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A.A. Hussain, L. Guo, J. Marshall; Identification of a Matrix Metalloproteinase (MMP)-based Survelliance System for Maintenance of Structural Integrity of Ageing Human Bruch's Membrane . Invest. Ophthalmol. Vis. Sci. 2003;44(13):4227.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Ageing of Bruch's membrane is associated with a drastic reduction in its capacity for nutritional and waste transport. Underlying mechanisms for reduced porosity include deposition of debris, oxidative damage, and greater cross-linking due to both disulphide bridging and formation of advanced glycation end-products (AGE's). MMP's have been identified within Bruch's and constitute the degradative arm of a remodelling process for maintaining the structural integrity of the membrane. Having demonstrated release of these enzymes by the retinal pigment epithelium (RPE), the present investigation was designed to (a) determine the molecular weight exclusion limit (MWEL) of Bruch's membrane and hence the penetration potential of MMP's and (b) to evaluate the dynamics of the MMP-surveillance process through Bruch's membrane. Methods: MWEL was determined by measuring the rate of diffusion of various FITC-dextrans (4.4-150kDa) across the Bruch's-choroid complex held in standard Ussing-type chambers. MMP dynamics were determined by quantifying the rate of release of endogenous MMP's 2&9 following application of a hydrostatic pressure head to Bruch's surface whilst the preparation was held in an open modified Ussing chamber. The ensuing fluid transport across the preparation was collected at timed intervals onto pre-weighed filter paper discs and following elution with Tris-HCl buffer, the amount of released MMP's was quantified by gelatin zymography. Results: The MWEL of Bruch's membrane was determined to be 70 ± 20 kDa (mean ± 1 S.D) and remained invariant with ageing of donor. The elution profile for MMP's 2&9 when plotted as amount against elution volume showed sigmoidal characteristics with 98% removal of the endogenous pool of these enzymes. X-50, representing the elution volume required to remove 50% of endogenous MMP's was determined to be 15 ± 0.5µl per 4mm disc of membrane. Conclusions: The MWEL showed that MMP's 2&9 were capable of diffusing freely across ageing Bruch's membrane. Surprisingly, the elution profiles showed that these MMP's were not bound to components of Bruch's membrane but after release from the RPE performed a surveillance function with a turnover in the endogenous pool every 22 ± 5 hours. The relevance of this MMP-surveillance system in maintaining structural and functional competence of both normally aged and age-related macular degeneration (AMD) affected Bruch's will be discussed.
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