Effects of Exogenous Nitric Oxide and Hyperoxia on Lung Fibroblast Viability and DNA Fragmentation

Abstract

Effective lung repair after acute injury requires elimination of proliferating mesenchymal and inflammatory cells without inducing an acute inflammatory response or disturbing concomitant repair of lung microvasculature. Previous studies have shown that endogenous NO regulates programmed cell death in fibroblasts and can modulate wound fibroblast synthetic function. We hypothesized that exposure of human lung fibroblasts to NO gas would decrease viability and induce apoptotic cell death. Primary cultures of normal human lung fibroblasts were exposed for 4 h to room air (RA), 80% oxygen, NO (at either 20 or 50 ppm) blended with RA, or NO blended with 80% O2, then incubated for 24 to 72 h. Cell viability was determined by fluorescence viability/cytotoxicity assay and DNA fragmentation by TUNEL assay. Peroxynitrite formation was assessed using immunoblotting for S-nitrosotyrosine. NO plus O2 induced significant cell death at 20 and 50 ppm NO when compared to either RA or O2 alone at both 24 and 72 h (p < 0.05). Incubation with superoxide dismutase (SOD), catalase (CAT) or SOD + CAT significantly decreased cell death in fibroblasts treated with NO20/O2 and NO50/O2 compared with controls (p < 0.05). NO20/O2 and NO50/O2 exposure significantly increased TUNEL mean fluorescence intensity (MFI), consistent with increased DNA fragmentation, compared to RA at 24 and 72 h (p < 0.05). Antioxidants decreased MFI in cells exposed to NO20/O2 (CAT and SOD + CAT) compared to controls at 24 h (p < 0.05). Western blot analysis for S-nitrosotyrosine showed increased signal intensity in fibroblasts exposed to NO at 20 and 50 ppm plus O2 compared to RA or O2 alone. Incubation with SOD + CAT reduced signal intensity for peroxynitrite in cells exposed to NO20/O2. We conclude that NO in hyperoxic conditions induces fibroblast cell death and DNA fragmentation, which could be partially mediated by peroxynitrite synthesis.