The Effect of Sodium Hyaluronate on the Expression of Gelatinases in Rabbit Corneal Epithelial Wound Healing
Introduction
Hyaluronan is an important component of the extracellular matrix and is thought to participate in migration, proliferation, and differentiation of cells in connective tissues.1, 2 Hyaluronan is also involved in the physiological function of cells in the cornea. It has been reported that sodium hyaluronate (Na-HA) enhanced epithelium migration,3 the proliferation of epithelial cells,4, 5 and the corneal epithelial wound healing in vivo.6, 7
Recently, it has been suggested that matrix metalloproteinases (MMPs) also play an important role during corneal wound healing.8 The MMPs from an enzyme family that shares some highly conserved domains and that has the activity of degrading various components of the extracellular matrix. The proteolytic activity of the family is inhibited by members of the tissue inhibitor of matrix metalloproteinases (TIMP) family. Moreover, the MMP family can be divided mainly into three main subfamilies: collagenases, stromelysins, and gelatinases. Gelatinases consist of 72 kDa type gelatinase (MMP-2) and 92 kDa type gelatinase (MMP-9), which can cleave gelatin and type IV, V, and VII collagens.9 There are reports of gelatinases in remodeling rabbit cornea; that is, MMP-2 was increased in the stroma and MMP-9 was detected in both the epithelium and the stroma. Consequently, it was assumed that MMP-2 participates in the prolonged process of collagen remodeling in the corneal stroma and the MMP-9 functions in controlling resynthesis of the subepithelial basement membrane.10 We expected participation of these gelatinases not only in such a remodeling but also in corneal epithelial cell migration.
Therefore, in order to understand the mechanism of the enhancement of corneal epithelial wound healing by Na-HA, we investigated the effect of Na-HA on the expression of gelatinases, MMP-2 and MMP-9, and their inhibitor, TIMP-1, in rabbit corneal epithelial wound healing.
Section snippets
Animals
Twenty-seven female Japanese white rabbits, weighing about 3.0 kg, were used in our experiments.
Test Substances
Sodium hyaluronate with a molecular weight of 64 × 104 was obtained from Seikagaku Corporation (Tokyo); it was purified from chicken combs and free from pyrogen. Sodium hyaluronate was dissolved with phosphate-buffered saline (PBS) at a concentration of 0.2% (w/v), and the solution was used as a test solution. Phosphate-buffered saline, a solvent of Na-HA, was used as a negative control solution.
Corneal Organ Culture
The
Rt-pcr
The MMP-2 and TIMP-1 mRNA were expressed constitutively in normal cornea and expressed stably at all measured times after corneal epithelial wounding in both eyes (Figures 1A,C). In order to confirm the reproducibility, the same experiments were independently carried out using the corneas derived from rabbits of a different group, and the results were the same. Equal intensities were observed in the PCR product for GAPDH at all measured times (Figure 1D), so that similar amounts of cDNA would
Discussion
The present study demonstrates the stable expression of MMP-2 and TIMP-1 mRNA and the temporal expression of MMP-9 mRNA during corneal epithelial wound healing. As shown in Figure 1, the level of MMP-9 mRNA expression showed a first peak at 6 to 24 hours and a second increase at 72 hours after the wounding. In the rabbit corneal epithelial defective model in this study, the healing rate of the corneal epithelium is almost constant and most of the wounded area is re-epithelialized for 72 hours
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