title: Nonlethal oxidant injury to human retinal pigment epithelium cells causes cell membrane blebbing but decreased MMP-2 activity.
abstract: OBJECTIVE
  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.
authors.affiliation: Bascom Palmer Eye Institute, Department of Ophthalmology, University of Miami School of Medicine, 1638 NW 10th Avenue, Miami, FL 33136, USA. mcastano@med.miami.edu
authors.forename: Maria E
authors.initials: ME
authors.lastname: Marin-Castaño
authors.forename: Karl G
authors.initials: KG
authors.lastname: Csaky
authors.forename: Scott W
authors.initials: SW
authors.lastname: Cousins
mesh_terms: D015153:Blotting, Western; D002452:Cell Count; D002462:Cell Membrane / Q000473:pathology*; D002470:Cell Survival; D002478:Cells, Cultured; D024141:Collagen Type IV / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D005089:Exocytosis / Q000187:drug effects*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D006861:Hydrogen Peroxide / Q000633:toxicity*; D020778:Matrix Metalloproteinase 2 / Q000235:genetics / Q000378:metabolism*; D016877:Oxidants / Q000633:toxicity*; D009195:Peroxidase / Q000633:toxicity*; D010857:Pigment Epithelium of Eye / Q000187:drug effects* / Q000201:enzymology / Q000473:pathology; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013997:Time Factors; D014162:Transfection; D015854:Up-Regulation
chemical_list: D024141:Collagen Type IV; D016877:Oxidants; D012333:RNA, Messenger; D049452:Green Fluorescent Proteins; D006861:Hydrogen Peroxide; D009195:Peroxidase; D020778:Matrix Metalloproteinase 2