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

Iomdina, E.N.; Tikhomirova, N.K.; Bessmertny, A.M.; Serebryakova, M.V.; Baksheeva, V.E.; Zalevsky, A.O.; Kotelin, V.I.; Kiseleva, O.A.; Kosakyan, S.M.; Zamyatnin Jr, A.A.; Philippov, P.P.; Zernii, E.Y.

Авторы: Iomdina Elena N., Tikhomirova Natalya K., Bessmertny Alexander M., Serebryakova Marina V., Baksheeva Viktoriia E., Zalevsky Arthur O., Kotelin Vladislav I., Kiseleva Olga A., Kosakyan Sbrui M., Zamyatnin Jr Andrey A., Philippov Pavel P., Zernii Evgeni Yu
Журнал: Molecular vision
Том: 26
Год издания: 2020
Издательство: MOLECULAR VISION
Местоположение издательства: C/O JEFF BOATRIGHT, LAB B, 5500 EMORY EYECENTER, 1327 CLIFTON RD, N E, ATLANTA, USA, GA, 30322
Первая страница: 623
Последняя страница: 640
Аннотация: 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.
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Alterations in proteome of human sclera associated with primary open-angle glaucoma involve proteins participating in regulation of the extracellular matrixстатья Исследовательская статья

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