A Single-Cell Transcriptome Atlas of the Human Retinal Pigment Epithelium

Human retinal pigment epithelium cells are arranged in a monolayer that plays an important supporting role in the retina. Although the heterogeneity of specific retinal cells has been well studied, the diversity of hRPE cells has not been reported. Here, we performed a single-cell RNA sequencing on 9,302 hRPE cells from three donors and profiled a transcriptome atlas. Our results identified two subpopulations that exhibit substantial differences in gene expression patterns and functions. One of the clusters specifically expressed ID3, a macular retinal pigment epithelium marker. The other cluster highly expressed CRYAB, a peripheral RPE marker. Our results also showed that the genes associated with oxidative stress and endoplasmic reticulum stress were more enriched in the macular RPE. The genes related to light perception, oxidative stress and lipid metabolism were more enriched in the peripheral RPE. Additionally, we provided a map of disease-related genes in the hRPE and highlighted the importance of the macular RPE and peripheral RPE clusters P4 and P6 as potential therapeutic targets for retinal diseases. Our study provides a transcriptional landscape for the human retinal pigment epithelium that is critical to understanding retinal biology and disease.

Circ_0000615 promotes high glucose-induced human retinal pigment epithelium cell apoptosis, inflammation and oxidative stress via miR-646/YAP1 axis in diabetic retinopathy

Background: Diabetic retinopathy (DR), a common complication of diabetes mellitus, is the major cause of visual impairment and blindness. Circ_0000615 was found to be elevated in retina samples of diabetic patients. Hence, the detailed effects and molecular mechanisms of circ_0000615 in DN progression were explored. Methods: The levels of circ_0000615, microRNA (miR)-646 and YAP1 (yes-associated protein 1) were detected using quantitative real-time polymerase chain reaction and Western blot assays. Cell viability, apoptosis, inflammation and reactive oxygen species (ROS) generation were determined using cell counting kit-8 assay, flow cytometry, caspase3 activity analysis, Western blot, enzyme-linked immunosorbent assay (ELISA) and Dichlorofluorescein diacetate (DCFH-DA) assay, respectively. The binding interaction between miR-646 and circ_0000615 or YAP1 was determined using dual-luciferase reporter, RNA immunoprecipitation (RIP), and RNA pull-down assays. Results: Circ_0000615 was elevated in high glucose (HG)-induced human retinal pigment epithelium (HRPE) cells. Knockdown of circ_0000615 attenuated HG-triggered HRPE cell apoptosis, inflammation, and ROS generation. Mechanistically, miR-646 was confirmed to be a target of circ_0000615, inhibition of miR-646 reversed the protective effects of circ_0000615 knockdown on HG-evoked HRPE cell dysfunction. MiR-646 was verified to target YAP1, overexpression of YAP1 abolished the impairment induced by miR-646 on HG-induced HRPE cell damage. Besides that, we confirmed that circ_0000615 could regulate YAP1 expression via miR-646. Conclusion: Circ_0000615 contributed to HG-induced HRPE cell dysfunction via miR-646/YAP1 axis, suggesting a novel insight into the pathogenesis of DR and a potential candidate for DR treatment.

Sodium iodate induces ferroptosis in human retinal pigment epithelium ARPE-19 cells

Sodium iodate (SI) is a widely used oxidant for generating retinal degeneration models by inducing the death of retinal pigment epithelium (RPE) cells. However, the mechanism of RPE cell death induced by SI remains unclear. In this study, we investigated the necrotic features of cultured human retinal pigment epithelium (ARPE-19) cells treated with SI and found that apoptosis or necroptosis was not the major death pathway. Instead, the death process was accompanied by significant elevation of intracellular labile iron level, ROS, and lipid peroxides which recapitulated the key features of ferroptosis. Ferroptosis inhibitors deferoxamine mesylate (DFO) and ferrostatin-1(Fer-1) partially prevented SI-induced cell death. Further studies revealed that SI treatment did not alter GPX4 (glutathione peroxidase 4) expression, but led to the depletion of reduced thiol groups, mainly intracellular GSH (reduced glutathione) and cysteine. The study on iron trafficking demonstrated that iron influx was not altered by SI treatment but iron efflux increased, indicating that the increase in labile iron was likely due to the release of sequestered iron. This hypothesis was verified by showing that SI directly promoted the release of labile iron from a cell-free lysate. We propose that SI depletes GSH, increases ROS, releases labile iron, and boosts lipid damage, which in turn results in ferroptosis in ARPE-19 cells.