Altered lung morphogenesis, epithelial cell differentiation and mechanics in mice deficient in the Wnt/ß-catenin antagonist Chibby.

The canonical Wnt/ß-catenin pathway plays crucial roles in various aspects of lung morphogenesis and regeneration/repair. Here, we examined the lung phenotype and function in mice lacking the Wnt/ß-catenin antagonist Chibby (Cby). In support of its inhibitory role in canonical Wnt signaling, expression of ß-catenin target genes is elevated in the Cby(-/-) lung. Notably, Cby protein is prominently associated with the centrosome/basal body microtubule structures in embryonic lung epithelial progenitor cells, and later enriches as discrete foci at the base of motile cilia in airway ciliated cells. At birth, Cby(-/-) lungs are grossly normal but spontaneously develop alveolar airspace enlargement with reduced proliferation and abnormal differentiation of lung epithelial cells, resulting in altered pulmonary function. Consistent with the Cby expression pattern, airway ciliated cells exhibit a marked paucity of motile cilia with apparent failure of basal body docking. Moreover, we demonstrate that Cby is a direct downstream target for the master ciliogenesis transcription factor Foxj1. Collectively, our results demonstrate that Cby facilitates proper postnatal lung development and function.

Transient in utero nicotine exposure stimulates mechanosensory-dependent lung development.

Nicotine receptors are present in the developing lung yet their function is unknown. The transient role of nicotine receptors in lung development has not been addressed. In this study, nicotine's direct effect on smooth muscle contraction, necessary for mechanosensory-dependent fetal lung development, is examined after transient nicotine stimulation to determine the relationship between nicotine exposure, smooth muscle contraction, and fetal lung development. Rat fetuses at 16 days' gestation were exposed in utero to 5 different concentrations of nicotine or control injected directly into the amniotic fluid. Specific concentrations of in utero nicotine increased the phosphorylated Western blot analysis and immunohistochemistry of muscle contraction proteins. Respiratory function tests on nicotine-exposed rat pups showed a statistically significant decrease in airway resistance earlier in life compared to control and an upward shift of the pressure-volume curve pointing towards a structural maturation of the in utero nicotine-exposed lung. These results are consistent with transient nicotine exposure during intrauterine life stimulating stretch-induced lung organogenesis by altering phosphorylation of muscle contraction proteins. The increase in smooth muscle phosphorylation may stimulate stretch-induced lung organogenesis, which affects lung development and accelerates lung maturation in rats.

Simulated transport alters surfactant homeostasis and causes dose-dependent changes in respiratory function in neonatal Sprague-Dawley rats.

OBJECTIVE: Forces transmitted to the neonate as a consequence of accelerations during transport have been associated with adverse neonatal outcomes including broncho-pulmonary dysplasia. In this study, we sought to determine the relationship between the duration of transport and respiratory performance in the rat model. METHODS: Four groups of Sprague-Dawley rat pups (10-12 pups/groups) were exposed to simulated medical transport on postnatal day of life 11 or 12. Each group was exposed to an average impulse of 27.4 m/s(2)/min for 0, 30, 60 or 90 min. During the exposure periods, impulse was monitored by computerized sampling using a digital accelerometer. Post-exposure, animals were immediately prepared, placed on mechanical ventilation and analyzed for elastance, tissue damping, airway resistance, ratio of damping to elastance (eta), hysteresivity, and inertance at positive end expiratory pressures (PEEPs) of 0, 3 and 6 cm(3) of H(2)O. Total phospholipid content and surfactant proteins A, B, and C mRNA levels in broncho-alveolar lavage fluid and lung tissue were obtained. RESULTS: Increased transport time resulted in a significant step-wise increase in airway resistance at all levels of PEEP (P<0.01). Static compliance decreased significantly after 60 min at PEEPs of 3 and 6 cm H(2)O (P<0.01). Eta significantly decreased with greater transport time at a PEEP of 6 cm H(2)O (P<0.05). Tissue damping increased with duration of transport time across all PEEP levels, but only exhibited statistical significance at a PEEP of 0 cm H(2)O (P<0.05). No differences were seen in hysteresivity or inertance. Compared with controls, transport was associated with significant reductions in total phospholipid content and mRNA levels of surfactant proteins B and C. CONCLUSION: Rat pups experienced significant deterioration of respiratory function with increasing duration of simulated transport.

