Alterations in proteome of human sclera associated with primary open-angle glaucoma involve proteins participating in regulation of the extracellular matrix.

Purpose: Primary open-angle glaucoma (POAG) is a common ocular disease, associated with abnormalities in aqueous humor circulation and an increase in intraocular pressure (IOP), leading to progressive optical neuropathy and loss of vision. POAG pathogenesis includes alterations of the structural properties of the sclera, especially in the optic nerve head area, contributing to the degeneration of the retinal ganglion cells. Abnormal sclera biomechanics hinder adequate compensation of IOP fluctuations, thus aggravating POAG progression. The proteomic basis of biomechanical disorders in glaucomatous sclera remains poorly understood. This study is aimed at revealing alterations in major scleral proteins, associated with POAG, at different stages of the disease and with different IOP conditions. Methods: Samples of sclera were collected from 67 patients with POAG during non-penetrating deep sclerectomy and from nine individuals without POAG. Scleral proteins were extracted with a strong lysis buffer, containing a combination of an ionic detergent, a chaotropic agent, and a disulfide reducing agent, and were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The major scleral proteins were selected, subjected to in-gel digestion, and identified using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF)/TOF mass spectrometry (MS), coupled with tandem mass spectrometry (MS/MS). The specific POAG-associated alterations of the selected proteins were analyzed with SDS-PAGE and confirmed with western blotting of the scleral extracts, using the respective antibodies. The group of POAG-associated proteins was analyzed using Gene Ontology and genome-wide association study enrichment and protein-protein interaction network prediction. Results: A total of 11 proteins were identified, among which six proteins, namely, vimentin, angiopoietin-related protein 7, annexin A2, serum amyloid P component, serum albumin, and thrombospondin-4, were found to be upregulated in the sclera of patients with advanced and terminal POAG. In the early stages of the disease, thrombospondin-4 level was, on the contrary, reduced when compared with the control, whereas the concentration of vimentin varied, depending on the IOP level. Moreover, angiopoietin-related protein 7 manifested as two forms, exhibiting opposite behavior: The common 45 kDa form grew with the progression of POAG, whereas the 35 kDa (apparently non-glycosylated) form was absent in the control samples, appeared in patients with early POAG, and decreased in concentration over the course of the disease. Functional bioinformatics analysis linked the POAG-associated proteins with IOP alterations and predicted their secretion into extracellular space and their association with extracellular vesicles and a collagen-containing extracellular matrix. Conclusions: POAG is accompanied by alterations of the scleral proteome, which represent a novel hallmark of the disease and can reflect pathological changes in scleral biochemistry and biomechanics. The potential mechanisms underlying these changes relate mainly to the structure of the extracellular matrix, protein glycosylation, and calcium binding, and may involve fibroblast cytoskeleton regulation, as well as oxidative and inflammatory responses.

Comparative lipidomic analysis of inflammatory mediators in the aqueous humor and tear fluid of humans and rabbits.

INTRODUCTION: Ocular inflammation is a key pathogenic factor in most blindness-causing visual disorders. It can manifest in the aqueous humor (AH) and tear fluid (TF) as alterations in polyunsaturated fatty acids (PUFAs) and their metabolites, oxylipins, lipid mediators, which are biosynthesized via enzymatic pathways involving lipoxygenase, cyclooxygenase or cytochrome P450 monooxygenase and specifically regulate inflammation and resolution pathways. OBJECTIVES: This study aimed to establish the baseline patterns of PUFAs and oxylipins in AH and TF by their comprehensive lipidomic identification and profiling in humans in the absence of ocular inflammation and comparatively analyze these compounds in the eye liquids of rabbits, the species often employed in investigative ophthalmology. METHODS: Ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was used for qualitative and quantitative characterization of lipid compounds in the analyzed samples. RESULTS: A total of 28 lipid compounds were identified, including phospholipid derivatives and PUFAs, as well as 22 oxylipins. Whereas the PUFAs included arachidonic, docosahexaenoic and eicosapentaenoic acids, the oxylipins were derived mainly from arachidonic, linoleic and α-linolenic acids. Remarkably, although the concentration of oxylipins in AH was lower compared to TF, these liquids showed pronounced similarity in their lipid profiles, which additionally exhibited noticeable interspecies concordance. CONCLUSION: The revealed correlations confirm the feasibility of rabbit models for investigating pathogenesis and trialing therapies of human eye disorders. The identified metabolite patterns suggest enzymatic mechanisms of oxylipin generation in AH and TF and might be used as a reference in ocular inflammation studies.