Dataset Information

Nitration of Tyrosines In Complement Factor H Domains Alters Its Immunological Activity and Mediates A Pathogenic Role In Age Related Macular Degeneration

ABSTRACT: Nitrosative stress has been implicated in the pathogenesis of age related macular degeneration (AMD). Tyrosine nitration is a unique type of post translational modification that occurs in the setting of inflammation and nitrosative stress. To date, the significance and functional implications of tyrosine nitration of complement factor H (CFH), a key complement regulator in the eye has not been explored, and is examined in this study in the context of AMD pathogenesis. Sections of eyes from deceased individuals with AMD (n = 5) demonstrated the presence of immunoreactive nitrotyrosine CFH. We purified nitrated CFH from retinae from 2 AMD patients. Mass spectrometry of CFH isolated from AMD eyes revealed residues in domains critical for binding to heparan sulphate glycosaminoglycans (GAGs), lipid peroxidation by-products and complement (C) 3b were nitrated. Functional studies revealed that nitrated CFH did not bind to lipid peroxidation products, nor to the GAG of perlecan nor to C3b. There was loss of cofactor activity for Factor I mediated cleavage of C3b with nitrated CFH compared to non-nitrated CFH. CFH inhibits, but nitrated CFH significantly potentiates, the secretion of the pro-inflammatory and angiogenic cytokine IL-8 from monocytes that have been stimulated with lipid peroxidation by-products. AMD patients (n = 30) and controls (n = 30) were used to measure plasma nitrated CFH using a novel ELISA. AMD patients had significantly elevated nitrated CFH levels compared to controls (p = 0.0117). These findings strongly suggest that nitrated CFH contributes to AMD progression, and is a target for therapeutic intervention.

INSTRUMENT(S): LTQ FT

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Eye

DISEASE(S): Macular Degeneration

SUBMITTER: Jason Wong

LAB HEAD: Steven Krilis

PROVIDER: PXD005613 | Pride | 2017-08-29

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	InVitroNitration_CFH.RAW	Raw
	InVitroNitration_CFH.mzid.gz	Mzid
	InVitroNitration_CFH.pride.mztab.gz	Mztab
	InVivoNitration_CFH_lowband.mzid.gz	Mzid
	InVivoNitration_CFH_lowband.pride.mztab.gz	Mztab

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Publications

Nitration of tyrosines in complement factor H domains alters its immunological activity and mediates a pathogenic role in age related macular degeneration.

Krilis Matthew M Qi Miao M Madigan Michele C MC Wong Jason W H JWH Abdelatti Mahmoud M Guymer Robyn H RH Whitelock John J McCluskey Peter P Zhang Peng P Qi Jian J Hunyor Alex P AP Krilis Steven A SA Giannakopoulos Bill B

Oncotarget 20170701 30

Nitrosative stress has been implicated in the pathogenesis of age related macular degeneration (AMD). Tyrosine nitration is a unique type of post translational modification that occurs in the setting of inflammation and nitrosative stress. To date, the significance and functional implications of tyrosine nitration of complement factor H (CFH), a key complement regulator in the eye has not been explored, and is examined in this study in the context of AMD pathogenesis.Sections of eyes from deceas ...[more]

PMID: 28159936

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Project description:Purpose: Age-related degeneration (AMD) is a major cause of blindness in developed countries. The molecular pathogenesis of early events in AMD is poorly understood. We investigated differential gene expression in samples of human retinal pigment epithelium (RPE)/choroid from early AMD and control maculas using exon-based arrays. Methods: Gene expression levels in nine early AMD and nine control human donor eyes were assessed using Affymetrix Human Exon ST 1.0 arrays. Two controls did not pass quality control and were removed. Differentially expressed genes were annotated using DAVID, and gene set enrichment analysis (GSEA) was performed on RPE-specific and endothelium-associated gene sets. CFH genotype was also assessed and differential expression was analyzed with respect to high AMD risk (YH/HH) and low AMD risk (YY) genotypes. Results: Seventy-five genes were identified as differentially expressed (raw p-value < 0.01; >50% fold change, mean log2 expression level in AMD or control M-bM-^IM-% median of all average gene expression values); however, no genes were significant (adj. p-value < 0.01) after correction for multiple hypothesis testing. Of 52 genes with decreased expression in AMD (fold change < 0.5; raw p-value < 0.01), 18 genes were identified by DAVID analysis as associated with vision or neurological processes. GSEA of RPE-associated and endothelium-associated genes revealed a significant decrease in genes typically expressed by endothelial cells in the early AMD group compared to controls, consistent with previous histologic and proteomic studies. Analysis with respect to CFH genotype indicated decreased expression of ADAMTS9 in eyes with high-risk genotypes (fold change = -2.61; raw p-value = 0.0008). Conclusions: GSEA results suggest that RPE transcripts are preserved or elevated in early AMD, concomitant with loss of endothelial cell marker expression. These results are consistent with the notion that choroidal endothelial cell dropout occurs early in the pathogenesis of AMD. Using AltAnalyze (ver. 2.0.7 beta), we analyzed nine early AMD and nine control eyes using Affymetrix Human Exon ST 1.0 arrays. Following initial processing in AltAnalyze, two control arrays were identified as potential outliers by tests implement in arrayQualityMetrics, a package for R.

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.