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Find video protocols related to scientific articles indexed in Pubmed.
TGF? signaling inhibits goblet cell differentiation via SPDEF in conjunctival epithelium.
Development
PUBLISHED: 11-05-2014
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The ocular surface epithelia, including the stratified but non-keratinized corneal, limbal and conjunctival epithelium, in concert with the epidermal keratinized eyelid epithelium, function together to maintain eye health and vision. Abnormalities in cellular proliferation or differentiation in any of these surface epithelia are central in the pathogenesis of many ocular surface disorders. Goblet cells are important secretory cell components of various epithelia, including the conjunctiva; however, mechanisms that regulate goblet cell differentiation in the conjunctiva are not well understood. Herein, we report that conditional deletion of transforming growth factor ? receptor II (Tgfbr2) in keratin 14-positive stratified epithelia causes ocular surface epithelial hyperplasia and conjunctival goblet cell expansion that invaginates into the subconjunctival stroma in the mouse eye. We found that, in the absence of an external phenotype, the ocular surface epithelium develops properly, but young mice displayed conjunctival goblet cell expansion, demonstrating that TGF? signaling is required for normal restriction of goblet cells within the conjunctiva. We observed increased expression of SAM-pointed domain containing ETS transcription factor (SPDEF) in stratified conjunctival epithelial cells in Tgfbr2 cKO mice, suggesting that TGF? restricted goblet cell differentiation directly by repressing Spdef transcription. Gain of function of Spdef in keratin 14-positive epithelia resulted in the ectopic formation of goblet cells in the eyelid and peripheral cornea in adult mice. We found that Smad3 bound two distinct sites on the Spdef promoter and that treatment of keratin 14-positive cells with TGF? inhibited SPDEF activation, thereby identifying a novel mechanistic role for TGF? in regulating goblet cell differentiation.
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Foxm1 Regulates Resolution of Hyperoxic Lung Injury in Newborns.
Am. J. Respir. Cell Mol. Biol.
PUBLISHED: 10-03-2014
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Current treatments for inflammation associated with bronchopulmonary dysplasia fail to show clinical efficacy. Foxm1, a transcription factor of the Forkhead box family, is a critical mediator of lung development and carcinogenesis, but its role in bronchopulmonary dysplasia-associated pulmonary inflammation is unknown. Immunohistochemistry and RNA analysis were used to assess Foxm1 in lung tissue from hyperoxia-treated mice and patients with bronchopulmonary dysplasia. LysM-Cre/Foxm1-/- mice, in which Foxm1 was deleted from myeloid-derived inflammatory cells, including macrophages, monocytes and neutrophils, were exposed to neonatal hyperoxia causing lung injury and remodeling. Measurements of lung function and flow cytometry were used to evaluate effects of Foxm1 deletion on pulmonary inflammation and repair. Increased Foxm1 expression was observed in pulmonary macrophages of hyperoxia-exposed mice and lung tissue from patients with bronchopulmonary dysplasia. After hyperoxia, deletion of Foxm1 from the myeloid cell lineage decreased numbers of interstitial macrophages (CD45+CD11b+Ly6C-Ly6G-F4/80+CD68-) and impaired alveologenesis and lung function. The exaggerated bronchopulmonary dysplasia-like phenotype observed in hyperoxia-exposed LysM-Cre/Foxm1-/- mice was associated with increased expression of neutrophil-derived myeloperoxidase, proteinase 3 and cathepsin-g, all of which are critical for lung remodeling and inflammation. Our data demonstrate that Foxm1 influences pulmonary inflammatory responses to hyperoxia, inhibiting neutrophil-derived enzymes and enhancing monocytic responses that limit alveolar injury and remodeling in neonatal lungs.
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SPDEF inhibits prostate carcinogenesis by disrupting a positive feedback loop in regulation of the Foxm1 oncogene.
PLoS Genet.
PUBLISHED: 09-01-2014
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SAM-pointed domain-containing ETS transcription factor (SPDEF) is expressed in normal prostate epithelium. While its expression changes during prostate carcinogenesis (PCa), the role of SPDEF in prostate cancer remains controversial due to the lack of genetic mouse models. In present study, we generated transgenic mice with the loss- or gain-of-function of SPDEF in prostate epithelium to demonstrate that SPDEF functions as tumor suppressor in prostate cancer. Loss of SPDEF increased cancer progression and tumor cell proliferation, whereas over-expression of SPDEF in prostate epithelium inhibited carcinogenesis and reduced tumor cell proliferation in vivo and in vitro. Transgenic over-expression of SPDEF inhibited mRNA and protein levels of Foxm1, a transcription factor critical for tumor cell proliferation, and reduced expression of Foxm1 target genes, including Cdc25b, Cyclin B1, Cyclin A2, Plk-1, AuroraB, CKS1 and Topo2alpha. Deletion of SPDEF in transgenic mice and cultures prostate tumor cells increased expression of Foxm1 and its target genes. Furthermore, an inverse correlation between SPDEF and Foxm1 levels was found in human prostate cancers. The two-gene signature of low SPDEF and high FoxM1 predicted poor survival in prostate cancer patients. Mechanistically, SPDEF bound to, and inhibited transcriptional activity of Foxm1 promoter by interfering with the ability of Foxm1 to activate its own promoter through auto-regulatory site located in the -745/-660 bp Foxm1 promoter region. Re-expression of Foxm1 restored cellular proliferation in the SPDEF-positive cancer cells and rescued progression of SPDEF-positive tumors in mouse prostates. Altogether, SPDEF inhibits prostate carcinogenesis by preventing Foxm1-regulated proliferation of prostate tumor cells. The present study identified novel crosstalk between SPDEF tumor suppressor and Foxm1 oncogene and demonstrated that this crosstalk is required for tumor cell proliferation during progression of prostate cancer in vivo.
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Repair and regeneration of the respiratory system: complexity, plasticity, and mechanisms of lung stem cell function.
Cell Stem Cell
PUBLISHED: 08-09-2014
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Respiratory disease is the third leading cause of death in the industrialized world. Consequently, the trachea, lungs, and cardiopulmonary vasculature have been the focus of extensive investigations. Recent studies have provided new information about the mechanisms driving lung development and differentiation. However, there is still much to learn about the ability of the adult respiratory system to undergo repair and to replace cells lost in response to injury and disease. This Review highlights the multiple stem/progenitor populations in different regions of the adult lung, the plasticity of their behavior in injury models, and molecular pathways that support homeostasis and repair.
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The molecular era of surfactant biology.
Neonatology
PUBLISHED: 05-30-2014
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Advances in the physiology, biochemistry, molecular and cell biology of the pulmonary surfactant system transformed the clinical care and outcome of preterm infants with respiratory distress syndrome. The molecular era of surfactant biology provided genetic insights into the pathogenesis of pulmonary disorders, previously termed 'idiopathic', that affect newborn infants, children and adults. Knowledge related to the structure and function of the surfactant proteins and their roles in alveolar homeostasis has provided new diagnostic, prognostic and therapeutic tools to advance our understanding of the causes and treatments of acute and chronic lung diseases. Severe lung disease in newborn infants and older patients is caused by mutations in genes regulating alveolar epithelial cells and surfactant homeostasis. Mutations in genes encoding the surfactant proteins, transcription factors critical for alveolar morphogenesis and surfactant clearance, are now known to play important roles in the pathogenesis of chronic lung diseases. Identification of the genes underlying the diseases of alveolar homeostasis is useful for the diagnosis of lung disease before and after birth.
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Sox17 is required for normal pulmonary vascular morphogenesis.
Dev. Biol.
PUBLISHED: 01-10-2014
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The SRY-box containing transcription factor Sox17 is required for endoderm formation and vascular morphogenesis during embryonic development. In the lung, Sox17 is expressed in mesenchymal progenitors of the embryonic pulmonary vasculature and is restricted to vascular endothelial cells in the mature lung. Conditional deletion of Sox17 in splanchnic mesenchyme-derivatives using Dermo1-Cre resulted in substantial loss of Sox17 from developing pulmonary vascular endothelial cells and caused pulmonary vascular abnormalities before birth, including pulmonary vein varices, enlarged arteries, and decreased perfusion of the microvasculature. While survival of Dermo1-Cre;Sox17?/? mice (herein termed Sox17?/?) was unaffected at E18.5, most Sox17?/? mice died by 3 weeks of age. After birth, the density of the pulmonary microvasculature was decreased in association with alveolar simplification, biventricular cardiac hypertrophy, and valvular regurgitation. The severity of the postnatal cardiac phenotype was correlated with the severity of pulmonary vasculature abnormalities. Sox17 is required for normal formation of the pulmonary vasculature and postnatal cardiovascular homeostasis.
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Foxa3 induces goblet cell metaplasia and inhibits innate antiviral immunity.
Am. J. Respir. Crit. Care Med.
PUBLISHED: 01-08-2014
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Goblet cell metaplasia accompanies common pulmonary disorders that are prone to recurrent viral infections. Mechanisms regulating both goblet cell metaplasia and susceptibility to viral infection associated with chronic lung diseases are incompletely understood.
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Epithelial SCAP/INSIG/SREBP signaling regulates multiple biological processes during perinatal lung maturation.
PLoS ONE
PUBLISHED: 01-01-2014
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Pulmonary surfactant is required for lung function at birth and throughout postnatal life. Defects in the surfactant system are associated with common pulmonary disorders including neonatal respiratory distress syndrome and acute respiratory distress syndrome in children and adults. Lipogenesis is essential for the synthesis of pulmonary surfactant by type II epithelial cells lining the alveoli. This study sought to identify the role of pulmonary epithelial SREBP, a transcriptional regulator of cellular lipid homeostasis, during a critical time period of perinatal lung maturation in the mouse. Genome wide mRNA expression profiling of lung tissue from transgenic mice with epithelial-specific deletions of Scap (Scap(?/?), resulting in inactivation of SREBP signaling) or Insig1 and Insig2 (Insig1/2(?/?), resulting in activation of SREBP signaling) was assessed. Differentially expressed genes responding to SREBP perturbations were identified and subjected to functional enrichment analysis, pathway mapping and literature mining to predict upstream regulators and transcriptional networks regulating surfactant lipid homeostasis. Through comprehensive data analysis and integration, time dependent effects of epithelial SCAP/INSIG/SREBP deletion and defined SCAP/INSIG/SREBP-associated genes, bioprocesses and downstream pathways were identified. SREBP signaling influences epithelial development, cell death and cell proliferation at E17.5, while primarily influencing surfactant physiology, lipid/sterol synthesis, and phospholipid transport after birth. SREBP signaling integrated with the Wnt/?-catenin and glucocorticoid receptor signaling pathways during perinatal lung maturation. SREBP regulates perinatal lung lipogenesis and maturation through multiple mechanisms by interactions with distinct sets of regulatory partners.
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Uterine deletion of Gp130 or Stat3 shows implantation failure with increased estrogenic responses.
Mol. Endocrinol.
