Changes in expression of platelet-derived growth factor and its receptors in the lungs of newborn rats exposed to air or 60% O(2).

PDGF-related gene expression has been well characterized during fetal rat lung development and adult rat lung injury, but not during normal postnatal lung growth or injury. Lung expression of the mRNA for PDGF-A, -B, -alpha R, and -beta R and immunoreactive PDGF-AA, -BB, -alpha R, and -beta R were assessed in rat pups raised in air or 60% O(2) for up to 14 d after birth. Expression of mRNA and immunoreactive ligand did not correlate for pups raised in air. Immunoreactive PDGF-alpha R and -beta R, but not PDGF-AA and -BB, were evident throughout the lung at birth. Both PDGF-AA and -BB were evident in airway epithelium, PDGF-BB in alveolar epithelial cells and PDGF-AA was widely distributed in parenchymal tissue at 4 d. PDGF-alpha R was localized to airway epithelium, and PDGF-beta R to subendothelial perivascular regions and to airway and alveolar epithelium at 4 d. Immunoreactive PDGF ligands all declined after 4 d. Intraperitoneal injection of neutralizing antibodies or truncated soluble receptors to PDGF-BB reduced lung DNA synthesis in air. Exposure to 60% O(2) significantly increased mRNA for PDGF-B, -beta R, and -alpha R, but not PDGF-A, relative to air-exposed lung at various time points after birth. PDGF-A, -B, and -alpha R immunoreactivities in these lungs were reduced and delayed, consistent with a global inhibition of lung growth. Pups exposed to 60% O(2) had a similar distribution of PDGF-beta R to that seen in air, except that at 14 d PDGF-beta R was distributed throughout the lung parenchyma. We conclude that PDGF ligands and receptors are important for normal postnatal lung growth and that their expression is delayed by O(2) exposure.

Endothelin-1 and O2-mediated pulmonary hypertension in neonatal rats: a role for products of lipid peroxidation.

We hypothesized that reactive O2 species, or their intermediary products, generated during exposure to elevated O2 lead to pathologic endothelin-1 expression in the newborn lung. Endothelin-1 expression and 8-isoprostane content (an in vivo marker of lipid peroxidation) were examined and found to be elevated (p < 0.05) in the lungs of newborn rats with abnormal lung morphology and pulmonary hypertension, as assessed by right ventricular hypertrophy, after a 14-d exposure to 60% O2. The antioxidant and lipid hydroperoxide scavenger, U74389G (10 mg/kg), given by daily i.p. injection prevented O2-dependent right ventricular hypertrophy (p < 0.05 compared with vehicle-treated controls), but had no effect on abnormal lung morphology. Additionally, we observed that 8-isoprostane caused marked endothelin-1 mRNA up-regulation in vitro in primary rat fetal lung cell cultures. We conclude that reactive O2 species, or their bioactive intermediaries, are causative in O2-mediated pulmonary hypertension and endothelin-1 up-regulation. It is likely that the bioactive lipid peroxidation product, 8-isoprostane, plays a key role in pathologic endothelin-1 expression and pulmonary hypertension during oxidant stress.

Effect of the 21-aminosteroid U74389G on oxygen-induced free radical production, lipid peroxidation, and inhibition of lung growth in neonatal rats.

Bronchopulmonary dysplasia is a chronic pneumopathy of preterm infants, with significant associated mortality and morbidity, for which there is no effective preventive therapy. Pulmonary O2 toxicity is thought to be a major contributor to the development of bronchopulmonary dysplasia, and antioxidant interventions hold significant promise for therapy. The relative importance of specific reactive oxygen species in the development of O2-mediated lung injury is unknown. In this study, we tested the effect of a synthetic 21-aminosteroid, U74389G, on 95% O2-induced free radical production, lipid peroxidation, and inhibition of postnatal lung growth in a neonatal rat model. Lipid peroxidation products, as measured by total 8-isoprostane and aldehydes, and hydroxyl radical formation, assessed using salicylate metabolites, in rat lungs and serum were significantly increased after exposure to 95% O2. These changes could be completely or partially attenuated by U74389G. However, U74389G did not improve the survival rate or lung wet-to-dry weight ratio. Expression of proliferating cell nuclear antigen, a marker for DNA synthesis, was examined by immunohistochemistry. Four- or 7-d-old control rat lungs had active DNA synthesis, which was inhibited by exposure to 95% O2. U74389G had a protective effect against 95% O2-mediated inhibition of DNA synthesis. Air-exposed animals treated with U74389G had a modest reduction in lung DNA synthesis, consistent with a role for hydroxyl radicals or lipid hydroperoxides as second messengers in the normal regulation of lung growth.

