Department of Chemistry, Case Western Reserve University, Cleveland, OH, 44106, USA.
Department of Ophthalmology and Visual Sciences, Case Western Reserve University, Cleveland, OH, 44106, USA.
Exp Eye Res. 2019 Apr;181:325-345. doi: 10.1016/j.exer.2018.09.012. Epub 2018 Oct 5.
Oxidative cleavage of docosahexaenoate (DHA) in retinal pigmented epithelial (RPE) cells produces 4-hydroxy-7-oxohept-5-enoic acid (HOHA) esters of 2-lysophosphatidylcholine (PC). HOHA-PC spontaneously releases a membrane-permeant HOHA lactone that modifies primary amino groups of proteins and ethanolamine phospholipids to produce 2-(ω-carboxyethyl)pyrrole (CEP) derivatives. CEPs have significant pathological relevance to age-related macular degeneration (AMD) including activation of CEP-specific T-cells leading to inflammatory M1 polarization of macrophages in the retina involved in "dry AMD" and TLR2-dependent induction of angiogenesis that characterizes "wet AMD". RPE cells accumulate DHA from shed rod photoreceptor outer segments through phagocytosis and from plasma lipoproteins secreted by the liver through active uptake from the choriocapillaris. As a cell model of light-induced oxidative damage of DHA phospholipids in RPE cells, ARPE-19 cells were supplemented with DHA, with or without the lipofuscin fluorophore A2E. In this model, light exposure, in the absence of A2E, promoted the generation HOHA lactone-glutathione (GSH) adducts, depletion of intracellular GSH and a competing generation of CEPs. While DHA-rich RPE cells exhibit an inherent proclivity toward light-induced oxidative damage, photosensitization by A2E nearly doubled the amount of lipid oxidation and expanded the spectral range of photosensitivity to longer wavelengths. Exposure of ARPE-19 cells to 1 μM HOHA lactone for 24 h induced massive (50%) loss of lysosomal membrane integrity and caused loss of mitochondrial membrane potential. Using senescence-associated β-galactosidase (SA β-gal) staining that detects lysosomal β-galactosidase, we determined that exposure to HOHA lactone induces senescence in ARPE-19 cells. The present study shows that products of light-induced oxidative damage of DHA phospholipids in the absence of A2E can lead to RPE cell dysfunction. Therefore, their toxicity may be especially important in the early stages of AMD before RPE cells accumulate lipofuscin fluorophores.
二十二碳六烯酸（DHA）在视网膜色素上皮（RPE）细胞中的氧化断裂会产生 2-溶血磷脂酰胆碱（PC）的 4-羟基-7-氧庚酸（HOHA）酯。HOHA-PC 会自发释放一种具有膜通透性的 HOHA 内酯，该内酯会修饰蛋白质和乙醇胺磷脂中的伯氨基，生成 2-（ω-羧乙基）吡咯（CEP）衍生物。CEP 与年龄相关性黄斑变性（AMD）有显著的病理相关性，包括激活 CEP 特异性 T 细胞，导致视网膜中巨噬细胞的炎症 M1 极化，涉及“干性 AMD”，以及 TLR2 依赖性血管生成，其特征为“湿性 AMD”。RPE 细胞通过吞噬作用从脱落的杆状光感受器外段中积累 DHA，并通过肝脏分泌的脂蛋白从脉络膜毛细血管中主动摄取 DHA。作为 RPE 细胞中 DHA 磷脂光诱导氧化损伤的细胞模型，ARPE-19 细胞补充了 DHA，有或没有脂褐素荧光染料 A2E。在这个模型中，在没有 A2E 的情况下，光照会促进 HOHA 内酯-谷胱甘肽（GSH）加合物的生成、细胞内 GSH 的耗竭以及 CEP 的竞争生成。虽然富含 DHA 的 RPE 细胞表现出对光诱导氧化损伤的固有倾向，但 A2E 的光敏化作用使脂质氧化的量增加了近一倍，并将光敏感性的光谱范围扩展到了更长的波长。将 ARPE-19 细胞暴露于 1µM HOHA 内酯 24 小时会导致溶酶体膜完整性的大量（50%）损失，并导致线粒体膜电位丧失。使用衰老相关的β-半乳糖苷酶（SA β-半乳糖苷酶）染色来检测溶酶体β-半乳糖苷酶，我们发现 HOHA 内酯诱导了 ARPE-19 细胞的衰老。本研究表明，在没有 A2E 的情况下，DHA 磷脂的光诱导氧化损伤产物可导致 RPE 细胞功能障碍。因此，它们的毒性在 AMD 的早期阶段，即在 RPE 细胞积累脂褐素荧光染料之前，可能尤为重要。
