Healthy Retinal Pigment Epithelial Cells Research Articles

Dielectrophoretic characterization of peroxidized retinal pigment epithelial cells as a model of age-related macular degeneration

BackgroundAge-related macular degeneration (AMD) is a prevalent ocular pathology affecting mostly the elderly population. AMD is characterized by a progressive retinal pigment epithelial (RPE) cell degeneration, mainly caused by an impaired antioxidative defense. One of the AMD therapeutic procedures involves injecting healthy RPE cells into the subretinal space, necessitating pure, healthy RPE cell suspensions. This study aims to electrically characterize RPE cells to demonstrate a possibility using simulations to separate healthy RPE cells from a mixture of healthy/oxidized cells by dielectrophoresis.MethodsBPEI-1 rat RPE cells were exposed to hydrogen peroxide to create an in-vitro AMD cellular model. Cell viability was evaluated using various methods, including microscopic imaging, impedance-based real-time cell analysis, and the MTS assay. Healthy and oxidized cells were characterized by recording their dielectrophoretic spectra, and electric cell parameters (crossover frequency, membrane conductivity and permittivity, and cytoplasm conductivity) were computed. A COMSOL simulation was performed on a theoretical microfluidic-based dielectrophoretic separation chip using these parameters.ResultsIncreasing the hydrogen peroxide concentration shifted the first crossover frequency toward lower values, and the cell membrane permittivity progressively increased. These changes were attributed to progressive membrane peroxidation, as they were diminished when measured on cells treated with the antioxidant N-acetylcysteine. The changes in the crossover frequency were sufficient for the efficient separation of healthy cells, as demonstrated by simulations.ConclusionsThe study demonstrates that dielectrophoresis can be used to separate healthy RPE cells from oxidized ones based on their electrical properties. This method could be a viable approach for obtaining pure, healthy RPE cell suspensions for AMD therapeutic procedures.

Isolation, Culture, and Genetic Engineering of Mammalian Primary Pigment Epithelial Cells for Non-Viral Gene Therapy.

Age-related macular degeneration (AMD) is the most frequent cause of blindness in patients >60 years, affecting ~30 million people worldwide. AMD is a multifactorial disease influenced by environmental and genetic factors, which lead to functional impairment of the retina due to retinal pigment epithelial (RPE) cell degeneration followed by photoreceptor degradation. An ideal treatment would include the transplantation of healthy RPE cells secreting neuroprotective factors to prevent RPE cell death and photoreceptor degeneration. Due to the functional and genetic similarities and the possibility of a less invasive biopsy, the transplantation of iris pigment epithelial (IPE) cells was proposed as a substitute for the degenerated RPE. Secretion of neuroprotective factors by a low number of subretinally-transplanted cells can be achieved by Sleeping Beauty (SB100X) transposon-mediated transfection with genes coding for the pigment epithelium-derived factor (PEDF) and/or the granulocyte macrophage-colony stimulating factor (GM-CSF). We established the isolation, culture, and SB100X-mediated transfection of RPE and IPE cells from various species including rodents, pigs, and cattle. Globes are explanted and the cornea and lens are removed to access the iris and the retina. Using a custom-made spatula, IPE cells are removed from the isolated iris. To harvest RPE cells, a trypsin incubation may be required, depending on the species. Then, using RPE-customized spatula, cells are suspended in medium. After seeding, cells are monitored twice per week and, after reaching confluence, transfected by electroporation. Gene integration, expression, protein secretion, and function were confirmed by qPCR, WB, ELISA, immunofluorescence, and functional assays. Depending on the species, 30,000-5 million (RPE) and 10,000-1.5 million (IPE) cells can be isolated per eye. Genetically modified cells show significant PEDF/GM-CSF overexpression with the capacity to reduce oxidative stress and offers a flexible system for ex vivoanalyses and in vivo studies transferable to humans to develop ocular gene therapy approaches.

Inhibitory effect of nintedanib on VEGF secretion in retinal pigment epithelial cells induced by exposure to a necrotic cell lysate.

