David Warburton - US grants

The incidence of respiratory distress syndrome (RDS) is increased almost sixfold in infants of diabetic mothers (IDM), even when suitable corrections are made for gestational age. IDMs experience hyperinsulinemia both in utero and in the postnatal period as a result of intrauterine hyperglycemia, particularly when maternal glucose homeostasis is poorly controlled. We propose to examine the hypothesis that hyperinsulinemia secondary to hyperglycemia alters fetal lung maturation using both in vivo and in vitro studies. In vivo studies: Exogenous glucose will be infused into chronically catheterised fetal lambs from which tracheal fluid can be continuously collected. Surface activity of the tracheal fluid will be assayed on a surface balance and surfactant concentration and flux in tracheal fluid will be quantified. Lipid analysis of the tracheal fluid will be carried out in order to delineate surfactant composition. Respiratory surface area and the stability index of lungs will be derived from pressure-volume curves of excised lungs. Insulin, cortisol, beta-sympathomimetic, and thyroid hormone receptors will be assayed in nuclear and cytoplasmic fractions of whole lung homogenates. In vitro studies: Alveolar type II cells (greater than 95% purity) and lung fibroblasts (greater than 95% purity) will be separated from organotypic cultures of fetal rabbit lungs (26-31 days gestation) and grown in monolayer culture using Warburton's modification of Douglas' and Sevanian's methods. These preparations will be used to simulate and study the effects of the IDM's fetal environment on interactions between lung cells in three groups of experiments: 1. Effects of fibroblast conditioned media (FCM) prepared with insulin, cortisol, isoxuprine and/or propranolol, and thyroid hormone exposed fibroblasts on growth (3H thymidine DNA incorporation), metabolic rate (14CO2 production) and surfactant synthesis and release (3H choline and 3H myo-inositol incorporation) by alveolar type II cells in comparison with directly treated type II cells. 2. Effects of above FCM on cortisol, insulin, beta-sympathominetic and thyroid hormone receptors in alveolar type II cells. 3. Elaboration of extracellular matrix by lung fibroblasts, its composition and its effects on alveolar type II cell growth, metabolic rate and surfactant production will be studied. In addition, modification of extracellular matrix production by insulin, glucose and cortisol will be studied.

A major problem in developmental biology is to determine when and how time and position-restricted instructions are signalled and received during secondary embryonic inductions such as branching morphogenesis. Morphogenesis of embryonic mouse lungs cultured in vitro under serumless, chemically defined conditions is similar to lung development in vivo: branching morphogenesis and cytodifferentiation of type II pneumocytes occurs, suggesting the hypothesis that endogenous growth factors serve as instructive epigenetic signals. We hypothesize that EGF and TGF-alpha serve as local autocrine and paracrine epigenetic signals to regulate the three-dimensional timing and positional information for epithelial branching during lung morphogenesis in the mouse embryo and that these signals are transduced via activation of the EGF receptor. The Specific Aims of this project are designed to test this hypothesis: Aim 1. Localization: to detect and localize in time and space expression of EGF and TGF-alpha at the mRNA and protein levels during branching morphogenesis in embryonic (E. 11/12) mouse lung both in vivo and in culture in chemically-defined serum free medium. Aim 2. Signal transduction: to characterize and localize EGF receptor tyrosine phosphorylation signal transduction pathway. Aim 3. Inhibitory strategies: to investigate the developmental effects of EGF receptor activation during lung morphogenesis in vitro using inhibitory strategies with tyrphostins, antibodies, or antisense cRNA probes. Future goals of this research include identification of specific genes regulated by EGF receptor activation during mouse lung morphogenesis in vitro. The proposed studies will characterize novel EGF receptor mediated autocrine and paracrine molecular developmental mechanisms which regulate branching morphogenesis in the mouse embryo lung, will provide fundamental insights into epithelial-mesenchymal interaction, and may lead to novel preventive and therapeutic strategies for lung immaturity, lung injury and lung repair.

DESCRIPTION (Adapted from applicant's abstract) The hypothesis is that keratinocyte growth factor (KGF) and laminin together with telomerase direct the fate of AEC between proliferation, differentiation, and/or apoptosis both during lung development and repair after lung injury through tyrosine phosphorylation cascades targeting cyclin D and retinoblastoma protein (pRb). Characterization of the molecular signaling mechanisms determining the proliferative versus apoptotic versus differentiated "survival" fate of AEC will identify new targets for the design of novel rational therapeutic approaches to amelioration of alveolar injury and augmentation of repair in both neonatal and adult human alveolar injury.

