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Background In utero nicotine exposure disrupts the critical homeostatic pulmonary epithelial-mesenchymal paracrine signaling pathway, resulting in the transdifferentiation of alveolar intersitial fibroblasts (AIFs) to myofibroblasts (MYFs). AIF-to-MYF transdifferentiation is characterized by down-regulation of the peroxisome proliferator-activated receptor γ (PPAR-γ) signaling pathway and up-regulation of the Wingless/Int (Wnt) signaling pathway. Although effects of in utero nicotine exposure on PPAR-γ signaling are known, there is no information regarding its effect on Wnt signaling. We hypothesize that in utero nicotine exposure up-regulates AIF Wnt signaling, and in utero nicotine-induced lung injury can be prevented by specific molecular targeting to down-regulate Wnt signaling and/or up-regulate PPAR-γ signaling. Objectives To determine (1) how in utero nicotine exposure affects AIF Wnt signaling and (2) if Wnt signaling antagonists and/or PPAR-γ agonists can prevent in utero nicotine-induced AIF-to-MYF transdifferentiation. Methods WI38 cells, a human embryonic pulmonary fibroblast cell line, were cultured using standard methods. At near-confluence, the cells were treated twith nicotine (10−9 M) with or without a Wnt pathway antagonist (Wnt inhibitory factor [WIF-1]), or a PPAR-γ agonist (rosiglitazone [RGZ] 10−5M) for 24 hours to 7 days. Then the expression of various intermediates in Wnt and PPAR-γ signaling was determined at the mRNA (RT-PCR) and protein (Western blot) levels. Results Nicotine treatment up-regulated Wnt pathway signaling intermediates such as LEF-1, β-catenin, its downstream target α-smooth muscle actin (α-SMA), and down-regulated PPAR-γ pathway signaling intermediates such as PPAR-γ and adipocyte differentiation-related protein. Concomitant treatment with WIF-1 or RGZ completely inhibited the nicotine-induced up-regulation of the expression of LEF-1 and its downstream target α-SMA. Conclusions By either down-regulating AIF Wnt signaling and/or by up- regulating AIF PPAR-γ signaling, prohomeostatic AIFs can be maintained, preventing MYF transdifferentiation, thereby potentially preventing in utero nicotine-induced lung injury. Supported by grants from TRDRP ((14RT-0073 and 15IT-0250), Philip Morris USA Inc. and Philip Morris International, and NIH (HL55268 and HL075405).

Background Secretogogues, including β2-adrenergic agonists, bind to receptors on alveolar type II cells (ATII) and stimulate surfactant synthesis and secretion. Our laboratory has established an essential role for parathyroid hormone-related protein (PTHrP) signaling in alveolar development and surfactant synthesis. However, the effect of β2-adrenergic agonists such as albuterol, a common treatment of acute asthma exacerbations, is unknown. We hypothesize that albuterol will up-regulate the intermediary steps in alveolar type II cells that mediate the PTHrP signaling pathway. Further, levalbuterol (R-albuterol) will positively affect the PTHrP signaling pathway, whereas S-albuterol will have an inhibitory effect. Objective To determine the effects of albuterol on ATII PTHrP signaling pathway. Methods Lung ATII (either A549 or fetal rat at embryonic day 21) cells were cultured under standard conditions. At near-confluence, the cells were treated with R-albuterol, S-albuterol, or racemic-albuterol (50:50 mixture of R- and S-albuterols) (1 × 10−9 to 1 × 10−4 M for each) for 24 hours. The molecular markers of PTHrP signaling were then analyzed at mRNA (RT-PCR) and protein (Western blot) levels. The specific markers examined included PTHrP, leptin receptor, surfactant proteins A and B, and [3H]choline uptake for surfactant phospholipid synthesis. Results In general, albuterol treatment stimulated alveolar type II cell PTHrP signaling (increased PTHrP and leptin receptor expression), which was accompanied by an increase in surfactant protein B and surfactant phospholipid synthesis. Further, R-albuterol and racemic (R + S) albuterol, in general, had a stimulatory effect on PTHrP signaling, whereas, in contrast, S-albuterol had no effect or inhibitory effects. There were no significant differences in the effects of R- versus racemic (R + S) albuterol in stimulating either PTHrP signaling or surfactant phospholipid synthesis. Conclusions The β2-adrenergic agonist albuterol stimulates PTHrP signaling in pulmonary ATII. Further, R- and racemic (R + S)-albuterol are equally efficacious in stimulating PTHrP signaling and surfactant synthesis in alveolar type II cells, whereas S-albuterol may be inhibitory. Supported by grants from Sepracor Inc. and the NIH (HL55268 and HL075405).

