Dataset Information

Human RPE LC-MSMS

ABSTRACT: AMD is a complex disorder related to environmental factors and genetic polymorphisms. We explored the interplay between the two factors by comparing the differences between HTRA1/ARMS2 high-risk and low-risk AMD alleles after exposure to smokers’ serum using iTRAQ-based proteomics. Primary human retinal pigment epithelial (hRPE) cells were obtained from donor eyes and screened for ARMS2/HTRA1 AMD risk genotypes, wild type allele as control. A gene ontology annotation analysis and KEGG metabolic pathway analysis were employed to obtain a global functional view of differentially expressed (DE) proteins. Significant DE proteins were screened and validated by real-time PCR, immunofluorescence and western blot. The relationship between HTRA1 and caveolin-1 protein was explored by HTRA1-overexpressing lentivirus-transduced ARPE-19 cells. A total of 400 DE proteins were detected for the high-risk allele after cigarette sera stimulation, which was much higher than the low-risk allele. The caveolin-1 protein was significantly up-regulated when HTRA1-overexpressing cells were exposed to smokers’ serum and the smoking effect was stronger than AMD high-risk allele. There were interplays between AMD high-risk allele and cigarette smoking. Smokers’ sera also accelerated the phagocytosis of hRPE cells. In addition, caveolin-1 may play an important role in the disease process, but its molecular mechanism requires further study.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Primary Cell, Epithelial Cell

DISEASE(S): Age Related Macular Degeneration

SUBMITTER: Bincui Cai

LAB HEAD: Xiaorong Li

PROVIDER: PXD009309 | Pride | 2022-02-22

REPOSITORIES: Pride

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Publications

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

Cai Bincui B Zhang Zhe Z Sun Shuo S Lin Tingting T Ke Yifeng Y Li Zhiqing Z Yang Jin J Li Xiaorong X

Translational vision science & technology 20220201 2

<h4>Purpose</h4>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).<h4>Methods</h4>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 collecte ...[more]

PMID: 35138344

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Project description:Background Age-related macular degeneration (AMD) is the leading cause of irreversible blindness in the elderly population worldwide. Recent studies have demonstrated strong genetic associations between AMD and single nucleotide polymorphisms (SNPs) within genes such as CFH and HTRA1. However, we have identified monozygotic twins discordant for AMD phenotypes (one with disease, the other without disease), suggesting that an epigenetic mechanism may also contribute to the pathogenesis of AMD. Methods We identified two twin pairs with phenotypic discordance of AMD. We obtained genomic DNA from their peripheral blood mononuclear cells (PBMCs) and subjected them to DNA methylation-chip analysis (MeDIP-chip) that profiled genome-wide DNA methylation patterns on promoters of all genes and microRNAs. We next utilized the Methyl-Profiler DNA methylation assays to detect the methylation status of selected promoters. Flow cytometry and quantitative real-time PCR were used to detect the expression of the selected gene in peripheral blood cells, as well as in retinal and choroidal tissues of AMD patients and non-AMD controls. Results Our MeDIP-chip analysis identified 256 genes with hypo-methylated promoters only in the twins with AMD and 744 genes with hyper-methylated promoters only in the twins with AMD. Importantly, the promoter region of IL17RC was associated with hypo-methylated CpG sites only in the twins with AMD but not in the twins without AMD. We also found the association of the hypo-methylated IL17RC promoter with AMD in 7 pairs of siblings with discordant AMD pathology (P=0.003), as well as an independent patient cohort (95 neovascular wet and 107 geographic atrophy dry AMD patients as well as 96 non-AMD controls (95% CI, 0.01-0.10, P=9.8x10-8 for wet AMD vs. control; 95% CI, 0.05-0.32, P=2.2x10-5 for dry AMD vs. control)). Interestingly, we did not find an association between the levels of methylation on the IL17RC promoter with the previously identified genetic risk alleles in CFH, HTRA1, and ARMS2. We demonstrated an elevated expression of the IL-17RC protein in CD14+ monocytes in the peripheral blood of AMD patients as compared to non-AMD controls. These IL-17RC+ monocytes have elevated expression of CXCR1, CXCR2, and CXCR4. In addition, IL17RC was expressed only in the retinal and choroidal tissues from AMD patients but not from age-matched controls. Conclusions We show an association of the hypo-methylated IL17RC promoter with AMD, which resulted in the elevated expression of IL-17RC in peripheral blood cells as well as the retinal and choroidal tissues of AMD patients. Our study suggests that the hypo-methylated IL17RC promoter and elevated expression of IL-17RC can potentially serve as biomarkers for the diagnosis of AMD, while IL-17RC can be a new therapeutic target for AMD. In addition, our results strongly suggest an epigenetic control mechanism of AMD pathogenesis. The Affymetrix MOE430 2.0 GeneChip was used to detect gene expression patterns within the whole eye of C57B/6 normal mice.

Dataset Information

Human RPE LC-MSMS

Publications

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

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