BA: (1966) Yale University, New Haven, CT
MD: (1970) Columbia University, New York, NY
Residency: (1972) Columbia Presbyterian Hospital, New York, NY
Fellowship: (1977) Royal Victoria Hospital, Montrel, Canada
Academic & Professional Titles:
Kenan Professor of Medicine
Director, Cystic Fibrosis/Pulmonary Research & Treatment Center
Office: 7008 Marisco Hall
The Boucher lab continues its major interest in the functions of airway epithelia in health and disease. In general terms, the lab focuses on five interrelated areas of research:
1. Airway surface liquid (ASL) physiology
The Boucher lab is interested in the physiology of airway surface liquid at the microscopic and the integrated (alveolar/airway) macroscopic levels.
a. "Microscopic": The goal of the lab has been to understand the composition, organization, and physiology of the different components of the airway surface liquid (ASL) compartment. Recent advances using well-differentiated airway epithelial cultures that secrete mucin, effect coordinated ciliary beat regulation, and regulate airway surface liquid salt and water composition have allowed initiation of detailed studies of the topography as well as physiology of ASL. We interface these cultures with the confocal microscope, which allows us to study ASL as native "thin films" that exhibit both mucus and defined periciliary liquid layers. The use of confocal microscopy to study this layer has been complemented by the development of techniques to rapidly fix, without distortion, the ASL compartment, using perfluorocarbon/osmium techniques.
b. Function of ASL: We postulate that the major function of the airway surface liquid compartment is to effect mechanical clearance of deposited materials from airway surfaces We have been able to study the integrated activities of the airway surface liquid in the air-liquid interface culture model system. The airway surface liquid moves in a rotational (counterclockwise) fashion in this culture system. The rate of rotational mucus transport can be quantitated and approaches the velocities that are achieved in vivo. Many key aspects of airway surface liquid physiology have been tested using this system. For example, the contribution of the epithelium to local salt and water concentrations and volumes, respectively, has been studied. The air-liquid interface culture system has been utilized for measuring the composition and rate of volume absorption across airway epithelial surfaces. Airway surface liquid on normal airway surfaces is isotonic, and the epithelium mediates amiloride-sensitive isotonic volume absorption. In addition, this approach of employing well-differentiated cultures and confocal microscopy has been utilized to measure the permeability of the epithelium to water. These studies established that the airway epithelia are relatively water-permeable, which has major implications for the ability to hydrate inspired air and has therapeutic implications with regard to regulation of ASL volume on airway surfaces.
c. Macroscopic: There are several major questions with respect to the integrated physiology of surface liquid amongst the different regions of the lung. The first question is whether the periciliary liquid moves with the mucus layer in a cephalad fashion via the activities of cilia. This question relates to the magnitude of the volume burden that would be imposed on successive proximal airway surfaces as liquid moves from the very large to the very small areas of the small versus large airways, respectively. In a series of studies in a well-differentiated cell culture system, we used a column of photo-released FITC dextran to compare the relative movement of the mucus and periciliary liquid layers. These studies demonstrate that the periciliary liquid layer moved at a rate similar to that of the mucus layer, reflecting apparent frictional interactions between the mucus and the periciliary liquid layer. Biophysical modeling of these interactions is currently underway.
A major question relates to the site of the initial secretion of the liquids that move up airway surfaces. The lab is currently investigating the hypotheses that (1) the liquid is secreted in the distal portions of the bronchioles versus (2) that liquids are secreted on alveolar surfaces and move onto airway surfaces by a "wicking" phenomenon that reflects the forces generated by the ciliary movement of the airway surface liquid compartment.
2. Airway epithelial Na+ transport
The lab has had a long-term interest in the sites and regulation of Na+ transport in the conducting airways. These studies focus both on normal physiology and ion transport dysfunction in disease.
a. Normal regulation of airway epithelial Na+ transport: The airway epithelial Na+ channels (ENaC) appear to be expressed relatively homogeneously throughout the adult lung. There appears to be little hormonal regulation of ENaC expression to account for any major homeostatic regulation of Na+ transport in adult airways. Consequently, most efforts have focused on regulation of the activation status of ENaC channels. A critical question and area of interest in the lab with regard to airway surface liquid physiology is how the ENaC channel senses the volume of airway surface liquid and appropriately regulates the rate of Na+ absorption.
b. Role of Na+ transport in airways disease: A consistent interest of the laboratory has been the role of excessive Na+ transport in the pathogenesis of CF airways disease. Recent data have strongly indicated that excessive Na+ transport drives NaCl and water (isotonic volume) absorption across CF airways epithelium. As in normals, the absorption of NaCl appears to be isotonic in CF. A major breakthrough in the lab has been to directly link the excessive volume absorption by the CF airway epithelium to the defect in mucus clearance. It appears that volume depletion in CF is expressed not only in the dessication of the mucus layer but also in the depletion of the periciliary liquid layer. Thus, the pathophysiologic correlate of this phenomenon is that mucus plaques become adherent to the CF airway surface and cannot be cleared either via ciliary or by cough-dependent mechanisms. Importantly, we have been able to show, via either volume replacement directly or drug-induced volume replacement, that the defect in mucus transport that characterizes CF airways under these conditions is reversible. Thus, these observations set the paradigm for therapeutic strategies designed to clear the airways of the adherent, thickened mucus plaques that we postulate are the nidus of CF airways infection.
