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Age-dependent variation in metalloproteinase activity of isolated human Bruch's membrane and choroid
1Department of Ophthalmology, the Guy's, King's and St. Thomas' Hospitals Medical and Dental School, King's College London, United Kingdom. l.guo@umds.ac.uk
Purpose:
To characterize and determine the effect of aging on the matrix metalloproteinase (MMP) component of the extracellular matrix-remodeling mechanism of isolated human Bruch's-choroid.
Methods:
Immunohistochemical techniques and western blot analysis were used to detect and localize various members of the MMP family of proteolytic enzymes in the Bruch's-choroid complex. Gelatin substrate zymography was used to detect and quantify the levels of MMP-2 and -9 in homogenates of Bruch's-choroid from both macular and peripheral regions of the human fundus. Aging alterations in these enzymes were quantified by densitometric analysis of photographic negatives of the zymography gels.
Results:
Intact preparations of Bruch's-choroid showed the presence of inactive forms of two gelatinases (MMP-2, 65 kDa, and MMP-9, 92 kDa), interstitial collagenase (MMP-1, 52 kDa) and stromelysin (MMP-3, 57 kDa). MMP-1 and -3 were localized primarily to Bruch's membrane. MMP-9 was distributed evenly in Bruch's membrane with some patchy presence in the choroidal mass. Distribution of MMP-2 was similar to that of MMP-9, but the staining in Bruch's was much fainter. On gelatin zymography, an active form of MMP-2 (58-kDa species) was frequently observed in peripheral samples but only occasionally in macular regions. The levels of MMP-2 and -9 increased with aging in both the macular and the peripheral regions of the fundus (P < 0.05). MMP-2 levels were lower in macular regions than in the periphery but no such variation was observed with MMP-9. Both these inactive gelatinases could be activated in vitro.
Conclusions:
A matrix-degrading mechanism essential for extracellular remodeling was shown to be present in Bruch's membrane. In macular regions, increasing levels of inactive forms of metalloproteinase and scarcity of active forms of MMP-2 suggests possible involvement of impaired extracellular degradation in both aging and macular degeneration.
Insights
Aging increases matrix metalloproteinases (MMPs) in the Bruch's-choroid, potentially impairing extracellular degradation. This suggests a role for MMPs in aging and macular degeneration.
Area of Science:
- Ophthalmology
- Biochemistry
- Extracellular Matrix Biology
Background:
- The Bruch's-choroid complex is crucial for retinal health and is implicated in age-related diseases.
- Matrix metalloproteinases (MMPs) are key enzymes involved in extracellular matrix remodeling.
Purpose of the Study:
- To characterize matrix metalloproteinases (MMPs) in human Bruch's-choroid.
- To investigate the impact of aging on MMP levels and activity in the Bruch's-choroid.
Main Methods:
- Immunohistochemistry and Western blot to detect and localize MMPs.
- Gelatin zymography to quantify MMP-2 and MMP-9 levels.
- Densitometric analysis to assess age-related changes in MMPs.
Main Results:
- Inactive forms of MMP-2, MMP-9, MMP-1, and MMP-3 were detected in Bruch's-choroid.
- MMP-2 and MMP-9 levels significantly increased with aging in both macular and peripheral regions.
- Active MMP-2 was more prevalent in peripheral samples than macular samples.
Conclusions:
- A matrix-degrading mechanism involving MMPs is present in Bruch's membrane.
- Increased inactive MMPs and reduced active MMP-2 in macular regions suggest impaired extracellular degradation in aging and macular degeneration.