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ABSTRACT: Gastrin-releasing peptide (GRP), secreted by pulmonary neuroendocrine cells, mediates oxidant-induced lung injury in animal models. Considering that GRP blockade abrogates pulmonary inflammation and fibrosis in hyperoxic baboons, we hypothesized that ionizing radiation triggers GRP secretion, contributing to inflammatory and fibrotic phases of radiation-induced lung injury (RILI). Using an established C57BL/6 mouse model of RILI, with chronic fibrosis developing ≥20 weeks after high-dose thoracic radiation (15 Gy), we injected small molecule 77427 i.p. approximately 1 hour after radiation then twice weekly for up to 20 weeks. Sham controls were anesthetized and placed in the irradiator without radiation. Lung paraffin sections were immunostained and quantitative image analyses performed. Mice exposed to radiation plus PBS had increased interstitial CD68(+) macrophages 4 weeks after radiation and pulmonary neuroendocrine cells hyperplasia 6 weeks after radiation. At 10 weeks after radiation, radiation plus PBS controls had significantly increased pSmad2/3(+) nuclei/cm(2). GRP blockade with 77427 treatment diminished CD68(+), GRP(+), and pSmad2/3(+) cells. Finally, interstitial fibrosis was evident 20 weeks after radiation by immunostaining for α-smooth muscle actin and collagen deposition. Treatment with 77427 abrogated interstitial α-smooth muscle actin and collagen. Sham mice given 77427 did not differ significantly from PBS controls. Our data are the first to show that GRP blockade decreases inflammatory and fibrotic responses to radiation in mice. GRP blockade is a novel RILI-mitigating agent that could be clinically useful in humans exposed to radiation therapeutically or unintentionally.
American Journal Of Pathology 02/2013; · 4.89 Impact Factor
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Rui Lin,
Simone Degan,
Barbara S Theriot,
Bernard M Fischer,
Ryan T Strachan,
Jiurong Liang,
Richard A Pierce, Mary E Sunday,
Paul W Noble,
Monica Kraft,
Arnold R Brody,
Julia K L Walker
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ABSTRACT: Inhalation of a β-adrenoceptor agonist (β-agonist) is first-line asthma therapy, used for both prophylaxis against, and acute relief of, bronchoconstriction. However, repeated clinical use of β-agonists leads to impaired bronchoprotection and, in some cases, adverse patient outcomes. Mechanisms underlying this β(2) -adrenoceptor dysfunction are not well understood, due largely to the lack of a comprehensive animal model and the uncertainty as to whether or not bronchorelaxation in mice is mediated by β(2) -adrenoceptors. Thus, we aimed to develop a mouse model that demonstrated functional β-agonist-induced β(2) -adrenoceptor desensitization in the context of allergic inflammatory airway disease.
We combined chronic allergen exposure with repeated β-agonist inhalation in allergen-treated BALB/C mice and examined the contribution of β(2) -adrenoceptors to albuterol-induced bronchoprotection using FVB/NJ mice with genetic deletion of β(2) -adrenoceptors (KO). Associated inflammatory changes - cytokines (ELISA), cells in bronchoalevolar lavage and airway remodelling (histology) and β(2) -adrenoceptor density (radioligand binding) - were also measured. KEY RESULTS β(2) -Adrenoceptors mediated albuterol-induced bronchoprotection in mice. Chronic treatment with albuterol induced loss of bronchoprotection, associated with exacerbation of the inflammatory components of the asthma phenotype.
This animal model reproduced salient features of human asthma and linked loss of bronchoprotection with airway pathobiology. Accordingly, the model offers an advanced tool for understanding the mechanisms of the effects of chronic β- agonist treatment on β-adrenoceptor function in asthma. Such information may guide the clinical use of β-agonists and provide insight into development of novel β-adrenoceptor ligands for the treatment of asthma.
British Journal of Pharmacology 10/2011; 165(7):2365-77. · 4.41 Impact Factor
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Jennifer L Ingram,
Molly J Huggins,
Tony D Church,
Yuejuan Li,
Dave C Francisco,
Simone Degan,
Rafael Firszt,
Denise M Beaver,
Njira L Lugogo,
Ying Wang, Mary E Sunday,
Paul W Noble,
Monica Kraft
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ABSTRACT: Invasive cell phenotypes have been demonstrated in malignant transformation, but not in other diseases, such as asthma. Cellular invasiveness is thought to be mediated by transforming growth factor (TGF)-β1 and matrix metalloproteinases (MMPs). IL-13 is a key T(H)2 cytokine that directs many features of airway remodeling through TGF-β1 and MMPs.
