Proteomics Reveals Mechanisms of Delayed Keratoconus Progression: A Study of Corneas Following Two Light-Activated Crosslinking Treatments

PURPOSE: This study aims to elucidate on changes in biological pathways in rabbit corneas induced by two methods of light-activated corneal stiffening: topical application of riboflavin with dextran (RF-D) or WST11 with dextran (WST-D) followed by ultraviolet A (UVA) or near-infrared (NIR) illumination, respectively. METHODS: Rabbit corneas were mechanically de-epithelialized, then left untreated (N = 3) or treated with either RF-D/UVA (N = 3) or WST-D/NIR (N = 3). After one week, quantitative proteomics was performed on untreated, RF-D/UVA- and WST-D/NIR-treated corneas. Pathway enrichment analysis was performed to identify the biological processes associated with the treatments. To identify the abundance and spatial distribution of lipids in the untreated, WST-D/NIR- and RF-D/UVA-treated corneal stroma, lipid mass spectrometry imaging was performed together with hematoxylin and eosin staining. RESULTS: Between RF-D/UVA- and WST-D/NIR-treated corneas, 37 and 39 proteins, respectively, were differentially expressed compared to untreated corneas (P < 0.05). Pathway enrichment analysis showed the effect of RF-D/UVA treatment on cell metabolism and terminal differentiation of keratocytes, while WST-D/NIR modified extracellular matrix regulation and the mitogen-activated protein kinase signaling cascade. When comparing the RF-D/UVA and WST-D/NIR treatment, 74 proteins were differentially expressed, affecting cellular metabolism and respiration, complement activation, the activation of matrix metalloproteinases, and lipoprotein metabolism. The lipid profile for the RF-D/UVA- and WST-D/NIR-treated stromas were similar, whereas differences were observed comparing both treatments to untreated corneal stroma. CONCLUSIONS: Proteomics indicated a metabolic shift from oxidative phosphorylation to glycolysis and hypoxia after RF-D/UVA treatment. In contrast, WST-D/NIR stiffening maintained normal respiration and involved extracellular matrix remodeling.