The role of nonenzymatic glycation and carbonyls in collagen cross-linking for the treatment of keratoconus

Abstract

Purpose: Corneal cross-linking (CXL) is a treatment for keratoconus that eliminates the need for keratoplasty in most patients. However, its molecular mechanisms remain under study. Advanced glycation end products (AGEs) have been suggested by many studies as the causative strengthening agent during CXL, though no studies to date have directly tested this hypothesis.

Methods: Corneas of young rabbits and sharks were pretreated with pyridoxal hydrochloride and copper ions before CXL. Two known inhibitors of AGE formation, aminoguanidine and rifampicin, were applied during CXL in the treatment solution. Tensile strength tests were conducted after these experiments to detect diminished or accentuated corneal stiffening after CXL. SDS-PAGE was performed on type I collagen cross-linked in the absence and presence of AGE inhibitors.

Results: Pretreatment with pyridoxal hydrochloride resulted in significantly higher corneal stiffening after CXL. AGE inhibitors significantly diminished cross-linking as detected by both tensile strength measurements using whole corneas and gel electrophoresis of in vitro cross-linking of type I collagen in solution, in the presence and absence of the inhibitors. Rifampicin inhibited CXL more significantly than aminoguanidine in gel electrophoresis and tensile strength tests, confirming recent findings on its efficacy as an AGE inhibitor.

Conclusions: Data presented here suggest that CXL is carbonyl dependent and involves the formation of AGE cross-links. Six possible cross-linking mechanisms are discussed.

Figures

Figure 1.

Results of histochemical staining for the presence of carbonyl groups with Brady's reagent containing 2,4-DNP in rabbit corneas. Rinsing time in 1× PBS is displayed in upper left corners of the boxes. A positive stain for carbonyl groups is the presence of red-violet color complexes in the tissue. Complete elution of bound pyridoxal was seen after 4 hours of rinsing. n = 3.

Figure 2.

Treatments are described on the x-axes. Pyridoxal-hydrochloride (PL HCl) and pyridoxal phosphate (PLP) denote pretreatment with that chemical. Tensile strength of 20 mM PL HCl solution-pretreated and control rabbit corneas at pH 7.4. Pretreatment with PL HCl gave 156% increases in strength over CXL treatment in rabbits and 131% in sharks. Corneas were cut into 2-mm-wide strips after the final treatment step and were mounted in the apparatus for determining tissue strength. Controls were not statistically different; n = 8 corneal strips (4 corneas) per group. Error bars are expressed as SEM.

Figure 3.

Tensile strengths of rabbit and shark corneas undergoing CXL in the presence of the AGE inhibitors Rif and AG. Treatments are described on the x-axis. Rifampicin shows greater ability to suppress cross-linking than AG in both sharks and rabbits; n = 8 corneal strips (4 corneas) per group. Error bars are expressed as SEM.

Figure 4.

Electrophoretic analysis of AGE inhibitors. 9 μg collagen/lane. Lane 1, molecular weight marker; lane 2, collagen alone; lane 3, collagen + CXL; lane 4, collagen + CXL + AG; lane 5, collagen + CXL + AG + 3 mM glucose; lane 6, collagen + CXL + Rif; lane 7, collagen + CXL + Rif + 3 mM glucose; lane 8, collagen + CXL + 3 mM glucose.

Figure 5.

Diagram of proposed cross-linking mechanisms at the molecular level in the corneal stroma. Top: stromal structure before singlet-oxygen generation; bottom: six proposed chemical modifications to the stroma. Star: where singlet-oxygen reacts; Dotted line: possible places where modification could react. The six possible mechanisms are as follows: modification of sugar residue on GAG chain, which then reacts with a collagen molecule (1A) or a proteoglycan core protein (1B); modification of amino acid of a proteoglycan core protein, which then reacts with collagen (2A) or an adjacent proteoglycan core protein (2B); modification of an amino acid of a collagen molecule, which then reacts with a proteoglycan core protein (3A) or an adjacent collagen molecule (3B).