We hypothesized that, in human asthma, IL-13 stimulates increased airway fibroblast invasiveness via TGF-β1 and MMPs in asthma compared with normal controls.
Fibroblasts were cultured from endobronchial biopsies in 20 subjects with mild asthma (FEV(1): 90 ± 3.6% pred) and 17 normal control subjects (FEV(1): 102 ± 2.9% pred) who underwent bronchoscopy. Airway fibroblast invasiveness was investigated using Matrigel chambers. IL-13 or IL-13 with TGF-β1 neutralizing antibody or pan-MMP inhibitor (GM6001) was added to the lower chamber as a chemoattractant. Flow cytometry and immunohistochemistry were performed in a subset of subjects to evaluate IL-13 receptor levels.
IL-13 significantly stimulated invasion in asthmatic airway fibroblasts, compared with normal control subjects. Inhibitors of both TGF-β1 and MMPs blocked IL-13-induced invasion in asthma, but had no effect in normal control subjects. At baseline, in airway tissue, IL-13 receptors were expressed in significantly higher levels in asthma, compared with normal control subjects. In airway fibroblasts, baseline IL-13Rα2 was reduced in asthma compared with normal control subjects.
IL-13 potentiates airway fibroblast invasion through a mechanism involving TGF-β1 and MMPs. IL-13 receptor subunits are differentially expressed in asthma. These effects may result in IL-13-directed airway remodeling in asthma.
American Journal of Respiratory and Critical Care Medicine 03/2011; 183(12):1625-32. · 11.08 Impact Factor
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ABSTRACT: Gastrin-releasing peptide (GRP) is synthesized by pulmonary neuroendocrine cells in inflammatory lung diseases, such as bronchopulmonary dysplasia (BPD). Many BPD infants develop asthma, a serious disorder of intermittent airway obstruction. Despite extensive research, early mechanisms of asthma remain controversial. The incidence of asthma is growing, now affecting >300 million people worldwide. To test the hypothesis that GRP mediates asthma, we used two murine models: ozone exposure for air pollution-induced airway hyperreactivity (AHR), and ovalbumin (OVA)-induced allergic airway disease. BALB/c mice were given small molecule GRP blocking agent 77427, or GRP blocking antibody 2A11, before exposure to ozone or OVA challenge. In both models, GRP blockade abrogated AHR and bronchoalveolar lavage (BAL) macrophages and granulocytes, and decreased BAL cytokines implicated in asthma, including those typically derived from Th1 (e.g., IL-2, TNFα), Th2 (e.g., IL-5, IL-13), Th17 (IL-17), macrophages (e.g., MCP-1, IL-1), and neutrophils (KC = IL-8). Dexamethasone generally had smaller effects on all parameters. Macrophages, T cells, and neutrophils express GRP receptor (GRPR). GRP blockade diminished serine phosphorylation of GRPR with ozone or OVA. Thus, GRP mediates AHR and airway inflammation in mice, suggesting that GRP blockade is promising as a broad-spectrum therapeutic approach to treat and/or prevent asthma in humans.
