Adult Retinal Pigment Epithelium Research Articles

Oxidative and carbonyl stress induced AMD and Codonopsis lanceolata ameliorates AMD via controlling oxidative and carbonyl stress

Age-related macular degeneration (AMD) is one of the leading causes of blindness. AMD is currently incurable; the best solution is to prevent its occurrence. To develop drugs for AMD, it is crucial to have a model system that mimics the symptoms and mechanisms in patients. It is most important to develop safer and more effective anti-AMD drug. In this study, the dose of A2E and the intensity of blue light were evaluated to establish an appropriate atrophic in vitro model of AMD and anti-AMD effect and therapeutic mechanism of Codonopsis lanceolata. The experimental groups included a control group an AMD group treated with A2E and blue light, a lutein group treated with 25 μM lutein after AMD induction, and three groups treated with different doses of C. lanceolata (10, 20, and 50 μg/mL) after AMD induction. Intrinsic apoptotic pathway (Bcl-2 family), anti-oxidative system (Keap1/Nrf2/HO-1 antioxidant response element), and anti-carbonyl effect (4-hydroxynonenal [4-HNE]) were evaluated using immunofluorescence, MTT, TUNEL, FACS, and western blotting analyses. A2E accumulation in the cytoplasm of ARPE-19 cells depending on the dose of A2E. Cell viability of ARPE-19 cells according to the dose of A2E and/or blue light intensity. The population of apoptotic or necrotic cells increased based on the A2E dose and blue light intensity. Codonopsis lanceolata dose-dependently prevented cell death which was induced by A2E and blue light. The antiapoptotic effect of that was caused by activating Keap1/Nrf2/HO-1 pathway, suppressing 4-HNE, and modulating Bcl-2 family proteins like increase of antiapoptotic proteins such as Bcl-2 and Bcl-XL and decrease of proapoptotic protein such as Bim. Based on these findings, 30 μM A2E and 20 mW/cm2 blue light on adult retinal pigment epithelium-19 cells was an appropriate condition for AMD model and C. lanceolata shows promise as an anti-AMD agent.

Artemisinin Confers Cytoprotection toward Hydrogen Peroxide-Induced Cell Apoptosis in Retinal Pigment Epithelial Cells in Correlation with the Increased Acetylation of Histone H4 at Lysine 8.

Increased oxidative stress is one of the critical pathologies inducing age-related macular degeneration (AMD), characterized by retinal pigment epithelial (RPE) cell damage and death. The unbalanced acetylation and deacetylation of histones have been implicated in AMD pathogenesis or hydrogen peroxide (H2O2)-induced cell damage. Therefore, strategies aimed at controlling the balance between acetylation and deacetylation may effectively protect RPE cells from oxidative damage. Artemisinin is an antimalarial lactone drug derived from Artemisia annua, with antioxidant activity known to modulate histone acetylation in the brain, but its effect on the retina is unknown. In this study, we aimed to investigate whether Artemisinin exerts a cytoprotective effect on oxidative stress-induced apoptosis in RPE cells by regulating histone acetylation. We hypothesized that Artemisinin confers cytoprotection toward H2O2-induced apoptosis in RPE cells through this mechanism. In the present study, we found that Artemisinin at a sub-clinic dosage of 20 μM inhibited the H2O2-induced cell viability decrease and B-cell lymphoma 2 (Bcl-2) protein level decrease and attenuated the H2O2-induced decrease in the histone H4 lysine (Lys) 8 acetylation [Acetyl-H4 (Lys 8)] level in the retinal RPE cell line D407. As expected, histone deacetylase inhibitor Trichostatin A at the concentration of 250 nM increased the Acetyl-H4 (Lys 8) level in D407 cells and attenuated the H2O2-induced cell viability decrease and apoptosis. Similar findings were obtained using adult RPE (ARPE)19 cells, another human RPE cell line, and primary human RPE cell cultures. In conclusion, these results confirmed our hypothesis and indicated that Artemisinin attenuated H2O2-induced apoptosis in apparent correlation with the increase in the Acetyl-H4 (Lys 8) level, which is associated with gene transcription and cell survival. By modulating histone acetylation, Artemisinin may restore the balance between acetylation and deacetylation and enhance the resistance and survival of RPE cells under oxidative stress. Our study provides novel mechanistic insights into the effect of Artemisinin on histone acetylation and apoptosis in RPE cells and supports the potential application of Artemisinin in the prevention and/or treatment of AMD.

