Peroxisome proliferator-activated receptor-g agonist treatment increases septation and angiogenesis and decreases airway hyperresponsiveness in a model of experimental neonatal chronic lung disease.

Chronic lung disease (CLD) affects premature newborns requiring supplemental oxygen and results in impaired lung development and subsequent airway hyperreactivity. We hypothesized that the maintenance of peroxisome proliferator-activated receptor gamma (PPARgamma) signaling is important for normal lung morphogenesis and treatment with PPARgamma agonists could protect against CLD and airway hyperreactivity (AHR) following chronic hyperoxic exposure. This was tested in an established hyperoxic murine model of experimental CLD. Newborn mice and mothers were exposed to room air (RA) or moderate hyperoxia (70% oxygen) for 10 days and fed a standard diet or chow impregnated with the PPARgamma agonist rosiglitazone (ROSI) for the duration of study. Following hyperoxic exposure (HE) animals were returned to RA until postnatal day (P) 13 or P41. The accumulation of ROSI in neonatal and adult tissue was confirmed by mass spectrometry. Analyses of body weight and lung histology were performed on P13 and P41 to localize and quantitate PPARgamma expression, determine alveolar and microvessel density, proliferation and alpha-smooth muscle actin (alpha-SMA) levels as a measure of myofibroblast differentiation. Microarray analyses were conducted on P13 to examine transcriptional changes in whole lung. Pulmonary function and airway responsiveness were analyzed at P55. ROSI treatment during HE preserved septation and vascular density. Key array results revealed ontogeny groups differentially affected by hyperoxia including cell cycle, angiogenesis, matrix, and muscle differentiation/contraction. These results were further confirmed by histological evaluation of myofibroblast and collagen accumulation. Late AHR to methacholine was present in mice following HE and attenuated with ROSI treatment. These findings suggest that rosiglitazone maintains downstream PPARgamma effects and may be beneficial in the prevention of severe CLD with AHR.

Vascular release of nonheme iron in perfused rabbit lungs.

In this study, we hypothesized that the lung actively releases excess iron into the circulation to regulate iron homeostasis. We measured nonheme iron (NHFe) in the perfusate of control isolated perfused rabbit lungs and lungs with ischemia-reperfusion (I/R) ventilated with normoxic (21% O(2)) or hypoxic (95% N(2)) gas mixtures. Some were perfused with bicarbonate-free (HEPES) buffer or treated with the anion exchange inhibitor DIDS. The control lungs released approximately 0.25 microg/ml of NHFe or 20% of the total lung NHFe into the vascular space that was not complexed with ferritin, transferrin, or lactoferrin or bleomycin reactive. The I/R lungs released a similar amount of NHFe during ischemia and some bleomycin-detectable iron during reperfusion. NHFe release was attenuated by approximately 50% in both control and ischemic lungs by hypoxia and by >90% in control lungs and approximately 60% in ischemic lungs by DIDS and HEPES. Reperfusion injury was not affected by DIDS or HEPES but was attenuated by hypoxia. These results indicate that biologically nonreactive nonheme iron is released rapidly by the lung into the vascular space via mechanisms that are linked to bicarbonate exchange. During prolonged ischemia, redox-active iron is also released into the vascular compartment by other mechanisms and may contribute to lung injury.

Secretion of extracellular superoxide dismutase in neonatal lungs.

Extracellular superoxide dismutase (EC-SOD), the only known enzymatic scavenger of extracellular superoxide, may modulate reactions of nitric oxide (NO) in the lungs by preventing reactions between superoxide and NO. The regulation of EC-SOD has not been examined in developing lungs. We hypothesize that EC-SOD plays a pivotal role in the response to increased oxygen tension and NO in the neonatal lung. This study characterizes rabbit EC-SOD and investigates the developmental regulation of EC-SOD activity, protein expression, and localization. Purified rabbit EC-SOD was found to have several unique biochemical attributes distinct from EC-SOD in other species. Rabbit lung EC-SOD contains predominantly uncleaved subunits that do not form disulfide-linked dimers. The lack of intersubunit disulfide bonds may contribute to the decreased heparin affinity and lower EC-SOD content in rabbit lung. EC-SOD activity in rabbit lungs is low before birth and increases soon after gestation. In addition, the enzyme is localized intracellularly in preterm and term rabbit lungs. Secretion of active EC-SOD into the extracellular compartment increases with age. The changes in EC-SOD localization and activity have implications for the neonatal pulmonary response to oxidative stress and the biological activity of NO at birth.