PUBLISHED: 07-24-2013
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Leukemia inhibitory factor (LIF), a downstream target of estrogen, is essential for implantation in mice. LIF function is thought to be mediated by its binding to LIF receptor (LIFR) and recruitment of coreceptor GP130 (glycoprotein 130), and this receptor complex then activates signal transducer and activator of transcription (STAT)1/3. However, the importance of LIFR and GP130 acting via STAT3 in implantation remains uncertain, because constitutive inactivation of Lifr, Gp130, or Stat3 shows embryonic lethality in mice. To address this issue, we generated mice with conditional deletion of uterine Gp130 or Stat3 and show that both GP130 and STAT3 are critical for uterine receptivity and implantation. Implantation failure in these deleted mice is associated with higher uterine estrogenic responses prior to the time of implantation. These heightened estrogenic responses are not due to changes in ovarian hormone levels or expression of their nuclear receptors. In the deleted mice, estrogen-responsive gene, Lactoferrin (Ltf), and Mucin 1 protein, were up-regulated in the uterus. In addition, progesterone-responsive genes, Hoxa10 and Indian hedgehog (Ihh), were markedly down-regulated in STAT3-inactivated uteri. These changes in uteri of deleted mice were reflected by the failure of differentiation of the luminal epithelium, which is essential for blastocyst attachment.
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Foxa2 Regulates Leukotrienes to Inhibit Th2-mediated Pulmonary Inflammation.
Am. J. Respir. Cell Mol. Biol.
PUBLISHED: 07-05-2013
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Foxa2 is a member of the Forkhead family of nuclear transcription factors that is highly expressed in respiratory epithelial cells of the developing and mature lung. Foxa2 is required for normal airway epithelial differentiation, and its deletion causes goblet-cell metaplasia and Th2-mediated pulmonary inflammation during postnatal development. Foxa2 expression is inhibited during aeroallergen sensitization and after stimulation with Th2 cytokines, when its loss is associated with goblet-cell metaplasia. Mechanisms by which Foxa2 controls airway epithelial differentiation and Th2 immunity are incompletely known. During the first 2 weeks after birth, the loss of Foxa2 increases the production of leukotrienes (LTs) and Th2 cytokines in the lungs of Foxa2 gene-targeted mice. Foxa2 expression inhibited 15-lipoxygenase (Alox15) and increased Alox5 transcription, each encoding key lipoxygenases associated with asthma. The inhibition of the cysteinyl LT (CysLT) signaling pathway by montelukast inhibited IL-4, IL-5, eotaxin-2, and regulated upon activation normal T cell expressed and presumably secreted expression in the developing lungs of Foxa2 gene-targeted mice. Montelukast inhibited the expression of genes regulating mucus metaplasia, including Spdef, Muc5ac, Foxa3, and Arg2. Foxa2 plays a cell-autonomous role in the respiratory epithelium, and is required for the suppression of Th2 immunity and mucus metaplasia in the developing lung in a process determined in part by its regulation of the CysLT pathway.
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Molecular determinants of lung development.
Ann Am Thorac Soc
PUBLISHED: 04-24-2013
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Development of the pulmonary system is essential for terrestrial life. The molecular pathways that regulate this complex process are beginning to be defined, and such knowledge is critical to our understanding of congenital and acquired lung diseases. A recent workshop was convened by the National Heart, Lung, and Blood Institute to discuss the developmental principles that regulate the formation of the pulmonary system. Emerging evidence suggests that key developmental pathways not only regulate proper formation of the pulmonary system but are also reactivated upon postnatal injury and repair and in the pathogenesis of human lung diseases. Molecular understanding of early lung development has also led to new advances in areas such as generation of lung epithelium from pluripotent stem cells. The workshop was organized into four different topics, including early lung cell fate and morphogenesis, mechanisms of lung cell differentiation, tissue interactions in lung development, and environmental impact on early lung development. Critical points were raised, including the importance of epigenetic regulation of lung gene expression, the dearth of knowledge on important mesenchymal lineages within the lung, and the interaction between the developing pulmonary and cardiovascular system. This manuscript describes the summary of the discussion along with general recommendations to overcome the gaps in knowledge in lung developmental biology.
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Orphan G protein-coupled receptor GPR116 regulates pulmonary surfactant pool size.
Am. J. Respir. Cell Mol. Biol.
PUBLISHED: 04-18-2013
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Pulmonary surfactant levels within the alveoli are tightly regulated to maintain lung volumes and promote efficient gas exchange across the air/blood barrier. Quantitative and qualitative abnormalities in surfactant are associated with severe lung diseases in children and adults. Although the cellular and molecular mechanisms that control surfactant metabolism have been studied intensively, the critical molecular pathways that sense and regulate endogenous surfactant levels within the alveolus have not been identified and constitute a fundamental knowledge gap in the field. In this study, we demonstrate that expression of an orphan G protein-coupled receptor, GPR116, in the murine lung is developmentally regulated, reaching maximal levels 1 day after birth, and is highly expressed on the apical surface of alveolar type I and type II epithelial cells. To define the physiological role of GPR116 in vivo, mice with a targeted mutation of the Gpr116 locus, Gpr116(?exon17), were generated. Gpr116(?exon17) mice developed a profound accumulation of alveolar surfactant phospholipids at 4 weeks of age (12-fold) that was further increased at 20 weeks of age (30-fold). Surfactant accumulation in Gpr116(?exon17) mice was associated with increased saturated phosphatidylcholine synthesis at 4 weeks and the presence of enlarged, lipid-laden macrophages, neutrophilia, and alveolar destruction at 20 weeks. mRNA microarray analyses indicated that P2RY2, a purinergic receptor known to mediate surfactant secretion, was induced in Gpr116(?exon17) type II cells. Collectively, these data support the concept that GPR116 functions as a molecular sensor of alveolar surfactant lipid pool sizes by regulating surfactant secretion.
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Wntless is required for peripheral lung differentiation and pulmonary vascular development.
Dev. Biol.
PUBLISHED: 02-21-2013
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Wntless (Wls), a gene highly conserved across the animal kingdom, encodes for a transmembrane protein that mediates Wnt ligand secretion. Wls is expressed in developing lung, wherein Wnt signaling is necessary for pulmonary morphogenesis. We hypothesize that Wls plays a critical role in modulating Wnt signaling during lung development and therefore affects processes critical for pulmonary morphogenesis. We generated conditional Wls mutant mice utilizing Shh-Cre and Dermo1-Cre mice to delete Wls in the embryonic respiratory epithelium and mesenchyme, respectively. Epithelial deletion of Wls disrupted lung branching morphogenesis, peripheral lung development and pulmonary endothelial differentiation. Epithelial Wls mutant mice died at birth due to respiratory failure caused by lung hypoplasia and pulmonary hemorrhage. In the lungs of these mice, VEGF and Tie2-angiopoietin signaling pathways, which mediate vascular development, were downregulated from early stages of development. In contrast, deletion of Wls in mesenchymal cells of the developing lung did not alter branching morphogenesis or early mesenchymal differentiation. In vitro assays support the concept that Wls acts in part via Wnt5a to regulate pulmonary vascular development. We conclude that epithelial Wls modulates Wnt ligand activities critical for pulmonary vascular differentiation and peripheral lung morphogenesis. These studies provide a new framework for understanding the molecular mechanisms underlying normal pulmonary vasculature formation and the dysmorphic pulmonary vasculature development associated with congenital lung disease.
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Spdef null mice lack conjunctival goblet cells and provide a model of dry eye.
Am. J. Pathol.
PUBLISHED: 02-07-2013
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Goblet cell numbers decrease within the conjunctival epithelium in drying and cicatrizing ocular surface diseases. Factors regulating goblet cell differentiation in conjunctival epithelium are unknown. Recent data indicate that the transcription factor SAM-pointed domain epithelial-specific transcription factor (Spdef) is essential for goblet cell differentiation in tracheobronchial and gastrointestinal epithelium of mice. Using Spdef(-/-) mice, we determined that Spdef is required for conjunctival goblet cell differentiation and that Spdef(-/-) mice, which lack conjunctival goblet cells, have significantly increased corneal surface fluorescein staining and tear volume, a phenotype consistent with dry eye. Microarray analysis of conjunctival epithelium in Spdef(-/-) mice revealed down-regulation of goblet cell-specific genes (Muc5ac, Tff1, Gcnt3). Up-regulated genes included epithelial cell differentiation/keratinization genes (Sprr2h, Tgm1) and proinflammatory genes (Il1-?, Il-1?, Tnf-?), all of which are up-regulated in dry eye. Interestingly, four Wnt pathway genes were down-regulated. SPDEF expression was significantly decreased in the conjunctival epithelium of Sjögren syndrome patients with dry eye and decreased goblet cell mucin expression. These data demonstrate that Spdef is required for conjunctival goblet cell differentiation and down-regulation of SPDEF may play a role in human dry eye with goblet cell loss. Spdef(-/-) mice have an ocular surface phenotype similar to that in moderate dry eye, providing a new, more convenient model for the disease.
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Foxm1 transcription factor is required for lung fibrosis and epithelial-to-mesenchymal transition.
EMBO J.
PUBLISHED: 01-04-2013
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Alveolar epithelial cells (AECs) participate in the pathogenesis of pulmonary fibrosis, producing pro-inflammatory mediators and undergoing epithelial-to-mesenchymal transition (EMT). Herein, we demonstrated the critical role of Forkhead Box M1 (Foxm1) transcription factor in radiation-induced pulmonary fibrosis. Foxm1 was induced in AECs following lung irradiation. Transgenic expression of an activated Foxm1 transcript in AECs enhanced radiation-induced pneumonitis and pulmonary fibrosis, and increased the expression of IL-1?, Ccl2, Cxcl5, Snail1, Zeb1, Zeb2 and Foxf1. Conditional deletion of Foxm1 from respiratory epithelial cells decreased radiation-induced pulmonary fibrosis and prevented the increase in EMT-associated gene expression. siRNA-mediated inhibition of Foxm1 prevented TGF-?-induced EMT in vitro. Foxm1 bound to and increased promoter activity of the Snail1 gene, a critical transcriptional regulator of EMT. Expression of Snail1 restored TGF-?-induced loss of E-cadherin in Foxm1-deficient cells in vitro. Lineage-tracing studies demonstrated that Foxm1 increased EMT during radiation-induced pulmonary fibrosis in vivo. Foxm1 is required for radiation-induced pulmonary fibrosis by enhancing the expression of genes critical for lung inflammation and EMT.
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Diffuse lung disease in children: Summary of a scientific conference.
Pediatr. Pulmonol.
PUBLISHED: 01-03-2013
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A multi-disciplinary scientific conference focused on diffuse and interstitial lung diseases in children was held in La Jolla, CA in June 2012. The conference brought together clinicians (including Pediatric and Adult Pulmonologists, Neonatologists, Pathologists, and Radiologists), clinical researchers, basic scientists, government agency representatives, patient advocates, as well as children affected by diffuse lung disease (DLD) and their families, to review recent scientific developments and emerging concepts in the pathophysiology of childhood DLD. Invited speakers discussed translational approaches, including genetics and proteomics, epigenetics and epigenomics, models of DLD, including animal models and induced pluripotent stem cells, and regenerative medicine approaches. The presentations of the invited speakers are summarized here. Pediatr Pulmonol. © 2013 Wiley Periodicals, Inc.
