Responses of cultured human keratocytes and myofibroblasts to ethyl pyruvate: a microarray analysis of gene expression

Abstract

Purpose: Ethyl pyruvate (EP) has pharmacologic effects that remediate cellular stress. In the organ-cultured murine lens, EP ameliorates oxidative stress, and in a rat cataract model, it attenuates cataract formation. However, corneal responses to EP have not been elucidated. In this study, the potential of EP as a therapeutic agent in corneal wound healing was determined by examining its effects on the transition of quiescent corneal stromal keratocytes into contractile myofibroblasts.

Methods: Three independent preparations of cultured human keratocytes were treated with TGF-beta1, to elicit a phenotypic transition to myofibroblasts in the presence or absence of 10 or 15 mM EP. Gene expression profiles of the 12 samples (keratocytes +/- EP +/- TGF-beta1 for three preparations) were produced by using gene microarrays.

Results: TGF-beta1-driven twofold changes in at least two of three experiments defined a group of 1961 genes. Genes showing twofold modulation by EP in at least two experiments appeared exclusively in myofibroblasts (857 genes), exclusively in keratocytes (409 genes), or in both phenotypes (252 genes). Analysis of these three EP-modulated groups showed that EP (1) inhibited myofibroblast proliferation with concomitant modulation of some cell cycle genes, (2) augmented the NRF2-mediated antioxidant response in both keratocytes and myofibroblasts, and (3) modified the TGF-beta1-driven transition of keratocytes to myofibroblasts by inhibiting the upregulation of a subset of profibrotic genes.

Conclusions: These EP-induced phenotypic changes in myofibroblasts indicate the potential of EP as a therapeutic agent in corneal wound healing.

Figure 1.

EP-induced changes in cell proliferation. Effect of EP on the growth of human corneal stromal cells. Corneal stromal cells in P2 growing in DMEM/F12 with 10% FBS (i.e., fibroblasts) were subcultured into the desired number of 35-mm tissue culture dishes. After 24 hours of incubation, the medium was replaced with DMEM/F12 medium containing TGF-β1+1% FBS (i.e., conversion to myofibroblast). The cells were then incubated in this medium, with or without EP.

Figure 2.

EP-induced changes in the expression of Ki67. Human corneal stromal cells were doubled stained with phalloidin and Ki67 nuclear proliferative antigen. The cells were cultured in medium containing 8 ng/mL of TGF-β1 and 1% FBS, with or without 15 mM EP for 2 days. The cells were then double stained with anti-Ki67 antibodies (right) and phalloidin (left). There were only a few Ki67-positive cells in the EP-treated cultures.

Figure 3.

Signature gene distribution within subsets of the cell cycle: Genes from the data of Mizuno et al.which are not EP modulated (: 187 occurrences of 170 genes) and those that are EP modulated (■): 97 occurrences of 82 genes). G₁ class genes showed no EP modulation except at the M/G₁ or G₁/S interfaces.

Figure 4.

EP induced changes in the NRF2 pathway. Nuclear effects of the NRF2 transcription factor, which upregulates ARE-dependent genes in three functional groups. All genes in large, bold letters were upregulated by EP treatment (Table 4), except c-MAP (bold italics), which was downregulated.