1985 — 1987 |
Sheppard, Dean |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Role of Inflammation in Induced Airway Hyperreactivity @ University of California San Francisco
The proposed research is part of a long term effort to determine the mechanisms underlying the exaggerated bronchoconstrictor responsiveness that characterizes asthma. This project will combine methods of physiology, pharmacology and morphometry to study the mechanisms by which toluene diisocyanate (TDI), a widely used chemical that commonly causes asthma in exposed workers, induces both airway inflammation and airway hyperresponsiveness in guinea pigs. An advantage of this proposal is the long term collaborative interaction with a large group of scientists interested in the neurohumoral control of the lungs and airways. The general hypothesis we will test is that inflammation is a major mechanism of airway hyperresponsiveness. Thus, we will study the role of various aspects of the inflammatory response, including increased vascular permeability, chemical mediators of inflammation, and tissue infiltration with inflammatory cells, in the increase in airway responsiveness caused by exposure to TDI. First, we will use inhibitors of various steps in the metabolism of arachidonic acid to study the roles of prostaglandins, thromboxanes and leukotrienes in this response. By performing acetylcholine dose response curves and morphologic examination of sections of trachea and lung in treated and untreated animals exposed to air and to TDI, we will examine the effects of these inhibitors simultaneously on airway hyperresponsiveness and on morphologic evidence of airway injury and inflammation. Next, we will study the roles of polymophonuclear leukocytes (PMNs) in mediating the effects of TDI by performing similar studies in animals that have been depleted of PMNs or depleted and then repleted prior to exposure. We will perform similar studies in animals that have been depleted of eosinophils. In a parallel set of experiments we will study the role of inflammatory cells and mediators of inflammation in the increase in airway vascular permeability to Evans blue dye caused by exposure to TDI. Finally, we will study the effects of TDI on the in vitro behavior of airway smooth muscle both in the presence of and in the absence of PMNs eosinophils and airway epithelial cells. These studies should provide important insights into the mechanisms by which exposure to airborne chemicals can cause inflammation, edema, and hyperresponsiveness of the airways.
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1 |
1988 — 1992 |
Sheppard, Dean |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Role of Inflammation of Induced Airway Hyperreactivity @ University of California San Francisco
This application is part of a long-term effort to determine the relationship(s) between airway inflammation and airway hyperresponsiveness, a central feature of asthma. The proposed experiments will focus on the role of tachykinins, a class of low molecular weight peptides that are present in the airways of several mammals, including humans. Tachykinins play an important role in several inflammatory processes and have recently been found to be required for the induction of airway hyperresponsiveness by toluene diisocyanate (TDI), a widely used chemical that is a well known cause of occupational asthma. TDI exposure also potentiates the effects of exogenously administered tachykinins, at least in part by inhibition of neutral endopeptidase, an enzyme that contributes to local tachykinin metabolism. In the proposed experiments, we will first determine whether stimuli known to induce release of tachykinins into the airways can themselves induce airway hyperresponsiveness in guinea pigs, and whether this effect requires simultaneous inhibition of neutral endopeptidase. In similar experiments we will evaluate the effects of exogenous administration of specific tachykinins. Next we will determine whether tachykinins contribute to the increase in airway responsiveness caused by two other inflammatory stimuli, ozone and inhaled antigen, by evaluating the effects of tachykinin antagonists, tachykinin depletion and neutral endopeptidase inhibition on these responses. We will also determine whether these stimuli, like TDI, themselves lead to inhibition of airway neutral endopeptidase activity. To determine the mechanisms by which tachykinins contribute to airway hyperresponsiveness we will evaluate the effects of tachykinins and of TDI in augmenting acetylcholine release from efferent nerves and in increasing airway epithelial permeability. We will also evaluate the role of platelet activating factor in these responses. In addition, we will evaluate the mechanisms by which TDI inhibits airway neutral endopeptidase activity, by evaluating the effects of TDI on purified neutral endopeptidase and on neutral endopeptidase activity in tracheal explants. In these experiments we will focus on possible roles of toxic oxygen metabolites and proteolytic enzymes in inactivating neutral endopeptidase. Finally, we will pursue any positive in vitro experiments with studies of neutral endopeptidase inactivation by in vivo exposure of guinea pigs to TDI. These experiments will help to clarify the role of an important class of inflammatory mediators (tachykinins) in the induction of airway hyperresponsiveness and will also further our understanding of the links between hyperresponsiveness and inflammation.
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1 |
1992 — 1999 |
Sheppard, Dean |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Function of a Novel Airway Epithelial Adhesion Receptor @ University of California San Francisco
Airway epithelial injury plays an important role in a number of common airway diseases. Interactions with extracellular matrix are central to tissue repair. We have thus begun to characterize airway epithelial cell surface receptors for matrix proteins. Given the unique environment in which these cells function, we hypothesized that they might possess novel adhesion receptors. The homology based polymerase chain reaction (PCR) was used to amplify DNA fragments encoding 3 novel subunits of members of the integrin family of adhesion receptors from airway epithelial cells. One of these, beta6, pairs with the known integrin alpha subunit, alpha/v, to form one of the principal fibronectin-binding proteins in airway epithelial cells. This proposal will further characterize the cellular distribution, extracellular ligand(s), regulation, and functional significance of this novel integrin heterodimer (alpha/v beta6). PCR and immunofluorescence microscopy will be used to characterize the distribution of alpha/v beta6, and its relationship to other alpha/v -containing integrins. The extracellular ligand(s) of alpha/v beta6 will be determined by affinity chromatography and the functional significance of alpha/v beta6 in cell attachment to these ligands and in cell migration into wounds will be assessed. Previous data from this laboratory has shown that airway epithelial cells respond in a highly polarized fashion to the cytokine transforming growth factor beta1 (TGFbeta1), a known stimulus to the regulation of other integrins. In this proposal, the polarity of regulation of alpha/v beta6 and other alpha/v-containing integrins will be examined by Northern blotting and by selective biotinylation of either the apical or basolateral surface. In vivo regulation of alpha/v-containing integrins will be assessed by tissue immunofluorescence in a model of airway epithelial injury and repair. The effects of heterologous expression of alpha/v beta6 will be examined in both fibroblasts (NIH 3T3), and epithelial cells (HeLa), to determine the role of this protein in expression of the epithelial phenotype and the importance of other epithelial characteristics for full functional expression. Finally, the role of the unique cytoplasmic domain of beta6 will be examined by affinity chromatography, and by expression of mutants containing cytoplasmic deletions and chimeric proteins composed of the beta6 extracytoplasmic domain and the cytoplasmic domain of the alternative alpha/v partners, beta3 and beta5. Through these studies we hope to learn more about the functional significance of one of the principal adhesion receptors on airway epithelial cells as a first step toward determining the role of adhesion receptors in the deranged repair processes that characterize the development of airway diseases.
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1 |
1993 — 1997 |
Sheppard, Dean |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Airway Epithelial Receptors For Extracellular Matrix @ University of California San Francisco |
1 |
1995 — 1999 |
Sheppard, Dean |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
In Vivo Functions of Pulmonary Integrins @ University of California San Francisco
The integrin alphavbeta6 is a receptor for the extracellular matrix proteins fibronectin and tenascin. This receptor is highly expressed in the epithelium of the respiratory tract and the skin during development, after injury or inflammation, and in malignant tumors, but is not expressed at either site in normal, healthy adults. In vitro, heterologous expression of alphavbeta6 in tumor cells increases cell adhesion to fibronectin and tenascin and also increases the capacity of these cells to proliferate. However, nothing is known about what role, if any, this receptor plays in vivo. To examine the in vivo function of alphavbeta6, we are producing mice that are deficient in receptor expression using standard homologous recombination in embryonic stem cells. In parallel, we are producing mice that overexpress either functional receptor, or a mutant version engineered to be incapable of binding ligand, under the control of highly active promoters that target expression to the airway epithelium (CC10), to alveolar type II cells and bronchiolar epithelium (SPC), or to basal keratinocytes (K14). Examination of the growth and development of mice deficient in beta6 protein should provide important clues to what role, if any, this receptor plays in epithelial differentiation and organogenesis. In addition, we propose to produce cutaneous wounds and respiratory epithelial injuries in these mice to determine the role of alphavbeta6 in epithelial repair in the lungs and skin. We also propose to examine the effects of overexpression of intact or mutant beta6, and of knockout of the beta6 gene, on the development of spontaneous and chemically-induced lung tumors. These studies should provide important information about the in vivo function of this integrin in lung (and cutaneous) development, in the repair of lung (and cutaneous) injury, and in the development, growth and/or progression of lung tumors.
