Apolipoprotein E Containing High Density Lipoproteins can Function as Preferential Acceptors for Retinal Pigment Epithelium Cell Digested Photoreceptor Outer Segment Lipids

Abstract

Abstract: : Purpose: To identify factors involved in the basal export pathway of digested photoreceptor outer segment (POS) lipids in human retinal pigment epithelium (RPE) cells in culture. Methods: Primary cultures of human RPE cells were grown at confluence on laminin coated Transwell (Costar) plates for at least one week at confluence prior to RNA isolation or drug treatment. Apolipoprotein E expression was assessed by incubation of RPE cells in medium containing 10^-7 M thyroid hormone (T₃) for 36 hours prior to RNA and protein analysis. RNA expression levels were determined by reverse transcriptase-polymerase chain reaction (RT-PCR) of total cellular RNA using primer sets specific to human apo E. Apical and basal secretion of apo E was assessed by western blotting of concentrated cell culture medium. POS lipid transport was assessed using [¹⁴C]-labeled POS. RPE cells grown on Transwell plates were incubated with medium containing [¹⁴C]-POS in the apical chamber and medium containing either 1 mg/ml human low density lipoprotein (LDL), 1 mg/ml human high density lipoprotein (HDL), or 100% human plasma in the basal chamber. After 36 hrs lipoproteins were purified from basal medium by centrifugation and resolved by polyacrylamide gel electrophoresis. [¹⁴C]-Lipids in LDL, HDL and in plasma lipoproteins was quantified by scintillation counting of gel slices. [¹⁴C]-Lipid composition of labeled POS and of HDL and LDL bound lipids was determined by thin layer chromatography (TLC). Results: Western blotting of apical and basal cell culture medium revealed that RPE cells secrete apo E preferentially from the basal surface. When cells were treated with T₃, basal secretion consistently increased. Apo E mRNA levels were also increased by treatment with T₃ by 1.5 to 2-fold. [¹⁴C]-Labeled POS lipids bound to both HDL and LDL. However, when plasma was used a basal acceptor, [¹⁴C]-labeled POS lipids bound preferentially to HDL. Furthermore [¹⁴C]-labeled POS lipids bound preferentially to an HDL fraction that is enriched with apo E. TLC revealed that phospholipids were the most abundantly labeled lipids in both the labeled POS and in HDL. Conclusions: The results are consistent with a role for apo E and HDL in trafficking of digested POS lipids from the RPE cell basal surface and into the circulation.

Keywords: age-related macular degeneration • retinal pigment epithelium • lipids