Necrosis is a form of cell death that results in rupture of the plasma membrane and the release of cellular contents, and it can give rise to sterile inflammation in the retina and other tissues. The secretion of vascular endothelial growth factor (VEGF) by retinal pigment epithelial (RPE) cells contributes to retinal homeostasis as well as to pathological angiogenesis. We have now examined the effect of a necrotic cell lysate prepared from human RPE cells (NLR) on the release of VEGF by healthy RPE cells. We found that NLR markedly increased the release of VEGF from RPE cells and that this effect was attenuated by nintedanib, a multiple receptor tyrosine kinase inhibitor, whereas it was unaffected by inhibitors of NF-κB signaling or of caspase-1. NLR also induced the phosphorylation of extracellular signal–regulated kinase (Erk) and signal transducer and activator of transcription 3 (Stat3) in a manner sensitive to inhibition by nintedanib, although inhibitors of Erk and Stat3 signaling pathways did not affect NLR-induced VEGF secretion. In addition, nintedanib attenuated the development of choroidal neovascularization in mice. Our results have thus shown that a necrotic lysate of RPE cells induced VEGF secretion from healthy RPE cells and that this effect was mediated by receptor tyrosine kinase signaling. They therefore suggest that VEGF secretion by healthy RPE cells is a potential therapeutic target for retinal diseases associated with sterile inflammation and pathological angiogenesis.

Effects of Ranibizumab, Bevacizumab, and Aflibercept on Senescent Retinal Pigment Epithelial Cells.

PurposeAnti-vascular endothelial growth factor (VEGF) agents have been used for the last 10 years, but their safety profile, including cytotoxicity against various ocular cells such as retinal pigment epithelial (RPE) cells, remains a serious concern. Safety studies of VEGF agents conducted to date have primarily relied on healthy RPE cells. In this study, we assessed the safety of three anti-VEGF agents, namely, ranibizumab, bevacizumab, and aflibercept, on senescent RPE cells.MethodsSenescent human induced pluripotent stem cell-derived RPE cells were generated by continuous replication and confirmed with senescence biomarkers. The viability, proliferation, protein expression, and phagocytosis of the senescent RPE cells were characterized 3 days after anti-VEGF treatment with clinical doses of ranibizumab, bevacizumab, or aflibercept.ResultsClinical doses of ranibizumab, bevacizumab, or aflibercept did not decrease the viability or alter proliferation of senescent RPE cells. In addition, the anti-VEGF agents did not induce additional senescence, impair the protein expression of zonula occludens-1 and RPE65, or reduce the phagocytosis capacity of senescent RPE cells.ConclusionsClinical dosages of ranibizumab, bevacizumab, or aflibercept do not induce significant cytotoxicity in senescent RPE cells.

Retinal Pigment Epithelial Cells Suppress Phagolysosome Activation in Macrophages.

PurposeThe eye is an immune-privileged microenvironment that has adapted several mechanisms of immune regulation to prevent inflammation. One of these potential mechanisms is retinal pigment epithelial cells (RPE) altering phagocytosis in macrophages.MethodsThe conditioned media of RPE eyecups from eyes of healthy mice and mice with experimental autoimmune uveitis (EAU) were used to treat primary macrophage phagocytizing pHrodo bacterial bioparticles. In addition, the neuropeptides were depleted from the conditioned media of healthy RPE eyecups and used to treat phagocytizing macrophages. The conditioned media from healthy and EAU RPE eyecups were assayed for IL-6, and IL-6 was added to the healthy conditioned media, and neutralized in the EAU conditioned media. The macrophages were treated with the conditioned media and assayed for fluorescence. The macrophages were imaged, and the fluorescence intensity, relative to active phagolysosomes, was measured. Also, the macrophages were assayed using fluorescent viability dye staining.ResultsThe conditioned media from healthy, but not from EAU RPE eyecups suppressed phagolysosome activation. Depletion of the neuropeptides alpha-melanocyte–stimulating hormone and neuropeptide Y from the healthy RPE eyecup conditioned media resulted in macrophage death. In the EAU RPE eyecup conditioned media was 0.96 ± 0.18 ng/mL of IL-6, and when neutralized the conditioned media suppressed phagolysosome activation.ConclusionsThe healthy RPE through soluble molecules, including alpha-melanocyte–stimulating hormone and neuropeptide Y, suppresses the activation of the phagolysosome in macrophages. In EAU, the IL-6 produced by the RPE promotes the activation of phagolysosomes in macrophages. These results demonstrate that under healthy conditions, RPE promotes an altered pathway of phagocytized material in macrophages with implications on antigen processing and clearance.

