The Effect of Ethyl Pyruvate on the Corneal Wound Healing Phenotype

Abstract

Purpose: : Ethyl pyruvate (EP) is an aliphatic ester that is a stable derivative of pyruvic acid, an endogenous metabolite previously shown to exhibit anti-oxidative and anti-inflammatory effects. In this study, we determine the efficacy of EP in attenuating fibrosis using in vitro models of human corneal stromal wound healing.

Methods: : Primary cultures of corneal stromal keratocytes were obtained from human donor rims. Cells were arrested in G₀ via starvation in serum-free media and then activated with TGF-β1 in 1% serum or β-FGF to induce the wound-healing phenotype. The expression of specific proteins was evaluated by immunocytochemical and western blotting techniques, and the relative mRNA levels were determined by quantitative real-time RT-PCR. Additionally, we performed 3 sets of microarray analysis to identify gene transcripts that are significantly altered due to treatment with EP.

Results: : At a concentration of 10-15mM, EP treatment of activated keratocytes suppressed the expression of fibrotic markers including tenascin C, fibronectin and α-smooth muscle actin. EP also inhibited activated G₀-arrested corneal fibroblasts from progression beyond the G1/S phase. Microarray data revealed that EP treatment led to a downregulation of 117 genes, 68 of which are related to cell cycle regulation, DNA metabolism, cell proliferation and migration. Genes that showed robust (≥ 5 fold) upregulation included those that inhibit fibroblast proliferation, promote cell-cycle arrest, or lend protection against oxidative damage

Conclusions: : EP treatment of human corneal stromal cells attenuated the expression of certain fibrotic markers, inhibits proliferation of activated human stromal cells into myofibroblasts and suppresses other phenotypic characteristics of myofibroblasts. Microarray analysis suggests that EP functions by inhibiting genes that promote cell cycle progression and cytokinesis. Taken together, these results suggest that EP could ameliorate the fibrosis associated with the corneal wound healing response.

Keywords: cornea: stroma and keratocytes • wound healing • inflammation