Abstract
Keratoconus (KC) is an ectatic corneal disease characterized by progressive thinning and irregular astigmatism, and a leading indication for corneal transplantation. KC-associated changes have been demonstrated for the entire cornea, but the pathological thinning and mechanical weakening is usually localized. We performed quantitative proteomics using Sequential Windowed Acquisition of All Theoretical Fragment Ion Mass Spectrometry (SWATH-MS) to analyze epithelial and stromal changes between the topographically-abnormal cone and topographically-normal non-cone regions of advanced KC corneas, compared to age-matched normal corneas. Expression of 20 epithelial and 14 stromal proteins was significantly altered (≥2 or ≤0.5-fold) between cone and non-cone in all 4 KC samples. Ingenuity pathway analysis illustrated developmental and metabolic disorders for the altered epithelial proteome with mitochondrion as the significant gene ontology (GO) term. The differential stromal proteome was related to cellular assembly, tissue organization and connective tissue disorders with endoplasmic reticulum protein folding as the significant GO term. Validation of selected protein expression was performed on archived KC, non-KC and normal corneal specimens by immunohistochemistry. This is the first time to show that KC-associated proteome changes were not limited to the topographically-thinner and mechanically-weakened cone but also non-cone region with normal topography, indicating a peripheral involvement in KC development.
Highlights
Keratoconus (KC) is an asymmetric corneal ectatic disorder characterized by progressive focal thinning, that leads to myopia and irregular astigmatism with impaired visual acuity[1,2]
KC2 had ~2-fold more epithelial and stromal proteins extracted from cone than in non-cone region (Supplemental Fig. S1)
This study investigated the epithelial and stromal proteomes of 2 topographically divergent regions of KC cornea, i.e. cone with thin ectatic topography and non-cone with normal topography, using label-free SWATH-MS
Summary
Keratoconus (KC) is an asymmetric corneal ectatic disorder characterized by progressive focal thinning, that leads to myopia and irregular astigmatism with impaired visual acuity[1,2]. Linkage and genome-wide association studies have identified possible loci and gene variants, yet they remain to be validated in larger cohorts[1,13,14,15,16] External factors, such as contact lens wear, eye rubbing and ultraviolet (UV) light exposure, potentially induce corneal microtrauma and trigger the production of inflammatory mediators[17]. Tear film studies using proteomics and bioinformatics have shown altered proteins belonging to families of proteinases, inflammatory cytokines, cell adhesion molecules, glycoproteins and transporters in samples from KC patients, compared to controls[29,30,35,36,37,38,39]. A recent report on saliva proteomics has identified significant hormonal metabolite changes (including IL16, myoinositol and 1-methyl-histidine) associated with pro-inflammatory processes in KC versus healthy control subjects[43]. Recent studies on isolated KC epithelia and stroma have illustrated altered expression of cytokeratins and cytoskeleton, matrix components and regulatory proteins, suggesting that various degenerative pathways in association with inflammation, changes of innate immune functions, oxidative stress, abnormal mitochondrial functions, and cell death occur in KC25,44–47
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