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1 |
1996 — 2000 |
Sheppard, Dean |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. P50Activity Code Description: To support any part of the full range of research and development from very basic to clinical; may involve ancillary supportive activities such as protracted patient care necessary to the primary research or R&D effort. The spectrum of activities comprises a multidisciplinary attack on a specific disease entity or biomedical problem area. These grants differ from program project grants in that they are usually developed in response to an announcement of the programmatic needs of an Institute or Division and subsequently receive continuous attention from its staff. Centers may also serve as regional or national resources for special research purposes. |
Mechanisms of Persistent Airway Inflammation in Asthma @ University of California San Francisco
Airway inflammation is a central feature of chronic asthma. However, the mechanisms underlying the persistence of inflammation in asthma are poorly understood. In this SCOR, each project will examine the molecular mechanisms of recruitment or phenotypic differentiation of cells that, by virtue of their long life span in the airway wall, are likely to contribute to the persistence of airway inflammation in asthma. One project will examine the mechanisms by which naive CD-4+ T cells differentiate into pathogenic effector cells. Specifically, this project will identify the temporal and cellular requirements for IL-4 priming, the role of costimulatory signals delivered through the CD28-B7 and CD40-CD40L pathways, and the potential to tolerize T cells and abrogate pathologic airway responses to antigen through the use of altered ligands. A second project will utilize the same model, and blocking antibodies and/or knockout mice, to directly examine the contributions of members of the selectin family to the recruitment of leukocytes into the airways and to the subsequent physiologic derangements of airway function. Additional experiments will identify the signals and pathways by which selectins activate integrins, and will identify and characterize the extra lymphoid endothelial ligand for L-selectin. Antibodies directed at two distinct families of leukocyte integrins inhibit airway hyperresponsiveness, an effect that does not depend solely on inhibition of leukocyte recruitment. A third project will examine the mechanisms by which anti-integrin antibodies could directly and indirectly affect airway leukocytes. These studies should provide insight into how remodeled airways can provide pathogenic signals to leukocytes that perpetuate airway dysfunction in asthma. A fourth project will examine the effects of signals from the extracellular matrix on the production of chemokines and other cytokines from airway epithelial cells. These studies will utilize anti-integrin antibodies and recombinant matrix protein fragments to specifically trigger signals through each of the integrins expressed on airway epithelial cells. They will then utilize transgenic mice expressing mutant integrins or integrin ligands to examine the significance of these effects on two different models of airway inflammation and hyperresponsiveness. A fifth project will examine the expression of the two principal chymotrypsin-like mast cell proteases in normal and inflamed human airways, and will utilize the regulatory regions of the genes encoding each of these proteases to examine the DNA sequences and nuclear proteins responsible for regulation of protease expression. Finally, a sixth project will examine the integrated responses of all of the cells in the airway wall of human subjects to challenge with experimental or community acquired viral infection or inhaled allergen. By examining many of the endpoints utilized in each of the other five projects, this project will insure rapid application of an basic observations made to the direct study of human asthma.
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1 |
1996 — 2005 |
Sheppard, Dean |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. P50Activity Code Description: To support any part of the full range of research and development from very basic to clinical; may involve ancillary supportive activities such as protracted patient care necessary to the primary research or R&D effort. The spectrum of activities comprises a multidisciplinary attack on a specific disease entity or biomedical problem area. These grants differ from program project grants in that they are usually developed in response to an announcement of the programmatic needs of an Institute or Division and subsequently receive continuous attention from its staff. Centers may also serve as regional or national resources for special research purposes. |
Modulation of Airway Inflammation by Epithelial Integrins @ University of California San Francisco
The influx and activation of inflammatory cells in the airway wall is dependent, in large part, on the local release of inflammatory cytokines. Although much attention has focused on cytokines derived from leukocytes, it is now clear that airway epithelial cells, the most numerous Cell type in the airway wall, are themselves capable of synthesizing and secreting several cytokines that could profoundly influence airway inflammation. In non-epithelial cells, regulation of cytokine gene expression is critically dependent on signals provided from the extracellular matrix via the integrin family of transmembrane receptors. The existence of similar regulation of cytokine gene expression in airway epithelial cells could help to explain the coexistence of airway remodeling and persistent airway inflammation in asthma. We have recently generated lines of mice expressing a null mutation in the beta-6 integrin subunit, a subunit that is restricted in its expression to epithelia, especially in the lung and skin. Mice expressing this mutation develop and reproduce normally, but all have inflammatory cell infiltrates around hair follicles and scattered throughout the lungs and airways, consistent with a role for integrin- derived signals in regulation of inflammatory cell recruitment and/or activation in these sites. In the proposed studies, we will systematically examine the effects of various components of the normal extracellular matrix and of matrix proteins enriched in injured and inflamed airways, on the constitutive and stimulated synthesis and secretion of cytokines known to be synthesized by airway epithelial cells. Once we have determined the effects of various matrix proteins, we will examine the role of various integrins in these responses. For these experiments, we will use blocking anti-integrin antibodies, recombinant fragments of matrix proteins designed to be uniquely recognized by specific airway epithelial integrins, and murine airway epithelial cells derived from mice expressing mutations in integrin genes. Finally, to determine the significance of integrin and matrix-derived cytokine regulation in vivo, we will examine the influx of inflammatory cells, the production of selected cytokines, and the alterations in in vivo airway responsiveness produced when mice deficient in integrins or integrin ligands are challenged acutely or chronically by either immunologic or non-immunologic inflammatory stimuli. Through these studies, we hope to determine how alterations in airway epithelial integrins and/or their ligands could contribute to the chronic airway inflammation that characterizes persistent asthma.
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1 |
1996 — 2005 |
Sheppard, Dean |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. P50Activity Code Description: To support any part of the full range of research and development from very basic to clinical; may involve ancillary supportive activities such as protracted patient care necessary to the primary research or R&D effort. The spectrum of activities comprises a multidisciplinary attack on a specific disease entity or biomedical problem area. These grants differ from program project grants in that they are usually developed in response to an announcement of the programmatic needs of an Institute or Division and subsequently receive continuous attention from its staff. Centers may also serve as regional or national resources for special research purposes. |
Core--Animal @ University of California San Francisco
The small animal core will provide a centralized facility for the measurement of pulmonary resistance and compliance in anesthetized and ventilated mice, and for the measurement of responsiveness to intravenous acetylcholine. The core will also assist in broncholeveolar lavage and in the inflation, perfusion, and embedding of mouse lungs. The core facility will be equipped with two complete plethysmographic systems, including plethysmographs, pressure transducers, amplifiers, pulmonary mechanics analyzers, and chart recorders, and will be staffed by a full time staff research associate under the direct supervision of the principal investigator. The core will be extensively utilized by 4 of the 6 projects in this proposal, and the existence of a core facility will insure both quality control and rapid dissemination of technical information among these 4 projects. By utilizing existing equipment, and a newly constructed, dedicated small animal laboratory, this centralized facility will obviate the needs to train additional personnel in each project lab, to duplicate expensive equipment, and to set aside space in each laboratory suitable for experiments with live animals.
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1 |
2000 — 2007 |
Sheppard, Dean |
U01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
The Nhlbi-Bay Area Functional Genomics Consortium @ University of California San Francisco
The NHLBI-Bay Area Functional Genomics Consortium will use gene-trap vectors to inactivate thousands of genes in mouse embryonic stem (ES) cells and make them freely available for the purpose of generating knockout mice. In preliminary studies, custom gene-trap vectors have been used to trap more than 500 mouse genes, some completely novel, and many corresponding to ESTs of unknown function. Approximately 150 of the "trapped" ES cell clones have been transmitted through the germline, and studies of the knockout mice have already led to the identification of completely novel genes that are important in cardiopulmonary development and disease. The Consortium involves several leading San Francisco Bay Area research institutions: The J. David Gladstone Institutes, the University of California, San Francisco, and the University of California, Berkeley. The Consortium is organized into nine Components: (1) Gene Trapping in Embryonic Stem Cells, (2) Computational Methods for Predicting Gene Function, (3) In Situ Hybridization, (4) Gene Expression Profiling and Analysis, (5) Mouse Resource for Pulmonary Disease, (6) Mouse Resource for Lipid Metabolism and Atherogenesis, (7) Mouse Resource for Cardiopulmonary Development, (8) Cardiopulmonary Genomics Education, and (9) Administration. The major objective of the Consortium (corresponding to Component 1) is to use custom gene-trap vectors to inactivate at least 2,500 genes per year in ES cells. Each "trapped" ES cell line will be posted on the Consortium's website (genetrap.org) and will be distributed freely to the research community for the purpose of producing knockout mice. A second objective (corresponding to Components 2-4) is to assess which of the ES cell lines is likely to be valuable for understanding cardiopulmonary development and common cardiopulmonary diseases. To achieve this objective, the investigators will use computational approaches, expression profiling with DNA microarrays, and in situ hybridization studies. A third objective (corresponding to Components 5-7) is to select a few ES cell clones for the production of knockout mice, for the purpose of understanding genes involved in cardiopulmonary development and disease. The Consortium's resources will be distributed freely to any interested investigator and should provide a catalyst for many different NHLBI-funded research programs.