Adult onset lung disease following transient disruption of fetal stretch-induced differentiation.

One of the mechanisms by which adult disease can arise from a fetal origin is by in utero disruption of organogenesis. These studies were designed to examine respiratory function changes in aging rats following transient disruption of lung growth at 16 days gestation. Fetuses were treated in utero with a replication deficient adenovirus containing the cystic fibrosis conductance transmembrane regulator (CFTR) gene fragment cloned in the anti-sense direction. The in utero-treated rats demonstrated abnormal lung function beginning as early as 30 days of age and the pathology progressed as the animals aged. The pulmonary function abnormalities included decreased static compliance as well as increased conducting airway resistance, tissue damping, and elastance. Pressure volume (PV) curves demonstrated a slower early rise to volume and air trapping at end-expiration. The alterations of pulmonary function correlated with lung structural changes determined by morphometric analysis. These studies demonstrate how transient disruption of lung organogensis by single gene interference can result in progressive change in lung function and structure. They illustrate how an adult onset disease can arise from subtle changes in gene expression during fetal development.

Transient in utero disruption of cystic fibrosis transmembrane conductance regulator causes phenotypic changes in alveolar type II cells in adult rats.

BACKGROUND: Mechanicosensory mechanisms regulate cell differentiation during lung organogenesis. We have previously demonstrated that cystic fibrosis transmembrane conductance regulator (CFTR) was integral to stretch-induced growth and development and that transient expression of antisense-CFTR (ASCFTR) had negative effects on lung structure and function. In this study, we examined adult alveolar type II (ATII) cell phenotype after transient knock down of CFTR by adenovirus-directed in utero expression of ASCFTR in the fetal lung. RESULTS: In comparison to (reporter gene-treated) Controls, ASCFTR-treated adult rat lungs showed elevated phosphatidylcholine (PC) levels in the large but not in the small aggregates of alveolar surfactant. The lung mRNA levels for SP-A and SP-B were lower in the ASCFTR rats. The basal PC secretion in ATII cells was similar in the two groups. However, compared to Control ATII cells, the cells in ASCFTR group showed higher PC secretion with ATP or phorbol myristate acetate. The cell PC pool was also larger in the ASCFTR group. Thus, the increased surfactant secretion in ATII cells could cause higher PC levels in large aggregates of surfactant. In freshly isolated ATII cells, the expression of surfactant proteins was unchanged, suggesting that the lungs of ASCFTR rats contained fewer ATII cells. Gene array analysis of RNA of freshly isolated ATII cells from these lungs showed altered expression of several genes including elevated expression of two calcium-related genes, Ca2+-ATPase and calcium-calmodulin kinase kinase1 (CaMkk1), which was confirmed by real-time PCR. Western blot analysis showed increased expression of calmodulin kinase I, which is activated following phosphorylation by CaMkk1. Although increased expression of calcium regulating genes would argue in favor of Ca2+-dependent mechanisms increasing surfactant secretion, we cannot exclude contribution of alternate mechanisms because of other phenotypic changes in ATII cells of the ASCFTR group. CONCLUSION: Developmental changes due to transient disruption of CFTR in fetal lung reflect in altered ATII cell phenotype in the adult life.

Small interfering peptide (siP) for in vivo examination of the developing lung interactonome.

