Abstract
Purpose:
Keratoconus (KC) is a degenerative disorder of the cornea where structural changes cause it to thin, protrude and assume a more conical shape. Prevalence of the disease ranges from 4-600/100,000 people and can result in severe vision loss. The exact cause is uncertain, and patients with advanced KC require surgery to maintain vision. One of the major clinical problems with treating the disease is there are no methods for early detection. Also, there currently are no models available to investigate and understand the root causes of the disease.
Methods:
In vitro, human corneal fibroblasts (HCF), keratocytes, and keratoconus cells (HKC) were isolated and cultured. Cells were stimulated with a stable Vitamin C (VitC) derivative for 4 weeks, allowing them to secrete a self-assembled matrix. In vivo, human tears were collected from healthy and KC individuals. All samples were processed for metabolomic and proteomic analyses using LC-MS/MS. In vitro samples were also processed for indirect-immunofluorescence and transmission electron microscopy.
Results:
We identified more than 250 different metabolites of which >50 were differentially regulated between groups. Two of them, Lactate and Arginine, have been previously linked to corneal edema and thinning. In vitro, Lactate levels were elevated 4 fold in HKCs when compared to keratocytes and 2 fold when compared to HCFs; however, Arginine levels were significantly reduced in both HCFs and HKCs as compared to keratocytes. In addition, Glutathione levels were reduced when compared to keratocytes, significantly in HCFs and 2 fold in HKCs. In vivo, these metabolites were regulated similarly, Lactate levels increased and Arginine and Glutathione decreased in KC patients. We also identified more than 200 proteins in human tears, some of which may serve as new KC defect markers (i.e. Gross cystic disease fluid protein 15 (GCDFP-15) decreased by 2 fold and Lactate dehydrogenase isozyme (LDH) was significantly elevated in KC patients). Critically, our proteomics and metabolomics agree and are cross-validating since LDH converts pyruvate to lactate.
Conclusions:
Overall, we have developed a novel in vitro model that allows for the study of proteins and metabolites, both in vitro and in vivo, which may help understand the root problem of KC and may allow for identification of markers of KC disease.
Keywords: 574 keratoconus •
663 proteomics •
519 extracellular matrix