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S. Elliot, P. Catanuto, S. Cousins; Protection From Oxidant–mediated Loss of MMP–2 Activation in RPE Cells Requires Overexpression of Both MMP–14 and TIMP–2 . Invest. Ophthalmol. Vis. Sci. 2005;46(13):1001.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Age–related macular degeneration (AMD) demonstrates accumulation of specific lipid–rich deposits and extracellular matrix molecules under the retinal pigmented epithelium (RPE). Metalloproteinases (MMP) are crucial regulators of basement membrane and extracellular matrix turnover. Accordingly, loss of RPE MMP activity leads to excessive accumulation of collagen and other extracellular matrix, a potential mechanism for deposit formation. We have previously shown that MMP–2 activity, but not pro–MMP–2 protein, decreases following RPE oxidative injury, indicating that oxidant injury disrupts the enzymatic cleavage of pro–MMP–2. Activation of MMP–2 requires the formation of a trimolecular complex of pro–MMP–2, MMP–14 and Tissue inhibitor of metalloproteinases (TIMP) –2. Therefore we investigated the impact of oxidant injury on the interaction between these three molecules. Methods:Confluent cultures of 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. For over–expression studies either a plasmid containing full length MMP–14 or an adenoviral vector expressing TIMP–2 were used to transfect cells prior to injury. Results: Oxidant injury of control ARPE cells resulted in the decrease of both MMP–14 and TIMP–2 activity and protein. ARPE were successfully transfected with either MMP–14 and TIMP–2 as shown by Western blot analysis. Over–expresson of either MMP–14 and TIMP–2 alone prevented the injury–induced decrease of each observed in control cells. However, over–expression of each single molecule failed to prevent the injury–induced decrease of MMP–2 activity. On the other hand, over–expression of MMP–14 together with the addition of exogenous TIMP–2 did prevent the loss of MMP–2 activation. Conclusions:Loss of MMP–2 activity after oxidant injury is caused by downregulation of MMP–14 and TIMP–2. Over–expression of either MMP–14 or TIMP–2 alone prior to oxidant injury is not enough to prevent loss of MMP–2 activity. All three components of the trimolecular complex must be present to preserve normal MMP–2 activity after oxidant injury.
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