Laura Cerny

Peroxisome proliferator-activated receptor gamma agonists enhance lung maturation in a neonatal rat model.

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 d. Then the pups were killed 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.

Mechanism for nicotine-induced up-regulation of Wnt signaling in human alveolar interstitial fibroblasts

ABSTRACT Nicotine exposure alters normal homeostatic pulmonary epithelial-mesenchymal paracrine signaling pathways, resulting in alveolar interstitial fibroblast (AIF)-to-myofibroblast (MYF) transdifferentiation. Since the AIF versus MYF phenotype is determined by the expression of peroxisome proliferator-activated receptor γ (PPARγ) and Wingless/Int (Wnt) signaling, respectively, the authors 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 protein kinase C (PKC) signaling, the authors 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 × 10−6 M) for either 30 minutes or 24 hours, with or without 30-minute pretreatment with calphostin C (1 × 10−7 M), a pan-PKC inhibitor. Then the authors examined the activation of PKC (p-PKC) and Wnt signaling (p-GSK-3β, β-catenin, LEF-1, and fibronectin). Furthermore, activation of nicotinic acetylcholine receptor (nAChR)-α3 and -α7 and whether a PPARγ agonist, rosiglitazone (RGZ), 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 downstream targets of Wnt signaling at 24 hours. 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.

130 INVOLVEMENT OF CYCLOOXYGENASE 2 IN LIPOPOLYSACCHARIDE-INDUCED PRENATAL LUNG INFLAMMATION IS INDEPENDENT OF THE PARATHYROID HORMONE-RELATED PROTEIN SIGNALING PATHWAY.

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.

341 THE SAFETY AND EFFICACY OF THE PEROXISOME PROLIFERATOR-ACTIVATED RECEPTOR γ AGONIST ROSIGLITAZONE FOR LUNG DIFFERENTIATION IN A NEONATAL RAT MODEL.

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.

PREVENTION OF PERINATAL NICOTINE EXPOSURE-INDUCED ALTERATIONS IN PULMONARY FUNCTION BY PEROXISOME PROLIFERATOR-ACTIVATED RECEPTOR γ AGONISTS.: 124

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).

275 PREVENTION OF CHRONIC LUNG DISEASE IN THE PREMATURE INFANT: “AN INDIAN FLAVOR.”

Background Despite numerous advances in the treatment and prevention of respiratory distress syndrome (RDS) in the premature infant, chronic lung disease (CLD), a sequela of RDS remains extremely common. Its pathogenesis, treatment, and prevention remain incompletely understood. Exposure to hyperoxia, inflammation, and ventilation by the premature lung are the principal causative factors. Because of the well-known antioxidant and anti-inflammatory properties of curcumin [1,7-bis (4′-hydroxy-3′-methoxyphenyl)-1,6-heptadiene-3,5-dione], the naturally occurring pigment derived from the Indian spice tumeric, we hypothesized that it would be useful in preventing CLD of the newborn. Objectives To determine the effects of curcumin on the expression of the well-established markers of lung alveolar homeostasis and disease that have been previously validated in our laboratory. Design/Methods Lung explants, isolated fetal rat lung fibroblasts, or alveolar type II cells at embryonic (e) day 19.5 (term = e22) from Sprague-Dawley rat fetuses were treated in culture with curcumin (0-100 μM) for up to 48 h. The explants were maintained in Waymouths medium and isolated fibroblasts and ATII cells were cultured in DMEM at 37°C/5% CO2. Subsequently, [3 H]choline incorporation into saturated phosphatidylcholine and [3 H]triolein uptake, both markers of surfactant phospholipid synthesis, were examined. Further, mRNA and proteins were extracted, and using RT-PCR and Western hybridization, the expression of the well-characterized markers of lung alveolar health and disease were analyzed. These included the expression of peroxisome proliferator-activated receptorγ, adipocyte differentiation-related protein, surfactant protein-B, and α smooth muscle actin. Results Our data suggest that curcumin augments the expression of some but not all alveolar fibroblast and alveolar type II specific markers that are known to be critical in maintaining alveolar homeostasis. The studies to determine whether curcumin prevents hyperoxia- and infection-induced lung injury are in progress. Conclusions From these data, we conclude that curcumin administration may stimulate specific molecular pathways, which may help in preventing hyperoxia- and inflammation-induced CLD in 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.

108 EPITHELIAL-MESENCHYMAL TRANSITION IS NOT CRUCIAL IN THE PATHOGENESIS OF THE CHRONIC LUNG DISEASE OF THE PREMATURE INFANT.

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 Dulbeccos 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.