Research

 

1.- Thy-1 

Fig. 1. Thy-1 molecule and proposed soluble forms. Thy-1 is initially generated as a 161 AA pro form. The initial 19 AA signal peptide is removed, and the terminal 29 AA are replaced with a GPI anchor, generating the mature form, which is anchored to the outer leaflet of the cell membrane by the diacyl group of the GPI anchor. Post-translational modifications are not shown. Shed Thy-1 could be generated either by cleavage of the GPI anchor by GPLD1, or by undefined proteases acting at as-yet undetermined cleavage sites.

 

Over the past 15 years, our laboratory has studied the regulation of the fibroblast phenotype in the context of lung fibrogenesis. We have made significant contributions in defining the importance of fibroblast heterogeneity, cell-matrix interactions, and epigenetic mechanisms of phenotype regulation. Throughout, we have taken advantage of the phenotypic heterogeneity associated with differential expression of the thymocyte differentiation antigen-1 (Thy-1), a glycosylphosphatidylinositol (GPI)-linked cell surface glycoprotein (Fig. 1) expressed on numerous cell types, which regulates signals affecting cell adhesion, migration, differentiation, and survival. We have demonstrated that Thy-1 is a fibrosis-suppressor molecule which modulates critical aspects of the fibrogenic phenotype, including proliferation, cytokine and growth factor expression and responsiveness, migration, myofibroblastic differentiation, and cell survival. We have reported that Thy-1 inhibits lipid raft-associated signaling via the Src-family kinase (SFK) and focal adhesion kinase (FAK) pathways, promoting fibroblast adhesion and limiting migration. Recent work in our lab and others has demonstrated that Thy-1 interacts with av integrins and syndecan 4 at the cell surface, modulating cell-cell and cell-matrix interactions, and mechanical coupling to inhibit TGF-beta activation and myofibroblastic differentiation. We recently performed microarray analysis of transcriptional differences in sorted Thy-1(-) and Thy-1(+) mouse lung fibroblasts, confirming many of our previously published findings that place Thy-1 near the center of a molecular network regulating fibrogenesis (Fig.2). 

 

Fig. 2:Partial Thy-1 Interactome: Data from analysis of Agilent whole mouse oligo genome microarray analysis of Thy-1(-) vs Thy-1(+) lung fibroblasts (n=3 each). Data were imported into Ingenuity Pathway Analysis software, which generates networks based on published studies. Red indicates >2-fold higher expression in Thy-1+ cells, green in Thy-1- cells. Molecules found associated with Thy-1 in our prior studies are indicated in bold font.

Many of the molecules in this network (TGFb, CTGF, MMP7, MMP9, SERPINE1 [PAI-1], PTEN) are known to be critical in pulmonary fibrosis. An important implication from this analysis and our prior work is that Thy-1 is a broad modulator of the fibroblast phenotype, not simply a point along a single pathway. Thus, understanding the mechanisms by which Thy-1 alters fibroblast phenotype offers the possibility of broadly targeting myofibroblast differentiation and survival, which could be a significant therapeutic breakthrough for pulmonary fibrosis.

 

2.-Summary of Research Projects

Targeting the Apoptosis-Resistant Pulmonary Myofibroblast (R01 HL111169-01A1)

A final common pathway for the destructive remodeling which characterizes fibrosis is the apoptosis-resistant myofibroblast, which both creates and responds to an altered mediator and matrix microenvironment, thus perpetuating fibrogenesis. Halting the development of the myofibroblast phenotype, or reversing it once established, offers the best hope of successfully treating IPF. The effects of Thy-1 on the myofibroblast phenotype are broad, including: decreased expression of a number of muscle-specific proteins and myogenic transcription factors, inhibition of contractility, and promotion of apoptosis. Furthermore, some of the functions of Thy-1 can be recapitulated by the administration of soluble Thy-1, and preliminary data demonstrate that soluble Thy-1 reverses established fibrosis in a mouse model, indicating that Thy-1 itself can be used or modified for therapeutic benefit. The overall hypothesis of this project is that Thy-1-mediated signaling broadly suppresses myofibroblastic transformation and promotes apoptosis, restoring homeostatic function in lung fibroblasts. Improved understanding of the molecular mechanisms involved will uncover novel strategies for reversing the pathogenic myofibroblast phenotype in IPF and other fibrotic disorders.

