Oxidative stress in retinal pigment epithelium cells increases exosome secretion and promotes angiogenesis in endothelial cells

The retinal pigment epithelium (RPE), a monolayer located between the photoreceptors and the choroid, is constantly damaged by oxidative stress, particularly because of reactive oxygen species (ROS). As the RPE, because of its physiological functions, is essential for the survival of the retina, any sustained damage may consequently lead to loss of vision. Exosomes are small membranous vesicles released into the extracellular medium by numerous cell types, including RPE cells. Their cargo includes genetic material and proteins, making these vesicles essential for cell‐to‐cell communication. Exosomes may fuse with neighbouring cells influencing their fate. It has been observed that RPE cells release higher amounts of exosomes when they are under oxidative stress. Exosomes derived from cultured RPE cells were isolated by ultracentrifugation and quantified by flow cytometry. VEGF receptors (VEGFR) were analysed by both flow cytometry and Western blot. RT‐PCR and qPCR were conducted to assess mRNA content of VEGFRs in exosomes. Neovascularization assays were performed after applying RPE exosomes into endothelial cell cultures. Our results showed that stressed RPE cells released a higher amount of exosomes than controls, with a higher expression of VEGFR in the membrane, and enclosed an extra cargo of VEGFR mRNA. Angiogenesis assays confirmed that endothelial cells increased their tube formation capacity when exposed to stressed RPE exosomes.     

Retinal pigment epithelium-derived transforming growth factor-β2 inhibits the angiogenic response of endothelial cells by decreasing vascular endothelial growth factor receptor-2 expression

Transforming growth factor-β (TGF-β) is a multifunctional cytokine that is known to modulate various aspects of endothelial cell (EC) biology. Retinal pigment epithelium (RPE) is important for regulating angiogenesis of choriocapillaris and one of the main cell sources of TGF-β secretion, particularly TGF-β2. However, it is largely unclear whether and how TGF-β2 affects angiogenic responses of ECs. In the current study, we demonstrated that TGF-β2 reduces vascular endothelial growth factor receptor-2 (VEGFR-2) expression in ECs and thereby inhibits vascular endothelial growth factor (VEGF) signaling and VEGF-induced angiogenic responses such as EC migration and tube formation. We also demonstrated that the reduction of VEGFR-2 expression by TGF-β2 is due to the suppression of JNK signaling. In coculture of RPE cells and ECs, RPE cells decreased VEGFR-2 levels in ECs and EC migration. In addition, we showed that TGF-β2 derived from RPE cells is involved in the reduction of VEGFR-2 expression and inhibition of EC migration. These results suggest that TGF-β2 plays an important role in inhibiting the angiogenic responses of ECs during the interaction between RPE cells and ECs and that angiogenic responses of ECs may be amplified by a decrease in TGF-β2 expression in RPE cells under pathologic conditions.     

Mouse retinal progenitor cell (RPC) cocultivation with retinal pigment epithelial cell culture affects features of RPC differentiation

We provide evidence that coculturing of retinal progenitor cells (RPC) with retinal pigment epithelial cells significantly biases the standard in vitro RPC differentiation patterns. In particular, in cocultivation experiments RPCs lost the ability to differentiate spontaneously and displayed approximately 2.1-2.4-fold increase in immunoreactivity to the neural stem cell marker nestin and approximately 1.6-1.7-fold increase in rod photoreceptor cell rhodopsin marker immunoreactivity. The data suggest the influence of the intercellular interaction networks on RPC differentiation.     

Bone morphogenetic protein-4 enhances vascular endothelial growth factor secretion by human retinal pigment epithelial cells

Retinal pigment epithelial (RPE) cells secrete vascular endothelial growth factor (VEGF), a cytokine known to promote angiogenesis. Results from RNase protection assays (RPAs) show that RPE from non-diabetic human donors and from adult retinal pigment epithelium-19 (ARPE-19) cells expressed significant bone morphogenetic protein-4 (BMP-4) message. In addition, ARPE-19 cells cultured in high glucose (25 mM), compared to those in physiological glucose (5.5 mM) released significantly more BMP-4 into the conditioned media (CM). However, the effect of BMP-4 on the release of VEGF by ARPE-19 cells has not been studied. Accordingly, ARPE-19 cells were treated with BMP-4 to determine VEGF secretion. BMP-4 and VEGF levels in the CM and cell lysates were measured by enzyme-linked immunosorbent assay (ELISA). Cells treated with exogenous BMP-4 had higher VEGF in the CM and this treatment effect was dose- and time-dependent, while cell lysates had low levels of VEGF. Addition of cycloheximide (CHX) or actinomycin-D (ACT) significantly reduced VEGF secretion from cells treated with BMP-4, suggesting that the BMP-4-induced secretion of VEGF requires new RNA and protein synthesis. Our results suggest that BMP-4 may play a role in the regulation of ocular angiogenesis associated with diabetic retinopathy (DR) by stimulating VEGF release from RPE cells.     

