Current research projects and grant support Projects
- Interstitial lung diseases (ILD) include a devastating group of fibrotic parenchymal diseases with high morbidity and mortality, for which there are limited effective therapies. Pulmonary fibrosis (PF) develops in ILD patients in response to alveolar epithelial injury and the subsequent activation and accumulation of pro-fibrotic fibroblasts, which deposit collagen and other extracellular matrix (ECM) components. The accumulation and persistence of pro-fibrotic fibroblasts and the deposition of ECM leads to progressive fibrosis resulting in declining gas exchange in the alveolar-capillary units. The inhalation of silicate dust, cigarette smoke and toxic chemicals are known risk factors for developing fibrotic lung disease and these exposures have disproportionally affected US veteran’s, coal miners and construction workers. PF is generally believed to be irreversible. Consequently, it becomes increasingly important to identify molecular pathways that are targetable for therapeutic intervention. In this project we are seeking to address this unmet need by investigating the central hypothesis that the development of pro-fibrotic fibroblast resistance to apoptosis contributes to progressive fibrotic disease. Furthermore, we propose that expression of the anti-apoptotic gene Bcl-2 plays a central role in mediating the persistence of pro-fibrotic fibroblasts. We are testing this central hypothesis by 1) in vivo ablation of pro-fibrotic lung fibroblasts to induce the resolution of persistent pulmonary fibrosis initiated by the intratracheal instillation of silica particles; 2) using a genetic approach to determine if conditional deletion of the anti-apoptotic gene Bcl-2 in pro-fibrotic fibroblasts leads to the apoptosis of fibrotic lung fibroblasts and the resolution of persistent fibrosis in a model of silica-induced pulmonary fibrosis; and 3) by treating mice with a small molecule inhibitor to reduce Bcl-2 activity, prevent fibroblast survival and promote the resolution of persistent fibrosis in vivo. Mice are followed using micro-CT imaging to monitor disease development, progression and resolution. The proposed studies will provide new understanding about the targeting of pro-fibrotic fibroblasts for death and how this may aid in the resolution of fibrosis. Furthermore, the outcome of this work should significantly impact our understanding of the mechanisms that control the resolution of fibrosis and its persistence in other organs and tissues.
- A key feature of interstitial lung diseases (ILDs), including idiopathic pulmonary fibrosis (IPF), is the excessive deposition of extracellular matrix (ECM) and scar tissue. Fibroblasts persist in fibrotic lungs and continue to lay down matrix, contributing to a progressive and persistent phenotype in patients. Matrix metalloproteinases (MMPs) are enzymes that cleave and break down ECM during wound repair. However, in pulmonary fibrosis, many MMPs, including MMP-9, have increased expression which has resulted in them being classified as pro-fibrotic. However, we interpret this published data as an unsuccessful attempt to activate an anti-fibrotic pathway. Our preliminary data in MMP-9 deficient mice suggest that MMP-9 is necessary for fibrosis resolution. We therefore asked if the upregulation of pro-MMP-9 is: 1) an attempt by the lungs to initiate repair through matrix degradation, and 2) if MMP-9 activation is inhibited in IPF. Plasmin is a major activator of MMP-9, but its upstream activator urokinase plasminogen activating enzyme (uPA) is inhibited in IPF, reducing the efficacy of this pathway. In addition, beneficial pro-inflammatory cytokine singling through TNF-a is dampened in the TGF-b rich pro-fibrotic environment, further preventing the upregulation of uPA or pro-MMP-9. Due to the poor quality and significantly reduced life expectancy associated with ILDs, it is becoming increasingly important to identify molecular pathways that are targetable for therapeutic intervention. We are seeking address this unmet need by investigating the central hypothesis that MMP-9 activation by plasmin is necessary for fibrosis resolution and that this pathway can be induced through beneficial TNF-a signaling. We are testing this 1) using genetic approaches to determine if conditional deletion of MMP-9 in fibroblasts is sufficient to prevent fibrosis resolution in a spontaneously resolving fibrosis model and if exogenous TNF-a is sufficient to activate the uPA/plasminogen/MMP-9 pathway, inducing resolution in a non-resolving fibrosis model; 2) through the generation of a non-cleavable MMP-9 mutant and through pharmacological inhibition of MMP-9 in vivo; and 3 by treating mice with persistent fibrosis with recombinant urokinase to induce plasmin and MMP-9 activation as a mechanism of fibrosis resolution. The proposed studies will provide a novel understanding about how activating an anti-fibrotic pathway (uPA/plasminogen/MMP-9) may contribute to the resolution of fibrosis. Furthermore, the outcomes of these pre-clinical, therapeutic studies will significantly impact our understanding of the mechanisms that control fibrosis resolution.
