May 2007
Volume 48, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2007
Ambient Oxygen Concentration Modulates Hemoglobin (Hbg) Synthesis and Release from Human Retinal Pigment Epithelium (RPE)
Author Affiliations & Notes
  • T. H. Tezel
    Ophthalmology & Visual Sciences, University of Louisville, Louisville, Kentucky
  • L. Geng
    Ophthalmology & Visual Sciences, University of Louisville, Louisville, Kentucky
  • S. Schaal
    Ophthalmology & Visual Sciences, University of Louisville, Louisville, Kentucky
  • E. Bodek
    Ophthalmology & Visual Sciences, University of Louisville, Louisville, Kentucky
  • H. J. Kaplan
    Ophthalmology & Visual Sciences, University of Louisville, Louisville, Kentucky
  • Footnotes
    Commercial Relationships T.H. Tezel, None; L. Geng, None; S. Schaal, None; E. Bodek, None; H.J. Kaplan, None.
  • Footnotes
    Support NEI (1 K08 EY0416120-01, THT) Career Development Award (THT) from Research to Prevent Blindness, Inc, NYC, NY.
Investigative Ophthalmology & Visual Science May 2007, Vol.48, 2196. doi:
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      T. H. Tezel, L. Geng, S. Schaal, E. Bodek, H. J. Kaplan; Ambient Oxygen Concentration Modulates Hemoglobin (Hbg) Synthesis and Release from Human Retinal Pigment Epithelium (RPE). Invest. Ophthalmol. Vis. Sci. 2007;48(13):2196.

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

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Abstract

Purpose:: To determine the modulation of Hbg synthesis and release from human RPE the by ambient oxygen concentration.

Methods:: 12 human donor eyes (46-66 years old) were obtained from the eye bank within 24 hours of death. After removal of the anterior segment and sensory retina, eye cups were rinsed three times with PBS and the suprachoroidal space was sealed with cyanoacrylate glue. Eye cups were then filled with 2 ml of culture medium and kept in a hypoxic (5% O2) chamber for 16 hours. Fellow eyes maintained in normoxic conditions (21% O2) were used as controls. At the end of the incubation supernatants were collected and the amount of Hbg released from the RPE was determined using Hbg-ELISA. The cell count and viability of the remaining RPE were determined before mRNA and protein extraction for RT-PCR and western blot analysis. Results were corrected for viable RPE and consitutive Hbg; the latter was determined in control eye cups under identical conditions without RPE cells. Immunoelectron microscopy for human Hbg was used to confirm the results.

Results:: No significant change was observed in RPE viability under hypoxic conditions within the experimental period (82% vs 90%, respectively). Each human RPE cell contains 156.3 ± 82.5 ng of Hbg and releases it to the extracellular space at a rate of 0.028 ± 0.017 pg/hr. Hypoxia upregulates Hbg expression in RPE cells and results in a 2.7 ± 1.0-fold increase in the amount of released Hbg. Immunoelectron microscopy revealed clusters of Hbg within cytoplasmic vesicules. The main exocytosis site was the RPE basal membrane resulting in deposition of Hbg within Bruch’s membrane.

Conclusions:: Human RPE actively modulates Hbg expression depending on the ambient oxygen concentration. The polarized release of Hbg towards Bruch’s membrane supports the role of Hbg as a reservoir and/or natural transporter of oxygen to the outer retina. Defects in this mechanism may cause the outer retinal hypoxia in the macula in several diseases, including age-related macular degeneration.

Keywords: age-related macular degeneration • retinal pigment epithelium • hypoxia 
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