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Inhibition of the growth factor MDK/midkine by a novel small molecule compound to treat non-small cell lung cancer.
PLoS ONE
PUBLISHED: 01-01-2013
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Midkine (MDK) is a heparin-binding growth factor that is highly expressed in many malignant tumors, including lung cancers. MDK activates the PI3K pathway and induces anti-apoptotic activity, in turn enhancing the survival of tumors. Therefore, the inhibition of MDK is considered a potential strategy for cancer therapy. In the present study, we demonstrate a novel small molecule compound (iMDK) that targets MDK. iMDK inhibited the cell growth of MDK-positive H441 lung adenocarcinoma cells that harbor an oncogenic KRAS mutation and H520 squamous cell lung cancer cells, both of which are types of untreatable lung cancer. However, iMDK did not reduce the cell viability of MDK-negative A549 lung adenocarcinoma cells or normal human lung fibroblast (NHLF) cells indicating its specificity. iMDK suppressed the endogenous expression of MDK but not that of other growth factors such as PTN or VEGF. iMDK suppressed the growth of H441 cells by inhibiting the PI3K pathway and inducing apoptosis. Systemic administration of iMDK significantly inhibited tumor growth in a xenograft mouse model in vivo. Inhibition of MDK with iMDK provides a potential therapeutic approach for the treatment of lung cancers that are driven by MDK.
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Rapamycin decreases airway remodeling and hyperreactivity in a transgenic model of noninflammatory lung disease.
J. Appl. Physiol.
PUBLISHED: 09-08-2011
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Airway hyperreactivity (AHR) and remodeling are cardinal features of asthma and chronic obstructive pulmonary disease. New therapeutic targets are needed as some patients are refractory to current therapies and develop progressive airway remodeling and worsening AHR. The mammalian target of rapamycin (mTOR) is a key regulator of cellular proliferation and survival. Treatment with the mTOR inhibitor rapamycin inhibits inflammation and AHR in allergic asthma models, but it is unclear if rapamycin can directly inhibit airway remodeling and AHR, or whether its therapeutic effects are entirely mediated through immunosuppression. To address this question, we utilized transforming growth factor-? (TGF-?) transgenic mice null for the transcription factor early growth response-1 (Egr-1) (TGF-? Tg/Egr-1(ko/ko) mice). These mice develop airway smooth muscle thickening and AHR in the absence of altered lung inflammation, as previously reported. In this study, TGF-? Tg/Egr-1(ko/ko) mice lost body weight and developed severe AHR after 3 wk of lung-specific TGF-? induction. Rapamycin treatment prevented body weight loss, airway wall thickening, abnormal lung mechanics, and increases in airway resistance to methacholine after 3 wk of TGF-? induction. Increases in tissue damping and airway elastance were also attenuated in transgenic mice treated with rapamycin. TGF-?/Egr-1(ko/ko) mice on doxycycline for 8 wk developed severe airway remodeling. Immunostaining for ?-smooth muscle actin and morphometric analysis showed that rapamycin treatment prevented airway smooth muscle thickening around small airways. Pentachrome staining, assessments of lung collagen and fibronectin mRNA levels, indicated that rapamycin also attenuated fibrotic pathways induced by TGF-? expression for 8 wk. Thus rapamycin reduced airway remodeling and AHR, demonstrating an important role for mTOR signaling in TGF-?-induced/EGF receptor-mediated reactive airway disease.
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Maternal synchronization of gestational length and lung maturation.
PLoS ONE
PUBLISHED: 07-14-2011
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Among all mammals, fetal growth and organ maturation must be precisely synchronized with gestational length to optimize survival at birth. Lack of pulmonary maturation is the major cause of infant mortality in preterm birth. Whether fetal or maternal genotypes influence the close relationship between the length of gestation and lung function at birth is unknown. Structural and biochemical indicators of pulmonary maturity were measured in two mouse strains whose gestational length differed by one day. Shorter gestation in C57BL/6J mice was associated with advanced morphological and biochemical pulmonary development and better perinatal survival when compared to A/J pups born prematurely. After ovarian transplantation, A/J pups were born early in C57BL/6J dams and survived after birth, consistent with maternal control gestational length. Expression of genes critical for perinatal lung function was assessed in A/J pups born after ovarian transfer. A subset of mRNAs important for perinatal respiratory adaptation was selectively induced in the A/J pups born after ovarian transfer. mRNAs precociously induced after ovarian transfer indicated an important role for the transcription factors C/EBP? and CREB in maternally induced lung maturation. We conclude that fetal lung maturation is determined by both fetal and maternal genotypes. Ovarian transfer experiments demonstrated that maternal genotype determines the timing of birth and can influence fetal lung growth and maturation to ensure perinatal survival.
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Integrin ?6?4 defines a novel lung epithelial progenitor cell: a step forward for cell-based therapies for pulmonary disease.
J. Clin. Invest.
PUBLISHED: 06-23-2011
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The many challenges associated with lung transplantation provide a strong rationale for the development of cell- and tissue-based therapies for patients with respiratory failure caused by the loss of lung tissue that is associated with chronic pulmonary disease, injury, or resection. In this issue of the JCI, Chapman et al. take an important step forward in the development of regenerative medicine for the treatment of lung disease by identifying a novel integrin ?6?4-expressing alveolar epithelial cell that serves as a multipotent progenitor during repair of the severely injured lung.
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Cell plasticity in lung injury and repair: report from an NHLBI workshop, April 19-20, 2010.
Proc Am Thorac Soc
PUBLISHED: 06-10-2011
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In April 2010, a NIH workshop was convened to discuss the current state of understanding of lung cell plasticity, including the responses of epithelial cells to injury, with the objectives of summarizing what is known, what the field needs to know, and how to get there. The proximal stimulus for this workshop is the body of recent evidence suggesting that plasticity is a prominent but incompletely characterized property of lung epithelial cells, and that a focus on understanding this aspect of epithelial cell biology in particular, may be an important window into disease pathobiology and pathogenesis. In addition to their many vital functions in maintaining tissue homeostasis, epithelial cells have emerged as both a central target of disease initiation and an active contributor to disease progression, making a workshop to investigate the role of cell plasticity in lung injury and repair timely. The workshop was organized around four major themes: lung epithelial cell plasticity, signaling control of plasticity, fibroblast plasticity and crosstalk, and translation to human disease. Although this breakdown was recognized to be somewhat artificial, it was felt that this approach would promote cross-fertilization among groups that ordinarily do not communicate and lend itself to the generation of new approaches. The summary reports of individual group discussions below are followed by consensus priorities and recommendations of the workshop participants.
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Intersections between pulmonary development and disease.
Am. J. Respir. Crit. Care Med.
PUBLISHED: 06-07-2011
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Recent advances in cellular, molecular, and developmental biology have revolutionized our concepts regarding the process of organogenesis that have important implications for our understanding of both lung formation and pulmonary disease pathogenesis. Pulmonary investigators have long debated whether developmental processes are recapitulated during normal repair of the lung or in the setting of chronic pulmonary diseases. Although the cellular events involved in lung morphogenesis and those causing pulmonary disease are likely to include processes that are distinct, there is increasing evidence that the pathogenesis of many lung disorders involves the same genetic machinery that regulates cell growth,specification, and differentiation during normal lung development.
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Notch and basal cells take center stage during airway epithelial regeneration.
Cell Stem Cell
PUBLISHED: 06-01-2011
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Airway epithelium is maintained by basal cells, but the mechanisms responsible for repair are poorly characterized. In this issue of Cell Stem Cell, Rock et al. (2011) show that Notch signals regulate differentiation, but not self-renewal of basal cells after injury, and that this role appears conserved in mouse and human lung.
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Airway epithelial transcription factor NK2 homeobox 1 inhibits mucous cell metaplasia and Th2 inflammation.
Am. J. Respir. Crit. Care Med.
PUBLISHED: 05-13-2011
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Airway mucous cell metaplasia and chronic inflammation are pathophysiological features that influence morbidity and mortality associated with asthma and other chronic pulmonary disorders. Elucidation of the molecular mechanisms regulating mucous metaplasia and hypersecretion provides the scientific basis for diagnostic and therapeutic opportunities to improve the care of chronic pulmonary diseases.
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GM-CSF modulates pulmonary resistance to influenza A infection.
Antiviral Res.
PUBLISHED: 04-21-2011
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Alveolar type II epithelial or other pulmonary cells secrete GM-CSF that regulates surfactant catabolism and mucosal host defense through its capacity to modulate the maturation and activation of alveolar macrophages. GM-CSF enhances expression of scavenger receptors MARCO and SR-A. The alveolar macrophage SP-R210 receptor binds the surfactant collectin SP-A mediating clearance of respiratory pathogens. The current study determined the effects of epithelial-derived GM-CSF in host resistance to influenza A pneumonia. The results demonstrate that GM-CSF enhanced resistance to infection with 1.9×10(4) ffc of the mouse-adapted influenza A/Puerto Rico/8/34 (PR8) H1N1 strain, as indicated by significant differences in mortality and mean survival of GM-CSF-deficient (GM(-/-)) mice compared to GM(-/-) mice in which GM-CSF is expressed at increased levels. Protective effects of GM-CSF were observed both in mice with constitutive and inducible GM-CSF expression under the control of the pulmonary-specific SFTPC or SCGB1A1 promoters, respectively. Mice that continuously secrete high levels of GM-CSF developed desquamative interstitial pneumonia that impaired long-term recovery from influenza. Conditional expression of optimal GM-CSF levels at the time of infection, however, resulted in alveolar macrophage proliferation and focal lymphocytic inflammation of distal airways. GM-CSF enhanced alveolar macrophage activity as indicated by increased expression of SP-R210 and CD11c. Infection of mice lacking the GM-CSF-regulated SR-A and MARCO receptors revealed that MARCO decreases resistance to influenza in association with increased levels of SP-R210 in MARCO(-/-) alveolar macrophages. In conclusion, GM-CSF enhances early host resistance to influenza. Targeting of MARCO may reinforce GM-CSF-mediated host defense against pathogenic influenza.
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GM-CSF in the lung protects against lethal influenza infection.
Am. J. Respir. Crit. Care Med.
PUBLISHED: 04-07-2011
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Alveolar macrophages contribute to host defenses against influenza in animal models. Enhancing alveolar macrophage function may contribute to protection against influenza.
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Conditional disruption of mouse Klf5 results in defective eyelids with malformed meibomian glands, abnormal cornea and loss of conjunctival goblet cells.
Dev. Biol.
