Abstract
Purpose :
Exosomes are small extracellular vesicles that are released by most cells including Retinal Pigment Epithelial (RPE) cells. Exosome cargo has garnered significant recent interest, but how exosome release is governed remains largely unknown. While it has been suggested that exosome release can be regulated by Ca++, our studies have shown that stimulation of a G-protein coupled receptor that signals through Ca++, GPR143, halts the release of exosomes. Here we explore controlled exosome release using in situ isolates of RPE and the potential effects of dopamine receptor signaling. As a control we included ciliary body (CB) tissue preparations, which has known responses to dopaminergic signaling.
Methods :
Eye-cups were prepared from human and porcine eyes; the RPE were incubated for 30 minutes in serum-free DMEM. CB tissue isolates from porcine eyes were also incubated for 30 minutes. Conditioned medium (CM) was collected and exosomes were isolated by differential ultracentrifugation and analyzed by nanoparticle tracking analysis and/or unbiased SDS-PAGE protein staining. To test calcium signaling in RPE, a Ca++ ionophore, ionomycin, was included in the DMEM incubated eye-cups. Furthermore, to test the effects of dopaminergic signaling in both RPE and CB, the tissue was incubated in DMEM ± dopaminergic agents.
Results :
In 8 separate experiments our control, representing the constitutive release of exosomes, varied in a bimodal fashion; as we observed two significantly different basal exosome levels, suggesting exosomes in fresh tissue isolates exhibit different basal rates of release [176±42 vs. 434±28 exosome protein/min. p<.003]. Additionally, dopaminergic control of exosome release was complex and dependent upon the basal state [increased to 163±27 vs. decreased to 63±11 % of control, p<.01, n=11]. Unexpectedly, ionomycin, the Ca++ ionophore often used to stimulate exosome release in cultured cells, exhibited a pronounced decrease in exosome release in in situ human RPE.
Conclusions :
Our data show for the first time that release of exosomes in situ is not upregulated simply by Ca++, considering ionomycin did not increase exosome release in the RPE. We also show that the constitutive release of exosomes in both RPE and CB is bimodal, and the basal state of the tissue may be governed by an endogenous system that is not only conserved in our tissue isolates but also sensitive to stimulation of the dopaminergic system.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.