Browsing by Subject "Exosomes"

Age-related macular degeneration (AMD) is the leading cause of irreversible blindness in the elderly in developed countries. Oxidative stress is implicated in the pathobiology of AMD and initially targets the retinal pigmented epithelium (RPE). A major function of the RPE is to process photoreceptor outer segments and its polarity is responsible for the directional secretion of proteins, lipoproteins, and extracellular vesicles (including exosomes). Dysfunction of the RPE leads to the development of AMD. There are no non-imaging biomarkers currently available to detect AMD. A potential approach to detect AMD is through analysis of exosomes, which are nanovesicles that can be purified from biofluids (e.g., plasma and urine). Exosomes are known to be secreted during homeostasis and diseased states. Exosomes transfer their cargo containing protein and genetic material (mostly microRNAs) to other cells, resulting in activation of signaling pathways. The role of RPE-derived exosomes in AMD has been largely understudied. Our lab has established and published a protocol for isolating exosomes and identifying their cargo from primary porcine RPE grown on Transwells. To test the role RPE-derived exosomes at early stages of AMD, we used polarized primary RPE cells and exposed them to subtoxic chronic oxidative stress. Unbiased proteomic analyses of the content of basolateral exosomes isolated from RPE cultures under oxidative stress revealed changes to a number of desmosome and hemidesmosome proteins, in which we identified a stress profile. We also identified significant changes in proteins accumulating in the basal sub-RPE extracellular matrix during oxidative stress. To further interrogate the role of exosomes in RPE function, we analyzed the miRNA cargo from exosomes secreted apically and basally from control and chronic oxidative stressed primary RPE cells. We identified a set of miRNAs associated with RPE homeostasis. Unbiased miRNA analysis of secreted exosomes revealed differences based on polarity, exosome isolation method, and oxidative stress. Our data supports that proteins and miRNAs in exosomes contribute to RPE homeostasis and function in a polarized manner. Our findings suggest that exosomes are early indicators of RPE dysfunction prior to overt morphological changes, supporting further studies of RPE exosomal cargo as biomarkers for AMD.