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Abstract Background Transforming growth factor-beta 1 (TGF-β1) has been implicated in hyperoxia-induced cell death and impaired alveolarization in the developing lung. In addition, the c-JunNH2-terminal kinase (JNK) pathway has been shown to have a role for TGF-β1-mediated effects. We hypothesized that the JNK pathway is an important regulator of hyperoxia-induced pulmonary responses in the developing murine lung. Results We used cultured human lung epithelial cells, fetal rat lung fibroblasts and a neonatal TGF-β1 transgenic mouse model. We demonstrate that hyperoxia inhibits cell proliferation, activates cell death mediators and causes cell death, and promotes myofibroblast transdifferentiation, in a dose-dependent manner. Except for fibroblast proliferation, the effects were mediated via the JNK pathway. In addition, since we observed increased expression of TGF-β1 by epithelial cells on exposure to hyperoxia, we used a TGF-β1 transgenic mouse model to determine the role of JNK activation in TGF-β1 induced effects on lung development and on exposure to hyperoxia. We noted that, in this model, inhibition of JNK signaling significantly improved the spontaneously impaired alveolarization in room air and decreased mortality on exposure to hyperoxia. Conclusions When viewed in combination, these studies demonstrate that hyperoxia-induced cell death, myofibroblast transdifferentiation, TGF-β1- and hyperoxia-mediated pulmonary responses are mediated, at least in part, via signaling through the JNK pathway.

Speakers in this symposium presented examples of respiratory regulation that broadly illustrate principles of evolution from whole organ to genes. The swim bladder and lungs of aquatic and terrestrial organisms arose independently from a common primordial "respiratory pharynx" but not from each other. Pathways of lung evolution are similar between crocodiles and birds but a low compliance of mammalian lung may have driven the development of the diaphragm to permit lung inflation during inspiration. To meet the high oxygen demands of flight, bird lungs have evolved separate gas exchange and pump components to achieve unidirectional ventilation and minimize dead space. The process of "screening" (removal of oxygen from inspired air prior to entering the terminal units) reduces effective alveolar oxygen tension and potentially explains why nonathletic large mammals possess greater pulmonary diffusing capacities than required by their oxygen consumption. The "primitive" central admixture of oxygenated and deoxygenated blood in the incompletely divided reptilian heart is actually co-regulated with other autonomic cardiopulmonary responses to provide flexible control of arterial oxygen tension independent of ventilation as well as a unique mechanism for adjusting metabolic rate. Some of the most ancient oxygen-sensing molecules, i.e., hypoxia-inducible factor-1alpha and erythropoietin, are up-regulated during mammalian lung development and growth under apparently normoxic conditions, suggesting functional evolution. Normal alveolarization requires pleiotropic growth factors acting via highly conserved cell-cell signal transduction, e.g., parathyroid hormone-related protein transducing at least partly through the Wingless/int pathway. The latter regulates morphogenesis from nematode to mammal. If there is commonality among these diverse respiratory processes, it is that all levels of organization, from molecular signaling to structure to function, co-evolve progressively, and optimize an existing gas-exchange framework.

The physiological development and homeostasis of the lung alveolus is determined by the expression of peroxisome proliferator-activated receptor-[gamma] (PPAR-[gamma]) by the interstitial lipofibroblast. We have recently shown (Dasgupta C et al., Am J Physiol Lung Cell Mol Physiol 296: L103l-L1041, 2009.) that PPAR-[gamma] agonists administered postnatally accelerate lung maturation and prevent hyperoxia-induced lung injury. However, whether the same occurs antenatally is not known. The objective of this study was to test the hypothesis that the potent PPAR-[gamma] agonist rosiglitazone (RGZ), administered antenatally, enhances fetal lung maturation and protects against hyperoxia-induced neonatal lung injury. Sprague-Dawley rat dams were administered either diluent or RGZ (3 mg/kg), at late gestation, to determine its effect on lung maturation and on hyperoxia (95% [O.sub.2] exposure for 24 h)-induced neonatal lung injury. The lungs were examined for the expression of specific markers of alveolar development (surfactant proteins A and B, cholinephosphate cytidylyltransferase-[alpha], leptin receptor, triglyceride uptake, and [[sup.3]H]choline incorporation into saturated phosphatidylcholine) and injury/repair, in particular, the markers of transforming growth factor-[beta] signaling (activin receptor-like kinase-5, SMAD3, lymphoid enhancer factor-1, fibronectin, and calponin). Overall, antenatal RGZ accelerated lung maturation and blocked the inhibition of alveolar sacculation and septal wall thinning by hyperoxia. RGZ specifically stimulated the development of the alveolar epithelial type II cell, the lipofibroblast, and the vasculature. The increased expression of the transforming growth factor-[beta] intermediates, such as SMAD3 and lymphoid enhancer factor-1, implicated in hyperoxic lung injury, was also blocked by antenatal RGZ treatment. In conclusion, PPAR-[gamma] agonists can enhance fetal lung maturation and can effectively prevent hyperoxia-induced neonatal lung injury. lung development; peroxisome proliferator-activated receptor-[gamma]; surfactant; lipofibroblast; hyperoxia doi: 10.1152/ajplung.00240.2010.

