Reversal of Corneal Myofibroblasts and Fibroblasts to Keratocytes with 17-N-allylamino-17-demethoxygeldanamycin (17AAG)

Abstract

Purpose: Myofibroblast persistence within the corneal wound space is highly associated with stromal haze, a condition which compromises corneal transparency and visual acuity. Transforming growth factor-β1 (TGF-β1) is a critical signaling molecule in the differentiation of corneal fibroblasts to myofibroblasts and a logical target for pharmacologic intervention. 17-N-allylamino-17-demethoxygeldanamycin (17AAG) is a heat shock protein 90 (HSP90) inhibitor that has been shown to block the profibrotic effects of TGF-β1. The purpose of this study was to determine the effect of 17AAG on rabbit corneal fibroblast and myofibroblast differentiation in the absence and presence of TGF-β1.

Methods: Primary rabbit corneal fibroblasts were seeded in 6-well plates and cultured with media containing 0 or 10 ng/ml TGF-β1 for 72 h. Cells were treated for 72 h with DMSO (control) or 500 nM 17AAG. RNA was then harvested and quantitative PCR was performed to determine mRNA expression of myofibroblast phenotypic markers α-smooth muscle actin (α-SMA) and type III collagen (COL3A1) as well as rabbit corneal crystallins aldehyde dehydrogenase 1A1 (ALDH1A1) and keratocan (KERA) which decrease with injury-induced stromal haze. Immunocytochemistry was used to determine α-SMA protein expression. The effects of treatment on cellular morphology were photodocumented.

Results: In the presence or absence of TGF-β1, α-SMA and COL3A1 mRNA expression was dramatically decreased following treatment with 17AAG in comparison to DMSO-treated RCFs. Expression of corneal crystallins ALDH1A1 and KERA was markedly increased in RCFs treated with 17AAG versus DMSO regardless of TGF-β1 treatment. Consistent with previous studies, RCFs treated with TGF-β1 increased expression of α-SMA and COL3A1 and decreased expression of ALDH1A1 and KERA. RCFs treated with 17AAG had markedly decreased α-SMA protein expression with cells exhibiting a stellate morphology consistent with a keratocyte phenotype.

Conclusions: The HSP90 inhibitor, 17AAG, induces reversal of the fibroblast and myofibroblast phenotype back to the keratocyte phenotype likely via a TGF-β dependent mechanism. The data also suggest that 17AAG could have an effect on dysregulated wound healing processes in the cornea through modulating stromal cell differentiation.

Keywords: 484 cornea: stroma and keratocytes • 765 wound healing • 488 crystallins