The Involvement of Rho–Kinase Pathway and its Regulation in Cytokine–induced Collagen Gel Contraction by Hyalocytes

Abstract

Abstract: : Purpose: To investigate the involvement of Rho–kinase pathway in collagen gel contraction by hyalocytes. Methods: An in vitro type I collagen gel contraction assay was performed to evaluate the effect of PDGF–BB and TGF–ß2 on the collagen gel contraction by hyalocytes. The effect of both cytokines on the phosphorylation of myosin light chain (MLC) was analyzed by western blotting. To confirm the involvement of the Rho–kinase in the collagen gel contraction by hyalocytes, adenovirus–mediated transfer of dominant–negative Rho–kinase (AdDNRhoK) was performed. Hydroxyfasudil, a specific Rho–kinase inhibitor, was also used to evaluate its therapeutic potential for the treatment of vitreo–retinal interface diseases. Results: Significant collagen gel contraction was observed after the treatment of PDGF–BB and TGF–ß2. While PDGF–BB–dependent maximal contraction was detected within 24 hrs, TGF–ß2 –dependent contraction was much stronger (p<0.01) and occurred in a time–dependent manner at least up to 5 days. Transient and weak MLC–phosphorylation by PDGF–BB was observed around 4 hrs after stimulation. However, TGF–ß2–induced MLC–phosphorylation was in a time–dependent manner and was maintained at least up to 24 hrs. AdDNRhoK demonstrated significant inhibition of collagen gel contraction induced by both cytokines. Hydroxyfasudil dose dependently (0.03–20 µM) prohibited the phosphorylation of MLC. Finally, hydroxyfasudil inhibited collagen gel contraction at a corresponding concentration to that which inhibited MLC phosphorylation. Conclusions: Rho–kinase–mediated MLC phosphorylation appears to be involved in PDGF–BB–and TGF–ß2–induced collagen gel contraction by hyalocytes. Hydroxyfasudil might have therapeutic potential for the treatment of vitreo–retinal disorders associated with the tractional force between the vitreo–retinal interfaces.

Keywords: vitreous • proliferative vitreoretinopathy