Proceedings of the National Academy of Sciences 02/2011; 108(5):2100-5. · 9.68 Impact Factor
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Amy M Pastva,
Julia K L Walker,
Lee A Maddox,
Sambuddho Mukherjee,
Charles Giamberardino,
Bethany Hsia,
Erin Potts,
Hongmei Zhu,
Simone Degan, Mary E Sunday,
Barbara L Lawson,
Thomas R Korfhagen,
David A Schwartz,
Jerry P Eu,
William M Foster,
Timothy J McMahon,
Loretta Que,
Jo Rae Wright
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ABSTRACT: Surfactant protein A (SP-A) mediates innate immune cell responses to LPS, a cell wall component of gram-negative bacteria that is found ubiquitously in the environment and is associated with adverse health effects. Inhaled LPS induces lung inflammation and increases airway responsiveness (AR). However, the role of SP-A in mediating LPS-induced AR is not well-defined. Nitric oxide (NO) is described as a potent bronchodilator, and previous studies showed that SP-A modulates the LPS-induced production of NO. Hence, we tested the hypothesis that increased AR, observed in response to aerosolized LPS exposure, would be significantly reduced in an SP-A-deficient condition. Wild-type (WT) and SP-A null (SP-A(-/-)) mice were challenged with aerosolized LPS. Results indicate that despite similar inflammatory indices, LPS-treated SP-A(-/-) mice had attenuated AR after methacholine challenge, compared with WT mice. The attenuated AR could not be attributed to inherent differences in SP-D concentrations or airway smooth muscle contractile and relaxation properties, because these measures were similar between WT and SP-A(-/-) mice. LPS-treated SP-A(-/-) mice, however, had elevated nitrite concentrations, inducible nitric oxide synthase (iNOS) expression, and NOS activity in their lungs. Moreover, the administration of the iNOS-specific inhibitor 1400W completely abrogated the attenuated AR. Thus, when exposed to aerosolized LPS, SP-A(-/-) mice demonstrate a relative airway hyporesponsiveness that appears to be mediated at least partly via an iNOS-dependent mechanism. These findings may have clinical significance, because recent studies reported associations between surfactant protein polymorphisms and a variety of lung diseases.
American Journal of Respiratory Cell and Molecular Biology 03/2010; 44(2):175-84. · 5.13 Impact Factor
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ABSTRACT: Trachealess (Trh) is a PAS domain transcription factor regulating Drosophila tracheogenesis. No other Trh homolog has been associated with a respiratory phenotype. Seeking homolog(s) regulating lung development, we screened murine genomic DNA using trh oligonucleotides, identifying only Npas3. Npas3 mRNA peaks in lung from E10.5 to E13.5, verified by sequencing, with immunostaining in airway epithelial cells. Npas3-null mice have reduced lung branching morphogenesis but are viable prenatally. Npas3-null newborns die in respiratory distress, with diminished alveolarization, decreased Shh, Fgf9, Fgf10, and Bmp4 mRNAs, and increased Spry2, consistent with reduced FGF signaling. Exogenous FGF10 rescues branching morphogenesis in Npas3-null lungs. In promoter reporter assays, NPAS3 directly upregulates Shh and represses Spry2. Npas3(+/-) mice have a milder lung phenotype, surviving postnatally, but develop emphysema and airways hyperreactivity. Therefore, absence of a developmentally expressed transcription factor can alter downstream gene expression and multiple signaling pathways in organogenesis. NPAS3 haploinsufficiency may also lead to emphysema and asthma.
Proceedings of the National Academy of Sciences 08/2009; 106(28):11691-6. · 9.68 Impact Factor
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Julie G Ledford,
Hisatsugu Goto,
Erin N Potts,
Simone Degan,
Hong Wei Chu,
Dennis R Voelker, Mary E Sunday,
George J Cianciolo,
William M Foster,
Monica Kraft,
Jo Rae Wright
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ABSTRACT: The lung is constantly challenged during normal breathing by a myriad of environmental irritants and infectious insults. Pulmonary host defense mechanisms maintain homeostasis between inhibition/clearance of pathogens and regulation of inflammatory responses that could injure the airway epithelium. One component of this defense mechanism, surfactant protein-A (SP-A), exerts multifunctional roles in mediating host responses to inflammatory and infectious agents. SP-A has a bacteriostatic effect on Mycoplasma pneumoniae (Mp), which occurs by binding surface disaturated phosphatidylglycerols. SP-A can also bind the Mp membrane protein, MPN372. In this study, we investigated the role of SP-A during acute phase pulmonary infection with Mp using mice deficient in SP-A. Biologic responses, inflammation, and cellular infiltration, were much greater in Mp infected SP-A(-/-) mice than wild-type mice. Likewise, physiologic responses (airway hyperresponsiveness and lung compliance) to Mp infection were more severely affected in SP-A(-/-) mice. Both Mp-induced biologic and physiologic changes were attenuated by pharmacologic inhibition of TNF-alpha. Our findings demonstrate that SP-A is vital to preserving lung homeostasis and host defense to this clinically relevant strain of Mp by curtailing inflammatory cell recruitment and limiting an overzealous TNF-alpha response.
