Nordgaard Curtis L, Berg Kristin M, Kapphahn Rebecca J, Reilly Cavan, Feng Xiao, Olsen Timothy W, Ferrington Deborah A
Department of Ophthalmology, University of Minnesota, Minneapolis, USA.
Invest Ophthalmol Vis Sci. 2006 Mar;47(3):815-22. doi: 10.1167/iovs.05-0976.
Age-related macular degeneration (AMD) is characterized clinically by changes in the retinal pigment epithelium (RPE), formation of drusen between the RPE and the underlying vasculature, geographic atrophy, and choroidal neovascularization. Later clinical stages are accompanied by impaired central vision. A limited understanding of the molecular events responsible for AMD has constrained the development of effective treatments. A proteomics approach was used to investigate the underlying mechanisms of AMD and to identify proteins exhibiting significant changes in expression with disease onset and progression.
Human donor eyes were categorized into one of four progressive stages of AMD. Proteins from the RPE were resolved and quantified by two-dimensional (2-D) gel electrophoresis. Proteins exhibiting significant expression changes at different disease stages were identified by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry. 2-D and semiquantitative one dimensional (1-D) Western blot analyses were used to determine whether changes identified by the proteomic analysis were specific for a protein subpopulation or representative of the entire protein population.
Proteins were identified from several critical pathways that changed at early and late disease stages, indicating potential causal mechanisms and secondary consequences of AMD, respectively. Proteins involved in protecting from stress-induced protein unfolding and aggregation, mitochondrial trafficking and refolding, and regulating apoptosis changed early in the disease process. Late-stage changes occurred in proteins that regulate retinoic acid and regeneration of the rhodopsin chromophore.
These results provide the first direct evidence of AMD stage-specific changes in human RPE protein expression and provide a basis for functional investigation of AMD that may ultimately suggest new therapeutic strategies.
年龄相关性黄斑变性（AMD）在临床上的特征为视网膜色素上皮（RPE）改变、RPE与下方脉管系统之间形成玻璃膜疣、地图样萎缩以及脉络膜新生血管。疾病后期会伴有中心视力受损。对导致AMD的分子事件了解有限，限制了有效治疗方法的开发。本研究采用蛋白质组学方法来探究AMD的潜在机制，并鉴定在疾病发生和进展过程中表达有显著变化的蛋白质。
将人类供体眼分为AMD的四个进展阶段之一。通过二维（2-D）凝胶电泳对RPE中的蛋白质进行分离和定量。通过基质辅助激光解吸电离飞行时间（MALDI-TOF）质谱鉴定在不同疾病阶段有显著表达变化的蛋白质。二维和半定量一维（1-D）蛋白质印迹分析用于确定蛋白质组分析所鉴定的变化是特定于蛋白质亚群还是代表整个蛋白质群体。
从几个关键途径中鉴定出了在疾病早期和晚期发生变化的蛋白质，分别表明了AMD的潜在因果机制和继发后果。参与保护细胞免受应激诱导蛋白解折叠和聚集、线粒体运输和重折叠以及调节细胞凋亡的蛋白质在疾病过程早期发生变化。晚期变化发生在调节视黄酸和视紫红质发色团再生的蛋白质中。
这些结果首次直接证明了人类RPE蛋白质表达中存在AMD阶段特异性变化，并为AMD的功能研究提供了基础；最终可能会由此提出新的治疗策略。
