An in vitro study of scarring formation mediated by human Tenon fibroblasts: Effect of Y‐27632, a Rho kinase inhibitor

Abstract

Scar formation is the most common cause for failure of glaucoma filtration surgery because of increased fibroblast proliferation and activation. We have now examined the effect of Y‐27632, a Rho‐associated protein kinase (ROCK) inhibitor, on postsurgical scarring formation in human Tenon fibroblasts (HTFs). Collagen gel contraction assay was used to compare contractility activity of Y‐27632 with several antiglaucoma drugs. Immunofluorescence and western blotting were used to examine expression of scar formation–related factors. We found that Y‐27632 inhibited collagen gel contraction, as well as α‐smooth muscle actin and vimentin expression; these were promoted by treatment with latanoprost, timolol, or transforming growth factor (TGF)–β. To investigate the effect of Y‐27632 in postsurgical scarring, we mimicked TGF‐β secretion by stimulating HTFs with TGF‐β prior to Y‐27632 treatment. HTFs cultured in the presence of TGF‐β significantly increased gel contraction. In contrast, when HTFs were treated with 10μM Y‐27632, contraction was significantly inhibited. Furthermore, Y‐27632 reduced TGF‐β–induced phosphorylation of mitogen‐activated protein kinase signalling. These results suggest that ROCK inhibitors may inhibit fibrosis by inhibiting transdifferentiation of Tenon fibroblasts into myofibroblasts and by inhibiting TGF‐β signalling after surgery through mitogen‐activated protein kinase pathway suppression. These results implicate that ROCK inhibitors may improve outcomes after filtering surgery with a potential antiscarring effect, while latanoprost and timolol may induce fibrosis. Significance of the study Scar formation is the primary cause of failure after glaucoma filtration surgery. A ROCK inhibitor, Y‐27632, has been introduced as a novel potential antiglaucoma treatment to reduce intraocular pressure. The aim of our study was to elucidate the effect of Y‐27632 on scarring formation after glaucoma filtration surgery, in direct comparison with other antiglaucoma drugs. Our findings thus suggested that Y‐27632 may inhibit fibrosis and improve outcome after glaucoma filtration surgery through inhibition of transdifferentiation of Tenon fibroblasts into myofibroblasts, and the TGF‐β and MAPK signalling after surgery, while latanoprost and timolol may induce fibrosis.

AR12286 Alleviates TGF-β-Related Myofibroblast Transdifferentiation and Reduces Fibrosis after Glaucoma Filtration Surgery

“…AR12286 reduced the phosphorylated SMAD2 expression that is induced by TGF-β1. These findings differ from findings reported by Meyer-ter-Vehn et al (2006) and Ibrahim et al (2019) [ 3 , 41 ]. Meyer-ter-Vehn et al explored the p38-dependence of TGF-β-induced myofibroblast transdifferentiation and provided a mechanism for the effect of H1152 on α-SMA expression.…”

“…Compared with treatment using other ROCK inhibitors, AR12286 treatment requires lower concentrations (250 and 500 nM). A study reported that 5 to 10 μM ROCK inhibitors were required for myofibroblast differentiation [ 35 , 41 ]. However, drug concentrations may prove to be a limitation of AR12286.…”

Rosiglitasone and ROCK Inhibitors Modulate Fibrogenetic Changes in TGF-β2 Treated Human Conjunctival Fibroblasts (HconF) in Different Manners

“…Therapeutic possibilities for Rip will be also expected for the treatment of corneal endothelial dystrophy and inflammation of retinal pigment epithelium [ 53 , 54 ]. Furthermore, previous studies, using 2D cultured HconH cells [ 55 ] as well as human tenon fibroblasts [ 56 ], have demonstrated that Rip and other pan-ROCK-is also induce beneficial effects toward conjunctival fibrosis formation. Here, the findings reported herein reveal additional proof of the effects of ROCK-is, Rip and KD025 toward conjunctival fibrogenetic changes by a direct measuring stiffness of the 3D HconF cells.…”

Investigational Rho Kinase Inhibitors for the Treatment of Glaucoma

“…It has also been shown that in vitro administration of Y-27632 protects against post-surgical scarring by reduction of collagen gel contraction, unlike other anti-glaucoma drugs, eg timolol, latanoprost, which may induce fibrosis. 31 This agent prevented fibrosis through inhibition of both the transdifferentiation of Tenon fibroblasts into activated myofibroblasts and TGF-β 1 signaling through suppression of the mitogen‐activated protein kinase (MAPK) pathway. 31 …”

“… 31 This agent prevented fibrosis through inhibition of both the transdifferentiation of Tenon fibroblasts into activated myofibroblasts and TGF-β 1 signaling through suppression of the mitogen‐activated protein kinase (MAPK) pathway. 31 …”