Abstract
Purpose :
Exosomes are extracellular vesicles that are released by the retinal pigment epithelium (RPE). They participate in inter-tissue communication, as carriers of mRNA, miRNA and proteins. The delivery of exosome-cargo by stressed tissue to downstream cells has been shown to promote angiogenesis, especially in cancer metastasis. Exosome-mediated communication may play a role in the pathobiology of Age-related Macular Degeneration (AMD), in which abnormal angiogenesis is the major cause of vision loss. Here we explore the release of exosomes from in situ RPE, relative to postmortem changes over time.
Methods :
Paired eyecups were prepared from bovine eyes; the RPE were incubated for 30 minutes in serum-free DMEM. Eyes were dissected at three different postmortem time points: Time 0; paired eyes dissected at the same time and within 1hr of animal harvest, Time 15; paired eyes dissected 15 hours apart, Time 24; paired eyes dissected 24 hours apart from each other. Between dissection time points, the eyes were stored at 4°C on ice. Conditioned medium from the in situ preps was collected and exosomes were isolated by differential ultracentrifugation and analyzed by unbiased SDS-PAGE protein staining and/or direct protein quantitation by UV absorption. Two-tailed t-tests with Holms-Bonferroni correction was used for statistical analysis.
Results :
In 13 separate experiments done in triplicate, the rate of RPE exosome release increased significantly with postmortem time. We observed a significant increase in purified exosome protein at Time 24 and Time 15 when compared to their fellow eye at Time 0 [T24; 153% increase, P<0.01, n=12. T15; 157% increase, P<0.001, n=36]. As a control we tested paired eyes dissected at the same time, and they did not differ in purified exosome protein at any time postmortem.
Conclusions :
Studies have shown that apical and basal RPE exosomes each carry distinctive cargo, however; in situ RPE exosome function with respect to the activity in the retina and choroid remains unclear. Our study demonstrates that while RPE are viable for culture and experiments when kept on ice postmortem for 30 hours, changes are occurring. One of the changes is a significant increase in exosome release. RPE exosomes are likely a symptom of RPE pathology, in this case related to postmortem hypoxia. Reduction of RPE exosome release, as occurs immediately upon GPR143 activation by L-DOPA may point to the mechanism of action for L-DOPA protection from AMD.
This is a 2020 ARVO Annual Meeting abstract.