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A. Kumar, A. El-Osta, A. A. Hussain, J. Marshall; The Relative Distribution of Free and Bound Matrix Metalloproteinase (MMPs) Enzymes in Human Bruch’s Membrane. Invest. Ophthalmol. Vis. Sci. 2008;49(13):4535.
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Matrix metalloproteinases (MMPs), released by the retinal pigment epithelium (RPE) enter Bruch’s to form the degradative arm of a remodeling process for maintaining the structural integrity of the membrane. However, ageing is associated with accumulation of inactive MMPs and the increased thickness of Bruch’s suggests impaired degradation and remodeling. The present investigation was designed to assess the possibility that these MMP enzymes may become sequestered and therefore unable to carry out their functional role (activation and degradation) leading to the observed ageing of Bruch’s membrane.
Freshly isolated Bruch’s-choroid preparations (14 eyes, age range 45-86 years) were mounted in Ussing chambers and MMP elution was instigated by application of a hydrostatic pressure of 14-16mmHg. The ensuing fluid transported across the preparation was collected at timed intervals over a 6-8 hour period and the amount of released MMPs quantified by gelatin zymography and densitometry. At the end of the experiment, the bound/trapped MMP pool was extracted with SDS-sample buffer and quantified.
Most of the free MMP pool (MMPs 2&9) could be eluted from Bruch’s within 5-6 hours representing an elution volume of 350+/-80ul. Surprisingly, 60-80% of the total endogenous pool of MMPs remained bound to the matrix and could only be removed by harsh SDS extraction. Furthermore, polymeric forms of MMPs 2&9 (indicative of oxidative damage and cross-linking) were routinely observed in the membrane extracts.
The high degree of sequestration and/or entrapment of MMPs may explain the reduction in degradation capacity of ageing Bruch’s membrane leading to the accumulation of denatured collagens. The relevance of this finding to functional deterioration of Bruch’s in normal ageing and age-related diseases will be discussed.
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