Changes in tear protein profile in keratoconus disease

Abstract

Purpose To analyze tear protein profile variations in patients with keratoconus (KC) and to compare them with those of control subjects. Subjects and methods Tears from 12 normal subjects and 12 patients with KC were analyzed by two-dimensional gel electrophoresis (2-DE) and liquid chromatography-mass spectrometry (LC-MS). Analysis of the 2-DE gels was performed using Progenesis SameSpots software (Nonlinear Dynamics). Proteins exhibiting high variation in expression levels (P-value o0.05) were identified using matrix-assisted laser desorption/ionization-TOF spectrometry. For LC-MS analysis, a label-free quantification approach was used. Tears were digested with trypsin, subjected to data-independent acquisition (MS E ) analysis, and identified proteins were relatively quantified using ProteinLynx Global Server software (Waters). Results The 2-DE and LC-MS analyses revealed a significant decrease in the levels of members of the cystatin family and an increase in lipocalin-1 in KC patients. A 1.43-fold decrease was observed for cystatin-S by 2-DE, and 1.69-and 1.56-fold for cystatin-SN and cystatin-SA by LC-MS, respectively. The increase in lipocalin-1 was observed by both methods with fold changes of 1.26 in the 2-DE approach and 1.31 according to LC-MS. Significant protein upregulation was also observed for Ig-j chain C and Ig J chain proteins by 2-DE. Levels of lipophilin-C, lipophilin-A, and phospholipase A2 were decreased in tears from KC patients according to LC-MS. Serum albumin was found to be increased in KC patients according to LC-MS. Conclusion The results show differences in the tear protein profile of KC and control subjects. These changes are indicative of alterations in tear film stability and in interactions with the corneal surface in KC patients.

Keratoconus: an inflammatory disorder?

“…They found a relative increase in the abundance levels of cathepsin B and decreased levels of polymeric immunoglobulin receptor, α-fibrinogen, cystatin SN, and cystatin S in the tears of subjects with keratoconus, meaning that tear proteins differentially expressed in keratoconus included increased proteases and decreased protease inhibitors. 95 The findings of a lower level of cystatins by Balasubramanian et al 95 confirmed the ones previously reported in 2011 by Acera et al, 96 who compared tears from 12 normal subjects and 12 patients with keratoconus, using two-dimensional gel electrophoresis and liquid chromatography-mass spectrometry, and found a significant decrease in the levels of members of the cystatin family in keratoconus patients. Cystatins are inhibitors of cysteine proteases, also known as thiol proteases, which are enzymes that degrade proteins.…”

“…19,20,106,[108][109][110]144 An important decrease in the level of protease inhibitors such as cystatins (inhibitors of cysteine proteases) and TIMP-1 (inhibitor of MMPs) have also been reported. 95,96,101,[129][130][131] The increased activity of several proteolytic enzymes results in higher concentrations of ROS, RNS, cytotoxic aldehydes (CAs) and peroxynitrates (Ps) (which decreases the activity of TIMP-1 and increase MMP-2), 63,143,144,146,149 and given the lower production of SOD 143 possibly related to IL-1α, 158 an environment with high oxidative stress and low pH is formed, causing an increase in the activation of the caspases (caspase-9 and -12), mitochondrial dysfunction (MD), and DNA damage, 156 which eventually lead to increased apoptosis. All of these could probably be the result of a complex interaction of both genetic predisposition and environmental triggering factors, such as eye rubbing and contact lenses wear (the 'two-hit hypothesis') in keratoconus.…”

“…98 Cystatins, inhibitors of those proteolitic enzymes, are divided into type 1 known as intracellular (A and B), type 2 known as extracellular (C, D, E/M, F, G, S, SN, and SA) and type 3 known as intravascular (L-and H-kininogen). 99,100 The decreased levels of cystatins found in tears from eyes with keratoconus could be a sign of an increase in the degradation of tear proteins, which would be correlated with the decrease in the concentration of total protein in tears from keratoconus patients found by Acera et al 96 In concordance with findings by Balasubramanian et al 95 in tears, lysosomal cathepsin B and cathepsin G have been found to be elevated in keratocytes of keratoconic corneas, localized beneath compromised regions of Bowman's layer and the stroma of morphologically compromised regions, compared with normal tissue. 101 Jun et al 103 evaluated Type-1 helper T cell cytokines (IL-12, IFN-γ, and TNF-α), Type-2 helper T cell cytokines (IL-4, IL-10, and IL-13), and T-helper 17 cell cytokines (IL-17) in serum and tears of patients with keratoconus, to determine whether an altered inflammatory response can contribute to the keratoconus etiology or not.…”

“…103 Whether the altered protein composition of tears in eyes suffering from keratoconus is an effect of events in the corneal epithelium or has a direct influence in the development of the disease remains to be elucidated. 96 Cheung et al 86 recently analyzed ex vivo the modulation of the healing process of corneas with keratoconus and of non-keratoconic corneas. The affected corneas had higher levels of IL-1α, IGF-1 (insulin-like growth factor 1), TNF-α, and TGF-B1 (TGFβ-1) than normal corneas, and lower levels of HGF and B-NGF (β-nerve growth factor).…”

Diabetic retinopathy: Proteomic approaches to help the differential diagnosis and to understand the underlying molecular mechanisms

“…Electrophoresis [86], protein arrays [87] and various mass spectrometry-related techniques [85,88] have been applied. Various forms of chemical labeling [39,43], SRM/MRM-based targeted proteomics and label free quantification have been used for tear studies but no PRM or SWATH analyses have been carried out so far [89,90].…”