Inhibition of matrix metalloproteases by a chemical cross-linker to halt the corneal degradation in keratoconus

Abstract

The need for better and simpler alternative crosslinking strategies to treat keratoconus (KC) is becoming essential as there is only a single approved way to treat it. Recently, conventional UV-A Riboflavin crosslinking is proven to have some disadvantages such as causing damage to the corneal endothelium and inducing keratocyte apoptosis. A chemical cross-linker (CXL) using carbodiimide chemistry and an octanedioic acid spacer is found effective in stiffening the cornea and has the potential to be developed as an alternative therapy to halt KC progression. In order to investigate the molecular changes induced by the cross-linker, we have analyzed the effect of the cross-linker on the activity of matrix metalloproteases (MMPs) in epithelial and stromal layers of KC corneas and in vitro cellular systems to determine its role in stiffening the KC cornea. At well-optimized concentration, KC corneal buttons were treated with the CXL and the stiffening of the cornea was measured. The collagen fibril assembly in the stroma was analyzed using transmission electron microscopy and the activity of MMPs 2 and 9 were visualized using gelatin zymography. KC corneal fibroblasts in culture and tumor necrosis factor-α (TNF-α) induced human corneal epithelial (HCE) cell line were treated with CXL and secretion of MMPs 1, 2, 3 and 9 were analyzed by enzyme-linked immunosorbent assay (ELISA). We found that the CXL stiffened the KC corneas comparable to the normal corneas, with very less cytotoxicity. The collagen fiber assembly was reorganized in an orderly fashion and fibril density and diameter increased after CXL treatment. The activity of MMPs and cathepsin G in the epithelial and stromal layers of KC tissues decreased post-treatment. Secretion and activity of MMPs from the corneal epithelial and stromal cells after CXL treatment were significantly reduced while the epithelial lysyl oxidase activity increased. The CXL, intended to stop the KC progression, modified the extracellular matrix collagen assembly in the stroma and decreased the secretion of a group of metalloproteases and their activity. We have demonstrated a set of molecular changes effected by the CXL, which might aid in the stiffening of the KC cornea.