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1 |
2000 — 2013 |
Sheppard, Dean |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. R37Activity Code Description: To provide long-term grant support to investigators whose research competence and productivity are distinctly superior and who are highly likely to continue to perform in an outstanding manner. Investigators may not apply for a MERIT award. Program staff and/or members of the cognizant National Advisory Council/Board will identify candidates for the MERIT award during the course of review of competing research grant applications prepared and submitted in accordance with regular PHS requirements. |
In Vivo Function of Pulmonary Integrins @ University of California San Francisco
DESCRIPTION (provided by applicant): This competitive renewal application is to continue on-going studies of the functions of the epithelial integrin, alphavbeta6. Based on analysis of beta6 knockout mice, alphavbeta6 plays a critical role in regulating pulmonary responses to injury through spatially and temporally regulated activation of the cytokine, transforming growth factor beta (TGFbeta). Alphavbeta6-mediated TGFbeta-activation is required for induction of pulmonary edema and pulmonary fibrosis in response to bleomycin, and homeostatically regulates macrophage protease expression, protecting the lungs from protease-mediated emphysema. Lung injury increases expression of alphavbeta6, but multiple lines of evidence suggest that under resting conditions alphavbeta6 is expressed but does not activate TGFbeta, and that alphavbeta6-mediated TGFbeta-activation is itself "activated" by extracellular signals. The studies proposed in this application will address the critical question of how this process is regulated. Preliminary evidence suggests that known downstream targets of integrin signaling, the focal adhesion kinase (FAK) and the small GTPases, Rac1 and RhoA activate this pathway in a model culture system. Furthermore, IL-1beta, a toll-like/IL-1 receptor (TIR) ligand, activates this process in polarized alveolar and airway epithelial cells. We will therefore determine the range of TIR ligands involved, the roles of known components of the TIR signaling pathway, and how this pathway connects to FAK, Rac1 and RhoA. Preliminary results suggest that at least one structural alteration in the beta6 subunit, that mimics a mutant known to "activate" integrins in leukocytes and platelets, also enhances alphavbeta6-mediated TGFbeta-activation. We will therefore examine a series of beta6 mutants targeting each of the steps associated with conformation-dependent activation of integrins on non-adherant cells to determine the extent to which activation of alphavbeta6-mediated TGFbeta-activation mimics this process of "inside-out" conformational change of other integrins. Finally, we will utilize lines of mice already available to us expressing null mutations of components of the TIR signaling pathway, and mice we will generate predicted to either inducibly activate this pathway in alveolar epithelial cells or inducibly express maximally "activated" integrin, to examine the relevance of our in vitro findings to each of the in vivo roles we have identified for alphavbeta6. The proposed studies should provide important general insights into the mechanisms underlying affinity and avidity modulation of integrins in adherant cells. They should also identify important targets that could be used to design treatments to intervene in acute lung injury, pulmonary fibrosis and emphysema.
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1 |
2000 — 2003 |
Sheppard, Dean |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Role of a Neutrophil Integrin in Lung Inflammation @ University of California San Francisco
Neutrophils contribute to the tissue injury central to a number of common lung diseases, including acute lung injury, cystic fibrosis and chronic bronchitis. A key step in the development of neutrophil-mediated tissue injury is the recruitment of neutrophils to sites of extravascular injury. Although several of the critical receptors involved in the recruitment of neutrophils have been identified, considerable evidence suggests that unidentified receptors must participate in neutrophil recruitment, especially recruitment into the lung. Recently, we have identified the integrin alpha9beta1 on human neutrophils, and have found that this integrin together with its close structural relative, alpha4beta1, is critical for neutrophil migration across activated endothelial monolayers in vitro. The central issues addressed in this application are the mechanisms by which alpha9beta1 in the distinct steps of rolling, stable adhesion and endothelial transmigration, we will utilize blocking monoclonal antibodies and neutrophils derived from the bone marrow of alpha9 null chimeric mice. To determine the in vivo significance of alpha9beta1 on neutrophils we will examine neutrophil sequestration, extra-vascular emigration and neutrophil-mediated tissue injury in the lungs and peritoneal cavity in guinea pigs treated with alpha9beta1 blocking antibody and in chimeric mice with alpha9 null neutrophils. We will then utilize cell lines stably transfected with a variety of deletion and chimeric mutant versions of alpha9 to determine the role of specific sequences in the alpha9 and alpha4 cytoplasmic domains in adhesion, migration and endothelial transmigration. Finally we will utilize ch8imeric and mutant forms of alpha9 with defined functional properties to determine the roles of rapid spatial redistribution of integrins and cytoskeletal associations in integrin-mediated migration, both in leukocyte and non-leukocyte model systems. These studies should provide insight into key steps in neutrophil recruitment and could lead to the development of novel interventions for the treatment of diseases characterized by neutrophil-mediated tissue injury.
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1 |
2000 |
Sheppard, Dean |
U09Activity Code Description: To provide the chairman of an initial review group funds for operation of the review group. |
Scientific Review and Evaluation Grant @ U.S. Phs Public Advisory Groups |
0.907 |
2001 — 2003 |
Sheppard, Dean |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
In Vivo Function of Pulmonary Intergrins @ University of California San Francisco
DESCRIPTION (Adapted from the Applicant's): This is a competitive renewal application to study the mechanisms whereby the epithelial integrin avb6 regulates pulmonary inflammation and fibrosis as well as airway remodeling. The investigators have shown that a homozygous null mutation of the b6 subunit gene leads to an accumulation of activated macrophages and lymphocytes in the lungs and airways of affected mice and to induction of the macrophage metalloelastase (MME) gene, yet the mice are protected from bleomycin-induced pulmonary fibrosis. Having shown that avb6 binds to a specific sequence within the latency-associated protein (LAP) of TGFb1, the investigators propose to use the b6 subunit knockout mouse (b6-/-) model to study the in vivo role of avb6 in lung inflammation and fibrosis. There are 3 specific aims. The first aim is to determine the role of TGFb1 activation in the induction of lung inflammation and protection from pulmonary fibrosis in beta6-/- mice. For this, transgenic mice expressing full-length and mutant forms of the b6 subunit will be used to determine whether TGFb1 activation or other effects of avb6 can prevent lung inflammation or induce pulmonary fibrosis. Also, they will evaluate whether avb6 acts upstream or downstream of TGFb1 activation, by infecting wild type and b6-/- mice with adenoviruses expressing either active or latent TGFb1 or transgenes that are considered to induce pulmonary fibrosis by acting upstream of TGFb. The second aim is to determine the significance of MME induction in the beta-/- phenotype. For this purpose, they will examine macrophage phenotypic features of double knockout (MME-/- b6-/-) mice generated by cross breeding of MME-/- and b-/- mice. The third aim is to determine the role of avb6 induction in airway remodeling. This will be accomplished by cross breeding b6-/- mice with transgenic mice that develop sub-epithelial fibrosis due to overexpression of IL-6, IL-11 or IL-13.