Identification and Potential Roles of Human MicroRNAs in Ebola Virus Infection and Disease Pathogenesis.

Ebola virus (EBOV) is a highly pathogenic virus that causes a severe illness called Ebola virus disease (EVD). EVD has a high mortality rate and remains a significant threat to public health. Research on EVD pathogenesis has traditionally focused on host transcriptional responses. Limited recent studies, however, have revealed some information on the significance of cellular microRNAs (miRNAs) in EBOV infection and pathogenic mechanisms, but further studies are needed. Thus, this study aimed to identify and validate additional known and novel human miRNAs in EBOV-infected adult retinal pigment epithelial (ARPE) cells and predict their potential roles in EBOV infection and pathogenic mechanisms. We analyzed previously available small RNA-Seq data obtained from ARPE cells and identified 23 upregulated and seven downregulated miRNAs in the EBOV-infected cells; these included two novel miRNAs and 17 additional known miRNAs not previously identified in ARPE cells. In addition to pathways previously identified by others, these miRNAs are associated with pathways and biological processes that include WNT, FoxO, and phosphatidylinositol signaling; these pathways were not identified in the original study. This study thus confirms and expands on the previous study using the same datasets and demonstrates further the importance of human miRNAs in the host response and EVD pathogenesis during infection.

Inhibition of Connective Tissue Growth Factor Expression in Adult Retinal Pigment Epithelial-19 Cells by Blocking Yes-Associated Protein/Transcriptional Coactivator with PDZ-Binding Motif Activity.

Purpose: To investigate the effect of yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ) on connective tissue growth factor (CTGF) expression in adult retinal pigment epithelial (ARPE)-19 cells. We also studied the inhibitory effect of K-975, a new pan-transcriptional enhanced associate domain (TEAD) inhibitor, and luteolin, a plant-derived flavonoid on CTGF expression. Methods: ARPE-19 cells were transfected with either YAP or TAZ overexpression plasmid or treated with transforming growth factor (TGF)-β2. The cells were cultured either with or without K-975 or luteolin. The expression of YAP, TAZ, and CTGF was examined using real-time PCR. Results: ARPE-19 cells overexpressing YAP or TAZ exhibited significantly increased CTGF expression. This increase was attenuated by K-975 or luteolin alone. TGF-β2 treatment significantly raised the expression of not just YAP and TAZ, but also CTGF in ARPE-19 cells. TGF-β2 treatment-enhanced CTGF expression was considerably lowered by the addition of K-975 or luteolin. Conclusions: Overexpression of YAP or TAZ and treatment with TGF-β2 led to an increase in the expression of CTGF in ARPE-19 cells. These increases were attenuated by treatment with K-975 and luteolin. These findings suggest that YAP and TAZ may be related to the expression of CTGF in ARPE-19 cells and that K-975 and luteolin can be explored as potential therapeutic agents for preventing CTGF production in vitreoretinal fibrosis.

Diabetes and high-glucose could upregulate the expression of receptor for activated C kinase 1 in retina.

Diabetic retinopathy (DR) is a major ocular complication of diabetes mellitus, leading to visual impairment. Retinal pigment epithelium (RPE) injury is a key component of the outer blood retinal barrier, and its damage is an important indicator of DR. Receptor for activated C kinase 1 (RACK1) activates protein kinase C-ε (PKC-ε) to promote the generation of reactive oxygen species (ROS) in RPE cells, leading to apoptosis. Therefore, we hypothesize that the activation of RACK1 under hypoxic/high-glucose conditions may promote RPE cell apoptosis by modulating PKC-ε/ROS, thereby disrupting the barrier effect of the outer blood retinal barrier and contributing to the progression of DR. To investigate the role and associated underlying mechanisms of RACK1 in the development of early DR. In this study, Sprague-Dawley rats and adult RPE cell line-19 (ARPE-19) cells were used as in vivo and in vitro models, respectively, to explore the role of RACK1 in mediating PKC-ε in early DR. Furthermore, the impact of RACK1 on apoptosis and barrier function of RPE cells was also investigated in the former model. Streptozotocin-induced diabetic rats showed increased apoptosis and up-regulated expression of RACK1 and PKC-ε proteins in RPE cells following a prolonged modeling. Similarly, ARPE-19 cells exposed to high glucose and hypoxia displayed elevated mRNA and protein levels of RACK1 and PKC-ε, accompanied by an increases in ROS production, apoptosis rate, and monolayer permeability. However, silencing RACK1 significantly downregulated the expression of PKC-ε and ROS, reduced cell apoptosis and permeability, and protected barrier function. RACK1 plays a significant role in the development of early DR and might serve as a potential therapeutic target for DR by regulating RPE apoptosis and barrier function.