The Journal of Immunology 07/2009; 182(12):7818-27. · 5.79 Impact Factor
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ABSTRACT: Pseudomonas aeruginosa has the capacity to invade lung epithelial cells by co-opting the intrinsic endocytic properties of lipid rafts, which are rich in cholesterol, sphingolipids, and proteins, such as caveolin-1 and -2. We compared intratracheal Pseudomonas infection in wild type and caveolin-deficient mice to investigate the role of caveolin proteins in the pathogenesis of Pseudomonas pneumonia. Unlike wild type mice, which succumb to pneumonia, caveolin-deficient mice are resistant to Pseudomonas. We observed that Pseudomonas invasion of lung epithelial cells is dependent on caveolin-2 but not caveolin-1. Phosphorylation of caveolin-2 by Src family kinases is an essential event for Pseudomonas invasion. Our studies also reveal the existence of a distinct signaling mechanism in lung epithelial cells mediated by COOH-terminal Src kinase (Csk) that negatively regulates Pseudomonas invasion. Csk migrates to lipid raft domains, where it decreases phosphorylation of caveolin-2 by inactivating c-Src. Whereas Pseudomonas co-opts the endocytic properties of caveolin-2 for invasion, there also exists in these cells an intrinsic Csk-dependent cellular defense mechanism aimed at impairing this activity. The success of Pseudomonas in co-opting lipid raft-mediated endocytosis to invade lung epithelial cells may depend on the relative strengths of these counteracting signaling activities.
Journal of Biological Chemistry 03/2009; 284(15):9955-64. · 4.77 Impact Factor
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ABSTRACT: Gastrin-releasing peptide (GRP) is produced by pulmonary neuroendocrine cells (PNECs), with highest numbers of GRP-positive cells present in fetal lung. Normally GRP-positive PNECs are relatively infrequent after birth, but PNEC hyperplasia is frequently associated with chronic lung diseases. To address the hypothesis that GRP mediates chronic lung injury, we present the cumulative evidence implicating GRP in bronchopulmonary dysplasia (BPD), the chronic lung disease of premature infants who survive acute respiratory distress syndrome. The availability of well-characterized animal models of BPD was a critical tool for demonstrating that GRP plays a direct role in the early pathogenesis of this disease. Potential mechanisms by which GRP contributes to injury are analyzed, with the main focus on innate immunity. Autoreactive T cells may contribute to lung injury late in the course of disease. A working model is proposed with GRP triggering multiple cell types in both the innate and adaptive immune systems, promoting cascades culminating in chronic lung disease. These observations represent a paradigm shift in the understanding of the early pathogenesis of BPD, and suggest that GRP blockade could be a novel treatment to prevent this lung disease in premature infants.
Annals of the New York Academy of Sciences 12/2008; 1144:136-47. · 3.15 Impact Factor
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ABSTRACT: The adult human bronchial tree is covered with a continuous layer of epithelial cells that play a critical role in maintaining the conduit for air, and which are central to the defenses of the lung against inhaled environmental concomitants. The epithelial sheet functions as an interdependent unit with the other lung components. Importantly, the structure and/or function of airway epithelium is deranged in major lung disorders, including chronic obstructive pulmonary disease, asthma, and bronchogenic carcinoma. Investigations regarding the airway epithelium have led to many advances over the past few decades, but new developments in genetics and stem cell/progenitor cell biology have opened the door to understanding how the airway epithelium is developed and maintained, and how it responds to environmental stress. This article provides an overview of the current state of knowledge regarding airway epithelial stem/progenitor cells, gene expression, cell-cell interactions, and less frequent cell types, and discusses the challenges for future areas of investigation regarding the airway epithelium in health and disease.
Proceedings of the American Thoracic Society 10/2008; 5(7):772-7.
