Gary W. Hoyle - US grants

Interstitial lung diseases are characterized by inflammation and fibrosis that can be caused by toxic agents such as dusts, particles, drugs, or infectious agents. Regardless of the initiating agent, pulmonary fibrosis induced by inhaled toxicants shares common features, which include inflammation, proliferation of interstitial cells, and deposition of extracellular matrix. We have been studying the molecular mechanisms of pulmonary fibrosis induced by asbestos, a model fibrogenic agent. Such materials upregulate a variety of genes in lung, but the genes that are the key, initiating mediators of pulmonary fibrosis have not been identified. We have studied platelet-derived growth factor (PDGF) as a candidate mediator of pulmonary fibrosis. PDGF is the most potent mesenchymal cell mitogen known, and has also been implicated as an inducer of extracellular matrix secretion. We have found that the levels of PDGF in the lungs of laboratory rodents increase dramatically after inhalation of a fibrogenic dose of chrysotile asbestos. A particularly high level of asbestos-induced PDGF expression is found in epithelial cells. We propose a model in which the fibroproliferative response caused by inhalation of a fibrogenic agent is driven by PDGF released from the overlying epithelial cells. The central hypothesis to be tested here is that epithelial-derived PDGF is a key mediator of interstitial pulmonary fibrosis. The hypothesis will be tested in transgenic mice that overexpress PDGF subunit genes or a dominant-negative PDGF mutant from the lung epithelial-specific surfactant protein C (SPC) gene promoter. The SPC promoter will be used to overexpress genes encoding the PDGF A and PDGF B genes singly and in combination to assess the potential of PDGF isoforms to initiate pulmonary fibrosis in the absence of a fibrogenic agent. These transgenic mice will be exposed to asbestos to examine the combined effect of PDGF overexpression and asbestos-induced lung injury. Transgenic mice will also be generated that express a dominant-negative form of PDGF to determine whether interference with epithelial-derived PDGF function prevents asbestos-induced lung injury. The proposed experiments will allow a determination of which features of pulmonary fibrosis can be initiated by PDGF and which require other triggering events. Identification of PDGF as a key mediator of fibrosis would serve to establish this factor as a target for therapeutic intervention in interstitial lung disease.

DESCRIPTION (provided by applicant): Chlorine gas is a highly toxic respiratory irritant that is considered a chemical threat agent. The goal of the research proposed in this renewal application is to develop a medical countermeasure to treat lung injury induced by chlorine inhalation. During the previous U01 award, a mouse model of chlorine gas inhalation was developed, a detailed characterization of lung injury following chlorine exposure was performed, signaling pathways representing possible targets for therapeutic intervention were investigated, strategies for manipulating signaling pathways of interest were tested, and compounds that significantly inhibited lung injury or inflammation in chlorine-exposed mice were identified. Rolipram, a type 4 phosphodiesterase inhibitor, was shown to stimulate alveolar fluid transport, decrease pulmonary edema, and inhibit airway hyperreactivity. Triptolide, a diterpenoid anti-inflammatory compound, inhibited chlorine-induced lung inflammation. In the present application, formulations of rolipram and triptolide will be developed for intramuscular (i.m.) injection, a preferred delivery route for potential mass casualty situations. In addition, methylprednisolone, an FDA-approved anti-inflammatory compound, will be evaluated as an alternative to triptolide. The overall hypothesis to be tested is that i.m. injection of an encapsulated formulation of rolipram in combination with an anti-inflammatory compound will represent the most effective countermeasure for chlorine-induced lung injury. Countermeasure formulations will be tested in mice to determine efficacy against chlorine-induced acute lung injury, to assess longer term effects on airway remodeling and airway hyperreactivity, and to measure the length of effective post-exposure treatment window. The most effective countermeasure formulation will be evaluated in rabbits to demonstrate efficacy in a non-rodent animal model. An optimized countermeasure formulation with efficacy against chlorine-induced lung injury in mice and rabbits will be subject to additional studies necessary for progression toward regulatory approval, including collection of pharmacokinetic/pharmacodynamic and toxicity data and production of the countermeasure using current good manufacturing practices. Public Health Relevance: The goal of the proposed experiments is to develop novel ways to treat acute lung injury caused by chlorine gas inhalation. The research will focus on medical countermeasures that can be easily and rapidly administered in a mass casualty situation. This type of research is important because of concerns that U.S. civilians could be adversely affected by the accidental or intentional release of highly toxic chemicals.

