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1.  Sex-Specific Perinatal Nicotine-Induced Asthma in Rat Offspring 
Recently, we have suggested that down-regulation of homeostatic mesenchymal peroxisome proliferator–activated receptor γ signaling after in utero nicotine exposure might contribute to asthma. Here, we have exploited an in vivo rat model of asthma to determine if the effects of perinatal nicotine exposure on offspring pulmonary function and mesenchymal markers of airway contractility in both tracheal and lung parenchymal tissue are sex specific, and whether the protection afforded by the peroxisome proliferator–activated receptor γ agonist, rosiglitazone (RGZ), against the perinatal nicotine-induced effect on offspring lung is also sex specific. Pregnant rat dams received placebo, nicotine, or nicotine plus RGZ daily from Embryonic Day 6 until Postnatal Day 21, at which time lung resistance, compliance, tracheal contractility, and the expression of structural and functional mesenchymal markers of pulmonary contractility were determined. Compared with control animals, perinatal nicotine exposure caused a significant increase in airway resistance and a decrease in airway compliance after a methacholine challenge in both male and female offspring, with more pronounced changes in the males. In contrast to this, the effects of perinatal nicotine exposure on acetylcholine-induced tracheal constriction, along with the expression of its mesenchymal markers, were observed exclusively in the male offspring. Concomitant treatment with RGZ normalized the nicotine-induced alterations in pulmonary function in both sexes, as well as the male-specific effects on acetylcholine-induced tracheal constriction, along with the affected mesenchymal markers. These data suggest that perinatal nicotine exposure causes sex-specific perinatal cigarette smoke exposure–induced asthma, providing a powerful phenotypic model for unequivocally determining the underlying nature of the cell molecular mechanism for this disease.
doi:10.1165/rcmb.2011-0344OC
PMCID: PMC3547089  PMID: 23002101
nicotine; asthma; pregnancy; peroxisome proliferator–activated receptor γ; cigarette smoke
2.  Neonatology Specialty Grand Challenge 
doi:10.3389/fped.2013.00013
PMCID: PMC3860889  PMID: 24400259
3.  Postnatal Rosiglitazone Administration to Neonatal Rat Pups Does Not Alter the Young Adult Metabolic Phenotype 
Neonatology  2011;101(3):217-224.
Background
Rosiglitazone (RGZ), a peroxisome proliferator-activated receptor-γ (PPARγ) agonist, significantly enhances lung maturation without affecting blood biochemical and metabolic profiles in the newborn period. However, whether this exposure to RGZ in neonatal life alters the adult metabolic phenotype is not known.
Objective
To determine the effects of early postnatal administration of RGZ on the young adult metabolic phenotype.
Methods
Newborn rat pups were administered either saline or RGZ for the first 7 days of life. At 11–14 weeks, glucose and insulin tolerance tests and deuterium labeling were performed. Blood and tissues were analyzed for various metabolic parameters.
Results
Overall, there was no effect of early postnatal RGZ administration on young adult body weight, glucose and insulin tolerance, plasma cholesterol and triglyceride profiles, insulin, glucagon, cardiac troponin, fatty acid synthesis, or tissue adipogenic differentiation.
Conclusions
Treatment with RGZ in early neonatal life does not alter later developmental metabolic programming or lead to an altered metabolic phenotype in the young adult, further re-enforcing the safety of PPARγ agonists as a novel lung-protective strategy.
doi:10.1159/000331772
PMCID: PMC3388271  PMID: 22076469
Rosiglitazone; Lung development; Peroxisome proliferator-activated receptor-γ; Fetal programming
4.  EFFECTS OF MATERNAL FOOD RESTRICTION ON FETAL LUNG EXTRACELLULAR MATRIX DEPOSITION AND LONG TERM PULMONARY FUNCTION IN AN EXPERIMENTAL RAT MODEL 
Pediatric Pulmonology  2011;47(2):162-171.
