Abstract
Purpose: :
To examine the outcomes of Rho-kinase inhibition on the proliferation, migration, and contractility of the RPEs as well as the integrity of the cell-cell junctions and the extent of differentiation.
Methods: :
Confluent primary bovine RPEs were maintained in low serum medium (2% v/v) for at least 8 weeks to induce differentiation whereas a subset of the cells were subcultured further until passage 5. The specific pharmacological inhibitor H-1152 was applied at the concentrations of 0.1-10 µM to inhibit the Rho-kinase activity. Cell proliferation was determined by the MTT test and Ki-67 immunostaining. Live-Dead and phalloidin stainings were performed to detect the cell viability and the organization of the actin cytoskeleton, respectively. The expression and localization of beta-Catenin, vimentin, and fibronectin were analyzed by immunocytochemistry and Western blot. The wound healing assay was performed on differentiated RPEs to test the effects of H-1152 on serum-induced migration. To examine the contractility, the RPEs were seeded onto the nerve fiber layer of porcine retinal explants and cultured with TGF-beta 2 and/or H-1152. The contraction of the underlying retina and the deposition of fibronectin were assessed by immunohistochemistry on the frozen sections and retinal mounts.
Results: :
H-1152 exerted a significant anti-proliferative activity (p<0.05) in response to serum and PDGF-CC with no loss of viability. The RPEs incubated with TGF-beta 2 or 10% serum exhibited a dedifferentiated morphology with prominent levels of stress fibers, vimentin, and fibronectin as well as weakly formed cell-cell junctions with an increase in cytoplasmic and nuclear beta-catenin. However, in the presence of H-1152, the majority of the cells acquired a more differentiated phenotype characterized by the circumferential actin filaments, a reduction in the levels of vimentin and fibronectin, and the localization of beta-catenin mainly to the cell-cell junctions. H-1152 also reduced the extent of migration and retinal contraction together with a decrease in fibronectin deposition.
Conclusions: :
Inhibition of Rho-kinase arises as a novel alternative for the treatment of the ocular complications marked by the dedifferentiation and enhanced fibrotic activity of the RPEs.
Keywords: retinal pigment epithelium • differentiation • proliferative vitreoretinopathy