Abstract
Purpose :
Drusen seen in Age Related Macular Degeneration (AMD) are filled with lipid particles. Phospholipid Transfer Protein (PLTP) and Cholesterol Ester Transfer Protein (CETP) are main players of lipid homeostasis systemically, however their role in lipid metabolism and drusen formation remains unknown. In this pilot study, we aim to characterize PLTP and CETP expression and activity levels in human retinal pigment epithelium (RPE) cells.
Methods :
Immortalized human ARPE-19 cells were grown at 37°C to 70-80% confluence in Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12 (DMEM:F12) with 5% Fetal Bovine Serum (FBS) and 1% Penicillin/Streptomycin in culture flasks before conducting experiments. PLTP and CETP protein expression levels and activity were measured on RPE cell lysate and media via western blot analysis and activity assay kits. Monolayers of cultured ARPE-19 cells were fixed and immunostained with anti-PLTP and anti-CETP antibodies to examine cellular localization of these proteins.
Results :
The presence of PLTP in ARPE-19 cells was confirmed via western blot. PLTP activity in ARPE-19 cell lysate was found to be similar to its activity in WT mouse plasma. Activity assays also detected CETP activity in ARPE-19 cell lysate. Immunohistochemistry (IHC) analysis of PLTP and CETP in ARPE-19 cells was successful in visualizing both proteins but insufficient to determine possible protein localization to a cellular compartment.
Conclusions :
Our findings showed that PLTP and CETP are both expressed in RPE cells. PLTP activity is measurable in RPE cells and is comparable to its activity in plasma when adjusted for protein concentration. CETP activity is present but it is low. Our data suggest that RPE cells possess the machinery for lipid production and that the study of these proteins in RPE physiology and their role in AMD pathogenesis is prudent.
This is a 2021 ARVO Annual Meeting abstract.