Abstract
Purpose :
Exosomes, vesicles derived from the endolysosomal pathway, participate in intercellular communication and disease processes. Although retinal pigment epithelial (RPE) cells secrete exosomes, little is known about their composition, function and fate. Here, we analyzed the size, morphology and protein repertoires of exosomes released at the apical and basolateral surfaces of polarized primary RPE, and investigated how this is modulated by cholesterol accumulation due to vitamin A dimers.
Methods :
Exosomes were purified from the apical and basolateral media of primary porcine RPE cultured on Transwell filters by density gradient ultracentrifugation. Transmission electron microscopy and Nanosight particle tracking were performed to analyze the morphology, size distribution and number of exosomes. Exosomal proteins were analyzed by immunoblotting and mass spectrometry. Live-cell spinning disk microscopy was used to follow internalization of exosomes by RPE cells.
Results :
Exosomes secreted by polarized primary RPE have cup-shaped morphologies under TEM and mean diameters of 100 nm. Exosomes released apically have flotillin-1, whereas those released basally have Hsp70. In RPE with the vitamin A dimer A2E, Nanosight tracking showed that significantly more exosomes are released apically compared to controls. We have previously shown that vitamin A dimers increase ceramide levels by abnormally activating acid sphingomyelinase. Ceramide is a cone-shaped lipid that induces inward budding of membranes and increases the biogenesis of exosomes. Immunoblotting and mass spectrometric analysis revealed that exosomes released apically from cells with A2E contain pro-inflammatory proteins. Live-cell imaging showed that naïve RPE monolayers take up RPE-derived exosomes.
Conclusions :
Primary adult RPE monolayers secrete exosomes in a polarized manner with a distinct repertoire of proteins packaged into exosomes destined for apical or basolateral release. Vitamin A dimers can promote retinal pathology by inducing secretion of pro-inflammatory exosomes, which can then be taken up by neighboring cells in the retina. This transfer could induce or amplify inflammatory and/or immune responses and spread pathogenic stimuli. Current studies are focused on identifying promising approaches to modulate exosome release and decrease inflammation in the outer retina.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.