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Teri J Franks,
Thomas V Colby,
William D Travis,
Rubin M Tuder,
Herbert Y Reynolds,
Arnold R Brody,
Wellington V Cardoso,
Ronald G Crystal,
Christopher J Drake,
John Engelhardt, [......],
Sem H Phan,
Scott H Randell,
Mary C Rose,
Troy Stevens,
Julie Serge, Mary E Sunday,
Judith A Voynow,
Brant M Weinstein,
Jeffrey Whitsett,
Mary C Williams
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ABSTRACT: The purpose of the workshop was to identify still obscure or novel cellular components of the lung, to determine cell function in lung development and in health that impacts on disease, and to decide promising avenues for future research to extract and phenotype these cells. Since robust technologies are now available to identify, sort, purify, culture, and phenotype cells, progress is now within sight to unravel the origins and functional capabilities of lung cells in developmental stages and in disease. The Workshop's agenda was to first discuss the lung's embryologic development, including progenitor and stem cells, and then assess the functional and structural cells in three main compartments of the lung: (1) airway cells in bronchial and bronchiolar epithelium and bronchial glands (basal, secretory, ciliated, Clara, and neuroendocrine cells); (2) alveolar unit cells (Type 1 cells, Type 2 cells, and fibroblasts in the interstitium); and (3) pulmonary vascular cells (endothelial cells from different vascular structures, smooth muscle cells, and adventitial fibroblasts). The main recommendations were to: (1) characterize with better cell markers, both surface and nonsurface, the various cells within the lung, including progenitor cells and stem cells; (2) obtain more knowledge about gene expression in specific cell types in health and disease, which will provide insights into biological and pathologic processes; (3) develop more methodologies for cell culture, isolation, sorting, co-culture, and immortalization; and (4) promote tissue banks to facilitate the procurement of tissue from normal and from diseased lung for analysis at all levels.
Proceedings of the American Thoracic Society 10/2008; 5(7):763-6.
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ABSTRACT: Murine lung development begins at embryonic day (E) 9.5. Normal lung structure and function depend on the patterns of localization of differentiated cells. Pulmonary mesenchymal cell lineages have been relatively unexplored. Importantly, there has been no prior evidence of clonality of any lung cells. Herein we use a definitive genetic approach to demonstrate a common origin for proximal and distal pulmonary mesenchymal cells. A retroviral library with 3,400 unique inserts was microinjected into the airway lumen of E11.5 lung buds. After 7-11 days of culture, buds were stained for placental alkaline phosphatase (PLAP). Most PLAP+ cells are peribronchial smooth muscle cells, initially localized laterally near the hilum, then migrating down airways to the subpleural region. Laser-capture microdissection and polymerase chain reaction confirm the clonal identities of PLAP+ cells proximally and distally. Our observation of this fundamental process during lung development opens new avenues for investigation of maladaptive mesenchymal responses in lung diseases.
Developmental Dynamics 04/2008; 237(3):750-7. · 2.54 Impact Factor
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ABSTRACT: The ontogeny of the C-C chemokines eotaxin-1, eotaxin-2, and eotaxin-3 has not been fully elucidated in human lung. We explored a possible role for eotaxin in developing lung by determining the ontogeny of eotaxin-1 (CCL11), eotaxin-2 (CCL24), eotaxin-3 (CCL26), and the eotaxin receptor, CCR3. We tested discarded surgical samples of developing human lung tissue using quantitative RT-PCR (QRT-PCR) and immunostaining for expression of CCL11, CCL24, CCL26, and CCR3. We assessed possible functionality of the eotaxin-CCR3 system by treating lung explant cultures with exogenous CCL11 and analyzing the cultures for evidence of changes in proliferation and activation of ERK1/2, a signaling pathway associated with CCR3. QRT-PCR analyses of 22 developing lung tissue samples with gestational ages 10-23 wk demonstrated that eotaxin-1 mRNA is most abundant in developing lung, whereas mRNAs for eotaxin-2 and eotaxin-3 are minimally detectable. CCL11 mRNA levels correlated with gestational age (P < 0.05), and immunoreactivity was localized predominantly to airway epithelial cells. QRT-PCR analysis detected CCR3 expression in 16 of 19 developing lung samples. Supporting functional capacity in the immature lung, CCL11 treatment of lung explant cultures resulted in significantly increased (P < 0.05) cell proliferation and activation of the ERK signaling pathway, which is downstream from CCR3, suggesting that proliferation was due to activation of CCR3 receptors by CCL11. We conclude that developing lung expresses the eotaxins and functional CCR3 receptor. CCL11 may promote airway epithelial proliferation in the developing lung.