Intrauterine growth restriction (IUGR) increases the risk of respiratory compromise throughout postnatal life. However, the molecular mechanism(s) underlying the respiratory compromise in offspring following IUGR is not known. We hypothesized that IUGR following maternal food restriction (MFR) would affect extracellular matrix deposition in the lung, explaining the long-term impairment in pulmonary function in the IUGR offspring. Using a well-established rat model of MFR during gestation to produce IUGR pups, we found that at postnatal day 21, and at 9 months of age the expression and abundance of elastin and alpha smooth muscle actin (αSMA), two key extracellular matrix proteins, were increased in IUGR lungs when compared to controls (p<0.05, n = 6), as determined by both Western and immunohistochemistry analyses. Compared to controls, the MFR group showed no significant change in pulmonary resistance at baseline, but did have significantly decreased pulmonary compliance at 9 months (p<0.05 vs control, n=5). In addition, MFR lungs exhibited increased responsiveness to methacholine challenge. Furthermore, exposing cultured fetal rat lung fibroblasts to serum deprivation increased the expression of elastin and elastin-related genes, which was blocked by serum albumin supplementation, suggesting protein deficiency as the predominant mechanism for increased pulmonary elastin deposition in IUGR lungs. We conclude that accompanying the changes in lung function, consistent with bronchial hyperresponsiveness, expression of the key alveolar extracellular matrix proteins elastin and αSMA increased in the IUGR lung, thus providing a potential explanation for the compromised lung function in IUGR offspring.
doi:10.1002/ppul.21532
PMCID: PMC3258334  PMID: 22058072
intrauterine growth restriction; maternal food restriction; extracellular matrix; elastin; α smooth muscle actin; pulmonary function
5.  A Cell–Molecular Approach Predicts Vertebrate Evolution 
Molecular Biology and Evolution  2011;28(11):2973-2981.
In contrast to the conventional use of genes to determine the evolution of phenotypes, we have functionally integrated epithelial–mesenchymal interactions that have facilitated lung phylogeny and ontogeny in response to major geologic epochs. As such, this model reveals the underlying principles of lung physiology based on the evolutionary interactions between internal and external selection pressures, providing a novel understanding of lung biology. As a result, it predicts how cell–molecular changes in this process can cause disease and offers counterintuitive insights to diagnosis and treatment based on evolutionary principles.
doi:10.1093/molbev/msr134
PMCID: PMC3199438  PMID: 21593047
lung; phylogeny; ontogeny; ecology; mesenchymal–epithelial interaction; selection pressure
6.  Mechanism of Reduced Lung Injury by High Frequency Nasal Ventilation in a Preterm Lamb Model of Neonatal Chronic Lung Disease 
Pediatric research  2011;70(5):462-466.
The mechanism underlying the potentially beneficial effects of the “gentler” modes of ventilation on chronic lung disease (CLD) of the premature infant is not known. We have previously demonstrated that alveolar Parathyroid Hormone-related Protein-Peroxisome Proliferator-Activated Receptorγ (PTHrP-PPARγ) signaling is critically important in alveolar formation, and this signaling pathway is disrupted in hyperoxia- and/or volutrauma-induced neonatal rat lung injury. Whether the same paradigm is also applicable to CLD, resulting from prolonged intermittent mandatory ventilation (IMV), and whether differential effects of the mode of ventilation on the PTHrP-PPARγ signaling pathway explain the potential benefits of the “gentler” modes of ventilation are not known. Using a well-established preterm lamb model of neonatal CLD, we tested the hypothesis that ventilatory support using high-frequency nasal ventilation (HFNV) promotes alveolar PTHrP-PPARγ signaling, whereas IMV inhibits it. Preterm lambs managed by HFNV or IMV for 21 days following preterm delivery at 132-day gestation were studied by Western hybridization and immunofluorescence labeling for key markers of alveolar homeostasis and injury/repair. In lambs managed by IMV, the abundance of key homeostatic alveolar epithelial-mesenchymal markers was reduced, whereas it was significantly increased in the HFNV group, providing a potential molecular mechanism by which “gentler” modes of ventilation reduce neonatal CLD.
doi:10.1203/PDR.0b013e31822f58a1
PMCID: PMC3189277  PMID: 21814155
7.  Perinatal nicotine exposure induces asthma in second generation offspring 
BMC Medicine  2012;10:129.
Background
By altering specific developmental signaling pathways that are necessary for fetal lung development, perinatal nicotine exposure affects lung growth and differentiation, resulting in the offsprings' predisposition to childhood asthma; peroxisome proliferator-activated receptor gamma (PPARγ) agonists can inhibit this effect. However, whether the perinatal nicotine-induced asthma risk is restricted to nicotine-exposed offspring only; whether it can be transmitted to the next generation; and whether PPARγ agonists would have any effect on this process are not known.
