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The Dual Effect of Rho-Kinase Inhibition on Trabecular Meshwork Cells Cytoskeleton and Extracellular Matrix in an In Vitro Model of Glaucoma
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International audience. The trabecular meshwork (TM) is the main site of drainage of the aqueous humor, and itsdysfunction leads to intraocular pressure elevation, which is one of the main risk factors of glaucoma.We aimed to compare the effects on cytoskeleton organization and extracellular matrix (ECM) oflatanoprost (LT) and a Rho-kinase inhibitor (ROCKi) on a transforming growth factor beta2 (TGF- 2)-induced glaucoma-like model developed from primary culture of human TM cells (pHTMC). TheTGF- 2 stimulated pHTMC were grown and incubated with LT or a ROCKi (Y-27632) for 24 h.The expression of alpha-smooth muscle actin ( SMA) and fibronectin (FN), and phosphorylationof the myosin light chain (MLC-P) and Cofilin (Cofilin-P) were evaluated using immunofluorescenceand Western blot. The architectural modifications were studied in a MatrigelTM 3D culture.TGF- 2 increased the expression of SMA and FN in pHTMC and modified the cytoskeleton withcross-linked actin network formation. LT did not alter the expression of SMA but decreased FNdeposition. The ROCKi decreased TGF- 2-induced SMA and FN expression, as well as MLC-P andCofilin-P, and stimulated the cells to recover a basal cytoskeletal arrangement. In the preliminary3D study, pHTMC organized in a mesh conformation showed the widening of the TM under theeffect of Y-27632. By simultaneously modifying the organization of the cytoskeleton and the ECM,with fibronectin deposition and overexpression, TGF- 2 reproduced the trabecular degenerationdescribed in glaucoma. The ROCKi was able to reverse the TGF- 2-induced cytoskeletal and ECMrearrangements. LT loosened the extracellular matrix but had no action on the stress fibers.
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