Ethyl pyruvate ameliorates endotoxin-induced corneal inflammation.

PURPOSE: The purpose of this study was to evaluate the anti-inflammatory effect of ethyl pyruvate (EP) in a mouse model of lipopolysaccharide (LPS)-induced corneal inflammation. METHODS: LPS was injected intrastromally into the corneas of C57BL/6 mice followed by treatment with a solution of 2.5% EP in 0.2% hydroxypropyl methylcellulose (HPMC) every 90 minutes during the course of 12 hours. Prednisolone acetate 1% solution (PRED FORTE) was used as a positive control. Mice were sacrificed after 3 days, and corneas were examined by in vivo confocal microscopy and analyzed for infiltrated cells by flow cytometry. Gr-1, TNF-α, and pNF-κB-p65 were detected immunohistochemically, and TNF-α, IL-6, and IL-1ß levels were quantified by ELISA. RESULTS: LPS-induced haze in mice corneas was decreased by 2-fold upon EP treatment; however, it was not changed upon PRED FORTE treatment. Flow cytometry and immunohistochemistry showed infiltration of leukocytes in the LPS-treated corneas; among the infiltrated cells, neutrophils (Gr-1+ and CD11b+) and macrophages (F4/80+ and CD11b+) were 3403.4- and 4.5-fold higher in number, respectively, than in vehicle-treated control corneas. EP or PRED FORTE treatment of LPS-injected corneas decreased the number of neutrophils 7.5- and 7.2-fold and macrophages by 5.6- and 3.5-fold, respectively. Both EP and PRED FORTE decreased TNF-α and IL-6 expression considerably, and to a lesser extent IL-1ß expression, in the LPS-treated corneas. CONCLUSIONS: The present study demonstrated that EP reduces LPS-induced inflammation in the cornea and thus may have a potential therapeutic application in the inhibition of corneal inflammation.

Initial in vitro investigation of the human immune response to corneal cells from genetically engineered pigs.

PURPOSE: To compare the in vitro human humoral and cellular immune responses to wild-type (WT) pig corneal endothelial cells (pCECs) with those to pig aortic endothelial cells (pAECs). These responses were further compared with CECs from genetically engineered pigs (α1,3-galactosyltransferase gene-knockout [GTKO] pigs and pigs expressing a human complement-regulatory protein [CD46]) and human donors. METHODS: The expression of Galα1,3Gal (Gal), swine leukocyte antigen (SLA) class I and class II on pCECs and pAECs, with or without activation by porcine IFN-γ, was tested by flow cytometry. Pooled human serum was used to measure IgM/IgG binding to and complement-dependent cytotoxicity (CDC) to cells from WT, GTKO, and GTKO/CD46 pigs. The human CD4(+) T-cell response to cells from WT, GTKO, GTKO/CD46 pigs and human was tested by mixed lymphocyte reaction (MLR). RESULTS: There was a lower level of expression of the Gal antigen and of SLA class I and II on the WT pCECs than on the WT pAECs, resulting in less antibody binding and reduced human CD4(+) T-cell proliferation. However, lysis of the WT pCECs was equivalent to that of the pAECs, suggesting more susceptibility to injury. There were significantly weaker humoral and cellular responses to the pCECs from GTKO/CD46 pigs compared with the WT pCECs, although the cellular response to the GTKO/CD46 pCECs was greater than to the human CECs. CONCLUSIONS: These data provide the first report of in vitro investigations of CECs from genetically engineered pigs and suggest that pig corneas may provide an acceptable alternative to human corneas for clinical transplantation.

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

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.