Beneficial Effects of Fibroblast Growth Factor-1 on Retinal Pigment Epithelial Cells Exposed to High Glucose-Induced Damage: Alleviation of Oxidative Stress, Endoplasmic Reticulum Stress, and Enhancement of Autophagy.

Diabetic retinopathy (DR) severely affects vision in individuals with diabetes. High glucose (HG) induces oxidative stress in retinal cells, a key contributor to DR development. Previous studies suggest that fibroblast growth factor-1 (FGF-1) can mitigate hyperglycemia and protect tissues from HG-induced damage. However, the specific effects and mechanisms of FGF-1 on DR remain unclear. In our study, FGF-1-pretreated adult retinal pigment epithelial (ARPE)-19 cells were employed to investigate. Results indicate that FGF-1 significantly attenuated HG-induced oxidative stress, including reactive oxygen species, DNA damage, protein carbonyl content, and lipid peroxidation. FGF-1 also modulated the expression of oxidative and antioxidative enzymes. Mechanistic investigations showed that HG induced high endoplasmic reticulum (ER) stress and upregulated specific proteins associated with apoptosis. FGF-1 effectively alleviated ER stress, reduced apoptosis, and restored autophagy through the adenosine monophosphate-activated protein kinase/mammalian target of the rapamycin signaling pathway. We observed that the changes induced by HG were dose-dependently reversed by FGF-1. Higher concentrations of FGF-1 (5 and 10 ng/mL) exhibited increased effectiveness in mitigating HG-induced damage, reaching statistical significance (p < 0.05). In conclusion, our study underscores the promising potential of FGF-1 as a safeguard against DR. FGF-1 emerges as a formidable intervention, attenuating oxidative stress, ER stress, and apoptosis, while concurrently promoting autophagy. This multifaceted impact positions FGF-1 as a compelling candidate for alleviating retinal cell damage in the complex pathogenesis of DR.

Identifying biomarkers of heterogeneity and transplantation efficacy in retinal pigment epithelial cells.

Transplantation of retinal pigment epithelial (RPE) cells holds great promise for patients with retinal degenerative diseases, such as age-related macular degeneration. In-depth characterization of RPE cell product identity and critical quality attributes are needed to enhance efficacy and safety of replacement therapy strategies. Here, we characterized an adult RPE stem cell-derived (RPESC-RPE) cell product using bulk and single-cell RNA sequencing (scRNA-seq), assessing functional cell integration in vitro into a mature RPE monolayer and in vivo efficacy by vision rescue in the Royal College of Surgeons rats. scRNA-seq revealed several distinct subpopulations in the RPESC-RPE product, some with progenitor markers. We identified RPE clusters expressing genes associated with in vivo efficacy and increased cell integration capability. Gene expression analysis revealed lncRNA (TREX) as a predictive marker of in vivo efficacy. TREX knockdown decreased cell integration while overexpression increased integration in vitro and improved vision rescue in the RCS rats.

Single-cell transcriptomics reveals maturation of transplanted stem cell–derived retinal pigment epithelial cells toward native state

Transplantation of stem cell-derived retinal pigment epithelial (RPE) cells is considered a viable therapeutic option for age-related macular degeneration (AMD). Several landmark Phase I/II clinical trials have demonstrated safety and tolerability of RPE transplants in AMD patients, albeit with limited efficacy. Currently, there is limited understanding of how the recipient retina regulates the survival, maturation, and fate specification of transplanted RPE cells. To address this, we transplanted stem cell-derived RPE into the subretinal space of immunocompetent rabbits for 1 mo and conducted single-cell RNA sequencing analyses on the explanted RPE monolayers, compared to their age-matched invitro counterparts. We observed an unequivocal retention of RPE identity, and a trajectory-inferred survival of all invitro RPE populations after transplantation. Furthermore, there was a unidirectional maturation toward the native adult human RPE state in all transplanted RPE, regardless of stem cell resource. Gene regulatory network analysis suggests that tripartite transcription factors (FOS, JUND, and MAFF) may be specifically activated in posttransplanted RPE cells, to regulate canonical RPE signature gene expression crucial for supporting host photoreceptor function, and to regulate prosurvival genes required for transplanted RPE's adaptation to the host subretinal microenvironment. These findings shed insights into the transcriptional landscape of RPE cells after subretinal transplantation, with important implications for cell-based therapy for AMD.