Background Peroxisome proliferator-activated receptor γ (PPAR-γ), a member of the retinoid X-receptor heterodimer family of ligand-activated nuclear receptors, plays a critical role in modulating lung injury/repair. Recently, using both in vitro and in vivo models of acute neonatal lung injury, our laboratory has shown that PPAR-γ agonists can prevent nicotine- and short-term hyperoxia-induced lung injury. These studies clearly suggest the potential therapeutic usefulness of PPAR-γ agonists in preventing neonatal lung injury. However, there is no information on the effects of systemically administered PPAR-γ agonists on lung differentiation and metabolic profile in the developing system. Objectives To determine the effects of systemically administered PPAR-γ agonist rosiglitazone (RGZ) on lung differentiation, serum electrolytes, blood glucose, and blood gases in a developing neonatal rat model. Methods Newborn Sprague-Dawley rat pups were divided into the following six groups: control and RGZ 0.1 mg/kg, 0.3 mg/kg, 1 mg/kg, 3 mg/kg, or 8 mg/kg groups. The diluent (saline) or RGZ was administered IP in 100 μL volumes once daily for either 1 day or 7 days. The pups were sacrificed and the lungs were collected and processed either immediately for choline incorporation and triolein uptake or later for protein (Western hybridization) and mRNA (RT-PCR) analysis for markers of lung maturation. Results Systemically administered RGZ significantly increased the expression of surfactant proteins B and C, cholinephosphate cytidyltransferase, PPAR-γ, and adipocyte differentiation-related protein in a dose-dependent manner, both at day 1 and day 7. This was also accompanied by significantly increased choline incorporation and triolein uptake, the functional markers of surfactant phospholipid synthesis. There were no significant effects on serum electrolytes, blood glucose, and blood gases except at the highest dose of RGZ (8 mg/kg), which resulted in significantly lower blood glucose values. Conclusions Systemically administered RGZ significantly enhances lung differentiation without significantly affecting the serum metabolic profile. We speculate that PPAR-γ agonists may be safe and effective agents for enhancing repair of lung injury in neonates. Supported by grants from NIH (HL55268 and HL075405), TRDRP (14RT-0073 and 15IT-0250), and Philip Morris USA Inc. and Philip Morris International.

