PURPOSE: To demonstrate the production of hemoglobin by human retinal pigment epithelium (RPE). METHODS: Proteomic analysis using 10 donor eyes identified hemoglobin as a major constituent of soluble human RPE proteome. Western blot analysis, RT-PCR, and immunocytochemistry were used to confirm the RESULTS: The presence of erythrocyte-specific proteins within primary human RPE cytosol was investigated to rule out phagocytosis as the source of hemoglobin. ELISA was used to determine the rate of hemoglobin secretion from human RPE cells. Globin mRNA expression of human RPE was studied in comparison with a human erythroblast cell line and a spontaneously transformed human RPE cell line (ARPE-19). results. Hemoglobin is a regular constituent of soluble human RPE proteome. RT-PCR and Western blot analysis confirmed the presence of hemoglobin in human RPE. No other erythrocyte-specific proteins were detected within human RPE cytosol. Hemoglobin expression persisted up to seven passages in vitro. Human RPE globin expression exceeded that in human erythroblast and ARPE-19 cells. Immunohistochemistry revealed the presence of hemoglobin within RPE and Bruch's membrane. The hemoglobin release rate was calculated to be 1.9+/-1.2 attomoles per cell per hour. CONCLUSIONS: Hemoglobin expression by human RPE brings a new perspective to the understanding of oxygen transport to the outer retina. Malfunction of RPE-hemoglobin production may underlie the pathophysiology of ocular diseases characterized by subfoveal hypoxia and VEGF upregulation, such as age-related macular degeneration and diabetic retinopathy. Pharmacologic modulations of local hemoglobin production in RPE cells will create new opportunities to interfere with the course of these diseases.
PURPOSE: To demonstrate the production of hemoglobin by human retinal pigment epithelium (RPE). METHODS: Proteomic analysis using 10 donor eyes identified hemoglobin as a major constituent of soluble human RPE proteome. Western blot analysis, RT-PCR, and immunocytochemistry were used to confirm the RESULTS: The presence of erythrocyte-specific proteins within primary human RPE cytosol was investigated to rule out phagocytosis as the source of hemoglobin. ELISA was used to determine the rate of hemoglobin secretion from human RPE cells. Globin mRNA expression of human RPE was studied in comparison with a human erythroblast cell line and a spontaneously transformed human RPE cell line (ARPE-19). results. Hemoglobin is a regular constituent of soluble human RPE proteome. RT-PCR and Western blot analysis confirmed the presence of hemoglobin in human RPE. No other erythrocyte-specific proteins were detected within human RPE cytosol. Hemoglobin expression persisted up to seven passages in vitro. Human RPE globin expression exceeded that in human erythroblast and ARPE-19 cells. Immunohistochemistry revealed the presence of hemoglobin within RPE and Bruch's membrane. The hemoglobin release rate was calculated to be 1.9+/-1.2 attomoles per cell per hour. CONCLUSIONS: Hemoglobin expression by human RPE brings a new perspective to the understanding of oxygen transport to the outer retina. Malfunction of RPE-hemoglobin production may underlie the pathophysiology of ocular diseases characterized by subfoveal hypoxia and VEGF upregulation, such as age-related macular degeneration and diabetic retinopathy. Pharmacologic modulations of local hemoglobin production in RPE cells will create new opportunities to interfere with the course of these diseases.
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