June 2017
Volume 58, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2017
Polarized secretion of pro-inflammatory exosomes by the retinal pigment epithelium is selectively regulated by vitamin A dimers and membrane cholesterol
Author Affiliations & Notes
  • Aparna Lakkaraju
    Ophthalmology & Visual Sciences, University of Wisconsin-Madison, Madison, Wisconsin, United States
  • Colin Germer
    Ophthalmology & Visual Sciences, University of Wisconsin-Madison, Madison, Wisconsin, United States
  • Li Xuan Tan
    Ophthalmology & Visual Sciences, University of Wisconsin-Madison, Madison, Wisconsin, United States
  • Gulpreet Kaur
    Ophthalmology & Visual Sciences, University of Wisconsin-Madison, Madison, Wisconsin, United States
  • Footnotes
    Commercial Relationships   Aparna Lakkaraju, None; Colin Germer, None; Li Xuan Tan, None; Gulpreet Kaur, None
  • Footnotes
    Support  NIH grant R01EY023299, BrightFocus Foundation grant M2015350, Macular Society UK, Research to Prevent Blindness Foundation, Retina Research Foundation Rebecca Meyer Brown Professorship
Investigative Ophthalmology & Visual Science June 2017, Vol.58, 1049. doi:
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    • Get Citation

      Aparna Lakkaraju, Colin Germer, Li Xuan Tan, Gulpreet Kaur; Polarized secretion of pro-inflammatory exosomes by the retinal pigment epithelium is selectively regulated by vitamin A dimers and membrane cholesterol. Invest. Ophthalmol. Vis. Sci. 2017;58(8):1049.

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      © ARVO (1962-2015); The Authors (2016-present)

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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.

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