Effects of the MMP Inhibitor, GM6001, on TGF–ß– Induced Gene Expression in a Rat Subcapsular Cataract Model

Abstract

Abstract: : Purpose: We have shown that Matrix Metalloproteinase (MMP) activity is involved in anterior subcapsular cataract (ASC) formation in that co–treatment with the broad MMP inhibitor GM6001 suppresses TGF–ß–induced cataractous changes including epithelial to mesenchymal transition (EMT) of the lens epithelium (ARVO 2004; 1039). Here, we further examine the effects of the GM6001 inhibitor on mRNA expression of candidate genes involved in ASC and EMT. Methods: Rat lenses were cultured with TGF–ß, TGF–ß + GM6001, or left untreated (control) for six days. Cryostat sections of the lenses were then subjected to laser capture microdissection (LCM) to isolate cells from cataractous plaques and normal lens epithelium. Real–Time Quantitative PCR (RT–QPCR) was carried out to quantify changes in mRNA expression for candidate genes. Results: Following 6 days of treatment with TGF–ß distinct anterior subcapsular plaques had formed in the rat lens. Unlike the lens epithelium from untreated lenses, cells isolated from the plaques were found to express α smooth muscle actin (α–SMA) mRNA and substantially higher levels of MMP–9 mRNA. In contrast, E–cadherin mRNA expression was substantially suppressed in the plaque cells as compared to normal lens epithelium. Lenses co–treated with TGF–ß and GM6001 did not exhibit plaques, and the epithelium from these lenses exhibited a substantial decrease in α–SMA and MMP–9 mRNA levels approaching the levels of controls. Interestingly, the epithelium of GM6001 treated lenses expressed a 7–fold increase in E–cadherin mRNA expression over that of controls. Conclusions: Using LCM and RT–QPCR we have shown a differential expression of E–cadherin, αSMA, and MMP–9 mRNA in cells isolated from subcapsular plaques versus normal lens epithelium. The fact that co–treatment with GM6001 resulted in an elevation in E–Cadherin mRNA levels above that of controls suggests that GM6001 may stabilize E–cadherin and thereby protect lens epithelial cells from undergoing EMT.

Keywords: cataract • cytokines/chemokines • gene/expression