Background In utero exposure to maternal smoking is associated with adverse pulmonary effects, including reduced lung function and increased incidence of asthma, with both earlier onset and greater severity. However, the mechanisms underlying these adverse pulmonary effects are unknown, and there is no effective preventive or therapeutic intervention. Recently, we suggested that down-regulation of homeostatic mesenchymal peroxisome proliferator-activated receptor γ (PPAR-γ signaling following in utero nicotine (Nic) exposure might be a contributor to chronic lung diseases such as asthma. Furthermore, by up-regulating PPAR-γ, we might be able to prevent Nic-induced adverse pulmonary effects. Objective To determine (1) the change in the compliance (C) and resistance (R) of the respiratory system and bronchial reactivity following in utero Nic exposure and (2) if administration of a PPAR-γ agonist blocks the effects of in utero Nic exposure on C and bronchial reactivity. Methods Pregnant Sprague-Dawley rat dams received placebo (diluent), Nic (1 or 2 mg/kg), Nic + a PPAR-γ agonist rosiglitazone (RGZ) (3 mg/kg), or Nic (2 mg/kg) + RGZ (3 mg/kg) + a PPAR-γ antagonist GW9662 (0.25 mg/kg), IP in 100 μL volumes daily from e6 until term. Pups were delivered spontaneously and breast-fed ad libitum. At postnatal days 8 to 14, the pups were studied for C and R of the respiratory system, at baseline and following methacholine (MC) challenge (up to 2 mg/mL dose). Measurements were made on anesthetized ventilated (MiniVent, Harvard Apparatus, Holliston, MA) pups at 8 mL/kg tidal volume in a whole-body plethysmograph. Results Compared with control, with perinatal Nic exposure, there was a significant decrease in C without any change in R under basal conditions. There was a significant decrease in C and a significant increase in R following MC challenge. Concomitant treatment with RGZ completely blocked the Nic-induced alterations in pulmonary C and R under both basal and MC-challenge conditions. Treatment with GW9662 partially blocked RGZ-mediated effects on R and C. Conclusion Alterations in pulmonary function in a rodent model of perinatal Nic exposure are completely blocked by concomitant administration of RGZ. We speculate that perinatal Nic exposure significantly affects both alveolar and airway PPAR-γ signaling. Supported by grants from TRDRP ((14RT-0073 and 15IT-0250), Philip Morris USA Inc. and Philip Morris International, and NIH (HL55268 and HL075405).

Background Prenatal inflammation results in a paradoxical decrease in respiratory distress syndrome but an increase in bronchopulmonary dysplasia. We have implicated epithelial-mesenchymal signaling in mediating this response. Cyclooxygenase 2 (COX-2) and its metabolites are known mediators of lung inflammation, yet their role in parathyroid hormone-related protein (PTHrP)-mediated epithelial-mesenchymal paracrine signaling is not known. We hypothesize that COX-2 plays an integral role in inflammation-induced lung injury via PTHrP-driven signaling. Objectives (1) Determine the dose response and time course of COX-2 expression and prostaglandin E 2 synthesis in lipopolysaccharide (LPS)-induced lung injury; (2) determine the specificity of COX-2 in mediating inflammation-induced effects on PTHrP signaling by examining (a) the effect of the nonselective COX inhibitor indomethacin on the expression of PTHrP signaling pathway-related markers and (b) the effect of the PTHrP signaling pathway agonist PGJ 2 on COX-2 expression. Design/Methods Fetal rat lung explants (FRLE), alveolar type II cells (ATII), and lipofibroblasts (LF) from embryonic day 19.5 Sprague-Dawley rat fetuses were treated with LPS (0-50 μg/mL) with or without I (1 or 10 μM) and PGJ 2 (5-50 μM) for up to 72 hours. FRLE, LF, and ATII cells were maintained in standard culture media. Using RT-PCR and Western hybridization, the expression of COX-2 and markers of the PTHrP pathway were analyzed, including PTHrP, PTHrP receptor, peroxisome proliferator-activated receptor γ, adipocyte differentiation-related protein, surfactant protein B, and cholinephosphate cytidyltransferase α. Results LPS treatment of FRLE, ATII, and LF increased COX-2 expression in a time- and dose-dependent manner. Increased expression of COX-2 and markers of the PTHrP signaling pathway were blocked by pretreatment with indomethacin. Treatment with PGJ 2 blocked the effect of LPS on PTHrP signaling pathway markers but did not block the increase in COX-2 expression. Conclusion COX-2 may play an integral role in LPS-induced prenatal lung inflammation and its modulation may attenuate the consequent lung injury. COX-2 does not seem to be centrally involved in LPS-induced effects on PTHrP-driven epithelial-mesenchymal paracrine signaling. We speculate that in combination, COX-2 inhibitors and PTHrP pathway agonists might have an additive effect on blocking LPS-induced lung injury. NIH grants HL55268 and HL075405.

