Analysis of the Aqueous Humor Proteome in Patients With Age-Related Macular Degeneration

Abstract

Purpose Age-related macular degeneration (AMD) is associated with altered gene and protein expression in the retina. We characterize the aqueous humor (AH) proteome in AMD to gain insight into the pathogenesis of the disease and identify potential biomarkers. Methods AH was collected from age and gender matched neovascular AMD (nvAMD; n = 10) patients and controls ( n = 10). AH was pooled to create two samples (nvAMD and control), followed by intensity-based label-free quantification (MS1). Functional and bioinformatic analysis were then performed. A validation set (20 controls, 15 atrophic AMD and 15 nvAMD) was tested via multiplex ELISA for nine differentially expressed proteins according to the MS1 findings. Results MS1 identified 674 proteins in the AH. 239 proteins were upregulated in nvAMD (nvAMD/control > 2, peptide tags (PT) > 2), and 86 proteins were downregulated (nvAMD/control < 0.5, PT > 2). Functional analysis of proteins upregulated in AMD demonstrated enrichment for platelet degranulation (enrichment score (ES):28.1), negative regulation of endopeptidase activity (ES:18.8), cellular protein metabolic process (ES:11.8), epidermal growth factor-like domain (ES:10.3), sushi/SCR/CCP (ES:10.1), and complement/coagulation cascades (ES:9.2). AMD protein clusters were upregulated for 3/6 (χ2 < 0.05 compared to randomization). Validation via ELISA confirmed MS1 in 2/9 proteins (Clusterin and Serpin A4, P < 0.05), while 3/9 showed differential expression between aAMD and nvAMD (Clusterin, Serpin A4, and TF P < 0.05). Receiver operating characteristic curve calculation identified the area under the curve of 0.82 for clusterin as a biomarker for distinction of AMD. Conclusions AH proteomics in AMD patients identified several proteins and functional clusters with altered expression. Further research should confirm if these proteins may serve as biomarkers or therapeutic target for the disease.

Retinal pigment epithelium extracellular vesicles are potent inducers of age‐related macular degeneration disease phenotype in the outer retina

“…Clusterin and fibronectin are known cleavage targets of HTRA1, hence their abundance in the high‐risk EVs may constitute favourable environment for extracellular debris formation (Lin et al., 2018). As other proteins specifically enriched in the apical or basal high risk RPE EVs (SLC2A1, RELN, TPI1, EEF2, COL1A2, SQSTM1 and LGALS3BP) have been associated in the literature with AMD, this further validates our findings (Hongisto et al., 2017; Kim et al., 2014; Kim et al., 2018; Rinsky et al., 2021; Viiri et al., 2013; Xu et al., 2019; Zuo et al., 2012).…”

Recent Advances in Proteomic-based Approaches to Study Age-related Macular Degeneration: A Systematic Review

“…As previously described for vitreous humour, aqueous clusterin has recently been proposed as a biomarker for AMD progression by Rinsky et al, [95]. Clusterin was first detected as overrepresented in the aqueous humour of nAMD patients vs controls (n=10 in both cases) and later validated by ELISA in a larger cohort including nAMD patients (n = 15), aAMD patients (n = 15) and controls (n = 20).…”

Recent Advances in Proteomics-Based Approaches to Studying Age-Related Macular Degeneration: A Systematic Review

“…As previously described for vitreous humor, aqueous clusterin has recently been proposed as a biomarker for AMD progression by Rinsky et al [ 66 ]. Clusterin was first detected as being overrepresented in the aqueous humor of nAMD patients vs. controls (n = 10 in both cases), which was later validated using ELISA in a larger cohort including nAMD patients (n = 15) and aAMD patients (n = 15) and controls (n = 20).…”