PUBLISHED: 04-05-2011
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Members of the Krüppel-like family of transcription factors regulate diverse developmental processes in various organs. Previously, we have demonstrated the role of Klf4 in the mouse ocular surface. Herein, we determined the role of the structurally related Klf5, using Klf5-conditional null (Klf5CN) mice derived by mating Klf5-LoxP and Le-Cre mice. Klf5 mRNA was detected as early as embryonic day 12 (E12) in the cornea, conjunctiva and eyelids, wherein its expression increased during development. Though the embryonic eye morphogenesis was unaltered in the Klf5CN mice, postnatal maturation was defective, resulting in smaller eyes with swollen eyelids that failed to separate properly. Klf5CN palpebral epidermis was hyperplastic with 7-9 layers of keratinocytes, compared with 2-3 in the wild type (WT). Klf5CN eyelid hair follicles and sebaceous glands were significantly enlarged, and the meibomian glands malformed. Klf5CN lacrimal glands displayed increased vasculature and large number of infiltrating cells. Klf5CN corneas were translucent, thicker with defective epithelial basement membrane and hypercellular stroma. Klf5CN conjunctiva lacked goblet cells, demonstrating that Klf5 is required for conjunctival goblet cell development. The number of Ki67-positive mitotic cells was more than doubled, consistent with the increased number of Klf5CN ocular surface epithelial cells. Co-ablation of Klf4 and Klf5 resulted in a more severe ocular surface phenotype compared with Klf4CN or Klf5CN, demonstrating that Klf4 and Klf5 share few if any, redundant functions. Thus, Klf5CN mice provide a useful model for investigating ocular surface pathologies involving meibomian gland dysfunction, blepharitis, corneal or conjunctival defects.
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Ectopic respiratory epithelial cell differentiation in bronchiolised distal airspaces in idiopathic pulmonary fibrosis.
Thorax
PUBLISHED: 03-21-2011
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Bronchiolisation of distal airspaces is an unexplained feature of idiopathic pulmonary fibrosis (IPF). The authors sought to identify mechanisms driving the differentiation of mucus cells during the bronchiolisation process.
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Cell-specific conditional deletion of Pten in the uterus results in differential phenotypes.
Gynecol. Oncol.
PUBLISHED: 02-28-2011
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Endometrial cancer (EMC) is the most common gynecological malignancy. The etiology and the cell types that are conducive to EMC are not completely understood, provoking further studies. Our objective was to determine whether deletion of Pten specifically in the uterine stroma and myometrium induces cancer or manifests different phenotypes.
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Kruppel-like factor 5 is required for formation and differentiation of the bladder urothelium.
Dev. Biol.
PUBLISHED: 02-03-2011
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Kruppel-like transcription factor 5 (Klf5) was detected in the developing and mature murine bladder urothelium. Herein we report a critical role of KLF5 in the formation and terminal differentiation of the urothelium. The Shh(GfpCre) transgene was used to delete the Klf5(floxed) alleles from bladder epithelial cells causing prenatal hydronephrosis, hydroureter, and vesicoureteric reflux. The bladder urothelium failed to stratify and did not express terminal differentiation markers characteristic of basal, intermediate, and umbrella cells including keratins 20, 14, and 5, and the uroplakins. The effects of Klf5 deletion were unique to the developing bladder epithelium since maturation of the epithelium comprising the bladder neck and urethra was unaffected by the lack of KLF5. mRNA analysis identified reductions in Ppar?, Grhl3, Elf3, and Ovol1expression in Klf5 deficient fetal bladders supporting their participation in a transcriptional network regulating bladder urothelial differentiation. KLF5 regulated expression of the mGrhl3 promoter in transient transfection assays. The absence of urothelial Klf5 altered epithelial-mesenchymal signaling leading to the formation of an ectopic alpha smooth muscle actin positive layer of cells subjacent to the epithelium and a thinner detrusor muscle that was not attributable to disruption of SHH signaling, a known mediator of detrusor morphogenesis. Deletion of Klf5 from the developing bladder urothelium blocked epithelial cell differentiation, impaired bladder morphogenesis and function causing hydroureter and hydronephrosis at birth.
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CTGF disrupts alveolarization and induces pulmonary hypertension in neonatal mice: implication in the pathogenesis of severe bronchopulmonary dysplasia.
Am. J. Physiol. Lung Cell Mol. Physiol.
PUBLISHED: 01-14-2011
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The pathological hallmarks of bronchopulmonary dysplasia (BPD), one of the most common long-term pulmonary complications associated with preterm birth, include arrested alveolarization, abnormal vascular growth, and variable interstitial fibrosis. Severe BPD is often complicated by pulmonary hypertension characterized by excessive pulmonary vascular remodeling and right ventricular hypertrophy that significantly contributes to the mortality and morbidity of these infants. Connective tissue growth factor (CTGF) is a multifunctional protein that coordinates complex biological processes during tissue development and remodeling. We have previously shown that conditional overexpression of CTGF in airway epithelium under the control of the Clara cell secretory protein promoter results in BPD-like architecture in neonatal mice. In this study, we have generated a doxycycline-inducible double transgenic mouse model with overexpression of CTGF in alveolar type II epithelial (AT II) cells under the control of the surfactant protein C promoter. Overexpression of CTGF in neonatal mice caused dramatic macrophage and neutrophil infiltration in alveolar air spaces and perivascular regions. Overexpression of CTGF also significantly decreased alveolarization and vascular development. Furthermore, overexpression of CTGF induced pulmonary vascular remodeling and pulmonary hypertension. Most importantly, we have also demonstrated that these pathological changes are associated with activation of integrin-linked kinase (ILK)/glucose synthesis kinase-3? (GSK-3?)/?-catenin signaling. These data indicate that overexpression of CTGF in AT II cells results in lung pathology similar to those observed in infants with severe BPD and that ILK/GSK-3?/?-catenin signaling may play an important role in the pathogenesis of severe BPD.
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Epithelial EGF receptor signaling mediates airway hyperreactivity and remodeling in a mouse model of chronic asthma.
Am. J. Physiol. Lung Cell Mol. Physiol.
PUBLISHED: 12-17-2010
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Increases in the epidermal growth factor receptor (EGFR) have been associated with the severity of airway thickening in chronic asthmatic subjects, and EGFR signaling is induced by asthma-related cytokines and inflammation. The goal of this study was to determine the role of EGFR signaling in a chronic allergic model of asthma and specifically in epithelial cells, which are increasingly recognized as playing an important role in asthma. EGFR activation was assessed in mice treated with intranasal house dust mite (HDM) for 3 wk. EGFR signaling was inhibited in mice treated with HDM for 6 wk, by using either the drug erlotinib or a genetic approach that utilizes transgenic mice expressing a mutant dominant negative epidermal growth factor receptor in the lung epithelium (EGFR-M mice). Airway hyperreactivity (AHR) was assessed by use of a flexiVent system after increasing doses of nebulized methacholine. Airway smooth muscle (ASM) thickening was measured by morphometric analysis. Sensitization to HDM (IgG and IgE), inflammatory cells, and goblet cell changes were also assessed. Increased EGFR activation was detected in HDM-treated mice, including in bronchiolar epithelial cells. In mice exposed to HDM for 6 wk, AHR and ASM thickening were reduced after erlotinib treatment and in EGFR-M mice. Sensitization to HDM and inflammatory cell counts were similar in all groups, except neutrophil counts, which were lower in the EGFR-M mice. Goblet cell metaplasia with HDM treatment was reduced by erlotinib, but not in EGFR-M transgenic mice. This study demonstrates that EGFR signaling, especially in the airway epithelium, plays an important role in mediating AHR and remodeling in a chronic allergic asthma model.
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Interference with intraepithelial TNF-? signaling inhibits CD8(+) T-cell-mediated lung injury in influenza infection.
Viral Immunol.
PUBLISHED: 12-15-2010
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CD8(+) T-cell-mediated pulmonary immunopathology in respiratory virus infection is mediated in large part by antigen-specific TNF-? expression by antiviral effector T cells, which results in epithelial chemokine expression and inflammatory infiltration of the lung. To further define the signaling events leading to lung epithelial chemokine production in response to CD8(+) T-cell antigen recognition, we expressed the adenoviral 14.7K protein, a putative inhibitor of TNF-? signaling, in the distal lung epithelium, and analyzed the functional consequences. Distal airway epithelial expression of 14.7K resulted in a significant reduction in lung injury resulting from severe influenza pneumonia. In vitro analysis demonstrated a significant reduction in the expression of an important mediator of injury, CCL2, in response to CD8(+) T-cell recognition, or to TNF-?. The inhibitory effect of 14.7K on CCL2 expression resulted from attenuation of NF-?B activity, which was independent of I?-B? degradation or nuclear translocation of the p65 subunit. Furthermore, epithelial 14.7K expression inhibited serine phosphorylation of Akt, GSK-3?, and the p65 subunit of NF-?B, as well as recruitment of NF-?B for DNA binding in vivo. These results provide insight into the mechanism of 14.7K inhibition of NF-?B activity, as well as further elucidate the mechanisms involved in the induction of T-cell-mediated immunopathology in respiratory virus infection.
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Klf5 regulates lineage formation in the pre-implantation mouse embryo.
Development
PUBLISHED: 10-27-2010
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Kruppel-like transcription factors (Klfs) are essential for the induction and maintenance of pluripotency of embryonic stem cells (ESCs), yet little is known about their roles in establishing the three lineages of the pre-implantation embryo. Here, we show that Klf5 is required for the formation of the trophectoderm (TE) and the inner cell mass (ICM), and for repressing primitive endoderm (PE) development. Although cell polarity appeared normal, Klf5 mutant embryos arrested at the blastocyst stage and failed to hatch due to defective TE development. Klf5 acted cell-autonomously in the TE, downstream of Fgf4 and upstream of Cdx2, Eomes and Krt8. In the ICM, loss of Klf5 resulted in reduced expression of pluripotency markers Oct4 and Nanog, but led to increased Sox17 expression in the PE, suggesting that Klf5 suppresses the PE lineage. Consistent with this, overexpression of Klf5 in transgenic embryos was sufficient to suppress the Sox17(+) PE lineage in the ICM. Klf5 overexpression led to a dose-dependent decrease in Sox17 promoter activity in reporter assays in cultured cells. Moreover, in chimeric embryos, Klf5(-/-) cells preferentially contributed to the Sox17(+) PE lineage and Cdx2 expression was not rescued in Klf5(-/-) outer cells. Finally, outgrowths from Klf5(-/-) embryos failed to form an ICM/pluripotent colony, had very few Oct4(+) or Cdx2(+) cells, but showed an increase in the percentage of Sox17(+) PE cells. These findings demonstrate that Klf5 is a dynamic regulator of all three lineages in the pre-implantation embryo by promoting the TE and epiblast lineages while suppressing the PE lineage.
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Conditional depletion of airway progenitor cells induces peribronchiolar fibrosis.
Am. J. Respir. Crit. Care Med.
PUBLISHED: 09-24-2010
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The respiratory epithelium has a remarkable capacity to respond to acute injury. In contrast, repeated epithelial injury is often associated with abnormal repair, inflammation, and fibrosis. There is increasing evidence that nonciliated epithelial cells play important roles in the repair of the bronchiolar epithelium after acute injury. Cellular processes underlying the repair and remodeling of the lung after chronic epithelial injury are poorly understood.
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Sox2 activates cell proliferation and differentiation in the respiratory epithelium.
