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Sasha Rosen, Anita Ghosh, Evan B Stubbs, Vidhya R Rao, Simon Kaja; Loxl1 knockdown precipitates phenotypes of reactive astrocytosis and elastinopathy in optic nerve head astrocytes. Invest. Ophthalmol. Vis. Sci. 2018;59(9):3526.
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Exfoliation glaucoma is a common type of open-angle glaucoma that has been linked to single nucleotide polymorphisms in the lysyl oxidase-like 1 (LOXL1) gene. Genetic studies have identified a putative mechanism by which non-coding variants in LOXL1 can result in reduced tissue expression levels of Loxl1 protein in multiple tissues, including the optic nerve head. The purpose of this study was to determine the effect of Loxl1 down-regulation on optic nerve head astrocytes.
Knockdown of Loxl1 expression in primary adult rat optic nerve head astrocyte (ONHA) cultures was achieved by transfection of rat Loxl1 gene-specific siRNA using Lipofectamine® RNAiMAX Reagent (Thermo Fisher). Quantitative PCR, immunoblotting and immunocytochemistry were performed using previously validated rat specific primers and antibodies. In some experiments, reactive astrocytosis was induced experimentally by culturing primary ONHA in a humidified hyperbaric (20 - 25 mm Hg) atmosphere of 95% Air/5% CO2 for 20 h.
siRNA-mediated Loxl1 knockdown in ONHA elicited a statistically significant increase in GFAP immunoreactivity (n = 5, P < 0.001), suggestive of reactive astrocytosis. Given the critical role for calcium signaling in maintenance and integrity of the extracellular matrix (ECM) and reactive astrocytosis, we quantified the expression of pore-forming CaVα subunits of voltage-gated calcium channels and ECM proteins in response to Loxl1 knockdown. Pan-CaVα expression was significantly increased (n = 3, P < 0.01) and this effect appeared to be largely due to an increase in expression of the CaV-α1A pore-forming subunit of the CaV2.1 (P/Q-type) channel (1.8-fold increase, n = 3, P < 0.01). At the same time, expression of the ECM proteins elastin, fibulin-2 and fibulin-4 were decreased, suggestive of cellular elastinopathy. Intriguingly, induction of reactive astrocytosis by exposure to a hyperbaric environment showed similarly decreased Loxl1 protein expression (n = 3, P < 0.05) and the ensuing changes in CaVα and ECM protein expression.
Loxl1 knockdown in ONHAs elicits cellular phenotypes consistent with induction of reactive astrocytosis and elastinopathy. LOXL1 variants associated with exfoliation glaucoma may sensitize the optic nerve head to the deleterious consequences of elevated IOP, thereby initiating the clinical progression of exfoliation glaucoma.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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