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Qitao Zhang, Feriel K. Presswalla, David N Zacks, Debra A Thompson, Jason Miller; Lipid Handling by RPE Predicts Certain Features of Age-Related Macular Degeneration. Invest. Ophthalmol. Vis. Sci. 2017;58(8):2293. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
The mechanisms and efficiency of lipid processing in the retinal pigment epithelium (RPE) are predicted to have profound effects on the deposition of intracellular lipofuscin and extracellular drusen and reticular pseudodrusen (RSD) that are hallmarks of dry age-related macular degeneration (AMD). In this study, cultured RPE, with and without lipofuscin-like material, were used to track lipid droplet (LD) formation, ketone body (KB) production, and secretion of drusen lipids in response to outer segment (OS) challenge.
Highly mature cultures of polarized primary human fetal RPE were maintained on transwells and fed photo-oxidized bovine OS daily for 4 weeks to generate cells laden with lipofuscin-like material, which were compared to cells without lipofuscin-like material. Following acute challenge with non-oxidized OS, the buildup and dissipation of LDs and KBs were tracked by ADRP staining and a fluorometric assay, respectively. The secretion of apolipoprotein E (APOE) and apolipoprotein B (APOB) into apical and basolateral chambers of the culture system was evaluated by western analysis. Cholesterol was assayed using a fluorometric assay.
Primary human fetal RPE cultures responded to an OS bolus by temporary formation of LDs, which quickly dissipated. We hypothesize that dissipated lipid is partly shunted into KB production while other lipid is secreted in the form of APOE. The comparable levels of APOE secreted apically and basolaterally in our cultures is consistent with the presence of APOE in both apical RSD and basolateral drusen. The levels of esterified vs. unesterified cholesterol, as well as APOB, were different between the apical and basolateral chambers, again consistent with the compositional differences between RSD and drusen seen in AMD patients. APOB secretion appeared to be constitutive, as it did not change in response to OS challenge. Both APOE and APOB secretion required the presence of lipid in the culture media, and was absent in cultures grown in standard media without serum. Surprisingly, lipofuscin has only limited influence on RPE processing of OS lipid.
Our findings suggest that degradation and redistribution of OS lipid accounts for some, but not all, of the compositional features of drusen and RSD seen in AMD patients. While APOE-based secretion is responsive to OS ingestion, APOB-based secretion is constitutive and dependent on external lipid.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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