A Pilot Application of an iTRAQ-Based Proteomics Screen Estimates the Effects of Cigarette Smokers’ Serum on RPE Cells With AMD High-Risk Alleles

PURPOSE: The aim of this study was to explore whether there are interactions between genetic (ARMS2/HTRA1) and environmental factors (cigarette smoking) in the pathogenesis of age-related macular degeneration (AMD). METHODS: Primary human retinal pigment epithelial (hRPE) cells were obtained from four donors’ eyes with AMD high-risk ARMS2/HTRA1 alleles, and two donors’ eyes with wild-type alleles were used as controls. The pooled serum from 32 smokers and 35 nonsmokers were collected and used separately to treat hRPE cells. The isobaric tag for relative and absolute quantitation (iTRAQ)-based proteomics was used to identify associated proteins and comparing the differences between AMD high-risk and low-risk HTRA1/ARMS2 alleles after exposure to smokers’ serum. RESULTS: After stimulation with the smokers’ serum, 400 differentially expressed proteins (DEPs) were detected in the high-risk allele cells. Several DEPs are involved in neuronal protein degeneration and oxidative stress pathways. The smokers’ serum stimulation or HTRA1 overexpression can both upregulate caveolin-1, which was one of the DEPs. Besides, the smokers’ serum enhanced the phagocytosis of cultured human RPE cells. CONCLUSIONS: The study confirmed the AMD high-risk alleles, HTRA1, and cigarette smoking can promote AMD development by regulating caveolin-1 expression. TRANSLATIONAL RELEVANCE: AMD high-risk alleles and environmental risk factors can promote the occurrence and development of AMD by regulating caveolin-1 expression, upregulation of which will induce apoptotic cell death in response to cellular stress in early AMD conditions.