All-trans retinoic acid regulates the expression of MMP-2 and TGF-β2 via RDH5 in retinal pigment epithelium cells.

To investigate the effect of all-trans retinoic acid (ATRA) on retinol dehydrogenase 5 (RDH5), matrix metalloproteinase-2 (MMP-2) and transforming growth factor-β2 (TGF-β2) transcription levels, and the effect of RDH5 on MMP-2 and TGF-β2 in retinal pigment epithelium (RPE) cells. After adult RPE cell line-19 (ARPE-19 cells) intervened with gradient concentrations of ATRA (0-20 µmol/L) for 24h, flow cytometry was used to detect the proliferation and apoptosis of cells in each group, and quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect RDH5, MMP-2 and TGF-β2 mRNA expression. Then, after ARPE-19 cells transfected with three different siRNA targets for 48h, the RDH5 knockdown efficiency of each group and expression of MMP-2 and TGF-β2 mRNA within them was detected by qRT-PCR. Flow cytometry results showed that ATRA could inhibit the proliferation of RPE cells and promote the apoptosis of RPE cells, and the difference of apoptosis was statistically significant when the ATRA concentration exceeded 5 µmol/L and compared with the normal control group (P=0.027 and P=0.031, respectively). qRT-PCR results showed that ATRA could significantly inhibit the expression level of RDH5 mRNA (P<0.001) and promote the expression of MMP-2 and TGF-β2 mRNA (P=0.03 and P<0.001, respectively) in a dose-dependent manner, especially when treated with 5 µmol/L ATRA. The knockdown efficiency of RDH5 siRNA varies with different targets, among which RDH5 siRNA-435 had the highest knockdown efficiency, i.e., more than 50% lower than that of the negative control group (P=0.02). When RDH5 was knocked down for 48h, the results of qRT-PCR showed that the expressions of MMP-2 and TGF-β2 mRNA were significantly up-regulated (P<0.001). ATRA inhibits the expression of RDH5 and promotes MMP-2 and TGF-β2, and further RDH5 knockdown significantly upregulates MMP-2 and TGF-β2. These findings suggest that RDH5 may be involved in an epithelial-mesenchymal transition of RPE cells mediated by ATRA.

Comparison of vegetable oils on the uptake of lutein and zeaxanthin by ARPE-19 cells.

To compare the effect of vegetable oils on the uptake of lutein and zeaxanthin by adult retinal pigment epithelial (ARPE)-19 cells in vitro. ARPE-19 cells were cultured in Dulbecco's Modified Eagle Medium-F-12 supplemented with 10% foetal bovine serum and 1% penicillin-streptomycin in a humidified 5% CO2 incubator maintained at 37°C. Cells were treated with 247 µmol/L lutein, 49 µmol/L zeaxanthin and 1% (v/v) of either coconut oil, corn oil, peanut oil, olive oil, sunflower oil, soybean oil, castor oil, or linseed oil for 48h. Lutein and zeaxanthin concentration in the cells were quantified by high performance liquid chromatography. Among the oils tested, the highest lutein and zeaxanthin uptake was observed with coconut oil while the lowest was observed with linseed oil. ARPE-19 uptake of lutein and zeaxanthin are found to be dependent on the type of oils.

Single-cell RNA sequencing reveals transcriptional changes of human choroidal and retinal pigment epithelium cells during fetal development, in healthy adult and intermediate age-related macular degeneration.

