Nonlethal oxidant injury to human retinal pigment epithelium cells causes cell membrane blebbing but decreased MMP-2 activity.
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 H(2)O(2) (100 microM). Sustained (>6 hours) or transient (up to 6 hours) exposure to MPO/H(2)O(2) 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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Related Subject Headings
- Up-Regulation
- Transfection
- Time Factors
- Reverse Transcriptase Polymerase Chain Reaction
- RNA, Messenger
- Pigment Epithelium of Eye
- Peroxidase
- Oxidants
- Ophthalmology & Optometry
- Matrix Metalloproteinase 2
Citation
Published In
DOI
ISSN
Publication Date
Volume
Issue
Start / End Page
Location
Related Subject Headings
- Up-Regulation
- Transfection
- Time Factors
- Reverse Transcriptase Polymerase Chain Reaction
- RNA, Messenger
- Pigment Epithelium of Eye
- Peroxidase
- Oxidants
- Ophthalmology & Optometry
- Matrix Metalloproteinase 2