Aims To evaluate the expression of β-galactoside-binding proteins galectin (Gal)-1 and Gal-3 in patients with keratoconus (KC) and postcorneal collagen cross-linking (CXL) treatment in vitro.
Methods Tear fluid, cornea samples and conjunctival impression cytology specimens from control and KC patients were used to evaluate Gal-1 and Gal-3 expressions. Primary keratocytes were isolated by collagenase digestion from surgically removed corneas of five normal or KC human corneal buttons and cultured in Dulbecco’s modified eagle medium/Ham’s F12 medium supplemented with 2% fetal bovine serum. These cells were evaluated under two experimental conditions: control and submitted to the application of ultraviolet A light and riboflavin 0.1% (CXL) for 30 min.
Results Patients with KC displayed increased levels of Gal-1 and Gal-3 in conjunctival epithelial cells compared with control. Furthermore, KC corneas were associated with intense expression of Gal-1 in the stroma, released by keratocytes. Ultrastructural analysis of keratocytes showed a marked increase of endogenous Gal-3 levels, but not Gal-1, in KC. In vitro, CXL induced significant release of Gal-1 in keratocyte supernatants (116±18 ng/mL, P<0.05) and decreased inflammatory biomarkers as interleukin (IL)-6, IL-8, matrix metalloproteinase (MMP)-2 and MMP-9. Gal-3 levels were not detected in the keratocyte supernatants.
Conclusion Gal-1 and Gal-3 represent new interesting KC biomarkers as revealed by their different expression patterns in KC and control corneal samples. CXL has an immunosuppressive effect on keratocytes by reducing the release of cytokines and MMPs and increased expression of anti-inflammatory protein Gal-1.
- impression cytology
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Contributors FECA and JLC performed the experiments, contributed equally to this paper and are considered cofirst authors. FECA, JLC, LR, RMH, MSdS, MC and JÁPG contributed to the sample collection and data analysis/interpretation. CDG conceived the study and wrote the manuscript. All authors have been reviewed and approved the final version of manuscript.
Funding The authors would like to thank Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP – Grant 2015/09858-3). FECA and JLC were PhD fellows from Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES).
Competing interests None declared.
Patient consent Obtained.
Ethics approval Ethics approval was obtained from the Federal University of São Paulo Ethics Committee (CEP number 0586/2015).
Provenance and peer review Not commissioned; externally peer reviewed.