A. K. Tanswell

Alpha 1-antitrypsin protects neonatal rats from pulmonary vascular and parenchymal effects of oxygen toxicity.

ABSTRACT: We investigated whether α1-antitrypsin (α1-AT) might protect neonatal rats from the pulmonary parenchymal and vascular effects resulting from hyperoxic exposure. Neonatal rats born into and maintained in hyperoxia (60% fraction of inspired oxygen) or room air were injected with a loading dose of α1AT (72 mg/kg) followed by 36 mg/kg every 72 h or with vehicle during the first 14 d of life. At the end of the experimental period, we measured body weight, lung compliance, lung volume, alveoli per mm2, and total number of alveoli and assessed right ventricular hypertrophy and vascular changes consisting of medial hypertrophy, muscular extension into peripheral, normally nonmuscular arteries, and number of peripheral arteries relative to alveoli. Our data show that α1-AT treatment prevented the reduced lung compliance observed in the untreated hyperoxia- exposed neonatal rats, as well as the right ventricular hypertrophy and the associated vascular changes of medial hypertrophy of muscular arteries and muscularization of distal arteries. Reduced lung compliance in the hyperoxic but α1-AT-untreated rats was associated with a reduction in lung elastin compared with room-air or α1-AT-treated rats. In room-air rats, α1-AT treatment increased lung compliance but also reduced the number of arteries relative to the number of alveoli, a feature that was not, however, associated with right ventricular hypertrophy. Our data suggest that supplemental α1-AT might restore the imbalance in elastolytic activity induced by hyperoxia and thereby alleviate the toxic effects on lung parenchymal and vascular development.

Growth of distal fetal rat lung epithelial cells in a defined serum-free medium.

SummaryFetal rat distal lung epithelial cells, in contrast to adult type II pneumocytes, will divide readily in culture in the presence of 10% (vol:vol) fetal bovine serum. The presence of serum makes purification of uncontaminated cell-derived growth factors difficult and modifies cellular responses to oxidant injury. We report the development of a defined serum-free medium that will support growth of fetal distal lung epithelial cells in primary culture. Initial studies used a low-serum (2%; vol:vol) to determine the effect of basal media, substrata, and various additives. Subsequent studies demonstrated growth on a poly-d-lysine substratum under serum-free culture conditions in Dulbecco’s modified minimal essential medium with insulin (50 µg/ml), endothelial cell growth supplement (20 µg/ml), bovine pituitary extract (100 µg/ml), bovine serum albumin (50 µg/ml), selenous acid (4 ng/ml), reduced glutathione (500 ng/ml), soybean trypsin inhibitor (100 µg/ml), transferrin (5 µg/ml), HEPES buffer (2.6 mg/ml), and cholera toxin (5 µg/ml). Growth was enhanced by reducing the gas phase oxygen concentration from 21 to 3%. The undefined components of this medium, bovine pituitary extract and endothelial cell growth supplement, could be replaced by platelet-derived growth factor (20 ng/ml) with prostaglandin E1 (25 nM). The response of fetal distal lung epithelial cells to known growth factors differs substantially from that observed with type II pneumocytes from adult lung and is similar in many, though not all, respects to the responses reported for proximal airway cells from adult lung.

Hormonal and Local Factors Influence Antioxidant Enzyme Activity of Rat Fetal Lung Cells in Vitro

Monolayer cultures of fetal rat mixed lung cells respond to sublethal concentrations (50%) of oxygen by a reduced growth rate. Exposure to 95% O2 causes growth arrest and cell loss. In the presence of serum the addition of dexamethasone (5.5 nM), tri-iodothyronine (5.5 nM), or insulin (5 microU/ml) appeared to increase the cytotoxicity of 95% O2. Under growth-arrested conditions, in the absence of serum or elevated O2 concentrations, all three agents influence cellular antioxidant enzyme activities. Dexamethasone (0.055 nM) increased CuZn superoxide dismutase activity by 72% and glutathione peroxidase activity by 94%. Triiodothyronine (5.5 nM) increased CuZn superoxide dismutase activity 93%. Insulin (5 microU/ml) increased CuZn superoxide dismutase activity 90%, and catalase activity 58%. Dexamethasone, but not tri-iodothyronine or insulin, seems to have a protective effect against subsequent acute hyperoxia under serum-free conditions. Local non-hormonal factors may also influence lung cell responses to acute increases in oxygen concentrations, since cells acutely exposed to 50% or 95% O2 release a transferable factor(s) into their culture medium which increases antioxidant enzyme activities of non-hyperoxic lung cells.

Circulating Factors That Modify Lung Cell DNA Synthesis Following Exposure to Inhaled Oxidants. III. Effect of Plasma on Lung Pneumocyte and Fibroblast DNA Synthesis Following Exposure of Adult Rats to 85% Oxygen

Previous studies, in which adult rats were exposed to 1 ppm ozone for 2 weeks, demonstrated the appearance in plasma of separate factors that stimulated DNA synthesis by cultured pneumocytes and lung fibroblasts in a dose-dependent and cell-specific fashion. Both factors had isoelectric points of 6.45-6.75, but differed by molecular mass. The pneumocyte factor had an estimated weight of 38 +/- 3 kDa, while the fibroblast factor had an estimated molecular weight of 32 +/- 2 kDa. To determine whether the appearance of these factors in plasma is specific for ozone injury or whether they appear in response to other oxidant injuries, adult rats were exposed to 85% O2 or air for up to 2 weeks. Animals were sacrificed at 3, 5, 7, or 14 days after the onset of exposure. Plasma samples were subjected to sequential preparative electrofocusing and high-performance liquid chromatography (HPLC). Heat-inactivated plasma fractions, with an isoelectric point of 6.45-6.75, contained a factor of 32 +/- 2 kDa, which enhanced lung fibroblast DNA synthesis at a single time point on day 5 of 85% O2 exposure, and a factor of 38 +/- 3 kDa, which enhanced pneumocyte DNA synthesis on days 5, 7, and 14 of 85% O2 exposure. Of the known growth factors, those most likely to have these physical characteristics are platelet-derived growth factor (PDGF) and insulin-like growth factor-1. Additional groups of animals were exposed to air or 85% O2 for 5 days for plasma collection. Animals exposed to 85% O2 had a 60% increase of plasma immunoreactive PDGF and a 90% increase of plasma immunoreactive IGF-1, compared with values for control animals exposed to air.