Patricia L. Ramsay

Physiological Regulation of Uteroglobin/CCSP Expression

Abstract: Uteroglobin/CCSP is expressed specifically in the Clara cells. This allows the gene to be used as a marker to identify the elements regulating the physiologic and cell‐specific expression of this gene. The regulation of UG/CCSP by IFN‐γ was shown to be at the level of the proximal promoter by the upregulation of HNF3β. This has allowed the determination of the factors responsible for the expression of UG/CCSP.

Dexamethasone Enhancement of Hyperoxic Lung Inflammation in Rats Independent of Adhesion Molecule Expression

Infants and adults on oxygen often are treated with glucocorticoids in an attempt to reduce lung inflammatory injury. However, glucocorticoids hasten the development of hyperoxic lung injury in some animal models. The purpose of this study was to test the hypothesis that dexamethasone alters the lung inflammatory responses to hyperoxia exposure. We studied male Sprague-Dawley rats, placing them in >95% oxygen immediately after administration of 0, 0.1, 1, or 10 mg/kg of dexamethasone. At 0, 24, or 48 hr of exposure to hyperoxia, extravascular lung water contents were measured, and lung inflammatory responses were assessed by lung myeloperoxidase activities, lung neutrophil counts, and lung expression of E-Selectin and intercellular adhesions molecule-1 (ICAM-1). Dexamethasone, independent of exposure to hyperoxia, led to marked increases in lung neutrophil counts, without increases in lung myeloperoxidase activities or increases in the expression of the adhesion molecules. Hyperoxia exposure also enhanced lung neutrophil accumulation, and extravascular lung water increased earlier in animals exposed to hyperoxia and dexamethasone than in those exposed to hyperoxia alone. In conclusion, the increase in lung neutrophils in dexamethasone-treated rats without enhanced expression of E-Selectin or intracellular adhesions molecule-1 suggests that dexamethasone leads to lung neutrophil accumulation by its effect on neutrophils. The more rapid development of hyperoxic lung injury associated with earlier lung neutrophil accumulation suggests that dexamethasone-induced lung neutrophil sequestration primes the lung for the development of hyperoxic lung injury and supports further the conclusion that lung inflammation contributes significantly to the development of hyperoxic lung injury.

CoASH and CoASSG Levels in Lungs of Hyperoxic Rats as Potential Biomarkers of Intramitochondrial Oxidant Stresses

Coenzyme A (CoASH) is compartmentalized preferentially in the mitochondria, and CoASH and its mixed disulfide with glutathione (CoASSG) undergo thiol/disulfide exchange reactions with glutathione (GSH) and glutathione disulfide (GSSG) in vitro. We measured CoASH and CoASSG in freeze-clamped lung tissues from Fischer-344 and Sprague-Dawley rats maintained in room air or exposed to >95% O2 for 48 h to test the hypothesis that oxidant stresses on lung thiol status would be observed in the CoASH/CoASSG redox couple, suggesting oxidant stress responses in the mitochondria. Lung tissue concentrations of CoASSG in the Fischer-344 rats declined from 0.89 ± 0.15 to 0.51 ± 0.13 nmol/g of lung after 48 h of hyperoxia. CoASH levels declined from 6.40 ± 0.84 to 3.0 ± 0.65 nmol/g of lung, and acetyl CoA levels also were lower in the lungs of animals exposed to hyperoxia. CoASH/CoASSG ratios were lower in animals exposed to hyperoxia, satisfying our previously defined criteria for an oxidant stress on this thiol/disulfide redox couple, but absolute CoASSG levels were not increased, as would be expected for oxidant stresses driven simply by increases in reactive oxygen species or other oxidants. Pulmonary edema was observed in the hyperoxic rats and accounted for some of the declines in CoASH concentrations, but CoASH contents per total lung also declined. Lung mitochondrial succinate dehydrogenase activities were not diminished in rats exposed to hyperoxia, indicating that the decreases in CoASH concentrations are not attributable to general destruction of lung mitochondria. Lung GSSG contents were greater in the hyperoxia animals, but GSH/GSSG ratios, which are dominated by extramitochondrial pools, did not decrease in these animals. The mechanisms responsible for, and the possible pathophysiologic consequences of, the decreases in lung CoASH concentrations are not evident from the data available at the present time, but the loss of more than half the tissue contents of CoASH is likely to generate additional metabolic effects that could have significant pathophysiologic consequences.

