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A. Lakkaraju, L.W. Leung, J. Hu, D. Bok, E. Rodriguez–Boulan; Altered cholesteryl ester metabolism in retinal pigment epithelial cells containing the lipofuscin component A2E . Invest. Ophthalmol. Vis. Sci. 2004;45(13):1796.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: To investigate the effect of A2E on cholesteryl ester metabolism and identify determinants of cholesterol homeostasis in RPE cells. Methods: d407 cells were exposed to 50 µM A2E for 6h and the degradation of Bodipy–labeled cholesteryl ester (Bodipy–CE) was followed over time after a 1h pulse. Undegraded Bodipy–CE recovered from cells was quantified by thin layer chromatography. To get a true measure of Bodipy–CE accumulation, FR179254, an ACAT inhibitor, was used to inhibit cholesterol re–esterification. Changes in intralysosomal pH were evaluated by Lysosensor Blue (LysoB) staining. To study the expression of candidate receptors involved in CE internalization, human fetal RPE (hfRPE) and ARPE–19 cells were immunostained with antibodies to LDL receptor–related protein (LRP) and scavenger receptor B1 (SR–BI) and imaged by confocal microscopy. Results: (i) In d407 cells, A2E colocalized with the lysosomal marker Lamp–1. Bodipy–CE fluorescence, initially seen on the plasma membrane and intracellularly, partially colocalized with A2E after a 2h chase, indicating that Bodipy–CE trafficked to lysosomes. (ii) After 24h, 22% of the initial amount of Bodipy–CE was recovered intact from control cells while 38% was recovered from A2E–treated cells. A2E significantly increased the amount of undegraded Bodipy–CE recovered from d407 cells irrespective of ACAT inhibition. (iii) For a qualitative estimate of lysosomal pH alterations caused by A2E, LysoB staining intensities were compared. No differences were observed in LysoB fluorescence between control and A2E–loaded cells. Quantitative ratiometric fluorescence measurements of lysosomal pH using Lysosensor Yellow/Blue are ongoing. (iv) Immunofluorescence experiments showed that LRP expression was nonpolar in filter–grown hfRPE and ARPE–19 cells while SR–BI was predominantly apical in hfRPE and mostly intracellular in ARPE–19cells. Conclusions: Our results indicate that (i) A2E alters cholesteryl ester metabolism in d407 cells; (ii) accumulation of cholesteryl esters in A2E–treated cells is not due to increased cholesterol re–esterification; (iii) the presence of A2E in d407 lysosomes does not cause gross changes in lysosomal pH; and (iv) human RPE cells demonstrate robust expression and differential polarity of LRP and SR–BI, the receptors likely involved in cholesteryl ester internalization. Current work is focused on elucidating the mechanisms involved in A2E–induced cholesteryl ester accumulation in the RPE.
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