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Y. Lee, A. A. Hussain, J. Marshall; Compositional Analyses and Age-Related Changes of High Molecular Weight Gelatinases of Human Bruch’s Membrane and Their Changes After Exposure to EGTA. Invest. Ophthalmol. Vis. Sci. 2010;51(13):5959.
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The structural and functional changes of ageing Bruch’s membrane is associated with accumulation of inactive matrix metalloproteinase (MMP) and reduction of active forms. The gelatinases of the MMP degradation system comprises MMPs 2 and 9, and two high molecular weight species (HMW 1 and 2) have not characterised of their role with age-related changes. This study determines age-related changes of HMW species and characterise the effect of metal chelation with EGTA on them.
Gelatinse zymography is followed by densitometric scanning to quantify the HMW species. Gel filtration chromatography fractionated the MMPs by their molecular weights. To assess the changes of HMW with metal chelation, human Bruch’s choroid preparations were mounted in Ussing chambers and perfused with phosphate-buffered saline (PBS) at 300mm H2O. After 6-8 hours, the perfusate was switched to 10mM EGTA and eluted fractions collected for timed intervals.
The substantial amount of HMW 1 and 2 (80 and 87% respectively) was bound to the matrix. Ageing was related with a significant changes of HMW 1 and 2 (P <0.005 and P<0.05 respectively). One single large macromolecular MMP complex (LMMC) contains HMW 1 and 2, MMP9 and MMP2 on gel filtration. Activation of HMW 1 and 2 shows their composition of hetero-polymers of MMP 2 and 9.
The age-related increase of HMW species and formation of LMMC results in sequestration of MMPs and causes reduction of free pool for activation. Exposure to the heavy metal chelator EGTA changes the level of HMW species. Decrease of HMW species of the free and bound pool by metal chelation may promote activation of MMP degradation systems in the ageing Bruch’s membrane.
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