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R.P. Kirwan, M.O. Leonard, A.F. Clark, C.J. O'Brien; Effect of Hypoxic Stress on Extra–Cellular Matrix Gene Expression in Lamina Cribrosa Cells From the Human Optic Nerve Head . Invest. Ophthalmol. Vis. Sci. 2005;46(13):2373.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Blood flow studies show reduced perfusion of the optic nerve head in glaucoma. Immuno–histochemistry studies have identified elevated HIF protein (hypoxia inducible transcription factor) in POAG optic nerve head tissue. Marked extra–cellular matrix (ECM) remodeling in the lamina cribrosa (LC) layer has also been demonstrated in POAG. LC cells may sense reduced optic nerve head vascular perfusion in POAG and respond by inducing ECM synthesis and degradation. We, therefore, investigated the effect of hypoxic stress on ECM gene expression profiles and VEGF protein secretion in LC cells in–vitro. Methods: Primary cultures of LC cells were generated from the optic nerve head tissue of three normal human donors (n=3). Confluent cells were exposed to 24 hours of hypoxia (1% O2) or normoxia (21% O2). Pooled RNA was hybridised to Affymetrix U133A microarrays and the expression data was normalised using Robust Multi–chip Analysis (RMA). Genes up or down regulated by more than 0.6 signal log ratios were retained for further analysis. The filtered data was separated in functional groups using NIH DAVID software. Promoter sequences of hypoxia sensitive ECM genes in our system were interrogated for HIF transcription factor (TF) binding sites using Genomatix MatInspector software. VEGF protein release in LC cell media supernatants was measured using ELISA. Results: Hypoxic stress induced differential expression of 1884 genes (1242 up regulated, 642 down regulated). Of these, 64 were members of the ECM gene category. Up regulated ECM genes included: Collagens IV, VIII, XIII, XVI, decorin, LTBP4, fibulin 1, THBS2, tenascin XB and VEGF. Down regulated ECM genes included: MMP–16, LOX, collagen V, TIMP3, and periostin. VEGF protein release was also significantly (*p<0.05, n=3) increased in the media of LC cells exposed to hypoxia. We identified putative HIF TF binding sites in the promoters of collagen XVI, fibulin 1, LTBP4, THBS2 and tenascin XB. Conclusions: Exposure of LC cells to hypoxic stress induces the expression of ECM components and modulators, which we believe facilitate matrix remodeling in the lamina cribrosa. We have also identified putative HIF TF binding sites in the promoters of hypoxia sensitive ECM genes in LC cells, which may yield novel therapeutic avenues in POAG management.
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