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1 |
2001 — 2005 |
Sheppard, Dean |
P50Activity Code Description: To support any part of the full range of research and development from very basic to clinical; may involve ancillary supportive activities such as protracted patient care necessary to the primary research or R&D effort. The spectrum of activities comprises a multidisciplinary attack on a specific disease entity or biomedical problem area. These grants differ from program project grants in that they are usually developed in response to an announcement of the programmatic needs of an Institute or Division and subsequently receive continuous attention from its staff. Centers may also serve as regional or national resources for special research purposes. |
Interactions of Lymphocytes, Cytokines and Airway Cells @ University of California San Francisco
leukocyte activation /transformation; asthma; inflammation;
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1 |
2004 — 2008 |
Sheppard, Dean |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Integrin-Mediated Development of the Thoracic Duct @ University of California San Francisco
DESCRIPTION (provided by applicant): This competitive renewal application is to continue on-going studies of novel functions of the integrin, alpha9beta1. Based on analysis of alpha9 knockout mice, alpha9beta1 plays a critical role in development of the thoracic duct and other lymphatic vessels. One clue to the mechanism by which this integrin might contribute to lymphatic development comes from our identification of the lymphangiogenic growth factors, VEGFC and VEGFD as putative alpha9beta1 ligands. During the current funding period a unique mechanism by which alpha9beta1 enhances cell migration was also identified, and this effect was shown to be mediated by specific sequences in the alpha9 cytoplasmic domain. A single protein, the enzyme spermine/spermidine acetyltransferase, was found to bind to the alpha9 cytoplasmic domain and to specifically modulate alpha9-dependent enhancement of cell migration. This application proposes to evaluate each of these clues in more detail. The significance of binding of the extracellular domains of alpha9beta1 to VEGFC and D will be examined by assessing cell migration, proliferation, and early steps in integrin and growth factor receptor signaling in response to recombinant forms of each growth factor in mock- and alpha9-transfected cells. The importance of co-ligation of the canonical receptor for these growth factors, VEGFR3 will be assessed by performing all of these studies in the presence or absence of co-expression of VEGFR3. The alpha9 expressing cells critical for lymphatic development will be evaluated in mice homozygous for a conditional alpha9 null allele that will be used to inactivate this gene in specific cell types that might contribute to developing lymphatics. Stable cell lines co-expressing wild type and mutant forms of alpha9beta1 along with wild type or mutant forms of SSAT will be used to map the interaction sites in each protein and determine the importance of SSAT binding and enzymatic activity for alpha9beta1-mediated enhancement of cell migration. In vitro binding of recombinant versions of each protein, co-immunoprecipitation and double staining immunofluorescence will be used to determine whether this interaction is direct, occurs in living mammalian cells and results in co-localization to informative cellular compartments. Finally, the in vivo significance of alpha9-mediated migration and interactions with SSAT will be determined utilizing mice expressing knock-in mutations of the alpha9 cytoplasmic domain specifically designed to eliminate enhanced migration and/or SSAT binding. The proposed studies should provide important information about the role this widely expressed integrin plays in lymphatic development and cell migration and could ultimately lead to the design of novel interventions in diseases affected by each of these processes.
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1 |
2006 — 2009 |
Sheppard, Dean |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Regulation of Pulmonary Vascular Permeability by Integrin Alphavbeta5 @ University of California San Francisco
[unreadable] DESCRIPTION (provided by applicant): Acute lung injury is a common disorder with no known, effective, pharmacologic treatment. One of the central contributing factors to the development of acute lung injury is an increase in the permeability of the pulmonary vasculature. Mice homozygous for a null mutation in the integrin [unreadable]5 subunit gene, generated in the principal investigator's laboratory, are protected from pulmonary edema in a model of ventilator induced lung injury, and a blocking monoclonal antibody against the av[unreadable]5 integrin protects wild type mice in this same model and also protects rats from pulmonary edema induced by unilateral lung ischemia and reperfusion. Blockade or genetic ablation of the av[unreadable]5 integrin protects cultured endothelial cells from increases in monolayer permeability and from both the formation of rhoA-induced actin stress fibers and the phosphorylation of components of the endothelial adherens junction. In the current proposal, we will explore the molecular pathways linking the av[unreadable]5 integrin to regulated increases in vascular permeability in more detail. We will use dominant negative and constitutively active constructs, siRNA knockdown and endothelial cells from knockout mice to test the hypotheses that this integrin regulates vascular permeability through interaction with the small GTPases, Ga12 and Ga13 and the known G protein activator integrin associated protein (IAP). We will then examine the in vivo roles for each of these proteins by studying ventilator-induced lung injury in mice expressing global or conditional knockouts of each of these proteins. Because our preliminary data suggest that av[unreadable]5 is a central component of a multi-protein complex that contains Ga13, and many of the components of the rhoA dependent signaling pathway, we will examine which components of this complex depend on av[unreadable]5 for assembly and which regions of the [unreadable]5 subunit are required by reconstituting endothelial cells from [unreadable]5 knockout mice with wild type, truncated and chimeric forms of this integrin. Finally, to determine whether the pathways we are studying are broadly relevant to non-cardiogenic pulmonary edema, we will further examine the role of av[unreadable]5 and any other component found to be critical in three additional in vivo models - intratracheal bleomycin, intratracheal endotoxin and the combination of low dose endotoxin and moderate volume ventilation. Through the proposed studies we hope to determine whether av[unreadable]5 or other components of this pathway are attractive targets for intervention in acute lung injury. [unreadable] [unreadable] [unreadable]
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1 |
2006 — 2009 |
Sheppard, Dean |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Novel Leukocyte Integrins @ University of California San Francisco
DESCRIPTION (provided by applicant): Receptors for the Fc domain of IgG (FcgammaR) and for the third component of complement are critical for pathogen elimination in host defense against infection and in generation of an inflammatory response. In preliminary, we have found that specific functions of both FcgammaRs and the integrin complement receptor aMa2 are lost in dendritic cells and macrophages that lack glycan phosphatidylinositol (GPI) anchored proteins because of a targeted deletion of the enzyme, Pig-a, which is required for synthesis of the GPI anchor. GPI-anchored proteins are not uniformly distributed in the plasma membrane, but are found in lipid rafts, domains enriched in cholesterol and sphingolipids. In unactivated cells raft domains are small patches where, on the cytoplasmic face, some signaling molecules accumulate. Cell activation by a variety of means leads to lipid raft coalescence, which in turn may lead to activation of signaling cascades because of the increased concentration of signaling molecules in the enlarged raft domains. Ligated FcgammaRs and alphaMbeta2 can cluster together with aggregated lipid rafts, suggesting a potential role for GPI-anchored proteins in targeting these receptors to rafts. Raft association may be a critical step in transmembrane signaling by these receptors. The purpose of this application is to understand the molecular basis for the requirement for GPI anchored proteins in the function of FcgammaRs and aMa2, to determine the molecular mechanisms by which these receptors are targeted to lipid rafts, and to test the hypothesis that their targeting to the raft compartment allows communication of the ligated receptors with intracellular tyrosine kinase signaling cascades.
|
1 |
2007 |
Sheppard, Dean |
R13Activity Code Description: To support recipient sponsored and directed international, national or regional meetings, conferences and workshops. |
Conference On Fibronectin, Integrins &Related Molecules @ Gordon Research Conferences
[unreadable] DESCRIPTION (provided by applicant): Research on cell interactions with extracellular matrices has exploded during the past decade. Fibronectin has been the prototype extracellular matrix molecule for much of this investigation and has been a major focus on this Gordon Conference since 1982. The discovery of integrins in the mid-1980s as receptors for fibronectin and other constituents of extracellular matrices expanded the scope of the conference. The emerging understanding over the last five years that plasma membrane and cytosolic molecular partners regulate integrin function and specificity has further expanded the scope of the conference. Together, fibronectin, integrins, and their binding partners control many basic biological processes including cell adhesion, cell shape, organization of the cytoskeleton and the extracellular matrix, cell motility, regulation of morphogenesis and tissue interactions, immune cell trafficking, regulation of cell activation state and response to growth factors, induction and resolution of inflammation, hemostasis, and wound repair. Important new insights have been made in recent years, including: [unreadable] [unreadable] Determination of the first crystal structure of the extracellular domain of an integrin. This work provides a platform for detail molecular dissection of integrin function. [unreadable] [unreadable] Molecular mechanisms involved in regulation of the activation state of integrins, critical to their ability to bind ligand and transduce signals. [unreadable] [unreadable] The nature and regulation of signaling complexes assembled at adhesion sites, including cytoskeletal components, receptor and non-receptor tyrosine kinases, and other elements of signaling cascades. [unreadable] New families of ligands including growth factors, neuronal and immune adhesion molecules, and disintegrins, and new families of physically-associated molecules including growth factor and other receptors, tetraspanins, caveolin, and multiple cytosolic partners, that have broadened the context within which integrins function. [unreadable] [unreadable] A central role for integrin function and dysfunction in a variety of physiologic and pathologic states, which make these molecules focused therapeutic targets. [unreadable] [unreadable] The conference outlined in this application is directed at communicating these exciting new developments and stimulating discussion among participants from different disciplines. This cross-pollination is a most effective way of stimulating new waves of insight and information. [unreadable] [unreadable]
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0.915 |
2008 — 2015 |
Sheppard, Dean |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. U19Activity Code Description: To support a research program of multiple projects directed toward a specific major objective, basic theme or program goal, requiring a broadly based, multidisciplinary and often long-term approach. A cooperative agreement research program generally involves the organized efforts of large groups, members of which are conducting research projects designed to elucidate the various aspects of a specific objective. Substantial Federal programmatic staff involvement is intended to assist investigators during performance of the research activities, as defined in the terms and conditions of award. The investigators have primary authorities and responsibilities to define research objectives and approaches, and to plan, conduct, analyze, and publish results, interpretations and conclusions of their studies. Each research project is usually under the leadership of an established investigator in an area representing his/her special interest and competencies. Each project supported through this mechanism should contribute to or be directly related to the common theme of the total research effort. The award can provide support for certain basic shared resources, including clinical components, which facilitate the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence. |
Administration @ University of California, San Francisco
PROJECT SUMMARY (See instructions): The administrative core will be responsible for all administrative activities of the tPPG and will insure that the program operates according to its objectives and NIH guidelines. The core will be responsible for coordinating all fiscal management and personnel management. The core will also coordinate and schedule regular monthly research conferences, visits by external advisors, meetings of internal advisors and the annual tPPG retreat. The core will also be responsible for the timely renewal and modification of animal research, human research, bio-safety and readiation safety approvals, for insuring the timely training of all new and existing personnel in safe and ethical lab practices and for the coordination and preparation of the annual progress reports. The core will act as the intermediary for all components of the program and to the funding agency to effectively administer and integrate all components of the program and to provide clear, effective and succinct communication.
