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Chris Hodge, Jing Jing You, Li Wen, Athena Roufas, Michele Madigan, John W. McAvoy, Gerard Sutton; WNT Signalling Pathway-Associated Proteins in Control and Keratoconus (KC) Tears and Corneas. Invest. Ophthalmol. Vis. Sci. 2012;53(14):4221.
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To investigate the Wnt pathway-associated secreted frizzled-related proteins (SFRPs) in tears and corneas from control and keratoconus (KC) patients.
Basal tear samples were collected from control and KC patients (n=15) using capillary micropipettes, and prepared as tryptic digests. A targeted multiple reaction monitoring (MRM) mass spectrometric approach was used to detect SFRP1. Three peptides, each with three transition ions, were selected for the MRM analysis. Western blot was used to confirm and relatively quantify the expression of SFRP1 in tears using an external loading control. SFRP1, 3 and 5 immunoreactivity in control (n=4) and KC (n=10) corneas was studied using immunoflurorescence and confocal microcopy.
MRM analysis successfully detected SFRP-1 in tears from both groups. SFRP-1 was detected in tears with immunoblotting at reduced levels in KC, compared to control patients. Heterogeneous epithelial SFRP1 expression was seen in all KC corneas compared to low expression in controls. SFRP3 and SFRP5 showed unique patterns of immunolabelling. SFRP3 localised to epithelial cell membranes and limbal vessels in control corneas, cytoplasmic and more central expression was seen in KC. SFRP5 was expressed strongly in corneal stroma (KC and control).
This is the first study to report SFRP1 in tears from control and KC patients. MRM analysis could detect the low abundance SFRP1 in tears in rapid fashion, indicating its potential application in tear biomarker research. The pathogenesis of KC remains poorly understood, however our current study showing regulation of SFRPs in tears and cornea in KC, and earlier observations of SFRP1 gene expression in corneal tissue, further indicate a role for the Wnt signalling pathway in the pathogenesis of KC.
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