Central Theme: to understand the molecular basis of specific transcriptional factor and peptide growth factor signaling mechanisms that instruct lung morphogenesis, then to understand their role in the perturbation of lung morphogenesis by injury and repair, and then to use this information to devise and evaluate novel rational therapeutic approaches. Project 1. Positive and negative regulators of lung development: will determine the role of the Nkx 2.1 family of transcriptional factors as key temporo-spatial developmental cues for normal lung morphogenesis, and will determine how Nkx 2.1 is positively and negatively regulated by peptide growth factor signaling. Project 2. TGF-beta signaling in lung morphogenesis, injury and repair: will further determine the role of endogenous TGF-beta peptide growth factor signaling in lung morphogenesis, and of excessive TGF-beta peptide expression and signaling in lung injury and repair, including its role in regulating epithelial apoptosis. Project 3. TGF-beta signaling and apoptosis: will determine the specific molecular signal transduction mechanism by which TGF-beta mediates activation of apoptosis and will relate these findings to lung injury and repair. Project 4. TGR-beta3 in lung morphogenesis, injury and repair: will further determine the specific functional roles of TGF-beta3 peptides in lung development, injury and repair/healing using novel regulatable transgenic/null mutant strategies. Project 5. TGR-beta rational therapeutics: will devise and test novel rationally based approaches to perturbing TGF-beta ligand-receptor interactions, based on functional predictions from computer modeling of ligand-receptor interactions and establish their utility in modulating excess TGF-beta signaling in lung injury and repair.

Hypothesis: (1) excess TGF-beta signaling disrupts the orderly temporo-spatial molecular cues that normally instruct lung morphogenesis and cytodifferentiation as seen following exposure of the developing lung to elevated ambient oxygen; therefore (2) modulationof excess TGF-beta signaling will ameliorate lung injury and augment repair of the developing lung. Speicifc Aims: 1. To determine the impact of increased ambient oxygen on TGF-beta production, activation and signaling in vivo in postnatal and adult murine lung. 2. To determine the spatial effects of over-expression of TGF-beta peptides ineither latent or active (mutated) forms using TGF-beta recombinant adenoviral vectors in embryonic mouse lung in ex vivo culture. 3. To detemine the effects of TGF-beta peptide over-expression on alveolarizationin neonatal mouse lung in vivo byintra-nasal or intratracheal administration of TGF-beta recombinant adenoviral vectors. 4. To determine the in vivo role of epithelial TGF-beta receptor signal transduction molecules onlung morphogenesis, injury and repair by transgenic expression of active versus C-terminally truncated negative Smads using the hSP-C lung epithelium specific promoter. 5. To determine whether strategies to down-modulate TGF-beta signaling including immunoperturbation, competing peptides and speicifc antisnse oligodeoxynucleotides can ameliorate the adverse impact of excess TGF-beta signaling. Significance to Human Health: the propsoed studies will determine whether excess TGF-beta signaling plays an adverse role in developing lung and will determine the feasibility of novel therapeutic strategies tomodulate TGF-beta signaling. Prevention andtreatment of chronic lung disease in premature infants may become possible.

DESCRIPTION (provided by applicant): FGF signaling is essential for lung morphogenesis. During years 06-10 of this grant, we have shown that the murine and indeed the human sprouty gene family function as important, highly conserved, inducible negative modulators of FGF signaling in respiratory branching morphogenesis. Recently, we and others have further discovered that tyrosine phosphorylation is critical for Sprouty function. Now our new preliminary data show that the tyrosine phosphatase Shp2, as well as FRS2, which are both required for FGF signaling, reversibly bind mSpry2. Hypothesis: Shp2 protein tyrosine phosphatase plays a critical role in finely modulating mSpry2 function, in a supra-molecular assembly with FRS2, within the FGF signaling complex, which is essential for lung morphogenesis. Aim 1: To determine and compare the spatial and sub-cellular co-distribution of mSpry2 with Shp2, FRS2 and other key FGF signaling elements during lung morphogenesis. Aim 2: To determine the functional role of Shp2 and FRS2 in lung morphogenesis in culture. Aim 3: To determine the relative functional contribution of mSpry2, Shp2 and FRS2 in lung morphogenesis by complementation of FGF10 hypomorphism. Aim 4: To determine the role of tyrosine phosphorylation in protein-protein interaction and supra-molecular assembly of mSPRY2 with SHP2 and FRS2. Aim 5: To determine the functional importance of Shp2 in lung morphogenesis in vivo.