Despite tremendous technological and therapeutic advances, bronchopulmonary dysplasia (BPD) remains a leading cause of respiratory morbidity in very low birth weight infants, and there are no effective preventive and/or therapeutic options. We have previously reported that hyperoxia-induced neonatal rat lung injury might be prevented by rosiglitazone (RGZ). Here, we characterize I) perturbations in wingless/Int (Wnt) and transforming growth factor (TGF)-[beta] signaling, and 2) structural aberrations in lung morphology following 7-day continuous in vivo hyperoxia exposure to neonatal rats. We also tested whether treatment of neonatal pups with RGZ, concomitant to hyperoxia, could prevent such aberrations. Our study revealed that hyperoxia caused significant upregulation of Wnt signaling protein markers lymphoid enhancer factor 1 (Lef-1) and [beta]-catenin and TGF-[beta] pathway transducers phosphorylated Smad3 and Smad7 proteins in whole rat lung extracts. These changes were also accompanied by upregulation of myogenic marker proteins [alpha]-smooth muscle actin ([alpha]-SMA) and calponin but significant downregulation of the lipogenic marker peroxisome proliferator-activated receptor-[gamma] (PPAR[gamma]) expression. These molecular perturbations were associated with reduction in alveolar septal thickness, radial alveolar count, and larger alveoli in the hyperoxia-exposed lung. These hyperoxia-induced molecular and morphological changes were prevented by systemic administration of RGZ, with lung sections appearing near normal. This is the first evidence that in vivo hyperoxia induces activation of both Wnt and TGF-[beta] signal transduction pathways in lung and of its near complete prevention by RGZ. Hyperoxia-induced arrest in alveolar development, a hallmark of BPD, along with these molecular changes strongly implicates these proteins in hyperoxia-induced lung injury. Administration of PPAR[gamma] agonists may thus be a potential strategy to attenuate hyperoxia-induced lung injury and subsequent BPD. bronchopulrnonary dysplasia; peroxisome proliferator-activated receptor-[gamma]; lung development; lung fibroblast

Chorioamnionitis alters lung development, resulting in a paradoxical decrease in the incidence of respiratory distress syndrome but an increase in the incidence of bronchopulmonary dysplasia (BPD). The mechanism(s) underlying this disparity in the pulmonary outcomes is not known. We hypothesized that specific alterations in alveolar epithelial-mesenchyreal interactions might explain this apparent disparity in the pulmonary outcome following chorioamnionitis. We determined the effects of lipopolysaccharide (LPS) on parathyroid hormone-related protein (PTHrP)-driven epithelial-mesenchymal interactions that are essential for normal lung development and homeostasis. Lung explants from embryonic day 19.5 Sprague-Dawley rat fetuses were treated with LPS with or without a PTHrP pathway agonist, prostaglandin [J.sub.2] ([PGJ.sub.2]). LPS treatment affected the production of proinflammatory cytokines and the expression of the key markers of the epithelial-mesenchymal paracrine interactions in a time-dependent manner. At 6 h, there was a significant increase in the expression of PTHrP and the other key markers of alveolar homeostasis without any significant effect on [alpha]-smooth muscle actin ([alpha]SMA). In contrast, at 72 h, there was a significant decrease in the expression of PTHrP and the other key markers of alveolar homeostasis accompanied by a significant increase in [alpha]SMA expression. The cytokine and molecular changes at 72 h were completely prevented by the concomitant treatment with [PGJ.sub.2]. We speculate that these data provide a likely mechanism for the acute stimulation of lung differentiation, accompanied paradoxically by BPD following chorioamnionitis, and suggest that by specifically targeting PTHrP signaling, the inflammation-induced molecular injury that is known to result in BPD can be prevented. bronchopulmonary dysplasia; chorioamnionitis; chronic lung disease; parathyroid hormone-related protein doi:10.1152/ajplung.00319.2006

