Purchase this article with an account.
R.P. Kirwan, M.O. Leonard, A.F. Clark, C.J. O'Brien; Identification of the Differential Extracellular Matrix and Global Gene Expression Profiles Between Normal and Glaucomatous Human GFAP–Negative Lamina Cribrosa Cells . Invest. Ophthalmol. Vis. Sci. 2006;47(13):1824.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
The lamina cribrosa (LC) of normal and POAG human optic nerve heads demonstrate marked disparity in extracellular matrix (ECM) architecture and composition. Our objectives were to establish, (1) an ECM/fibrotic gene focused baseline transcription differential between GFAP–negative LC cells from normal and POAG donors and, (2) whether the in–vitro stimuli of mechanical stretch, hypoxia or TGF–ß, can induce glaucomatous transcription profiles in normal LC cells.
GFAP–negative LC cell lines were generated from normal (n=3) and POAG (n=3) human donors. Both cell types were grown under un–stimulated cell culture conditions (21% O2, 37oC) until confluent. RNA from normal and POAG cells were pooled and hybridised to separate Affymetrix arrays. Arrays were normalised using robust multichip average (RMA) and selected gene targets were validated using TaqMan PCR. Arrays were also prepared from additional normal LC cells (n=3) stimulated with 24 hours of hypoxia (1% O2), mechanical strain (15%, 1Hz), or TGF–ß1 (10ng/ml). Using the normalised RMA values from all 5 arrays, global unsupervised average linkage hierarchical cluster analysis was performed was using Cluster v3.
Baseline transcription in normal versus POAG LC cells showed marked differences in ECM/fibrotic gene expression. ECM/fibrotic genes up–regulated in POAG cells included: periostin, cartilage linking protein 1, CTGF, versican, lumican, collagens I, III, IV, V, and XIV, lysyl oxidase. ECM/fibrotic genes dowregulated in POAG cells included MMP1, decorin and fibulin 1. Six of these genes were independently validated by TaqMan PCR, and the changes were significant (*p<0.05) and consistent with the array data. Global cluster analysis also demonstrated that mechanical stretch, hypoxia and TGF–ß1 induce glaucomatous patterns of transcription in otherwise normal LC cells.
POAG LC cells in–vitro demonstrate a classically pro–fibrotic transcription profile which may be pathologically characteristic of POAG LC cells in–vivo. We believe this underlines a potential role for LC cells in optic nerve head ECM remodeling. Hypoxia, mechanical stretch and TGF–ß1 also induce glaucomatous gene clusters in normal LC cells and are therefore potentially useful glaucoma–like stimuli.
This PDF is available to Subscribers Only