Abstract
Abstract: :
Purpose: We have previously demonstrated an increase in hydraulic conductivity (HC) across Bruch's membrane from normal donor tissue following incubation with retinal pigment epithelial (RPE) cell derived matrix metalloproteinase-2 (MMP-2) or MMP-9. This study was therefore undertaken to investigate any similar effects of the metalloproteinases on the fluid transport of AMD affected Bruch's membrane, and identify potential substrates for the MMPs in this tissue. Methods: Matrix metalloproteinase-2 or -9 was collected in the media of cultured human RPE cells. Bruch's-choroid tissue was isolated from peripheral regions of AMD affected donors, and mounted in modified Ussing chambers, thereby isolating the Bruch's membrane surface from that of the choroid. Bruch's membrane was then exposed to either active MMP-2 or active MMP-9 for 20hrs at 37ºC. In control experiments, AMD tissue was incubated with latent MMPs. Hydraulic conductivity of each tissue was determined both before and after treatment, and concurrent tissue samples were collected for transmission electron microscopy analysis. Results: The baseline (control) hydraulic conductivity of AMD affected tissue was lower then that for normal tissue in agreement with previous reports. Both MMP-2 and MMP-9 significantly increased the HC of Bruch's membrane from AMD affected donor tissue, from control values of 2x10-10msec-1Pa-1 to 16 x10-10msec-1Pa-1 for MMP-2 and 13 x10-7msec-1Pa-1 for MMP-9. While the increase in HC following MMP-2 incubation was much higher in normal compared to AMD tissue, the increase in HC for MMP-9 treated samples was always higher in AMD tissue than in age-matched controls. On electron microscopy, only MMP-9 appeared to visually alter Bruch's membrane structure, demonstrating an apparent reduction in debris material in the collagenous layers. Conclusions: Matrix metalloproteinases have the ability to improve fluid transport across AMD affected Bruch's membrane, with MMP-9 having a more marked effect. These data may explain the limited success of sub-threshold laser grid treatments in AMD suffering patients, where some debris regression is observed following treatment, and we suggest a regime of repetitive treatment for effective long-term removal.
Keywords: 308 age-related macular degeneration • 333 Bruch's membrane • 399 enzymes/enzyme inhibitors