To understand the role of reactive oxygen species in mechanosensory control of lung development a new approach to interfere with protein-protein interactions by means of a short interacting peptide was developed. This technology was used in the developing rodent lung to examine the role of NADPH oxidase (NOX), casein kinase 2 (CK2), and the cystic fibrosis transmembrane conductance regulator (CFTR) in stretch-induced differentiation. Interactions between these molecules was targeted in an in utero system with recombinant adeno-associated virus (rAAV) containing inserted DNA sequences that express a control peptide or small interfering peptides (siPs) specific for subunit interaction or phosphorylation predicted to be necessary for multimeric enzyme formation. In all cases only siPs with sequences necessary for a predicted normal function were found to interfere with assembly of the multimeric enzyme. A noninterfering control siP to nonessential regions or reporter genes alone had no effect. Physiologically, it was shown that siPs that interfered with the NOX-CFTR-CK2 complex that we call an "interactonome" affected markers of stretch-induced lung organogenesis including Wnt/beta-catenin signaling.

CFTR and Wnt/beta-catenin signaling in lung development.

BACKGROUND: Cystic fibrosis transmembrane conductance regulator (CFTR) was shown previously to modify stretch induced differentiation in the lung. The mechanism for CFTR modulation of lung development was examined by in utero gene transfer of either a sense or antisense construct to alter CFTR expression levels. The BAT-gal transgenic reporter mouse line, expressing beta-galactosidase under a canonical Wnt/beta-catenin-responsive promoter, was used to assess the relative roles of CFTR, Wnt, and parathyroid hormone-related peptide (PTHrP) in lung organogenesis. Adenoviruses containing full-length CFTR, a short anti-sense CFTR gene fragment, or a reporter gene as control were used in an intra-amniotic gene therapy procedure to transiently modify CFTR expression in the fetal lung. RESULTS: A direct correlation between CFTR expression levels and PTHrP levels was found. An inverse correlation between CFTR and Wnt signaling activities was demonstrated. CONCLUSION: These data are consistent with CFTR participating in the mechanicosensory process essential to regulate Wnt/beta-Catenin signaling required for lung organogenesis.

The Peter Pan paradigm.

Genetic and environmental agents that disrupt organogenesis are numerous and well described. Less well established, however, is the role of delay in the developmental processes that yield functionally immature tissues at birth. Evidence is mounting that organs do not continue to develop postnatally in the context of these organogenesis insults, condemning the patient to utilize under-developed tissues for adult processes. These poorly differentiated organs may appear histologically normal at birth but with age may deteriorate revealing progressive or adult-onset pathology. The genetic and molecular underpinning of the proposed paradigm reveals the need for a comprehensive systems biology approach to evaluate the role of maternal-fetal environment on organogenesis."You may delay, but time will not" Benjamin Franklin, USA Founding Father.

Cystic fibrosis transmembrane conductance regulator (CFTR) dependent cytoskeletal tension during lung organogenesis.

There is growing evidence for the role of CFTR (cystic fibrosis transmembrane conductance regulator) in lung development and differentiation. The mechanism by which the chloride channel could affect lung organogenesis, however, is unknown. In utero CFTR gene transfer in the fetal lungs of mice, rats, and non-human primates was shown previously to alter lung structure and function. A study of the genes altered in the fetal rat lung following CFTR overexpression was initiated in an effort to determine the molecular mechanism for CFTR-dependent differentiation. In utero gene transfer with recombinant adenoviruses carrying either a reporter gene or CFTR resulted in the increased expression of a number of genes upon microarray analysis. The majority of the genes overexpressed in the CFTR-treated lungs were primarily associated with muscle structure and function. Histological and biochemical characterization of these proteins including myosin heavy chain, heat shock protein 27, and isoforms of myosin light chain showed that CFTR overexpression had a profound effect on smooth muscle contraction-related proteins. The CFTR-dependent regulation of smooth muscle contraction related proteins was shown to be related to chloride and extracellular ATP and was dependent upon the PI3 Kinase and Phospholipase C pathways. The changes in smooth muscle proteins were consistent with CFTR-dependent contractions of the embryonic airway smooth muscle. An assay was developed using fluorescent polystyrene beads to show that CFTR did indeed increase amniotic fluid flow into the fetal lung. Increased amniotic fluid pressure was shown previously to be associated with stretch-induced differentiation of the lung. Evaluation of neonatal respiratory function showed that CFTR-dependent muscle contractions and increased amniotic fluid pressure resulted in accelerated maturation of the neonatal rat lung. In addition, these CFTR-dependent changes were shown to be sufficient to reverse the lung phenotype of the CFTR knockout mouse. Mechanical forces influence lung development through pulmonary distension. CFTR overexpression in the fetal lung altered differentiation and development in the lung. These results are consistent with CFTR influencing lung development by regulating the muscle contractions associated with cytoskeletal tension and stretch induced differentiation. Deficiency of CFTR altering lung development would contribute significantly to the Cystic Fibrosis disease phenotype.

