Gadolinium chloride inhibits pulmonary macrophage influx and prevents O(2)-induced pulmonary hypertension in the neonatal rat.

Newborn rats exposed to 60% O(2) for 14 d demonstrated a bronchopulmonary dysplasia-like lung morphology and pulmonary hypertension. A 21-aminosteroid antioxidant, U74389G, attenuated both pulmonary hypertension and macrophage accumulation in the O(2)-exposed lungs. To determine whether macrophage accumulation played an essential role in the development of pulmonary hypertension in this model, pups were treated with gadolinium chloride (GdCl(3)) to reduce lung macrophage content. Treatment of 60% O(2)-exposed animals with GdCl(3) prevented right ventricular hypertrophy (p < 0.05) and smooth muscle hyperplasia around pulmonary vessels, but had no effect on morphologic changes in the lung parenchyma. In addition, GdCl(3) inhibited 60% O(2)-mediated increases in endothelin-1, 8-isoprostane, and nitrotyrosine residues. Organotypic cultures of fetal rat distal lung cells were subjected to cyclical mechanical strain to assess the potential role of GdCl(3)-induced blockade of stretch-mediated cation channels in these effects. Mechanical strain caused a moderate increase of endothelin-1 (p < 0.05), which was unaffected by GdCl(3), but had no effect on 8-isoprostane or nitric oxide synthesis. A critical role for endothelin-1 in O(2)-mediated pulmonary hypertension was confirmed using the combined endothelin receptor antagonist SB217242. We concluded that pulmonary macrophage accumulation, in response to 60% O(2), mediated pulmonary hypertension through up-regulation of endothelin-1.

Targeting transgene expression for cystic fibrosis gene therapy.

We have developed an expression cassette for cystic fibrosis (CF) gene therapy using control elements from the human cytokeratin 18 gene (KRT18, also known as K18). KRT18 is naturally expressed in a spatial pattern similar to that of CFTR, the gene mutated in CF. We delivered a KRT18-driven lacZ plasmid complexed with cationic liposomes intravenously to mice and examined expression in various tissues. We found expression in nasal and bronchial epithelium, airway submucosal glands, gall bladder, and kidneys. Expression was low in pancreas and gut, and absent from liver and alveolar lung. This is consistent with the expression pattern reported for a K18lacZ transgenic mouse. Following delivery of a cytomegalovirus (CMV) major immediate-early promoter/enhancer-driven lacZ plasmid, we found expression in bronchi, submucosal glands, alveolar cells, liver, and kidney. We did not detect expression in nose, pancreas, gall bladder, or gut. Using fluorescently labeled plasmid delivered by means of liposomes, we identified the liver, alveolar lung, and kidneys as the major plasmid deposition sites. Our data demonstrate that a KRT18-driven expression vector delivered systemically can target gene expression to CF-affected tissues, despite an uneven distribution of plasmid DNA. A KRT18-based vector may be a useful alternative to viral promoter-based vectors in clinical gene therapy trials to treat CF.

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.

A human epithelium-specific vector optimized in rat pneumocytes for lung gene therapy.

Gene therapy vectors based on mammalian promoters offer the potential for increased cell specificity and may be less susceptible than viral promoters to transcription attenuation by host cytokines. The human cytokeratin 18 (K18) gene is naturally expressed in the lung epithelia, a target site for gene therapies to treat certain genetic pediatric lung diseases. Our original vector based on the promoter and 5' control elements of K18 offered excellent epithelial cell specificity but relatively low expression levels compared with viral promoters. In the present study, we found that adding a stronger SV40 poly(A) signal boosted primary rat lung epithelial cell expression but greatly reduced cell specificity. Addition of a 3' portion of the K18 gene to our vector as a 3' untranslated region (UTR) improved epithelial cell-specific expression by reducing expression in lung fibroblasts. The effect of the 3' UTR was not related to gross differences in cell-specific splicing. A deletion variant of this UTR further increased lung epithelial cell expression while retaining some cell specificity. These data illustrate the possibilities for using 3' UTR to regulate cell-specific transgene expression. Our improved K18 vector should prove useful for pediatric lung gene therapy applications.

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.