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Updated: May 1, 2026
Assessment of Oxidative Damage in the Primary Mouse Ocular Surface Cells/Stem Cells in Response to Ultraviolet-C UV-C Damage
Published on: February 15, 2020
In vitro model suggests oxidative stress involved in keratoconus disease
D Karamichos1, A E K Hutcheon2, C B Rich3
1Department of Ophthalmology, University of Oklahoma Health Sciences Center, USA.
Abstract:
Keratoconus (KC) affects 1:2000 people and is a disorder where cornea thins and assumes a conical shape. Advanced KC requires surgery to maintain vision. The role of oxidative stress in KC remains unclear. We aimed to identify oxidative stress levels between human corneal keratocytes (HCKs), fibroblasts (HCFs) and keratoconus cells (HKCs). Cells were cultured in 2D and 3D systems. Vitamin C (VitC) and TGF-β3 (T3) were used for 4 weeks to stimulate self-assembled extracellular matrix (ECM). No T3 used as controls. Samples were analyzed using qRT-PCR and metabolomics. qRT-PCR data showed low levels of collagen I and V, as well as keratocan for HKCs, indicating differentiation to a myofibroblast phenotype. Collagen type III, a marker for fibrosis, was up regulated in HKCs. We robustly detected more than 150 metabolites of the targeted 250 by LC-MS/MS per condition and among those metabolites several were related to oxidative stress. Lactate levels, lactate/malate and lactate/pyruvate ratios were elevated in HKCs, while arginine and glutathione/oxidized glutathione ratio were reduced. Similar patterns found in both 2D and 3D. Our data shows that fibroblasts exhibit enhanced oxidative stress compared to keratocytes. Furthermore the HKC cells exhibit the greatest level suggesting they may have a myofibroblast phenotype.
Insights
Oxidative stress is elevated in keratoconus cells (HKCs), suggesting a myofibroblast phenotype. This finding may clarify the role of oxidative stress in keratoconus progression and disease mechanisms.
Area of Science:
- Ophthalmology
- Cell Biology
- Biochemistry
Background:
- Keratoconus (KC) is a corneal disorder leading to thinning and a conical shape, often requiring surgery.
- The specific role of oxidative stress in the pathogenesis of keratoconus remains incompletely understood.
- Understanding cellular differences in oxidative stress is crucial for developing targeted therapies.
Purpose of the Study:
- To investigate and compare oxidative stress levels in human corneal keratocytes (HCKs), human corneal fibroblasts (HCFs), and keratoconus cells (HKCs).
- To analyze the impact of extracellular matrix (ECM) stimulation on oxidative stress markers.
- To explore potential phenotypic differences between HCKs, HCFs, and HKCs.
Main Methods:
- Cell culture in 2D and 3D systems with Vitamin C and TGF-β3 stimulation.
- Analysis of gene expression using qRT-PCR for collagen types and keratocan.
- Metabolomic profiling using LC-MS/MS to quantify over 150 metabolites, including oxidative stress markers.
Main Results:
- HKCs showed reduced collagen I, V, and keratocan, alongside upregulated collagen type III, indicating a myofibroblast phenotype.
- Elevated lactate levels, lactate/malate, and lactate/pyruvate ratios were observed in HKCs.
- Reduced arginine and glutathione/oxidized glutathione ratios were detected in HKCs, consistent with increased oxidative stress.
Conclusions:
- Fibroblasts exhibit higher oxidative stress compared to keratocytes.
- Keratoconus cells (HKCs) display the most significant oxidative stress, supporting a potential myofibroblast phenotype.
- These findings highlight the role of oxidative stress in keratoconus pathogenesis and suggest a distinct cellular phenotype in affected corneas.
