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THE ROLE OF MATRIX METALLEOPROTEINASES (MMP) AND THEIR INHIBITORS (TIMP) IN AGE RELATED MACULAR DEGENERATION Age Related Macular Degeneration (AMD) is the most common cause of blindness in adults over 60. Drusen, a plaque like deposit found between Bruch's membrane (the inner layer of the choroid) and the Retinal Pigment Epithelium (RPE) is thought to contribute to the damage of photoreceptors in the macula, thus causing degeneration of eyesight. The purpose of this experiment is to explore the expression of matrix metalleoproteinases (MMPs) and tissue inhibitors of metalleoproteinases (TIMPs) in RPE cells. MMPs are enzymes that break down the Extra Cellular Matrix (ECM), promote angiogenisis (the formations of new blood vessels) and connective tissue turnover. TIMPs regulate ECM turnover and neovascularization by inhibiting MMPs. Under normal conditions TIMPs and MMPs are functionally balanced to maintain an ideal rate of ECM turnover and angiogenisis. When the functions of MMPs and TIMPs become unbalanced (an event thought to occur during the aging process) the result can be excess ECM digestion and neovascularization, resulting in a build up of ECM deposits. In the present study, we exposed human fetal RPE to oxidative stress (incubation in 100mM hydrogen peroxide) in order to simulate aspects of the aging process. The expression of MMP 1,-2,-3,-7,-9 and TIMP-1,-2,-3,-4 were monitored with and without oxidative stress using immunohistochemistry. Each TIMP and MMP was labeled with a primary antibody and a flourescently labeled secondary antibody. The results were analyzed under a confocal microscope. Out of all the proteins analyzed, TIMP -1,-2,-3,-4 and MMP -7 and -9 possessed visible and comparable staining in both the control and the H202 cells. All visibly labeled TIMPs and MMPs exhibited slightly less staining under oxidative stress. These results were inconsistent with previously collected Array data (except for TIMP-4, which showed a decrease under oxidative stress). Because of this inconsistency the next step in the project was to alter the incubation H202 periods to 6, 10, 24, and 48 hours. The stressed cells and growth media will then be analyzed by immunohistochemistry and the ELISA method (Enzyme-Linked Immunosorbent Assays). The ELISA will detect the proteins that have been excreted into the media, and therefore may not of appeared in the initial immunohistochemistry experiments. With this information we will be able to determine more precisely the cellular expression and secretion of TIMP?s and MMP?s under varying levels of oxidative stress.
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