Changes to the tear film proteome in keratoconus

Abstract

Keratoconus (KC) is a progressive degenerative disease of the eye which causes an irregularly shaped cornea leading to severe impairment of vision. Despite basic and clinical studies KC remains a poorly understood disease. This project was designed to investigate the levels of proteins, proteases and cytokines in the tears of people with KC. Basal tears were collected from normal controls (C); people with KC and from people who had undergone corneal collagen cross-linking (CXL) for the treatment of KC. Corneal curvature of each subject was mapped. Proteomic technologies including gel electrophoresis, ELISA, mass spectrometry, antibody arrays and activity assays were used to examine the changes in tear proteins, proteases and cytokines between the different subject groups. There was approximately 2-fold decrease in total protein levels, lactoferrin, secretory IgA between KC and C tears, but the level of albumin was not significantly reduced. The tear protein changes correlated to the severity of the disease. Increased levels of cathepsin B (2.7-fold) and decreased levels of cystatin S (2.1-fold) and cystatin SN (2-fold), polymeric immunoglobulin receptor (9.4-fold), fibrinogen alpha chain (8.2-fold) were observed in KC compared to C. Keratin type-1 cytoskelatal-14 and keratin type-2 cytoskeletal-5 was present only in the tears of KC. Tears of people with KC had 1.9 times higher levels of proteolytic activity and over-expression of several matrix metalloproteinases (MMP) -1, -3, -7, -13 and interleukins (IL) -4, -5, -6, -7, -8 and tumour necrosis factor (TNF) -α, -β compared to tears from C. No significant difference in MMPs were observed between the C and CXL groups, although the expression levels of TNF-α was 1.5 times increased in CXL compared to C. The activity of tear proteases in CXL were not significantly different compared to either KC or C subjects. The tears of people with KC appear to have an altered tear protein profile, higher levels of proteases and cytokines that might reflect the pathological events in KC corneas. The novel findings reported in this thesis might lead the way to the development of non-invasive diagnostic or prognostic tests for KC or determine the success of CXL using tear proteomics.