Am. J. Respir. Cell Mol. Biol.
PUBLISHED: 09-20-2010
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Sox2, a transcription factor critical for the maintenance of embryonic stem cells and induction of pluripotent stem cells, is expressed exclusively in the conducting airway epithelium of the lung, where it is required for differentiation of nonciliated, goblet, and ciliated cells. To determine the role of Sox2 in respiratory epithelial cells, Sox2 was selectively and conditionally expressed in nonciliated airway epithelial cells and in alveolar type II cells in the adult mouse. Sox2 induced epithelial cell proliferation within 3 days of expression. Epithelial cell proliferation was associated with increased Ki-67 and cyclin D1 staining. Expression of cell cycle genes, including FoxM1, Ccna2 (Cyclin A2), Ccnb2 (Cyclin B2), and Ccnd1 (Cyclin D1), was increased. Consistent with a role in cell proliferation, Sox2 activated the transcription of FoxM1 in vitro. In alveoli, Sox2 caused hyperplasia and ectopic differentiation of epithelial cells to those with morphologic and molecular characteristics of conducting airway epithelium. Sox2 induced the expression of conducting airway epithelial specific genes, including Scgb1a1, Foxj1, Tubb3, and Cyp2f2. Although prolonged expression of Sox2 caused cell proliferation and epithelial hyperplasia, Sox2 did not induce pulmonary tumors. Sox2 induces proliferation of respiratory epithelial cells and, subsequently, partially reprograms alveolar epithelial cells into cells with characteristics of the conducting airways.
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A systems approach to mapping transcriptional networks controlling surfactant homeostasis.
BMC Genomics
PUBLISHED: 07-26-2010
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Pulmonary surfactant is required for lung function at birth and throughout life. Lung lipid and surfactant homeostasis requires regulation among multi-tiered processes, coordinating the synthesis of surfactant proteins and lipids, their assembly, trafficking, and storage in type II cells of the lung. The mechanisms regulating these interrelated processes are largely unknown.
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Kruppel-like factor 5 is not required for K-RasG12D lung tumorigenesis, but represses ABCG2 expression and is associated with better disease-specific survival.
Am. J. Pathol.
PUBLISHED: 07-16-2010
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K-RAS mutations are found in approximately 30% of lung cancers. The transcription factor Krüppel-like Factor 5 (KLF5) has been shown to mediate cellular transformation signaling events downstream of oncogenic RAS in other cancers, but a role for KLF5 in lung tumorigenesis has not been defined. We show here that knockdown of KLF5 expression significantly decreased anchorage-independent growth, but did not affect proliferation of human lung adenocarcinoma cells. Moreover, Klf5 is not required for lung tumor formation in an inducible oncogenic K-Ras(G12D) mouse model of lung tumorigenesis, and non-small cell lung cancer patients expressing high levels of KLF5 (21/258) have a significantly better disease-specific survival than those with intermediate to no KLF5 expression. Further, KLF5 knockdown in K-RAS-mutant human lung cancer cells resulted in a fivefold increase in ATP-binding cassette, subfamily G (WHITE), member 2 (ABCG2), an anthracycline drug transporter, which lead to significantly increased resistance to doxorubicin treatment, a chemotherapeutic agent clinically used to treat lung cancer. In summary, while KLF5 is not required for oncogenic mutant K-Ras-induced lung tumorigenesis, KLF5 regulation of ABCG2 expression may be important for chemotherapeutic resistance and patient survival.
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Hereditary pulmonary alveolar proteinosis: pathogenesis, presentation, diagnosis, and therapy.
Am. J. Respir. Crit. Care Med.
PUBLISHED: 07-09-2010
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We identified a 6-year-old girl with pulmonary alveolar proteinosis (PAP), impaired granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor function, and increased GM-CSF.
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Signaling pathways in the epithelial origins of pulmonary fibrosis.
Cell Cycle
PUBLISHED: 07-03-2010
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Pulmonary fibrosis complicates a number of disease processes and leads to substantial morbidity and mortality. Idiopathic pulmonary fibrosis (IPF) is perhaps the most pernicious and enigmatic form of the greater problem of lung fibrogenesis with a median survival of three years from diagnosis in affected patients. In this review, we will focus on the pathology of IPF as a model of pulmonary fibrotic processes, review possible cellular mechanisms, review current treatment approaches and review two transgenic mouse models of lung fibrosis to provide insight into processes that cause lung fibrosis. We will also summarize the potential utility of signaling pathway inhibitors as a future treatment in pulmonary fibrosis. Finally, we will present data demonstrating a minimal contribution of epithelial-mesenchymal transition in the development of fibrotic lesions in the transforming growth factor-alpha transgenic model of lung fibrosis.
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A nonredundant role for mouse Serpinb3a in the induction of mucus production in asthma.
J. Allergy Clin. Immunol.
PUBLISHED: 06-10-2010
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Asthma is a major public health burden worldwide. Studies from our group and others have demonstrated that SERPINB3 and SERPINB4 are induced in patients with asthma; however, their mechanistic role in asthma has yet to be determined.
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Foxa2 programs Th2 cell-mediated innate immunity in the developing lung.
J. Immunol.
PUBLISHED: 05-05-2010
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After birth, the respiratory tract adapts to recurrent exposures to pathogens, allergens, and toxicants by inducing the complex innate and acquired immune systems required for pulmonary homeostasis. In this study, we show that Foxa2, expressed selectively in the respiratory epithelium, plays a critical role in regulating genetic programs influencing Th2 cell-mediated pulmonary inflammation. Deletion of the Foxa2 gene, encoding a winged helix/forkhead box transcription factor that is selectively expressed in respiratory epithelial cells, caused spontaneous pulmonary eosinophilic inflammation and goblet cell metaplasia. Loss of Foxa2 induced the recruitment and activation of myeloid dendritic cells and Th2 cells in the lung, causing increased production of Th2 cytokines and chemokines. Loss of Foxa2-induced expression of genes regulating Th2 cell-mediated inflammation and goblet cell differentiation, including IL-13, IL-4, eotaxins, thymus and activation-regulated chemokine, Il33, Ccl20, and SAM pointed domain-containing Ets transcription factor. Pulmonary inflammation and goblet cell differentiation were abrogated by treatment of neonatal Foxa2(Delta/Delta) mice with mAb against IL-4Ralpha subunit. The respiratory epithelium plays a central role in the regulation of Th2-mediated inflammation and innate immunity in the developing lung in a process regulated by Foxa2.
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Increased expression of FoxM1 transcription factor in respiratory epithelium inhibits lung sacculation and causes Clara cell hyperplasia.
Dev. Biol.
PUBLISHED: 04-05-2010
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Foxm1 is a member of the Forkhead Box (Fox) family of transcription factors. Foxm1 (previously called Foxm1b, HFH-11B, Trident, Win, or MPP2) is expressed in multiple cell types and plays important roles in cellular proliferation, differentiation and tumorigenesis. Genetic deletion of Foxm1 from mouse respiratory epithelium during initial stages of lung development inhibits lung maturation and causes respiratory failure after birth. However, the role of Foxm1 during postnatal lung morphogenesis remains unknown. In the present study, Foxm1 expression was detected in epithelial cells of conducting and peripheral airways and changing dynamically with lung maturation. To discern the biological role of Foxm1 in the prenatal and postnatal lung, a novel transgenic mouse line that expresses a constitutively active form of FoxM1 (FoxM1 N-terminal deletion mutant or FoxM1-?N) under the control of lung epithelial-specific SPC promoter was produced. Expression of the FoxM1-?N transgene during embryogenesis caused epithelial hyperplasia, inhibited lung sacculation and expression of the type II epithelial marker, pro-SPC. Expression of FoxM1-?N mutant during the postnatal period did not influence alveologenesis but caused focal airway hyperplasia and increased proliferation of Clara cells. Likewise, expression of FoxM1-?N mutant in conducting airways with Scgb1a1 promoter was sufficient to induce Clara cell hyperplasia. Furthermore, FoxM1-?N cooperated with activated K-Ras to induce lung tumor growth in vivo. Increased activity of Foxm1 altered lung sacculation, induced proliferation in the respiratory epithelium and accelerated lung tumor growth, indicating that precise regulation of Foxm1 is critical for normal lung morphogenesis and development of lung cancer.
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Review: The intersection of surfactant homeostasis and innate host defense of the lung: lessons from newborn infants.
Innate Immun
PUBLISHED: 03-29-2010
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The study of pulmonary surfactant, directed towards prevention and treatment of respiratory distress syndrome in preterm infants, led to the identification of novel proteins/genes that determine the synthesis, packaging, secretion, function, and catabolism of alveolar surfactant. The surfactant proteins, SP-A, SP-B, SP-C, and SP-D, and the surfactant lipid associated transporter, ABCA3, play critical roles in surfactant homeostasis. The study of their structure and function provided insight into a system that integrates the biophysical need to reduce surface tension in the alveoli and the innate host defenses required to maintain pulmonary structure and function after birth. Alveolar homeostasis depends on the intrinsic, multifunctional structures of the surfactant-associated proteins and the shared transcriptional regulatory modules that determine both the expression of genes involved in surfactant production as well as those critical for host defense. Identification of the surfactant proteins and the elucidation of the genetic networks regulating alveolar homeostasis have provided the basis for understanding and diagnosing rare and common pulmonary disorders, including respiratory distress syndrome, inherited disorders of surfactant homeostasis, and pulmonary alveolar proteinosis.
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Patient-derived granulocyte/macrophage colony-stimulating factor autoantibodies reproduce pulmonary alveolar proteinosis in nonhuman primates.
Am. J. Respir. Crit. Care Med.
PUBLISHED: 03-11-2010
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Granulocyte/macrophage colony-stimulating factor (GM-CSF) autoantibodies (GMAb) are strongly associated with idiopathic pulmonary alveolar proteinosis (PAP) and are believed to be important in its pathogenesis. However, levels of GMAb do not correlate with disease severity and GMAb are also present at low levels in healthy individuals.
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Pulmonary pathology in thyroid transcription factor-1 deficiency syndrome.
Am. J. Respir. Crit. Care Med.
PUBLISHED: 03-04-2010
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Thyroid transcription factor-1 (TTF-1) deficiency syndrome is characterized by neurologic, thyroidal, and pulmonary dysfunction. Children usually have mild-to-severe respiratory symptoms and occasionally die of respiratory failure. Herein, we describe an infant with a constitutional 14q12-21.3 haploid deletion encompassing the TTF-1 gene locus who had cerebral dysgenesis, thyroidal dysfunction, and respiratory insufficiency. The clinical course was notable for mild hyaline membrane disease, continuous ventilatory support, and symmetrically distributed pulmonary cysts by imaging. He developed pneumonia and respiratory failure and died at 8 months. Pathologically, the lungs had grossly visible emphysematous changes with "cysts" up to 2 mm in diameter. The airway generations and radial alveolar count were diminished. In addition to acute bacterial pneumonia, there was focally alveolar septal fibrosis, pneumocyte hypertrophy, and clusters of airspace macrophages. Ultrastructurally, type II pneumocytes had numerous lamellar bodies, and alveolar spaces contained fragments of type II pneumocytes and extruded lamellar bodies. Although immunoreactivity for surfactant protein SP-A and ABCA3 was diminished, that for SP-B and proSP-C was robust, although irregularly distributed, corresponding to the distribution of type II pneumocytes. Immunoreactivity for TTF-1 protein was readily detected. In summation, we document abnormal airway and alveolar morphogenesis and altered expression of surfactant-associated proteins, which may explain the respiratory difficulties encountered in TTF-1 haploinsufficiency. These findings are consistent with experimental evidence documenting the important role of TTF-1 in pulmonary morphogenesis and surfactant metabolism.
