Abstract
Purpose :
Keratoconus (KC) is a multifactorial, degenerative and progressive disease characterized by thinning, steepening and protrusion of the cornea. Although the origins are unknown, KC develops during puberty and progresses until the fourth decade of life. Prolactin-induced protein (PIP) is a 17kDa glycoprotein that has been deemed a potential KC biomarker. This study aimed to investigate the impact of PIP on the metabolic modulation of KC- and Healthy donor-derived corneal stromal cells.
Methods :
Human corneal fibroblasts from healthy (HCF) and keratoconus (HKC) donors were isolated and grown on polycarbonate membranes, supplemented with stable 0.5nM Vitamin C (3D in vitro model). Cells were further stimulated with PIP (50ng/ml, 100ng/ml, 200ng/ml, 350ng/ml or 500ng/ml) in EMEM media, supplemented with 10% FBS and 1% antibiotic-antimycotic solutions. Cultures with no PIP served as controls. At four weeks, all constructs were collected, metabolites were extracted, and processed for mass spectrometry (metabolomics).
Results :
We identified 265 metabolites, with 37 significantly modulated in HCFs (6 upregulated and 31 downregulated) and 39 in HKCs (3 upregulated and 36 downregulated). In HCFs, the top 5 most impacted by PIP metabolomic pathways were: Pantothenate and Coenzyme A biosynthesis, Warburg effect, purine, thiamine and pyrimidine metabolism. In HKCs, the top 5 pathways were: Warburg effect, pyruvate metabolism, purine metabolism, starch and sucrose metabolism, and citric acid cycle. Based on upregulated-only metabolites by PIP, the top 3 pathways in HCFs were: Oxidation of Branched Chain Fatty acids, Thiamine metabolism, and Beta Oxidation of Very Long Fatty Acids. In HKCs the top 3 were: Spermidine and Spermine Biosynthesis, Methionine Metabolism and Glycine and Serine Metabolism. Based on downregulated-only metabolites by PIP, the top 3 pathways for HCF were: purine metabolism, Warburg effect and pyrimidine metabolism; whereas HKCs showed: Warburg effect, pyruvate metabolism and purine metabolism.
Conclusions :
The present study revealed a significant impact on critical metabolomic pathways in both HCFs and HKCs. Interestingly, PIP appears to downregulate most of the metabolites tested in both cell types, consistent with the observed significance. Future studies may further unravel PIP’s role in KC cellular metabolism.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.