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M. L. Bajenaru, Y. Wang, D. Ivanov, G. Dvoriantchikova, E. Hernandez, J. L. Goldberg, V. I. Shestopalov, M. E. Fini; Laminin-integrin Signaling in Retinal Ganglion Cells (rgc) Survival After Retinal Ischemia-reperfusion Injury. Invest. Ophthalmol. Vis. Sci. 2009;50(13):138.
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RGC survival and neurite outgrowth are promoted by neurotrophins, and extracellular matrix molecules (ECM) such as laminin. Degradation of laminin in the ECM of RGC in experimental animal models relevant to retinal disease contributes to RGC death. Integrins are the major ECM receptors. The purpose of this study is to investigate the laminin-integrin signaling in RGC survival after retinal ischemia-reperfusion injury (RIRI).
Studies were performed in a rat RIRI model and in the RGC-5 cell line, exposed to oxidative stress. RIRI was induced in Sprague-Dawley rats by unilateral cannulation of the interior chamber and elevation of the intraocular pressure (IOP) to 110 mm Hg for 60 min. RGC loss was quantified in the retina after retrograde labeling with Fluorogold, and DiA. RGC apoptosis was determined by TUNEL and Annexin V staining. RGC-5 cells were plated without, or on laminin substrate and treated with high concentrations (10 mM) of glutamate, typical to glutamate oxidative stress for 24 hours. Cell survival was examined by the MTT assay. Expression of β1 integrin, focal adhesion kinase (FAK), was analyzed by immunofluorescence in retinal sections, and RGC-5 cells, and western blotting in retinal, and RGC-5 extracts. RGC-5 cells were treated with β1 activating, and blocking antibodies, and pharmacological inhibitors, genistein, PP2 for FAK, and LY294002 for PI3K/Akt.
We have previously shown that laminin degradation in the ECM of RGC correlated with RGC loss apparent at 1 day and 45 % 5 days post-RIRI. Also, laminin confered 25% resistance to RGC-5 cells exposed to oxidative stress. In this study we detected a two-fold decrease in β1 integrin expression, FAK and Akt dephosphorylation, and reduced expression of the anti-apoptotic protein bcl-xL in RGC by western blotting, and immunohistochemistry in the retina 1 day post-RIRI and in RGC-5 24 hours after oxidative stress. Immunohistochemical analysis showed a decrease in β1 integrin, talin, paxillin, dephosphorylation of FAK at focal adhesion (FA) contacts, and ultimately disassembly of FA in RGC-5 cells as a result of oxidative stress. Treatment with β1 activating antibodies HUTS-21, 9EG-7 mimic the protective effect of laminin against oxidative stress in RGC-5, while FAK and Akt inhibitors abolish it.
We demonstrated that laminin protective effect is mediated by the β1 integrin survival pathway in RGC and identified FAK, Akt, paxillin, and talin as critical downstream regulators. This pathway was disrupted in RGC after retinal ischemia injury.
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