Primary cilia in retinal pigment epithelium development and diseases

Retinal pigment epithelium (RPE) is a highly polarized epithelial monolayer lying between the photoreceptor layer and the Bruch membrane. It is essential for vision through participating in many critical activities, including phagocytosis of photoreceptor outer segments, recycling the visual cycle-related compounds, forming a barrier to control the transport of nutrients, ions, and water, and the removal of waste. Primary cilia are conservatively present in almost all the vertebrate cells and acts as a sensory organelle to control tissue development and homeostasis maintenance. Numerous studies reveal that abnormalities in RPE lead to various retinal diseases, such as age-related macular degeneration and diabetic macular oedema, but the mechanism of primary cilia in these physiological and pathological activities remains to be elucidated. Herein, we summarize the functions of primary cilia in the RPE development and the mutations of ciliary genes identified in RPE-related diseases. By highlighting the significance of primary cilia in regulating the physiological and pathological processes of RPE, we aim to provide novel insights for the treatment of RPE-related retinal diseases.     

The retinal pigment epithelium as a gateway for monocyte trafficking into the eye

The choroid plexus epithelium within the brain ventricles orchestrates blood‐derived monocyte entry to the central nervous system under injurious conditions, including when the primary injury site is remote from the brain. Here, we hypothesized that the retinal pigment epithelium (RPE) serves a parallel role, as a gateway for monocyte trafficking to the retina following direct or remote injury. We found elevated expression of genes encoding leukocyte trafficking determinants in mouse RPE as a consequence of retinal glutamate intoxication or optic nerve crush (ONC). Blocking VCAM‐1 after ONC interfered with monocyte infiltration into the retina and resulted in a local pro‐inflammatory cytokine bias. Live imaging of the injured eye showed monocyte accumulation first in the RPE, and subsequently in the retina, and peripheral leukocytes formed close contact with the RPE. Our findings further implied that the ocular milieu can confer monocytes a phenotype advantageous for neuroprotection. These results suggest that the eye utilizes a mechanism of crosstalk with the immune system similar to that of the brain, whereby epithelial barriers serve as gateways for leukocyte entry.     

Sirtuin 6 protects human retinal pigment epithelium cells from LPS-induced inflammation and apoptosis partly by regulating autophagy

ABSTRACT

Lipopolysaccharides (LPS)-induced retinal inflammation is an important factor in retinal diseases. This study was aimed to investigate the effect of Sirt6 on LPS-induced retinal injury. ARPE-19 cells were incubated with LPS to induce inflammation. The cell viability was determined using CCK-8 assay. The mRNA level and protein expression of corresponding genes was detected using qRT-PCR and western blot, respectively. The production of inflammatory cytokines was measured using ELISA kit. The levels of oxidative stress-related factors were measured using their detection kits. Cell apoptosis was observed using TUNEL assay. The results showed that Sirt6 was downregulated after LPS treatment. Sirt6 strengthened LPS-induced autophagy by promoting the expression of LC3II/I, beclin1 and ATG5. Sirt6 treatment significantly inhibited LPS-induced inflammation, oxidative stress and cell apoptosis, which was then partly abolished by 3 MA. These results suggest Sirt6 to be an important regulator for LPS-induced inflammation, oxidative stress, and apoptosis partly by regulating cell autophagy.     