Developmental changes in respiratory mechanics in the neonatal rat.

The rat lung undergoes rapid changes during the first several weeks of life. Respiratory function on neonatal rats was performed to determine if physiologic alterations coincides with classically described lung growth, and morphologic and morphometric changes. The authors examined respiratory mechanics during normal postnatal lung development in the rat utilizing both a forced oscillation technique, which partitions the mechanical properties of the lung tissue from the airways, as well as pressure-volume (PV) curves to assess lung elasticity. Respiratory function testing was performed on each postnatal days of life 10 to 20. The PV curves were characterized by a leftward shift over days 10 to 16, followed by a shift back to the right over days 17 to 20. Both tissue damping (G) and elastance (H) decreased from days of life 10 to 15, and then equilibrated from days 15 to 21. Hysteresivity (=G/H) at positive end-expiratory pressure (PEEP) = 1 and 3 cm H2O declined significantly with age. The effect of PEEP was significantly greater in the younger animals than in the older animals. The respiratory function assessments coincide well with the classically described periods of lung growth. These studies provide a normal baseline for correlating changes in structural with respiratory function.

Improvement of pulmonary hypoplasia associated with congenital diaphragmatic hernia by in utero CFTR gene therapy.

Congenital diaphragmatic hernia (CDH) may be an ideal candidate disease for in utero gene therapy as disrupted fetal lung growth plays a significant role in disease outcome. We previously demonstrated that transient in utero overexpression of CFTR during fetal development resulted in lung epithelial proliferation and differentiation. We hypothesized that gene therapy with CFTR would improve the pulmonary hypoplasia associated with congenital diaphragmatic hernia (CDH). CDH was induced by the herbicide 2,4-dichlorophenyl-4-nitrophyl ether (nitrofen) following maternal ingestion at either 10 or 13 days gestation. In utero gene transfer of the CFTR gene was subsequently performed at 16 days gestation. Examination of the fetuses at 22 days gestation revealed little improvement in the CFTR-treated lungs following induction of hernias with nitrofen at 10 days gestation. However, the CFTR gene treatment significantly improved internal surface area, saccular density, overall saccular number, and amount of saccular air space in the lungs that were treated with nitrofen at 13 days gestation. RT-PCR demonstrated that gene transfer occurred following treatment at 13 days gestation but not in the lungs treated with nitrofen at 10 days gestation, despite gene transfer at the same gestational age (16 days) in both groups. As disruption of lung development correlates with the gestational stage at which nitrofen exposure occurs, these results confirmed previous findings that in utero gene transfer efficiency depends on the stage of lung development. Lung development may be significantly delayed in human CDH to allow for successful gene transfer later in gestation, providing a substantial therapeutic window.

Developmental paradigm for early features of cystic fibrosis.

Cystic fibrosis (CF) is a progressive disease in which the lung is perceived to be normal at birth and is injured by recurrent infection. However, there is increasing evidence that the lung is functionally and structurally abnormal prior to the appearance of clinical infection. The cystic fibrosis transmembrane regulator (CFTR) is highly expressed in fetal tissues, and this review examines the role of CFTR in regulatory cascades during lung development. Early changes in the CF lung are examined from a perspective of disrupted fetal development, explaining a number of paradoxes seen with the disease.