AJP Lung Cellular and Molecular Physiology 02/2008; 294(2):L214-24. · 3.66 Impact Factor
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ABSTRACT: The incidence of bronchopulmonary dysplasia (BPD), a chronic lung disease of newborns, is paradoxically rising despite medical advances. We demonstrated elevated bombesin-like peptide levels in infants that later developed BPD. In the 140-day hyperoxic baboon model of BPD, anti-bombesin antibody 2A11 abrogated lung injury. Objectives: To test the hypothesis that bombesin-like peptides mediate BPD in extremely premature baboons (born at Gestational Day 125 and given oxygen pro re nata [PRN], called the 125-day PRN model), similar to "modern-day BPD."
The 125-day animals were treated with 2A11 on Postnatal Day 1 (P1), P3, and P6. On P14 and P21, lungs were inflation-fixed for histopathologic analyses of alveolarization. Regulation of angiogenesis by bombesin was evaluated using cultured pulmonary microvascular endothelial cells.
In 125-day PRN animals, urine bombesin-like peptide levels at P2-3 are directly correlated with impaired lung function at P14. Gastrin-releasing peptide (the major pulmonary bombesin-like peptide) mRNA was elevated eightfold at P1 and remained high thereafter. At P14, 2A11 reduced alveolar wall thickness and increased the percentage of secondary septa containing endothelial cells. At P21, 2A11-treated 125-day PRN animals had improved alveolarization according to mean linear intercepts and number of branch points per millimeter squared. Bombesin promoted tubulogenesis of cultured pulmonary microvascular endothelial cells, but cocultured fetal lung mesenchymal cells abrogated this effect.
Early bombesin-like peptide overproduction in 125-day PRN animals predicted alveolarization defects weeks later. Bombesin-like peptide blockade improved septation, with the greatest effects at P21. This could have implications for preventing BPD in premature infants.
American Journal of Respiratory and Critical Care Medicine 12/2007; 176(9):902-12. · 11.08 Impact Factor
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ABSTRACT: Drosophila trachealess (Trl), master regulator of tracheogenesis, has no known functional mammalian homolog. We hypothesized that genes similar to trachealess regulate lung development. Quantitative (Q)RT-PCR and immunostaining were used to determine spatial and temporal patterns of npas1 gene expression in developing murine lung. Immunostaining for alpha-smooth muscle actin demonstrated myofibroblasts, and protein gene product (PGP)9.5 identified neuroendocrine cells. Branching morphogenesis of embryonic lung buds was analyzed in the presence of antisense or sense oligodeoxynucleotides (ODN). Microarray analyses were performed to screen for changes in gene expression in antisense-treated lungs. QRT-PCR was used to validate the altered expression of key genes identified on the microarrays. We demonstrate that npas1 is expressed in murine embryonic lung. npas1 mRNA peaks early at Embryonic Day (E)10.5-E11.5, then drops to low levels. Sequencing verifies the identity of npas1 transcripts in embryonic lung. NPAS1 immunostaining occurs in nuclei of parabronchial mesenchymal cells, especially at the tracheal bifurcation. Arnt, the murine homolog of Tango (the heterodimerization partner for Trl) is also expressed in developing lung but at constant levels. npas1- or arnt-antisense ODN inhibit lung branching morphogenesis, with altered myofibroblast development and increased pulmonary neuroendocrine cells. On microarrays, we identify > 50 known genes down-regulated by npas1-antisense, including multiple genes regulating cell migration and cell differentiation. QRT-PCR confirms significantly decreased expression of the neurogenic genes RBP-Jk and Tle, and three genes involved in muscle development: beta-ig-h3, claudin-11, and myocardin. Npas1 can regulate myofibroblast distribution, branching morphogenesis, and neuroendocrine cell differentiation in murine embryonic lung.
