Future Research Directions in Idiopathic Pulmonary
NHLBI Workshop Summary
Summary of a National Heart, Lung, and Blood
Institute Working Group
Published in Am J Respir Crit Care Med Vol 166. pp
236-246, 2002 Internet address: www.atsjournals.org
Ronald G. Crystal, Peter B. Bitterman, Brooke
Mossman, Marvin I. Schwarz, Dean Sheppard, Laura Almasy, Harold A.
Chapman, Scott L. Friedman, Talmadge E. King Jr., Leslie A. Leinwand, Lance
Liotta, George R. Martin, David A. Schwartz, Gregory S. Schultz, Carston
R. Wagner, and Robert A. Musson
Division of Pulmonary and Critical Care Medicine,
Weill Medical College of Cornell University, New York, New York; Department of
Medicine, University of Minnesota Medical School, Minneapolis, Minnesota;
Department of Pathology, University of Vermont, Burlington, Vermont; Pulmonary
Sciences and Critical Care Medicine, University of Colorado Health Sciences
Center, Denver, Colorado; Department of Medicine, University of California, San
Francisco; Department of Genetics, Southwest Foundation for Biomedical
Research, San Antonio, Texas; Pulmonary and Critical Care Division, University
of California at San Francisco, San Francisco, California; Division of Liver
Diseases, Mount Sinai School of Medicine, New York, New York; Division of
Pulmonary and Critical Care Medicine, Department of Medicine, San Francisco
General Hospital, University of California at San Francisco, San Francisco,
California; Department of Molecular, Cellular, and Developmental Biology,
University of Colorado, Boulder, Colorado; Cell and Cancer Biology Branch,
Center for Cancer Research, National Cancer Institute, Bethesda, Maryland;
FibroGen Inc., San Francisco, California; Department of Medicine, Duke
University, Durham, North Carolina; Department of Obstetrics and Gynecology,
University of Florida, Gainesville, Florida; Department of Medicinal Chemistry,
University of Minnesota, Minneapolis, Minnesota; and Division of Lung Diseases,
National Heart, Lung, and Blood Institute, Bethesda, Maryland
Idiopathic pulmonary fibrosis
(IPF) is an insidious inflammatory fibroproliferative disease whose
cause and course before diagnosis are unknown, and for which existing
treatments are of limited benefit. The National Heart, Lung, and Blood
Institute convened a working group to develop specific recommendations
for future IPF research. Inflammatory and immune processes are involved
in IPF pathogenesis, and current therapeutic strategies are aimed at
suppressing the inflammation. Recent data suggest that the
molecular processes underlying the fibrogenesis may provide new
opportunities for therapeutic intervention. Specific areas of future
research recommended by the working group include studies to
elucidate the etiology of IPF, to develop novel diagnostic
techniques and molecular diagnostics, to establish a program for
identification of molecular targets for IPF treatment and identification
and generation of agonists or antagonists that inhibit
fibrogenesis, to foster investigations that couple the use of new
technologies (e.g., laser capture microdissection, microarrays, and mass
spectroscopic analysis of proteins) with data from the human
genome project, to establish a national consortium of Clinical
Centers of Excellence to conduct coordinated clinical and laboratory
studies of well-characterized patients and patient-derived
materials, and to stimulate research to develop animal models of
persistent and progressive pulmonary fibrosis for evaluation of new
Keywords: lung diseases,
interstitial; pulmonary fibrosis; National Institutes of Health (United States)
Idiopathic pulmonary fibrosis (IPF)
is a chronic diffuse interstitial lung disease of unknown cause, characterized
pathologically by inflammation and fibrosis of the lung parenchyma (1). The
disease is limited to the lung. Epidemiology data on IPF are scant and
variable. The data suggest that it is among the most common chronic
interstitial lung diseases, accounting for a majority of new interstitial lung
disease cases. The incidence has been estimated at 10.7 cases per 100,000 per
year for males and 7.4 cases per 100,000 per year for females (2). The
prevalence of IPF has been estimated at 29 of 100,000 for males and 27 of
100,000 for females. In the United States, IPF has been reported as an
infrequent cause of death, but it is likely that death certificates underreport
the diagnosis of IPF (3). IPF is estimated to have a 50 to 70% mortality at 5
years after the diagnosis (1, 4-6). Not only is the mortality associated with
IPF likely to be grossly underreported, but also the considerable morbidity
from this chronic disease is not defined by epidemiologic data.
In the past, the primary focus of
research in IPF has been on the inflammatory component of the disease. Much has
been learned about the role for various inflammatory cells and mediators in the
injury to the alveoli that characterizes IPF and the contribution of the
inflammatory process to the fibrosis of the lung parenchyma. More recently, the
role of fibrogenesis per se has emerged as an important component of the
pathogenesis of the disease.
Because of the considerations
outlined previously here and the paucity of effective treatment for most
patients with IPF, the National Heart, Lung, and Blood Institute (NHLBI)
convened a working group to discuss potential directions for future research.