Age-related macular degeneration (AMD) is the most prevalent cause of blindness in the developed world. Vision loss in the advanced stages of the disease is caused by atrophy of retinal photoreceptors, overlying retinal pigment epithelium (RPE) and choroidal endothelial cells. The molecular events that underline the development of these cell types from in utero to adult as well as the progression to intermediate and advanced stages AMD are not yet fully understood. We performed single-cell RNA-sequencing (RNA-Seq) of human fetal and adult RPE-choroidal tissues, profiling in detail all the cell types and elucidating cell type-specific proliferation, differentiation and immunomodulation events that occur up to midgestation. Our data demonstrate that progression from the fetal to adult state is characterized by an increase in expression of genes involved in the oxidative stress response and detoxification from heavy metals, suggesting a better defence against oxidative stress in the adult RPE-choroid tissue. Single-cell comparative transcriptional analysis between a patient with intermediate AMD and an unaffected subject revealed a reduction in the number of RPE cells and melanocytes in the macular region of the AMD patient. Together these findings may suggest a macular loss of RPE cells and melanocytes in the AMD patients, but given the complex processing of tissues required for single-cell RNA-Seq that is prone to technical artefacts, these findings need to be validated by additional techniques in a larger number of AMD patients and controls.

Role of Adrenomedullin 2/Intermedin in the Pathogenesis of Neovascular Age-Related Macular Degeneration

Adrenomedullin 2 (AM2; also known as intermedin) is a member of the adrenomedullin (AM) peptide family. Similarly to AM, AM2 partakes in a variety of physiological activities. AM2 has been reported to exert protective effects on various organ disorders; however, its significance in the eye is unknown. We investigated the role of AM2 in ocular diseases. The receptor system of AM2 was expressed more abundantly in the choroid than in the retina. In an oxygen-induced retinopathy model, physiological and pathologic retinal angiogenesis did not differ between AM2-knockout (AM2-/-) and wild-type mice. In contrast, in laser-induced choroidal neovascularization, a model of neovascular age-related macular degeneration, AM2-/- mice had enlarged and leakier choroidal neovascularization lesions, with exacerbated subretinal fibrosis and macrophage infiltration. Contrary to this, exogenous administration of AM2 ameliorated the laser-induced choroidal neovascularization–associated pathology and suppressed gene expression associated with inflammation, fibrosis, and oxidative stress, including that of VEGF-A, VEGFR-2, CD68, CTGF, and p22-phox. The stimulation of human adult retinal pigment epithelial (ARPE) cell line 19 cells with TGF-β2 and TNF-α induced epithelial-to-mesenchymal transition (EMT), whereas AM2 expression was also elevated. The induction of EMT was suppressed when the ARPE-19 cells were pretreated with AM2. A transcriptome analysis identified 15 genes, including mesenchyme homeobox 2 (Meox2), whose expression was significantly altered in the AM2-treated group compared with that in the control group. The expression of Meox2, a transcription factor that inhibits inflammation and fibrosis, was enhanced by AM2 treatment and attenuated by endogenous AM2 knockout in the early phase after laser irradiation. The AM2 treatment of endothelial cells inhibited endothelial to mesenchymal transition and NF-κB activation; however, this effect tended to be canceled following Meox2 gene knockdown. These results indicate that AM2 suppresses the neovascular age-related macular degeneration–related pathologies partially via the upregulation of Meox2. Thus, AM2 may be a promising therapeutic target for ocular vascular diseases.

Self-Healing Thermosensitive Hydrogel for Sustained Release of Dexamethasone for Ocular Therapy.

The aim of this study was to develop an injectable hydrogel delivery system for sustained ocular delivery of dexamethasone. To this end, a self-healing hydrogel consisting of a thermosensitive ABA triblock copolymer was designed. The drug was covalently linked to the polymer by copolymerization of methacrylated dexamethasone with N-isopropylacrylamide (NIPAM) and N-acryloxysuccinimide (NAS) through reversible addition-fragmentation chain transfer (RAFT) polymerization, using poly(ethylene glycol) (PEG) functionalized at both ends with a chain transfer agent (CTA). Hydrogel formation was achieved by mixing aqueous solutions of the formed thermosensitive polymer (with a cloud point of 23 °C) with cystamine at 37 °C, to result in covalent cross-linking due to the reaction of the N-hydroxysuccimide (NHS) functionality of the polymer and the primary amines of cystamine. Rheological analysis showed both thermogelation and covalent cross-linking at 37 °C, as well as the self-healing properties of the formed network, which was attributed to the presence of disulfide bonds in the cystamine cross-links, making the system injectable. The release of dexamethasone from the hydrogel occurred through ester hydrolysis following first-order kinetics in an aqueous medium at pH 7.4 over 430 days at 37 °C. Based on simulations, administration of 100 mg of hydrogel would be sufficient for maintaining therapeutic levels of dexamethasone in the vitreous for at least 500 days. Importantly, dexamethasone was released from the hydrogel in its native form as determined by LC-MS analysis. Cytocompatibility studies showed that at clinically relevant concentrations, both the polymer and the cross-linker were well tolerated by adult retinal pigment epithelium (ARPE-19) cells. Moreover, the hydrogel did not show any toxicity to ARPE-19 cells. The injectability of the hydrogel, together with the long-lasting release of dexamethasone and good cytocompatibility with a retinal cell line, makes this delivery system an attractive candidate for treatment of ocular inflammatory diseases.