American Journal of Respiratory Cell and Molecular Biology 04/2007; 36(4):427-34. · 5.13 Impact Factor
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ABSTRACT: The notch gene family encodes transmembrane receptors that regulate cell differentiation by interacting with surface ligands on adjacent cells. Previously, we demonstrated that tumor necrosis factor-alpha (TNF) induces neuroendocrine (NE) cell differentiation in H82, but not H526, undifferentiated small cell lung carcinoma lines. We now test the hypothesis that TNF mediates NE cell differentiation in part by altering Notch gene expression. First, using RT-PCR, we determined that TNF treatment of H82, but not H526, transiently decreases notch-1 mRNA in parallel with induction of gene expression for the NE-specific marker DOPA decarboxylase (DDC). Second, we treated H82 and H526 with notch-1 antisense vs. sense oligodeoxynucleotides. Using quantitative RT-PCR and Western analyses we demonstrate that DDC mRNA and protein are increased in H82 by notch-1 antisense, whereas notch-1 mRNA and activated Notch-1 protein are decreased. mRNA for Hes1, a transcription factor downstream from activated Notch, is also decreased by Notch-1 antisense in H82 but not H526. After 7 days of Notch-1 antisense treatment, neural cell adhesion molecule (NCAM) immunoreactivity is induced in H82 but not H526. Third, we generated transgenic mice bearing notch-1 driven by the neural/NE-specific calcitonin promoter, which express activated Notch-1 in developing lung epithelium. Newborn NotchCal mouse lungs have high levels of hes1 mRNA, reflecting increased activated Notch, compared with wild-type. NotchCal lungs have decreased CGRP-positive NE cells, decreased protein gene product 9.5 (PGP9.5)-positive NE cells, and decreased gastrin-releasing peptide (GRP), CGRP, and DDC mRNA levels compared with normal littermates. Cumulatively, these observations provide further support for a role for Notch-1 signaling in regulating pulmonary NE cell differentiation.
AJP Lung Cellular and Molecular Physiology 03/2007; 292(2):L500-9. · 3.66 Impact Factor
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ABSTRACT: Bronchopulmonary dysplasia (BPD), a chronic lung disease of newborns triggered by oxygen and barotrauma, is characterized by arrested alveolarization. Increased levels of bombesin-like peptides shortly after birth mediate lung injury: anti-bombesin antibody 2A11 protects against BPD in two baboon models. The role of adaptive immunity in BPD has not been explored previously.
Our goal was to test the hypothesis that thymic architecture and/or T-cell function is altered with BPD, leading to autoimmunity and immunodeficiency.
Thymic structure was analyzed by histopathology of thymic architecture and immunohistochemistry for thymic maturation markers (terminal deoxynucleotidyl transferase, proliferating cell nuclear antigen, CD4, and CD8). Thymic cortical epithelial cells (nurse cells) were studied using HLA-DR and protein gene product 9.5 as markers. Functional analysis was performed with "mixed lymphocyte reaction" of thymocyte or splenocyte responder cells with autologous lung cells as the stimulators.
2A11 treatment attenuates thymic cortical involution in BPD animals, sustaining terminal deoxynucleotidyl transferase-positive prothymocytes and thymocyte proliferation. BPD animals have increased CD4(+) cells in thymic cortex and lung interstitium, which are reduced by 2A11. Conversely, cortical protein gene product 9.5/HLA-DR-positive thymic nurse cells are depleted in BPD animals, but are preserved by 2A11-treatment. Whereas fetal thymocytes and splenocytes respond to phythemagglutinin/ionomycin and to a lesser extent, to autologous lung, BPD thymocytes and splenocytes are phythemagglutinin/ionomycin-unresponsive, and yet react strongly to autologous lung. The 2A11 normalizes these responses.
These observations suggest that bombesin-like peptides mediate premature thymic maturation and thymic nurse-cell depletion, leading to autoreactive T cells that could contribute to lung injury.
American Journal of Respiratory and Critical Care Medicine 08/2006; 174(1):75-83. · 11.08 Impact Factor
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ABSTRACT: Bombesin-like peptides promote fetal lung development. Normally, levels of mammalian bombesin (gastrin-releasing peptide [GRP]) drop postnatally, but these levels are elevated in newborns that develop bronchopulmonary dysplasia (BPD), a chronic lung disease characterized by arrested alveolarization. In premature baboons with BPD, antibombesin antibodies reduce lung injury and promote alveolarization.
The present study tests whether exogenous bombesin or GRP given perinatally alters alveolar development in newborn mice.
Mice were given peptides intraperitoneally twice daily on Postnatal Days 1-3. On Day 14 lungs were inflation-fixed for histopathologic analyses of alveolarization.