DESCRIPTION (provided by applicant): Co-morphogenesis of the lung vasculature in correct apposition to the epithelium is essential for the eventual transition to air breathing and is adversely impacted in lethal forms of human lung hypoplasia and neonatal lung injury. As yet the mechanisms that drive this coordinated process are incompletely understood. Null mutation reveals that FGF10 signaling is absolutely required for epithelial morphogenesis in the lung. Now our published and preliminary results show that VEGF signaling enhances both vascular and epithelial morphogenesis. However VEGFR2 is expressed in the embryonic mesenchyme and not the epithelium, suggesting indirect stimulation of the epithelium. Hypothesis: FGF and VEGF signaling functionally co-regulate lung epithelial and vascular morphogenesis. Aim 1. To determine the precise temporospatial relationship between the key elements in the FGF10 and VEGF signaling pathways during embryonic lung morphogenesis. Aim 2. To determine the functional impact of VEGF signaling on lung epithelial and vascular morphogenesis in vivo using novel inducible and reversible gain and loss of function in mice. Aim 3. To determine the function of FGF10 signaling to regulate VEGF signaling during lung epithelial and vascular morphogenesis in vivo. Aim 4. To determine and compare the functional role of key FGF10 and VEGF signaling elements and to determine whether the effects of VEGF on the epithelium are direct or indirect in serumless, chemically defined embryonic lung organ culture system.

DESCRIPTION (provided by applicant): Hypothesis: The TGFbeta signaling pathway may function as a final common pathway to mediate the negative impact of neonatal lung injury on alveolar formation and gas exchange. Aim 1. Candidate gene induction: to determine the impact of neonatal hyperoxia and/or LP5 endotoxin, on time and space-specific expression and activity of TGFbeta ligand, cognate receptor and cognate Smad (2,3 and 4) expression in neonatal mouse lung. Aim 2. Candidate gene function: to compare the impact of neonatal hyperoxia and/or LP5 endotoxin, versus local overexpression of IL1-beta, or TNF-alpha, with that of TGF-beta1 using recombinant adenoviral vectors to determine which of them also phenocopies BPD by abrogation of alveolarization. Aim 3. Smad3 as a common downstream gene: to discover which effects of neonatal hyperoxia and/or LPS endotoxin injury, IL1beta, TNFalpha, and/or TGFbeta1 signaling in neonatal lung are transduced through a common TGF-alpha/Smad3 signaling pathway, using the Smad3 null mouse as a test model. Aim 4. Protection: to determine whether the negative sequelae of neonatal hyperoxia and/or LP5 endotoxin injury on alveolar formation can be blocked or ameliorated, either upstream or downstream within the TGF-beta pathway using recombinant viruses expressing Decorin or Smad7. Health Significance: we postulate that blocking key upstream inducers of TGFabeta signaling, correctly modulating TGFbeta signaling and/or inhibiting the downstream effects of TGFbeta could prevent or ameliorate alveolar hypoplasia/BPD.

DESCRIPTION (provided by applicant): In the first cycle of this P01, our collective and synergistic efforts have clearly established the validity of our central hypothesis that "excessive TGFB signaling plays a key role in the pathobiology of the neonatal chronic lung injury termed BPD". The logical extension of these findings "what's upstream and downstream of TGF in chronic neonatal lung injury and are there any useful therapeutic targets in this pathway?" is a key question that will be the focus of this revised renewal application. Thus, we propose a research program to address the following 5 inter-related, mutually reinforcing and synergistic aspects of this key question. Project 1. Warburton and Gauldie. Excess TGFB in neonatal lung injury and repair. Project 2. Minoo. BPD: interactions between inflammation and morphogenesis. Project 3. Derynck. Non-Smad mechanisms of TGFB signaling Project 4. Groffen. Negative regulation of lung inflammation by Abr/Bcr. Project 5. Bellusci. FGFR2b pathway in Bronchopulmonary Dysplasia.

