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.