Bombesin had multiple effects on Day 14 lung, when alveolarization was about half complete. First, bombesin induced alveolar myofibroblast proliferation and increased alveolar wall thickness compared with saline-treated control animals. Second, bombesin diminished alveolarization in C57BL/6 (but not Swiss-Webster) mice. We used receptor-null mice to explore which receptors might mediate these effects. Compared with wild-type littermates, bombesin-treated GRP receptor (GRPR)-null mice had increased interstitial fibrosis but reduced defects in alveolarization. Neuromedin B (NMB) receptor-null and bombesin receptor subtype 3-null mice had the same responses as their wild-type littermates. GRP had the same effects as bombesin, whereas neither NMB nor a synthetic bombesin receptor type 3 ligand had any effect. All effects of GRP were abrogated in GRPR-null mice.
Bombesin/GRP can induce features of BPD, including interstitial fibrosis and diminished alveolarization. GRPR appears to mediate all effects of GRP, but only part of the bombesin effect on alveolarization, suggesting that novel receptors may mediate some effects of bombesin in newborn lung.
American Journal of Respiratory and Critical Care Medicine 07/2006; 173(12):1377-85. · 11.08 Impact Factor
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ABSTRACT: A single course of antenatal steroids is widely used during preterm labor to promote fetal lung maturation. However, little is known regarding efficacy and safety of multiple courses of antenatal steroids. In animal models and clinical trials, treatment with glucocorticoids can inhibit growth. The present study of single- vs multiple-course steroids in pregnant ewes analyzes the effects of steroids vs placebo on fetal lung histopathology. Single-course groups received dexamethasone (Dex) 6 mg or normal saline every 12 hr for 48 hr at 104-106 days of gestation (term = 150 days). Multiple-course groups received the first course at 76-78 days; this was repeated weekly for 5 weeks. At 108 days, lungs were analyzed using immunohistochemistry for alpha-smooth muscle actin, a myofibroblast marker and proliferating cell nuclear antigen. Cell injury/death was evaluated using TdT-mediated dUTP digoxigenin nick end labeling (TUNEL) analysis. Although fetal growth was restricted by either single or multiple courses of Dex, alveolar development was accelerated as measured by mean linear intercepts. Alveolar walls were thinner, developing septa were longer, and septal myofibroblasts were increased for both Dex groups compared with controls. Cell proliferation increased following multiple steroid courses, especially in the distal parenchyma, with a corresponding decrease in apoptosis. These observations suggest that Dex promotes alveolarization, whether given in single or multiple courses.
Journal of Histochemistry and Cytochemistry 01/2006; 53(12):1469-79. · 2.72 Impact Factor
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ABSTRACT: The p66(Shc) adapter protein antagonizes mitogen-activated protein, or MAP, kinase, mediates oxidative stress, and is developmentally regulated in fetal mouse lungs.
To determine if p66(Shc) is similarly regulated in primates and in bronchopulmonary dysplasia (BPD), which results from oxidative injury to immature lungs.
Normal and injured lungs from humans and baboons were evaluated by Western analysis and immunohistochemistry.
In baboons, p66(Shc) decreased 80% between 125 and 175 days' gestation (p = 0.025), then doubled after term delivery at 185 days (p = 0.0013). In the hyperoxic 140-day fetal baboon BPD model, p66(Shc) expression persisted, and its localization shifted from the epithelium of gestational controls to the mesenchyme of diseased lungs, coincident with expression of proliferating cell nuclear antigen and cleaved poly(adenyl ribose) polymerase, a marker of apoptosis. Treatment with the antibombesin antibody 2A11 attenuated BPD, reduced cell proliferation, increased p66(Shc) expression 10.5-fold, and preserved epithelial p66(Shc) localization. p66(Shc) also decreased during normal human lung development, falling 87% between 18 and 24 weeks' gestation (p = 0.02). p66(Shc) was expressed throughout 18-week human lungs, became restricted to scattered epithelial cells by 24 weeks, and localized to isolated mesenchymal cells after term delivery. In contrast, p66(Shc) remained prominent in the epithelium of lungs with acute injury or mild BPD, and in the mesenchyme of lungs with severe disease. p66(Shc) localized to tissues expressing proliferating cell nuclear antigen and cleaved poly(adenyl ribose) polymerase.
p66(Shc) expression, cell proliferation, and apoptosis are concomitantly altered during lung development and in BPD.
American Journal of Respiratory and Critical Care Medicine 07/2005; 171(12):1384-94. · 11.08 Impact Factor
