Proteomic Analysis of Aqueous Humor in Central Retinal Artery Occlusion: Unveiling Novel Insights Into Disease Pathophysiology

Abstract

Purpose: Central retinal artery occlusion (CRAO) is an ocular emergency that results from acute blockage of the blood supply to the retina and leads to a sudden vision loss. Other forms of ischemic retinopathies include diabetic retinopathy (DR), which involves chronic retinal ischemia and remains the leading cause of blindness in working-age adults. This study is the first to conduct a proteomic analysis of aqueous humor (AH) from patients with CRAO with a comparative analysis using vitreous humor (VH) samples from patients with DR.

Methods: AH samples were collected from 10 patients with CRAO undergoing paracentesis and 10 controls undergoing cataract surgery. VH samples were collected from 10 patients with DR and 10 non-diabetic controls undergoing pars plana vitrectomy (PPV). Samples were analyzed using mass spectrometry.

Results: Compared with controls, AH levels of 36 differentially expressed proteins (DEPs) were identified in patients with CRAO. Qiagen Ingenuity Pathway Analysis (IPA) revealed 11 proteins linked to ophthalmic diseases. Notably, enolase 2, a glycolysis enzyme isoform primarily expressed in neurons, was upregulated, suggesting neuronal injury and enzyme release. Additionally, clusterin, a protective glycoprotein, was downregulated. ELISA was conducted to confirm proteomics data. VH samples from patients with DR exhibited changes in a distinct set of proteins, including ones previously reported in the literature.

Conclusions: The study provides novel insights into CRAO pathophysiology with multiple hits identified. Proteomic results differed between DR and CRAO studies, likely due to the different pathophysiology and disease duration.

Translational relevance: This is the first proteomic analysis of CRAO AH, with the potential to identify future therapeutic targets.

Figure 1.

Schematic of sample collection and processing. AH from patients with CRAO, and VH from patients with DR were collected along with respective control samples, aliquoted and stored in a −80°C freezer for later proteomic analysis using an Orbitrap Eclipse Tribrid mass spectrometer. Bioinformatic analysis and ELISA were then performed.

Figure 2.

CRAO aqueous humor (AH) proteome. (A) Volcano plot of the differentially expressed proteins in CRAO AH as compared to control AH at logFC > |1|, and q value < 0.05. (B) Quantification of enolase 2 in plasma samples from controls and patients with CRAO using ELISA. (C) Quantification of clusterin in AH samples from controls and patients with CRAO using ELISA. (D) Quantification of clusterin in plasma samples from controls and patients with CRAO using ELISA.

Figure 3.

DR vitreous humor (VH) proteome. (A) Volcano plot of the differentially expressed proteins in DR VH as compared to control VH at logFC > |1|, and p value < 0.05. (B) Quantification of clusterin in VH samples from controls and patients with DR using ELISA. (C) Quantification of enolase 2 in plasma samples from controls and patients with DR using ELISA.

Figure 4.

Shared proteins between the CRAO AH and DR VH datasets. (A) Venn diagram of the number of proteins shared between AH and VH proteomic analyses. (B) Venn diagram of the number of differentially expressed proteins (p < 0.05) shared between AH and VH proteomic analyses. (C) Quantification of IGFBP6 in plasma samples from controls and patients with CRAO using ELISA. (D) Quantification of IGFBP6 in plasma samples from controls and patients with DR using ELISA. (E) Heat map of the diseases and functions activated or inhibited in the CRAO AH and DR VH proteomic analyses.

Figure 5.

Network analyses of CRAO AH and DR VH. (A) CRAO AH network shows molecules involved in ophthalmic disease, hereditary disorders, organismal injury, and abnormalities with Akt as a central mediator. (B) DR VH network shows molecules involved in hematological disease, hereditary disorders, organismal injury, and abnormalities converging to CRP.