Serum starvation of ARPE-19 changes the cellular distribution of cholesterol and Fibulin3 in patterns reminiscent of age-related macular degeneration

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.

Extracellular vesicle-mediated long-range communication in stressed retinal pigment epithelial cell monolayers

“…1E,F). Finally, as the ExoQuick-TC method used here for vesicle isolation might also isolate lipoprotein particles [39] and RPE cells release a range of modified lipoprotein particles, [40] ELISA measurements were performed to determine the ratio between syntenin-1 (exosome marker) [41] and ApoB (modified lipoprotein particle and secreted by ARPE-19 cells) [42]. All samples contained syntenin-1, whereas no ApoB was detected (data not shown).…”

FBLN3 inhibits the invasion and metastasis of colorectal cancer through the AKT/mTOR pathway

“…Many oncogenes, anti-oncogenes and pathways participate in the tumorigenesis process and metastasis. FBLN3, which is also called epidermal growth factor-containing fibulin-like extracellular matrix protein 1 (EFEMP1), is related to many diseases, such as age-related macular degeneration [7], knee osteoarthritis [8], asbestos-related diseases [9], rheumatoid arthritis [10], mesothelioma [11], glioblastoma [12], osteosarcoma [13], ovarian cancer [14] and cervical cancer [15]. Many studies have reported that the fibulin family, which consists of 7 secreted extracellular glycoproteins, is involved in cell morphology maintenance, cell growth, adhesion and movement.…”

Directional ABCA1-mediated cholesterol efflux and apoB-lipoprotein secretion in the retinal pigment epithelium

“…The RPE expresses cluster of differentiation 36, a scavenger receptor for modified LDL, including oxLDL and acLDL, on both the basolateral side and the apical side and can avidly take-up modified LDL (33,74,75). RPE cells express enzymes and transporters required for cholesterol esterification, catabolism to hydroxycholesterol, release in apoB-lipoprotein and HDL, and enhanced diffusion to extracellular acceptors (21,37,76). It is important to understand cholesterol metabolism in the RPE, especially its release in apoB-lipoprotein and HDL.…”