Background Exposure to nicotine disrupts specific alveolar epithelial-mesenchymal paracrine signaling pathways, resulting in the transdifferentiation of pulmonary alveolar interstitial fibroblasts (AIFs) to myofibroblasts (MYFs). We have suggested that AIF-to-MYF transdifferention is a central phenomenon that contributes to the altered pulmonary development and function in infants born to mothers who smoke during pregnancy. Further, we have shown that modulation of specific parathyroid hormone-related protein (PTHrP)-driven signaling can almost completely prevent nicotine-induced AIF-to-MYF transdifferentiation. However, once this process has occurred, whether it can be reversed is not known. Objectives To determine whether nicotine-induced AIF-to-MYF can be reversed by specifically targeting the PTHrP-mediated cAMP-dependent PKA signaling pathway. Methods WI38 cells, a human embryonic pulmonary fibroblast cell line, cultured in MEM + 10%FBS at 37°C, were plated in 6-well plates, 60 mm, and 100 mm dishes, as needed. At near confluence, the cells were initially treated with nicotine (10-9-10-6 M) for 7 days and AIF-to-MYF was confirmed by determining the down-regulation of the key lipogenic marker, peroxisome proliferator activated receptor γ (PPARγ) and up-regulation of the key myogenic marker, α-smooth muscle actin (αSMA). Subsequently, the cells were treated with prostaglandin J2 (PGJ2) (10 μM), PTHrP (5 × 10-7 M), or dibutryl cAMP (10-4 M) for 7 days and the expression of PTHrP receptor, PPARγ, adipocyte differentiation related protein (ADRP), calponin, αSMA was determined at mRNA (RT-PCR) and protein levels (Western analysis and immunohistochemistry). Simultaneous studies to determine the functional status of these fibroblasts were performed by measuring triglyceride uptake. Results Nicotine-induced AIF-to-MYF transdifferentiation was almost completely reversed by treatment with PTHrP, dibutryl cAMP, or PGJ2, as determined at the mRNA, protein, and functional assays. Conclusions Using a specific molecular approach and targeting specific molecular intermediates in PTHrP-driven cAMP-mediated PKA signaling pathway, these data, for the first time, demonstrate the reversibility of nicotine-induced AIF-to-MYF transdifferentiation, suggesting not only the possibility of prevention but also the potential for reversal of nicotine-induced lung injury. This clearly has significant potential therapeutic implications for both in utero and postnatal nicotine-induced lung injury. Supported by Philip Morris USA Inc. and Philip Morris International.

Background Although the expression of the glycophosphatidylinositol-linked protein Thy-1 has been suggested to be important in alveolar fibroblast proliferation and myofibroblast (MYF) differentiation, its role in chronic lung disease of the premature infant is not known. We have previously demonstrated that hyperoxia augments spontaneous transdifferentiation of alveolar lipofibroblasts (LIFs) to MYFs when passaged in culture. We hypothesized that Thy-1 is differentially expressed by LIFs and MYFs and that exposure to hyperoxia would specifically affect Thy-1 expression by alveolar interstitial fibroblasts. Objectives To characterize the expression of Thy-1 and other well-established markers of LIFs and MYFs phenotypes in spontaneously passaged alveolar interstitial fibroblasts and to determine the effect of hyperoxia on their expression. Design/Methods Fetal rat lung fibroblasts (FRLF) from embryonic (e) day 19 (term = e22) Sprague-Dawley rat fetuses were isolated, maintained, and passaged in Minimum Essential Medium + 10% fetal bovine serum in 21% O 2 /5% CO 2 at 37°C. At passage (P) 1, 5, and 10, cells were also exposed to 95% O 2 /5% CO 2 for 24 h, following which the expression of the well-characterized intermediates of the parathyroid hormone-related protein (PTHrP receptor and peroxisome proliferator-activated receptorγ) and Wnt (αsmooth muscle actin, calponin, and SMAD3) signaling pathways were determined by Western hybridization. Thy-1 expression was established by Western hybridization, flow cytometry, and semiquantitative RT-PCR. These data were also complemented by genome-wide microarray analysis of the RNA extracted from normoxia- and hyperoxia-exposed FRLFs. Results Normoxia-exposed P1 FRLFs were Thy-1 (-), whereas P5 and P10 cells expressed Thy-1 robustly. Exposure to hyperoxia increased the expression of Thy-1 at all passages. Exposure to hyperoxia, in general, decreased the expression of the PTHrP signaling pathway intermediates while concomitantly increasing the expression of the Wnt signaling intermediates. Cluster analysis of the microarray data suggests that hyperoxia decreases the expression of the cholesterol and fatty acid synthesis genes while increasing the expression of the fatty acid degradation enzymes- and Wnt-related genes. Conclusions These data suggest an association of the Thy-1 (+) phenotype with the canonical Wnt signaling pathway and a possible role for Thy-1 expression in fibroblast to MYF transdifferentiation.

