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C.–M. Li, N. Dashti, C.A. Curcio; Cultured Human Retinal Pigment Epithelium (RPE) Contains, Synthesizes, and Secretes Neutral Lipid . Invest. Ophthalmol. Vis. Sci. 2004;45(13):1805.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: To evaluate the potential for intraocular biosynthesis and secretion of lipoprotein by human RPE cells, we determined esterified cholesterol (EC) mass and composition in ARPE–19 cells and medium and assessed de novo synthesis and secretion of neutral lipid using methods established for human hepatoma cells. Lipoprotein particles consist of a neutral lipid core of triacylglycerols (TG) and EC surrounded by phospholipids (PL), unesterified cholesterol (UC), and apolipoproteins. Apolipoprotein B (apoB)–containing lipoprotein assembly involves TG and EC transfer to nascent apoB. Genes for apoB and microsomal triglyceride transfer protein, required for this process, are expressed in RPE. Methods:ARPE–19 cells were incubated for 24 hr with DMEM/F12 medium containing oleic acid (OA)/fatty acid–free bovine serum albumin (FAF–BSA) or with [3H]OA/FAF–BSA for metabolic labeling. Lipids were extracted from cells and medium. Total cholesterol (TC) and UC were measured by fluorometric enzymatic assay. Concentrations of 6 species of cholesteryl esters were determined with electrospray ionization mass spectrometry and referenced to internal and external standards. Cellular and medium lipids were separated by thin layer chromatography. Bands and intervening blanks were scraped for liquid scintillation counting. Results:1) In ARPE–19 cells: a) the concentrations of TC, UC, and EC cells were 23, 21 and 2 µg/mg cell protein, respectively; b) EC accounted for 8.7% of TC; and c) the concentrations of cholesteryl palmitate, cholesteryl linoleate, cholesteryl oleate, cholesteryl stearate, cholesteryl arachidonate, and cholesteryl docosohexanoate were 52, 35, 87, 16, 38, and 25 ng/mg cell protein, respectively. 2) In culture medium of OA–supplemented ARPE–19 cells, the concentrations of the 6 above cholesteryl esters were 2– to 5– fold higher than in non–conditioned medium. 3) Metabolic labeling studies with [3H]OA demonstrated a very high level of radioactivity in TG, EC and PL, both in the medium and cells. Conclusions:These results indicate that ARPE–19 cells take up and metabolize OA, secrete EC, and exhibit de novo synthesis and secretion of TG, EC, and PL. Further experiments are necessary to determine if these secretory products constitute the neutral lipid core of lipoprotein particles. Nevertheless, these data support the hypothesis that an RPE–secreted apoB–lipoprotein with neutral lipid core is a plausible mechanism for producing abundant EC and UC in aging Bruch's membrane and age–related maculopathy–associated drusen and basal deposits.
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