Justilien Verline, Pang Ji-Jing, Renganathan Kutralanathan, Zhan Xianquan, Crabb John W, Kim So Ra, Sparrow Janet R, Hauswirth William W, Lewin Alfred S
Department of Molecular Genetics, University of Florida, Gainesville, Florida, USA.
Invest Ophthalmol Vis Sci. 2007 Oct;48(10):4407-20. doi: 10.1167/iovs.07-0432.
To test the hypothesis that oxidative injury to the retinal pigment epithelium (RPE) may lead to retinal damage similar to that associated with the early stages of age-related macular degeneration (AMD).
A ribozyme that targets the protective enzyme manganese superoxide dismutase (MnSOD) was expressed in RPE-J cells, and adeno-associated virus (AAV) expressing the ribozyme gene was injected beneath the retinas of adult C57BL/6 mice. The RPE/choroid complex was examined for SOD2 protein levels and protein markers of oxidative damage using immunoblot analysis and LC MS/MS-identification of proteins and nitration sites. Lipids were extracted from retinal tissue and analyzed for the bis-retinoid compounds A2E and iso-A2E. The mice were analyzed by full-field electroretinography (ERG) for light response. Light and electron microscopy were used to measure cytological changes in the retinas.
The treatment of RPE-J cells with Rz432 resulted in decreased MnSOD mRNA and protein as well as increased levels of superoxide anion and apoptotic cell death. When delivered by AAV, Rz432 reduced MnSOD protein and increased markers of oxidative damage, including nitrated and carboxyethylpyrrole-modified proteins in the RPE-choroid of mice. Ribozyme delivery caused a progressive loss of electroretinograph response, vacuolization, degeneration of the RPE, thickening of Bruch's membrane, and shortening and disorganization of the photoreceptor outer and inner segments. Progressive thinning of the photoreceptor outer nuclear layer resulted from apoptotic cell death. Similar to the eyes of patients with AMD, ribozyme-treated eyes exhibited increased autofluorescence and elevated levels of A2E and iso-A2E, major bis-retinoid pigments of lipofuscin.
These results support the hypothesis that oxidative damage to the RPE may play a role in some of the key features of AMD.
验证视网膜色素上皮（RPE）的氧化损伤可能导致与年龄相关性黄斑变性（AMD）早期阶段相关的视网膜损伤这一假说。
在RPE-J细胞中表达靶向保护性酶锰超氧化物歧化酶（MnSOD）的核酶，并将表达核酶基因的腺相关病毒（AAV）注射到成年C57BL/6小鼠的视网膜下。使用免疫印迹分析以及蛋白质和硝化位点的液相色谱-质谱/质谱鉴定，检测RPE/脉络膜复合体中的SOD2蛋白水平和氧化损伤的蛋白质标志物。从视网膜组织中提取脂质，并分析双视黄醛化合物A2E和异A2E。通过全视野视网膜电图（ERG）分析小鼠的光反应。使用光学和电子显微镜测量视网膜中的细胞学变化。
用Rz432处理RPE-J细胞导致MnSOD mRNA和蛋白水平降低，以及超氧阴离子水平升高和凋亡性细胞死亡。当通过AAV递送时，Rz432降低了MnSOD蛋白水平，并增加了氧化损伤的标志物，包括小鼠RPE-脉络膜中硝化和羧乙基吡咯修饰的蛋白质。核酶递送导致视网膜电图反应逐渐丧失、空泡化、RPE变性、布鲁赫膜增厚以及光感受器外段和内段缩短和紊乱。光感受器外核层的逐渐变薄是由凋亡性细胞死亡引起的。与AMD患者的眼睛相似，经核酶处理的眼睛表现出自发荧光增加以及脂褐素的主要双视黄醛色素A2E和异A2E水平升高。
这些结果支持RPE的氧化损伤可能在AMD的一些关键特征中起作用这一假说。
