Prolonged exposure to supraphysiological oxygen concentrations results in the generation of reactive oxygen species, which can cause significant lung injury in critically ill patients. Supplementation with human recombinant antioxidant enzymes (AOE) may mitigate hyperoxic lung injury, but it is unclear which combination and concentration will optimally protect pulmonary epithelial cells. First, stable cell lines were generated in alveolar epithelial cells (MLE12) overexpressing one or more of the following AOE: Mn superoxide dismutase (MnSOD), CuZnSOD, or glutathione peroxidase 1. Next, A549 cells were transduced with 50-300 particles/cell of recombinant adenovirus containing either LacZ or each of the three AOE (alone or in combination). Cells were then exposed to 95% O(2) for up to 3 days, with cell number and viability determined daily. Overexpression of either MnSOD (primarily mitochondrial) or CuZnSOD (primarily cytosolic) reversed the growth inhibitory effects of hyperoxia within the first 48 h of exposure, resulting in a significant increase in viable cells (P < 0.05), with 1.5- to 3-fold increases in activity providing optimal protection. Protection from mitochondrial oxidation was confirmed by assessing aconitase activity, which was significantly improved in cells overexpressing MnSOD (P < 0.05). Data indicate that optimal protection from hyperoxic injury occurs in cells coexpressing MnSOD and glutathione peroxidase 1, with prevention of mitochondrial oxidation being a critical factor. This has important implications for clinical trials in preterm infants receiving SOD supplementation to prevent acute and chronic lung injury.

We examined the ability of the human surfactant protein B (SP-B) promoter to confer cell specificity of transgene expression in an adenoviral vector. Using similar replication-deficient adenoviruses (rAd), we compared lacZ reporter gene expression driven by the human SP-B promoter (rAd.SPBlacZ) with the ubiquitously expressed Rous sarcoma virus promoter (rAd.RSVlacZ). rAd.SPBlacZ expressed lacZ in H-441 and A549 lung epithelial cell lines and not in HeLa cells whereas rAd.RSVlacZ expressed in all three cell lines. In primary human fetal lung fibroblasts, beta-galactosidase activity from rAd.RSVlacZ transduction increased in a dose-dependent manner whereas activity from rAd.SPBlacZ remained low. In mixed cell cultures prepared from human fetal lung explants that contained fibroblasts and type II cells, X-Gal staining localized rAd.SPBlacZ expression to only type II cells whereas rAd.RSVlacZ expressed in both cell types. In 24-wk gestation human fetal tissue explants infected ex vivo, the RSV promoter directed lacZ expression in lung, trachea, heart, liver, and esophagus, whereas with the SP-B promoter lacZ was expressed only in lung, specifically in air space-lining cells. This specificity was maintained in vivo. lacZ expression was undetectable in lung and other tissues after intravenous administration of rAd.SPBlacZ whereas rAd.RSV-lacZ expressed primarily in liver. After intratracheal instillation of rAd.SPBlacZ into mice, X-Gal staining localized expression to type II and Clara cells. In contrast, rAd.RSVlacZ expressed in all pulmonary epithelial cell types. Our results indicate that the SP-B promoter may be useful in targeting type II and Clara cells for gene therapy of conditions such as inherited deficiency of SP-B.

We compared lung delivery methods of recombinant adenovirus (rAd): (1) rAd suspended in saline, (2) rAd suspended in saline followed by a pulse-chase of a perfluorochemical (PFC) liquid mixture, and (3) a PFC-rAd suspension. Cell uptake, distribution, and temporal expression of rAd were examined using A549 cells, a murine model using luciferase bioluminescence, and histological analyses. Relative to saline, a 4X increase in transduction efficiency was observed in A549 cells exposed to PFC-rAd for 2–4 h. rAd transgene expression was improved in alveolar epithelial cells, and the level and distribution of luciferase expression when delivered in PFC-rAd suspensions consistently peaked at 24 h. These results demonstrate that PFC-rAd suspensions improve distribution and enhance rAd-mediated gene expression which has important implications in improving lung function by gene therapy.