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1 |
2008 — 2012 |
Sheppard, Dean |
U19Activity Code Description: To support a research program of multiple projects directed toward a specific major objective, basic theme or program goal, requiring a broadly based, multidisciplinary and often long-term approach. A cooperative agreement research program generally involves the organized efforts of large groups, members of which are conducting research projects designed to elucidate the various aspects of a specific objective. Substantial Federal programmatic staff involvement is intended to assist investigators during performance of the research activities, as defined in the terms and conditions of award. The investigators have primary authorities and responsibilities to define research objectives and approaches, and to plan, conduct, analyze, and publish results, interpretations and conclusions of their studies. Each research project is usually under the leadership of an established investigator in an area representing his/her special interest and competencies. Each project supported through this mechanism should contribute to or be directly related to the common theme of the total research effort. The award can provide support for certain basic shared resources, including clinical components, which facilitate the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence. |
Mechanisms of Initiation and Persistence of Allergic Asthma @ University of California, San Francisco
DESCRIPTION (provided by applicant): This Asthma and Allergic Diseases Center Grant application is an effort to continue a long-standing fruitful collaboration among 4 UCSF faculty members with a long-standing interest in the mechanisms underlying allergic airway inflammation and asthma. The central goals of this application are to determine critical mechanisms underlying the initiation and persistence of allergic airway inflammation and airway hyperresponsiveness. The application includes two projects that will utilize murine models and a third that will examine the relevance of molecular targets and pathways identified in these models to asthma prevalence, severity and drug responsiveness in humans. The Center Principal Investigator and leader of Project 1, Dean Sheppard, has identified critical roles for activation of transforming growth factor ? (TGF??) by two different integrins (?v?6 and ?v?8) in airway hyperresponsiveness in a chronic model of allergic asthma and in modulation of cognate immune responses. This proposal will take advantage of a series of lines of genetically modified mice to directly examine the role of TGF?? in these effects, to determine the critical cells types responsible, and to identify the molecular mechanisms underlying these responses. Project 2 is based on recent data from Richard Locksley, the project leader, that chitin, a prominent structural component of fungi, parasites and crustaceans, activates macrophages and primes innate immune cells for initiation of type 2 immune responses. Work proposed in this project will utilize a series of novel reporter lines to examine the critical chitin-responsive cells and the mechanisms and relevance of this pathway for initiation of allergic inflammation. Project 3, co-led by Esteban Burchard and John Fahy will follow-up on preliminary observations about genetic associations between sequence variants in chitin-degrading enzymes and TGF?? and allergic sensitization and asthma to more deeply interrogate associations and gene-gene interactions for sequence variants in chitinases and multiple components of the TGF? activation and signaling pathways, including the integrin subunits examined in Project 1. This project will also evaluate the functional significance of associated genes using bronchoalveolar lavage samples and tissue from asthmatic patients and healthy control subjects. These projects will be supported by a Physiology and Tissue Analysis core that will provide extensive support for all 3 projects, and by a centralized Administrative core. Lay summary - This Center will evaluate the mechanisms underlying initiation and persistence of asthma. By identifying novel pathways and molecular targets and testing their relevance to asthma, asthma severity and drug response in humans, the work in the Center should provide clues for the development of new treatments for this common and often devastating disease. PROJECT 1: ?v Integrins in Cognate Immunity and Airway Hyperresponsiveness (SHEPPARD, D) PROJECT 1 DESCRIPTION (provided by applicant): Mice lacking the epithelial integrin, ?v?6, that we have shown activates latent TGF-?, are protected from the persistent airway hyperresponsiveness (AHR) that follows chronic allergen challenge. Surprisingly, this protection is not associated with any decrease in sub-epithelial airway fibrosis, a central TGF-? -dependent feature of this model. Mice with leukocyte specific knockout of the related integrin, ?v?8, which also activates TGF-? show evidence of enhanced adaptive immunity. In this proposal, we will determine whether these altered responses in ?v?6 subunit knockout mice are a direct consequence of loss of the ?v?6 integrin and/or of TGF-? activation from conducting airway epithelial cells using rescue mice expressing either the wild type integrin or active TGF-? in airway epithelial cells. We will evaluate the effects of ?v?6 antibodies and a TGF-? RII-lg chimera on these same endpoints to further confirm the importance of this pathway and evaluate the feasibility of targeting this pathway for therapeutic intervention. To determine the mechanisms by which loss of ?v?6 protects from induction of AHR, we will evaluate the relationship between airway responsiveness and expression of a small number of candidate genes identified as linked to this phenotype in preliminary experiments utilizing expression microarrays. We will also determine the cellular distribution of expression by immunostaining and/or in situ hybridization, and will evaluate functional significance using commercially available lines of mice expressing null mutations of specific candidates. Because the cytokine IL-13 is known to play a central role in induction of AHR in multiple models, and because two of the most promising candidates identified by microarrays, leukotrienes C4 synthase and interleukin-18 have been suggested to be upstream of IL-13 induction in the airways, we will also examine the cellular sources of IL-13 in chronically challenged wild type and ?v?6 knockout mice. Finally, we will determine how loss of leukocyte ?v?8 leads to enhancement of adaptive immunity and examine the relevance of this pathway to allergic airway inflammation and its consequences. Lay summary - This project will examine how a single growth factor, transforming growth factor ?, can either contribute to development of chronic asthma or inhibit allergic sensitization and its consequences, depending on where and how this growth factor is activated.
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1 |
2008 — 2012 |
Sheppard, Dean |
U19Activity Code Description: To support a research program of multiple projects directed toward a specific major objective, basic theme or program goal, requiring a broadly based, multidisciplinary and often long-term approach. A cooperative agreement research program generally involves the organized efforts of large groups, members of which are conducting research projects designed to elucidate the various aspects of a specific objective. Substantial Federal programmatic staff involvement is intended to assist investigators during performance of the research activities, as defined in the terms and conditions of award. The investigators have primary authorities and responsibilities to define research objectives and approaches, and to plan, conduct, analyze, and publish results, interpretations and conclusions of their studies. Each research project is usually under the leadership of an established investigator in an area representing his/her special interest and competencies. Each project supported through this mechanism should contribute to or be directly related to the common theme of the total research effort. The award can provide support for certain basic shared resources, including clinical components, which facilitate the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence. |
Av Integrina in Cognate Immunity and Airway Hyperresponsiveness @ University of California, San Francisco
Mice lacking the epithelial integrin, avpe, that we have shown activates latent TGFp, are protected from the persistent airway hyperresponsiveness (AHR) that follows chronic allergen challenge. Surprisingly, this protection is not associated with any decrease in sub-epithelial airway fibrosis, a central TGFp-dependent feature of this model. Mice Iwith leukocyte specific knockout of the related integrin, avp8, which also activates TGFp show evidence of enhanced adaptive immunity. In this proposal, we will determine whether these altered responses in P6 subunit knockout mice are a direct consequence of loss of the avp6 integrin and/or of TGFp activation from conducting airway epithelial cells using "rescue" mice expressing either the wild type integrin or active TGFp in airway epithelial cells. We will evaluate the effects of avpe antibodies and a TGFpRII-lg chimera on these same endpoints to further confirm the importance of this pathway and evaluate the feasibility of targeting this pathway for therapeutic intervention. To determine the mechanisms by which loss of avp6 protects from induction of AHR, we will eavluate the relationship between airway responsiveness and expression of a small number of candidate genes identified as linked to this phenotype in preliminary experiments utilizing expression microarrays. We will also determine the cellular distribution of expression by immunostaining and/or in situ hybridization, and will evaluate functional significance using commercially available lines of mice expressing null mutations of specific candidates. Because the cytokine IL-13 is known to play a central role in induction of AHR in multiple models, and because two of the most promising candidates identified by microarrays, leuotriene C4 synthase and interleukin-18 have been suggested to be upstream of IL-13 induction in the airways, we willl also examine the cellular sources of IL- 13 in chronically challenged wild type and P6 knockout mice. Finally, we will determine how loss of leukocyte avp8 leads to enhancement of adaptive immunity and examine the relevance of this pathway to allergic airway inflammation and its consequences. Lay summary- This project will examine how a single growth factor, transforming growth factor p, can either contribute to development of chronic asthma or inhibit allergic sensitization and its consequences, depending on where and how this growth factor is activated.
