Article ID | Journal | Published Year | Pages | File Type |
---|---|---|---|---|
8451881 | Experimental Cell Research | 2017 | 9 Pages |
Abstract
Retinal pigment epithelium (RPE) has been implicated as key source of cholesterol-rich deposits at Bruch's membrane (BrM) and in drusen in aging human eye. We have shown that serum-deprivation of confluent RPE cells is associated with upregulation of cholesterol synthesis and accumulation of unesterified cholesterol (UC). Here we investigate the cellular processes involved in this response. We compared the distribution and localization of UC and esterified cholesterol (EC); the age-related macular degeneration (AMD) associated EFEMP1/Fibulin3 (Fib3); and levels of acyl-coenzyme A (CoA): cholesterol acyltransferases (ACAT) ACAT1, ACAT2 and Apolipoprotein B (ApoB) in ARPE-19 cells cultured in serum-supplemented and serum-free media. The results were compared with distributions of these lipids and proteins in human donor eyes with AMD. Serum deprivation of ARPE-19 was associated with increased formation of FM dye-positive membrane vesicles, many of which co-labeled for UC. Additionally, UC colocalized with Fib3 in distinct granules. By day 5, serum-deprived cells grown on transwells secreted Fib3 basally into the matrix. While mRNA and protein levels of ACTA1 were constant over several days of serum-deprivation, ACAT2 levels increased significantly after serum-deprivation, suggesting increased formation of EC. The lower levels of intracellular EC observed under serum-deprivation were associated with increased formation and secretion of ApoB. The responses to serum-deprivation in RPE-derived cells: accumulation and secretion of lipids, lipoproteins, and Fib3 are very similar to patterns seen in human donor eyes with AMD and suggest that this model mimics processes relevant to disease progression.
Keywords
apoBRPEPVDFCFHHDLSfMBRMAMDACATPFAFCSBSAhigh-density lipoproteinbovine serum albuminApolipoprotein Bretinal pigment epitheliumComplement factor Hpolyvinylidene difluorideAgingserum free mediumfetal calf serumage-related macular degenerationendoplasmic reticulumBruch’s membraneSerum deprivationparaformaldehydecholesterolesterified cholesterolunesterified cholesterolBlind
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Authors
Dinusha Rajapakse, Katherine Peterson, Sanghamitra Mishra, Graeme Wistow,