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/H 2O2 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.
ASJC Scopus subject areas
- Sensory Systems
- Cellular and Molecular Neuroscience