Pathophysiologic consequences following inhibition of a CFTR-dependent developmental cascade in the lung.

BACKGROUND: Examination of late gestation developmental genes in vivo may be limited by early embryonic lethality and compensatory mechanisms. This problem is particularly apparent in evaluating the developmental role of the cystic fibrosis transmembrane conductance regulator (CFTR) gene in the cystic fibrosis (CF) phenotype. A previously described transient in utero knockout (TIUKO) technology was used to address the developmental role of CFTR in the rat lung. RESULTS: Rat fetuses transiently treated with antisense cftr in utero developed pathology that replicated aspects of the human CF phenotype. The TIUKO CF rat developed lung fibrosis, chronic inflammation, reactive airway disease, and the CF Antigen (MRP8/14), a marker for CF in human patients, was expressed. CONCLUSIONS: The transient in utero antisense technology can be used to evaluate genes that exhibit either early lethality or compensating gene phenotypes. In the lung CFTR is part of a developmental cascade for normal secretory cell differentiation. Absence of CFTR results in a constitutive inflammatory process that is involved in some aspects of CF pathophysiology.

Activation of Src kinase Lyn by the Kaposi sarcoma-associated herpesvirus K1 protein: implications for lymphomagenesis.

The K1 gene of Kaposi sarcoma-associated herpesvirus (KSHV) encodes a transmembrane glycoprotein bearing a functional immunoreceptor tyrosine-based activation motif (ITAM). Previously, we reported that the K1 protein induced plasmablastic lymphomas in K1 transgenic mice, and that these lymphomas showed enhanced Lyn kinase activity. Here, we report that systemic administration of the nuclear factor kappa B (NF-kappaB) inhibitor Bay 11-7085 or an anti-vascular endothelial growth factor (VEGF) antibody significantly reduced K1 lymphoma growth in nude mice. Furthermore, in KVL-1 cells, a cell line derived from a K1 lymphoma, inhibition of Lyn kinase activity by the Src kinase inhibitor PP2 decreased VEGF induction, NF-kappaB activity, and the cell proliferation index by 50% to 75%. In contrast, human B-cell lymphoma BJAB cells expressing K1, but not the ITAM sequence-deleted mutant K1, showed a marked increase in Lyn kinase activity with concomitant VEGF induction and NF-kappaB activation, indicating that ITAM sequences were required for the Lyn kinase-mediated activation of these factors. Our results suggested that K1-mediated constitutive Lyn kinase activation in K1 lymphoma cells is crucial for the production of VEGF and NF-kappaB activation, both strongly implicated in the development of KSHV-induced lymphoproliferative disorders.

The "Goldilocks effect" in cystic fibrosis: identification of a lung phenotype in the cftr knockout and heterozygous mouse.

BACKGROUND: Cystic Fibrosis is a pleiotropic disease in humans with primary morbidity and mortality associated with a lung disease phenotype. However, knockout in the mouse of cftr, the gene whose mutant alleles are responsible for cystic fibrosis, has previously failed to produce a readily, quantifiable lung phenotype. RESULTS: Using measurements of pulmonary mechanics, a definitive lung phenotype was demonstrated in the cftr-/- mouse. Lungs showed decreased compliance and increased airway resistance in young animals as compared to cftr+/+ littermates. These changes were noted in animals less than 60 days old, prior to any long term inflammatory effects that might occur, and are consistent with structural differences in the cftr-/- lungs. Surprisingly, the cftr+/- animals exhibited a lung phenotype distinct from either the homozygous normal or knockout genotypes. The heterozygous mice showed increased lung compliance and decreased airway resistance when compared to either homozygous phenotype, suggesting a heterozygous advantage that might explain the high frequency of this mutation in certain populations. CONCLUSIONS: In the mouse the gene dosage of cftr results in distinct differences in pulmonary mechanics of the adult. Distinct phenotypes were demonstrated in each genotype, cftr-/-, cftr +/-, and cftr+/+. These results are consistent with a developmental role for CFTR in the lung.