The aims of this study are: Aim 1: To define the mechanisms by which Thy-1- inhibits myofibroblastic differentiation of lung fibroblasts; Aim 2: To define the mechanisms by which Thy-1 promotes myofibroblast apoptosis; and Aim 3: To harness Thy-1-modulated signaling to reverse the apoptosis-resistant myofibroblast phenotype in vivo. Significance: Because Thy-1 modulates multiple signaling pathways critical to fibrogenesis, capitalizing on its function is more attractive than targeting a single pathway, which has been shown to be an ineffective approach in IPF. Our study will define mechanisms by which Thy-1 suppresses profibrotic differentiation of lung fibroblasts, and translate that knowledge into innovative therapeutic interventions for fibrotic lung disease.

 

 

Regulation of Fibroblast Phenotype in Lung Fibrosis (2R01 HL082818-06A1)

In studying the molecular regulation of Thy-1, we have found that in response to stress, fibroblasts release membrane-originating extracellular vesicles (EV) containing membrane-bound Thy-1. In bronchoalveolar lavage fluid (BALF) from IPF patients, the level of membrane-associated Thy-1 correlates with numbers of fibroblastic foci on biopsy (Fig.3), suggesting that Thy-1+ EV may be useful biomarkers of disease activity. In other fields such as cancer and immunology, EV such as exosomes are increasingly appreciated as critical in cell-cell communication; they usually contain non-coding RNA and mRNA that are taken up by recipient cells and alter their phenotypes. Also, EV have been found to be excellent biomarkers in many diseases. Recently, supernatants from mesenchymal stem cells (MSC), which contain EV, have been shown to promote repair of neonatal lung injury, and our preliminary data indicate that a single infusion of MSC-EV can reverse fibrosis, whereas fibroblast EV potentiate fibrosis, indicating that EV transmit potent signaling programs modulating lung injury and repair.

Hypothesis: Extracellular vesicles (EV) from activated fibroblasts sustain and amplify, whereas MSC-derived EV inhibits, profibrotic cellular phenotypes in pulmonary fibrosis.

The aims of this study are: Aim 1: To define the molecular characteristics of EV released by mesenchymal cells in response to profibrotic stimuli; Aim 2. To define the role of EV in the phenotypic modification of lung cells; and Aim 3: To define the role of EV in fibrosis.

Impact: Defining the roles of EV in lung fibrosis, and the mechanisms by which they modulate cell phenotypes, will significantly impact fibrosis research by identifying novel molecular and cellular paradigms for pathogenesis and progression, bringing added precision to biomarker studies, and uncovering novel therapeutic targets. Modifying EV-mediated signaling, harnessing EV as therapeutics, or in the future, engineering of EV, may allow precise, targeted modulation of specific cellular phenotypes, a groundbreaking approach which transcends simple molecular targeting.

 

Fig. 3: EV from activated fibroblasts (left), can modulate the phenotype of surrounding fibroblasts and amplify fibroproliferation. MSC release EV which can promote resolution of fibrosis (right). It is likely that other cell types modulate remodeling and are themselves altered by EV.

 

 

MicroRNA and Early Disease in Cystic Fibrosis (Pediatrics Department Project Pilot Grants)

Although Cystic Fibrosis (CF) was first described in the 1930's, and the CF gene, the cystic fibrosis transmembrane conductance regulator (CFTR), was discovered in 1989, we still don't fully understand determinants of disease severity or life expectancy. Although prognosis has improved in the past two decades, there is still a precipitous decline in lung function starting in mid-adolescence which shortens life expectancy for most affected individuals. Even among patients with the same genetic mutation, disease course varies widely. It is thought that a combination of modifier genes and early life events shape the course of an individual's illness. MicroRNAs (miRNAs) have emerged as powerful and predictable regulators of patterns of gene expression with major effects on disease phenotypes. They are useful as biomarkers, clues to critical disease mechanisms, and therapeutic targets. The role of miRNAs in CF pathogenesis has not been explored. The purpose of this project is to define miRNA signatures in young children with CF which correlate with indicators of clinical severity and which may provide additional insight into CF pathogenesis and possibly lay the groundwork for novel therapy.

