Rajapakse Dinusha, Peterson Katherine, Mishra Sanghamitra, Wistow Graeme
Section on Molecular Structure and Functional Genomics, National Eye Institute, National Institutes of Health, Building 6 Room 106, Bethesda, MD, USA.
Exp Cell Res. 2017 Dec 15;361(2):333-341. doi: 10.1016/j.yexcr.2017.10.036. Epub 2017 Oct 31.
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.
视网膜色素上皮（RPE）被认为是衰老人眼中布鲁赫膜（BrM）和玻璃膜疣处富含胆固醇沉积物的关键来源。我们已经表明，汇合的RPE细胞血清剥夺与胆固醇合成上调和未酯化胆固醇（UC）积累有关。在这里，我们研究了参与这种反应的细胞过程。我们比较了UC和酯化胆固醇（EC）的分布和定位；与年龄相关性黄斑变性（AMD）相关的EFEMP1/纤维连接蛋白3（Fib3）；以及在补充血清和无血清培养基中培养的ARPE-19细胞中酰基辅酶A（CoA）：胆固醇酰基转移酶（ACAT）ACAT1、ACAT2和载脂蛋白B（ApoB）的水平。将结果与患有AMD的人类供体眼中这些脂质和蛋白质的分布进行了比较。ARPE-19细胞的血清剥夺与FM染料阳性膜泡形成增加有关，其中许多与UC共标记。此外，UC与Fib3在不同颗粒中共定位。到第5天，在Transwell上生长的血清剥夺细胞将Fib3基础分泌到基质中。虽然在血清剥夺的几天内ACTA1的mRNA和蛋白质水平保持恒定，但血清剥夺后ACAT2水平显著增加，表明EC形成增加。在血清剥夺条件下观察到的细胞内EC水平较低与ApoB形成和分泌增加有关。RPE衍生细胞对血清剥夺的反应：脂质、脂蛋白和Fib3的积累和分泌与患有AMD的人类供体眼中看到的模式非常相似，表明该模型模拟了与疾病进展相关的过程。
