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R. Rogers, S. Ackloo, M. Dharsee, J. G. Flanagan; Biomarkers in Response to Biomechanical Stress From Human Optic Nerve Head Astrocytes. Invest. Ophthalmol. Vis. Sci. 2010;51(13):2184.
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© ARVO (1962-2015); The Authors (2016-present)
To determine the differential expression profile of proteins following biomechanical stress applied to normal human optic nerve head (ONH) astrocytes.
Human ONH astrocytes were isolated and grown from donor tissue (Eye Bank of Canada) in DMEM/F12 (10 % FBS; penicillin/ streptomycin) at 37°C in a 5 % CO2 humidified incubator. Cells were seeded onto collagen I and IV 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 then subjected to 12 % cyclic (1 Hz, sinusoid) equi-axial stretch for 2 hours using the Flexercell FX-4000 Tension Plus System. Control cells were serum-deprived and incubated without stretch for the duration of the experiment. Nano LC- MS/MS and database searching with ProteinPilot (Applied Biosystems, USA) was used to identify proteins and their differential expression. Pathway analysis using Ingenuity Pathway Analysis (Ingenuity Systems, USA) was used to explore the perturbed pathways.
A 12% stretch applied for 2 hours resulted in the differential regulation of 48 proteins for the astrocytes. Potential biomarkers for glaucoma include, but are not limited to:Upregulated - Phosphatase and actin regulator 2 (PHACTR2), sterol regulatory element binding transcription factor 2 (SREBF2), postmeiotic segregation increased 1 (PMS1), kinesin family member 14 (KIF14), cartilage intermediate layer protein, nucleotide pyrophosphohydrolase (CILP).Downregulated - High mobility group AT-hook 2 (HMGA2), crystallin, alpha B (CRYAB), CDK5 regulatory subunit associated protein 2 (CDK5RAP2), membrane associated guanylate kinase (MAGI1) and helicase with zinc finger (HELZ).
Human ONH astrocytes up-regulated proteins involved in: Protein binding, transcription regulation, DNA repair, intracellular transport and division, and ECM reorganization. The down-regulated proteins are involved in: Cellular differentiation, molecular aggregating chaperones, neuronal differentiation, scaffolding of cell-to-cell junctions and RNA unwinding activity.
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