title: Proteases, proteolysis and inflammatory molecules in the tears of people with keratoconus.
abstract: OBJECTIVE
  To investigate the expression of proteases, proteolytic activity and cytokines in the tear film of people with keratoconus.
  
  
  METHODS
  Basal tears from people with keratoconus, from individuals who had undergone corneal collagen cross-linking for the treatment of keratoconus, and from normal controls were collected using a capillary tube. Corneal curvature of each subject was mapped. The total protein in tears was estimated. Levels and activity of proteases in the tears were analysed using specific antibody arrays and activity assays.
  
  
  RESULTS
  The total tear protein level was significantly reduced in keratoconus (4.1 ± 0.9 mg/ml) compared with normals (6.7 ± 1.4 mg/ml) (p < 0.0001) or subjects who had undergone corneal collagen cross-linking (5.7 ± 2.3 mg/ml) (p < 0.005). Significantly (p < 0.05) increased tear expression of matrix metalloproteinases (MMP) -1, -3, -7, -13, interleukins (IL) -4, -5, -6, -8 and tumour necrosis factor (TNF) -α, -β were evident in keratoconus. Tear IL-6 was the only cytokine significantly (p < 0.05) increased in tears of keratoconus subjects compared with the collagen cross-linked group. No significant difference in tear proteases were observed between the normal and the cross-linked groups, although the expression of TNF-α was significantly (p < 0.05) increased in the cross-linked group compared with the controls. Elevated gelatinolytic (87.5 ± 33.6 versus 45.8 ± 24.6 FIU, p < 0.0001) and collagenolytic (6.1 ± 3.2 versus 3.6 ± 2.0 FIU, p < 0.05) activities were observed in tears from keratoconus compared with normal subjects. The activity of tear gelatinases (69.6 ± 22.2 FIU) and collagenases (5.7 ± 3.3 FIU) in the collagen cross-linked group was not significantly different compared with either keratoconus or normals.
  
  
  CONCLUSIONS
  Tears of people with keratoconus had 1.9 times higher levels of proteolytic activity and over expression of several MMPs and cytokines compared with tears from controls. Further investigations are required to study the possible implications of these changes and whether they can be used to monitor disease progression or determine the success of corneal collagen cross-linking.
authors.affiliation: Brien Holden Vision Institute, Sydney, NSW, Australia. sivaramanbala10@gmail.com
authors.forename: Sivaraman Arumugam
authors.initials: SA
authors.lastname: Balasubramanian
authors.forename: Sujatha
authors.initials: S
authors.lastname: Mohan
authors.forename: David Cecil
authors.initials: DC
authors.lastname: Pye
authors.forename: Mark Duncan Perry
authors.initials: MD
authors.lastname: Willcox
mesh_terms: D000328:Adult; D003094:Collagen / Q000378:metabolism; D017364:Collagenases / Q000378:metabolism; D003319:Corneal Stroma / Q000378:metabolism; D019781:Corneal Topography; D003432:Cross-Linking Reagents / Q000627:therapeutic use; D016207:Cytokines / Q000378:metabolism*; D005136:Eye Proteins / Q000378:metabolism*; D005260:Female; D018093:Gelatinases / Q000378:metabolism; D006801:Humans; D007640:Keratoconus / Q000188:drug therapy / Q000378:metabolism*; D008297:Male; D010447:Peptide Hydrolases / Q000378:metabolism*; D017319:Photosensitizing Agents / Q000627:therapeutic use; D059748:Proteolysis; D012256:Riboflavin / Q000627:therapeutic use; D013666:Tears / Q000378:metabolism*
chemical_list: D003432:Cross-Linking Reagents; D016207:Cytokines; D005136:Eye Proteins; D017319:Photosensitizing Agents; C042434:tear proteins; D003094:Collagen; D010447:Peptide Hydrolases; D017364:Collagenases; D018093:Gelatinases; D012256:Riboflavin