Awardees and Abstracts

2003 Awardees

Senior Awards

Gökhan Hotamisligil, M.D., Ph.D.
Harvard School of Public Health
Nutrition
Novel Pathways Controlling Inflammatory Responses in Asthma
Lee Niswander, Ph.D.
Sloan-Kettering Institute/HHMI (Now at University of Colorado Health Sciences Center)
Developmental Biology
Lung Development and Disease: Genetic and Experimental Approaches
Hidde L. Ploegh, Ph.D.
Harvard Medical School (Now at Massachusetts Institute of Technology)
Pathology
Application of a New Cell Biological and Chemical Strategy to the Study of Asthma
Danuta Radzioch, Ph.D.
McGill University
Experimental Medicine and Human Genetics
Genetic Dissection of Susceptibility to Allergic Asthma
Kevan Shokat, Ph.D.
University of California, San Francisco
Cellular and Molecular Pharmacology
A New Therapeutic Strategy for Asthma: Inhibition of Cortisol Metabolism in the Lung
Jonathan S. Stamler, M.D.
Duke University/HHMI (Now at Case Western Reserve University)
Medicine and Biochemistry
The Role of S-Nitrosothiols in the Pathogenesis of Asthma

Junior Awards

Richard Bond, Ph.D.
University of Houston
Pharmacological and Pharmaceutical Sciences
Effects of Beta-Adrenoceptor Inverse Agonists Treatment on Murine Models of Asthma
Robert Brenner, Ph.D.
University of Texas HSC at San Antonio
Physiology
Knockout Studies of the BK Potassium Channel Beta1 Subunit in Airway Smooth Muscle
William C. Sha, M.D., Ph.D.
University of California, Berkeley
Molecular and Cell Biology
Regulation of Effector Responses in Asthma by B7h-ICOS Costimulatory Molecules
Ivaylo Stoilov, M.D.
University of Connecticut Health Center (Now at Envision Pharma)
Surgery
Asthma and the Cytochrome p450 System of the Lung
Raul M. Torres, Ph.D.
University of Colorado/National Jewish Health
Immunology
Regulation of GTP-binding Proteins in Asthma and Airway Hypersensitivity

2003 Awards Project Abstracts

Richard Bond, Ph.D. — 2003 Junior Award

University of Houston

Effects of Beta-Adrenoceptor Inverse Agonists Treatment on Murine Models of Asthma

We propose to investigate whether the chronic use of ß-adrenoceptor (ßAR) inverse agonists is a viable strategy for the treatment of asthma. Traditionally, ßAR agonists are used to increase signaling and induce bronchodilation. While ßAR agonist therapy is effective for acute treatment, with chronic use their effectiveness is significantly reduced. Very recently, a large clinical trial using a long-acting ßAR agonist, salmeterol, was stopped due to possible increased mortality and adverse events. Recently, a paradigm shift has occurred in the therapeutic use of ßAR drugs in congestive heart failure (CHF). As in asthma, ßAR agonists are used in CHF to acutely increase signaling. The chronic use of ßAR agonists in CHF leads to increased mortality. By contrast, some ßAR inverse agonists, once contraindicated in heart failure, are now the most successful drugs ever used to decrease mortality in CHF. ßAR inverse agonists are currently contraindicated in asthma. We suggest there are many parallels between the treatment of asthma and CHF with ßAR ligands, and that ßAR inverse agonists may be useful in the treatment of asthma. We have obtained preliminary data consistent with this hypothesis in mouse model of asthma where chronic treatment with ß2AR inverse agonists significantly reduces the effects of a bronchoconstrictor. The long-term goal of these studies is to provide a mechanistic basis for the potential use of ß AR inverse agonists in treating asthma. Our central hypothesis is that agonists and inverse agonists have reciprocal effects on cellular signaling and therefore chronic use inverse agonists increases signaling.

Robert Brenner, Ph.D. — 2003 Junior Award

University of Texas HSC at San Antonio

Knockout Studies of the BK Potassium Channel Beta1 Subunit in Airway Smooth Muscle

Although airway smooth muscle (ASM) contraction is initiated by calcium release from intracellular stores, there is considerable evidence that the control of membrane potential by potassium channels is an important factor in regulating ASM constriction. It is hypothesized that depolarization or block of potassium channels allows recruitment of voltage-dependent calcium channels (VDCCs) and increased contraction. The large conductance calcium-activated (BK type) potassium channel is activated by calcium and voltage, and so this channel is an ideal negative-feedback regulator of VDCCs. BK channel opening hyperpolarizes membranes, deactivates VDCCs and thereby opposes contraction. Previously, we had shown that the BK channel accessory beta1 subunit is required for BK channel function in vascular smooth muscle and bladder smooth muscle. Knockout of the beta1 subunit results in BK channels that fail to open, increased calcium influx and increased tone. Although potassium channels have been implicated in controlling ASM constriction, as of yet no mouse model has been utilized to evaluate their roles. BK channels are abundantly expressed in ASM, and have been implicated in mediating airway relaxation by b-adrenergic agonists. Our preliminary data demonstrate that BK channel beta1 subunits are expressed in ASM. Patch clamp recordings of BK channels in ASM of beta1 knockout mice have a dramatically reduced open probability. We propose to utilize the beta1 knockout to determine how BK channels regulate calcium signaling in ASM cells and determine the relevance of BK channel function in physiological studies of tracheal contraction in vitro and airway responsiveness of unrestrained mice.

