Benzalkonium chloride-induced myofibroblastic transdifferentiation of Tenon\u2019s capsule fibroblasts is inhibited by coculture with corneal epithelial cells or by interleukin-10

Chiemi Yamashiro,Makiko Wakuta,Manami Ota,Kazuhiro Tokuda,Tadahiko Ogata,Kazuhiro Kimura,Yuka Kobayashi,Takuya Yoshimoto,Sho-Hei Uchi,Shinichiro Teranishi,Fumiaki Higashijima,Makoto Hatano,Atsushige Ashimori,Masaaki Kobayashi

doi:10.1038/s41598-021-94852-8

Abstract

Benzalkonium chloride (BAC) is used as a preservative in eyedrops but induces subconjunctival fibrosis that can result in failure of glaucoma surgery. Tenon’s capsule fibroblasts in subconjunctival tissue interact with the corneal epithelium through tear fluid. With the use of a coculture system, we have now investigated the effect of human corneal epithelial (HCE) cells on myofibroblastic transdifferentiation of human Tenon fibroblasts (HTFs) induced by BAC (5 × 10−6%). Immunofluorescence and immunoblot analyses revealed that the BAC-induced expression of α smooth muscle actin (αSMA) in HTFs was suppressed by coculture of these cells with HCE cells (p < 0.01). The concentration of interleukin-10 (IL-10) in culture supernatants of BAC-treated HTFs was increased by coculture with HCE cells (17.26-fold, vs. coculure, p < 0.001). Immunofluorescence and immunoblot analyses also showed that exogenous IL-10 (300 pg/ml) suppressed the BAC-induced expression of αSMA by 43.65% (p < 0.05) as well as the nuclear translocation of myocardin-related transcription factor-A (MRTF-A) by 39.32% (p < 0.01) in HTFs cultured alone. Our findings suggest that corneal epithelial cells may protect against subconjunctival fibrosis by maintaining IL-10 levels and preventing the MRTF-A-dependent transdifferentiation of HTFs into myofibroblasts.

Highlights

Benzalkonium chloride (BAC) is used as a preservative in eyedrops but induces subconjunctival fibrosis that can result in failure of glaucoma surgery
We have examined the effect of BAC on the transdifferentiation of human Tenon’s capsule fibroblasts (HTFs) into myofibroblasts in a coculture system with simian virus 40 (SV40)immortalized human corneal epithelial (HCE) cells
We found that the amount of lactate dehydrogenase (LDH) in the medium of human Tenon fibroblasts (HTFs) (p = 0.014) or HCE (p < 0.001) cells was increased by exposure to BAC in a concentration-dependent manner, with this effect being significant at BAC concentrations of ≥ 10 × 1 0−6% (Fig. 1)

Summary

Introduction

Benzalkonium chloride (BAC) is used as a preservative in eyedrops but induces subconjunctival fibrosis that can result in failure of glaucoma surgery. With the use of a coculture system, we have investigated the effect of human corneal epithelial (HCE) cells on myofibroblastic transdifferentiation of human Tenon fibroblasts (HTFs) induced by BAC (5 × 1 0−6%). Immunofluorescence and immunoblot analyses showed that exogenous IL-10 (300 pg/ml) suppressed the BAC-induced expression of αSMA by 43.65% (p < 0.05) as well as the nuclear translocation of myocardin-related transcription factor-A (MRTF-A) by 39.32% (p < 0.01) in HTFs cultured alone. Glaucoma filtration surgery involves the formation of a filtering bleb composed of subconjunctival tissue that contributes to a sustained lowering of intraocular pressure. BAC was shown to shorten bleb survival time after rabbit glaucoma filtration surgery as well as to increase the expression of αSMA in the filtration b leb[18]

Methods

Results

Conclusion