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Conditional deletion of Abca3 in alveolar type II cells alters surfactant homeostasis in newborn and adult mice.
Am. J. Physiol. Lung Cell Mol. Physiol.
PUBLISHED: 02-26-2010
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ATP-binding cassette A3 (ABCA3) is a lipid transport protein required for synthesis and storage of pulmonary surfactant in type II cells in the alveoli. Abca3 was conditionally deleted in respiratory epithelial cells (Abca3(?/?)) in vivo. The majority of mice in which Abca3 was deleted in alveolar type II cells died shortly after birth from respiratory distress related to surfactant deficiency. Approximately 30% of the Abca3(?/?) mice survived after birth. Surviving Abca3(?/?) mice developed emphysema in the absence of significant pulmonary inflammation. Staining of lung tissue and mRNA isolated from alveolar type II cells demonstrated that ?50% of alveolar type II cells lacked ABCA3. Phospholipid content and composition were altered in lung tissue, lamellar bodies, and bronchoalveolar lavage fluid from adult Abca3(?/?) mice. In adult Abca3(?/?) mice, cells lacking ABCA3 had decreased expression of mRNAs associated with lipid synthesis and transport. FOXA2 and CCAAT enhancer-binding protein-?, transcription factors known to regulate genes regulating lung lipid metabolism, were markedly decreased in cells lacking ABCA3. Deletion of Abca3 disrupted surfactant lipid synthesis in a cell-autonomous manner. Compensatory surfactant synthesis was initiated in ABCA3-sufficient type II cells, indicating that surfactant homeostasis is a highly regulated process that includes sensing and coregulation among alveolar type II cells.
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Surfactant protein-d inhibits lung inflammation caused by ventilation in premature newborn lambs.
Am. J. Respir. Crit. Care Med.
PUBLISHED: 02-04-2010
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Premature newborns frequently require manual ventilation for resuscitation during which lung injury occurs. Although surfactant protein (SP)-D regulates pulmonary inflammation, SP-D levels are low in the preterm lung. Commercial surfactants for treatment of respiratory distress syndrome do not contain SP-D.
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Fatal familial lung disease caused by ABCA3 deficiency without identified ABCA3 mutations.
J. Pediatr.
PUBLISHED: 01-07-2010
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To test the hypothesis that some functionally significant variants in the gene encoding member A3 of the ATP Binding Cassette family (ABCA3) are not detected using exon-based sequencing approaches.
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Inhibition of PI3K by PX-866 prevents transforming growth factor-alpha-induced pulmonary fibrosis.
Am. J. Pathol.
PUBLISHED: 12-30-2009
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Transforming growth factor-alpha (TGFalpha) is a ligand for the epidermal growth factor receptor (EGFR). EGFR activation is associated with fibroproliferative processes in human lung disease and animal models of pulmonary fibrosis. EGFR signaling activates several intracellular signaling pathways including phosphatidylinositol 3-kinase (PI3K). We previously showed that induction of lung-specific TGFalpha expression in transgenic mice caused progressive pulmonary fibrosis over a 4-week period. The increase in levels of phosphorylated Akt, detected after 1 day of doxycycline-induced TGFalpha expression, was blocked by treatment with the PI3K inhibitor, PX-866. Daily administration of PX-866 during TGFalpha induction prevented increases in lung collagen and airway resistance as well as decreases in lung compliance. Treatment of mice with oral PX-866 4 weeks after the induction of TGFalpha prevented additional weight loss and further increases in total collagen, and attenuated changes in pulmonary mechanics. These data show that PI3K is activated in TGFalpha/EGFR-mediated pulmonary fibrosis and support further studies to determine the role of PI3K activation in human lung fibrotic disease, which could be amenable to targeted therapy.
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Developmental aspects of the upper airway: report from an NHLBI Workshop, March 5-6, 2009.
Proc Am Thorac Soc
PUBLISHED: 09-11-2009
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The upper airway serves three important functions: respiration, swallowing, and speech. During development it undergoes significant structural and functional changes that affect its size, shape, and mechanical properties. Abnormalities of the upper airway require prompt attention, because these often alter ventilatory patterns and gas exchange, particularly during sleep when upper airway motor tone and ventilatory drive are diminished. Recognizing the relationship of early life events to lung health and disease, the National Heart, Lung, and Blood Institute (NHLBI), with cofunding from the Office of Rare Diseases (ORD), convened a workshop of extramural experts, from many disciplines. The objective of the workshop was: (1) to review the state of science in pediatric upper airway disorders; (2) to make recommendations to the Institute to fill knowledge gaps; (3) to prioritize new research directions; and (4) to capitalize on scientific opportunities. This report provides recommendations that could facilitate translation of basic research findings into practice to better diagnose, treat, and prevent airway compromise in children.
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Sox2 is required for maintenance and differentiation of bronchiolar Clara, ciliated, and goblet cells.
PLoS ONE
PUBLISHED: 08-05-2009
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The bronchioles of the murine lung are lined by a simple columnar epithelium composed of ciliated, Clara, and goblet cells that together mediate barrier function, mucociliary clearance and innate host defense, vital for pulmonary homeostasis. In the present work, we demonstrate that expression of Sox2 in Clara cells is required for the differentiation of ciliated, Clara, and goblet cells that line the bronchioles of the postnatal lung. The gene was selectively deleted in Clara cells utilizing Scgb1a1-Cre, causing the progressive loss of Sox2 in the bronchioles during perinatal and postnatal development. The rate of bronchiolar cell proliferation was decreased and associated with the formation of an undifferentiated, cuboidal-squamous epithelium lacking the expression of markers of Clara cells (Scgb1a1), ciliated cells (FoxJ1 and alpha-tubulin), and goblet cells (Spdef and Muc5AC). By adulthood, bronchiolar cell numbers were decreased and Sox2 was absent in extensive regions of the bronchiolar epithelium, at which time residual Sox2 expression was primarily restricted to selective niches of CGRP staining neuroepithelial cells. Allergen-induced goblet cell differentiation and mucus production was absent in the respiratory epithelium lacking Sox2. In vitro, Sox2 activated promoter-luciferase reporter constructs for differentiation markers characteristic of Clara, ciliated, and goblet cells, Scgb1a1, FoxJ1, and Agr2, respectively. Sox2 physically interacted with Smad3 and inhibited TGF-beta1/Smad3-mediated transcriptional activity in vitro, a pathway that negatively regulates proliferation. Sox2 is required for proliferation and differentiation of Clara cells that serve as the progenitor cells from which Clara, ciliated, and goblet cells are derived.
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SAM pointed domain ETS factor (SPDEF) regulates terminal differentiation and maturation of intestinal goblet cells.
Exp. Cell Res.
PUBLISHED: 07-06-2009
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Background and Aims: SPDEF (also termed PDEF or PSE) is an ETS family transcription factor that regulates gene expression in the prostate and goblet cell hyperplasia in the lung. Spdef has been reported to be expressed in the intestine. In this paper, we identify an important role for Spdef in regulating intestinal epithelial cell homeostasis and differentiation. Methods: SPDEF expression was inhibited in colon cancer cells to determine its ability to control goblet cell gene activation. The effects of transgenic expression of Spdef on intestinal differentiation and homeostasis were determined. Results: In LS174T colon cancer cells treated with Notch/gamma-secretase inhibitor to activate goblet cell gene expression, shRNAs that inhibited SPDEF also repressed expression of goblet cell genes AGR2, MUC2, RETLNB, and SPINK4. Transgenic expression of Spdef caused the expansion of intestinal goblet cells and corresponding reduction in Paneth, enteroendocrine, and absorptive enterocytes. Spdef inhibited proliferation of intestinal crypt cells without induction of apoptosis. Prolonged expression of the Spdef transgene caused a progressive reduction in the number of crypts that expressed Spdef, consistent with its inhibitory effects on cell proliferation. Conclusions: Spdef was sufficient to inhibit proliferation of intestinal progenitors and induce differentiation into goblet cells; SPDEF was required for activation of goblet cell associated genes in vitro. These data support a model in which Spdef promotes terminal differentiation into goblet cells of a common goblet/Paneth progenitor.
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Conditional overexpression of connective tissue growth factor disrupts postnatal lung development.
Am. J. Respir. Cell Mol. Biol.
PUBLISHED: 06-18-2009
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Connective tissue growth factor (CTGF) is a member of an emerging family of immediate-early gene products that coordinates complex biological processes during development, differentiation, and tissue repair. Overexpression of CTGF is associated with mechanical ventilation with high tidal volume and oxygen exposure in newborn lungs. However, the role of CTGF in postnatal lung development and remodeling is not well understood. In the present study, a double-transgenic mouse model was generated with doxycycline-inducible overexpression of CTGF in respiratory epithelial cells. Overexpression of CTGF from Postnatal Days 1-14 resulted in thicker alveolar septa and decreased secondary septal formation. This is correlated with increased myofibroblast differentiation and disorganized elastic fiber deposition in alveolar septa. Overexpression of CTGF also decreased alveolar capillary network formation. There were increased alpha-smooth muscle actin expression and collagen deposition, and dramatic thickening in the peribronchial/peribronchiolar and perivascular regions in the double-transgenic lungs. Furthermore, overexpression of CTGF increased integrin-linked kinase expression, activated its downstream signaling target, Akt, as well as increased mRNA expression of fibronectin. These data demonstrate that overexpression of CTGF disrupts alveologenesis and capillary formation, and induces fibrosis during the critical period of alveolar development. These histologic changes are similar to those observed in lungs of infants with bronchopulmonary dysplasia.
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Deletion of Forkhead Box M1 transcription factor from respiratory epithelial cells inhibits pulmonary tumorigenesis.
