Quantitative Proteomic Analysis of Non-Human Primate (NHP) Peripapillary (PP) Sclera in Early Experimental Glaucoma (EEG)

Abstract

Purpose: To study the molecular mechanisms of scleral remodeling in NHP EEG and discover candidate biomarkers, we quantified proteomic changes in the PP-sclera of Rhesus Macaques with laser-induced, unilateral EEG.

Methods: A PP-scleral donut (6-10 mm around the optic nerve head) was isolated from the EEG and control eyes of 4 adult NHP (3 high IOP (IOP Max >28 mm Hg, and 1 low IOP (IOP Max < 20 mm Hg). Protein was extracted in SDS and digested with trypsin. Peptides were labeled with iTRAQ tags, fractionated by cation exchange chromatography, and analyzed by LC MS/MS. Proteins were identified using the Swiss-Protein human database; iTRAQ tags were quantified by the weighted averaged method. Proteins were significantly altered if average ratios (EEG/ control) determined with ≥ 2 unique peptides in the 3 high IOP animals (or in the low IOP animal) were at least 1 standard deviation from the mean with p values ≤ 0.05. Proteomic analysis of EEG and POAG plasma was performed following immunodepletion. Bioinformatics analyses utilized Ingenuity Pathway Analysis.

Results: In the high IOP PP-sclera, 44 significantly altered proteins were identified within 1002 quantified. Altered proteins were often different in the low IOP sclera. Significantly increased proteins in high IOP NHPs included myocilin, fibronectin, major vault protein and endoplasmin. Significantly decreased proteins in high IOP NHPs included tubulin beta-2A, cadherin-13, LIM domain-binding protein 3, and seven mitochondrial proteins. Many of the altered proteins were detected in EEG plasma, in POAG plasma, and in the human plasma atlas, supporting the possibility of monitoring these proteins for biomarker validation.

Conclusions: Top canonical pathways associated with altered scleral proteins in high IOP NHP EEG included protein ubiquitination and mitochondrial dysfunction. Top networks included cell morphology, maintenance, interactions and signaling, skeletal and muscular disorders, connective tissue development and function. While additional animals must be studied, proteomic differences appear to exist in PP-sclera from low and high IOP EEG NHPs. Altered proteins provide insights into the molecular mechanisms underlying early PP-scleral remodeling in EEG and candidate blood-borne biomarkers.

Keywords: 708 sclera • 568 intraocular pressure • 663 proteomics