Increased soluble E-Selectin is associated with lung inflammation, and lung injury in hyperoxia-exposed rats

The pulmonary damage caused by prolonged exposure to high oxygen concentrations is accompanied by lung inflammation, which may contribute to the expression of hyperoxic lung injury. In turn, adhesion molecules are crucial for initiating inflammatory responses. The goal of the present study was to investigate the association of contents of soluble adhesion molecules in plasma or alveolar fluids of hyperoxic rats with lung expression of adhesion molecules, lung inflammation and lung injury. We exposed adult Sprague-Dawley rats to > 95% oxygen for up to 60 h and measured the contents of intercellular adhesion molecule-I (ICAM-I) and E-Selectin in plasma and lung tissue expression of the same molecules, and we assessed lung myeloperoxidase (MPO) activties and lung water contents as indices of lung inflammation and injury, respectively. We also assessed ICAM-I content in lavage samples, because ICAM-I may be shed from the alveolar epithelium. Lung water was elevated at 60 h of hyperoxia-exposure, and this effect was preceded by increases in lung MPO activities. Lung ICAM-I expression was more than doubled at 48 h, although soluble ICAM-I contents were not elevated in plasma or lavage. Soluble E-Selectin was increased by more than 50% at 24 h of hyperoxia-exposure, while lung expressions of E-Selectin were not increased until 48 h. The sequence of the events observed in the present studies suggests that E-Selectin contributes to lung inflammation in hyperoxia and the acceleration of lung injury immediately following the inflammatory response suggests a pivotal role for inflammation in this injury.

Dexamethasone enhances P-selectin mRNA expression in hyperoxic rat lungs.

Abstract.Objective and Design: To test the hypothesis that glucocorticoid administration would diminish the lung expression of P-selectin mRNA in hyperoxia-exposed rats.¶Animals: Adult male Sprague-Dawley rats were divided into 6 separate groups containing 10 to 13 animals per group.¶Treatment: Rats were dosed with 1 mg/kg of dexamethasone or vehicle only, ip. Immediately after dosing, animals were placed in >95% oxygen. Some animals were maintained in room air and are presented as 0 h of exposure to hyperoxia. Another group of animals was dosed with 10 mg/kg lipopolysaccharide (LPS) ip immediately after dosing with either dexamethasone or vehicle as above.¶Methods: At 24 or 48 h, lung samples were obtained, and lung weight to body weight ratios calculated. In the LPS studies, samples were obtained 4 h after LPS dosing. In a subset of animals, lung sections were hybridized for P-selectin mRNA. All data except for hybridizations were analyz ed with three-way ANOVA, with subsequent post-hoc testing. P-selectin hybridizations were quantified by counting the number of positive vessels per high-powered field, and subsequently analyzed by unpaired Students t-test. Immunohistochemical analyses for P-selectin expression were also performed to determine whether changes in P-selectin mRNA were associated with differences in protein expression. All data are expressed as means ± SEM.¶Results: Rats dosed with dexamethasone had higher lung/body weight ratios after 24 and 48 h of exposure to hyperoxia than did similarly exposed rats dosed only with vehicle (at 48 h, 0.87 ± 0.04 versus 0.65 ± 0.06, respectively, P<0.05). The higher ratios in hyperoxic animals dosed with dexamethasone were associated with much higher levels of lung expression for P-selectin mRNA than was observed in similarly exposed rats dosed with vehicle alone (at 48 h, 3.93 ± 1.02, versus 0.20 ± 0.06, respectively, P<0.01). In contrast dexamethasone dosing lead to lower lung P-selectin mRNA expression in animals exposed to LPS (1.23 ± 1.08 in dexamethasone dosed animals versus 6.80 ± 0.92 in vehicle only dosed animals). Consistent with the mRNA data, P-selectin immunoreactivity increased as a function of hyperoxia-exposure time in animals dosed with dexamethasone, while immunoreactivity decreased as a function of hyperoxia-exposure time in animals dosed with vehicle only.¶Conclusions: Increased P-selectin mRNA combined with increased P-selectin protein expression in animals exposed to hyperoxia and dosed with dexamethasone suggests that enhanced expression of P-selectin may contribute to the greater lung injury and inflammation caused by hyperoxia in rats treated with dexamethasone.

Attenuation in the Postnatal Expression of the Clara Cell Secreted Protein Is Associated with the Development of Bronchopulmonary Dysplasia

Attenuation in the Postnatal Expression of the Clara Cell Secreted Protein Is Associated with the Development of Bronchopulmonary Dysplasia

Protein Oxidation and Nitrotyrosine formation in Tracheal Aspirate fluids of Mechanically Ventilated Neonates exposed to Hyperoxia and/or Inhaled Nitric Oxide † 1182