Methods
Time-mated Sprague Dawley rat dams received either placebo or nicotine (1 mg/kg, s.c.), once daily from day 6 of gestation to postnatal day (PND) 21. Following delivery, at PND21, generation 1 (F1) pups were either subjected to pulmonary function tests, or killed to obtain their lungs, tracheas, and gonads to determine the relevant protein markers (mesenchymal contractile proteins), global DNA methylation, histone 3 and 4 acetylation, and for tracheal tension studies. Some F1 animals were used as breeders to generate F2 pups, but without any exposure to nicotine in the F1 pregnancy. At PND21, F2 pups underwent studies similar to those performed on F1 pups.
Results
Consistent with the asthma phenotype, nicotine affected lung function in both male and female F1 and F2 offspring (maximal 250% increase in total respiratory system resistance, and 84% maximal decrease in dynamic compliance following methacholine challenge; P < 0.01, nicotine versus control; P < 0.05, males versus females; and P > 0.05, F1 versus F2), but only affected tracheal constriction in males (51% maximal increase in tracheal constriction following acetylcholine challenge, P < 0.01, nicotine versus control; P < 0.0001, males versus females; P > 0.05, F1 versus F2); nicotine also increased the contractile protein content of whole lung (180% increase in fibronectin protein levels, P < 0.01, nicotine versus control, and P < 0.05, males versus females) and isolated lung fibroblasts (for example, 45% increase in fibronectin protein levels, P < 0.05, nicotine versus control), along with decreased PPARγ expression (30% decrease, P < 0.05, nicotine versus control), but only affected contractile proteins in the male trachea (P < 0.05, nicotine versus control, and P < 0.0001, males versus females). All of the nicotine-induced changes in the lung and gonad DNA methylation and histone 3 and 4 acetylation were normalized by the PPARγ agonist rosiglitazone except for the histone 4 acetylation in the lung.
Conclusions
Germline epigenetic marks imposed by exposure to nicotine during pregnancy can become permanently programmed and transferred through the germline to subsequent generations, a ground-breaking finding that shifts the current asthma paradigm, opening up many new avenues to explore.
doi:10.1186/1741-7015-10-129
PMCID: PMC3568737  PMID: 23106849
nicotine; lung; epigenetic; asthma; multigenerational; gender difference
8.  Prenatal Rosiglitazone Administration to Neonatal Rat Pups Does Not Alter the Adult Metabolic Phenotype 
PPAR Research  2012;2012:604216.
Prenatally administered rosiglitazone (RGZ) is effective in enhancing lung maturity; however, its long-term safety remains unknown. This study aimed to determine the effects of prenatally administered RGZ on the metabolic phenotype of adult rats. Methods. Pregnant Sprague-Dawley rat dams were administered either placebo or RGZ at embryonic days 18 and 19. Between 12 and 20 weeks of age, the rats underwent glucose and insulin tolerance tests and de novo fatty acid synthesis assays. The lungs, liver, skeletal muscle, and fat tissue were processed by Western hybridization for peroxisome proliferator-activated receptor (PPAR)γ, adipose differentiation-related protein (ADRP), and surfactant proteins B (SPB) and C (SPC). Plasma was assayed for triglycerides, cholesterol, insulin, glucagon, and troponin-I levels. Lungs were also morphometrically analyzed. Results. Insulin and glucose challenges, de novo fatty acid synthesis, and all serum assays revealed no differences among all groups. Western hybridization for PPARγ, ADRP, SPB, and SPC in lung, liver, muscle, and fat tissue showed equal levels. Histologic analyses showed a similar number of alveoli and septal thickness in all experimental groups. Conclusions. When administered prenatally, RGZ does not affect long-term fetal programming and may be safe for enhancing fetal lung maturation.
doi:10.1155/2012/604216
PMCID: PMC3398645  PMID: 22829803
9.  PPARγ Signaling Mediates the Evolution, Development, Homeostasis, and Repair of the Lung 
PPAR Research  2012;2012:289867.