DESCRIPTION (provided by applicant): Acute high-level exposures to chemicals that damage the respiratory tract can cause life-threatening lung injury. Chlorine gas is a highly toxic respiratory irritant that is considered a chemical threat agent because of the possibility that it could be released in industrial accidents or terrorist attacks. Chlorine, like many pulmonary toxicants, produces injury to epithelial cells lining the respiratory tract. Loss of the respiratory epithelial cell barrier contributes to other sequelae observed in acute lung injury, including pulmonary edema, airway obstruction, inflammation, and respiratory infections. Efficient repair of respiratory epithelium and restoration of barrier function are essential for resolution of chlorine-induced lung injury. The epithelium of the respiratory tract is repaired following injury through the proliferation and differentiation of stem cells. One type of cell involved in the repair of airway epithelium is the basal cell. The low-affinity nerve growth factor receptor (NGFR) has been identified as a marker for basal cells, as well as for epithelial stem cells in other tissues, but the role of this protein in stem cell function is unknown. NGFR binds multiple neurotrophin ligands, including nerve growth factor (NGF), brain-derived neurotrophic factor, and neurotrophin 3. The hypothesis to be tested in the proposed studies is that NGFR controls the proliferation, survival, and differentiation of lung epithelial stem cells following chlorine-induced lung injury. In Specific Aim 1, basal cells will be purified from the trachea of normal mice and from mutant mice lacking functional NGFR. The ability of the cultured cells to survive, proliferate, and differentiate into other types of epithelial cells (ciliated cells and Clara cells) will be measured in the presence or absence of NGFR ligands. In Specific Aim 2, wild-type, NGFR knockout, and NGF transgenic mice will be exposed to chlorine gas according to a protocol established in the Principal Investigator's laboratory. Effects of neurotrophins will be tested by delivering ligands intranasally or by using NGF-overexpressing transgenic mice previously developed in the Principal Investigator's laboratory. The extent of tracheal epithelial repair will be measured at various times after chlorine exposure. In Specific Aim 3, the effect of neurotrophins on epithelial repair following chlorine exposure in rabbits will be investigated. Rabbits will be used for these studies because of the wider distribution of basal cells in the airways in this species which is more similar to that in humans. The proposed experiments will not only investigate mechanisms of epithelial repair, but will also foster the development of therapeutic agents that could be used to stimulate resolution of lung injury following inhalation of toxic chemicals. PUBLIC HEALTH RELEVANCE: This application is submitted in response to PAR-10-019, CounterACT Exploratory/Developmental Projects in Translational Research (R21). The proposed experiments are designed to understand how the lung repairs itself after inhalation of a toxic gas with the goal of developing therapy for lung injury based on stimulating lungs cells to accelerate repair. This type of research is sought through the Funding Opportunity Announcement because of concerns that U. S. civilians could be adversely affected by highly toxic chemicals released intentionally in terrorist attacks or unintentionally in industrial accidents or natural disasters.

DESCRIPTION (provided by applicant): Chlorine gas is a highly toxic respiratory irritant that is considered a chemical threat agent because of the possibility that it could be released in industrial accidents or terrorist attacks. Acute effects of chlorine inhalation include dyspnea, hypoxemia, pneumonitis, and pulmonary edema. Longer term consequences of chlorine inhalation include pulmonary function impairment and airway structural changes that have been observed in some exposed individuals. We have developed a mouse chlorine inhalation model in which animals develop pulmonary edema, inflammation, airway obstruction, and airway hyperreactivity within one day after exposure. In new studies related to the current application, we have characterized injury and repair of the lung at longer times after chlorine inhalation. We have observed that airways with a partial loss of epithelium are repaired quickly by the proliferation and differentiation of epithelial cells that survive chlorine exposure. In contrast, portions of airways with few surviving epithelial cells are repaired inefficiently, and fibroproliferative lesions develop at such sites within a week after chlorine exposure. This development of airway fibrosis is associated with impaired lung function, including increased respiratory system resistance and airway hyperreactivity to inhaled methacholine. In the current application, we propose to develop countermeasures that can be administered following a chemical attack or accidental release to prevent chlorine-induced airway disease. The hypothesis to be tested in the proposed studies is that inefficient repair of airway epithelium following chlorine lung injury leads to airway fibrosis and impaired lung function; agents that mimic anti-fibrotic effects of airway epithelium or stimulate airway repair will ameliorate chlorin- induced airway disease. Specific Aim 1 will characterize airway epithelial repair, airway fibrosis, and lung function impairment following chlorine exposure in mice. Specific Aims 2-5 will evaluate the efficacy of the following potential countermeasures for the treatment of chlorine-induced airway disease: the prostanoid receptor agonists butaprost and iloprost; the long-lasting ß-agonist formoterol and the type 4 phosphodiesterase inhibitor rolipram; stimulation of plasminogen activation with urokinase-type plasminogen activator or the plasminogen activator inhibitor-1 inhibitor tiplaxtinin; and manipulation of Wnt/ß-catenin signaling with lithium chlorie or pyrvinium. Specific Aim 6 will involve development of a ferret model of chlorine-induced airway disease and testing of countermeasures in this non-rodent species. The proposed studies are expected to identify a countermeasure for prevention of chronic airway disease that develops as a result of an acute high level exposure to chlorine.