Transcriptional comparison of adult human primary Retinal Pigment Epithelium, human pluripotent stem cell-derived Retinal Pigment Epithelium, and ARPE19 cells.

The therapeutic potential of pluripotent stem cells is great as they promise to usher in a new era of medicine where cells or organs may be prescribed to replace dysfunctional tissue. At the forefront are efforts in the eye to develop this technology as it lends itself to in vivo monitoring and sophisticated non-invasive imaging modalities. In the retina, retinal pigment epithelium (RPE) is the most promising replacement cell as it has a single layer, is relatively simple to transplant, and is associated with several eye diseases. However, after transplantation, the cells may transform and cause complications. This transformation may be partially due to incomplete maturation. With the goal of learning how to mature RPE, we compared induced pluripotent stem cell-derived RPE (iPSC-RPE) cells with adult human primary RPE (ahRPE) cells and the immortalized human ARPE-19 line. We cultured ARPE-19, iPSC-RPE, and ahRPE cells for one month, and evaluated morphology, RPE marker staining, and transepithelial electrical resistance (TEER) as quality control indicators. We then isolated RNA for bulk RNA-sequencing and DNA for genotyping. We genotyped ahRPE lines for the top age-related macular degeneration (AMD) and proliferative vitreoretinopathy (PVR) risk allele polymorphisms. Transcriptome data verified that both adult and iPSC-RPE exhibit similar RPE gene expression signatures, significantly higher than ARPE-19. In addition, in iPSC-RPE, genes relating to stem cell maintenance, retina development, and muscle contraction were significantly upregulated compared to ahRPE. We compared ahRPE to iPSC-RPE in a model of epithelial-mesenchymal transition (EMT) and observed an increased sensitivity of iPSC-RPE to producing contractile aggregates in vitro which resembles incident reports upon transplantation. P38 inhibition was capable of inhibiting iPSC-RPE–derived aggregates. In summary, we find that the transcriptomic signature of iPSC-RPE conveys an immature RPE state which may be ameliorated by targeting “immature” gene regulatory networks.

Inflammasome Activation in Retinal Pigment Epithelium from Human Donors with Age-Related Macular Degeneration.

Age-related macular degeneration (AMD), the leading cause of blindness in the elderly, is characterized by the death of retinal pigment epithelium (RPE) and photoreceptors. One of the risk factors associated with developing AMD is the single nucleotide polymorphism (SNP) found within the gene encoding complement factor H (CFH). Part of the innate immune system, CFH inhibits alternative complement pathway activation. Multi-protein complexes called inflammasomes also play a role in the innate immune response. Previous studies reported that inflammasome activation may contribute to AMD pathology. In this study, we used primary human adult RPE cell cultures from multiple donors, with and without AMD, that were genotyped for the Y402H CFH risk allele. We found complement and inflammasome-related genes and proteins at basal levels in RPE tissue and cell cultures. Additionally, treatment with rotenone, bafilomycin A, and ATP led to inflammasome activation. Overall, the response to priming and activation was similar, irrespective of disease state or CFH genotype. While these data show that the inflammasome is present and active in RPE, our results suggest that inflammasome activation may not contribute to early AMD pathology.