Epithelial-mesenchymal interactions mediated by soluble growth factors determine the evolution of vertebrate lung physiology, including development, homeostasis, and repair. The final common pathway for all of these positively adaptive properties of the lung is the expression of epithelial parathyroid-hormone-related protein, and its binding to its receptor on the mesenchyme, inducing PPARγ expression by lipofibroblasts. Lipofibroblasts then produce leptin, which binds to alveolar type II cells, stimulating their production of surfactant, which is necessary for both evolutionary and physiologic adaptation to atmospheric oxygen from fish to man. A wide variety of molecular insults disrupt such highly evolved physiologic cell-cell interactions, ranging from overdistention to oxidants, infection, and nicotine, all of which predictably cause loss of mesenchymal peroxisome-proliferator-activated receptor gamma (PPARγ) expression and the transdifferentiation of lipofibroblasts to myofibroblasts, the signature cell type for lung fibrosis. By exploiting such deep cell-molecular functional homologies as targets for leveraging lung homeostasis, we have discovered that we can effectively prevent and/or reverse the deleterious effects of these pathogenic agents, demonstrating the utility of evolutionary biology for the prevention and treatment of chronic lung disease. By understanding mechanisms of health and disease as an evolutionary continuum rather than as dissociated processes, we can evolve predictive medicine.
doi:10.1155/2012/289867
PMCID: PMC3390135  PMID: 22792087
10.  Lung Organogenesis 
Developmental lung biology is a field that has the potential for significant human impact: lung disease at the extremes of age continues to cause major morbidity and mortality worldwide. Understanding how the lung develops holds the promise that investigators can use this knowledge to aid lung repair and regeneration. In the decade since the “molecular embryology” of the lung was first comprehensively reviewed, new challenges have emerged—and it is on these that we focus the current review. Firstly, there is a critical need to understand the progenitor cell biology of the lung in order to exploit the potential of stem cells for the treatment of lung disease. Secondly, the current familiar descriptions of lung morphogenesis governed by growth and transcription factors need to be elaborated upon with the reinclusion and reconsideration of other factors, such as mechanics, in lung growth. Thirdly, efforts to parse the finer detail of lung bud signaling may need to be combined with broader consideration of overarching mechanisms that may be therapeutically easier to target: in this arena, we advance the proposal that looking at the lung in general (and branching in particular) in terms of clocks may yield unexpected benefits.
doi:10.1016/S0070-2153(10)90003-3
PMCID: PMC3340128  PMID: 20691848
11.  MECHANISM FOR NICOTINE-INDUCED UP-REGULATION OF WNT SIGNALING IN HUMAN ALVEOLAR INTERSTITIAL FIBROBLASTS 
Experimental lung research  2010;37(3):144-154.
Nicotine exposure alters normal homeostatic pulmonary epithelial-mesenchymal paracrine signaling pathways, resulting in alveolar interstitial fibroblast (AIF)-to-myofibroblast (MYF) transdifferentiation. Since the AIF vs MYF phenotype is determined by the expression of Peroxisome Proliferator-Activated Receptor (PPAR)γ and Wingless/Int (Wnt) signaling, respectively, we hypothesized that nicotine-induced AIF-to-MYF transdifferentiation is characterized by the down-regulation of PPARγ, and the up-regulation of the Wnt signaling pathway. As nicotine is known to activate PKC signaling, we also hypothesized that in AIFs, nicotine-induced up-regulation of Wnt signaling might be due to PKC activation. Embryonic human lung fibroblasts (WI38 cells) were treated with nicotine (1 × 10−6M) for either 30 minutes or 24 hours, with or without 30 minute pretreatment with calphostin C (1 × 10−7), a pan-PKC inhibitor. Then we examined the activation of PKC (p-PKC) and Wnt signaling (p-GSK-3β, β-catenin, LEF-1, and fibronectin). Furthermore, activation of nicotinic acetylcholine receptors (nAChR)-α3 and −α7, and whether a PPARγ agonist, Rosiglitazone, blocks nicotine-mediated Wnt activation were examined. Following nicotine stimulation, there was clear evidence for nAChR-α3 and −α7 up-regulation, accompanied by the activation of PKC and Wnt signaling, which was further accompanied by significant changes in the expression of the down-stream targets of Wnt signaling at 24h. Nicotine-mediated Wnt activation was almost completely blocked by pretreatment with either calphostin C or RGZ, indicating the central involvement of PKC activation and Wnt/PPARγ interaction in nicotine-induced up-regulation of Wnt signaling, and hence AIF-to-MYF transdifferentiation, providing novel preventive/therapeutic targets for nicotine-induced lung injury.
doi:10.3109/01902148.2010.490288
PMCID: PMC3062662  PMID: 21133803
Chronic lung disease; Lipofibroblast; Myofibroblast; Nicotine; Peroxisome Proliferator-Activated Receptorγ; Wnt Signaling
12.  A Role for Wnt Signaling Genes in the Pathogenesis of Impaired Lung Function in Asthma 
Rationale: Animal models demonstrate that aberrant gene expression in utero can result in abnormal pulmonary phenotypes.
