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Christian Schori, Sajad Hamid Ahanger, Kristina Evans, Edward Levine, Christian Grimm; Activating the hypoxic response in Müller glia cells: consequences for photoreceptor survival. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):5470.
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We previously showed that a rod-specific hypoxic response protects photoreceptors only marginally against damage, whereas systemic hypoxic preconditioning is strongly protective in vivo (Grimm et al., 2002, Nat Med;8:718-24; Lange et al., 2011, IOVS;52:5872-80). Since Müller cells (MCs) can protect photoreceptors in normoxia by the production of cytokines and growth factors, we are testing whether MCs might be responsible for the protection mediated by hypoxic preconditioning.
A conditionally immortalized mouse MC line (ciMC6) was either exposed to hypoxia (0.2% O2) or treated with siRNA to specifically inactivate the von Hippel Lindau (Vhl) gene. The cellular response to the treatments was analyzed by Western blotting and real-time PCR. MCs expressing GFP (GFP(+)) were isolated by FACS from dissociated retinas of Rlbp1-GFP mice (Vázquez-Chona et al., 2009, IOVS;50:3996-4003) subjected to hypoxia (6h, 7% O2) and analyzed by real-time PCR. Mice lacking Vhl in MCs were generated by the Cre-LoxP system using a tamoxifen-inducible Rlbp1-CreER mouse line (Levine et al., unpublished).
Hypoxia, or siRNA-mediated inactivation of Vhl in ciMC6 cells stabilized hypoxia-inducible factor 1A (HIF1A) in vitro and upregulated expression of hypoxia response genes such as adrenomedullin (Adm), vascular endothelial growth factor (Vegf) and others. Pools of GFP(+) cells isolated from hypoxic retinas were strongly enriched with MCs and showed increased expression of Adm, Vegf and erythropoietin (Epo). Hypoxia-induced expression of Vegf was detected in GFP(+) but not in GFP(-) cell pools where it was below detection threshold. Adm and Epo were upregulated by hypoxia in both pools, as was the expression of fibroblast growth factor 2. The consequences of the MC specific inactivation of Vhl are currently under investigation.
Müller cells respond to reduced oxygen levels or to an inactivation of Vhl by an increased expression of hypoxia responsive genes in vitro and in vivo. Therefore, Müller cells can appropriately react to reduced oxygen levels and may thus be part of the neuroprotective response induced by hypoxic preconditioning in vivo. Consequences of an artificial activation of this response by the inactivation of Vhl in MCs will be tested in the light damage paradigm and in a model of inherited retinal degeneration.
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