title: Regulation of MMP-9 activity in human tear fluid and corneal epithelial culture supernatant.
abstract: OBJECTIVE
  To evaluate human corneal epithelial culture supernatant and tear fluid for the presence of activators and inhibitors of matrix metalloproteinase (MMP)-9, MMP-3, and tissue inhibitor of metalloproteinase (TIMP)-1, respectively, and to evaluate the effect of MMP-3 on the activation of MMP-9 in these specimens.
  
  
  METHODS
  Unstimulated tear fluid was collected from patients with ocular rosacea and normal control subjects. Levels of MMP-9, MMP-3, and TIMP-1 were determined by enzyme-linked immunosorbent assay (ELISA) and/or immunoblot analysis. Supernatants from primary human corneal epithelial cultures and human tear fluid were incubated with MMP-3. Cultured epithelial cells and their supernatants were also treated with doxycycline before MMP-3 was added. Gelatin zymography was used to identify activated 82-kDa MMP-9. MMP-9 activity was assessed with a commercial MMP-9 activity assay system.
  
  
  RESULTS
  MMP-9 and TIMP-1 were detected at significantly higher concentrations in rosacea-affected than in normal tear fluids. MMP-3 was detected exclusively in the tear fluid of patients with ocular rosacea who had corneal epithelial disease. Treatment of the supernatant and tear fluid with MMP-3 resulted in two bands with molecular weights of 92 kDa and 82 kDa, representing pro-MMP-9 and activated MMP-9, respectively. Doxycycline added to the conditioned media did not affect activation of MMP-9 by MMP-3. However, 24-hour treatment of corneal epithelial cultures with doxycycline resulted in a lower concentration and activity of MMP-9 in their supernatants.
  
  
  CONCLUSIONS
  MMP-9 and TIMP-1 are produced by the human corneal epithelium and are present in tear fluid. MMP-3 alone is sufficient to activate MMP-9 on the ocular surface. Doxycycline does not directly inhibit this activation by MMP-3, but it decreases MMP-9 activity when added to corneal epithelial cultures.
authors.affiliation: Ocular Surface and Tear Center, Department of Ophthalmology, Bascom Palmer Eye Institute, Florida, USA.
authors.forename: L
authors.initials: L
authors.lastname: Sobrin
authors.forename: Z
authors.initials: Z
authors.lastname: Liu
authors.forename: D C
authors.initials: DC
authors.lastname: Monroy
authors.forename: A
authors.initials: A
authors.lastname: Solomon
authors.forename: M G
authors.initials: MG
authors.lastname: Selzer
authors.forename: B L
authors.initials: BL
authors.lastname: Lokeshwar
authors.forename: S C
authors.initials: SC
authors.lastname: Pflugfelder
mesh_terms: D015153:Blotting, Western; D002478:Cells, Cultured; D004318:Doxycycline / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D004797:Enzyme-Linked Immunosorbent Assay; D019573:Epithelium, Corneal / Q000187:drug effects / Q000201:enzymology* / Q000473:pathology; D005141:Eyelid Diseases / Q000201:enzymology / Q000209:etiology; D006801:Humans; D019278:Matrix Metalloproteinase 3 / Q000378:metabolism / Q000494:pharmacology; D020780:Matrix Metalloproteinase 9 / Q000378:metabolism*; D008537:Meibomian Glands / Q000201:enzymology / Q000473:pathology; D012393:Rosacea / Q000150:complications / Q000201:enzymology; D013666:Tears / Q000201:enzymology*; D019715:Tissue Inhibitor of Metalloproteinase-1 / Q000378:metabolism
chemical_list: D019715:Tissue Inhibitor of Metalloproteinase-1; D019278:Matrix Metalloproteinase 3; D020780:Matrix Metalloproteinase 9; D004318:Doxycycline