Objectives: We sought to identify genes that are differentially expressed during in utero airway development and test the hypothesis that variants in these genes influence lung function in patients with asthma.
Methods: Stage 1 (Gene Expression): Differential gene expression analysis across the pseudoglandular (n = 27) and canalicular (n = 9) stages of human lung development was performed using regularized t tests with multiple comparison adjustments. Stage 2 (Genetic Association): Genetic association analyses of lung function (FEV1, FVC, and FEV1/FVC) for variants in five differentially expressed genes were conducted in 403 parent-child trios from the Childhood Asthma Management Program (CAMP). Associations were replicated in 583 parent-child trios from the Genetics of Asthma in Costa Rica study.
Measurements and Main Results: Of the 1,776 differentially expressed genes between the pseudoglandular (gestational age: 7–16 wk) and the canalicular (gestational age: 17–26 wk) stages, we selected 5 genes in the Wnt pathway for association testing. Thirteen single nucleotide polymorphisms in three genes demonstrated association with lung function in CAMP (P < 0.05), and associations for two of these genes were replicated in the Costa Ricans: Wnt1-inducible signaling pathway protein 1 with FEV1 (combined P = 0.0005) and FVC (combined P = 0.0004), and Wnt inhibitory factor 1 with FVC (combined P = 0.003) and FEV1/FVC (combined P = 0.003).
Conclusions: Wnt signaling genes are associated with impaired lung function in two childhood asthma cohorts. Furthermore, gene expression profiling of human fetal lung development can be used to identify genes implicated in the pathogenesis of lung function impairment in individuals with asthma.
doi:10.1164/rccm.200907-1009OC
PMCID: PMC2822972  PMID: 19926868
asthma; lung development; lung function; genetic variation; gene expression
13.  Mechanisms of impaired nephrogenesis with fetal growth restriction: altered renal transcription and growth factor expression 
Objective
Maternal food restriction during pregnancy results in growth restricted newborns and reduced glomerular number, contributing to programmed offspring hypertension. We investigated whether reduced nephrogenesis may be programmed by dysregulation of factors controlling ureteric bud branching and mesenchyme to epithelial transformation.
Study Design
10 to 20 days gestation, Sprague Dawley pregnant rats (n=6/group) received ad libitum food; FR rats were 50% food restricted. At embryonic day 20, mRNA and protein expression of WT1, Pax2, FGF2, GDNF, cRET, WNT4, WNT11, BMP4, BMP7, and FGF7 were determined by real-time PCR and Western blotting.
Results
Maternal FR resulted in up-regulated mRNA expression for WT1, FGF2, and BMP7 whereas Pax2, GDNF, FGF7, BMP4, WNT4, and WNT11 mRNAs were down-regulated. Protein expression was concordant for WT1, GDNF, Pax2, FGF7, BMP4 and WNT4.
Conclusion
Maternal FR altered gene expression of fetal renal transcription and growth factors, and likely contributes to development of offspring hypertension.
doi:10.1016/j.ajog.2008.05.018
PMCID: PMC2932650  PMID: 18639218
Branching morphogenesis; Fetal programming; Hypertension; Maternal food restriction; Metanephric development
14.  The Effects of Smoking on the Developing Lung: Insights from a Biologic Model for Lung Development, Homeostasis, and Repair 
Lung  2009;187(5):281-289.