The new paradigm: retinal pigment epithelium cells generated from embryonic or induced pluripotent stem cells

Compared with neural crest‐derived melanocytes, retinal pigment epithelium (RPE) cells in the back of the eye are pigment cells of a different kind. They are a part of the brain, form an epithelial monolayer, respond to distinct extracellular signals, and provide functions that far exceed those of a light‐absorbing screen. For instance, they control nutrient and metabolite flow to and from the retina, replenish 11‐cis‐retinal by re‐isomerizing all‐trans‐retinal generated during photoconversion, phagocytose daily a portion of the photoreceptors’ outer segments, and secrete cytokines that locally control the innate and adaptive immune systems. Not surprisingly, RPE cell damage is a major cause of human blindness worldwide, with age‐related macular degeneration a prevalent example. RPE replacement therapies using RPE cells generated from embryonic or induced pluripotent stem cells provide a novel approach to a rational treatment of such forms of blindness. In fact, RPE‐like cells can be obtained relatively easily when stem cells are subjected to a two‐step induction protocol, a first step that leads to a neuroectodermal fate and a second to RPE differentiation. Here, we discuss the characteristics of such cells, propose criteria they should fulfill in order to be considered authentic RPE cells, and point out the challenges one faces when using such cells in attempts to restore vision.     

A novel in vitro retinal differentiation model by co-culturing adult human bone marrow stem cells with retinal pigmented epithelium cells

Human retinal pigment epithelium (HRPE) cells are important in maintaining the normal physiology within the neurosensory retina and photoreceptors. Recently, transplantation of HRPE has become a possible therapeutic approach for retinal degeneration. By negative immunoselection (CD45 and glycophorin A), in this study, we have isolated and cultivated adult human bone marrow stem cells (BMSCs) with multilineage differentiation potential. After a 2- to 4-week culture under chondrogenic, osteogenic, adipogenic, and hepatogenic induction medium, these BMSCs were found to differentiate into cartilage, bone, adipocyte, and hepatocyte-like cells, respectively. We also showed that these BMSCs could differentiate into neural precursor cells (nestin-positive) and mature neurons (MAP-2 and Tuj1-positive) following treatment of neural selection and induction medium for 1 month. Furthermore, the plasticity of BMSCs was confirmed by initiating their differentiation into retinal cells and photoreceptor lineages by co-culturing with HRPE cells. The latter system provides an ex vivo expansion model of culturing photoreceptors for the treatment of retinal degeneration diseases.     

Culture of ratretinal pigment epithelium

Summary A method of preparing monolayer cultures of retinal pigment epithelium from normal pigmented neonatal rats is described. Critical features include incubating the eyes in balanced salt solution and treating with trypsin before dissecting the eyes. The tissue also has been cultured from RCS rats with inherited retinal degeneration. Since the pigment epithelium has been shown to be the primary site of action of the gene for retinal dystrophy in the RCS rat, the method should be useful in studying the defect(s) associated with this mutation. This work was supported by grants from the National Retinitis Pigmentosa Foundation, Baltimore, Maryland, and the George Gund Foundation, Cleveland, Ohio. Breeding pairs of RCS-p⁺ rats were provided through National Institutes of Health Research Contract EY-3-2133 to Dr. R. L. Sidman.     

Muscarinic receptors binding in retinal pigment epithelium during rat development

[³H]Quinuclidinyl benzylate (³H-QNB) specific binding of the developing rat retinal pigment epithelium (RPE) and neural retina has been examined. The binding of³H-QNB to RPE was saturable and displaced by the antagonist pirenzepine. Scatchard analysis of³H-QNB binding showed two high affinity sites to RPE, with K_B=2.6nM and 45 nM. Specific³H-QNB binding membranes from neural retina exhibited a characteristic developmental profile. RPE showed a high density of³H-QNB binding sites through all developmental periods studied. The major onset of binding sites is at the time of RPE differentiation. Our data open the possibility of muscarinic receptors being involved in differentiation and/or proliferation of RPE.     

Apomorphine inhibits the growth-stimulating effect of retinal pigment epithelium on scleral cells in vitro

