title: Increased levels of catalase and cathepsin V/L2 but decreased TIMP-1 in keratoconus corneas: evidence that oxidative stress plays a role in this disorder.
abstract: OBJECTIVE
  The mRNA levels of antioxidant enzymes, matrix metalloproteinases, cathepsin V/L2, and tissue inhibitor of matrix metalloproteinases (TIMPs) were determined in keratoconus and normal corneas. Protein levels or enzyme activities were analyzed when RNA levels were different.
  
  
  METHODS
  A total of 25 physiologic (normal) and 32 keratoconus corneas were studied. mRNAs were analyzed by semiquantitative reverse transcription-polymerase chain reaction and Southern blot analysis. Proteins were assessed by immunohistochemistry and/or Western blot analysis. Catalase activity was measured in corneal extracts. Antioxidant enzymes examined were catalase, superoxide dismutase (SOD)-1, SOD3, glutathione reductase, glutathione S-transferase and aldehyde dehydrogenase 3A1. Degradative enzymes examined were cathepsin V/L2 and matrix metalloproteinase (MMP)-1, -2, -7, -9, and -14. Tissue inhibitor of matrix metalloproteinase (TIMP)-1, -2, and -3 were also examined.
  
  
  RESULTS
  Keratoconus corneas exhibited a 2.2-fold increase of catalase mRNA level (P < 0.01) and 1.8-fold of enzyme activity (P < 0.03); a 1.5-fold increase of cathepsin V/L2 mRNA (P < 0.03) and abnormal protein distribution; and a 1.8-fold decrease of TIMP-1 mRNA (P < 0.05) and 2.8-fold decrease of protein (P < 0.0001) compared with normal (physiologic) corneas. RNA levels for other antioxidant and degradative enzymes were similar between normal and keratoconus corneas.
  
  
  CONCLUSIONS
  Keratoconus corneas have elevated levels of cathepsins V/L2, -B, and -G, which can stimulate hydrogen peroxide production, which, in turn, can upregulate catalase, an antioxidant enzyme. In addition, decreased TIMP-1 and increased cathepsin V/L2 levels may play a role in the matrix degradation that is a hallmark of keratoconus corneas. The findings support the hypothesis that keratoconus corneas undergo oxidative stress and tissue degradation.
authors.affiliation: Department of Ophthalmology, University of California Irvine, Medical Center, 101 The City Drive, Orange, CA 92868, USA. mkenny@uci.edu
authors.forename: M Cristina
authors.initials: MC
authors.lastname: Kenney
authors.forename: Marilyn
authors.initials: M
authors.lastname: Chwa
authors.forename: Shari R
authors.initials: SR
authors.lastname: Atilano
authors.forename: Annie
authors.initials: A
authors.lastname: Tran
authors.forename: Marilee
authors.initials: M
authors.lastname: Carballo
authors.forename: Mehrnoosh
authors.initials: M
authors.lastname: Saghizadeh
authors.forename: Vasilis
authors.initials: V
authors.lastname: Vasiliou
authors.forename: Wakako
authors.initials: W
authors.lastname: Adachi
authors.forename: Donald J
authors.initials: DJ
authors.lastname: Brown
mesh_terms: D000444:Aldehyde Dehydrogenase / Q000235:genetics / Q000378:metabolism; D015153:Blotting, Western; D002374:Catalase / Q000235:genetics / Q000378:metabolism*; D002403:Cathepsins / Q000235:genetics / Q000378:metabolism*; D003315:Cornea / Q000201:enzymology*; D003546:Cysteine Endopeptidases / Q000235:genetics / Q000378:metabolism*; D019084:Fluorescent Antibody Technique, Indirect; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology; D005980:Glutathione Reductase / Q000235:genetics / Q000378:metabolism; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007640:Keratoconus / Q000201:enzymology* / Q000235:genetics; D020782:Matrix Metalloproteinases / Q000235:genetics / Q000378:metabolism; D018384:Oxidative Stress / Q000502:physiology*; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013482:Superoxide Dismutase / Q000235:genetics / Q000378:metabolism; D019715:Tissue Inhibitor of Metalloproteinase-1 / Q000235:genetics / Q000378:metabolism*
chemical_list: D012333:RNA, Messenger; D019715:Tissue Inhibitor of Metalloproteinase-1; D002374:Catalase; D013482:Superoxide Dismutase; D000444:Aldehyde Dehydrogenase; D005980:Glutathione Reductase; D005982:Glutathione Transferase; D002403:Cathepsins; D003546:Cysteine Endopeptidases; C117627:CTSV protein, human; D020782:Matrix Metalloproteinases