DESCRIPTION (provided by applicant): The Mongolia/ Southern California Environmental Health Research and Training Center (MSCEHRTC) will provide training and research opportunities in a partnership between Health Sciences University of Mongolia in Ulaanbaatar and University of Southern California in Los Angeles, California, the United States. The major focus of the MSCEHRTC is on the consequences of atmospheric pollution and the introduction of sustainable control technologies on environmental health across the lifespan in Mongolia. Air pollution is becoming an increasingly serious problem in Ulaanbaatar with ground level air pollution, measured in terms of particulate matter (PM), estimated to be >200 times maximum acceptable standards. The training and research objectives will be accomplished by in-country short- and long-term training opportunities as well as opportunities in the US for graduate degrees, visiting scholarships, and short term training at USC and Children's Hospital Los Angeles as well as at affiliated centers sponsored by NHLBI, NIEHS and Southern California Air Quality Management District. Funding for the program will include the $250,000 per year from the FIC as well as real dollar matches of >$25,000 per year each from The Saban Research Institute, The Saban Family Foundation, the Pasadena Guild Endowment, plus available competitive training scholarships from the USC Global Health Institute, Saban Research Institute, NIEHS Environmental Health Institute USC, California Institute for Regenerative Medicine the Ulan Bator Foundation, as well as Anonymous Private Philanthropy. The program will be sustainable because in response to evidence based approaches by ourselves and other collaborators, The Government of Mongolia has recently demonstrated its practical commitment to the reduction of air pollution through efforts funded under the Millennium Challenge Grant to distribute more efficient stoves to Ger dwellers. This effort is being implemented by several major Mongolian banks, the Ministry of Mining and Natural Resources, The Ministry of Energy and the National Agency for Meteorology and the Environment of Mongolia (NAMEM) and its National Office of Air Quality (NAQO) which is equipped to monitor air quality nationwide. To achieve sustainable improvements in pollution control we have enlisted the enthusiastic policy collaboration of Dr. Oyun who is a an Oxford-educated Chevening Scholar, Member of Mongolian Parliament, Leader of the Women's Caucus, former Foreign Minister and leading member of the Clean Air Group in the Grand Khuraltai. She is an expert advocate for sustainable policy, working within the Mongolian government structure, who lives in Ulaanbaatar and thus appreciates the importance of urgent evidence-based action on air pollution in her capital city. She will co-chair our international advisory committee and will act as a friend and mentor to recruit, retain and develop MSCEHRTC program graduates to affect sustainable evidence-based change on the consequences of air pollution across the lifespan at the national policy and implementation level in Mongolia.

DESCRIPTION (provided by applicant): The following Goals are designed to achieve the overarching Strategic Goal to construct a rich multiscale atlas of human alveolar development and to readily share the resulting materials with other research centers in the UO1 and with the broad research community. Goal 1. Make a digital map of alveolar development over time in vivo and in tissue pieces using novel micro- CT (uCT), micro MRI (uMRI) and Phase contrast X-ray (PCX) technology validated in mice and then in human lung tissue. Goal 2. Make a digital map of spatiotemporal gene expression during alveolar development over time using newly modified high throughput multiplex ISH with novel slice and dice, Vibra-SSIM confocal technology, in mouse and human lung tissue. Goal 3. Make a digital map of the fine structure of alveolar matrix during development in mouse and human tissue. Goal 4. Develop image-processing technology to meld digital multiscale images of alveolar structure with cell autonomous gene expression with extracellular matrix protein configuration into a readily navigable, annotated novel data resource for the scientific community. SOPs will be developed and process driven milestones will be set up to ensure deliverability and return on this significant scientific investment. Drs. Warburton, Shi and Driscoll are expert alveolar biologists while Drs. Fraser, Moats and Lansford are expert imaging scientists who are adept at recording, registering and assembling digital, multiscale combinatorial maps of organ development in Drosophila and Mouse and have invented many novel and highly innovative techniques to do so. Overall impact on human health: Human and mouse lung are fundamentally structurally different (reviewed in Warburton et al, 2010). Length of gestation, lobation, airway branch pattern, number of airway branches, proximo-distal airway epithelial differentiation and alveolar epithelial and capillary surface area as well as alveolar epithelial cell number differ in importan respects between mouse and human. Not least, alveolarization begins postnatally in mice, yet begins in utero in humans. We propose eventually to translate our novel concepts on alveolar development derived in mice rapidly to studies in vivo in humans. We hope that this fundamental knowledge will eventually be useful for the prevention and treatment of alveolar hypoplasia, such as occurs in bronchopulmonary dysplasia of the human premature neonate, as well as potentially to informing regeneration of alveolar tissue or amelioration or reversal of alveolar degeneration in older children, adolescents and adults with lung diseases. (End of Abstract)