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Y. Lee, A. A. Hussain, J. Marshall; Divalent Metal Ion Chelation is Associated With the Activation and Release of Matrix Metalloproteinase-2 From Human Bruch’s Membrane. Invest. Ophthalmol. Vis. Sci. 2009;50(13):3401.
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Ageing of Bruch’s membrane is associated with accumulation of inactive matrix metalloproteinase enzymes (Pro-MMPs 2&9) together with marked reduction of active forms. These changes are thought to diminish the rejuvenation potential of Bruch’s resulting in an age-related loss in the transport properties of the membrane. This study was designed to assess the role of divalent metal ions on the sequestration and activation status of the MMP system of Bruch’s membrane.
Donor human Bruch’s-choroid preparations were mounted Ussing chambers and perfused from the choroidal side with PBS at a hydrostatic pressure of 300mm H2O and eluted fractions collected at timed intervals. After 6-8 hours, the perfusate was switched to 10mM EGTA and eluted fractions collected for another 2 hours. At the end of the experiment, the exposed 6mm diameter of Bruch’s-choroid was removed. Tissue samples and eluted fractions were subjected to gelatine zymography and MMPs 2&9 quantified by densitometric analyses.
The releasable pool of MMPs was completely eluted from Bruch’s within 6-8 hours of perfusion with PBS. The level of active MMP-2 within the eluted fractions (as a percentage of total MMP-2 activity) was determined to be 12.6 ± 7.4 % (Mean ± SD, 12 donors, age range 28-84 years). Similarly, in the bound/sequestered pool within the membrane, the level of active MMP-2 was 13.0 ± 8.2 %. Exposure to EGTA was associated with further release of MMP-2 from the membrane and the percentage of active MMP-2 in the eluant was 69.6 ± 6.9 % and that in the tissue fraction 66.5 ± 17%. EGTA did not lead to further release of MMP-9 and was without effect on the active/inactive ratio for this MMP.
Removal of divalent metal ions from Bruch’s is associated with marked activation of MMP-2 and some release from the bound pool within the membrane. The age-related increased deposition of heavy metals in Bruch’s membrane may promote greater sequestration of MMP-2 species and may further disturb the activation kinetics of the MMP system. These results have a bearing on the progression of age-related macular degeneration.
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