Visual deprivation of the chicken eye causes axial elongation with high myopia. The cartilaginous layer of the myopic sclera shows an increase of mitotic activity. Previous studies reported that the in vivo administration of apomorphine, a dopamine nonselective agonist, effectively prevents visual-deprivation myopia. Because the retinal pigment epithelium (RPE) regulates growth of the sclera as we and others have shown previously, it is speculated that the RPE cells may play an important role in this preventive effect of apomorphine. In this study, to clarify the mechanism by which the administration of apomorphine inhibits the proliferation of scleral chondrocytes in vivo, we have investigated the effect of apomorphine on the proliferation of scleral chondrocytes with or without co-cultured RPE cells in vitro. We previously demonstrated that cell proliferation of scleral chondrocytes remarkably increases with co-cultured RPE cells. In this study, we found that apomorphine at concentrations of higher than 2×10⁻⁵ M dramatically reduced the growth-stimulatory effect of RPE cells on the scleral chondrocytes, whereas the inhibitory effect of apomorphine on the proliferation of scleral chondrocytes without RPE cells was very little. Our results strongly suggest that apomorphine may reduce the production and/or release of some humoral factors from RPE cells, which stimulate the growth of scleral cells. There is also a possibility that apomorphine reduces the reactivity of scleral cells to the humoral factors released from RPE cells. © 1997 John Wiley & Sons, Ltd.     

Molecular and pharmacological characterization of muscarinic receptors in retinal pigment epithelium: role in light-adaptive pigment movements

Muscarinic receptors are the predominant cholinergic receptors in the central and peripheral nervous systems. Recently, activation of muscarinic receptors was found to elicit pigment granule dispersion in retinal pigment epithelium isolated from bluegill fish. Pigment granule movement in retinal pigment epithelium is a light-adaptive mechanism in fish. In the present study, we used pharmacological and molecular approaches to identify the muscarinic receptor subtype and the intracellular signaling pathway involved in the pigment granule dispersion in retinal pigment epithelium. Of the muscarinic receptor subtype-specific antagonists used, only antagonists specific for M1 and M3 muscarinic receptors were found to block carbamyl choline (carbachol)-induced pigment granule dispersion. A phospholipase C inhibitor also blocked carbachol-induced pigment granule dispersion, and a similar result was obtained when retinal pigment epithelium was incubated with an inositol trisphosphate receptor inhibitor. We isolated M2 and M5 receptor genes from bluegill and studied their expression. Only M5 was found to be expressed in retinal pigment epithelium. Taken together, pharmacological and molecular evidence suggest that activation of an odd subtype of muscarinic receptor, possibly M5, on fish retinal pigment epithelium induces pigment granule dispersion.     

Ultrastructure of the retinal pigment epithelium in the young masu salmon Oncorhynchus masou

The ultrastructure of retinal pigment epithelium (RPE) cells in the masu salmon Oncorhynchus masou fry was studied. The physiological state of the pigment was dependent on the level of light/dark adaptation. Similar morphological properties were observed in the response of the RPE cells to light and a magnetic field.     

Analysis of a soluble lipid-protein complex carrying endogenous 11-cis retinaldehyde from bovine retinal pigment epithelium

Summary A soluble lipid-protein complex in bovine retinal pigment epithelium is shown to carry endogenous 11-cis retinaldehyde, in the extent of 15% of the total 11-cis retinaldehyde found in this tissue. The complex, analyzed with respect to its chemical composition, exhibits a lipid composition close resembling the lipid composition of the rod outer segment membrane; the SDS-PAGE evidences the presence of a number of protein bands, two of which of 34 and 27 kDa appear glycoproteins. Finally, the lipid-protein complex exhibits a discrete level of a Cathepsin D-like protease activity. From the above, the possibility is discussed that the soluble lipid-protein complex could represent some phagolysosomal inclusion occurring in the pigment epithelial cells upon rod outer segment phagocytosis.     

Gene replacement therapy restores RCBTB1 expression and cilium length in patient-derived retinal pigment epithelium

Biallelic mutations in the RCBTB1 gene cause retinal dystrophy. Here, we characterized the effects of RCBTB1 gene deficiency in retinal pigment epithelial (RPE) cells derived from a patient with RCBTB1-associated retinopathy and restored RCBTB1 expression in these cells using adeno-associated viral (AAV) vectors. Induced pluripotent stem cells derived from a patient with compound heterozygous RCBTB1 mutations (c.170delG and c.707delA) and healthy control subjects were differentiated into RPE cells. RPE cells were treated with AAV vectors carrying a RCBTB1 transgene. Patient-derived RPE cells showed reduced expression of RCBTB1. Expression of NFE2L2 showed a non-significant reduction in patient RPE cells compared with controls, while expression of its target genes (RXRA, IDH1 and SLC25A25) was significantly reduced. Trans-epithelial electrical resistance, surface microvillus densities and primary cilium lengths were reduced in patient-derived RPE cells, compared with controls. Treatment of patient RPE with AAV vectors significantly increased RCBTB1, NFE2L2 and RXRA expression and cilium lengths. Our study provides the first report examining the phenotype of RPE cells derived from a patient with RCBTB1-associated retinopathy. Furthermore, treatment of patient-derived RPE with AAV-RCBTB1 vectors corrected deficits in gene expression and RPE ultrastructure, supporting the use of gene replacement therapy for treating this inherited retinal disease.     

