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Tatiana Maria Suarez-Cortes, Javier Soria Esponera, Juan A Duran, Nerea Gonzalez, Arantxa Acera; Proteome analysis of human conjunctival epithelium in ocular surfaces diseases by impression cytology and 2D-DIGE approaches. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):348.
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© ARVO (1962-2015); The Authors (2016-present)
To comparatively analyze conjunctiva proteome changes in ocular surface diseases (OSD) such as dry eye (DE) and meibomian gland dysfunction (MGD) by 2D-Differential in gel electrophoresis (2D-DIGE). Three aspects were evaluated: i) proteins whose profiles are able to differentiate between study pathologies; ii) Correlation of protein alterations with squamous metaplasia (SM) degree; iii) Proteins involved in the OSD onset and progression when comparing to healthy state
A prospective case-controlled study was carried out including 62 patients. Patients with MGD, DE and healthy subjects (CT) were recruited at the Instituto Clínico Quirúrgico de Oftalmología (Spain). Conjunctival impression cytology (CIC) specimens for proteomic analyses and PAS-hematoxylin staining were collected. Protein extraction from CICs was done using DIGE Lysis Buffer. Total protein extracts were analyzed by 2D-DIGE, gel images were analyzed with Progenesis SameSpots Software. Protein spots of interest were automatically picked up from the gel and protein spots with greater expression changes were identified by MALDI-TOF/TOF MS. Functional enrichment analysis was performed with the bioinformatics tool DAVID
A conjunctival epithelium proteome map formed by 348 resolved spots.<br /> Higher expression of proteins S100A4, S100A8, Retinal Dehydrogenase-1, Peroxiredoxin-1, Annexin A1, α-enolase, Annexin A2 and Glutathione S-transferase-P was observed in DE group, whereas highest expression of Peroxiredoxin-6, actin cytoplasmic-1, Peroxiredoxin-2, and Heat shock protein HSP-90-α was observed in MGD.<br /> When comparing protein changes with SM degree, 5 different cytokeratins (CK1, CK4, CK8, CK10 and CK13) were found to be correlated.<br /> An overall proteome analysis of OSD revealed upregulation of 9 proteins in pathological groups (Annexin A1, α-enolase, Annexin A2, S100A8, cytokeratin, Peroxiredoxin-2 and Leukocyte elastase inhibitor) and downregulation of 2 proteins (Galectin-3 and Lipocalin-1).
DE group in comparison with MGD presented higher over expression of proteins related with defense response, tissular damage, wound healing and regulation of body fluid secretions. MGD is related with oxidative stress, stress response and antiapoptotic processes. There exists a correlation between SM degree and pairs of cytokeratins and proteins related with cytoskeleton and keratinization.
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