Abstract
Disruption of retinal pigment epithelial (RPE) barrier integrity and RPE migration are hallmark features in neovascular age-related macular degeneration (nAMD), but the underlying causes and pathophysiology are not completely well-defined. Herein, we aimed to evaluate the effect of bone morphogenetic proteins (BMPs) on the barrier function and migration of RPE. In particular, we investigated the role of BMP2 and BMP4 in these processes as our analysis of RNA-sequencing (seq) data from human donor eyes demonstrated that they are highly differentially expressed BMP members in macular RPE/choroid versus macular retina. We used electrical cell-substrate impedance sensing (ECIS) system to monitor precisely in real time the barrier integrity and migration of ARPE-19 after treatment with various concentrations of BMP2 or BMP4. Immunofluorescence was also used to assess the changes in the expression and the organization of the key tight junction protein, zona occludens (ZO)-1, in ARPE-19 cells under BMP2 or BMP4 treatment. This was followed by measuring the activity of matrix metalloproteinases (MMPs). Finally, RNA-seq and ELISA were used to determine the local and circulating levels of BMP2 and BMP4 in retinas and serum samples from nAMD donors. Our ECIS results showed that BMP4 but not BMP2 decreased the transcellular electrical resistance (TER) of ARPE-19 and increased their migration in comparison with control (vehicle-treated cells). Furthermore, immunofluorescence showed a disorganization of ZO-1 in BMP4-treated ARPE-19 not in BMP2-treated cells or vehicle-treated controls. This effect of BMP4 was associated with significant increases in the activity of MMPs, specifically MMP2. Lastly, these results were corroborated by additional findings that circulating but not local BMP4 levels were significantly higher in nAMD donor samples compared to controls. Collectively, our results demonstrated unreported effects of BMP4 on inducing RPE dysfunction and suggest that BMP4 but not BMP2 may represent a potential therapeutic target in nAMD.
Highlights
Age-related macular degeneration (AMD) is a progressive neurodegenerative disease causing vision loss and even blindness in geriatric population over age of 60 [1]
Given the fact that loss of monolayer integrity is closely related to the retinal pigment epithelial cell (RPE) dysfunction in neovascular AMD (nAMD), a functional assay was carried out in vitro to investigate whether BMP2 or BMP4 disrupts barrier function of ARPE-19 cells using a real-time monitoring of transepithelial electric resistance (TER) as an indicator of barrier integrity
Thereafter, monolayers of ARPE-19 cells were treated with BMP2 at different concentrations (0, 25, 50, 100, 200, or 400 ng/mL; Figure 1A–F, respectively) and the barrier integrity was monitored over a 100-h period as well as across 9 frequencies (250, 500, 1000, 2000, 4000, 8000, 16,000, 32,000, and 64,000 Hz, represented as log values on the x-axis)
Summary
Age-related macular degeneration (AMD) is a progressive neurodegenerative disease causing vision loss and even blindness in geriatric population over age of 60 [1]. AMD begins as a clinically undetectable disease with the thinning of macula, a light sensitive tissue in the center of the retina needed for seeing fine detail. As this early stage of the disease progresses, a noticeable accumulation of debris known as drusen becomes more visible upon ophthalmoscopic examination as yellow spots beneath the retina [2]. While many patients respond well to the anti-VEGF treatments for neovascular AMD, a subset of patients do not These modalities may be limited by their invasiveness and burden to the patient and treating ophthalmologist. There is an urgent need, to find innovative mechanistic avenues to treat or prevent AMD
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