Dry age-related macular degeneration (AMD) is a progressive state that causes blindness primarily due to the loss of retinal pigment epithelium (RPE). Damage to RPE cells that function both as a blood-retinal barrier and regulator of the overlying photoreceptor (PR) layer is closely associated with dry AMD. Early-stage dry AMD is characterized by yellowish deposits (drusen) below the Bruch membrane and disordered pigmentation in the macular RPE layers (Jager
The etiology of dry AMD is complex and remains unresolved. However, accumulating evidence shows that accelerated aging-like changes in the RPE play a fundamental role in the development of AMD. The lipofuscin fluorophore N-retinylidene-N-retinylethanolamine (A2E) undergoes photo-oxidation and generates oxygen adducts in the presence of blue light (BL) and oxygen; thus, it might contribute to RPE cell degeneration (Wielgus
Degeneration of the RPE induced by light is a popular model for studies of dry AMD
Here, we investigate the effects of long-term exposure to BL on RPE cells
Human retinal pigment epithelial cells (ARPE-19; American Type Culture Collection, Manassas, VA, USA) were grown in Dulbecco’s modified Eagle’s medium F-12 (DMEM/F-12; Welgene, Daegu, Korea) containing 10% fetal bovine serum at 37°C under a 5% CO2 atmosphere.
We seeded ARPE-19 cells (5×104 cells/well) in 6-well plates and incubated them with 5 μM A2E (AptaBio, Yongin, Korea) every other day and exposed them to BL (BL; 430 nm, 6,000 lux, 5 min/day) for 0 (D-0), 40 (D-40), 100 (D-100), and 120 (D-120) days. The device for irradiating the ARPE-19 cells with blue light is equipped with an LED on the top of the inside of the device to emit a blue light peak at 430 nm (Four Tech, Gunpo, Korea). The temperature inside the device was kept constant during blue light irradiation (5 min). The intensity of blue light was measured using a display color analyzer (CA-310, Konica Minolta Sensing, Inc., Tokyo, Japan). Total RNA was extracted at each time point using TRIzol (Invitrogen, Carlsbad, CA, USA).
Total RNA was processed to prepare an mRNA sequencing (mRNA-seq) library using TruSeq Stranded mRNA kits (Illumina Inc., San Diego, CA, USA) (Yang
Total RNA isolated from APRE-19 cells was reverse-transcribed using iScript cDNA synthesis kits (Bio-Rad Laboratories, Hercules, CA, USA) in a total volume of 20 μL containing 2 μL of RT products (Ul-Haq
Clinical transcriptome data for comparison with long-term exposure models
The RT-qPCR data were statistically analyzed using two-tailed Student t-tests. Values with
Studies of transcriptomes and related pathways in dry AMD have been limited to RPE cell lines exposed to A2E and BL for short periods or retinal tissues from patients with advanced AMD. However, because dry macular degeneration occurs slowly, current approaches are insufficient to understand changes in gene transcription as the disease progresses and thus have limited ability to identify the genes responsible for AMD pathogenesis. Therefore, we designed a long-term model to overcome these limitations and determine the molecular mechanisms involved in the pathogenesis of dry AMD. We applied a low concentration of A2E (5 μM) to ARPE-19 cells at 2-day intervals and exposed them to BL for 5 min/day for 120 days (Fig. 1A). To identify DEGs in this ARPE-19 model of dry AMD
The global expression profiles of A2E-laden RPE cells irradiated with BL for various periods would be valuable for elucidating the mechanism of dry AMD. The RNA-seq data from D-100 revealed 106 DEGs in RPE cells incubated with A2E and exposed to BL compared with control group (D-0). We analyzed pathways based on these findings using Enrichr (BioPlanet 2019) (Kuleshov
Genes with significant changes in expression at the early stage might affect PRE cell dysfunction and trigger other changes during the middle and late stages. Similarly, functional damage at an early stage induces cellular physiological changes at a later stage and consequently results in AMD lesions. Therefore, to further investigate the relationship between gene expression changes, we separately analyzed DEGs with increased or decreased expression during the early, middle, and late stages. Thus, the DEGs identified by long-term models of dry AMD
We identified cell biological pathways affected by DEGs at each time point using MsigDB (Fig. 3C). The results showed that genes transiently downregulated by BL in RPE cells incubated with A2E were significantly enriched in pathways associated with epithelial-mesenchymal transition (EMT), UV-response, and KRAS signaling. These genes consistently and significantly declined during the middle and late stages. The continuous decrease in the expression of genes involved in the EMT was consistent with the results of the DEG analysis (Fig. 2). Genes involved in glycolysis, angiogenesis, and mitotic spindles were selectively downregulated in the intermediate group. In contrast, genes involved in adipogenesis, hypoxia, apical junctions, and coagulation were affected in the intermediate and late groups. These results suggested that the BL effects on glycolysis, angiogenesis, and mitotic spindle precede those on adipogenesis, hypoxia, apical junctions, and coagulation. Additionally, genes involved in inflammatory pathways such as TNFα signaling through NF-κB, TGF-β signaling, and complement and cell cycle pathways, such as mTORC1 signaling, p53 pathway, and apoptosis, were significantly altered in the late group. Specifically, the genes corresponding to TNFα signaling through NF-κB were downregulated during the early stage (
We compared our RNA-seq results with gene expression profiles in retinas from AMD patients to validate the clinical relevance of our model. We analyzed retinal tissues donated by three patients (aged 78, 84, and 85 years, AMD) with a clinical diagnosis of atrophic AMD and by two normal donors (aged 78, 84 years, CTR) using a DNA microarray (Radeke
Clinically, dry AMD including early and geographic atrophy (GA), accounts for approximately 90% of all patients with AMD (Holz
The combination of BL illumination and A2E is toxic to RPE, ARPE-19, and primary porcine PRE cells
A genome-wide meta-analysis of AMD associations identified extracellular matrix tissue (
To our knowledge, this is the first study to identify genome-wide changes in gene expression in ARPE-19 cells after prolonged exposure to low doses of A2E and BL. Our findings not only provide a new perspective on the pathogenesis of dry AMD, but also provide a useful model for studying the mechanism of dysfunction induced by BL in RPE cells. The novel information about the different stages of the molecular pathogenesis of dry AMD provides new insights into identifying novel therapeutic targets to treat dry AMD by reducing lasting damage or by replacing, repairing, or regenerating damaged cells.
This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2021R1A2C1011132) to K.W.J. and National Natural Science Foundation of China (No. 81960667) to H.L.J.
The authors have declared that there is no conflict of interest.