H2O2 mediates O2 toxicity in cultured fetal rat distal lung epithelial cells.

It is unknown which of the reactive oxygen species is primarily responsible for the cytotoxicity of 95% O2 for rat distal fetal lung epithelial cells in vitro. Incubation of cells with 25 U/ml polyethylene glycol (PEG)-conjugated SOD and 50 U/ml PEG-catalase, but not PEG-SOD or SOD mimics alone, significantly reduced 95% O2-mediated cytotoxicity. Liposome-entrapped catalase, without SOD, also significantly reduced 95% O2-mediated cytotoxicity. Increased formation of lipid hydroperoxides, as assessed by the formation of 8-isoprostane and aldehydes, was attenuated by both 100 microM Trolox, a vitamin E analogue, and by 5 microM U74389G, an amino steroid. Trolox, but not U74389G, prevented an increase in cell-derived H2O2, hydroxyl radical and 95% O2-mediated cytotoxicity. An increase in hydroxyl radical formation, but not cell death, observed in 95% O2, was prevented by 0.1 microM phenanthrolene, a cell permeant iron chelator. DNA extracts of rat distal fetal lung epithelial cells maintained under serum-free conditions had an electrophoretic pattern consistent with some degree of apoptosis. However, no increase in laddering was seen with exposure to 95% O2. These data are consistent with hydrogen peroxide, but not lipid hydroperoxides or hydroxyl radical, being a critical effector of O2-mediated necrotic cell death in distal lung epithelial cells.

Liposome-mediated transfection of fetal lung epithelial cells: DNA degradation and enhanced superoxide toxicity.

Cationic liposomes, 1:1 (mol/mol) 1,2-dioleoyldimethylammonium chloride-1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, were used to transfect primary cultures of distal rat fetal lung epithelial cells with pCMV4-based plasmids. A DNA-to-lipid ratio of 1:10 to 1:15 (wt/wt) optimized DNA uptake over a 24-h exposure. At a fixed DNA-to-lipid ratio of 1:15, chloramphenicol acetyltransferase (CAT) reporter gene expression declined at lipid concentrations > 2.5 nmol/cm2 cell surface area, whereas DNA uptake remained concentration dependent. CAT expression peaked 48 h after removal of the liposome-DNA complex, declining thereafter. Reporter gene expression was increased, and supercoiled cDNA degradation was reduced by the addition of 0.2 mM nicotinamide and 10 microM chloroquine. Rat fetal lung epithelial cells transfected with two different expression cassettes had an increased susceptibility to superoxide-mediated cytotoxicity. This could be attributed to a nonspecific delivery of exogenous DNA or some other copurified factor. The DNA-dependent increase in superoxide-mediated cytotoxicity, but not basal levels of cytotoxicity, was inhibited by the addition of 0.2 mM nicotinamide and 10 microM chloroquine.

Alternative reducing agents for reductive methylation of amino groups in proteins.

Dimethylamine borane and trimethylamine borane were compared with sodium cyanoborohydride as reducing agents for the reductive methylation of amino groups in proteins. Dimethylamine borane performed nearly as well as cyanoborohydride; either reducing agent (15 mM) with 20 mM formaldehyde gave extensive methylation of turkey ovomucoid (5 mg/ml) in 2 h at 22 degrees. Trimethylamine borane gave equivalent levels of methylation only with a higher concentration of formaldehyde, suggesting that it is an even milder reducing agent than the other two under these conditions. With all three reducing agents, the pH optimum for methylation covered a range of pH 6-9. Methylations should be performed at the lowest possible pH to avoid side reactions of formaldehyde with the protein. Possible effects of these reducing agents on the disulfide bonds of proteins were studied. No reduction of disulfides of turkey ovomucoid was observed using each of the three reducing agents under conditions for methylation.