Hypothesis: MicroRNA profiling in younger (<12 yo) children with CF will uncover signatures associated with pathogenesis and disease severity.

Aim One: Compare miRNA profiles of pPhe508del -homozygous CF patients aged 6-12 by sequencing (mIR-seq) vs. age-matched controls

Aim Two: Compare miRNA profiles to measures of disease severity in childhood (positive sputum culture for mucoid Pseudomonas aeruginosa [PsA]; FEV1 < 80% predicted; hospitalized > 1/yr in past 2 years).

 

 

Targeting the Apoptosis-Resistant Pulmonary Myofibroblast

A final common pathway for the destructive remodeling which characterizes fibrosis is the apoptosis-resistant myofibroblast, which both creates and responds to an altered mediator and matrix microenvironment, thus perpetuating fibrogenesis. Halting the development of the myofibroblast phenotype, or reversing it once established, offers the best hope of successfully treating IPF. The effects of Thy-1 on the myofibroblast phenotype are broad, including: decreased expression of a number of muscle-specific proteins and myogenic transcription factors, inhibition of contractility, and promotion of apoptosis. Furthermore, some of the functions of Thy-1 can be recapitulated by the administration of soluble Thy-1, and preliminary data demonstrate that soluble Thy-1 reverses established fibrosis in a mouse model, indicating that Thy-1 itself can be used or modified for therapeutic benefit. The overall hypothesis of this project is that Thy-1-mediated signaling broadly suppresses myofibroblastic transformation and promotes apoptosis, restoring homeostatic function in lung fibroblasts. Improved understanding of the molecular mechanisms involved will uncover novel strategies for reversing the pathogenic myofibroblast phenotype in IPF and other fibrotic disorders.

Significance: Because Thy-1 modulates multiple signaling pathways critical to fibrogenesis, capitalizing on its function is more attractive than targeting a single pathway, which has been shown to be an ineffective approach in IPF. Our study will define mechanisms by which Thy-1 suppresses profibrotic differentiation of lung fibroblasts, and translate that knowledge into innovative therapeutic interventions for fibrotic lung disease.

(Supported by the NHLBI: R01 HL111169-01A1)

 

 

Targeted Therapeutics for Pulmonary Fibrosis

Idiopathic pulmonary fibrosis (IPF) is an incurable, fatal disease with increasing incidence and mortality. Despite recent coordinated attempts to rapidly translate biologically promising therapies into effective treatments, there have been no major clinical breakthroughs. It is likely that some therapies have failed because of poor penetration into the lung.

A staggering number of biological molecules have been implicated in pulmonary fibrosis. Our systems analysis of the literature indicates that Thy-1, mTOR and TGF-b1 occupy critical "nodes" regulating fibrogenesis in IPF. Our laboratory has defined the roles and mechanisms of Thy-1 as a molecule with broad fibrosis-suppressive effects. Lung fibroblasts normally express Thy-1 as a GPI-anchored protein on their cell surface, but its expression is lost during myofibroblastic differentiation. Thy-1 inhibits activation of TGF-b and myofibroblastic differentiation. We have found that forced expression of Thy-1, as well as exposure to soluble Thy-1, broadly affects the myofibroblast phenotype, including: decreased expression of muscle-specific proteins and myogenic transcription factors, inhibition of contractility, and promotion of apoptosis. Thus the potential use of Thy-1 as an antifibrotic agent needs to be tested in vivo. We are working with multiple collaborative partners to specifically target Thy-1 and other therapeutic molecules directly to sites of active fibrosis in the lung. The hypothesis is that lung-specific delivery of anti-fibrogenic therapies is feasible and will result in enhancement of their therapeutic efficacy.

Significance: These projects represent bold, innovative, breakthrough approaches to therapy in IPF. Efficacy and toxicity are both addressed by delivery of therapeutic agents directly and specifically to the pulmonary interstitium. Additionally, a framework infrastructure will be created and refined for testing additional agents and combinations in the future.