Cytotoxic effect of ZnS nanoparticles on primary mouse retinal pigment epithelial cells

The multiple properties of zinc sulphide nanoparticles (ZnS-NPs) are attracting great attention in the field of chemical and biological research. ZnS-NPs also find their application in biosensor and photocatalysis. Zinc is an important metal ion in retina and its deficiency leads to age-related macular degeneration. As of now, not much research is available on bio-interaction of ZnS as nanoform with retinal pigment epithelial (RPE) cells. RPE cells in the retina help in maintaining normal photoreceptor function and vision. To begin with, ZnS-NPs were synthesized and characterized using UV-visible spectra, X-ray diffraction, Fourier transform infrared spectrum, transmission electron microscopy and dynamic light scattering. Followed by the confirmation of nanoparticles, our study extended to investigate the impact of ZnS-NPs in primary mouse RPE (MRPE) cells at different concentrations. ZnS-NPs showed dose-dependent cytotoxicity in MRPE cells and no changes were observed in cells’ tight intactness at minimal concentration. In addition, exposure to ZnS-NPs increased cellular permeability in dose- and time-dependent manner in MRPE cells. The findings from DCFH-DA analysis revealed that ZnS-NPs-treated cells had elevated level of reactive oxygen species and partial activation of cell apoptosis was identified after exposure to ZnS-NPs at higher concentration. Furthermore, pre-treatment of the primary MRPE cells with ZnS-NPs led to phosphorylation of Akt (Ser 473), which indicates the crucial role of ZnS-NPs in regulating cell survival at minimal concentration. Altogether, this study enumerates requisite dose of using ZnS-NPs to maintain healthy RPE cells and contributes to future studies in development of therapeutic drug and drug carrier for ocular-related disorders.

Induction of necrotic cell death by oxidative stress in retinal pigment epithelial cells

Age-related macular degeneration (AMD) is a degenerative disease of the retina and the leading cause of blindness in the elderly. Retinal pigment epithelial (RPE) cell death and the resultant photoreceptor apoptosis are characteristic of late-stage dry AMD, especially geographic atrophy (GA). Although oxidative stress and inflammation have been associated with GA, the nature and underlying mechanism for RPE cell death remains controversial, which hinders the development of targeted therapy for dry AMD. The purpose of this study is to systematically dissect the mechanism of RPE cell death induced by oxidative stress. Our results show that characteristic features of apoptosis, including DNA fragmentation, caspase 3 activation, chromatin condensation and apoptotic body formation, were not observed during RPE cell death induced by either hydrogen peroxide or tert-Butyl hydroperoxide. Instead, this kind of cell death can be prevented by RIP kinase inhibitors necrostatins but not caspase inhibitor z-VAD, suggesting necrotic feature of RPE cell death. Moreover, ATP depletion, receptor interacting protein kinase 3 (RIPK3) aggregation, nuclear and plasma membrane leakage and breakdown, which are the cardinal features of necrosis, were observed in RPE cells upon oxidative stress. Silencing of RIPK3, a key protein in necrosis, largely prevented oxidative stress-induced RPE death. The necrotic nature of RPE death is consistent with the release of nuclear protein high mobility group protein B1 into the cytoplasm and cell medium, which induces the expression of inflammatory gene TNFα in healthy RPE and THP-1 cells. Interestingly, features of pyroptosis or autophagy were not observed in oxidative stress-treated RPE cells. Our results unequivocally show that necrosis, but not apoptosis, is a major type of cell death in RPE cells in response to oxidative stress. This suggests that preventing oxidative stress-induced necrotic RPE death may be a viable approach for late-stage dry AMD.

Retinal pigment epithelial (RPE) cells convert bone marrow derived macrophages into myeloid suppressor cells with novel phenotype

AbstractPurpose We have shown previously that macrophages/microglia accumulate in the subretinal space and express CD68 and Arginase‐1 in the aging eye. We hypothesize that Retinal Pigment Epithelial (RPE) cells may play an important role in regulating macrophage/microglial phenotype and function.Methods Bone marrow derived macrophages (BMDMs) and RPE were cultured from C57BL/6J mice and the phenotype was confirmed by CD11b and F4/80 (for BMDMs) and RPE65 and cytokeratin (for RPE cells) stainings. BMDMs were co‐cultured with RPE cells for different times. Macrophages were then isolated for phenotypic and functional assays.Results Co‐culture of BMDMs with RPE cells resulted in a time‐dependent down‐regulation of MHC‐II and the generation of CD11b+F4/80+Ly6G+ myeloid‐derived suppressor cells (MDSC). The MDSCs expressed high levels of IL‐6, IL‐1β, Arginase‐1, and complement inhibitor C1INH, but lower levels of IL‐12p40 and TNF‐a compared to naïve BMDMs. The expression levels of iNOS, TGF‐β and Ym1 did not change compared to naive BMDMs. Furthermore, MDSCs had reduced phagocytic activity and lower ability to stimulate T cell activation and proliferation. When RPE cells were pre‐treated with oxidized photoreceptor outer segments, the expression of IL‐1β and IL‐6 in BMDMs was increased and the expression of Arginase‐1 was decreased.Conclusion Our results suggest that healthy RPE cells can convert BMDMs into myeloid‐derived suppressor cells under in vitro culture conditions. RPE‐induced myeloid‐derived suppressor cells are CD11b+F4/80+Ly6G+MHC‐IIlowIL‐6+IL‐1b+Arg‐1+. This ability of RPE cells is reduced when suffering from oxidative insults.