There is extensive epidemiologic and experimental evidence from both animal and human studies that demonstrates detrimental long-term pulmonary outcomes in the offspring of mothers who smoke during pregnancy. However, the molecular mechanisms underlying these associations are not understood. Therefore, it is not surprising that that there is no effective intervention to prevent the damaging effects of perinatal smoke exposure. Using a biologic model of lung development, homeostasis, and repair, we have determined that in utero nicotine exposure disrupts specific molecular paracrine communications between epithelium and interstitium that are driven by parathyroid hormone-related protein and peroxisome proliferator-activated receptor (PPAR)γ, resulting in transdifferentiation of lung lipofibroblasts to myofibroblasts, i.e., the conversion of the lipofibroblast phenotype to a cell type that is not conducive to alveolar homeostasis, and is the cellular hallmark of chronic lung disease, including asthma. Furthermore, we have shown that by molecularly targeting PPARγ expression, nicotine-induced lung injury can not only be significantly averted, it can also be reverted. The concept outlined by us differs from the traditional paradigm of teratogenic and toxicological effects of tobacco smoke that has been proposed in the past. We have argued that since nicotine alters the normal homeostatic epithelial-mesenchymal paracrine signaling in the developing alveolus, rather than causing totally disruptive structural changes, it offers a unique opportunity to prevent, halt, and/or reverse this process through targeted molecular manipulations.
doi:10.1007/s00408-009-9158-2
PMCID: PMC2928656  PMID: 19641967
Nicotine; Lung development; Tobacco; Smoking; Chronic obstructive pulmonary disease; Chronic lung disease
15.  PEROXISOME PROLIFERATOR-ACTIVATED RECEPTOR (PPAR) γ AGONISTS ENHANCE LUNG MATURATION IN A NEONATAL RAT MODEL 
Pediatric research  2009;65(2):150-155.
The nuclear transcription factor Peroxisome Proliferator-Activated Receptor (PPAR) γ plays a central role in normal lung development. However, the effects of modulating PPARγ expression by exogenously administered PPARγ agonists on lung development and basic blood biochemical and metabolic profiles in a developing animal are not known. To determine these effects, newborn Sprague-Dawley rat pups were administered either diluent or rosiglitazone (RGZ), a potent PPARγ agonist, for either 1 or 7 days. Then the pups were sacrificed and the lungs were examined for specific markers of alveolar epithelial, mesenchymal, and vascular maturation, and lung morphometry. The effect of RGZ on a limited number of blood biochemical and metabolic parameters was also determined. Overall, systemically administered RGZ significantly enhanced lung maturation without affecting serum electrolytes, blood glucose, blood gases, plasma cholesterol, triglycerides, and serum cardiac troponin levels. The lung maturation effect of PPARγ agonists was also confirmed by another PPARγ agonist, the naturally occurring PPARγ ligand prostaglandin J2. We conclude that systemically administered RGZ significantly enhances lung maturation without significantly affecting the acute blood biochemical and metabolic profiles, providing rationale for further studying PPARγ agonists for enhancing lung maturation, and for promoting lung injury/repair in neonates.
doi:10.1203/PDR.0b013e3181938c40
PMCID: PMC2921215  PMID: 19262292
Bronchopulmonary Dysplasia; Chronic Lung Injury; Lung maturity; PPARγ; Rosiglitazone
16.  Effect of Maternal Food Restriction on Fetal Lung Lipid Differentiation Program 
Pediatric pulmonology  2009;44(7):635-644.
Summary
Although “fetal programming” has been extensively studied in many organs, there is only limited information on pulmonary effects in the offspring following intrauterine growth restriction (IUGR). We aimed to determine the effects of nutrient restriction on the lung structure and lung lipid differentiation programs in offspring using an animal mode of maternal food restriction (MFR). We utilized a rodent model of 50% MFR from day 10 of gestation to term and then using lung morphology, Western blotting, Real Time- RT-PCR and oil red O staining, lung structure and development of the offspring were examined at postnatal days (p) 1, p21, and 9 months (9M). At postnatal day 1, MFR pups weighed significantly less compared to control pups, but at p21 and 9M, they weighed significantly more. However, lung weight, expressed as a percentage of body weight between the two groups was not different at all time-points examined. The MFR group had significantly decreased alveolar number and significantly increased septal thickness at p1 and 9M, indicating significantly altered lung structure in the MFR offspring. Furthermore, although at p1, compared to the control group, lung lipid accumulation was significantly decreased in the MFR group, at 9M, it was significantly increased. There were significant temporal changes in the Parathyroid Hormone-related Protein/Peroxisome Proliferator-Activated Receptor gamma signaling pathway and surfactant synthesis. We conclude that MFR alters fetal lung lipid differentiation programming and lung morphometry by affecting specific epithelial-mesenchymal signaling pathways, offering the possibility for specific interventions to overcome these effects.