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1 |
2010 — 2011 |
Sheppard, Dean |
P41Activity Code Description: Undocumented code - click on the grant title for more information. |
Endothelial Alpha V Integrin Complexes Modulated Actin Cytoskeletal Organization @ University of California, San Francisco
This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Integrins are cell surface receptors that serve a number of basic cellular functions. One of these functions, which is relevant to the organ edema that occurs in inflammatory diseases like acute lung injury, is the regulation of passage of fluid and solute across the endothelium, the layer of cells lining blood vessels throughout the circulatory system. The Sheppard lab has recently demonstrated that alpha-v integrins modulate endothelial permeability by organizing the actin cytoskeleton. Specifically, alpha-v beta-3 integrin is necessary for rearrangement of actin into cortical distribution that increases vascular barrier strength in response to sphingosine 1-phosphate, a plasma borne barrier-enhancing molecule;on the other hand, alpha-v beta-5 is required for organization of the cytoskeleton into stress fibers that decrease barrier strength in response to thrombin, a blood borne mediator of increased blood vessel leakiness. Our objective is to identify the constituents of the protein complexes associated with these integrins. An ideal approach in this regard is to employ mass spectrometry, where biochemically isolated integrin complexes can be submitted to mass spectrometric sequencing for protein discovery. With this unbiased approach, the distinct actin binding and/or modulatory proteins associated with these two integrins may be identified and studied further to understand how they modulate actin organization. Armed with this powerful information, we may further dissect pathways mediating the opposing biological response of these two integrins and target specific points along their respective pathways as potential treatments for organ edema in the setting of sepsis and other acute inflammatory diseases.
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1 |
2011 — 2014 |
Sheppard, Dean |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Integrin-Mediated Regulation of Airway Smooth Muscle @ University of California, San Francisco
Project Summary/Abstract The ¿9¿1 integrin is highly expressed in airway smooth muscle. Mice we have generated lacking this integrin only in smooth muscle cells have marked in vivo airway hyperresponsiveness and lung slices from these mice have increased airway narrowing. We have previously shown that the ¿9 subunit cytoplasmic domain directly binds the enzyme spermine/spermidine acetyltransferase (SSAT), the rate limiting step in catabolism of higher order polyamines, and that this association and polyamine catabolism are important modulators of ¿9¿1 function. Pharmacologic stabilization of SSAT also augments ¿9¿1-dependent prevention of airway smooth muscle contraction. In the current application we will systematically evaluate the effects of ¿9¿1 on responses of airway smooth muscle to multiple contractile agonists and to isoproterenol-induced relaxation using 4 parallel experimental systems (in vivo AHR, tracheal ring contraction, airway narrowing in lung slices and shortening of airway smooth muscle cells). We will utilize a variety of mutant and chimeric constructs of ¿9 and SSAT, in vivo and in vitro studies with SSAT knockout mice, catalytically inactive SSAT mutants and genetic and pharmacologic inhibitors to thoroughly examine what contribution interaction of ¿9 with SSAT makes to this response. Force generatioin in smooth muscle depends on calcium-dependent actin-myosin cross-bridging and parallel actin polymerization, and we will systematically evaluate the effects of ¿9¿1 on each of these pathways. Because the two major cytosolic effects of higher order polyamines are prevention of potassium efflux through Kir channels and activation of the lipid kinase, PIP5K1¿, we will pay special attention to the roles of Kir channels and PIP5K1¿ in this process.The proposed studies will test the overall hypothesis that ligated ¿9¿1 normally serves as a brake on airway narowing by concentrating SSAT, catabolizing polyamines and thus inhibiting potassium efflux and/or PIP2 production, resulting in reduced calcium oscillations, decreased actin-myosin cross-bridging and/or impaired actin polymerization. Abnormalities in this pathway, either acquired or genetic, could contribute to diseases such as asthma that are characterized by enhanced airway narrowing.
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1 |
2012 — 2015 |
Sheppard, Dean |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Inhaled Inhibitors of Tgfb Activation by Integrins @ University of California, San Francisco
The avB6 integrin activates the fibrogenic cytokine, TGFB and is highly expressed in the epithelium of patients with Idiopathic Pulmonary Fibrosis. A blocking monoclonal antibody to this integrin effectively treats bleomycin and radiation-induced pulmonary fibrosis in mice, and a humanized version of this antibody is currently in phase 2 clinical trials. It would be desirably to target this pathway in patients, without the problems of antibody therapy or the risk of systemic side effects of blocking this integrin in unaffected organs. We and others have identified other av integrins that can activate TGFB, and these integrins might also play an important role in inducing fibrosis in the human lung. It would thus be desirable to be able to simultaneously target all of these integrins in patients with IPF. We have identified an absolute requirement for RhoA and its effector, Rho Kinase (ROCK) in avP6-mediated activation of TGFB. In the proposed studies we will first examine the effects of well-described small molecule inhibitors of ROCK and of of multiple av integrins that we have synthesized to inhibit pulmonary fibrosis in 4 different experiemental models in mice (induction of DNA damage and alveolar apoptosis by high dose bleomycin, induction of DNA damage combined with endoplasmic reticulum (ER) stress by low dose bleomycin and tunicamycin or by low dose bleomycin and expression of a surfactant protein C mutant, and induction of ER and lyosomal stress in a genetic model of the Hermansky Pudlak Syndrome). To minimize systemic toxicity, we will administer these drugs by aerosol and will then develop and test new small molecules based on these compounds that we have designed to increase hepatic metabolism and thus further reduce systemic toxicity. We will utilize BAL and blood samples from Core B to examine the potential of nriacrophage gene expression and SMAD2 phosphorylation, and secreted proteins identified as mechanistically informative biomarkers of this pathway in Core C to optimize a stategy for monitoring effective inhibition of this pathway in patients. Based on this work we expect to identify drugs to test in clinical trials in the second phase of this PPG and a greatly improved strategy for rapidly monitoring the effectiveness of these compounds in patients.
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1 |
2012 — 2021 |
Erle, David J Huang, Laurence Sheppard, Dean |
T32Activity Code Description: To enable institutions to make National Research Service Awards to individuals selected by them for predoctoral and postdoctoral research training in specified shortage areas. |
Multidisciplinary Training Program in Lung Disease @ University of California, San Francisco
ABSTRACT The proposed renewal is for a continuation of the present Multidisciplinary Training Program in Lung Disease (HL-07185), which provides training in basic and clinical sciences important to the respiratory system. The program draws on the basic and clinical research skills of the broad group of faculty at UCSF addressing questions directly relevant to lung biology and disease. The grant is to accommodate a total of thirteen postdoctoral trainees, including both MD's and PhD's. The main program is an actual research experience under the close supervision of a Preceptor and a review committee selected on the basis of the experimental approaches of the trainee. These approaches include disciplines applicable at the molecular, cellular, tissue, organ, whole animal, clinical, or population levels. In addition, considerable emphasis is placed on special educational opportunities that have been created to prepare individuals for a diverse range of careers in academic medicine. The educational program provides special courses, small group conferences and seminars in basic sciences, biostatistics, epidemiological study design, medical economics, and ethics of scientific conduct. Emphasis is placed on personal instruction specifically designed for individual trainees. New facilities and faculty are being added to further solidify the disciplines of modern biology, bioinformatics, and rigorous clinical research. Considerable emphasis has been placed on multidisciplinary interaction in research and training, and developing and implementing academic careers in areas of scientific need, especially in those previously underemphasized. Emerging areas of growth in our program include high- throughput genomics, microbial pathogenesis, immunology, stem cell biology, advanced microscopy, cancer biology, multi-institutional disease-specific clinical networks for research, health disparities and global lung health. An additional important area of emphasis is the recruitment and retention of underrepresented minorities and women in the program. The past and future success of our multidisciplinary program is predicated on a close collaboration among a widely diverse faculty, including clinical and bench-oriented scientists and translating this collaboration to a coordinated approach to research training and career development.