Hypoxia compared with normoxia alters the effects of nitric oxide in ischemia-reperfusion lung injury.

Because both the biosynthesis of nitric oxide (NO.) and its metabolic fate are related to molecular O2, we hypothesized that hypoxia would alter the effects of NO. during ischemia-reperfusion (IR) in the lung. In this study, buffer-perfused lungs from rabbits underwent either normoxic IR (AI), in which lungs were ventilated with 21% O2 during ischemia and reperfusion, or hypoxic IR (NI), in which lungs were ventilated with 95% N2 during ischemia followed by reoxygenation with 21% O2. Lung weight gain (WG) and pulmonary artery pressure (Ppa) were monitored continuously, and microvascular pressure (Pmv) was measured after reperfusion to calculate pulmonary vascular resistance. We found that both AI and NI produced acute lung injury, as shown by increased WG and Ppa during reperfusion. In AI, where perfusate PO2 was > 100 mmHg, the administration of the NO. synthase inhibitor N-nitro-L-arginine methyl ester (L-NAME) before ischemia worsened WG and Ppa. Pmv also increased, suggesting a hydrostatic mechanism involved in edema formation. The effects of L-NAME could be attenuated by giving L-arginine and exogenous NO. donors before ischemia or before reperfusion. Partial protection was also provided by superoxide dismutase. In contrast, lung injury in NI at perfusate PO2 of 25-30 mmHg was attenuated by L-NAME; this effect could be reversed by L-arginine. Exogenous NO. donors given either before ischemia or before reperfusion, however, did not increase lung injury. NO. production was measured by quantifying the total nitrogen oxides (NOx) accumulating in the perfusate. The average rate of NOx accumulation was greater in AI than in NI. We conclude that hypoxia prevented the protective effects of NO on AI lung injury. The effects of hypoxia may be related to lower NO. production relative to oxidant stress during IR and/or altered metabolic fates of NO.-mediated production of peroxynitrite by hypoxic ischemia.

Protection of perfused lung from oxidant injury by inhibitors of anion exchange.

Hyperoxic lung injury is enhanced in isolated perfused lungs (IPL) in the presence of L-arginine. Reactive O2 species such as superoxide anion (O2-.) produced during hyperoxia are known to react with nitric oxide to form the strong oxidant species peroxynitrite. The appearance of O2-. in red blood cell membranes in vitro and in buffer-perfused lung preparations can be inhibited by the stilbene compound 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) DIDS also inhibits anion exchange across the cell membrane regulated by a family of anion exchange proteins (AE). In this study, we hypothesized that anion exchange inhibitors would prevent lung injury from hyperoxia and L-arginine (O2 + L-Arg) by decreasing O2-. flux into the vascular space of the IPL. We found that both DIDS and a structurally distinct anion transport blocker, dipyridamole, protected the rabbit IPL from pulmonary hypertension and edema produced by O2 + L-Arg. The protective effect was associated with increased nitrite concentrations in the perfusate. Protection also was conferred when sodium bicarbonate in the perfusion buffer was replaced with either sodium thiosulfate or N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES). In lungs perfused with thiosulfate or HEPES-containing buffer, protection from O2 and L-arginine was also associated with diminished detection of reducing activity consistent with O2-. in the vascular space. Western blot analysis of lung protein and immunocytochemical staining of lung sections using antibodies against rabbit red blood cell AE1 and mouse gastric AE2 peptide showed that lung contains membrane protein antigenically similar to gastric AE2. These data suggest the possibility that inhibition of AE or other anion transporters may play an important role in mediating oxidative lung injury.