PLoS ONE
PUBLISHED: 06-08-2009
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The Forkhead Box m1 (Foxm1) protein is induced in a majority of human non-small cell lung cancers and its expression is associated with poor prognosis. However, specific requirements for the Foxm1 in each cell type of the cancer lesion remain unknown. The present study provides the first genetic evidence that the Foxm1 expression in respiratory epithelial cells is essential for lung tumorigenesis. Using transgenic mice, we demonstrated that conditional deletion of Foxm1 from lung epithelial cells (epFoxm1(-/-) mice) prior to tumor initiation caused a striking reduction in the number and size of lung tumors, induced by either urethane or 3-methylcholanthrene (MCA)/butylated hydroxytoluene (BHT). Decreased lung tumorigenesis in epFoxm1(-/-) mice was associated with diminished proliferation of tumor cells and reduced expression of Topoisomerase-2alpha (TOPO-2alpha), a critical regulator of tumor cell proliferation. Depletion of Foxm1 mRNA in cultured lung adenocarcinoma cells significantly decreased TOPO-2alpha mRNA and protein levels. Moreover, Foxm1 directly bound to and induced transcription of the mouse TOPO-2alpha promoter region, indicating that TOPO-2alpha is a direct target of Foxm1 in lung tumor cells. Finally, we demonstrated that a conditional deletion of Foxm1 in pre-existing lung tumors dramatically reduced tumor growth in the lung. Expression of Foxm1 in respiratory epithelial cells is critical for lung cancer formation and TOPO-2alpha expression in vivo, suggesting that Foxm1 is a promising target for anti-tumor therapy.
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C/EBP{alpha} is required for pulmonary cytoprotection during hyperoxia.
Am. J. Physiol. Lung Cell Mol. Physiol.
PUBLISHED: 05-22-2009
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A number of transcriptional pathways regulating fetal lung development are active during repair of the injured lung. We hypothesized that C/EBPalpha, a transcription factor critical for lung maturation, plays a role in protection of the alveolar epithelium following hyperoxic injury of the mature lung. Transgenic Cebpalpha(Delta/Delta) mice, in which Cebpalpha was conditionally deleted from Clara cells and type II cells after birth, were developed. While no pulmonary abnormalities were observed in the Cebpalpha(Delta/Delta) mice (7-8 wk old) under normal conditions, the mice were highly susceptible to hyperoxia. Cebpalpha(Delta/Delta) mice died within 4 days of exposure to 95% oxygen in association with severe lung inflammation, altered maturation of surfactant protein B and C, decreased surfactant lipid secretion, and abnormal lung mechanics at a time when all control mice survived. mRNA microarray analysis of isolated type II cells at 0, 2, and 24 h of hyperoxia demonstrated the reduced expression of number of genes regulating surfactant lipid and protein homeostasis, including Srebf, Scap, Lpcat1, Abca3, Sftpb, and Napsa. Genes influencing cell signaling or immune responses were induced in the lungs of Cebpalpha(Delta/Delta) mice. C/EBPalpha was required for the regulation of genes associated with surfactant lipid homeostasis, surfactant protein biosynthesis, processing and transport, defense response to stress, and cell redox homeostasis during exposure to hyperoxia. While C/EBPalpha did not play a critical role in postnatal pulmonary function under normal conditions, C/EBPalpha mediated protection of the lung during acute lung injury induced by hyperoxia.
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Foxa1 and Foxa2 function both upstream of and cooperatively with Lmx1a and Lmx1b in a feedforward loop promoting mesodiencephalic dopaminergic neuron development.
Dev. Biol.
PUBLISHED: 05-19-2009
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Mesodiencephalic dopaminergic neurons control voluntary movement and reward based behaviours. Their dysfunction can lead to neurological disorders, including Parkinsons disease. These neurons are thought to arise from progenitors in the floor plate of the caudal diencephalon and midbrain. Members of the Foxa family of forkhead/winged helix transcription factor, Foxa1 and Foxa2, have previously been shown to regulate neuronal specification and differentiation of mesodiencephalic progenitors. However, Foxa1 and Foxa2 are also expressed earlier during regional specification of the rostral brain. In this paper, we have examined the early function of Foxa1 and Foxa2 using conditional mutant mice. Our studies show that Foxa1 and Foxa2 positively regulate Lmx1a and Lmx1b expression and inhibit Nkx2.2 expression in mesodiencephalic dopaminergic progenitors. Subsequently, Foxa1 and Foxa2 function cooperatively with Lmx1a and Lmx1b to regulate differentiation of mesodiencephalic dopaminergic neurons. Chromatin immunoprecipitation experiments indicate that Nkx2.2 and TH genes are likely direct targets of Foxa1 and Foxa2 in mesodiencephalic dopaminergic cells in vivo. Foxa1 and Foxa2 also inhibit GABAergic neuron differentiation by repressing the Helt gene in the ventral midbrain. Our data therefore provide new insights into the specification and differentiation of mesodiencephalic dopaminergic neurons and identifies Foxa1 and Foxa2 as essential regulators in these processes.
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SPDEF is required for mouse pulmonary goblet cell differentiation and regulates a network of genes associated with mucus production.
J. Clin. Invest.
PUBLISHED: 05-04-2009
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Various acute and chronic inflammatory stimuli increase the number and activity of pulmonary mucus-producing goblet cells, and goblet cell hyperplasia and excess mucus production are central to the pathogenesis of chronic pulmonary diseases. However, little is known about the transcriptional programs that regulate goblet cell differentiation. Here, we show that SAM-pointed domain-containing Ets-like factor (SPDEF) controls a transcriptional program critical for pulmonary goblet cell differentiation in mice. Initial cell-lineage-tracing analysis identified nonciliated secretory epithelial cells, known as Clara cells, as the progenitors of goblet cells induced by pulmonary allergen exposure in vivo. Furthermore, in vivo expression of SPDEF in Clara cells caused rapid and reversible goblet cell differentiation in the absence of cell proliferation. This was associated with enhanced expression of genes regulating goblet cell differentiation and protein glycosylation, including forkhead box A3 (Foxa3), anterior gradient 2 (Agr2), and glucosaminyl (N-acetyl) transferase 3, mucin type (Gcnt3). Consistent with these findings, levels of SPDEF and FOXA3 were increased in mouse goblet cells after sensitization with pulmonary allergen, and the proteins were colocalized in goblet cells lining the airways of patients with chronic lung diseases. Deletion of the mouse Spdef gene resulted in the absence of goblet cells in tracheal/laryngeal submucosal glands and in the conducting airway epithelium after pulmonary allergen exposure in vivo. These data show that SPDEF plays a critical role in regulating a transcriptional network mediating the goblet cell differentiation and mucus hyperproduction associated with chronic pulmonary disorders.
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Strategic plan for pediatric respiratory diseases research: an NHLBI working group report.
Pediatr. Pulmonol.
PUBLISHED: 04-25-2009
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The Division of Lung Diseases of the National Heart, Lung and Blood Institute (NHLBI) recently held a workshop to identify gaps in our understanding and treatment of childhood lung diseases and to define strategies to enhance translational research in this field. Leading experts with diverse experience in both laboratory and patient-oriented research reviewed selected areas of pediatric lung diseases, including perinatal programming and epigenetic influences; mechanisms of lung injury, repair, and regeneration; pulmonary vascular disease (PVD); sleep and control of breathing; and the application of novel translational methods to enhance personalized medicine. This report summarizes the proceedings of this workshop and provides recommendations for emphasis on targeted areas for future investigation. The priority areas identified for research in pediatric pulmonary diseases included: (1) epigenetic and environmental influences on lung development that program pediatric lung diseases, (2) injury, regeneration, and repair in the developing lung, (3) PVD in children, (4) development and adaptation of ventilatory responses to postnatal life, (5) nonatopic wheezing: aberrant large airway development or injury? (6) strategies to improve assessment, diagnosis, and treatment of pediatric respiratory diseases, and (7) predictive and personalized medicine for children.
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Deletion of Scap in alveolar type II cells influences lung lipid homeostasis and identifies a compensatory role for pulmonary lipofibroblasts.
J. Biol. Chem.
PUBLISHED: 04-07-2009
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Pulmonary function after birth is dependent upon surfactant lipids that reduce surface tension in the alveoli. The sterol-responsive element-binding proteins (SREBPs) are transcription factors regulating expression of genes controlling lipid homeostasis in many tissues. To identify the role of SREBPs in the lung, we conditionally deleted the SREBP cleavage-activating protein gene, Scap, in respiratory epithelial cells (ScapDelta/Delta) in vivo. Prior to birth (E18.5), deletion of Scap decreased the expression of both SREBPs and a number of genes regulating fatty acid and cholesterol metabolism. Nevertheless, ScapDelta/Delta mice survived postnatally, surfactant and lung tissue lipids being substantially normalized in adult ScapDelta/Delta mice. Although phospholipid synthesis was decreased in type II cells from adult ScapDelta/Delta mice, lipid storage, synthesis, and transfer by lung lipofibroblasts were increased. mRNA microarray data indicated that SCAP influenced two major gene networks, one regulating lipid metabolism and the other stress-related responses. Deletion of the SCAP/SREBP pathway in respiratory epithelial cells altered lung lipid homeostasis and induced compensatory lipid accumulation and synthesis in lung lipofibroblasts.
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Sox17 regulates organ lineage segregation of ventral foregut progenitor cells.
Dev. Cell
PUBLISHED: 03-28-2009
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The ventral pancreas, biliary system, and liver arise from the posterior ventral foregut, but the cell-intrinsic pathway by which these organ lineages are separated is not known. Here we show that the extrahepatobiliary system shares a common origin with the ventral pancreas and not the liver, as previously thought. These pancreatobiliary progenitor cells coexpress the transcription factors PDX1 and SOX17 at E8.5 and their segregation into a PDX1+ ventral pancreas and a SOX17+ biliary primordium is Sox17-dependent. Deletion of Sox17 at E8.5 results in the loss of biliary structures and ectopic pancreatic tissue in the liver bud and common duct, while Sox17 overexpression suppresses pancreas development and promotes ectopic biliary-like tissue throughout the PDX1+ domain. Restricting SOX17+ biliary progenitor cells to the ventral region of the gut requires the notch effector Hes1. Our results highlight the role of Sox17 and Hes1 in patterning and morphogenetic segregation of ventral foregut lineages.
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Surfactant protein-D regulates the postnatal maturation of pulmonary surfactant lipid pool sizes.
J. Appl. Physiol.
PUBLISHED: 03-05-2009
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Surfactant protein (SP)-D plays an important role in host defense and pulmonary surfactant homeostasis. In SP-D-deficient (Sftpd(-/-)) mice, the abnormal large surfactant forms seen at the ultrastructural level are taken up inefficiently by type II cells, resulting in an over threefold increase in the surfactant pool size. The mechanisms by which SP-D influences surfactant ultrastructure are unknown. We hypothesized that SP-D binds to surfactant immediately after being secreted and influences surfactant ultrastructure conversion. In newborn and adult sheep lungs, immunogold-labeled SP-D was associated with both lamellated membranous lipid structures of newly secreted surfactant and with small aggregate surfactant but not with tubular myelin. Since SP-D preferentially binds to phosphatidylinositol (PI) in vitro, the postnatal changes in PI were assessed. PI content in the bronchoalveolar lavage fluid increased after birth and peaked at 2-5 days of age, a time of rapid conversion of surfactant forms that is associated with the peak of surfactant lipid pool size. SP-D selectively interacted with PI-rich liposomes in vitro, causing their lysis. Similarly, the abnormal surfactant ultrastructure in Sftpd(-/-) mice was corrected by the addition of SP-D or melittin, and both peptides caused lysis of lipid vesicles. The normal conversion of surfactant ultrastructure requires SP-D that preferentially interacts with PI-rich, newly secreted surfactant, causing lysis of surfactant lipid membranes, converting the lipid forms into smaller surfactant lamellated structures that are critical for surfactant uptake by type II cells and normal surfactant homeostasis. SP-D regulates the dramatic decreases in the surfactant pool size that occurs in the newborn period.