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1 |
2012 — 2016 |
Sheppard, Dean |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Targeting Epithelial Cells to Treat Pulmonary Fibrosis @ University of California, San Francisco
DESCRIPTION: This translational program project grant is designed to develop new effective therapies for idiopathic pulmonary fibrosis (IPF). The grant is entirely focused on developing new drugs that specifically target known pathways in alveolar epithelial cells that are critical fo the development of pulmonary fibrosis. The grant will also use a novel, mechanism-based approach to identify informative biomarkers from human cells and tissues that should provide early indicators of the effectiveness of each of the drugs we develop in subsequent early phase clinical trials in patients with IPF. The rationale for this grant is that IPF is a consequence of n-going epithelial stress and apoptosis, which leads to activation of extracellular latent TGFbeta by the alpha nu beta integrin on alveolar epithelial cells, which in turn induces progressive fibrosis at least in part through induction of TGFbeta signaling in epithelial cells. The proposal includes 3 projects which will each utilize multiple mouse models of pulmonary fibrosis to evaluate the efficacy of existing drug agents that target apoptosis induced by the unfolded protein response, integrin-mediated TGFbeta activation and TGFbeta signaling in epithelial cells, and to develop novel agents with improved potency and reduced systemic toxicity. By parallel analysis of the effects of these drug agents on human alveolar epithelail cells and human lung fragments, these projects will further asess the applicability of murine findings to humans. Each project will also take advantage of serial samples of blood and BAL cells and fluid from patients with IPF and healthy controls to characterize the utiltiy and reproducibility of putative biologically informative biomarkers. These projects will be supported by a human lung cell and tissue core, a longitudinal cohort core, a mediciinal chemistry core and a centralized administrative core. By performing extenisve pre-clinical analysis of the most promising candidate drugs, establishing their effectiveness in human lung, and developing read-outs of drug effectiveness that can be detected in blood, bronchoalveolar lavage fluid or alveolar macrophages, the work performed should lay the groundwork for clinical trials of the most promising candidates in a planned second phase of this program.
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1 |
2013 — 2017 |
Sheppard, Dean |
U19Activity Code Description: To support a research program of multiple projects directed toward a specific major objective, basic theme or program goal, requiring a broadly based, multidisciplinary and often long-term approach. A cooperative agreement research program generally involves the organized efforts of large groups, members of which are conducting research projects designed to elucidate the various aspects of a specific objective. Substantial Federal programmatic staff involvement is intended to assist investigators during performance of the research activities, as defined in the terms and conditions of award. The investigators have primary authorities and responsibilities to define research objectives and approaches, and to plan, conduct, analyze, and publish results, interpretations and conclusions of their studies. Each research project is usually under the leadership of an established investigator in an area representing his/her special interest and competencies. Each project supported through this mechanism should contribute to or be directly related to the common theme of the total research effort. The award can provide support for certain basic shared resources, including clinical components, which facilitate the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence. |
Il-13 and Il-17 Dynamics in the Asthmatic Airway @ University of California, San Francisco
This proposal seeks to renew an Asthma and Allergic Diseases Cooperative Research Center that has been focusing on the mechanisms of initiation and persistence of allergic asthma. During the course of these studies and earlier work done in our laboratories and multiple other labs, it has become clear that two cytokines, IL-13 and IL-17, make important contributions to the most critical functional endpoints in asthma (airway hyperresponsiveness and mucus metaplasia) through spatially and temporally restricted effects on airway epithelial cells and airway smooth muscle. In the current proposal, individual projects will focus on how each cytokine regulates airway epithelial cell differentiation and mucous metaplasia, how these cytokines work alone and in combination to regulate contractility of airway smooth muscle, and the dynamic behavior of the cells that generate these cytokines in the airway wall. Each of these projects will have a major focus on human asthma and will rely heavily on a Clinical Subject and Biospecimen Core that will provide basic characterization of airway physiology, evidence for IL-13 and IL-17 bioactivity, and samples of bronchoalveolar lavage fluid, epithelial brushings and airway biopsies for use in each of the 3 projects. These samples and clinical information will be obtained from subjects in 3 cohorts, ACE, RITA and SARP, that will enroll subjects for segmental allergen challenge, subjects with mild-to-moderate asthma undergoing a trial of inhaled corticosteroids and patients with severe asthma, respectively. Through the proposed studies we hope to gain new insights into the dynamic effects of IL-13 and IL-17 in asthma, to develop better tools to characterize subsets of patients with asthma and to improve the prospects for targeted therapy of this disease.
|
1 |
2013 — 2017 |
Sheppard, Dean |
U19Activity Code Description: To support a research program of multiple projects directed toward a specific major objective, basic theme or program goal, requiring a broadly based, multidisciplinary and often long-term approach. A cooperative agreement research program generally involves the organized efforts of large groups, members of which are conducting research projects designed to elucidate the various aspects of a specific objective. Substantial Federal programmatic staff involvement is intended to assist investigators during performance of the research activities, as defined in the terms and conditions of award. The investigators have primary authorities and responsibilities to define research objectives and approaches, and to plan, conduct, analyze, and publish results, interpretations and conclusions of their studies. Each research project is usually under the leadership of an established investigator in an area representing his/her special interest and competencies. Each project supported through this mechanism should contribute to or be directly related to the common theme of the total research effort. The award can provide support for certain basic shared resources, including clinical components, which facilitate the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence. |
Cytokine Regulation of Airway Smooth Muscle @ University of California, San Francisco
ROJECT SUMMARY (See instructions): We have found that mice that are unable to generate Thi7 cells and mice that are unable to respond to IL- 17 (due to global loss of IL-17 receptor C) are protected from airway hyperresponisveness in two models of allergic asthma. We also found that IL-17A directly increases the contractility of both murine and human airway smooth muscle through a pathway we characterized involving activation of NF-kB, increased expression of RhoA and enhancement of myosin phosphorylation. In addition, we found dramatic synergy between IL-17 and the Th2 cytokine IL-13 in enhancing airway smooth muscle contraction. In the proposed studies we will first determine the relative contributions of direct effects of IL-17 on airway smooth muscle or adjacent airway epithelium in vivo utilizing mice that specifically lack IL-17RC or the key IL-17 receptor signaling molecule, Act-1, only in smooth, only in lung epithelial cells or in both cell types, and by analyzing effects on contraction of human bronchi with or without the overlying epithelium. Because synergistic interactions between IL-17 and IL-13 might have special relevance to therapeutic strategies in asthma, we will also carefully disect the site(s) of signal amplification that underlie the synergistic effects of these cytokines on human airway smooth muscle, including smooth muscle cells cultured from patients with asthma. Finally, we will utilize the 3 cohorts of patients with asthma to be analyzed in the Clinical Subject and Biospecimen Core to determine the relationships between the signaling responses in airway smooth muscle cells, the number and location of IL-13 and IL-17 producing cells and airway hyperresponsiveness in patients with moderate or severe asthma and in responses to segmental allergen challenge. These studies should complement the studies proposed in Project 1 to study effects of the same cytokines on airway epithelial cells and the studies proposed in Project 3 to examine the dynamics of IL-13 and IL-17 production and signaling in asthmatic airways. Together, the proposed studies should provide important details about how these critical cytokines contribute to the initiation and persistence of allergic asthma and should identify novel steps for intervention.
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2014 — 2018 |
Degrado, William F (co-PI) [⬀] Sheppard, Dean |
UH2Activity Code Description: To support the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) UH3Activity Code Description: The UH3 award is to provide a second phase for the support for innovative exploratory and development research activities initiated under the UH2 mechanism. Although only UH2 awardees are generally eligible to apply for UH3 support, specific program initiatives may establish eligibility criteria under which applications could be accepted from applicants demonstrating progress equivalent to that expected under UH2. |
Treatment of Pulmonary Fibrosis With Inhibitors of Integrin Alphavbeta1. @ University of California, San Francisco
DESCRIPTION (provided by applicant): Pulmonary fibrosis is a currently untreatable condition with a high mortality rate. One central common step in the development and progression of pulmonary fibrosis is the differentiation and expansion of pathologic fibroblasts that are largely responsible for the excess production of collagen and other extracellular matrix components that characterize tissue fibrosis. Transforming growth factor beta (TGF?) is a critical driver of fibroblast differentiation and expansion. The applicants have identified a single integrin (?v?1) on the surface of fibroblasts that is responsible for fibroblast-mediated TGF? activation. They have taken advantage of extensive experience in developing integrin inhibitors to generate a small molecule that is the first potent and highly selective inhibitor of ?v?1 and have shown that this drug can inhibit bleomycin-induced pulmonary fibrosis when administered beginning 14 days after bleomycin, during the late fibrotic phase in this model. They now propose to chemically modify this lead compound to optimize its potency, bioavailability and tolerability, with the goal, in the first two years, of generating at least one lead drug that will be suitable fr oral or sub- cutaneous administration. Direct administration into the airways will be assessed, if necessary, as a back-up strategy. The applicants will also use labeled versions of lead compounds to assess the cell and tissue distribution of the target and develop assays for flow cytometry and potentially in vivo imaging. In the final 3 years of this two stage proposal the applicants will thoroughly evaluate the pharmacokinetics, stability, dose- response potency and toxicology of the most promising drug developed in the first two years, will scale up synthesis and generate GLP quality drug to perform 7 day and 28 day GLP- toxicity studies in rats and Beagle dogs to enable submission of an IND for first in man studies to the FDA. Because the results of this series of studies cannot be entirely predicted, the applicants will also continue a vigorous chemical modification, synthesis and evaluation pipeline to be sure that there are multiple additional promising candidates if the chosen lead compound fails at any step of the pre-clinical work-up. With this strategy there should be a high likelihood of generating an ?v?1-targeting drug suitable for clinical trials.
