Upregulation of Mmp-2 and -9 Release by Human Rpe Cells Mediates Bruch’s Membrane Permeability

Abstract

Purpose: : The Aging retina is characterized by accumulation of various lipid-rich extracellular matrix (ECM) deposits within Bruch’s membrane. Such changes interfere with the transport functions of Bruch’s membrane for the delivery of nutrients and removal of waste products to and from the RPE and retina. The production of active forms of matrix metalloproteinases (MMPs) mediates the degradation of abnormal collagens in aged and AMD affected Bruch’s and thereby rejuvenate the membrane and improve its transport characteristics. The purpose of the current study is therefore to assess the potential impacts of upregulation of MMPs release on improvement of Bruch’s membrane permeability.

Methods: : Human Bruch’s-Choroid (hBC) membranes and human RPE-Bruch’s-Choroid (RPE-BC) explants were clamped in our tailor-made organ culture system and maintained in DMEM/ Ham’s F-12, supplemented with 1% FCS at physiological conditions for 1-3 days to allow equilibration. The hRBC explants were then irradiated with Ellex 2RT nanosecond laser system. Human RPE cells from primary cultures were introduced onto the hBC explants. At different time intervals the presence of MMPs in the bathing medium was determined before and after cells seeding and laser treatment by standard gelatine zymography and densitometric analyses. Permeability was studied by using the organ culture system described above with a dye or a labeled dextran tracer. The rate of diffusion across the hBC preparations was determined by using standard colorimetric analysis.

Results: : A significant upregulation of activated MMPs 2&9 was observed following introduction of human primary RPE cells on the hBC explants. Transport studies show that the permeability of the hBC membranes to the labeled tracer was dramatically increased from 0.08±0.021 (OD₅₈₅ ) at 0 day (pre-seeding) to 0.43±0.015 (OD₅₈₅) at 3 days and 0.63±0.025 (OD₅₈₅) at 7 days post-seeding, a 4.98 folds and 7.33 folds increases, respectively (Mean ±SEM, p<0.001, n=4) following the growth of RPE cells on the hBC membrane.A similar pattern was observed on RPE-BC explants with 2RT laser injuries

Conclusions: : Enhancing the synthesis and release of active forms of MMPs 2&9 from RPE cells and entry of such active species into Bruch’s allows for improvement in the transport characteristics of the membrane

Keywords: aging • extracellular matrix • enzymes/enzyme inhibitors