Gökhan Hotamisligil, M.D., Ph.D. — 2003 Senior Award

Harvard School of Public Health

Novel Pathways Controlling Inflammatory Responses in Asthma

Asthma is a chronic inflammatory condition of the airways. While the prevalence of this disease is increasing at alarming rates worldwide, the therapeutic modalities are limited in variety and efficacy and the mechanisms underlying the disease are not clearly understood. Here, we propose a line of research involving the role of fatty acid binding proteins (FABPs) in lipid signaling and inflammatory responses and their contribution to the pathogenesis and treatment of asthma. In asthma, it is generally accepted that many cell types contribute to the overall inflammatory response. These include T cells, particularly the Th2 phenotype, eosinophils and mast cells as primary effectors leading to further activation and recruitment of other inflammatory cells, such as macrophages, and alterations of the bronchial epithelium function. We recently showed an unexpected role for FABPs in regulation of macrophage inflammatory responses. Interestingly, recent experiments have also detected aP2 and mal1 production in the bronchial epithelium during asthma and specifically in response to IL-4 and IL-13. We hypothesize that members of the fatty acid binding protein (FABP) family, aP2 and mal1, play an important role in the generation of inflammatory responses and airway hyperresponsiveness in asthma; blocking the activity of these proteins will result in protection from development of asthma and its chronic complications. This hypothesis will be tested in aP2-, mal1-, and combined aP2-mal1-deficient cells and mice

Lee Niswander, Ph.D. — 2003 Senior Award

Sloan-Kettering Institute/HHMI (Now at University of Colorado Health Sciences Center)

Lung Development and Disease: Genetic and Experimental Approaches

Embryonic development of the lung requires a coordinated series of cellular and molecular events that results in the elaborate branching pattern observed in the adult lung. Extensive interactions between the lung endoderm and mesenchyme induce the initial lung buds to form and to further elaborate the intricate branching pattern (Fig. 1a). Once the initial pattern is established, additional interactions are necessary to establish the aveoli and the vascular smooth muscle as well as the specific differentiated cell types required to facilitate efficient gas/air exchange. Furthermore, airway remodeling that occurs in asthmatic patients involves communication between different cell types in the lung in a process that is thought to be analogous to that which occurs between the endoderm and the mesenchyme during lung development11, 30. Relatively little is known of the molecular events that are required to make a fully functional lung. Moreover, the genetic basis of lung defects is not well understood. In this proposal we will identify genes involved in critical aspects of embryonic lung morphogenesis by expanding an on-going mutagenesis screen in mice (Aim 1). We will characterize the phenotype associated with mutations in these critical genes to determine the mechanism of protein action (Aim 2). Finally we will test the hypothesis that genes that control embryonic lung function are abnormally expressed in a mouse model of asthma and that mutations in these genes may confer asthma susceptibility (Aim 3). These studies will establish the key genetic, cellular and molecular events that regulate embryonic lung development and generate new animal models of lung development and disease. In addition, they have the potential to uncover new therapeutic targets for asthma treatment and repair of damaged lung tissue.

Hidde L. Ploegh, Ph.D. — 2003 Senior Award

Harvard Medical School (Now at Massachusetts Institute of Technology)

Application of a New Cell Biological and Chemical Strategy to the Study of Asthma

Class II Antigen presentation is pivotal to asthma pathophysiology. We propose to study presentation of aerosolized antigen (ovalbumin; OVA) in the mouse model of asthma. First, we shall generate mice that harbor antigen-specific B cells capable of isotope switching to the pathophysiologically relevant IgE. Second, the antigen-presenting cell (APC) itself will be investigated. A recently generated mouse, where all class II molecules are EGFP tagged, allows visualization of respiratory APC and even the class II molecules themselves. Finally, we shall develop a novel chemical strategy to visualize the fate of the offending antigen. Together, these approaches will allow unprecedented access to quantitative and qualitative aspects of antigen presentation in asthmatic disease.