The working group was charged with evaluating the current state of knowledge of
IPF, identifying critical gaps in our knowledge and understanding, recognizing
the most promising opportunities, and developing specific recommendations to be
used by the NHLBI in planning its promotion of future IPF research. This
article summarizes the meeting of that working group, which was held on June
26-27, 2001, in Bethesda, Maryland. Specific recommendations for future
research directions in IPF follow a description of the important issues that
were raised at the meeting.
OVERVIEW OF IPF
IPF is a progressive, and often
fatal, inflammatory, fibroproliferative lung disease. Despite significant
advances, IPF remains
a conundrum to clinicians in terms
of etiologic factors, diagnosis, and treatment. The typically late presentation
of IPF and the nonspecificity of its clinical features in comparison to a
number of other pulmonary diseases hamper the development of effective
preventive and therapeutic strategies.
Surgical lung biopsy specimens
from individuals with IPF typically have the underlying histologic appearance
of usual interstitial pneumonia (1). Usual interstitial pneumonia is the most
common of several idiopathic interstitial pneumonias (4, 7). The histologic
picture of usual interstitial pneumonia is distinct from the other idiopathic
interstitial pneumonias, such as nonspecific interstitial pneumonia, acute
interstitial pneumonia, and desquamative interstitial pneumonia (8, 9).
The key difference is the temporal heterogeneity of the specific histologic
features of usual interstitial pneumonia so that there are areas of
interstitial collagen accumulation (typically subpleural) in the same specimen
in which there is end-stage honeycomb lung and early active areas of
fibroblastic proliferation, known as fibroblastic foci. Other fibrosis-related
features sometimes observed in IPF include bands of smooth muscle and bone
formation. In contrast, in acute interstitial pneumonia and nonspecific
interstitial pneumonia, the histologic features tend to be uniform (temporally
homogenous). The fibroblastic foci are absent or inconspicuous, and the fibrous
connective tissue appears approximately the same age (1, 8).
Depending on the time in the
clinical course at which the biopsy is done, there are variable, patchy amounts
of inflammation in the lung biopsy specimens of individuals with IPF.
Assessment of biopsies, as well as cells recovered by bronchoalveolar lavage,
demonstrates variable increased numbers of alveolar macrophages, neutrophils,
lymphocytes, and eosinophils, many of which are activated and release a variety
of mediators relevant to the pathogenesis of IPF.
Fibroblastic foci are located
within the interstitial space directly beneath the alveolar epithelium and are
often observed at the interphase between collagenized and normal-appearing lung
(10, 11). It is hypothesized that the fibroblastic foci represent the leading
edge of the progressive fibrotic process. These fibroblastic foci are
characterized by fibroblast/myofibroblast migration and proliferation to sites
of injury, decreased myofibroblast apoptosis, and increased activity of and
response to fibrogenic cytokines such as transforming growth factor-ß1
(TGF-ß1), tumor necrosis factor-alpha, platelet-derived growth factor,
and insulin-like growth factor (12, 13). At these sites, there is an
inappropriate re-epithelialization and impaired remodeling of the
extracellular matrix (ECM). It is hypothesized that IPF is associated with
ongoing diffuse microscopic alveolar epithelial injury and abnormal wound
healing. Given the nonresponsiveness of many cases of IPF to current
antiinflammatory approaches to treatment, and in view of the evidence that the
myofibroblasts within fibroblastic foci proliferate, interact with the
epithelial repair process, produce collagen, and have contractile properties,
they represent potential new therapeutic targets for these patients (13-16).
The potential importance of these
fibroblastic foci in the pathogenesis of lung fibrosis is further supported by
contrasting them with the intra-airway fibrotic lesions (Masson's bodies)
found in bronchiolitis obliterans organizing pneumonia (13). The Masson's
bodies are similar in structure (myofibroblasts and ECM components) to the
fibroblastic foci of IPF (10, 12). However, the Masson's bodies resolve
spontaneously or respond to antiinflammatory therapy, whereas the fibroblastic
foci do not (17). The mechanisms that account for these differences are
unclear. Evidence suggests that in the fibroblastic foci of IPF, there is less
inflammation (12, 18), an absence of apoptosis that allows for continued
proliferation of myofibroblasts (19, 20), an imbalance in matrix
metalloproteinases and tissue inhibitors of matrix metalloproteinases (12, 18,
21, 22), delayed or absent re-epithelialization (14, 15), as well as diminished
vascularity (19, 20) when compared with bronchiolitis obliterans organizing
Over the past three decades, there
has been considerable insight into the fundamental biologic processes involved
in fibroproliferation. Critical ligands, receptors and signaling systems
regulating mesenchymal cell motility, proliferation, viability, connective
tissue metabolism, and differentiated state have been identified and assessed.
