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1.  Genetic Influences on Asthma Susceptibility in the Developing Lung 
Asthma is the leading serious pediatric chronic illness in the United States, affecting 7.1 million children. The prevalence of asthma in children under 4 years of age has increased dramatically in the last 2 decades. Existing evidence suggests that this increase in prevalence derives from early environmental exposures acting on a pre-existing asthma-susceptible genotype. We studied the origins of asthma susceptibility in developing lung in rat strains that model the distinct phenotypes of airway hyperresponsiveness (Fisher rats) and atopy (brown Norway [BN] rats). Postnatal BN rat lungs showed increased epithelial proliferation and tracheal goblet cell hyperplasia. Fisher pups showed increased lung resistance at age 2 weeks, with elevated neutrophils throughout the postnatal period. Diverse transcriptomic signatures characterized the distinct respiratory phenotypes of developing lung in both rat models. Linear regression across age and strain identified developmental variation in expression of 1,376 genes, and confirmed both strain and temporal regulation of lung gene expression. Biological processes that were heavily represented included growth and development (including the T Box 1 transcription factor [Tbx5], the epidermal growth factor receptor [Egfr], the transforming growth factor beta-1-induced transcript 1 [Tgfbr1i1]), extracellular matrix and cell adhesion (including collagen and integrin genes), and immune function (including lymphocyte antigen 6 (Ly6) subunits, IL-17b, Toll-interacting protein, and Ficolin B). Genes validated by quantitative RT-PCR and protein analysis included collagen III alpha 1 Col3a1, Ly6b, glucocorticoid receptor and Importin-13 (specific to the BN rat lung), and Serpina1 and Ficolin B (specific to the Fisher lung). Innate differences in patterns of gene expression in developing lung that contribute to individual variation in respiratory phenotype are likely to contribute to the pathogenesis of asthma.
PMCID: PMC3159089  PMID: 20118217
asthma susceptibility; lung development; developmental gene expression
2.  Modulation of Lgl1 by steroid, retinoic acid, and Vitamin D models complex transcriptional regulation during alveolarization 
Pediatric research  2010;67(4):375-381.
Alveolarization depends on circulating glucocorticoid (GC), retinoid (RA) and Vitamin D (VitD). Bronchopulmonary dysplasia (BPD), a leading cause of neonatal morbidity, is associated with arrested alveolarization. In hyperoxia-exposed rats displaying features of BPD, reduced levels of Lgl1 normalize during recovery. We show that GC (100nM) stimulates (7–115 fold) and VitD (100µM) suppresses (2 fold) Lgl1 expression. RA (all trans/9-cis, 10µM) effects are biphasic. From postnatal (PN) days 7–10, RA was stimulatory (2 fold) at 24h, after which effects were inhibitory (3–15 fold). Lgl1 promoter-luciferase reporter assays confirmed that these agents operated at the transcriptional level. Interestingly, the individual inhibitory effects of VitD and RA on GC induction of Lgl1 were abrogated when both agents were present, suggesting that steric hindrance may influence promoter accessibility. Analysis of the proximity (<50 base pairs) of binding sites for overlapping VitD and RA receptors to that of the GC receptor identified 81% of promoters in 66 genes (including Lgl1) important in human lung development compared to 48% in a random set of 1000 genes. Complex integration of the effects of GC, RA, and VitD on gene expression in the postnatal lung is likely to contribute to the timely advance of alveolarization without attendant inflammation.
PMCID: PMC3104505  PMID: 20057335
3.  Inflammatory cytokines, goblet cell hyperplasia and altered lung mechanics in Lgl1+/- mice 
Respiratory Research  2009;10(1):83.
Neonatal lung injury, a leading cause of morbidity in prematurely born infants, has been associated with arrested alveolar development and is often accompanied by goblet cell hyperplasia. Genes that regulate alveolarization and inflammation are likely to contribute to susceptibility to neonatal lung injury. We previously cloned Lgl1, a developmentally regulated secreted glycoprotein in the lung. In rat, O2 toxicity caused reduced levels of Lgl1, which normalized during recovery. We report here on the generation of an Lgl1 knockout mouse in order to determine whether deficiency of Lgl1 is associated with arrested alveolarization and contributes to neonatal lung injury.
An Lgl1 knockout mouse was generated by introduction of a neomycin cassette in exon 2 of the Lgl1 gene. To evaluate the pulmonary phenotype of Lgl1+/- mice, we assessed lung morphology, Lgl1 RNA and protein, elastin fibers and lung function. We also analyzed tracheal goblet cells, and expression of mucin, interleukin (IL)-4 and IL-13 as markers of inflammation.
