Abstract
Purpose: :
Glial cell activation in response to mechanical strain at the level of the lamina cribrosa of optic nerve head likely plays a role in the pathogenesis of glaucomatous optic neuropathy. We use selected reaction monitoring (SRM), (proteomics-based analysis) to investigate targeted peptides following equiaxial stretch of human lamina cribrosa cells
Methods: :
LC cells were isolated and grown from donor tissue in DMEM (4 mM L-glutamine;1g/L glucose;1.5 g/L sodium bicarbonate;10 % FBS; penicillin/ streptomycin) and at 37C,5 % CO2 humidified incubator. Cells were seeded onto collagen I coated BioFlex culture plates and grown to confluence. Cells were rinsed with DPBS and grown for 24 hours in serum-free media. The cells were subjected to 12 % cyclic stretch for 2 hours using the Flexercell Tension Plus System. Control cells were serum-deprived and incubated without stretch for the duration of the experiment. SRM was performed in triplicate, targeting 81 proteins of interest using the AB Sciex 5500 QTRAP. Of 81, 60 were from previous iTRAQ targeting, and 21 were from literature
Results: :
51 proteins sourced from the iTRAQ study and 15 proteins derived from the literature were quantified by SRM-MS. Proteins displaying significant expression changes included Protein S100-A6 (iTRAQ match fold-change (iTm) -1.3; SRM fold-change: -1.2), 40S ribosomal protein SA (iTm, -1.5; -1.3), Transforming Protein RhoA (iTm, -1.5; -1.3), Glial Fibrillary Acidic Protein (literature match fold-change (lit); -1.9), Myocilin (lit; 2.2)
Conclusions: :
LC cells react in vitro to mechanical strain by differentially regulating various proteins, some of which have been previously associated with glaucoma, but some that may provide novel biomarkers. Proteins responsible for cellular remodelling, such as S100-S6, are involved in cell cycle progression and differentiation. The Rho protein network is important in signal transduction and plasma membrane assembly to actin stress fibers. 40S protein is involved in signalling, differentiation, migration, and neurite outgrowth. GFAP has traditionally been associated with upregulation in the activation of glial cells. Myocilin has been connected to glaucoma previously and plays a the role in cytoskeletal function of various ocular tissues.
Keywords: proteomics • lamina cribrosa • glia