Danuta Radzioch, Ph.D. — 2003 Senior Award

McGill University

Genetic Dissection of Susceptibility to Allergic Asthma

Asthma represents a complex trait with a strong genetic component. Previous studies in our Centre and by others have demonstrated major gene effects underlying the traits of susceptibility or resistance to this disease as well as of several intermediate phenotypes related to certain components of the immune/inflammatory pathways that the host exposed to an allergen. Furthermore, the proposed studies will test the hypothesis that the immunization with M.bovis BCG or/and imidazoquinolines prior to antigenic challenge may slow down or totally inhibit the development of allergic asthma phenotype in asthma-susceptible individuals. Our tool to dissect such complex phenotypes is a unique, newly-developed gene-discovery platform of recombinant congenic strains (RCS) of mice derived from inbred progenitors that are either susceptible to asthma (A/J, abbreviated A) or resistant to asthma (C57BL/6J, abbreviated B). The first objective of the proposed studies is to identify chromosomal loci/genetic factors which control the differences in susceptibility to antigen-induced murine model of asthma. The second objective is to identify discrete mechanistic phenotypes affected by these loci and biological markers of susceptibility to this inflammatory disorder. A particular emphasis will be on the regulation of T helper subset selection, an immunological pathway of pivotal importance in the susceptibility or resistance to pathological inflammation. Finally, we will explore the possible protective effect of M. bovis BCG vaccine/and a potent immunoadjuvant immidazoquinoline against development of allergen induced asthma. The discovery of genetically linked, quantitative biomarkers of asthma will contribute not only to better understanding of the disease pathogenesis but also will facilitate the genetic analysis of this disease in humans.

William C. Sha, M.D., Ph.D. — 2003 Junior Award

University of California, Berkeley

Regulation of Effector Responses in Asthma by B7h-ICOS Costimulatory Molecules

The costimulatory ligand-receptor pair B7h-ICOS has been implicated as a critical regulator in the pathogenesis of asthma. However, in different experimental models of asthma, perturbation of B7h-ICOS signaling interactions resulted in strikingly different outcomes. Systemic blockade of B7h-ICOS interactions during the effector phase of T-cell responses inhibited multiple indices of allergic lung inflammation including cellular infiltrates and IgE production, suggesting that blockade of B7h-ICOS interactions might be an effective strategy in treatment of asthma. In contrast, local blockade of B7h-ICOS interactions in the lung during aerosol tolerization prevented generation of antigen-specific TR cells that control allergic lung inflammation, suggesting that clinical blockade of B7h-ICOS interactions could actually exacerbate asthma. Although B7h is expressed on multiple cell lineages important in asthma, including B cells, dendritic cells, and lung endothelial cells, how distinct effector responses of ICOS+ T cells are regulated is poorly understood with respect to when, where, and with which B7h-expressing cells these T cells interact in vivo. We will examine the pathogenesis of asthma in mouse models where expression of B7h is restricted to distinct cell lineages, using crosses of lineage-specific B7h transgenic mice to B7h-/- mice. Using two distinct antigen-sensitization protocols that induce allergic lung inflammation by either systemic intraperitoneal or local intranasal routes, we will compare the nature and significance of B7h-ICOS interactions during local and systemic sensitization, and determine the contribution of individual lineages of B7h-expressing cells to either induction or inhibition of asthma.

Kevan Shokat, Ph.D. — 2003 Senior Award

University of California, San Francisco

A New Therapeutic Strategy for Asthma: Inhibition of Cortisol Metabolism in the Lung

11β-hydroxysteroid dehydrogenase 2 (11β-HSD2) is the enzyme responsible for conversion of the anti-inflammatory hormone cortisol to cortisone in the lung and kidney, thus inactivating glucocorticoid response in these tissues. We propose to develop the first potent and selective small-molecule inhibitor of 11β-HSD2 as a potential new therapy for asthma. Our hypothesis is that inhibition of 11β-HSD2 in the lung will prevent conversion of the anti-inflammatory steroid cortisol to inactive cortisone in the lung epithelium, resulting in reduced bronchial tube swelling associated with asthma attacks. Vastly different levels of 11β-HSD2 are expressed in the lung compared to other organs, providing an avenue for potent inhibition in the lung without significant inhibition systemically. Interestingly, a steroidal natural product isolated from licorice, glycyrrizic acid, has been used to treat asthma since the 16th century and is a potent inhibitor of 11β-HSD2. Glycyrrizic acid binds to the steroid binding site of 11β-HSD2 which is common to several cortisol/cortisone metabolizing enzymes and is thus pleiotropic in its effects. We propose to generate inhibitors of 11β-HSD2 which bind to the NAD+ binding pocket to achieve highly specific and tight binding inhibitors. In support of this approach, our laboratory has discovered a potent and selective NADPH competitive inhibitor of carbonyl reductase 1, an enzyme in the short-chain dehydrogenase reductase family which includes 11β-HSD2. Using this inhibitor as a lead structure, we propose synthesize analogs which are specific for 11β-HSD2 as a potential alternative or adjunct to synthetic corticosteroid treatment for asthma.