Absence of Lgl1 was lethal prior to lung formation. Postnatal Lgl1+/- lungs displayed delayed histological maturation, goblet cell hyperplasia, fragmented elastin fibers, and elevated expression of TH2 cytokines (IL-4 and IL-13). At one month of age, reduced expression of Lgl1 was associated with elevated tropoelastin expression and altered pulmonary mechanics.
Our findings confirm that Lgl1 is essential for viability and is required for developmental processes that precede lung formation. Lgl1+/- mice display a complex phenotype characterized by delayed histological maturation, features of inflammation in the post-natal period and altered lung mechanics at maturity. Lgl1 haploinsufficiency may contribute to lung disease in prematurity and to increased risk for late-onset respiratory disease.
PMCID: PMC2760518  PMID: 19772569
4.  The angiogenic factor midkine is regulated by dexamethasone and retinoic acid during alveolarization and in alveolar epithelial cells 
Respiratory Research  2009;10(1):77.
A precise balance exists between the actions of endogenous glucocorticoids (GC) and retinoids to promote normal lung development, in particular during alveolarization. The mechanisms controlling this balance are largely unknown, but recent evidence suggests that midkine (MK), a retinoic acid-regulated, pro-angiogenic growth factor, may function as a critical regulator. The purpose of this study was to examine regulation of MK by GC and RA during postnatal alveolar formation in rats.
Newborn rats were treated with dexamethasone (DEX) and/or all-trans-retinoic acid (RA) during the first two weeks of life. Lung morphology was assessed by light microscopy and radial alveolar counts. MK mRNA and protein expression in response to different treatment were determined by Northern and Western blots. In addition, MK protein expression in cultured human alveolar type 2-like cells treated with DEX and RA was also determined.
Lung histology confirmed that DEX treatment inhibited and RA treatment stimulated alveolar formation, whereas concurrent administration of RA with DEX prevented the DEX effects. During normal development, MK expression was maximal during the period of alveolarization from postnatal day 5 (PN5) to PN15. DEX treatment of rat pups decreased, and RA treatment increased lung MK expression, whereas concurrent DEX+RA treatment prevented the DEX-induced decrease in MK expression. Using human alveolar type 2 (AT2)-like cells differentiated in culture, we confirmed that DEX and cAMP decreased, and RA increased MK expression.
We conclude that MK is expressed by AT2 cells, and is differentially regulated by corticosteroid and retinoid treatment in a manner consistent with hormonal effects on alveolarization during postnatal lung development.
PMCID: PMC2739515  PMID: 19698107
5.  Importin-13 genetic variation is associated with improved airway responsiveness in childhood asthma 
Respiratory Research  2009;10(1):67.
Glucocorticoid function is dependent on efficient translocation of the glucocorticoid receptor (GR) from the cytoplasm to the nucleus of cells. Importin-13 (IPO13) is a nuclear transport receptor that mediates nuclear entry of GR. In airway epithelial cells, inhibition of IPO13 expression prevents nuclear entry of GR and abrogates anti-inflammatory effects of glucocorticoids. Impaired nuclear entry of GR has been documented in steroid-non-responsive asthmatics. We hypothesize that common IPO13 genetic variation influences the anti-inflammatory effects of inhaled corticosteroids for the treatment of asthma, as measured by change in methacholine airway hyperresponsiveness (AHR-PC20).
10 polymorphisms were evaluated in 654 children with mild-to-moderate asthma participating in the Childhood Asthma Management Program (CAMP), a clinical trial of inhaled anti-inflammatory medications (budesonide and nedocromil). Population-based association tests with repeated measures of PC20 were performed using mixed models and confirmed using family-based tests of association.
Among participants randomized to placebo or nedocromil, IPO13 polymorphisms were associated with improved PC20 (i.e. less AHR), with subjects harboring minor alleles demonstrating an average 1.51–2.17 fold increase in mean PC20 at 8-months post-randomization that persisted over four years of observation (p = 0.01–0.005). This improvement was similar to that among children treated with long-term inhaled corticosteroids. There was no additional improvement in PC20 by IPO13 variants among children treated with inhaled corticosteroids.
IPO13 variation is associated with improved AHR in asthmatic children. The degree of this improvement is similar to that observed with long-term inhaled corticosteroid treatment, suggesting that IPO13 variation may improve nuclear bioavailability of endogenous glucocorticoids.
PMCID: PMC2724419  PMID: 19619331

Results 1-5 (5)