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C. Neumann, E. Lütjen-Drecoll, M. Birke; Effect of Osteopontin (OPN) on Cultured Human Optic Nerve (ON) Astrocytes and RGC5 Retinal Ganglion Cells. Invest. Ophthalmol. Vis. Sci. 2009;50(13):2787.
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© ARVO (1962-2015); The Authors (2016-present)
To identify transforming growth factor (TGF)-β2 responsive factors in cultured human optic nerve (ON) astrocytes and to analyze in vitro effects of Osteopontin (OPN) on cultured ON astrocytes.
ON astrocyte cultures were established from 11 donors aged 19 to 62 years without any history of eye disease. Cultures of passage 3-5 were treated with 1ng/ml recombinant human TGF-β2 for 72h and expression changes were analyzed by an Oligo GEArray®. Regulatory effects were confirmed and quantified by PCR, real time PCR (rtPCR) and western blot (WB). Expression of OPN receptors on ON astrocytes was assessed by PCR and Immunohistochemistry (IHC). The in vitro effect of OPN (200ng/ml) on extracellular matrix (ECM) component expression was analyzed by RT-PCR. Effects of OPN and TGF-β2 on astrocyte cell activity and proliferation were tested by MTS assay. Dose dependent effects of OPN on RGC5 cell proliferation after H2O2 treatment were also assessed by MTS assay.
TGF-β2 increased the expression of OPN by nine fold in microarrays. This upregulation confirmed and quantified by PCR (2x), rtPCR (6x) and WB (3x). ON astrocytes expressed OPN receptor CD44 and receptor subunits integrin (ITG) AV, ITGA4, ITGA5, ITGA6, ITGA9, ITGB1, ITGB3 and ITGB5. TGF-β2 had no regulatory effect on their expression. OPN treatment did not alter ECM component expression. In contrast to TGF-β2, OPN increased astrocyte activity and proliferation compared to untreated controls. RGC5 cells displayed a faster recovery after H2O2 challenge upon OPN treatment.
OPN might be involved in counteracting the degenerative effect of TGF-β2 on ON astrocytes. By its pro-proliferative effect, OPN could contribute to astrocytosis observed in glaucoma. Moreover, OPN might be a protective factor for RGCs after ischeamic stresses.
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