 

 

Epigenomic Alterations in Lung Alveolar Development and Remodeling

Bronchopulmonary dysplasia (BPD), a major cause of morbidity and mortality in premature infants, is characterized by impaired alveolar septation and varying degrees of lung fibrosis and vascular remodeling, which is mimicked in animal models of hyperoxia or hypoxia exposure. Idiopathic Pulmonary Fibrosis (IPF) is a chronic, progressive, and usually lethal pulmonary disorder of uncertain etiology in adulthood, with aberrant activation of pathways critical in lung development and response to hypoxia. The critical gene regulatory pathways involved in lung development are largely undetermined, but likely depend upon integration of numerous signals from multiple cross-talking pathways (e.g. Wnt, TGF-β, PTEN, Hedgehog, Retinoid pathways) affecting gene expression in a coordinated manner, leading to phenotypic reprogramming of lung parenchymal cells.

We and our collaborators have previously identified mechanisms underlying abnormal alveolarization, epigenetic alterations regulating phenotypic reprogramming within fibroblastic foci of IPF, and altered developmental gene expression profiles in IPF. Our preliminary analyses using global approaches have indicated that the TGF-β and IGF-1 pathways are associated with IPF as well as with developmental processes that may be relevant to BPD. Based on our preliminary results we hypothesize that the gene regulatory networks supporting stem cell and epithelial cell differentiation are inhibited and networks supporting mesenchymal cell proliferation are accentuated in human BPD or IPF, leading to impaired alveolar septation in BPD and a profibrogenic state in IPF.. Our approach is to determine the key miRNA, mRNA, and epigenetic regulatory controls during normal alveolar development in human and mouse lung using multiple state-of-the-art techniques and analytic methods, evaluate alterations in these controls during BPD and IPF, and then validate these alterations in mouse models. The goal is to define targets for novel interventions, such as epigenetic therapies or antagomirs to "silence" miRNA.

Significance: The computational models developed through these studies will identify regulatory "control points" in alveolarization, and molecular elements that predict disease severity, susceptibility, and therapeutic response. These experiments will also identify alterations in lung development pathways in reproducible animal models, and their correlation with those in lung disease, and permit the development and evaluation of novel therapeutic strategies.

 

 

Extracellular Vesicles Alter Cell Phenotype in Pulmonary Fibrosis

Our lab has found that in response to stress, lung fibroblasts release membrane-originating extracellular vesicles (EV) containing membrane-bound Thy-1. In bronchial lavage fluid (BALF) from IPF patients, the level of membrane-associated Thy-1 correlates with numbers of fibroblastic foci on biopsy, suggesting that Thy-1+ EV may be useful biomarkers of disease activity. In other fields such as cancer and immunology, EV such as exosomes are increasingly appreciated as critical to cell-cell communication; they often contain non-coding RNA and mRNA that be taken up by recipient cells and alter their phenotypes. Also, EV in urine and other body fluids have been found to be excellent biomarkers in many diseases. We therefore hypothesize that EV released from lung fibroblasts in IPF interact with alveolar epithelial cells to promote a profibrotic phenotype, and that analysis of EV in BALF of IPF patients will represent a novel method to monitor disease activity.

Significance: By accomplishing these aims, the proposed studies will define a novel pathway regulating the cellular phenotypic alterations characteristic of IPF, uncover new therapeutic targets and approaches, and possibly provide novel biomarkers for measuring disease activity or response to treatment.

(Supported by the Pulmonary Fibrosis Foundation)

 

 

MicroRNA and Early Disease in Cystic Fibrosis

Although Cystic Fibrosis (CF) was first described in the 1930's, and the CF gene, the cystic fibrosis transmembrane conductance regulator (CFTR), was discovered in 1989, we still don't fully understand determinants of disease severity or life expectancy. Although prognosis has improved in the past two decades, there is still a precipitous decline in lung function starting in mid-adolescence which shortens life expectancy for most affected individuals. Even among patients with the same genetic mutation, disease course varies widely. It is thought that a combination of modifier genes and early life events shape the course of an individual's illness. MicroRNAs (miRNAs) have emerged as powerful and predictable regulators of patterns of gene expression with major effects on disease phenotypes. They are useful as biomarkers, clues to critical disease mechanisms, and therapeutic targets. The role of miRNAs in CF pathogenesis has not been explored. Hypothesis: MicroRNA profiling in younger (<12 yo) children with CF will uncover signatures associated with pathogenesis and disease severity.

Significance: This project will define miRNA signatures in young children with CF which may correlate with indicators of clinical severity and which may provide additional insight into CF pathogenesis and possibly lay the groundwork for novel therapy.