Heterogeneity of Potassium Channels in Human Embryonic Stem Cell-Derived Retinal Pigment Epithelium

Human pluripotent stem cell (hPSC)-derived retinal pigment epithelium (RPE) is extensively used in RPE research, disease modeling, and transplantation therapies. For successful outcomes, a thorough evaluation of their physiological authenticity is a necessity. Essential determinants of this are the different ion channels of the RPE, yet studies evaluating this machinery in hPSC-RPE are scarce. We examined the functionality and localization of potassium (K+) channels in the human embryonic stem cell (hESC)-derived RPE. We observed a heterogeneous pattern of voltage-gated K+ (KV) and inwardly rectifying K+ (Kir) channels. Delayed rectifier currents were recorded from most of the cells, and immunostainings showed the presence of KV1.3 channel. Sustained M-currents were also present in the hESC-RPE, and based on immunostaining, these currents were carried by KCNQ1-KCNQ5 channel types. Some cells expressed transient A-type currents characteristic of native human fetal RPE (hfRPE) and cultured primary RPE and carried by KV1.4 and KV4.2 channels. Of the highly important Kir channels, we found that Kir7.1 is present both at the apical and basolateral membranes of the hESC- and fresh native mouse RPE. Kir currents, however, were recorded only from 14% of the hESC-RPE cells with relatively low amplitudes. Compared to previous studies, our data suggest that in the hESC-RPE, the characteristics of the delayed rectifier and M-currents resemble native adult RPE, while A-type and Kir currents resemble native hfRPE or cultured primary RPE. Overall, the channelome of the RPE is a sensitive indicator of maturity and functionality affecting its therapeutic utility.

Oleuropein Protects Human Retinal Pigment Epithelium Cells from IL-1β-Induced Inflammation by Blocking MAPK/NF-κB Signaling Pathways.

Proinflammatory mediators such as interleukin (IL)-1β cause retinal pigment epithelium (RPE) inflammation, which is related to visual deterioration, including age-related macular degeneration and diabetic retinopathy. Oleuropein is a polyphenol compound that shows potent anti-inflammatory, antioxidant, and anti-cancer activities, but its effects on IL-1β-induced inflammation have not been examined in the adult RPE cell line ARPE-19. Here, we assessed the ability of oleuropein to attenuate this inflammation in ARPE-19 cells. IL-1β induced secretion of the inflammatory cytokines IL-6, monocyte chemoattractant protein-1 (MCP)-1, and soluble intercellular adhesion molecule (sICAM)-1. As measured by enzyme-linked immunosorbent assay, oleuropein significantly inhibited levels of all three proteins and led to decreased monocyte adhesiveness to ARPE-19 cells. To clarify the underlying anti-inflammatory mechanisms, we used western blots to evaluate the effect of oleuropein on inactivation of the nuclear factor-kappa B (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways. The results showed that oleuropein significantly decreased levels of the inflammatory mediator cyclooxygenase-2 and increased anti-inflammatory protein HO-1 expression. We next examined if the anti-inflammatory activity of oleuropein arises via inactivated NF-κB. We found that suppressing phosphorylation of the JNK1/2 and p38 MAPK signaling pathways inhibited IL-6, MCP-1, and sICAM-1 secretion, implicating these pathways and NF-κB suppression in the effects of oleuropein. These results indicate that oleuropein shows potential for the prevention and treatment of inflammatory diseases of the retina.

Intravitreal connective tissue growth factor neutralizing antibody or bevacizumab alone or in combination for prevention of proliferative vitreoretinopathy in an experimental model