- Interstitial lung diseases (ILD) comprise a group of devastating and debilitating disorders with high morbidity and limited options for therapy. Idiopathic pulmonary fibrosis (IPF) is a restrictive, interstitial lung disease of unknown etiology in which progressive fibrosis of the alveolar-capillary units leads to respiratory failure and death. Pulmonary fibrosis develops in ILD patients due to repetitive injury to the alveolar epithelium and the ensuing activation of pro-fibrotic fibroblasts, which deposit collagen and extracellular matrix (ECM) components, leading to a continuous cycle of pro-fibrotic fibroblast activation, pro-fibrotic signaling, destruction of the airways, and tissue scarring. The pathways involved in the initiation of fibrosis are not well understood. Further, the interplay between macrophages and fibroblasts as it contributes to the fibrotic process is not well defined. Understanding the molecular mechanisms and signaling cascades that lead to the initiation and progression of fibrosis is essential for the identification of new therapeutic targets for IPF. The pulmonary epithelium, specifically AT2 cells, play an important role in the de novo synthesis of lipids in pulmonary surfactant that is required for normal lung function. Perturbations in the amount and/or type of lipid within surfactant are associated with the development of many chronic lung diseases, including pulmonary fibrosis. Surfactant lipids are regulated by a tight balance between the synthesis and clearance of lipids. This lipid monolayer allows for the surface-tension reducing properties of surfactant and for efficient gas exchange. Surfactant lipid clearance is mediated equally by the uptake and recycling of surfactant lipids by AT2 cells and the uptake and catabolism by alveolar macrophages. Given such a crucial role for lipids in pulmonary function it is not surprising that lipid trafficking and homeostasis are important for, and affect, multiple lung cell types. Cholesterol is absolutely essential for maintaining the biophysical properties of surfactant. Indeed, disruptions in genes that regulate cholesterol balance result in lung pathologies. We have hypothesized that dysregulated macrophage lipid handling results in the persistent secretion of specific inflammatory mediators that drive fibrosis. The overall goal of this application is to identify the lipid mediators and downstream signaling cascades that lead to the initiation, development, and resolution of fibrosis. Completion of these studies will further our understanding of the role of specific lipids in the pathogenesis.
- This project is part of a collaborative P01. The overall goal our project is to understand how increased misexpression of MUC5B in distal airways leads to honeycomb cyst formation, fibroblast accumulation, pro-fibrotic programming and ultimately fibrosis in response to distal airway and alveolar epithelial injury. Through a combination of in vivo studies in mouse models and in vitro studies with human lung epithelial cells and fibroblasts, we seek to fill this knowledge gap by testing the hypothesis that MUC5B misexpression and persistent ER stress in distal airway epithelial cells creates susceptibility to apoptosis in both alveolar epithelial cells and distal airway epithelial cells leading to fibrosis and honeycomb cyst formation, respectively. We are addressing mechanistic questions about: 1) how MUC5B misexpression by distal airway epithelial cells creates vulnerability to apoptosis induction leading to fibroblast accumulation, pro-fibrotic programming, fibrosis and honeycomb cyst formation, 2) the relative roles of alveolar and distal airway epithelial cell apoptosis in the development of fibrosis and honeycomb cysts, and 3) from a remedial perspective, if prevention of epithelial cell apoptosis mitigates the development of pulmonary fibrosis and honeycomb cyst formation. We will test these questions using an epithelial cell ablation strategy in the context of Muc5b misexpression and during prevention of alveolar and distal airway epithelial cell apoptosis even in the context of increased distal airway Muc5b misexpression. Completion of the proposed studies will innovate understanding of these understudied questions and provide novel insights into the unresolved question of how MUC5B contributes to the development of interstitial fibrosis and honeycomb cysts in the vulnerable lung.