IOVS Journal of Clinical Investigation
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(Investigative Ophthalmology and Visual Science. 2005;46:3331-3340.)
© 2005 by The Association for Research in Vision and Ophthalmology, Inc.
DOI:  10.1167/iovs.04-1224
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Nonlethal Oxidant Injury to Human Retinal Pigment Epithelium Cells Causes Cell Membrane Blebbing but Decreased MMP-2 Activity

Maria E. Marin-Castaño,1 Karl G. Csaky,2 and Scott W. Cousins1

1From the Bascom Palmer Eye Institute, Department of Ophthalmology, University of Miami School of Medicine, Miami, Florida; and the 2Laboratory of Immunology, National Eye Institute, Bethesda, Maryland.

PURPOSE. This study was undertaken to determine whether transient or sustained nonlethal oxidant injury can induce RPE cell membrane blebbing and alter RPE expression of matrix metalloproteinase (MMP)-2 and type IV collagen, two molecules that are necessary for regulation of the turnover of the RPE basal lamina.

METHODS. The ARPE-19 cell line stably expressing green fluorescent protein (GFP) targeted to the cell membrane was bleb injured by exposure to myeloperoxidase (MPO; 10 microunits) and H2O2 (100 µM). Sustained (>6 hours) or transient (up to 6 hours) exposure to MPO/H2O2 was evaluated. An MTS assay conversion and cell counts were used to detect cell viability. Supernatants and the cell homogenates were collected from cultured ARPE-19 to assess fluorescent GFP-derived blebs, MMP-2 protein by Western blot, MMP-2 activity by zymography, and type IV collagen accumulation by ELISA. Expression of MMP-2 was examined by real-time RT-PCR with total RNA.

RESULTS. Both sustained and transient exposure of RPE cells to nonlethal oxidant injury upregulated blebbing and increased pro-MMP2 protein, but downregulated the MMP-2 activity released into the supernatant in a time-dependent manner. Only sustained oxidant injury for 24 hours induced an increase in collagen type IV. After removal of transient oxidant exposure, blebbing resolved and RPE MMP-2 activity and protein recovered to normal levels within 48 hours.

CONCLUSIONS. Sustained or transient oxidant injury causes increased cell membrane blebbing but decreased activation of MMP-2. The findings lead to the hypothesis that blebs released in the absence of active MMP-2 may become trapped between the RPE and its basal lamina as sub-RPE deposits, possibly contributing to drusen formation in age-related macular degeneration. Also, the results lead to the postulation that oxidant injury disrupts the cell-specific surface proteases necessary to cleave and activate pro-MMP-2.




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