Background Although epithelial-mesenchymal transition (EMT) has been well described in other organs, eg, the kidney, it has only recently been described in the lung. Using the rat alveolar type II (ATII) cell line, RLE-6TN and adult rat ATIIs, Willis et al (Am J Pathol 2005;166:1321-32) have recently demonstrated that ATII cells when chronically exposed to transforming growth factor (TGF)-β undergo EMT, raising the possibility that epithelial cells can serve as a source of myofibroblasts in chronic fibrotic conditions. Whether EMT occurs in chronic lung disease of the premature infant, a condition characterized by myofibroblast proliferation, is not known. Objective To determine whether cultured fetal rat ATII cells undergo EMT upon treatment with cytokines that are known to be associated with CLD of the newborn. Design/Methods Sprague-Dawley fetal rat lung ATII cells were isolated at embryonic (e) day 19 and cultured in Dulbecco9s Minimum Essential Medium + 10% FBS at 37°C in 6-well plates, 60 mm, and 100 mm dishes, as needed. At near confluence, the cells were maintained in culture with and without recombinant human TGF-β1 (2.5 ng/mL), tumor necrosis factor (TNF)-α (1 ng/mL), and TGF-β1 (2.5 ng/mL) + TNF-α (1 ng/mL) up to 10 days. EMT was examined by determining the expression of well-established markers of the alveolar epithelial (surfactant protein-B and -C, CTP:cholinephosphate cytidylyltransferase α, and aquaporin-5) and mesenchymal (α-smooth muscle actin, calponin, type I collagen) phenotypes on day 1, 6, and 10 at mRNA (RT-PCR) and protein (Western hybridization) levels. ATII cell function was also simultaneously determined through [3 H]choline incorporation into saturated phosphatidylcholine. Results Based on the expression of well-established alveolar epithelial and mesenchymal markers, there was no evidence of either spontaneous or cytokine-stimulated EMT up to 10 days in culture in e19 rat ATII cells. Conclusions Unlike the pathogenesis of the pulmonary fibrosis in the adult, EMT is not a prominent feature of the CLD of the premature infant. We speculate that premature ATII cells are resistant to EMT, and fibroblast-to-myofibroblast transdifferentiation rather than EMT is crucial in the pathogenesis of the CLD of the premature infant. Supported in part by NIH grants HL75405A (V.K.R. and J.S.T.) and HL55268 (J.S.T. and V.K.R.), Philip Morris USA Inc. and Philip Morris International, Tobacco-Related Disease Research Program (14RT-0073), and March of Dimes.

