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Yuichi Kaji, Tomohiko Usui, Tetsuro Oshika, Masao Matsubara, Hidetoshi Yamashita, Makoto Araie, Toshinori Murata, Tatsuro Ishibashi, Ryoji Nagai, Seikoh Horiuchi, Shiro Amano; Advanced Glycation End Products in Diabetic Corneas. Invest. Ophthalmol. Vis. Sci. 2000;41(2):362-368.
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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
Nε-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-6-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
aminoguanidine 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.
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