Quantitative proteomic analysis of aqueous humor from patients with drusen and reticular pseudodrusen in age-related macular degeneration

Abstract

BackgroundTo identify novel biomarkers related to the pathogenesis of dry age-related macular degeneration (AMD), we adopted a human retinal pigment epithelial (RPE) cell culture model that mimics some features of dry AMD including the accumulation of intra- and sub-RPE deposits. Then, we investigated the aqueous humor (AH) proteome using a data-independent acquisition method (sequential window acquisition of all theoretical fragment ion mass spectrometry) for dry AMD patients and controls.MethodsAfter uniformly pigmented polarized monolayers of human fetal primary RPE (hfRPE) cells were established, the cells were exposed to 4-hydroxy-2-nonenal (4-HNE), followed by Western blotting, immunofluorescence analysis and ELISA of cells or conditioned media for several proteins of interest. Data-dependent acquisition for identification of the AH proteome and SWATH-based mass spectrometry were performed for 11 dry AMD patients according to their phenotypes (including soft drusen and reticular pseudodrusen [RPD]) and 2 controls (3 groups).ResultsIncreased intra- and sub-RPE deposits were observed in 4-HNE-treated hfRPE cells compared with control cultures based on APOA1, cathepsin D, and clusterin immunoreactivity. Additionally, the differential abundance of proteins in apical and basal chambers with or without 4-HNE treatment confirmed the polarized secretion of proteins from hfRPE cells. A total of 119 proteins were quantified in dry AMD patients and controls by SWATH-MS. Sixty-five proteins exhibited significantly altered abundance among the three groups. A two-dimensional principal component analysis plot was generated to identify typical proteins related to the pathogenesis of dry AMD. Among the identified proteins, eight proteins, including APOA1, CFHR2, and CLUS, were previously considered major components or regulators of drusen. Three proteins (SERPINA4, LUM, and KERA proteins) have not been previously described as components of drusen or as being related to dry AMD. Interestingly, the LUM and KERA proteins, which are related to extracellular matrix organization, were upregulated in both RPD and soft drusen.ConclusionsDifferential protein expression in the AH between patients with drusen and RPD was quantified using SWATH-MS in the present study. Detailed proteomic analyses of dry AMD patients might provide insights into the in vivo biology of drusen and RPD.Electronic supplementary materialThe online version of this article (10.1186/s12886-018-0941-9) contains supplementary material, which is available to authorized users.

The Constitutive Proteome of Human Aqueous Humor and Race Specific Alterations

“…Although proteins in AH are present in relatively low concentrations when compared to blood plasma, they are vital in the maintenance of anterior segment homeostasis [ 2 , 8 , 12 , 13 , 14 , 15 , 16 , 17 , 18 , 19 ]. Previous studies have shown significant alterations in several proteins in the AH obtained from eyes with glaucoma [ 12 , 13 , 14 , 15 , 20 , 21 ] and other eye disorders, including age-related macular degeneration [ 14 , 16 , 22 , 23 , 24 , 25 ].…”

Association of imaging biomarkers and local activation of complement in aqueous humor of patients with early forms of age-related macular degeneration

“…Furthermore, the presence of RPD has been associated with major AMD risk polymorphisms in ARMS2/HTRA1 genes [24,49,50]. RPD are located in the subretinal space and share compositional similarities with conventional drusen, yet their lipid composition is different than soft drusen [51][52][53][54][55]. Nevertheless, RPD appear also in other retinal diseases such as Sorsby fundus dystrophy and pseudoxanthoma elasticum, and a common pathogenetic pathway involving BrM and RPE interface is possible [56].…”

“…Nevertheless, RPD appear also in other retinal diseases such as Sorsby fundus dystrophy and pseudoxanthoma elasticum, and a common pathogenetic pathway involving BrM and RPE interface is possible [ 56 ]. In a recent study, RPD patients were reported to have different AH protein profiles in comparison with patients with soft drusen [ 55 ]. In the same study, it was speculated that RPD formation is linked to a dysfunctional RPE, which secretes proteins aberrantly towards the apical surface instead of onto basolateral surface leading to the accumulation of deposits in subretinal space [ 55 ].…”

“…In a recent study, RPD patients were reported to have different AH protein profiles in comparison with patients with soft drusen [ 55 ]. In the same study, it was speculated that RPD formation is linked to a dysfunctional RPE, which secretes proteins aberrantly towards the apical surface instead of onto basolateral surface leading to the accumulation of deposits in subretinal space [ 55 ].…”

Proteomic Study of Aqueous Humor and Its Application in the Treatment of Neovascular Glaucoma

“…Je-Hyun Baek et al used SWATH-MS technology to quantify differentially expressed proteins in the AH between patients with drusen and reticular pseudodrusen (RPD) of age-related macular degeneration (AMD). In three newly discovered dry AMD-related proteins, LUM and KERA were upregulated in both RPD and soft drusen, and these are correlated to the extracellular matrix organization (Baek et al, 2018). Chiang SY et al used MALDI-TOF MS and identified 11 AH proteins with a pathophysiological role in diabetic retinopathy.…”