doi:10.1002/ppul.21030
PMCID: PMC2919756  PMID: 19514059
Maternal food restriction; fetal lung development; lipid differentiation program; fetal programming; PTHrP; PPARγ
17.  Cell–cell signaling drives the evolution of complex traits: introduction—lung evo-devo 
Physiology integrates biology with the environment through cell–cell interactions at multiple levels. The evolution of the respiratory system has been “deconvoluted” (Torday and Rehan in Am J Respir Cell Mol Biol 31:8–12, 2004) through Gene Regulatory Networks (GRNs) applied to cell–cell communication for all aspects of lung biology development, homeostasis, regeneration, and aging. Using this approach, we have predicted the phenotypic consequences of failed signaling for lung development, homeostasis, and regeneration based on evolutionary principles. This cell–cell communication model predicts other aspects of vertebrate physiology as adaptational responses. For example, the oxygen-induced differentiation of alveolar myocytes into alveolar adipocytes was critical for the evolution of the lung in land dwelling animals adapting to fluctuating Phanarezoic oxygen levels over the past 500 million years. Adipocytes prevent lung injury due to oxygen radicals and facilitate the rise of endothermy. In addition, they produce the class I cytokine leptin, which augments pulmonary surfactant activity and alveolar surface area, increasing selection pressure for both respiratory oxygenation and metabolic demand initially constrained by high-systemic vascular pressure, but subsequently compensated by the evolution of the adrenomedullary beta-adrenergic receptor mechanism. Conserted positive selection for the lung and adrenals created further selection pressure for the heart, which becomes progressively more complex phylogenetically in tandem with the lung. Developmentally, increasing heart complexity and size impinges precociously on the gut mesoderm to induce the liver. That evolutionary-developmental interaction is significant because the liver provides regulated sources of glucose and glycogen to the evolving physiologic system, which is necessary for the evolution of the neocortex. Evolution of neocortical control furthers integration of physiologic systems. Such an evolutionary vertical integration of cell-to-tissue-to-organ-to-physiology of intrinsic cell–cell signaling and extrinsic factors is the reverse of the “top-down” conventional way in which physiologic systems are usually regarded. This novel mechanistic approach, incorporating a “middle-out” cell–cell signaling component, will lead to a readily available algorithm for integrating genes and phenotypes. This symposium surveyed the phylogenetic origins of such vertically integrated mechanisms for the evolution of cell–cell communication as the basis for complex physiologic traits, from sponges to man.
doi:10.1093/icb/icp017
PMCID: PMC2895351  PMID: 20607136
18.  Deconvoluting lung evolution: from phenotypes to gene regulatory networks 
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.
doi:10.1093/icb/icm069
PMCID: PMC2895359  PMID: 20607138
19.  The Effects of Volatile Salivary Acids and Bases on Exhaled Breath Condensate pH 
Rationale: Recent studies have reported acidification of exhaled breath condensate (EBC) in inflammatory lung diseases. This phenomenon, designated “acidopnea,” has been attributed to airway inflammation.
Objectives: To determine whether salivary acids and bases can influence EBC pH in chronic obstructive pulmonary disease (COPD).
Methods: Measurements were made of pH, electrolytes, and volatile bases and acids in saliva and EBC equilibrated with air in 10 healthy subjects and 10 patients.
Results: The average EBC pH in COPD was reduced (normal, 7.24 ± 0.24 SEM; range, 6.11–8.34; COPD, 6.67 ± 0.18; range, 5.74–7.64; p = 0.079). EBCs were well buffered by NH4+/NH3 and CO2/HCO3− in all but four patients, who had NH4+ concentrations under 60 μmol/L, and acetate concentrations that approached or exceeded those of NH4+. Saliva contained high concentrations of acetate (∼ 6,000 μmol/L) and NH4+ (∼ 12,000 μmol/L). EBC acetate increased and EBC NH4+ decreased when salivary pH was low, consistent with a salivary source for these volatile constituents. Nonvolatile acids did not play a significant role in determining pH of condensates because of extreme dilution of respiratory droplets by water vapor (∼ 1:12,000). Transfer of both acetic acid and NH3 from the saliva to the EBC was in the gas phase rather than droplets.
