Transient oxidative injury decreases MMP–14 and TIMP–2 resulting in decreased MMP–2 activity and extracellular matrix accumulation.

Abstract

Abstract: : Purpose:Age–related macular degeneration (AMD) demonstrates accumulation of specific lipid–rich deposits and extracellular matrix molecules under the retinal pigmented epithelium (RPE). Oxidant–mediated cellular injury and dysregulation of extracellular matrix (ECM) turnover by injured cells contributes to many age–related degenerative disorders. Accordingly, oxidant injury to the RPE has been implicated as a mechanism in AMD, although dysregulated turnover of Bruch’s membrane (BrM) by matrix metalloproteinases (e.g., MMP–2) produced by injured RPE has not been explored. We have previously shown that MMP–2 activity decreases following RPE oxidative injury. MMP–14 is a major activator of pro–MMP–2, requiring the formation of a trimolecular complex between of MMP–2, MMP–14, and TIMP–2. In this study we investigated if the oxidant–mediated decrease in MMP–2 activity is caused by injury–induced altered expression of MMP–14 and TIMP–2 , and to determine if the change is due to transcriptional and/or translational mechanisms. Methods:Confluent cultures of the human ARPE–19 cell line were oxidant injured by transient exposure to H₂O₂ and myeloperoxidase. Cells were collected for mRNA and protein, and 100ug of protein extract was immunoprecipitated for western analysis of MMP–2 and MMP–14. Supernatant from injured cells was utilized in activity assays for MMP–14. Zymography and reverse zymography were performed for anlaysis of MMP–2 and TIMP–2 respectively. Results:Western blotting indicated that both MMP–14 and TIMP–2 protein were expressed in RPE–19 cells and significantly decreased following oxidant injury. Both MMP–14 and TIMP–2 activity decreased athough the maximum decrease was at 20 hours for TIMP–2 and 48 hours for MMP–14. MMP–2 activity also decreased in a coordinate manner although MMP–2 protein did not. mRNA expression of MMP–14, TIMP–2 and MMP–2 was decreased following injury and rapidly recovered. Conclusions:Exposure of RPE–19 cells to transient injury decreases the key molecules required for MMP–2 activation. MMP–2 protein levels are not altered, suggesting that the decrease in the molecules required for MMP–2 activation causes decreased activity. This mechanism may have important implications for ECM accumulation seen in early dry AMD.

Keywords: retina • Bruch's membrane