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M. Birke, C. Neumann, U. Welge-Lüßen, A. Yu, E. Lütjen-Drecoll; Effect of TGF-ß2 on Elastin Expression and Components of the Proteolytic System in Human Optic Nerve Head Astrocytes. Invest. Ophthalmol. Vis. Sci. 2007;48(13):3660.
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© ARVO (1962-2015); The Authors (2016-present)
A severe elastosis, defined by overproduction and disorganization of elastic fibers in the lamina cribrosa (LC), and accumulation of ECM, characterized by thickened tissue septae in the optic nerve, is frequently described in POAG donor eyes. TGF-ß2 was shown to induce epxression of ECM components in vitro in human ONH astrocytes. Here we investigate the effect of TGF-ß2 on the expression of elastin and whether TGF-ß2 represses ECM degradation by modulating expression of proteolytic enzymes and their regulators in human ONH astrocytes.
ONH astrocytes were isolated from 8 donors aged 35 to 81 years and chracterized by immunofluourescence staining for smooth-muscle alpha actin (α-smA), vimentin (VIM), glial fibrillary acidic protein (GFAP), neural cell adhesion molecule 1 (NCAM-1), S100 and paired box gene 2 (PAX-2). Cultures of passage 3-5 were treated with 1ng/ml TGF-ß2 for 72h and effects on expression of elastin, MMPs-1, -2, -3, -7, -9, -13, tissue inhibitors of MMPs (TIMPs) -1, -2, -3, plasminogen activator inhibitor 1 (PAI-1), urokinase and tissue plasminogen activator (uPA, tPA) were analyzed by semiquantitative reverse transcription PCR (sqRT-PCR). Regulative effects were confirmed and quantified by northern blot and real time PCR. Changes in protein levels were analyzed by western blot.
TGF-ß2 treatment induced the expression of elastin, MMP-2, TIMP-1, -3 and PAI-1, whereas MMP-7 and tPA expression was repressed. MMP-9 and -13 were below detection. All other factors were not TGF-ß2 responsive. Quantification of fold inductions or fold repression, respectively, from northern blot and rtPCR gave inductions of 5.2±0.7 for elastin, 1.8±0.3 for MMP-2, 1.5±0.2 for TIMP-1, 1.5±0.1 for TIMP-3, 9.2±1.6 for PAI-1, and repressions of 0.4±0.1 for MMP-7 and 0.5±0.2 for tPA. Western blots qualitatively and quantitatively confirmed the TGF-ß2 dependent regulation of the factors on the protein level.
TGF-ß2 potently activated elastin expression and thereby could initiate or at least contribute to elastotic changes in the LC of POAG eyes. By activiation of PAI-1 and the concurrent repression of tPA, TGF-ß2 could reduce the overall activation of pro-MMPs, whereas the TGF-ß2 induced increase in TIMP-1 and -3 expression could inhibit preexisting MMP activity. Both effects would reduce ECM degradation and together with the activation of ECM-components lead to the described accumulation of elastin and other ECM components in the optic nerve of POAG eyes.
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