Early embryonic interaction of retinal pigment epithelium and mesenchymal tissue induces conversion of pigment epithelium to neural retinal fate in the silver mutation of the Japanese quail

The neural retina and retinal pigment epithelium (RPE) diverge from the optic vesicle during early embryonic development. They originate from different portions of the optic vesicle, the more distal part developing as the neural retina and the proximal part as RPE. As the distal part appears to make contact with the epidermis and the proximal part faces mesenchymal tissues, these two portions would encounter different environmental signals. In the present study, an attempt has been made to investigate the significance of interactions between the RPE and mesenchymal tissues that derive from neural crest cells, using a unique quail mutant silver (B/B) as the experimental model. The silver mutation is considered to affect neural crest-derived tissues, including the epidermal melanocytes. The homozygotes of the silver mutation have abnormal eyes, with double neural retinal layers, as a result of aberrant differentation of RPE to form a new neural retina. Retinal pigment epithelium was removed from early embryonic eyes (before the process began) and cultured to see whether it expressed any phenotype characteristic of neural retinal cells. When RPE of the B/B mutant was cultured with surrounding mesenchymal tissue, neural retinal cells were differentiated that expressed markers of amacrine, cone or rod cells. When isolated RPE of the B/B mutant was cultured alone, it acquired pigmentation and did not show any property characteristic of neural retinal cells. The RPE of wild type quail always differentiated to pigment epithelial cells. In the presence of either acidic fibroblast growth factor (aFGF) or basic FGF (bFGF), the RPE of the B/B mutant differentiated to neural retinal cells in the absence of mesenchymal tissue, but the RPE of wild type embryos only did so in the presence of 10–40 times as much aFGF or bFGF. These observations indicate that genes responsible for the B/B mutation are expressed in the RPE as well as in those cells that have a role in the differentiation of neural crest cells. They further suggest that development of the neural retina and RPE is regulated by some soluble factor(s) that is derived from or localized in the surrounding embryonic mesenchyme and other ocular tissues, and that FGF may be among possible candidates.     

MEK-ERK and heparin-susceptible signaling pathways are involved in cell-cycle entry of the wound edge retinal pigment epithelium cells in the adult newt

The onset mechanism of proliferation in mitotically quiescent retinal pigment epithelium (RPE) cells is still obscure in humans and newts, although it can be a clinical target for manipulating both retinal diseases and regeneration. To address this issue, we investigated factors or signaling pathways involved in the first cell-cycle entry of RPE cells upon retinal injury using a newt retina-less eye-cup culture system in which the cells around the wound edge of the RPE exclusively enter the cell cycle. We found that MEK-ERK signaling is necessary for their cell-cycle entry, and signaling pathways whose activities can be modulated by heparin, such as Wnt-, Shh-, and thrombin-mediated pathways, are capable of regulating the cell-cycle entry. Furthermore, we found that the cells inside the RPE have low proliferation competence even in the presence of serum, suggesting inversely that a loss of cell-to-cell contact would allow the cells to enter the cell cycle.     

Human retinal pigment epithelial cells

''Human retinal pigment epithelial cells'' is the first set of guidelines on human retinal pigment epithelial cells in China, jointly drafted and agreed upon by experts from the Chinese Society for Stem Cell Research. This standard specifies technical requirements, test methods, inspection rules, instructions for usage, labelling requirements, packaging requirements, storage requirements and transportation requirements and waste disposal requirements for human retinal pigment epithelial cells, which is applicable to quality control during the process of manufacturing and testing of human retinal pigment epithelial cells. It was originally released by the Chinese Society for Cell Biology on 9 January 2021. We hope that publication of these guidelines will promote institutional establishment, acceptance and execution of proper protocols and accelerate the international standardization of human retinal pigment epithelial cells for applications.