Hypoxia Protects Human CornealEndothelium from Tertiary Butyl Hydroperoxide and Paraquat-Induced Cell Death In Vitro

Abstract

To develop new and safe approaches to protect and preserve the function of the human corneal endothelium (HCE), we have established an in vitro model of cell loss using an HCE cell line and have examined the potential for hypoxia conditioning to protect HCE from death induced by two ROS generating cytotoxins (tertiary butyl hydroperoxide [tBHP] and paraquat [PQ]). Cell death was assessed by flow cytometric analysis of Annexin-V (An) and propidium iodide (PI) double staining and oligonucleosome production. Mitochondrial membrane potential (MMP) was measured with JC-1 fluorescent dye to determine possible associations between MMP preservation and cell survival. PQ and tBHP both induced HCE cell death in a dosedependent manner, with 48% and 32% of An1 cells observed with 60 lM tBHP and 500 lM PQ, respectively. In addition, this level of tBHP and PQ resulted in 66% and 49% decreases in MMP, respectively. Hypoxia (0.6% 6 0.1% oxygen) pretreatment (8 hrs) significantly reduced An1 cells caused by 60 lM tBHP to 15%, whereas hypoxia had no effect on the decreased MMP. Hypoxia pretreatments (24 hrs) slightly reduced An1 cells caused by 500 lM PQ to 25% and completely prevented the induced MMP reduction. Conversely, hypoxia posttreatment (24 hrs) resulted in a greater inhibition of cell death than pretreatment and yet failed to prevent the PQ-induced MMP collapse. In conclusion, these results suggest that hypoxia can sufficiently protect HCE cells against cell death caused by tBHP and PQ, although no direct link between hypoxia cell protection and MMP preservation was observed.