isolated from peripheral blood. Proliferation was measured by 3H-thymidine incorporation assay. In selected experiments, either RPE or T cells were pre-treated with blocking antibodies specific for cell surface molecules. For comparison, dendritic cells were used as superantigen presenting cells for T cells. This study showed that presentation of SEA by RPE cells to resting T cells was dependent on the presence of the molecules CD2, CD58 and CD18, CD54. The cycling status of T cells was decisive, thus resting T cells but not activated T cells were capable to proliferate in response to SEA presentation. Proliferation of T cells induced by adult RPE cells was comparable to the proliferation induced by dendritic cells at concentrations of SAg above 100ngml−1, but at concentrations of SAg below 10ngml−1 the response was significantly lower for SAg presented by RPE cells compared to dendritic cells. The results demonstrate that CD2–CD58 and CD18–CD54 interactions are critical for SAg presentation by RPE cells to T cells. The findings thus suggest that also presentation of peptides to resting T cells by RPE cells may be dependent upon these interactions.

Lack of FasL Expression in Cultured Human Retinal Pigment Epithelial Cells

Retinal pigment epithelial (RPE) cells have been proposed to play a part in maintaining the eye as an immune privileged organ. However, our knowledge of the implicated mechanism is still sparse. Fas ligand (FasL) expression of RPE cells is generally recognized to be essential for the immune privilege of the eye, but due to contradictory published results, it is unclear whether RPE cells express this molecule. The purpose of this study was to investigate the expression of FasL in RPE cells in vitro and in vivo. Cultured human fetal and adult RPE cells were examined by flow cytometry, Western blotting, RT-PCR and RNase Protection assay for FasL expression. Additionally, sections of ocular tissue were stained for FasL by immunohistochemistry. None of the used methods indicated FasL expression in cultured fetal or adult RPE cells of various passages. However, RPE cells in vivo, as judged from tissue sections, were positive for FasL, indicating a discrepancy between RPE cells in vitro and in vivo with regard to this molecule.