Background Maternal smoking during pregnancy results in lung hypoplasia characterized by a marked decrease in alveolar septation. However, the molecular mechanisms underlying this decrease in septation remain largely unknown. Since the interaction between Slit (ligand) and Robo (receptor), the human homologs of the Drosophila gene, is a critical repulsion cue for axon and myoblast migration during fetal development, and Sit/Robo expression in the lung has been shown to be developmentally regulated, we hypothesized that Slit/Robo interactions may play a critical role in pulmonary alveolar septation, and these genes would be specifically down regulated in lungs of the in utero nicotine-exposed fetuses. Objectives 1) To demonstrate the Slit/Robo expression in the developing rat lung and in response to in utero nicotine exposure and 2) to determine if nicotine-induced effects on lung Slit/Robo expression can be prevented by agonists of Peroxisome Proliferator Activated Receptor (PPAR)γ, an intervention that has been recently shown by us to prevent in utero nicotine-induced lung damage. Design/Methods Time-mated pregnant Sprague Dawley rat dams received placebo (diluent), nicotine (1 or 2 mg/kg) alone or nicotine (2 mg/kg) with a PPARγ agonist, rosiglitazone (RGZ) (3 mg/kg), i. p. in 100 μl volumes once daily from e6 of gestation until their sacrifice either on e20 or at term (e22). Lungs from e20 rat fetuses and PND 1 rat pups were either snap frozen or processed immediately for alveolar type II cell (ATII) or fibroblast isolation. The expressions of Robo1 and 2, and Slit2 and 3 were determined by RT-PCR from both lung tissue and isolated cells. Results Robo 1 and 2 and Slit 2 and 3 were abundantly expressed in the lung tissue at both e20 and PND1. The expression of Robo 1 and 2 was restricted to ATII cells and that of Slit 2 and 3 to fibroblasts. In utero nicotine exposure decreased Robo 2 and Slit 2 and 3 expression in a dose-dependent manner (p≤0.05 for all vs. controls), while the expression of Robo 1 was unaffected. Concomitant treatment with RGZ completely prevented the nicotine-induced decrease in Robo/Slit expression. Conclusions Slit and Robo are expressed in a spatially-specific pattern during perinatal lung development. In-utero nicotine exposure specifically down regulates lung Slit/Robo expression, tempting us to speculate that nicotine-induced failure in alveolar septation is due to its effect on Slit/Robo expression, which can be completely prevented by concomitant treatment with specific molecular interventions.

Background We have recently determined that disruption of pulmonary alveolar epithelial-mesenchymal paracrine loop, dictated by Peroxisome Proliferator Activated Receptor γ (PPARγ) expression by lung fibroblasts is a critical event in the pathobiology of bronchopulmonary dysplasia (BPD). In this study, we utilize a PPARγ ligand to reinforce the epithelial-mesenchymal paracrine loop to prevent lung injury in response to short-term hyperoxic ventilation in the developing rat. Objective To determine if stabilization of epithelial-mesenchymal paracrine loop by PPARγ ligand, rosiglitazone (RGZ), prevents hyperoxia-induced alveolar lung injury in a ventilated developing rat model. Design/Methods Two week old rat pups were anesthetized, and ventilated for 4h with 21% O2 (+ placebo), 95% O2 (+ placebo), or 95% O2 + 3 mg/kg RGZ in 100 μl i.p. administered 24h prior to ventilation. Peak Inspiratory/Positive End Expiratory Pressures were kept at 12/3 cm of water, and ventilatory rate was adjusted to keep PaCO2 (35-45 mm of Hg) similar among groups. At the end of 4h, bronchoalveolar lavage (BAL) was collected for Parathyroid Hormone-related Protein (PTHrP) determination using radioimmunoassay, animals sacrificed, and lung tissue analyzed for mRNA (RT-PCR) and protein (Western blotting) for alveolar type II cell [surfactant protein-B (SP-B), PTHrP, and leptin receptor], and fibroblast lipogenic [PTHrP receptor, PPARγ, and Adipocyte Differentiation Related Protein (ADRP), and leptin], and fibroblast myogenic [αsmooth muscle actin (αSMA)] markers. Results Hyperoxic ventilation alone decreased PTHrP in BAL (p ≤0.05), decreased PTHrP, PTHrP receptor, SP-B, leptin, leptin receptor, PPARγ, and ADRP mRNA expression (p≤0.05 for all), while increasing the expression αSMA mRNA (p≤0.05). These changes were completely prevented by prior administration of RGZ. Similarly, significant decreases in some but not all alveolar type II and fibroblast lipogenic proteins in response to hyperoxia were prevented by RGZ. Conclusions Our data provide clear evidence that PPARγ agonists can prevent hyperoxia-induced molecular lung injury that is known to lead to BPD. This is the first demonstration that reinforcing critically important epithelial-mesenchymal signaling pathways through the manipulation of the nuclear transcription factor, PPARγ, may be a novel and effective approach to prevent BPD.