Conclusions: EBC acidification in COPD can be affected by the balance of volatile salivary acids and bases, suggesting that EBC pH may not be a reliable marker of airway acidification. Salivary acidification may play an important role in acidopnea.
doi:10.1164/rccm.200507-1059OC
PMCID: PMC2662940  PMID: 16284109
acetate; ammonium; bicarbonate; buffer; exhaled breath condensate
20.  Dihydrotestosterone Inhibits Fetal Rabbit Pulmonary Surfactant Production 
Journal of Clinical Investigation  1982;69(3):611-616.
Males have a higher morbidity and mortality for neonatal respiratory distress syndrome (RDS) than females, and respond less well to hormone therapy designed to prevent RDS by stimulating fetal pulmonary surfactant production. We have shown that male fetuses exhibit delayed production of pulmonary surfactant. We tested the hypothesis that the sex difference in fetal pulmonary surfactant production is under hormonal control. Pulmonary surfactant was measured as the saturated phosphatidylcholine/sphingomyelin ratio (SPC/S) in the lung lavage of fetal rabbits at 26 d gestation. There was an association between the sex of neighboring fetuses and the SPC/S ratio of the female fetuses, such that with one or two male neighbors, respectively, females had decreasing SPC/S ratios (P < 0.05). We injected dihydrotestosterone (DHT) into pregnant does from day 12 through day 26 of gestation in doses of 0.1, 1.0, 10, and 25 mg/d, and measured the SPC/S ratio in fetal lung lavage on day 26. In groups with the normal sex difference in fetal serum androgen levels (controls, 0.1 mg DHT/d) the normal sex difference in the SPC/S ratio was also present (females > males, P = 0.03). In the 1-mg/d group there was no sex difference in androgen levels and the sex difference in the SPC/S ratio was also eliminated as the female values were lowered to the male level. Higher doses of DHT (10, 25 mg/d) further reduced the SPC/S ratios. We injected the anti-androgen Flutamide (25 mg/d) from day 12 through day 26 of gestation. This treatment eliminated the normal sex difference in the lung lavage SPC/S ratio by increasing the male ratios to that of the females. We conclude that androgens inhibit fetal pulmonary surfactant production. An understanding of the mechanism of the sex difference in surfactant production may allow development of therapy that is as effective in males as in females for preventing RDS.
PMCID: PMC371018  PMID: 6916770
21.  Evidence for Different Gestation-Dependent Effects of Cortisol on Cultured Fetal Lung Cells 
Journal of Clinical Investigation  1974;53(6):1518-1526.
The effect of cortisol (5.5 μM) on primary monolayer cultures of trypsin-dispersed lung cells from rabbit fetuses of 20-28 days gestation was monitored with respect to (a) cellular growth as determined by DNA content after 72 h, at which time all cultures were in the exponential phase of growth, and (b) cellular maturation as reflected by the incorporation of [14C]-palmitate into saturated lecithin and its release into the culture medium.
Cortisol significantly increased growth in cultures prepared from 20 day (control: 59.8±8.9 nmol DNA/flask; cortisol: 118.7±15.7, P < 0.001) and 22 day (control: 69.2±17.2; cortisol: 106.7±13.3, P < 0.001) fetuses but had no effect on the growth of cells from 24 or 26 day fetuses. At 28 days, the effect was reversed, cortisol reducing growth by a factor of two (control: 42.0±8.5; cortisol: 19.3±4.0, P < 0.001).
Incorporation of palmitate into lecithin was expressed as picomoles incorporated per micromole DNA per flask, thus correcting for differences in the number of cells. Cortisol had no effect on palmitate incorporation until day 26, at which time it caused a slight increase (control: 51.2±5.5: cortisol: 72.8±16.2, P < 0.01) which became very striking by day 28 (control: 19.7±3.1; cortisol; 286.8±47.0, P < 0.001). The proportion of recovered radiolabeled lecithin that was disaturated rose with gestational age from 72% at 20 days to 98% at 28 days. Saturated lecithin made up over 90% at the two gestational ages (26 and 28 days) where cortisol increased palmitate incorporation. In contrast, cortisol had no effect on the incorporation of palmitate into sphingomyelin at any of the gestational ages studied.
The results suggest that cortisol may increase fetal pulmonary cellular growth in early gestation while enhancing maturation and slowing growth as term approaches.
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PMCID: PMC302647  PMID: 4830219

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