Elevated O2 tensions are employed in the treatment of pulmonary insufficiency, and inhaled nitric oxide (NO) is used for treatment of pulmonary vascular disease. Although useful, both of these drugs are potentially reactive chemicals that may cause significant adverse effects, most probably through oxidation or nitration of critical tissue molecules. The purpose of the present study was to test the hypothesis that oxidized proteins, assessed by western analysis after derivatization with 2,4-dinitrophenylhydrazine (DNPH), or nitrotyrosine-containing proteins could function as biomarkers of the respective oxidations. Identification of the specific proteins and modifications would provide important insight into the mechanisms responsible and the biomarkers could be used to guide efforts to minimize the adverse effects of these therapies. Three groups of infants born at greater than 36 wk and mechanically ventilated were studied. Infants were ventilated with: 1) 60% O2; and 3) NO, which is preceded by 100% O2. Tracheal aspirate fluids were obtained by suctioning, at times indicated clinically, and aliquots were frozen at -70oC and analyzed by treatment with DNPH, separation by SDS-PAGE, and probed with anti-DNP antibodies. Anti-nitrotyrosine antibodies were employed in samples not treated with DNPH. The major fractions of both modes of protein oxidation were observed uniformly in proteins migrating at molecular weights >87 kDa. In infants ventilated with hyperoxia, the anti-nitrotyrosine-reactive proteins were higher than in infants ventilated with room air in initial samples, but levels in the former group fell during the first week of life, whereas in the latter group the levels did not change markedly. Surprisingly, infants exposed to inhaled NO studied to date showed no distinctly greater levels or differences in distribution of nitrated proteins. The highest levels were observed in the initial sample from each child, with decreases through the first week of life. In infants that required longer ventilation, increased levels of DNPH-reactive proteins were observed. These studies indicate that NO therapy in neonates does not necessarily increase protein oxidation or nitration markedly, and that prolonged mechanical ventilation may contribute significantly to protein oxidation in these infants.

Soluble E-Selectin (sE-Selectin) is Increased at 24 Hours in Premature Baboons that Develop Bronchopulmonary Dysplasia (BPD) 1728

Lung inflammation is a major factor in the development of BPD. Previously, we have found that serum levels of the inflammatory adhesion molecule, sE-Selectin, was higher on day of life one in premature infants that develop BPD than in infants that do not. The goal in this study was to test the hypothesis that sE-Selectin concentrations would increase early in the baboon model of BPD. We measured serum concentrations of sE-Selectin and sP-Selectin at delivery, and at 24, 72 and 120h after delivery in baboons delivered at 75% term gestation. The animals were maintained on mechanical ventilation and supplemental oxygen to maintain normal arterial blood gas values throughout the experimental period. We found that serum levels of sE-Selectin were higher at 24h than at birth and the levels remained increased through 120h. In contrast, we found no differences in sP-Selectin through 120h. These data are similar to findings in premature humans, and suggest that sE-Selectin may be useful as an indicator for developing BPD. In addition, the similarity of the premature human and baboon soluble adhesion molecule profiles in developing BPD, suggests that the mechanisms of lung inflammation may be similar, and that further studies in the baboon model of BPD may provide useful insights into mechanisms of lung inflammation in both the baboon BPD model, and in premature infants.

SOLUBLE E-SELECTIN: AN EARLY PROGNOSTIC INDICATOR FOR THE DEVELOPMENT OF BRONCHOPULMONARY DYSPLASIA |[dagger]| 1014

Bronchopulmonary dysplasia (BPD) continues to be a major complication in prematurely born infants. Early recognition of infants at greatest risk for BPD would allow intervention prior to the development of lung injury. We have reported that E-Selectin, an inflammatory adhesion molecule, increased in the lungs and serum (sE-Selectin) of animals exposed to hyperoxia. We tested the hypothesis that sE-Selectin would increase in infants at risk for the development of BPD. We measured sE-Selectin in cord blood and in samples on days 1, 3, and 7 from neonates born at ≤29 wks gestation. The infants were evaluated at one month of life to determine whether they met criteria for the diagnosis of BPD, characterized as mild (stage 1-2) or severe (stage 3-4) by the Northway radiographic stages of BPD. Table

THE EFFECTS OF DEXAMETHASONE (DEX) AND HYPEROXIA ON THE GLUTATHIONE (GSH) SYSTEM IN RATS. † 2065

Deficiencies in antioxidant defense mechanisms within the lungs of premature infants are generally accepted as predisposing to the development of bronchopulmonary dysplasia (BPD). Pharmacological management of premature infants often utilizes the glucocorticoid, dexamethasone. Investigators have shown, however, that DEX accelerates hyperoxia-induced lung injury, a critical factor in the development of BPD. The purpose of this study was to determine the effect of DEX and hyperoxia on the critical intracellular antioxidant GSH. We administered 1 mg/kg DEX or vehicle ip and immediately placed rats in>95% O2. At 0, 24, and 48 h of hyperoxia-exposure, lung, liver and plasma GSH, as well as lung and liver glutathione disulfide (GSSG) were measured. The data were analyzed by two way ANOVA to determine the effect of DEX and hyperoxia, and a modified t test when appropriate. Lung GSH increased in the hyperoxia-exposed rats independent of DEX treatment, while lung GSSG increased in both hyperoxia-exposed and DEX-treated rats. We found no differences in plasma GSH. The earlier increase in lung GSSG suggests that DEX increases the oxidant stress experienced by the lung, and that the possible deleterious effects of glucocorticoid treatment on the oxidant/antioxidant balance should be considered in infants exposed to hyperoxia.Table