Responses of Cultured Human Keratocytes and Myofibroblasts to Ethyl Pyruvate: A Microarray Analysis of Gene Expression

Pyruvic acid is the final product of the glycolytic pathway, the starting substrate for the tricarboxylic acid (TCA) cycle, and a scavenger of reactive oxygen species (ROS).1,2 Ethyl pyruvate (EP) is a membrane-permeant ester of pyruvate, and exogenous EP has the potential to augment intracellular pyruvate levels. In hypoxia, elevated intracellular pyruvate enables the cell to protect itself from ROS-mediated damage and to slough off excess reducing equivalents (by converting pyruvate to lactate). However, intracellular hydrolysis of EP is relatively slow, and several studies (for a review, see Fink 3) have shown that the intact ester also has direct pharmacologic effects. Using murine lens in organ culture, Varma et al.4 showed that EP ameliorates oxidative stress when present concurrently and can partly reverse deleterious effects when 2 hours are added to the stress period.5 Moreover, in intact rats fed a 30% galactose diet (a model for the development of sugar cataract) the concurrent application of EP eye drops attenuated cataract development up to 40 days.6 These authors point out that the reaction of ROS with glycated lens proteins is a major contributor to cataract formation, and so EP very likely protects against cataract development by decreasing ROS levels. Apart from the work of Varma et al.,4 the potential therapeutic effects of EP have been investigated predominantly in splanchnic systems (for a review, see Fink3). These studies focused mainly on rodent models of endotoxin (bacterial lipopolysaccharide [LPS]) induced damage (e.g., LPS infusion, bacterial peritonitis, or acute endotoxemia). The NF-κB pathway is prominent in mediating the proinflammatory effects seen in these models, and EP inhibits NF-κB-dependent signaling by directly targeting p65.7 Therefore EP is of obvious interest in the corneal response to bacterial infection. However, a separate clinical concern is corneal scarring absent infection. This scarring is largely driven by the TGF-β-mediated conversion of quiescent stromal keratocytes to myofibroblasts. Although TGF-β isoforms are absent from the corneal stroma in the normal human eye,8 increased local TGF-β2 is seen in patients with superior limbic keratoconjunctivitis.9 In the rabbit, antibodies against TGF-β1 decrease subepithelial collagen deposition (corneal haze) after excimer laser photorefractive keratectomy (PRK),10 and antibodies against TGF-β2 reduce subconjunctival scarring after glaucoma filtration surgery.11 In the rat, antibodies against TGF-β1 inhibit the increase in the number of stromal cells in the laser-ablated area 5 days after PRK,12 including the recruitment of highly reflective activated keratocytes. Myofibroblast transformation and consequent stromal fibrosis also are inhibited. Experiments in vitro suggest that in the cornea, stromal-to-epithelial signaling predominantly involves HGF and KGF (FGF7),13 whereas epithelial-to-stromal signaling is predominantly by TGF-β1, bFGF (FGF2), and EGF.14 Cultured corneal keratocytes undergo phenotype shifts to fibroblasts and myofibroblasts in response to FGF2 and TGFβ, respectively.15 In corneal fibroblasts expression of TGF-β1 and TGF-βRI (but not TGF-βRII or -RIII) is upregulated by exogenous TGF-β1.16 Exogenous FGF-2 decreases TGF-β1 mRNA levels,17 but TGF-β1 has no reciprocal effect on FGF-2. Relative to keratocytes, myofibroblasts show upregulation of α-smooth muscle actin and TGF-β receptors, and downregulation of connexin 43,18,19 they stain positively for integrin α5β1, myosin, and α-actinin, and form f-actin microfilament bundles (stress fibers) that co-localize with fibronectin. The previous reports in splanchnic models mentioned earlier suggest that EP should have therapeutic anti-inflammatory effects in the cornea; and in the intact rat, long-term (40 day) instillation of EP eye drops is well tolerated.6 However, before human trials are conducted, it is important to survey the responses to EP of both normal and activated human corneal stromal cells. In the present study, we use cultured human keratocytes to model the effects of EP on normal stroma, and TGF-β1–activated keratocytes to model the effects of EP on the TGF-β–driven scarring process that can occur in vivo. The present microarray analyses provide global surveillance of the resultant gene expression changes and an encouraging picture of the potential therapeutic uses of EP.