Galectin-1 and -3 as prosprective biomarkers in keratoconus patients

Abstract

Purpose : To evaluate the expression of galectin-1 (Gal-1) and -3 (Gal-3) in the keratoconus (KC) diagnosed patients and riboflavin and ultraviolet light effect on human keratocytes cultivated in vitro.

Methods : Tear fluid and conjunctival impression cytology specimens from control and KC patients with and without atopy (n = 10/group) were used to evaluate Gal-1 and Gal-3 expression by immunofluorescence and ELISA. Primary human keratocytes were isolated by digestion in collagenase from surgically removed corneas of five normal human corneal buttons and cultured in DMEM/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 UVA light and riboflavin 0.1% for 30 minutes called cross-linking (CXL). After 24 hours of CXL, expression of Gal-1 and Gal-3 in the and their supernatants was performed using immunofluorescence and ELISA; multiplex assay was also done to detect inflammatory biomarkers in supernatants. Student’s t-test was used for statistical analysis.

Results : Atopic KC patients exhibited increased levels of Gal-1 in the tear fluids (14.5±3 ng/mL, p>0.05) and epithelial cells of bulbar conjunctiva (130±3 arbitrary units, p<0.001) compared to control (9.8±0.5 and 87±1.6, respectively). On the other hand, non-atopic KC patients exhibited increased levels of Gal-3 in the tear fluid (70±16 ng/mL, p<0.05), but decreased endogenous expression in conjunctival epithelial cells. In in vitro experiments, keratocytes exhibited intense nuclear Gal-1 expression while Gal-3 was localized especially in the cytoplasm and both were strongly diminished after 24 hours of CXL. Further, CXL induced significant release of Gal-1 in the cell supernatants (116±18 ng/mL, p<0.05) and decreased inflammatory biomarkers as IL-6, IL-8, MMP-2 and MMP-9. Gal-3 levels was not detected in the keratocyte supernatants

Conclusions : Gal-1 and Gal-3 represent new interesting biomarkers as revealed by their different expression patterns in atopic and non-atopic KC patients. CXL has immunosuppressive effect on keratocytes by reducing the release of cytokines and MMPs and increased anti-inflammatory protein Gal-1.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.