Proteomic and Cytokine Profiling in Plasma from Patients with Normal-Tension Glaucoma and Ocular Hypertension

Abstract

Primary open-angle glaucoma (POAG) is subdivided depending on eye pressure. Patients with normal-tension glaucoma (NTG) have never had high intraocular pressure (IOP) measured while patients with ocular hypertension (OHT) have high eye pressure but no signs of glaucoma. Although IOP is considered to be a risk factor for all glaucoma patients, it is reasonable to assume that other risk factors such as inflammation play a role. We aimed to characterize the proteome and cytokine profile during hypoxia in plasma from patients with NTG (n = 10), OHT (n = 10), and controls (n = 10). Participants were exposed to hypoxia for two hours, followed by 30 min of normoxia. Samples were taken before ("baseline"), during ("hypoxia"), and after hypoxia ("recovery"). Proteomics based on liquid chromatography coupled with mass spectrometry (LC-MS) was performed. Cytokines were measured by Luminex assays. Bioinformatic analyses indicated the involvement of complement and coagulation cascades in NTG and OHT. Regulation of high-density lipoprotein 3 (HDL3) apolipoproteins suggested that changes in cholesterol metabolism are related to OHT. Hypoxia decreased the level of tumor necrosis factor-α (TNF-α) in OHT patients compared to controls. Circulating levels of interleukin-1β (IL-1β) and C-reactive protein (CRP) were decreased in NTG patients compared to controls during hypoxia. After recovery, plasma interleukin-6 (IL-6) was upregulated in patients with NTG and OHT. Current results indicate an enhanced systemic immune response in patients with NTG and OHT, which correlates with pathogenic events in glaucoma. Apolipoproteins may have anti-inflammatory effects, enabling OHT patients to withstand inflammation and development of glaucoma despite high IOP.

Keywords: Cytokines; Hypoxia; Immune response; Normal-tension glaucoma; Ocular hypertension; Proteomics.

Fig. 1

Heatmap showing the differential expression of proteins identified in plasma from patients with NTG (n = 10), OHT (n = 10), and controls (n = 10). Data in each group is pooled from the three time points (“baseline”, “hypoxia”, and “recovery”). Clustering is based on the control group and protein names are displayed as row labels. Up-regulated protein expression is displayed in red, downregulated expression in blue, and intermediate-regulated expression is in shades of red and blue. LFQ values refer to mean values. LFQ label-free quantification

Fig. 2

PCA score plots of the full dataset. PCA score plot of samples colored according to a test group (controls, OHT, and NTG patients), b time point (“baseline”, “hypoxia”, and “recovery”), and c sex (men and women). M men; NTG normal-tension glaucoma; OHT ocular hypertension; PCA principal component analysis; W women

Fig. 3

STRING network analysis of differentially expressed proteins in plasma from a patients with NTG compared to controls, b patients with OHT compared to controls, and c patients with NTG compared to patients with OHT. Each protein is represented as a node, and lines represent interactions between two nodes. Red nodes indicate more abundant proteins and blue nodes indicate less abundant proteins. Proteins are separated based on their involvement in biological pathways according to KEGG analysis

Fig. 4

Plasma levels of cytokines and CRP in patients with NTG (n = 10) and OHT (n = 10) compared to controls (n = 10) at baseline, hypoxia, and recovery. Plasma levels of a IL-1β, b IL-6, c TNF-α, d IFN-α, e IFN-γ, f TGF-β1, and g CRP. Mann–Whitney U-test was used to test differences between groups and Wilcoxon-signed rank test to test differences between time points. Data are presented as individual data points with median and the interquartile range. *p < 0.05. CRP C-reactive protein; IFN-α interferon-α; IFN-γ interferon-γ; IL-1β interleukin-1β; IL-6 interleukin-6; TGF-β1 transforming growth factor-β1; TNF-α tumor necrosis factor-α