Matrix metalloproteinase activity is enhanced during corneal wound repair in high glucose condition

Abstract

Purpose. (1) To investigate the effect of elevated extracellular glucose on migration, proliferation, and the activity of matrix metalloproteinases (MMPs) of SV40-transformed human corneal epithelial cells (HCEC). (2) To examine MMP activity in wounded corneal epithelium in diabetic rats. Methods. HCEC were cultured in media containing 5.5 mM or 31.2 mM D-glucose, or in a combination of 5.5 mM D-glucose and 25.7 mM D-mannitol on fibronectin/collagen I-coated 48-well plates. After reaching confluence (day 0), cells in the central part of the plate were wounded and the residual cells were cultured for 3 days. Migration and proliferation were evaluated by assessing the increasing amount of area covered by cells, and the day-3 to day-0 ratio of DNA levels, respectively. To determine MMP activity, cells were reacted with synthetic fluorogenic substrates specific to MMPs 1, 2, 3, 7, 9, and MMP activity was determined by a fluorometric kinetic assay. Diabetic rats were induced by streptozotocin injection. Corneal epithelium was scraped from limbus-to-limbus and allowed to heal. Normal rats were treated similarly to serve as controls. Healing epithelium was collected 24 hours later, and gelatin zymography was performed. Results. In the cell culture study, migration in 31.2 mM glucose was significantly slower than that in 5.5 mM, but proliferation in each concentration was similar. The osmotic effect of D-mannitol did not alter migration or proliferation. MMP activity in 31.2 mM was significantly higher than that in 5.5 mM. Zymography revealed enhanced activity of pro and active MMP-9 in healing corneal epithelium in diabetic rats. Conclusions. MMP activity was enhanced in healing corneal epithelium, both in in vitro and in vivo diabetic models, suggesting its involvement in diabetic keratopathy.