Hypoxia Preconditioning Protects Corneal Keratocytes From UV and FAS Mediated Apoptosis

Abstract

Abstract: : Purpose: Examine the mechanisms by which hypoxia preconditioning can protect corneal keratocytes from UV–induced or epithelial scrape–induced cell death. Method: Primary cultures of bovine corneal fibroblasts or keratocytes were established. Cells, incubated in 0.5% serum, were subjected to air, 0.5 or 1.5% oxygen or 100 uM Cobalt for 4–6 hrs. After 30 min normoxia, cells were UV–irradiated for 2 min. Two to four hours later, nuclei were stained with DAPI and/or TUNEL. Fibroblasts were also incubated in 0% serum with or without anti–Fas antibody for 24hrs. For epithelial scrape–induced keratocyte death, rabbit corneas were hypoxia preconditioned by contact lens wear (38% water, 0.2 mm thick) for 4 hrs, the epithelium scraped, and frozen sections were prepared for TUNEL assay four hours later. Results: UV exposure induced 40±8% apoptosis in control fibroblasts; 28±5% in 1.5% O₂ treated cells; and 7±2% apoptosis in 0.5% O₂ treated cells. Cobalt chloride treated cells showed 50% less apoptosis than control. Fibroblasts incubated for 24 hrs in 0% serum showed 12.5±3.6% apoptosis; with anti–Fas 25.9±3.3%; anti–Fas plus hypoxia or Cobalt pretreatment, 4.8±2.3 and 12.5±3.6% apoptosis, respectively. Cultured keratocytes (i.e., no serum) preconditioned with hypoxia showed 50% fewer TUNEL positive cells following UV exposure. Fibroblasts, incubated in air with media collected from hypoxic cultures, showed 7±4% UV induced apoptosis relative to 25±3% in controls. Western blots indicated a significant increase in nuclear transcription factor HIF1–α in hypoxia and Cobalt treated cells. Rabbit corneas preconditioned with 4 hrs of contact lens wear showed 10.3± 2.3% apoptosis whereas the control eye showed 20.6±3.2% following epithelial scrape wounding (n=2). Conclusions: Hypoxia preconditioning protects keratocytes from UV and FAS mediated apoptosis. This protection may involve secretion of protective factors induced by HIF1–α mediated gene expression.

Keywords: apoptosis/cell death • cornea: stroma and keratocytes • hypoxia