Parathyroid Hormone-related Protein (PTHrP) and its receptor are ubiquitously expressed in vertebrate phylogeny from the swim bladder of the fish through the lungs of amphibians, reptiles, mammals and birds. Structurally, PTHrP expression is necessary for lung development since the mouse knock out does not form alveoli (Rubin et al 2004), the key to the phylogenetic increase in lung surface area throughout phylogeny and ontogeny. Functionally, PTHrP/PTHrP receptor signaling is a stretch-regulated mechanism that up-regulates surfactant production to accommodate increased alveolar surface area, providing a rationale for the positive selection pressure on PTHrP signaling in ontogeny and phylogeny (Torday and Rehan, 2004). We hypothesize that the amplification of the molecular PTHrP signaling pathway for surfactant synthesis underlies the increased surface area for gas exchange through vertebrate phylogeny. To test this hypothesis we have examined the PTHrP signaling pathway from endoderm to mesoderm in the fish swim bladder (the structural origin of the lung), as well as the lungs of frogs, alligators, rats and chicks. PTHrP, its receptor and pulmonary surfactant are expressed in all of these tissues; PTHrP/PTHrP receptor signaling is up-regulated in the frog, chick and rat just before the onset of surfactant production. All of these tissues were cultured as explants and incubated with 5 × 10-7M PTHrP (1-34) for 24h. The tissues were incubated with 3H-choline chloride for the last 4h and subsequently analyzed for 3H-saturated phosphatidylcholine (SatPC) content (surfactant phospholipid). The alligator lung showed a 25% increase in de novo SatPC synthesis, and the rat lung showed a 200% increase, consistent with the hypothesized increase in PTHrP signaling through phylogeny. These experiments are an empiric test of the molecular basis for the ontogeny, phylogeny and ultimate evolution of the vertebrate lung.

Background Intrauterine lung inflammation alters lung development, resulting in a 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 outcome is not known. Since the spatio-temporal sequence of epithelial-mesenchymal interactions plays a key role in normal lung development and homeostasis, we hypothesize that specific alterations in these cell-cell interactions may explain this apparent disparity in the pulmonary outcome in response to prenatal lung inflammation. Objective To determine the effects of Lipopolysaccharide (LPS) on Parathyroid Hormone-related Protein (PTHrP)-driven epithelial-mesenchymal interactions that have been previously shown by us to be essential in the maintenance of lung homeostasis. Design/Methods Lung explants from embryonic (e) 19.5 (term = e22) Sprague Dawley rat pups were treated with LPS (0-50 ng/ml) with or without a PTHrP pathway agonist, Prostaglandin J2 (PGJ2) for up to 72h. The explants were maintained in Waymouth9s medium at 370C/5% CO2. At specific time points (6, 12, 24, 48, and 72h), mRNA and proteins were extracted and analyzed for the expression of the specific intermediates of PTHrP-driven epithelial-mesenchymal interactions, which included Peroxisome Proliferator Activated Receptorγ (PPARγ), Adipocyte Differentiation Related Protein (ADRP), leptin, leptin receptor, Surfactant Protein-B (SP-B), α smooth muscle actin (SMA) and elastin, in addition to PTHrP and PTHrP receptor by both RT-PCR and Western hybridization. Results LPS treatment affected the expression of the key markers of the epithelial-mesenchymal paracrine loop in a dose- and time-dependent manner. There were initially (6-24h) significant increases in the expressions of PTHrP, PPARγ, ADRP, leptin, and SP-B (p ≤0.05), without any significant effect on the expressions of αSMA and elastin. This was followed by later (48-72h) significant decreases in the expressions of PTHrP, PPARγ, leptin, and leptin receptor, which were accompanied by significant increases in the expression of αSMA and elastin (p ≤0.05), the key markers of BPD. These changes were completely prevented by the concomitant treatment with PGJ2. Conclusions These data provide an integrative mechanism for the acute stimulation of lung differentiation accompanied paradoxically by BPD following intrauterine inflammation. Further, treatment with specific agonists of epithelial-mesenchymal interactions can prevent inflammation-induced molecular lung injury that is known to result in BPD.