Small Extracellular Vesicle-Associated MiRNAs in Polarized Retinal Pigmented Epithelium.
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2024-11
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Abstract
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Oxidative stress in the retinal pigmented epithelium (RPE) has been implicated in age-related macular degeneration by impacting endocytic trafficking, including the formation, content, and secretion of extracellular vesicles (EVs). Using our model of polarized primary porcine RPE (pRPE) cells under chronic subtoxic oxidative stress, we tested the hypothesis that RPE miRNAs packaged into EVs are secreted in a polarized manner and contribute to maintaining RPE homeostasis.Methods
Small EVs (sEVs) enriched for exosomes were isolated from apical and basal conditioned media from pRPE cells grown for up to four weeks with or without low concentrations of hydrogen peroxide using two sEV isolation methods, leading to eight experimental groups. The sEV miRNA expression was profiled using miRNA-Seq with Illumina MiSeq, followed by quality control and bioinformatics analysis for differential expression using the R computing environment. Expression of selected miRNAs were validated using qRT-PCR.Results
We identified miRNA content differences carried by sEVs isolated using two ultracentrifugation-based methods. Regardless of the sEV isolation method, miR-182 and miR-183 were enriched in the cargo of apically secreted sEVs, and miR-122 in the cargo of basally secreted sEVs from RPE cells during normal homeostatic conditions. After oxidative stress, miR-183 levels were significantly decreased in the cargo of apically released sEVs from stressed RPE cells.Conclusions
We curated RPE sEV miRNA datasets based on cell polarity and oxidative stress. Unbiased miRNA analysis identified differences based on polarity, stress, and sEV isolation methods. These findings suggest that miRNAs in sEVs may contribute to RPE homeostasis and function in a polarized manner.Type
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Hernandez, Belinda J, Madison Strain, Maria Fernanda Suarez, W Daniel Stamer, Allison Ashley-Koch, Yutao Liu, Mikael Klingeborn, Catherine Bowes Rickman, et al. (2024). Small Extracellular Vesicle-Associated MiRNAs in Polarized Retinal Pigmented Epithelium. Investigative ophthalmology & visual science, 65(13). p. 57. 10.1167/iovs.65.13.57 Retrieved from https://hdl.handle.net/10161/31775.
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Scholars@Duke
W Daniel Stamer
My laboratory studies the disease of glaucoma, the second leading cause of blindness in the United States, affecting nearly 3 million people (70 million Worldwide). The primary risk factor for developing glaucoma is ocular hypertension (high intraocular pressure, IOP). IOP is a function of the regulated movement of aqueous humor into and out of the eye. Elevated IOP in glaucoma is a result of disease in the primary efflux route, the conventional outflow pathway, affecting proper homeostatic control of aqueous humor drainage.
Lowering IOP in glaucoma patients, whether or not they have ocular hypertension, is important because large clinical trials involving tens of thousands of patients repeatedly demonstrate that significant, sustained IOP reduction slows or halts vision loss. Unfortunately, current first-line medical treatments do not target the diseased conventional pathway and do not lower IOP sufficiently in most people with glaucoma. Therefore, finding new, more effective ways to medically control IOP by targeting the conventional pathway is a central goal the Stamer Laboratory.
Using molecular, cellular, organ and mouse model systems, my laboratory seeks to identify and validate novel drug targets in the human conventional outflow pathway to facilitate the development of the next generation of treatments for ocular hypertension and glaucoma.
Catherine Bowes Rickman
Dr. Bowes Rickman is a highly accomplished translational scientist whose research efforts over two decades have been focused on the molecular/cell biology and pathobiology of age-related macular degeneration (AMD). In an effort to better understand the pathophysiology of AMD, she has created a number of murine models that recapitulate many aspects of human AMD and point the way toward eventual treatments for AMD. Among many cited seminal contributions is her discovery of a connection between complement and lipoprotein metabolism and AMD and the development of a murine model that closely mirrors findings in humans. Using these models, Dr. Bowes Rickman has dissected disease mechanisms that contribute to AMD risk, and tested multiple novel therapeutic targets for its treatment.
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