July 2019
Volume 60, Issue 9
Open Access
ARVO Annual Meeting Abstract  |   July 2019
Exosome-mediated long-range communication in stressed retinal pigment epithelial cell monolayers – focus on up-take mechanisms.
Author Affiliations & Notes
  • Baerbel Rohrer
    Ophthalmology, Med Univ of South Carolina, Charleston, South Carolina, United States
    Division of Research, Ralph H. Johnson VA Medical Center, Charleston, South Carolina, United States
  • Masakii Ishii
    Ophthalmology, Med Univ of South Carolina, Charleston, South Carolina, United States
  • Crystal Nicholson
    Ophthalmology, Med Univ of South Carolina, Charleston, South Carolina, United States
    Division of Research, Ralph H. Johnson VA Medical Center, Charleston, South Carolina, United States
  • Carlene Brandon
    Ophthalmology, Med Univ of South Carolina, Charleston, South Carolina, United States
  • Bala Annamalai
    Ophthalmology, Med Univ of South Carolina, Charleston, South Carolina, United States
    Division of Research, Ralph H. Johnson VA Medical Center, Charleston, South Carolina, United States
  • Navjot Shah
    Ophthalmology, Med Univ of South Carolina, Charleston, South Carolina, United States
    Division of Research, Ralph H. Johnson VA Medical Center, Charleston, South Carolina, United States
  • Footnotes
    Commercial Relationships   Baerbel Rohrer, None; Masakii Ishii, None; Crystal Nicholson, None; Carlene Brandon, None; Bala Annamalai, None; Navjot Shah, None
  • Footnotes
    Support  This work was supported by the National Institutes of Health (EY019320), the Department of Veterans Affairs (I01 RX000444), and The SC SmartState Endowment.
Investigative Ophthalmology & Visual Science July 2019, Vol.60, 1916. doi:https://doi.org/
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      Baerbel Rohrer, Masakii Ishii, Crystal Nicholson, Carlene Brandon, Bala Annamalai, Navjot Shah; Exosome-mediated long-range communication in stressed retinal pigment epithelial cell monolayers – focus on up-take mechanisms.. Invest. Ophthalmol. Vis. Sci. 2019;60(9):1916. doi: https://doi.org/.

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

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Abstract

Purpose : RPE alterations in AMD occur in patches, potentially involving long-distance communication between damaged and healthy areas. We previously showed that exosomes can mediate this communication in polarized ARPE-19 and primary porcine RPE monolayers. Specifically, we showed that exosomes from oxidatively stressed donor cells reduced barrier function (transepithelial resistance, TER) in naïve recipient monolayers, when compared to control exosomes. This effect on TER was dependent on exosome uptake, which occurred rapidly with exosomes from oxidatively stressed donor cells. Here we extend this analysis to examine the molecular origin of this difference in uptake.

Methods : RPE cells were grown as monolayers in media supplemented with 1% FBS for >6 weeks followed by transfer to FBS-free media. Half the cultures were used to collect stress exosome-containing supernatants upon treatment with H2O2, the other half served as naïve recipient cells. In recipient monolayers, TER was used to monitor exosome-uptake-based activity, live-cell imaging to confirm uptake.

Results : Exosome uptake includes a variety of endocytic pathways, including clathrin-dependent endocytosis (CdE), and clathrin-independent mechanisms, such as lipid raft-mediated internalization (CiI). CdE was confirmed on the basis of dynasore treatment, an inhibitor of the dynamin GTPase required for CdE, which prevented TER loss. CiI was excluded as an uptake mechanism based on the lack of inhibition by filipin. Known ligand-receptor interactions involved in CdE of exosomes include annexins, integrins and proteoglycans. Annexin 2 on exosomes (annexin2 siRNA of donor cells), HSPG on exosomes and recipient cells (heparinase) and integrin-receptors on recipient cells (RGD peptide) were found to contribute to exosome-uptake-based activity of stress exosomes. Live-cell imaging is expected to confirm these results. Preliminary mass-spectrometry analysis suggests elevated levels of potential ligands on stress exosomes when compared to controls.

Conclusions : These findings demonstrate that exosomes from stressed cells differ from those of control cells in two profound ways. The former contain cargo to communicate stress messages to healthy RPE cells, and present ligands on their surface to promote uptake by recipient cells. We propose that exosomes could potentially contribute to RPE dysfunction in aging and disease.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.

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