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Venkata R M Chavali, Fiona La, Naqi Haider, Daniel J Rader, Nicholas N Lyssenko; Characterization of the lipid efflux pathways in RPE. Invest. Ophthalmol. Vis. Sci. 2017;58(8):1052.
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The RPE comprises of the outer blood-retinal barrier that is important to the maintenance of lipid homeostasis in the retina. Drusen deposits between the Bruch’s membrane and RPE are regarded as the hallmarks of AMD. They are predominantly composed of esterified cholesterol and other lipids, suggesting a role of lipid pathways in the formation of drusen and basal linear deposits. We propose to characterize the complete lipoprotein secretion profile in primary polarized hfRPE and also establish pathways required for the maintenance of basal lipid homeostasis in the RPE.
Polarized RPE cultures obtained from donor fetal eyes (hfRPE, passage 1-2) were grown on 12 well transwell plates. The hfRPE monolayers were characterized using apical and basal polarity markers, RT-PCR of RPE specific genes and TEER measurement. The media from the apical and basal compartments were collected from hfRPE monolayers after 48 hrs and complete fatty acid and cholesterol profiles were established from both conditioned media fractions using GC-MS and western blot analysis. Cholesterol efflux assays were carried out using [3H] labeled cholesterol, apoA1 and HDL as cholesterol acceptors, T0901317 (LXR agonist) and probucol (ABCA1 inhibitor).
Confluent and well-pigment hfRPE cultures on transwells obtained from 6 different donors eyes were evaluated for their basal fatty acid and cholesterol profiles and cholesterol efflux capacity. The apical/retinal side of hfRPE contained more apoE and cholesterol than the basal/choroid compartment, indicating that the RPE layer released more apoE and cholesterol, likely in apoE-HDL, to the retina. Cholesterol efflux to medium and apoAI was stronger on the basal/choroid side, while efflux to HDL was stronger on the retinal side. Our results suggest that ABCA1 was responsible for efflux to apoAI; while SR-BI, a membrane protein responsible for efflux to HDL.
Our experiments show that the RPE employs several cholesterol efflux pathways and that these pathways are active to different degrees on the choroid and retinal side.
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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