PURPOSE: Corneal complications are often associated with diabetes mellitus and can be vision threatening. Corneas in diabetic patients are exposed to increased glucose concentration despite cornea's avascular property, and this condition may contribute to the accumulation of advanced glycation end products (AGEs). The focus of this study was to examine the role of AGEs in the pathogenesis of diabetic keratopathy. METHODS: An anti-AGE monoclonal antibody (6D12), which recognizes a N(epsilon)-carboxymethyl lysine (CML)-protein adduct as an epitope, was prepared. Immunohistochemical localization of CML was examined in human age-matched diabetic and nondiabetic corneas (8 of each). In vitro, type I collagen-, type IV collagen-, or laminin-coated 96-well plates were glycated by glucose-phosphate. In some experiments, aminoguanidine was present in the incubation mixture. The amounts of CML-protein adducts in the extracellular matrix (ECM) were determined by enzyme-linked immunosorbent assay using 6D12. SV40-immortalized human corneal epithelial cells were seeded onto modified or unmodified ECM in 96-well plates and allowed to attach for 3 hours. Attached cells were fixed, and the areas of attached cells in each condition were measured. Attached cells without fixation were removed, and cell number was counted. RESULTS: In all of the 8 diabetic corneas, CML immunoreactivity was observed in the epithelial basement membrane, whereas CML immunoreactivity was not found in the corresponding area in 7 of 8 nondiabetic corneas. In vitro, nonenzymatic glycation of laminin on the culture dish attenuated adhesion and spreading of corneal epithelial cells. The presence of amninoguanidine in the incubation mixture during glycation inhibited CML formation and promoted the adhesion and spreading of corneal epithelial cells in a dose-dependent manner. CONCLUSIONS: The accumulation of AGEs on the basement membrane, particularly on laminin, may play a causative role in the corneal epithelial disorders of diabetic patients.
PURPOSE:Corneal complications are often associated with diabetes mellitus and can be vision threatening. Corneas in diabeticpatients are exposed to increased glucose concentration despite cornea's avascular property, and this condition may contribute to the accumulation of advanced glycation end products (AGEs). The focus of this study was to examine the role of AGEs in the pathogenesis of diabetic keratopathy. METHODS: An anti-AGE monoclonal antibody (6D12), which recognizes a N(epsilon)-carboxymethyl lysine (CML)-protein adduct as an epitope, was prepared. Immunohistochemical localization of CML was examined in human age-matched diabetic and nondiabetic corneas (8 of each). In vitro, type I collagen-, type IV collagen-, or laminin-coated 96-well plates were glycated by glucose-phosphate. In some experiments, aminoguanidine was present in the incubation mixture. The amounts of CML-protein adducts in the extracellular matrix (ECM) were determined by enzyme-linked immunosorbent assay using 6D12. SV40-immortalized human corneal epithelial cells were seeded onto modified or unmodified ECM in 96-well plates and allowed to attach for 3 hours. Attached cells were fixed, and the areas of attached cells in each condition were measured. Attached cells without fixation were removed, and cell number was counted. RESULTS: In all of the 8 diabetic corneas, CML immunoreactivity was observed in the epithelial basement membrane, whereas CML immunoreactivity was not found in the corresponding area in 7 of 8 nondiabetic corneas. In vitro, nonenzymatic glycation of laminin on the culture dish attenuated adhesion and spreading of corneal epithelial cells. The presence of amninoguanidine in the incubation mixture during glycation inhibited CML formation and promoted the adhesion and spreading of corneal epithelial cells in a dose-dependent manner. CONCLUSIONS: The accumulation of AGEs on the basement membrane, particularly on laminin, may play a causative role in the corneal epithelial disorders of diabeticpatients.
Authors: S Gadau; C Emanueli; S Van Linthout; G Graiani; M Todaro; M Meloni; I Campesi; G Invernici; F Spillmann; K Ward; P Madeddu Journal: Diabetologia Date: 2006-01-17 Impact factor: 10.122