Spatial cues for the enhancement of retinal pigment epithelial cell function in potential transplants

Retinal pigment epithelial (RPE) cellular morphology and function are vital to the health of the retina. In age-related macular degeneration, RPE dysfunction and changes in Bruch's membrane occur. Thus, a potential cure is a dual-layer biomimetic transplant consisting of a layer of healthy RPE cells cultured on a support membrane. In this study, we investigated human anterior lens capsule as a replacement for Bruch's membrane and also explored different seeding methods as ways of inducing the desired cellular morphology and function. Using in vitro assays, we demonstrated that RPE cells cultured on lens capsule exhibited epithelial characteristics, such as the presence of actin belts and the formation of tight junctions in the monolayer. Bovine photoreceptor outer segments were also incubated with the RPE cells in order to quantify the binding and ingestion activity of the RPE cells. With these assays, we determined that cells seeded by centrifugation appeared to possess the most epithelial-like morphology, with the shortest overall length and the smallest elongation. They also exhibited enhanced metabolic activity, with a 1.5-fold increase over conventional gravity seeding. Thus, the spatial cues provided by centrifugation may assist cells in assuming native RPE function. Therefore, a dual-layer transplant, with RPE cells organized by centrifugation onto lens capsule, appears promising in achieving native retinal function.

Optimal conditions for long-term photoreceptor cell rescue in RCS rats: The necessity for healthy RPE transplants

Retinal pigment epithelial (RPE) cells from normal Long-Evans, health Royal College of Surgeons (RCS) pigmented congenic (rdy +p +) and RCS pigmented dystrophic rats were transplanted into retinas of RCS non-pigmented dystrophic rats at post-natal days 17 and 26 (P17 and P26). When examined at P60, rescued photoreceptor cells were found in all transplanted groups. In addition, a small, but significant increase in the outer nuclear layer (ONL) thickness was detected in retinas injected with vehicle (sham control); however, the ONL thickness was reduced to control levels by 3–5 months after injections in both the sham controls and the group grafted with RCS pigmented dystrophic RPE cells. Likewise, large volumes of vehicle were injected into the subretinal space resulting in no long lasting beneficial effects. Also, there was no significant difference between the ONL thickness in retinas grafted with RCS congenic RPE and those grafted with Long-Evans RPE. When donor RPE cells from Long-Evans rats at neonatal and adult stages were transplanted into P26 RCS hosts, photoreceptor cell rescue could be found; however, the younger RPE cells affected a better rescue than those derived from adult eyes. In addition, when RPE cells from 6- to 9-day-old Long-Evans rats were transplanted into retinas of RCS dystrophic rats at 10, 17 and 26 days, significant rescue of photoreceptor cells was observed. Of these transplantation times, day 17 appeared to affect the best rescue of photoreceptor cells up to 1 yr. In contrast, little or no rescue was observed in the retinas of those RCS dystrophic rats when RPE cell transplantation was performed at P38, P43 and P48. The photoreceptor cell rescue was also found to be dependent on the concentration of RPE cells injected, which was maximal at 60000–120000 cells μl −1. These findings lead us to conclude that in order to affect long-term, up to 1 yr, rescue of photoreceptor cells in the RCS rat, young, healthy RPE cells are required and tranplantation needs to be performed at early stages of the disease process.

Dispersion of retinal pigment epithelial cells from experimental retinal holes

The effects of cryotherapy, transcleral massage, or directed irrigation on exposed retinal pigment epithelial (RPE) cells at the base of an experimental retinal hole were studied in living rabbit eyes. Cryotherapy or massage with or without vitrectomy failed to release RPE cells or result in preretinal membrane formation. Directed irrigation dispersed viable RPE cells into the vitreous and produced cellular precipitates with membranes on the inferior retina, although the membranes tended to regress within 6 weeks after surgery. These results suggest that healthy RPE cells at the base of retinal tears are not easily released into the vitreous and may not contribute to the formation of preretinal membranes found in proliferative vitreoretinopathy.