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T.H. Tezel, L. Geng, E. Bodek, H.J. Kaplan; Hemoglobin (Hgb) Expression in Human Retinal Pigment Epithelium (RPE): A New Perspective on Oxygen Transport to the Outer Retina . Invest. Ophthalmol. Vis. Sci. 2006;47(13):876.
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To investigate the expression of Hgb in human RPE and its possible role in oxygen transport to the outer retina.
Three different lines of human primary and serially cultured RPE were kept in normoxic (21% O2) and hypoxic (5% O2) conditions at 37°C for 16 hours. 30 µg of RPE protein were extracted and focused using pH 3–10 7.7cm IEF strips. Proteins were further separated in the second dimension on 4–12% Bis–Tris gels. After Sypro Ruby staining spots of interest were cut and peptide analysis was done using tandem mass spectrometry (LC/MS/MS). Western blotting and real–time PCR were used to confirm and analyze the changes in the Hgb, VEGF and HIF1–α expression. Quantification of protein expressions at different oxygen levels was done using Phoretix Expression 2D software.
Abundant amounts of Hgb side–chains are expressed in primary human RPE cells. Expression is confirmed by Western blotting and real–time PCR. However, RPE cells rapidly downregulate Hgb expression in the tissue culture environment. Hypoxia boosts Hb expression similar to VEGF and HIF1–α.
Hgb is expressed in human RPE profusely and its production is increased by hypoxia. A very likely role for Hgb is to act as a reservoir and natural transporter of oxygen to the outer retina; however, other possible roles merit further investigation – e.g. scavenging of NO, participation in oxidative damage and induction of RPE apoptosis and angiogenic signaling..
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