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R.P. Kirwan, J.K. Crean, C. Fenerty, A.F. Clark, C.J. O'Brien; Differential Gene Expression in Normal Primary Human Lamina Cribrosa Cells Exposed to Cyclical Mechanical Stretch: A Model for Glaucomatous Optic Neuropathy . Invest. Ophthalmol. Vis. Sci. 2003;44(13):1093.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: To create an in-vitro model for raised intraocular pressure (IOP) by exposing normal primary human lamina cribrosa cells to cyclical mechanical stretch. Methods: Lamina cribrosa cells were grown until confluent in Dulbecco’s medium. Cells were stretched in the Flexercell strain unit by 20% at a rate of 60 cycles per minute. Cells were removed at 12 and 24 hours. Total RNA was extracted using Trizol. Complimentary DNA (cDNA) was synthesized from pooled RNA by RT-PCR. Biotinylated cRNA was synthesized from the cDNA (24 hour time point) in an in-vitro transcription reaction. The cRNA was fragmented and hybridized to the human Affymetrix GenechipTM HG-U133A. Fluorescent intensities were globally normalized and filtered using the Affymetrix suite 5.0 software. Output from the microarray analysis was merged with GenBankTM descriptor and stored as a spreadsheet. Total RNA was also retained for real time PCR analysis. RNA was reverse transcribed to single stranded cDNA, and a TaqManTM probe for TGF-ß1 was added to the 12 and 24 hour cDNA samples. These were analyzed in a multiplex assay with the Prism 7700 sequence detection system. Results: Following 24 hours of mechanical stretch there was significant up or down-regulation of 334 genes: Components of the extracellular matrix (ECM): collagen VI, collagen V, elastin; Regulators of ECM remodeling: TIMP-4, MMP-23A; Cytoskeletal proteins: plectin-1, ß-actin, gelsolin, filamin-A; Transcription factors: EPAS-1, Nuclear Factor-ΚB; Protein kinases: MAP kinase-8, MAP-4 kinase-1, PKC-epsilon; Cell adhesion proteins: cadherin-8, cytohesin-1; Ion channels: K+ channel, Cl- channel. Real time PCR showed a significant increase in TGF-ß1 expression following 12 hours of mechanical stretch. Conclusions: This in-vitro technique induces gene expression of proteins that are also present in the laminæ cribrosæ of glaucomatous optic nerve heads. This suggests that mechanical stretch may be an appropriate model for raised IOP. It also suggests that the lamina cribrosa cell may be an important cell type in connective tissue remodeling in the optic nerve head.
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