Purchase this article with an account.
J.S. Austin, G.M. Gordon, M.E. Fini; Global Expression Analysis of the MMP–TIMP Gene Families in Corneal Epithelial Regeneration . Invest. Ophthalmol. Vis. Sci. 2005;46(13):2128.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Purpose: Matrix metalloproteinases (MMPs) are key regulators of tissue remodeling. Expression of the MMP gelatinase B (gelB, MMP–9) is induced at the migrating epithelial front and blocking its activity by "knock–out" of the MMP–9 gene results in faster epithelial resurfacing (Mohan et al., JBC, 2002). In contrast, broad–spectrum, synthetic MMP inhibitors retard the rate of epithelial resurfacing, suggesting role for other MMPs. The purpose of this study was to identify candidate MMPs, as well as members of the TIMP family of MMP inhibitors, involved in epithelial regeneration in the mouse cornea by global gene expression analysis. Methods: Corneal debridement was performed by demarcating a 1mm circular region of the cornea with a trephine and removing the corneal epithelium in that region with an algebrush. The corneal stromal integrity was visualized and ensured by topically applying 2% Fluorescein Disodium. The migrating corneal epithelium was harvested 24 hours later by scraping with a scalpel dipped in TRIzol reagent (Invitrogen), and frozen at –80°C. The unwounded epithelium from the contralateral eye was also harvested for comparison. Total RNA was purified from both sets of tissues the next day. Quantitative RT–PCR was performed using SYBR green as a probe. The PCR conditions were as follows: one step of denaturation for 2 min at 95°C followed by 50 cycles of amplification at 95°C for 15 sec, 60°C for 1 min. Results: In our first set of experiments, seven MMPs were assayed: MMP–1a and 1b, MMP–2, MMP–3, MMP–7, MMP–8, and MMP–10. The following MMPs were up–regulated in migrating epithelium: MMP–1a (2.9 fold), MMP3 (3.4–fold), MMP–7 (2.8–fold) and MMP–10 (2.7–fold). The mRNA for MMP–1b and MMP–8 was detected only in the samples from migrating epithelium. We are in the process of assaying the remaining sixteen members of the MMP family, eight members of the related ADAM family, the four TIMPs, and the related inhibitor, RECK. Conclusions: These preliminary data identify several MMPs which may be involved in corneal epithelial regeneration. The role of these enzymes will be assayed using gene knock–out and knock–down methodologies.
This PDF is available to Subscribers Only