We recently suggested that alveolar interstitial fibroblast-to-myofibroblast transdifferentiation may be a key mechanism underlying in utero nicotine-induced lung injury. However, the effects of in utero nicotine exposure on fetal alveolar type II (ATII) cells have not been fully determined. Placebo, nicotine (1 mg/kg), or nicotine (1 mg/kg) + the peroxisome proliferatoractivated receptor (PPAR)-[gamma] agonist prostaglandin [J.sub.2] ([PGJ.sub.2], 0.3 mg/kg) was administered intraperitoneally once daily to time-mated pregnant Sprague-Dawley rats from embryonic day 6 until their death on embryonic day 20. Fetal ATII cells were isolated, and ATII cell proliferation, differentiation (surfactant synthesis), and metabolism (metabolic profling with the stable isotope [[1,2.sup.-13][C.sub.2]]-D-glucose) were determined after nicotine exposure in utero or in vitro. In utero nicotine exposure significantly stimulated ATII cell proliferation, differentiation, and metabolism. Although the effects on ATII cell proliferation and metabolism were almost completely prevented by concomitant treatment with [PGJ.sub.2], the effects on surfactant synthesis were not. On the basis of in utero and in vitro data, we conclude that surfactant synthesis is stimulated by nicotine's direct effect on ATII cells, whereas cell proliferation and metabolism are affected via a paracrine mechanism(s) secondary to its effects on the adepithelial fibroblasts. These data provide evidence for direct and indirect effects of in utero nicotine exposure on fetal ATII cells that could permanently alter the 'developmental program' of the developing lung. More importantly, concomitant administration of PPAR-[gamma] agonists can effectively attenuate many of the effects of in utero exposure to nicotine on ATII cells. chronic lung disease; smoking; surfactant; fibroblast; peroxisome proliferator-activated receptor-[gamma]

We tested the hypothesis that in vitro nicotine exposure disrupts specific epithelial-mesenchymal paracrine signaling pathways and results in pulmonary interstitial lipofibroblast (LIF)-to-myofibroblast (MYF) transdifferentiation, resulting in altered pulmonary development and function. Studies were done to determine whether nicotine induces LIF-to-MYF transdifferentiation and to elucidate underlying molecular mechanism(s) involved and to determine whether nicotine-induced LIF-to-MYF transdifferentiation could be prevented by stimulating specific alveolar interstitial fibroblast lipogenic pathway. WI38 cells, a human embryonic pulmonary fibroblast cell line, were treated with nicotine with or without specific agonists of alveolar fibroblast lipogenic pathway, PTHrP, DBcAMP, or the potent PPAR[gamma] stimulant rosiglitazone (RGZ) for 7 days. Expression of key lipogenic and myogenic markers was examined by RT-PCR, Western hybridization, and immunohistochemistry. The effect of nicotine on triglyceride uptake by WI38 cells and PTHrP binding to its receptor was also determined. Finally, the effect of transfecting WI38 cells with a PPAR[gamma] expression vector on nicotine-induced LIF-to-MYF transdifferentiation was determined. Nicotine treatment resulted in significantly decreased expression of lipogenic and increased expression of myogenic markers in a dose-dependent manner, indicating nicotine-induced LIF-to-MYF transdifferentiation. This was accompanied by decreased PTHrP receptor binding to its receptor. The nicotine-induced LIF-to-MYF transdifferentiation was completely prevented by concomitant treatment with PTHrP, DBcAMP, RGZ, and by transiently overexpressing PPAR[gamma]. Our data suggest nicotine induces alveolar LIF-to-MYF transdifferentiation through a mechanism involving downregulation of lipogenic PTHrP-mediated, cAMP-dependent PKA signaling pathway, which can be prevented using specific molecular targets. Potential therapeutic implications of these observations against in utero nicotine-induced lung injury remain to be tested. chronic lung disease; lipofibroblast; myofibroblast; peroxisome proliferator-activated receptor [gamma]

Eligibility of ventilated preterm rabbit model to investigate extreme pulmonary immaturity at birth transition is unknown. By extending this model to early saccular stage of fetal lung development, we evaluated efficacy in survival, lung maturation, and underlying mechanisms of contemporary perinatal therapies. Pregnant New Zealand White rabbit does were given dexamethasone (DEX), or sham injection as control (NDEX), 48 and 24 h before delivery at gestational age (GA) of 25–28 days. At birth, newborn rabbits were anesthetized and randomly allocated to four groups receiving either surfactant or nonsurfactant for both DEX and NDEX, and mechanically ventilated within low tidal volumes. Ranges of time to maintain survival rate ≥ 50% in GA 25–28 days were 59–136, 138–259, 173–288, and 437 to ≥600 min, respectively, each across the four groups. The benefits of DEX and/or surfactant for survival were more obvious in GA 25–26 days, as judged by improved lung mechanics, lower lung injury scores, higher lung surfactant phospholipid pools, and surfactant protein mRNA expression, with DEX-surfactant combination being the most optimal for the outcome. In contrast, those of GA 27–28 days had variable but meaningful responses to the treatment. Cox regression analysis revealed GA, DEX, and surfactant being independently protective factors whereas pneumothorax was a risk factor. The extremely preterm rabbits at GA 25–26 days markedly responded to the perinatal therapies for longer survival, lung maturation and injury alleviation, and were relevant for study of preterm birth transition-associated morbidities and underlying mechanisms. [ABSTRACT FROM AUTHOR]