title: Repetitive nonlethal oxidant injury to retinal pigment epithelium decreased extracellular matrix turnover in vitro and induced sub-RPE deposits in vivo.
abstract: OBJECTIVE
  To determine the impact of repetitive nonlethal oxidant injury with hydroquinone (HQ) on regulation of cell membrane blebbing and molecules, which are essential in extracellular matrix turnover (ECM) maintenance, especially matrix metalloproteinase (MMP)-2, tissue inhibitor of MMP (TIMP)-2, and type IV collagen in cultured RPE. In addition, to determine whether chronic oral HQ causes induction of sub-RPE deposit formation in a mouse model.
  
  
  METHODS
  An ARPE-19 cell line stably expressing membrane-targeted green fluorescent protein (GFP) was challenged by exposure to HQ (100 microM). Repetitive acute (6 hours every 3 days for 4 weeks) or transient (6 hours followed by a recovery phase, every 5 days for 6 weeks) exposure to HQ were evaluated. An MTS assay, cell counts, and bromodeoxyuridine (BrdU) incorporation were used to detect cell viability and proliferation. Supernatants and cell homogenates were collected to assess MMP-2 and TIMP-2 activity by zymography and reverse zymography, proteins by Western blot, and type IV collagen accumulation by ELISA and immunostaining. Expression of MMP-2 and type IV collagen was examined by real-time RT-PCR on total RNA. Sixteen-month-old C57BL/6 female mice were fed a regular fat diet, with or without HQ (0.8%) in the drinking water, for 4 months. The eyes were removed for transmission electron microscopy of the retina and choroid after treatment. Semiquantitative grading of deposit severity was performed.
  
  
  RESULTS
  In vitro, high doses of HQ (400-250 microM) killed a significant fraction of RPE cells ( approximately 60% of control). Low doses (50-100 microM) were nonlethal but induced significant blebbing. Both nonlethal repetitive acute and transient exposure to HQ were associated with diminished MMP-2 activity and increased collagen type IV accumulation. In vivo, mice exposed to oral HQ demonstrated moderately thick basal laminar deposits and a variable degree of deposits within Bruch's membrane (BrM). These homogeneous sub-RPE deposits accumulated in the eyes, consistent with early laminar deposits.
  
  
  CONCLUSIONS
  In cultured RPE, nonlethal injury with HQ upregulated nonlethal blebbing and decreased ECM turnover. Similarly, in vivo exposure to oral HQ induced nonlethal bleb injury and sub-RPE deposits. These data support the hypothesis that HQ may regulate blebbing and molecules that influence ECM turnover. This study suggests that HQ may be another type of oxidant that causes injury to the RPE and may explain the association between environmental oxidants and early AMD.
authors.affiliation: Bascom Palmer Eye Institute, Department of Ophthalmology, University of Miami Miller School of Medicine, Miami, Florida 33136, USA. mcastano@med.miami.edu
authors.forename: Maria E
authors.initials: ME
authors.lastname: Marin-Castaño
authors.forename: Gary E
authors.initials: GE
authors.lastname: Striker
authors.forename: Oscar
authors.initials: O
authors.lastname: Alcazar
authors.forename: Paola
authors.initials: P
authors.lastname: Catanuto
authors.forename: Diego G
authors.initials: DG
authors.lastname: Espinosa-Heidmann
authors.forename: Scott W
authors.initials: SW
authors.lastname: Cousins
mesh_terms: D000818:Animals; D015153:Blotting, Western; D001973:Bromodeoxyuridine / Q000378:metabolism; D002452:Cell Count; D018929:Cell Culture Techniques; D049109:Cell Proliferation / Q000187:drug effects; D002829:Choroid / Q000187:drug effects / Q000648:ultrastructure; D024141:Collagen Type IV / Q000235:genetics / Q000378:metabolism*; D004032:Diet; D004797:Enzyme-Linked Immunosorbent Assay; D005109:Extracellular Matrix / Q000187:drug effects* / Q000378:metabolism / Q000648:ultrastructure; D005260:Female; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D006873:Hydroquinones / Q000633:toxicity*; D020778:Matrix Metalloproteinase 2 / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D009153:Mutagens / Q000633:toxicity*; D010857:Pigment Epithelium of Eye / Q000187:drug effects* / Q000378:metabolism / Q000648:ultrastructure; D012333:RNA, Messenger / Q000378:metabolism; D012160:Retina / Q000187:drug effects / Q000648:ultrastructure; D020133:Reverse Transcriptase Polymerase Chain Reaction; D019716:Tissue Inhibitor of Metalloproteinase-2 / Q000378:metabolism*
chemical_list: D024141:Collagen Type IV; D006873:Hydroquinones; D009153:Mutagens; D012333:RNA, Messenger; D019716:Tissue Inhibitor of Metalloproteinase-2; D049452:Green Fluorescent Proteins; D020778:Matrix Metalloproteinase 2; D001973:Bromodeoxyuridine; C031927:hydroquinone