Abstract
Keratoconus (KC) is a bilateral degenerative disease of the cornea characterized by corneal bulging, stromal thinning, and scarring. The etiology of the disease is unknown. In this study, we identified a new biomarker for KC that is present in vivo and in vitro. In vivo, tear samples were collected from age-matched controls with no eye disease (n = 36) and KC diagnosed subjects (n = 17). Samples were processed for proteomics using LC-MS/MS. In vitro, cells were isolated from controls (Human Corneal Fibroblasts-HCF) and KC subjects (Human Keratoconus Cells-HKC) and stimulated with a Vitamin C (VitC) derivative for 4 weeks, and with one of the three transforming growth factor-beta (TGF-β) isoforms. Samples were analyzed using real-time PCR and Western Blots. By using proteomics analysis, the Gross cystic disease fluid protein-15 (GCDFP-15) or prolactin-inducible protein (PIP) was found to be the best independent biomarker able to discriminate between KC and controls. The intensity of GCDFP-15/PIP was significantly higher in healthy subjects compared to KC-diagnosed. Similar findings were seen in vitro, using a 3D culture model. All three TGF-β isoforms significantly down-regulated the expression of GCDFP-15/PIP. Zinc-alpha-2-glycoprotein (AZGP1), a protein that binds to PIP, was identified by proteomics and cell culture to be highly regulated. In this study by different complementary techniques we confirmed the potential role of GCDFP-15/PIP as a novel biomarker for KC disease. It is likely that exploring the GCDFP-15/PIP-AZGP1 interactions will help better understand the mechanism of KC disease.
Highlights
Keratoconus (KC) is an ectatic eye disease associated with structural abnormalities in the cornea [1]
We have recently reported a 3D culture system for studying human keratoconus cells (HKC) [7,14]
We have reported findings that are in agreement with what is seen in vivo, such as increased oxidative stress levels in HKCs when compared to normal human corneal fibroblasts (HCF) [11]
Summary
Keratoconus (KC) is an ectatic eye disease associated with structural abnormalities in the cornea [1]. KC is characterized by a central or paracentral stroma thinning, corneal bulging, and scarring [1,2,3]. Other characteristics include the appearance of Fleischer’s ring, interruptions in Bowman’s layer, decreased keratocyte density, and severe changes in collagen gross organization [4]. KC typically appears in teens and progresses until the third or fourth decade of life [5]. The severity of the disease and how this is reached varies considerably between individuals. Vision quality is compromised and corneal transplantation is required in severe cases
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