Differential epithelial and stromal protein profiles in cone and non-cone regions of keratoconus corneas

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.

Comparison of tear proteomic and neuromediator profiles changes between small incision lenticule extraction (SMILE) and femtosecond laser-assisted in-situ keratomileusis (LASIK)

“…The SWATH-MS technology is emerging as a preferred tool for clinical proteomic studies with a large number of samples involved, especially when analyzing limited volume of samples for multiple time points [13] . Our group has previously used this method to document the tear protein changes in patients with thyroid eye diseases and keratoconus [17] , [18] . Our data showed that during the first month postoperatively, the complement activation and immunoglobulin production were the dominant responses shown in both SMILE and LASIK.…”

“…Tear proteins from finely cut Schirmer’s strip were extracted by ammonium bicarbonate (50 mM, 100 μl per strip; Sigma-Aldrich, St. Louis, MO, USA) at 1300 rpm, room temperature (23 °C) for 1 h, and the protein concentration was measured. Protein samples (100 μg) were applied for SWATH-MS and a spectral library was established as previously described [17] , [18] . The digested samples were reconstituted in loading buffer (0.1% formic acid, 2% acetonitrile in water), and iRT standard (Biognosys, Switzerland) was spiked.…”

“…The digested samples were reconstituted in loading buffer (0.1% formic acid, 2% acetonitrile in water), and iRT standard (Biognosys, Switzerland) was spiked. Sample analysis was performed using an Ultimate 3000 nanoLC system (Dionex, Thermo Fisher Scientific, MA, USA) coupled with AB Sciex 5600 triple TOF (AB Sciex, Framingham, MA, USA) as previously reported [18] , [19] .…”

Comparison of Corneal Biomechanical Properties between Post-LASIK Ectasia and Primary Keratoconus

“…In primary keratoconus, changes in the expression of corneal epithelium and stroma-specific genes at the apex of the cone result in focal weakening in keratoconus [ 16 ]. Also, changes in keratoconus-related proteome occur in the noncone regions of keratoconus corneas [ 17 ].…”

The utility of measures of anterior segment parameters of a Pentacam Scheimpflug tomographer in discriminating high myopic astigmatism from keratoconus

“…Keratoconic eyes are less resistant to deformation in post-LASIK ectasia due to focal weakening and significant reduction in corneal stiffness. This phenomenon results from variations in the expression of corneal epithelium and stroma-specific genes at the apex of the cone as well as changes in keratoconus-related proteome in non-cone regions of keratoconus corneas [ 9 – 11 ]. Accordingly, detecting keratoconus among refractive surgery candidates is crucial since removing tissue from an inherently weak cornea further weakens and threatens its integrity [ 12 ].…”