Long term physiologic modification using rAAV in utero gene-therapy.

BACKGROUND: Transfer of genes in utero via the amniotic fluid was shown previously with recombinant adeno-associated viruses (rAAV) to be highly efficient. Expression for over one year was demonstrated using reporter genes. In addition, it was shown previously that transgenes delivered by this method release protein into the general circulation. Given these results experiments were designed to test the hypothesis that in utero rAAV gene therapy could result in long term physiologic modification. METHODS: A rAAV recombinant expressing ciliary neurotrophic factor (cntf) and green fluorescent (gfp) in a polycistronic messenger was used to treat rat fetuses in utero. CNTF causes weight loss and decreased water consumption as a measurable physiologic effect. GFP was used as a marker of gene expression. RESULTS: In utero gene transfer with rAAV carrying human cntf and gfp resulted in long-term gene expression in rat. CNTF-specific physiologic effects of a decrease in weight and water intake were obtained. Expression of the GFP was documented in the treated animals at one year of age. CONCLUSION: Given this data, in utero gene therapy with rAAV into multipotential stem cells resulted in long term systemic physiologic modification of the treated animals by the transgene product. In utero rAAV gene therapy potentially could be used for gene replacement therapy in metabolic disorders.

Transient in utero knockout (TIUKO) of C-MYC affects late lung and intestinal development in the mouse.

BACKGROUND: Developmentally important genes often result in early lethality in knockout animals. Thus, the direct role of genes in late gestation organogenesis cannot be assessed directly. In utero delivery of transgenes was shown previously to result in high efficiency transfer to pulmonary and intestinal epithelial stem cells. Thus, this technology can be used to evaluate late gestation development. RESULTS: In utero gene transfer was used to transfer adenovirus with either an antisense c-myc or a C-MYC ubiquitin targeting protein to knockout out c-myc expression in late gestation lung and intestines. Using either antisense or ubiquitin mediated knockout of C-MYC levels in late gestation resulted in similar effects. Decreased complexity was observed in both intestines and lungs. Stunted growth of villi was evident in the intestines. In the lung, hypoplastic lungs with disrupted aveolarization were observed. CONCLUSIONS: These data demonstrated that C-MYC was required for cell expansion and complexity in late gestation lung and intestinal development. In addition they demonstrate that transient in utero knockout of proteins may be used to determine the role of developmentally important genes in the lungs and intestines.

In utero recombinant adeno-associated virus gene transfer in mice, rats, and primates.

BACKGROUND: Gene transfer into the amniotic fluid using recombinant adenovirus vectors was shown previously to result in high efficiency transfer of transgenes into the lungs and intestines. Adenovirus mediated in utero gene therapy, however, resulted in expression of the transgene for less than 30 days. Recombinant adenovirus associated viruses (rAAV) have the advantage of maintaining the viral genome in daughter cells thus providing for long-term expression of transgenes. METHODS: Recombinant AAV2 carrying green fluorescent protein (GFP) was introduced into the amniotic sac of fetal rodents and nonhuman primates. Transgene maintenance and expression was monitor. RESULTS: Gene transfer resulted in rapid uptake and long-term gene expression in mice, rats, and non-human primates. Expression and secretion of the reporter gene, GFP, was readily demonstrated within 72 hours post-therapy. In long-term studies in rats and nonhuman primates, maintenance of GFP DNA, protein expression, and reporter gene secretion was documented for over one year. CONCLUSIONS: Because only multipotential stem cells are present at the time of therapy, these data demonstrated that in utero gene transfer with AAV2 into stem cells resulted in long-term systemic expression of active transgene roducts. Thus, in utero gene transfer via the amniotic fluid may be useful in treatment of gene disorders.