The Dual Effect of Rho-Kinase Inhibition on Trabecular Meshwork Cells Cytoskeleton and Extracellular Matrix in an In Vitro Model of Glaucoma

Abstract

The trabecular meshwork (TM) is the main site of drainage of the aqueous humor, and its dysfunction 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) of latanoprost (LT) and a Rho-kinase inhibitor (ROCKi) on a transforming growth factor beta2 (TGF-β2)-induced glaucoma-like model developed from primary culture of human TM cells (pHTMC). The TGF-β2 stimulated pHTMC were grown and incubated with LT or a ROCKi (Y-27632) for 24 h. The expression of alpha-smooth muscle actin (αSMA) and fibronectin (FN), and phosphorylation of the myosin light chain (MLC-P) and Cofilin (Cofilin-P) were evaluated using immunofluorescence and Western blot. The architectural modifications were studied in a Matrigel^TM 3D culture. TGF-β2 increased the expression of αSMA and FN in pHTMC and modified the cytoskeleton with cross-linked actin network formation. LT did not alter the expression of αSMA but decreased FN deposition. The ROCKi decreased TGF-β2-induced αSMA and FN expression, as well as MLC-P and Cofilin-P, and stimulated the cells to recover a basal cytoskeletal arrangement. In the preliminary 3D study, pHTMC organized in a mesh conformation showed the widening of the TM under the effect of Y-27632. By simultaneously modifying the organization of the cytoskeleton and the ECM, with fibronectin deposition and overexpression, TGF-β2 reproduced the trabecular degeneration described in glaucoma. The ROCKi was able to reverse the TGF-β2-induced cytoskeletal and ECM rearrangements. LT loosened the extracellular matrix but had no action on the stress fibers.

Keywords: 3D culture; Matrigel; cytoskeleton; glaucoma; intraocular pressure; outflow; prostaglandin analog; rho-kinase inhibitor; trabecular meshwork.

Figure 1

Rho-kinase signaling pathway. The TGF-β receptor (TRF-β RI-II) activates its effector molecules, ROCKs (Rho-kinases ROCK1 and 2). ROCKs inhibit myosin light chain phosphatase (MLCP). Phosphorylation of the myosin light chain induces actin fiber contraction. ROCKs activate LIM kinases, which phosphorylate cofilin, leading to actin stabilization [27].

Figure 2

Cytoskeletal remodeling after TGF-β2 exposure. pHTMC were treated for 48 h with TMCM only (Vehicle) or with TGF-β2 (5 ng/mL). F-actin filaments were visualized by phalloidin staining (red) and alpha-SMA antibody (green), and nuclei were counterstained with DAPI (blue). Right: enlarged images from corresponding areas. Asterisks indicate CLANs.

Figure 3

(a) Immunofluorescence analysis of the effect of LT 0.5 µg/mL or the ROCK-inhibitor Y-27632 25 nM for 24 h on pHTMC. pHTMC were treated for 48 h with TMCM only (Vehicle) or with TGF-β2 5 ng/mL or with 24 h of TGF-β2 5 ng/mL followed by 24 h of TGF-β2 5 ng/mL along with LT 0.5 µg/mL or Y-27632 25 nM. Nuclei are stained with DAPI (blue). The green staining corresponds to the antibodies indicated. Scale bar = 30 µm. This figure shows the major effect of LT on the density of ECM with no effect on the cytoskeleton itself. The ROCKi modified both the cytoskeleton and the ECM relaxation. (b) Quantification, with Arrayscan, of the total positive labeling area related to the number of cells (µm²) in fold-change (mean ± SEM). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Figure 4

Protein expressions in pHTMC cultures after treatment with 5 ng/mL TGFβ2 for 48 h in the absence or presence of 25 nM Y27632 or 0.5 µg/mL LT for 24 h. (a) Representative Western blots of fibronectin and β-actin (mean ± SEM). (b) Densitometry of Western blot analysis of fibronectin normalized to β-actin.

Figure 5

Confocal microscopy images of the 3D cultured pHTMC. Analysis of the effect of the ROCK inhibitor Y27632 at 25 nM for 24 h and the effect of LT at 0.5 μg/mL on primary human pHTMC treated with TGF-β2 at 5 ng/mL for 48 h. The cells were treated with TMCM alone (vehicle), 5 ng/mL of TGF-β2 for 48 h, or with TGF-β2 (5 ng/mL) for 24 h followed by a combination of TGF-β2 at 5 ng/mL and with Y-27632 (25 nM) or LT (0.5 µg/mL) for 24 h. Actin fibers are stained in red by phalloidin, membranes with DiO (green), and nuclei with DAPI (blue). Magnification 200×. Scale bar = 30 µm.