Jonathan S. Stamler, M.D. — 2003 Senior Award

Duke University/HHMI (Now at Case Western Reserve University)

The Role of S-Nitrosothiols in the Pathogenesis of Asthma

Asthma is characterized by intermittent obstruction, hyperresponsivity and chronic inflammation. Levels of nitric oxide (NO) are increased in asthmatic airways, but it has been difficult to argue for a significant role of NO in the asthmatic response - either as ameliorative or pathogenic - because NO synthase (NOS) inhibitors exert only modest effects in patients with asthma, and mice deficient in NOS2 (inducible NOS) have no significant change in airway responsivity. S-nitrosothiols (SNO's) are stable endogenous molecules with bioactivity similar to NO. It has recently been shown that S-nitrosoglutathione (GSNO) (NO complexed to glutathione), a major source of NO-derived bronchodilator and antimicrobial activity, is substantially reduced in the airway lining fluid of asthmatics. Thus, NO production is increased in asthmatics, but overall NO bioactivity is decreased. This paradox is best explained by an augmentation of airway denitrosylating activity. We have recently identified an enzyme in mammalian cells, GSNO reductase, which is a primary regulator of cellular GSNO metabolism, and therefore a likely regulator of airway GSNO levels. Moreover, we have produced a GSNO reductase knock-out mouse which is hyporesponsive to asthma. In this grant we will test the hypothesis that dysregulation of SNO metabolism is important in the pathogenesis of bronchial hyperreactivity and airway inflammation in asthma.

Ivaylo Stoilov, M.D. — 2003 Junior Award

University of Connecticut Health Center (Now at Envision Pharma)

Asthma and the Cytochrome p450 System of the Lung

Cytochrome P450s are responsible for the metabolic transformation of nearly every known class of endogenous organic molecules as well as various xenobiotics entering the human body. Although the potential role of the P450 super-family in the etiology, pathogenesis and pharmacogenetics of asthma appears to be appreciated from a conceptual viewpoint, a very large number of known P450 genes have not yet been investigated in terms of lung expression, and those that have been studied have not yet been characterized extensively. Comparative expression profiling of the mouse P450 gene family, conducted in the PI's laboratory, found that as much as 60% of the known P450 genes could be expressed in the adult mouse lung. Such large number of P450s offers numerous metabolic routes to the xenobiotics entering the lung via the airways, some of which may produce toxic and/or asthmogenic compounds. We propose comprehensive study of the lung cytochrome P450 system. Our first objective will be to determine the normal P450 composition in the developing, adult and aging lung. Next we will evaluate the reaction of the lung P450 system in a mouse model of asthma. The data from these studies will be used to select lung P450s for the development of knock-out mouse models. Such models will make it possible to evaluate the role of the P450 system in sustaining lung homeostasis and the etiology and pathogenesis of asthma. Finally, we will establish a library of functionally characterized polymorphisms from human lung P450s for pharmacogenetic studies.

Raul M. Torres, Ph.D. — 2003 Junior Award

University of Colorado/National Jewish Health

Regulation of GTP-binding Proteins in Asthma and Airway Hypersensitivity

Inflammation and recruitment of eosinophils, mast cells, and lymphocytes to the lung are critical components in the pathophysiology of asthma. The precise mechanism(s) by which leukocytes are attracted to the lung are ill-defined, although chemokine-mediated cellular recruitment to the lung has been implicated and is consistent with the defined role of chemokines in coordinating leukocyte movement prior to, and during, an immune response. We have generated a mouse mutant whose hematopoietic cells lack an intracellular signaling molecule, lsc, which regulates the activity of both heterotrimeric Gasubunits and RhoA, and thus couples the two families of GTP-binding proteins in a common signal transduction pathway. Hematopoietic cells lacking lsc (lsc-/-) exhibit aberrant G-protein coupled receptor (GPCR) signaling as exemplified by an inability to fully desensitize particular chemokine receptors to chemokine stimulation. A consequence of dysregulated GPCR signaling is that an increased proportion of lsc-/- hematopoietic cells migrate towards particular chemoattractants. Importantly, upon challenge and allergen sensitization, lsc-deficient mice mount an antigen-specific IgE response but do not display typical hallmarks of allergen-induced asthma such as a predominance of Th2 cytokines in bronchoalveolar lavage, airway hyperresponsiveness, or goblet cell hyperplasia. These data together suggest that lsc regulation of GTP-binding proteins is essential for allergen-induced inflammation. In this proposal we wish to define the role of this hematopoietic signaling molecule in a mouse model of asthma.