Connective tissue growth factor (CTGF) is released by retinal pigment epithelial (RPE) cells and detectable in proliferative membranes (PrMs). This experimental study was performed to investigate the mRNA and protein levels of both CTGF and vascular endothelial growth factor A (VEGF-A) in a rabbit model of proliferative vitreoretinopathy (PVR). In addition, the effects of a single intravitreal injection of the safe dose of anti-CTGF or bevacizumab as monotherapy and in combination were evaluated. PVR was induced in the right eye of albino rabbits by intravitreal injection of cultured adult human RPE cells. Quantitative real-time reverse transcription PCR (qRT-PCR) and Western blot analysis of CTGF and VEGF-A were performed on whole eye tissue in the PVR model versus controls at different time points. In the next step, the PVR models were assigned to five groups. The monotherapy groups received a single intravitreal injection of 0.1 ml of anti-CTGF 100 μg/ml (final concentration of 6.6 μg/ml in the vitreous) or 0.03 ml of 25 mg/ml bevacizumab. In the combined group, the abovementioned amounts of anti-CTGF and bevacizumab were injected intravitreally from separate sites in one session. No antibody injection was performed in the control group. Intravitreal injection of 0.1 ml of control IgG (1 mg/ml of isotype matched) antibody was performed in the placebo group. After 2 weeks, histologic evaluation including, trichrome staining for collagen, immunostaining by anti-alpha-smooth muscle actin for myofibroblasts, and anti-collagen type-1 antibody on paraffin embedded anterior-posterior sections was done. In addition, fundus photography was performed for clinically equivalent PVR staging. Twenty-four hours following PVR induction, CTGF mRNA and protein levels increased five- and- three-fold compared to controls, respectively (P < 0.001). VEGF-A mRNA and protein levels decreased significantly after 72 h of PVR induction compared to controls (P < 0.05). Means of PrM thickness and myofibroblast cell counts significantly decreased in the anti-CTGF group (P < 0.001 and P < 0.05, respectively). The mean area of collagen type-1 fibers of PrM in the mono- and combination therapy groups that received intravitreal anti-CTGF was significantly reduced (P < 0.001); in addition, mild PVR (stage-1 and 2) formation occurred in comparison with moderate to severe PVR (stage-4 and higher) in other groups. In conclusion, we found that intravitreal injection of CTGF neutralizing antibody resulted in a reduction in PrM thickness, collagen fibers and myofibroblast density in the PVR model. CTGF inhibition may represent a potential therapeutic target for PVR.

SARS-CoV-2 infects human adult donor eyes and hESC-derived ocular epithelium.

The SARS-CoV-2 pandemic has caused unparalleled disruption of global behavior and significant loss of life. To minimize SARS-CoV-2 spread, understanding the mechanisms of infection from all possible routes of entry is essential. While aerosol transmission is thought to be the primary route of spread, viral particles have been detected in ocular fluid, suggesting that the eye may be a vulnerable point of viral entry. To this end, we confirmed SARS-CoV-2 entry factor and antigen expression in post-mortem COVID-19 patient ocular surface tissue and observed productive viral replication in cadaver samples and eye organoid cultures, most notably in limbal regions. Transcriptional analysis of ex vivo infected ocular surface cells and hESC-derived eye cultures revealed robust induction of NF-κB in infected cells as well as diminished type I/III interferon signaling. Together these data suggest that the eye can be directly infected by SARS-CoV-2 and implicate limbus as a portal for viral entry.

Protective effect of gypenoside LXXV from Gynostemma pentaphyllum against oxidative stress-induced retinal degeneration in vitro and in vivo

BackgroundCaring the eye from oxidative stress is very important in preventing the age-related macular degeneration (AMD), which is difficult to recover it once damaged. Particularly, the development of therapeutic anti-dry-AMD agent in a non-invasive manner is still on the horizon. PurposeIn the present study, we examined effect of gypenoside (Gyp) LXXV against dry-AMD in vitro and in vivo, to develop non-invasive anti-AMD agents based on a natural compound with anti-oxidative and anti-inflammatory activity. Study designGyp LXXV, a single ginseng ginsenoside compound, is orally administered to adult rabbits and mice that the dry-AMD was induced by asodium iodate or a light irradiation, respectively. The in vitro assay was performed using the retinal cells (ARPE-19) and macrophage cells (HMC-3). MethodsTo evaluate anti-oxidative capability and anti-inflammable efficacy of Gyp LXXV in retinal pigment epithelial (RPE) cells, real time PCR was employed using ARPE-19 cells and HMC-3 cells. To validate physiological effects of Gyp LXXV on dry-AMD, thickness measurement of RPE, electroretinogram (ERG), optical coherence tomography (OCT), and fundus examination were employed in either light irradiation- or sodium iodate-induced animal model. ResultsIt exhibited substantial alleviation of damaged retinal RPE tissues, via lowering the structural damage, the reactive oxygen species (ROS) level, and inflammatory cytokines in a dose-dependent manner, without showing any side effects. In addition, ROS-induced human adult RPE cells were treated with Gyp LXXV showed a protective effect against the ROS, confirming an excellent antioxidant efficacy with no potential cytotoxicity. ConclusionOur findings indicate that the oral route of Gyp LXXV exerts a potential therapeutic effect on dry-AMD. This work could guide the new therapeutic pathway of a phytochemical drug-based non-invasive treatment for dry-AMD in future clinical treatments.