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2018 |
Collard, Harold R Sheppard, Dean |
R13Activity Code Description: To support recipient sponsored and directed international, national or regional meetings, conferences and workshops. |
20th Biennial International Conference On Lung and Airway Fibrosis (Iclaf) @ University of California, San Francisco
PROJECT SUMMARY The Organizing Committee of the International Colloquium on Lung and Airway Fibrosis (ICLAF) is requesting support for its 2018 meeting to be held October 14-18, 2018, at the Asilomar Conference Grounds in Pacific Grove, California. The primary objective of this grant application is to enable junior scientists and scientists from groups traditionally underrepresented in science to participate in ICLAF 2018 through financial assistance and targeted outreach. ICLAF is a biennial scientific meeting dedicated to the science of lung fibrosis. Now in its 20th iteration, ICLAF 2018 will bring together scientists from clinical, translational and basic backgrounds to discuss original research, identify critical gaps in knowledge, and develop collaborative relationships and future research opportunities. Every effort will be made to attract a diverse group of attendees, with particular attention to junior investigators and investigators who are traditionally underrepresented in science. We believe events like ICLAF 2018 are essential to catalyzing scientific progress and discovery. Asilomar is a unique conference venue ? known as the Monterey Peninsula's ?Refuge by the Sea?, it is a 107-acre state beach and conference center that provides conference attendees with a retreat-like atmosphere conducive to scientific discussion and innovation. Insuring appropriate representation of groups traditionally underrepresented in science is a central priority of ICLAF 2018 and the primary objective behind this grant application. The majority of the ICLAF 2018 Organizing Committee (4 of 7) and nearly half of the overall conference leadership (20 of 46) consists of women and members of groups traditionally underrepresented in science. ICLAF 2018 is also committed to involving junior scientists in the leadership and content of the conference, with two junior scientists included on the Organizing Committee. ICLAF 2018 will feature eight scientific sessions, with each session consisting of two 30-minute ?invited lectures? and two 15-minute original research presentations. It will also feature original research posters, a junior investigator forum for presentation of research by trainees, and small group discussions identifying future priorities and challenges for our scientific community. ICLAF 2018 will aim to publish its proceedings in a major academic journal to allow the larger lung fibrosis community to review and comment on our presentations and discussions.
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1 |
2019 — 2021 |
Papa, Feroz R (co-PI) [⬀] Sheppard, Dean |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Interventional Targeting of the Ire1alpha-Tgfbeta Signaling Loop in Pulmonary Fibrosis @ University of California, San Francisco
PROJECT SUMMARY/ABSTRACT In this MPI RO1 application, we show that high endoplasmic reticulum (ER) stress and dysregulated unfolded protein response (UPR) signaling in lungs precedes development of fibrosis in murine models of pulmonary fibrosis (PF)?similar changes have been reported in lungs of human patients with idiopathic pulmonary fibrosis (IPF). We find that the pathognomonic features of these changes are due to hyperactive signaling by IRE1?, an ER transmembrane protein containing bifunctional kinase/endoribonuclease (RNase) catalytic activities. We have found that novel, small molecule UPR kinase inhibitors of IRE1?, called KIRAs (an acronym for kinase-inhibiting RNase attenuators) reduce IRE1?'s destructive UPR signaling and show potent anti- fibrotic effects in ER-stressed lungs of mice. Thus, even as IRE1? emerges as a potential therapeutic target for treating human patients suffering from IPF, the underlying mechanistic basis for the cytoprotective, anti-fibrotic, efficacy of the KIRA compounds needs to be understood. Through this application, we propose to understand the basis of the KIRA-mediated salutary effects on reducing lung epithelial injury, dysregulation, and death, and also on reducing collagen overproduction by activated fibroblasts. The project is enabled by the complementary skill sets of two labs (Papa and Sheppard) that together have found the UPR is wired through a signaling loop that leads to classical TGF-?-induced, pro-fibrotic signaling in lungs. Thus, the mechanistic understanding to be gained from the successful completion of the proposed studies promises to reveal new nodes and targets for rational disease modification in idiopathic pulmonary fibrosis, a currently incurable disease.
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2020 |
Sheppard, Dean |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Fibroblast Heterogeneity in Pulmonary Fibrosis @ University of California, San Francisco
ABSTRACT Pulmonary fibrosis, characterized by accumulation of extracellular matrix (ECM) proteins that impair normal function, is a major cause of disability and death. Although considerable research has been done to identify multiple potential lineages of cells that can give rise to the fibroblasts responsible for ECM production, it has been widely assumed that these cells are a relatively homogeneous population of cells called myofibroblasts, characterized by high expression of a smooth muscle actin (aSMA). However, limited information about the molecular characteristics of these cells in vivo has limited our understanding of the basic biology underlying fibrosis and hampered the development of effective therapies. To address this important gap, we have used single cell RNA sequencing (scRNAseq) of collagen producing cells to identify multiple distinct cell types that produce collagen in the normal and fibrotic murine and human lung. Using proximity ligation in situ hybridization (PLISH) we identified subsets of collagen-producing cells with distinct molecular signatures that were concentrated within the walls of conducting airways (peribronchial), surrounding bronchovascular bundles (adventitial) and diffusely distributed in gas exchanging regions (alveolar). After treatment with bleomycin, a distinct new subset emerged that expressed high levels of collagens and other ECM proteins and was uniquely marked by expression of collagen triple helix repeat containing protein 1 (cthrc1). scRNAseq of dissociated cells from normal and fibrotic human lungs also identified a population of cells marked by CTHRC1-expression that expressed the highest levels of collagens and other ECM proteins and was only seen in lungs from patients with pulmonary fibrosis. In the studies proposed here, we will first determine the geographic and temporal distribution and lineage of cthrc1+ cells in single and repeated dose bleomycin models using PLISH, adoptive transfer and novel ERcre lines we are developing to track cells derived from peribronchial, adventitial and alveolar fibroblasts. Next, we will evaluate the functional roles of cthrc1+ cells using adoptive transfer into normal or bleomycin-treated mice, in vitro studies of an array of behaviors associated with pathologic fibroblasts, and through deletion of these cells by crossing a novel cthrc1-ERcre line we have generated to mice expressing lox-stop-lox dta in the Rosa locus. Finally, we will examine the functional significance of each of the major collagen-producing cell populations we have identified in normal lungs at baseline and in models of alveolar and airway fibrosis, using a similar ablation strategy or through deletion of genes previously shown to contribute to tissue fibrosis. From these studies we hope to gain novel insights into the roles each of these unique fibroblast subsets plays in lung homeostasis and disease.
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2020 |
Papa, Feroz R (co-PI) [⬀] Sheppard, Dean |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Caraballo Diversity Supplement 093019 @ University of California, San Francisco
PROJECT SUMMARY/ABSTRACT In this MPI RO1 application, we show that high endoplasmic reticulum (ER) stress and dysregulated unfolded protein response (UPR) signaling in lungs precedes development of fibrosis in murine models of pulmonary fibrosis (PF)?similar changes have been reported in lungs of human patients with idiopathic pulmonary fibrosis (IPF). We find that the pathognomonic features of these changes are due to hyperactive signaling by IRE1?, an ER transmembrane protein containing bifunctional kinase/endoribonuclease (RNase) catalytic activities. We have found that novel, small molecule UPR kinase inhibitors of IRE1?, called KIRAs (an acronym for kinase-inhibiting RNase attenuators) reduce IRE1?'s destructive UPR signaling and show potent anti- fibrotic effects in ER-stressed lungs of mice. Thus, even as IRE1? emerges as a potential therapeutic target for treating human patients suffering from IPF, the underlying mechanistic basis for the cytoprotective, anti-fibrotic, efficacy of the KIRA compounds needs to be understood. Through this application, we propose to understand the basis of the KIRA-mediated salutary effects on reducing lung epithelial injury, dysregulation, and death, and also on reducing collagen overproduction by activated fibroblasts. The project is enabled by the complementary skill sets of two labs (Papa and Sheppard) that together have found the UPR is wired through a signaling loop that leads to classical TGF-?-induced, pro-fibrotic signaling in lungs. Thus, the mechanistic understanding to be gained from the successful completion of the proposed studies promises to reveal new nodes and targets for rational disease modification in idiopathic pulmonary fibrosis, a currently incurable disease.
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