July 2019
Volume 60, Issue 9
Open Access
ARVO Annual Meeting Abstract  |   July 2019
Photoreceptor Survival Factor, Clusterin, is Enriched in Human Retinal Pigment Epithelial Exosomes
Author Affiliations & Notes
  • Shinwu Jeong
    MDA Vision Research, USC Roski Eye Institute, Department of Ophthalmology, Keck School of Medicine of the University of Southern California, Los Angeles, California, United States
  • Andrew Vargas
    MDA Vision Research, USC Roski Eye Institute, Department of Ophthalmology, Keck School of Medicine of the University of Southern California, Los Angeles, California, United States
  • Kyra Yamamoto
    MDA Vision Research, USC Roski Eye Institute, Department of Ophthalmology, Keck School of Medicine of the University of Southern California, Los Angeles, California, United States
  • Eun-Jin Lee
    MDA Vision Research, USC Roski Eye Institute, Department of Ophthalmology, Keck School of Medicine of the University of Southern California, Los Angeles, California, United States
    Department of Biomedical Engineering, USC Viterbi School of Engineering of the University of Southern California, Los Angeles, California, United States
  • Cheryl Mae Craft
    MDA Vision Research, USC Roski Eye Institute, Department of Ophthalmology, Keck School of Medicine of the University of Southern California, Los Angeles, California, United States
    Department of Cell & Neurobiology, Keck School of Medicine of the University of Southern California, Los Angeles, California, United States
  • Footnotes
    Commercial Relationships   Shinwu Jeong, None; Andrew Vargas, None; Kyra Yamamoto, None; Eun-Jin Lee, None; Cheryl Craft, None
  • Footnotes
    Support  MDA Foundation; Dorie Miller Foundation; William Hansen Sandberg Foundation; Unrestricted Grant to the Department of Ophthalmology from Research to Prevent Blindness, New York, NY; P30EY029220 from NIH
Investigative Ophthalmology & Visual Science July 2019, Vol.60, 2365. doi:
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      Shinwu Jeong, Andrew Vargas, Kyra Yamamoto, Eun-Jin Lee, Cheryl Mae Craft; Photoreceptor Survival Factor, Clusterin, is Enriched in Human Retinal Pigment Epithelial Exosomes. Invest. Ophthalmol. Vis. Sci. 2019;60(9):2365.

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

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Abstract

Purpose : We have demonstrated both clusterin (CLU) and tissue inhibitor of matrix metalloproteinase (MMP)-1 (TIMP-1) promote rod photoreceptor (PR) survival in rodent models of Retinitis Pigmentosa1,2. Both proteins share in MMP inhibition with unique functional properties. To examine their essential role in PR survival and function, we explored the possibility of exosomal secretion of CLU and TIMP-1 and the expression and potential connectivity of their receptors in retinal pigment epithelial (RPE) cells.

Methods : Cultured primary human fetal RPE cells (provided by David Hinton, USC) and Long Evans rat retina were used for the experiments. Gene-specific RT-PCR, PAGE-SDS/immunoblot analysis and
immunofluorescence confocal microscopy were used to investigate the gene transcription, protein expression and localization, respectively. We examined CLU receptors: low-density lipoprotein receptor-related protein 2 (LRP2), apolipoprotein E receptor 2 (ApoER2) and very low density lipoprotein receptor (VLDLR); TIMP-1 receptors: LRP1 and CD63; and RPE-specific 65 kDa protein, RPE65. Exosomes were isolated from RPE culture conditioned media, by a conventional ultracentrifugation method or by Total Exosome Isolation Reagent (ThermoFisher Scientific).

Results : LRP1 and LRP2 were detected in rat RPE and retina outer nuclear layers. LRP1 and RPE65 were
localized towards basolateral side within the RPE layer, whereas LRP2, apically. In RPE cells, the genes of ApoER2 and VLDLR were constitutively expressed, determined by RT-PCR. Both endogenously and ectopically expressed CLU protein was enriched in exosomes from cultured RPE cells, whereas TIMP-1 was excluded from exosomes. CD63, also known as an exosome marker, was also enriched in the exosomes.

Conclusions : We found for the first time that CLU may be secreted in exosomes from the RPE cells, which express diverse receptors of CLU and TIMP-1. CLU exosomes may be important means for the paracrine cellular survival signaling, participating in transport of CLU and other regulatory proteins and nucleic acids between RPE and PR cells. Characterization of the function of exosomal CLU and its targeted receptors may identify novel mechanisms by which RPE cells communicate with PR cells in health and diseases in retina.
Reference: 1Vargas, Kim, Baral, Yu, Craft, Lee, PLOS ONE 12:e0182389; 2Kim, Vargas, Eom, Li, Yamamoto, Craft, Lee, PLOS ONE 13:e0197322

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

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