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J. G. Flanagan, R. Rogers, M. Dharsee, S. Ackloo; Biomarkers in Response to Biomechanical Stress From Human Optic Nerve Head Lamina Cribrosa Cells. Invest. Ophthalmol. Vis. Sci. 2010;51(13):2185.
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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) lamina cribrosa (LC) cells.
Human ONH LC cells were isolated and grown from donor tissue (Eye Bank of Canada) in DMEM (10 % FBS; penicillin/ streptomycin) at 37°C in a 5 % CO2 humidified incubator. Cells were seeded onto collagen I pre-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 68 proteins for the LC cells. Potential biomarkers for glaucoma include, but are not limited to:Upregulated - Inosine monophosphate dehydrogenase 2 (IMPDH2), non-POU domain containing, octamer-binding (NONO), ubiquitin-like modifier activating enzyme 3 (UBA3), ATPase, Na+/K+ transporting, alpha 3 polypeptide (ATP1A3), cytidine monophosphate N-acetylneuraminic acid synthetase (CMAS).Downregulated - Latent transforming growth factor beta binding protein 2 (LTBP2), calpain, small subunit 1 (CAPNS1), serpin peptidase inhibitor, clade B (ovalbumin), member B6 (SERPINB6), and neural precursor cell expressed, developmentally down-regulated 4 (NEDD4).
Human ONH LC cells up-regulated proteins involved in: Nucleotide biosynthesis, transcriptional regulation, targeting abnormal proteins, electrochemical gradient maintenance, and cell surface glycol-protein/lipid structure and function. The down-regulated proteins are involved in: microfibril cell adhesion, cystein proteases, peptidase inhibitor activity, and protein targeting to lysosomes.
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