CellSight researchers at the University of Colorado School of Medicine are offering the first evidence connecting drusen formation, or yellowish deposits that accumulate under the retina, with extracellular vesicles and age-related macular degeneration (AMD).
Published in the Journal of Extracellular Vesicles (2020 impact factor 25.8), Miguel Flores-Bellver, PhD, and Valeria Canto-Soler, PhD, highlight groundbreaking insight into mechanisms underlying early manifestations of AMD that could lead to early diagnosis and targeted treatment options of dry AMD.
Flores-Bellver’s research shows that retinal pigment epithelium (RPE) cells – a simple layer of cells located under the photoreceptor cells in the eye – release exosomes that contain both normal proteins and proteins that are associated with drusen, one of the key initial manifestations of AMD, in normal physiological conditions. Under stressful conditions, however, he found RPE cells release drusen-associated proteins about 20 times more, providing a potential biomarker of AMD.
“Miguel’s research shows that when RPE cells are exposed to an environment similar to that leading to AMD, they respond with a dramatic increase in the release of drusen-associated proteins via exosomes,” Canto-Soler, associate professor of ophthalmology, says. “It opens the possibility for early diagnosis of a person developing AMD by determining that their cells are secreting a lot more exosomes with these drusen-associated proteins than in normal conditions.”
For years, researchers have been searching for the origins of drusen-associated proteins as it relates to the symptoms and development of AMD. Now, this biomarker could be found in blood samples, tears, urine, or saliva.
Isolation and characterisation of induced-primary RPE-derived extracellular vesicles (EV)
Classical EV biomarker CD63 was identified in the cytoplasm of both, the RPE growing attached to the hRetOs (a) and the ipRPE monolayers (b). CD81, another EV indicator, showed cytoplasmic staining and prominent expression in punctate structures at the apical surface (c). Ultrastructural analysis of ipRPE monolayers revealed the presence of endosomes and multivesicular bodies (black arrowheads) intermingled with melanosomes in the apical side (d-d’’), and near the basal infoldings of the cells (d’’’, white arrowhead). EVs were isolated from either apical-A or basal-B conditioned media (CM) by differential ultracentrifugation as shown in the diagram (e). Typical clear rounded membrane vesicles within a size range of 50–200 nm were observed by TEM of EV pellets from both apical (f) and basal side (h). Nanoparticle tracking analysis revealed the size distribution for the apical (g) and basal (i) sides, the average concentration and, average size of EVs released by ipRPE monolayers (k). Western blots against well-known EV specific markers (TSG-101, Flotillin-1 and CD63) and an intracellular Golgi membrane protein (GM130) confirmed the purity of EV preparations obtained from ipRPE monolayers (l). Bar graphs represent mean ± SD. ***p < 0.0001. Scale bars, 10 μm (a and c); 20 μm (b); 500 nm (d–d’’’); 100 nm (f and h). n = 3
This discovery could help treat millions of Americans before they develop geographic atrophy, a later stage of dry AMD, because it is currently not possible to treat patient with early disease.
“Knowing that extracellular vesicles are releasing drusen-associated proteins presents an opportunity for novel diagnostic and therapeutical approaches,” Flores-Bellver says. “If we can define assays to measure these proteins released in these exosomes, we could potentially diagnose the disease early, perhaps even at the time of onset. Because of that, we may be able to treat AMD patients better. Also, we could think of developing new tools to target and modulate exosome release and protect the cells from the accumulation of drusen under the retina.”
“If we understand how drusen form and what cells or mechanisms contribute to their formation, we may be able to control the formation of drusen and slow down or even, perhaps prevent, some of the pathological events leading to AMD,” Canto-Soler adds.
“We started CellSight four years ago with the goal of developing stem cell-based diagnostics and therapies for retinal degenerative diseases,” Canto-Soler says. “This discovery opens a path for developing one of these potential diagnostic or therapeutic tools for patients with AMD.”