Chlorine gas is a highly toxic respiratory irritant that is considered a chemical threat agent because of the possibility that it could be released in industrial accidents or terrorist attacks. Acute effects of chlorine inhalation include dyspnea, hypoxemia, pneumonitis, and pulmonary edema. Longer term consequences of chlorine inhalation include both pulmonary function impairment and structural changes that have been observed in some exposed individuals. Our collaborator Dr. Erik Svendsen has documented pulmonary function abnormalities in a population of individuals who were exposed to chlorine as a result of a large accidental release that occurred in Graniteville, SC in 2005. The results point to a spectrum of respiratory disease states, but suggest that an important component involves small airway disease. An impediment to developing countermeasures for persistent chlorine-induced lung disease is the lack of a relevant animal model for efficacy testing. Our preliminary data indicate that rabbits exposed to chlorine and examined 7 days later exhibit inflammation of the small airways and develop sporadic obliterative lesions characteristic of bronchiolitis obliterans (BO). These animals exhibited normal baseline lung resistance but exhibited hyperreactivity to inhaled methacholine. Based on these preliminary data, we propose to expand on these studies to develop a model in which the obliterative changes to small airways are more widespread resulting in increased baseline lung resistance as has been observed in humans exposed to chlorine gas. We will then assess the efficacy of a potential countermeasure when administered following chlorine exposure for the prevention of small airway disease. The hypotheses to be tested are: 1) Increasing post-exposure monitoring time and/or chlorine dose will produce widespread small airway BO lesions that result in significant increases in baseline lung resistance; and 2) Treatment with inhaled corticosteroid plus ß- adrenergic agonist after chlorine exposure will inhibit the development of BO lesions and abnormal lung function. Specific Aim 1 will be to develop an animal model of persistent chlorine-induced lung disease that replicates the disease states observed in chlorine-exposed humans. Specific Aim 2 will be to evaluate a currently available therapy (inhaled corticosteroid plus long-acting ß-adrenergic agonist) in a rabbit model of persistent chlorine-induced lung disease. These exploratory studies will generate valuable information toward development of an appropriate animal model for countermeasure testing and will generate initial data regarding efficacy of a type of treatment currently used for irritant-induced asthma that could be readily adopted as a countermeasure.

The University of Louisville Center for Integrated Environmental Health Science (CIEHS) will promote research toward understanding how lifestyle factors interact with exposure to environmental toxicants in human health and disease. The CIEHS Pilot Project Program will further this overall goal by providing support for short-term projects leading to data collection enabling new external grant applications. In this way, the Pilot Project Program will enhance capacity for novel research in environmental health science at the University of Louisville. The Program will publicize the availability of pilot project funds, solicit and coordinate the review of pilot project applications, make funding recommendations, monitor the progress of research projects, assess usage of center cores by pilot projects, and track deliverables. A mix of investigators will be targeted, including new early stage investigators, community partners, and established researchers who can apply their expertise in novel ways relevant to environmental health. Results obtained through pilot projects will be disseminated via the annual Research!Louisville event that showcases research to the university, local, and regional communities. Because the ultimate goal is to develop new, self-sustaining research projects, an emphasis will be placed on facilitating and improving the quality of new grant applications pertaining to environmental exposures/lifestyle factors. Strategies for this include obtaining feedback from CIEHS members regarding pilot project findings and experimental design as well as performing rigorous internal review of grant proposals. Thus, the Program will assist investigators throughout the pilot project life cycle from initial idea to extramurally funded project and is expected to generate significant new research funding and enhance the capacity for research in the environmental health sciences at the University of Louisville.