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Ursula Schlotzer-Schrehardt, Angelika Mössner, Antonio Bergua, Friedrich E Kruse, Matthias Zenkel; In vitro model for pseudoexfoliation glaucoma: combined effects of LOXL1, TGF-beta1 and mechanical stress and strain. Invest. Ophthalmol. Vis. Sci. 2014;55(13):4233.
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There is clinical and experimental evidence that elevated intraocular pressure, increased levels of transforming growth factor (TGF)-beta1, and an elastinopathy of the lamina cribrosa resulting from a primary disturbance in lysyl oxidase-like 1 (LOXL1) regulation are involved in the pathogenesis of pseudoexfoliation (PEX) glaucoma. In this study, we evaluated the hypothesis that the combined effect of mechanical stress and strain, TGF-beta1 and dysregulated LOXL1 expression contributes to an abnormal elastin metabolism by optic nerve head (ONH) astrocytes using an in vitro model.
Primary human ONH astrocytes, LOXL1 deficient astrocytes (siRNA knockdown), and LOXL1 overexpressing astrocytes (stably transfected with LOXL1 constructs) were cultivated on BioFlex flexible culture plates with or without stimulation by TGF-beta1 (10 ng/ml). Cells were exposed to increased hydrostatic pressure (45 mm Hg gas pressure) for up to 24 hours using a custom-made, PC-controlled, open pressurized chamber system with dynamic humidified air flow and spacer-defined membrane extension designed to vary both pressure and strain. Controls included cells transfected with empty vector and cells cultivated under ambient pressure. Extracellular matrix-related gene expression was analyzed by real time PCR, and cell viability was assessed by trypan blue staining.
Increased hydrostatic pressure had no effect on cell viability during the entire culture period but induced differential expression of established stress-response genes (e.g. GFAP, TGF-beta1, IL-6, Hsp 27, NCAM) as well as extracellular matrix genes (e.g. elastin, fibrillin-1, fibulin-4, collagen type VI) relative to ambient pressure controls. Forced modulation of LOXL1 expression levels mainly affected elastin expression leading to a significant upregulation in LOXL1-deficient and a significant downregulation in LOXL1-overexpressing cells, respectively. In these cultures, hydrostatic pressure and TGF-beta1 caused an additional increase/decrease in elastin expression as early as 6 hours of cultivation.
The findings suggest that the combined effects of dysregulated LOXL1 expression, elevated pressure and TGF-beta1 affect elastin metabolism in the lamina cribrosa as a crucial determinant for a PEX-specific risk of glaucoma development.
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