March 2012
Volume 53, Issue 14
Free
ARVO Annual Meeting Abstract  |   March 2012
Oxidative Stress to ARPE-19 Cells by Treatment with Photosensitizers plus Light Decreases Protein Receptors that Mediate Outer Segment Phagocytosis
Author Affiliations & Notes
  • Magdalena M. Olchawa
    Biophysics/Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Krakow, Poland
  • Anja Herrnreiter
    Ophthalmology/Eye Institute, Medical College of Wisconsin, Milwaukee, Wisconsin
  • Christine Skumatz
    Ophthalmology/Eye Institute, Medical College of Wisconsin, Milwaukee, Wisconsin
  • Janice M. Burke
    Ophthalmology/Eye Institute, Medical College of Wisconsin, Milwaukee, Wisconsin
  • Tadeusz J. Sarna
    Biophysics/Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Krakow, Poland
  • Footnotes
    Commercial Relationships  Magdalena M. Olchawa, None; Anja Herrnreiter, None; Christine Skumatz, None; Janice M. Burke, None; Tadeusz J. Sarna, None
  • Footnotes
    Support  MNiSzW grant 2661/B/P01/2010/39; NIH grants R01EY019664, P30EY01931 and C06RR016511; RPB
Investigative Ophthalmology & Visual Science March 2012, Vol.53, 4775. doi:
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      Magdalena M. Olchawa, Anja Herrnreiter, Christine Skumatz, Janice M. Burke, Tadeusz J. Sarna; Oxidative Stress to ARPE-19 Cells by Treatment with Photosensitizers plus Light Decreases Protein Receptors that Mediate Outer Segment Phagocytosis. Invest. Ophthalmol. Vis. Sci. 2012;53(14):4775.

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

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Abstract

Purpose: : We previously showed that the specific phagocytosis by ARPE-19 cells of photoreceptor outer segments (POS) is transiently inhibited by light irradiation in the presence of photosensitizers merocyanine 540 or rose Bengal. Here we asked whether this outcome could be explained by treatment-induced changes in receptor proteins known to mediate POS phagocytosis.

Methods: : ARPE-19 cells pre-loaded with merocyanine 540 or rose Bengal were irradiated with green light to produce damage at the threshold of lethality as determined by a cell survival assay in which propidium iodide fluorescence is measured. Levels of the cytoskeletal protein actin and POS receptor proteins MerTK and integrin subunits αv and β5 were quantified by western blot analysis 30 min after and 24 hr after light irradiation with comparison to samples from paired cultures without photosensitizers. The intact integrin heterodimer αvβ5 was quantified by immunoprecipitation followed by blotting.

Results: : Borderline lethal light doses using both photosensitizers produced an immediate (at 30 min) decrease in the abundance of MerTK, the individual αv and β5 integrin subunits, and the integrin heterodimer. Light plus merocyanine 540, which was shown earlier to inhibit nonspecific phagocytosis of latex beads in addition to POS, also reduced levels of actin, a protein involved in internalization of particles regardless of type. Protein levels recovered after 24 hr, the time at which phagocytic function was previously shown to recover.

Conclusions: : After light irradiation in the presence of photosensitizers, POS receptor protein abundance and phagocytic function both show a coincident-in-time reduction then recovery suggesting that a diminution in receptor proteins contributes to the phagocytic defect. The additional inhibition by merocyanine-plus-light of nonspecific phagocytosis may result from more widespread effects of this photosensitizer on cytosolic proteins that participate in particle uptake. Overall the data imply that phagocytosis receptors in RPE cells are sensitive to oxidative modification, which raises the possibility that chronic oxidative stress in situ may reduce the efficiency of the RPE’s role in photoreceptor turnover, thereby contributing to retinal degenerations.

Keywords: retinal pigment epithelium • oxidation/oxidative or free radical damage • cell survival 
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