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Rapamycin prevents transforming growth factor-alpha-induced pulmonary fibrosis.
Am. J. Respir. Cell Mol. Biol.
PUBLISHED: 02-24-2009
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Transforming growth factor (TGF)-alpha is a ligand for the epidermal growth factor receptor (EGFR). EGFR activation is associated with fibroproliferative processes in human lung disease and animal models of pulmonary fibrosis. Overexpression of TGF-alpha in transgenic mice causes progressive and severe pulmonary fibrosis; however, the intracellular signaling pathways downstream of EGFR mediating this response are unknown. Using a doxycycline-regulatable transgenic mouse model of lung-specific TGF-alpha expression, we observed increased PCNA protein and phosphorylation of Akt and p70S6K in whole lung homogenates in association with induction of TGF-alpha. Induction in the lung of TGF-alpha caused progressive pulmonary fibrosis over a 7-week period. Daily administration of rapamycin prevented accumulation of total lung collagen, weight loss, and changes in pulmonary mechanics. Treatment of mice with rapamycin 4 weeks after the induction of TGF-alpha prevented additional weight loss, increases in total collagen, and changes in pulmonary mechanics. Rapamycin prevented further increases in established pulmonary fibrosis induced by EGFR activation. This study demonstrates that mammalian target of rapamycin (mTOR) is a major effector of EGFR-induced pulmonary fibrosis, providing support for further studies to determine the role of mTOR in the pathogenesis and treatment of pulmonary fibrosis.
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Genetic disorders of surfactant dysfunction.
Pediatr. Dev. Pathol.
PUBLISHED: 02-18-2009
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Mutations in the genes encoding the surfactant proteins B and C (SP-B and SP-C) and the phospholipid transporter, ABCA3, are associated with respiratory distress and interstitial lung disease in the pediatric population. Expression of these proteins is regulated developmentally, increasing with gestational age, and is critical for pulmonary surfactant function at birth. Pulmonary surfactant is a unique mixture of lipids and proteins that reduces surface tension at the air-liquid interface, preventing collapse of the lung at the end of expiration. SP-B and ABCA3 are required for the normal organization and packaging of surfactant phospholipids into specialized secretory organelles, known as lamellar bodies, while both SP-B and SP-C are important for adsorption of secreted surfactant phospholipids to the alveolar surface. In general, mutations in the SP-B gene SFTPB are associated with fatal respiratory distress in the neonatal period, and mutations in the SP-C gene SFTPC are more commonly associated with interstitial lung disease in older infants, children, and adults. Mutations in the ABCA3 gene are associated with both phenotypes. Despite this general classification, there is considerable overlap in the clinical and histologic characteristics of these genetic disorders. In this review, similarities and differences in the presentation of these disorders with an emphasis on their histochemical and ultrastructural features will be described, along with a brief discussion of surfactant metabolism. Mechanisms involved in the pathogenesis of lung disease caused by mutations in these genes will also be discussed.
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Type I insulin-like growth factor receptor induces pulmonary tumorigenesis.
Neoplasia
PUBLISHED: 02-10-2009
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Despite the type I insulin-like growth factor receptor (IGF-IR) being highly expressed in more than 80% of human lung tumors, a transgenic model of IGF-IR overexpression in the lung has not been created. We produced two novel transgenic mouse models in which IGF-IR is overexpressed in either lung type II alveolar cells (surfactant protein C [SPC]-IGFIR) or Clara cells (CCSP-IGFIR) in a doxycycline-inducible manner. Overexpression of IGF-IR in either cell type caused multifocal adenomatous alveolar hyperplasia with papillary and solid adenomas. These tumors expressed thyroid transcription factor 1 and Kruppel-like factor 5 in most tumor cells. Similar to our previous work with lung tumors that developed in the mouse mammary tumor virus-IGF-II transgenic mice, the lung tumors that develop in the SPC-IGFIR and CCSP-IGFIR transgenic mice expressed high levels of the cyclic adenosine monophosphate response element binding protein that was localized primarily to the nucleus. Although elevated IGF-IR expression can initiate lung tumor development, tumors can become independent of IGF-IR signaling as IGF-IR down-regulation in established tumors produced tumor regression in some, but not all, of the tumors. These findings implicate IGF-IR as an important initiator of lung tumorigenesis and suggest that the SPC-IGFIR and CCSP-IGFIR transgenic mice can be used to further our understanding of human lung cancer and the role IGF-IR plays in this disease.
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Pulmonary surfactant surface tension influences alveolar capillary shape and oxygenation.
Am. J. Respir. Cell Mol. Biol.
PUBLISHED: 02-06-2009
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Alveolar capillaries are located in close proximity to the alveolar epithelium and beneath the surfactant film. We hypothesized that the shape of alveolar capillaries and accompanying oxygenation are influenced by surfactant surface tension in the alveolus. To prove our hypothesis, surfactant surface tension was regulated by conditional expression of surfactant protein (SP)-B in Sftpb(-/-) mice, thereby inhibiting surface tension-lowering properties of surfactant in vivo within 24 hours after depletion of Sftpb. Minimum surface tension of isolated surfactant was increased and oxygen saturation was significantly reduced after 2 days of SP-B deficiency in association with deformation of alveolar capillaries. Intravascularly injected 3.2-mum-diameter microbeads through jugular vein were retained within narrowed pulmonary capillaries after reduction of SP-B. Ultrastructure studies demonstrated that the capillary protrusion typical of the normal alveolar-capillary unit was reduced in size, consistent with altered pulmonary blood flow. Pulmonary hypertension and intrapulmonary shunting are commonly associated with surfactant deficiency and dysfunction in neonates and adults with respiratory distress syndromes. Increased surfactant surface tension caused by reduction in SP-B induced narrowing of alveolar capillaries and oxygen desaturation, demonstrating an important role of surface tension-lowering properties of surfactant in the regulation of pulmonary vascular perfusion.
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Early growth response-1 suppresses epidermal growth factor receptor-mediated airway hyperresponsiveness and lung remodeling in mice.
Am. J. Respir. Cell Mol. Biol.
PUBLISHED: 02-02-2009
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Transforming growth factor (TGF)-alpha and its receptor, the epidermal growth factor receptor, are induced after lung injury and are associated with remodeling in chronic pulmonary diseases, such as pulmonary fibrosis and asthma. Expression of TGF-alpha in the lungs of adult mice causes fibrosis, pleural thickening, and pulmonary hypertension, in addition to increased expression of a transcription factor, early growth response-1 (Egr-1). Egr-1 was increased in airway smooth muscle (ASM) and the vascular adventitia in the lungs of mice conditionally expressing TGF-alpha in airway epithelium (Clara cell secretory protein-rtTA(+/-)/[tetO](7)-TGF-alpha(+/-)). The goal of this study was to determine the role of Egr-1 in TGF-alpha-induced lung disease. To accomplish this, TGF-alpha-transgenic mice were crossed to Egr-1 knockout (Egr-1(ko/ko)) mice. The lack of Egr-1 markedly increased the severity of TGF-alpha-induced pulmonary disease, dramatically enhancing airway muscularization, increasing pulmonary fibrosis, and causing greater airway hyperresponsiveness to methacholine. Smooth muscle hyperplasia, not hypertrophy, caused the ASM thickening in the absence of Egr-1. No detectable increases in pulmonary inflammation were found. In addition to the airway remodeling disease, vascular remodeling and pulmonary hypertension were also more severe in Egr-1(ko/ko) mice. Thus, Egr-1 acts to suppress epidermal growth factor receptor-mediated airway and vascular muscularization, fibrosis, and airway hyperresponsiveness in the absence of inflammation. This provides a unique model to study the processes causing pulmonary fibrosis and ASM thickening without the complicating effects of inflammation.
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Sox17 promotes cell cycle progression and inhibits TGF-beta/Smad3 signaling to initiate progenitor cell behavior in the respiratory epithelium.
PLoS ONE
PUBLISHED: 01-23-2009
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The Sry-related high mobility group box transcription factor Sox17 is required for diverse developmental processes including endoderm formation, vascular development, and fetal hematopoietic stem cell maintenance. Expression of Sox17 in mature respiratory epithelial cells causes proliferation and lineage respecification, suggesting that Sox17 can alter adult lung progenitor cell fate. In this paper, we identify mechanisms by which Sox17 influences lung epithelial progenitor cell behavior and reprograms cell fate in the mature respiratory epithelium. Conditional expression of Sox17 in epithelial cells of the adult mouse lung demonstrated that cell cluster formation and respecification of alveolar progenitor cells toward proximal airway lineages were rapidly reversible processes. Prolonged expression of Sox17 caused the ectopic formation of bronchiolar-like structures with diverse respiratory epithelial cell characteristics in alveolar regions of lung. During initiation of progenitor cell behavior, Sox17 induced proliferation and increased the expression of the progenitor cell marker Sca-1 and genes involved in cell cycle progression. Notably, Sox17 enhanced cyclin D1 expression in vivo and activated cyclin D1 promoter activity in vitro. Sox17 decreased the expression of transforming growth factor-beta (TGF-beta)-responsive cell cycle inhibitors in the adult mouse lung, including p15, p21, and p57, and inhibited TGF-beta1-mediated transcriptional responses in vitro. Further, Sox17 interacted with Smad3 and blocked Smad3 DNA binding and transcriptional activity. Together, these data show that a subset of mature respiratory epithelial cells retains remarkable phenotypic plasticity and that Sox17, a gene required for early endoderm formation, activates the cell cycle and reinitiates multipotent progenitor cell behavior in mature lung cells.
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Strategic plan for pediatric respiratory diseases research: an NHLBI working group report.
Proc Am Thorac Soc
PUBLISHED: 01-10-2009
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The Division of Lung Diseases of the National Heart, Lung, and Blood Institute (NHLBI) recently held a workshop to identify gaps in our understanding and treatment of childhood lung diseases and to define strategies to enhance translational research in this field. Leading experts with diverse experience in both laboratory and patient-oriented research reviewed selected areas of pediatric lung diseases, including perinatal programming and epigenetic influences; mechanisms of lung injury, repair, and regeneration; pulmonary vascular disease; sleep and control of breathing; and the application of novel translational methods to enhance personalized medicine. This report summarizes the proceedings of this workshop and provides recommendations for emphasis on targeted areas for future investigation. The priority areas identified for research in pediatric pulmonary diseases included: (1) epigenetic and environmental influences on lung development that program pediatric lung diseases; (2) injury, regeneration, and repair in the developing lung; (3) pulmonary vascular disease in children; (4) development and adaptation of ventilatory responses to postnatal life; (5) nonatopic wheezing: aberrant large airway development or injury?; (6) strategies to improve assessment, diagnosis, and treatment of pediatric respiratory diseases; and (7) predictive and personalized medicine for children.
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