3. Extracellular nucleotides
Our lab has had a major interest in the role of extracellular nucleotides in airways homeostasis. This interest spans all aspects of extracellular nucleotide biology/physiology.
a. Role of P2 receptors in airway epithelial function: the G-protein-coupled P2Y receptors appear to be highly expressed in airway epithelial cells and linked to the major functions of cells in the airway epithelium, e.g., salt and water transport and ciliary regulation in the ciliated cells, and mucin granule release in the "goblet" cells. Molecular studies in human airways and recent studies in P2Y2-targeted mice suggest that, at least with respect to Cl- secretion, the P2Y2 (ATP=UTP) receptor is the major receptor transducing extracellular nucleotide signals into regulation of airway epithelial Cl- transport.
b. Extracellular nucleotidases: Initial studies in the lab were designed to measure the rates of metabolism of nucleotides that may be deposited on the airway surfaces (e.g., UTP) for therapeutic purposes. However, analysis of these initial studies revealed a complex series of extracellular nucleotidases on airway surfaces that was heretofore unexpected. There are five different systems that may be expressed on airway surfaces. These studies are of interest to the lab, both for the potential relevance for the development of nucleotide-based therapies, and for the implications they have with respect to the control of extracellular nucleotide concentrations in response to endogenous nucleotide release.
c. Nucleotide release from cells: Nucleotides may be released from both goblet cells (co-packaged with mucin granules) and from ciliated cells themselves. A major effort in the lab is being focused on the signal transduction systems responsible for nucleotide release from ciliated cells. The most potent stimulus is that of stress or tactile deformation. In a recent series of studies, it was shown that tactile stimulation of polarized airway epithelial cells causes the release of ATP and UTP into the ipsilateral (side of stimulus) and contralateral surfaces of airway epithelial cells. The physiologic consequence of this release is to form "Ca2+ waves" that may integrate the activity of patches of airway epithelial cells in response to stimuli. Another implication of the result is that the contralateral release of nucleotides may mediate transepithelial signaling, i.e., permitting the submucosal space to "know" that stimuli are being presented to the lumen of cell.
4. Gene therapy
The lab has had a major interest in gene therapeutic approaches to CF lung diseases. The general concept of the lab has been to utilize the airway epithelial cell biologic expertise and more recently the gene targeting expertise afforded by our studies of G protein coupled receptors (GPCRs) located in the apical membrane, e.g., P2Y2-R, to increase the efficiency of gene transfer.
a. Barriers to gene transfer: Work in the lab over approximately the past five years has revealed that the major limitation to airways gene transfer is one of limited efficiency. This problem was first noted when we discovered that gene transfer to freshly excised in vivo airway epithelia was only effective when the epithelium was injured, reflecting a gross abrogation of multiple barriers to gene transfer, including the lumen-facing ciliated cells. This early recognition of these problems have led to a program to understand each of the potential barriers to gene transfer in series. Programs are underway to assess the epithelial barrier at each level, starting with the mucus barrier and ending with cytoplasmic nuclear translocation.
b. Vector targeting: An insight from the lab was that effective targeting of a vector to the apical membrane required a target that would both bind the vector and internalize it. Because none of the conventional targets that offered both functions were expressed on the apical membrane, e.g., growth factors, integrins, we identified a novel group of targets on this barrier that fulfilled these criteria. In general, this class of targets are the G protein-coupled receptors, which, as a part of their desensitization biology, sequester (internalize). The early focus of the lab has been on the P2Y2 receptor, which is expressed at relatively high levels on the airway epithelial cell and for which ~30% of the receptor can sequester in response to an agonist. Current strategies to redirect vectors to P2Y2 include development of monoclonal antibodies to external epitopes of P2Y2 for the generation of bi-specific antibody approaches and the modification of the native ligand, e.g., UTP, that will allow this molecule to be used as the cognate moiety of a molecular conjugate for targeting this particular receptor.
5. Clinical studies
The lab continues to provide translational concepts for testing in the clinical arena. Current areas of interest include:
Interactions with Na+ channel blockers and hypertonic saline: Work with the airway epithelial well-differentiated cell culture system, again interfaced with the confocal microscope, has indicated that the application of hypertonic saline to airway surfaces achieves only a very short-lived increase in airway surface liquid volume (the added salt osmotically driving water flow to the airway surface across the relatively large water-permeable airways epithelium). The short duration of action of hypertonic saline appears to reflect, in part, the rapid active absorption of Na+ (and Cl-/water) from the epithelial surface. The addition of amiloride to the culture system concurrent with the hypertonic saline greatly extends the actions of the osmotically active salt. This observation sets the groundwork for a series of studies focused on exploring the capacity of amiloride to extend the magnitude and duration of action of hypertonic saline when administered to human CF subjects. Thus, a series of studies is underway that will test both the acute and chronic (two week) effects of a regimen of hypertonic saline ± amiloride pretreatment of the mucus clearance and pulmonary function characteristics of the CF lung.