title: Proteomics analyses of activated human optic nerve head lamina cribrosa cells following biomechanical strain.
abstract: OBJECTIVE
  To determine protein regulation following activation of human, optic nerve head (ONH), lamina cribrosa (LC) cells in response to mechanical strain.
  
  
  METHODS
  LC cells were isolated and grown from donor tissue in specific media at 37°C and 5% CO(2) humidified incubator. Cells were grown to confluence on collagen I-coated flexible-bottom culture plates, rinsed with Dulbecco's phosphate-buffered saline, and left for 24 hours in serum-free media. They were subjected to 3% or 12% cyclic equiaxial stretch for 2 or 24 hours using a commercial strain-unit system. Control cells were serum-deprived and incubated without stretch for 24 hours. Nano liquid chromatography-mass spectrometry analysis with isobaric tags for relative and absolute quantitation labeling was used to determine protein regulation.
  
  
  RESULTS
  In all, 526 proteins were discovered at a 95% confidence limit. Analysis of associated pathways and functional annotation indicated that the LC cells reacted in vitro to mechanical strain by activating pathways involved in protein synthesis, cellular movement, cell-to-cell signaling, and inflammation. These pathways indicated consistent major protein hubs across all stretch/time conditions involving transforming growth factor-β1 (TGFβ1), tumor necrosis factor (TNF), caspase-3 (CASP3), and tumor protein-p53 (p53). Among proteins of particular interest, also found in multiple stretch/time conditions, were bcl-2-associated athanogene 5 (BAG5), nucleolar protein 66 (NO66), and eukaryotic translation initiation factor 5A (eIF-5A).
  
  
  CONCLUSIONS
  Pathway analysis identified major protein hubs (TGFβ1, TNF, CASP3, p53) and pathways all previously implicated in cellular activation and in the pathogenesis of glaucomatous optic neuropathy. Several specific proteins of interest (BAG5, NO66, eIF-5A) were identified for future investigation as to their role in ONH glial activation.
authors.affiliation: Institute of Medical Science, University of Toronto, Toronto, Ontario, Canada. ronan.rogers@utoronto.ca
authors.forename: Ronan
authors.initials: R
authors.lastname: Rogers
authors.forename: Moyez
authors.initials: M
authors.lastname: Dharsee
authors.forename: Suzanne
authors.initials: S
authors.lastname: Ackloo
authors.forename: John G
authors.initials: JG
authors.lastname: Flanagan
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism; D053148:Caspase 3 / Q000378:metabolism; D002853:Chromatography, Liquid / Q000379:methods; D002868:Chromosomal Proteins, Non-Histone / Q000378:metabolism; D049308:Dioxygenases; D056466:Histone Demethylases; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods; D009457:Neuroglia / Q000166:cytology / Q000378:metabolism*; D009898:Optic Disk / Q000166:cytology / Q000378:metabolism*; D010448:Peptide Initiation Factors / Q000378:metabolism; D040901:Proteomics; D016601:RNA-Binding Proteins / Q000378:metabolism; D013314:Stress, Mechanical; D016212:Transforming Growth Factor beta / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism; D000097574:Eukaryotic Translation Initiation Factor 5A
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D053148:Caspase 3; D002868:Chromosomal Proteins, Non-Histone; D049308:Dioxygenases; D056466:Histone Demethylases; D010448:Peptide Initiation Factors; D016601:RNA-Binding Proteins; D016212:Transforming Growth Factor beta; D014409:Tumor Necrosis Factor-alpha; D016159:Tumor Suppressor Protein p53; D000097574:Eukaryotic Translation Initiation Factor 5A; C496515:BAG5 protein, human; C492859:RIOX1 protein, human