Effects of an MMP Inhibitor, Ilomastat, on the Gene Expression Profile of Human Tenon’s Fibroblasts

Abstract

Purpose: : Matrix Metalloproteinases (MMPs) play a key role in wound healing. Following Glaucoma Filtration Surgery (GFS), Human Tenon’s Fibroblasts (HTFs) increase MMP production and these are extensively involved in scarring, which can lead to surgical failure. We have previously described the experimental use of an MMP inhibitor (MMPi), Ilomastat, to modulate wound healing. To further explore the effects induced by Ilomastat, we investigated its effect on the gene expression profile of HTFs in-vitro.

Methods: : HTFs were grown in the presence of either control media or Ilomastat (100 µM) for 72 hours. Total RNA derived from each treatment was subjected to microarray analysis using an Affymetrix Human Exon 1.0 ST array. LIMMA and PathVisio were used to perform data and pathway analysis. The Benjamini-Hochberg-corrected p-value cut off of 0.05 was used to select significantly different genes. Changes in selective gene expression were confirmed using RT-PCR. Ilomastat-induced effects on cell proliferation were evaluated using the MTT assay. Immunostaining of cells was undertaken to look at protein expression.

Results: : Microarray analysis of HTFs treated with Ilomastat revealed 1180 genes with changes in expression (p<0.05) with 36.2% of these upregulated and 63.8% downregulated. Pathways including cell cycle regulation and proliferation, interleukin signalling and focal adhesion were highly altered. Selective fold changes included TNC (↓4.26) MMP1 (↓3.97), IL-6(↓2.45) and FKBP5 (↑5.06). Cell proliferation was significantly reduced in the presence of Ilomastat (p<0.05).

Conclusions: : Ilomastat modulates the expression of genes encoding proteins known to have significant roles in the functional behaviour of HTFs. Although the key role of an MMPi is to inhibit MMPs, we have observed that Ilomastat may have a far greater effect on HTFs than the basic function of MMP inhibition. These may provide additional, significant, ways to optimise the inhibition of fibrosis and are currently being investigated.

Keywords: gene microarray • drug toxicity/drug effects • gene/expression