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Proteomics Reveals Mechanisms of Delayed Keratoconus Progression: A Study of Corneas Following Two Light-Activated
Demi H J Vogels1,2, Jurriaan Brekelmans1,3, Ronny Mohren4
1University Eye Clinic Maastricht, Maastricht University Medical Center, Maastricht, the Netherlands.
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.
Insights
Riboflavin with dextran/UVA treatment shifts corneal metabolism to glycolysis, while WST11 with dextran/NIR maintains respiration and remodels the extracellular matrix. Both methods alter protein and lipid profiles in rabbit corneas.
Area of Science:
- Ophthalmology
- Biochemistry
- Proteomics
Background:
- Corneal stiffening procedures aim to enhance structural integrity.
- Light-activated crosslinking methods are emerging for corneal treatments.
Purpose of the Study:
- To investigate the biological pathway alterations in rabbit corneas following two light-activated stiffening methods.
- To compare the effects of riboflavin with dextran/UVA and WST11 with dextran/NIR treatments on corneal biology.
Main Methods:
- Rabbit corneas underwent de-epithelialization and were treated with either RF-D/UVA or WST-D/NIR.
- Quantitative proteomics and lipid mass spectrometry imaging were performed after one week.
- Pathway enrichment analysis identified affected biological processes.
Main Results:
- RF-D/UVA treatment impacted keratocyte metabolism and differentiation; WST-D/NIR affected extracellular matrix regulation and MAPK signaling.
- Significant protein expression differences were observed between treated and untreated corneas, and between the two treatment groups.
- Lipid profiles showed similarities between treatments but differed from untreated corneas.
Conclusions:
- RF-D/UVA treatment induced a metabolic shift towards glycolysis and hypoxia.
- WST-D/NIR treatment promoted extracellular matrix remodeling while preserving normal respiration.
- Both methods induce distinct molecular changes in corneal tissue.
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