Many may be potential targets for therapeutic intervention. Unfortunately,
there is limited information regarding the differences between pathologic
fibroproliferation, in which critical structures are replaced by scar tissue
leading to organ dysfunction, and the physiologic fibroproliferation that is
thought to be essential for proper healing of both visceral and integumentary
wounds. These gaps in knowledge identify important research questions regarding
the etiology and pathogenesis of IPF:
Does epithelial injury
precede fibrosis, and is it necessary to trigger fibrogenesis?
Why does the epithelium die,
and why does it not regenerate normally?
What prevents normal repair
from going to completion?
What distinguishes affected
alveoli from normal alveoli?
What processes cause normal
fibroblasts/myofibroblasts to become abnormal or hyperproliferative?
How do fibroblastic foci
change temporally, and what factors regulate these changes?
Because tissue fibrosis is a
prominent feature of progressive diseases in a number of other organs,
important clues to the pathogenesis of pulmonary fibrosis are likely to come
from analysis of shared and divergent features of tissue fibrosis in other
In virtually every tissue,
myofibroblasts are widely accepted to play a significant role in fibrosis (23,
24). Previous studies have characterized these cell types as having a phenotype
intermediate between muscle and nonmuscle cells (24). Myofibroblasts are
contractile, and they express members of the myogenic regulatory family (MyoD,
myf5, and myogenin) and muscle structural proteins (25). A key marker for
myofibroblasts is alpha-smooth muscle actin (26). Cell culture models of
myofibroblasts, including hepatic stellate cells, kidney mesangial cells,
cardiac valvular interstitial cells, and pulmonary myofibroblasts, all express
one or more of these myogenic regulators (L. A. Leinwand, personal
communication). Consistent with the concept that these myogenic regulators are
functional are experiments demonstrating that a reporter construct with the
skeletal myosin light chain promoter is as active in liver and kidney
myofibroblasts as it is in differentiated skeletal muscle (25).
One intriguing feature of
myofibroblasts is their refractoriness to the cell cycle withdrawal that
normally accompanies expression and function of myogenic regulatory factors.
Because myofibroblast proliferation can become pathogenic, a better
understanding of the myofibroblast cell cycle would be useful.
The contractile properties of
myofibroblasts are thought to be very important for their function.
Contractility can be induced by a number of cytokines, including
endothelin-1 and TGF-ß (24).
appear to have greater contractility than their kidney, liver, or cardiac
counterparts (L.A. Leinwand, personal communication). The role that muscle
sarcomeric proteins play in contractility of myofibroblasts is unclear.
Experiments with dominant-negative mutants of sarcomeric myosin heavy chain and
troponin T suggest that sarcomeric protein function is required for
myofibroblast contractility. Although myofibroblasts from different organs
share many features, they are clearly diverse. Future research efforts should
be made to understand the signatures of myofibroblasts from different uninjured
and injured organs.
Molecular Mechanisms of
Significant advances in
understanding hepatic fibrosis have been made in the past decade, many of which
may be highly instructive for clarifying pulmonary fibrosis (27).
The liver as a model for the
pathogenesis of fibrosis provides several advantages that account for
this progress: (1) clear identification of the cellular target of organ
injury, typically the hepatocyte or epithelial compartment of liver due to
toxic or viral insult, (2) identification of activated stellate cells/
myofibroblasts as the cellular source of hepatic matrix in liver injury,
(3) reproducible methods to isolate and characterize hepatic stellate
cells, and (4) well-validated markers to identify stellate cells in
situ and track their response to liver injury.
Hepatic stellate cells are the
primary source of ECM in liver fibrosis after their -activation- into hepatic
myofibroblasts. These stellate cell-derived myofibroblasts bear remarkable
similarity to their counterparts in lungs, including the expression of
contractile proteins and responsiveness to proliferative, fibrogenic, and
contractile cytokines. Activated stellate cells/myofibroblasts synthesize a
large array of ECM molecules, including collagens, glycoproteins, and
proteoglycans. Sinusoidal endothelial cells make a small but important
contribution through their early production of cellular (EIIIA) fibronectin
during liver injury. They also can activate latent TGF-ß1, which enhances
fibrogenesis by stellate cells.
Activation of stellate cells
is a central event in the pathogenesis of hepatic fibrosis. The fundamental
features of stellate cell activation are similar regardless of the initial
cause of injury (e.g., alcohol, hepatitis C). Stellate cell activation occurs
in two general phases, an "initiation" or preinflammatory phase, followed by a
"perpetuation" phase in which inflammatory mediators and cytokines play an
important role. Release of cellular fibronectin by sinusoidal endothelial cells
and production of lipid peroxides by injured hepatocytes and Kupffer cells
contribute to initiation. Recent studies have defined many phenotypic features
of perpetuation, including (1) proliferation caused by induction of
platelet-derived growth factor receptors and upregulation of autocrine
platelet-derived growth factor as well as other mitogens, including hepatocyte
growth factor and insulin-like growth factor; (2) chemotaxis, also in
response to platelet-derived growth factor; (3) contractility caused by
upregulation of endothelin-1 production, a potent vasoconstrictor; (4)
fibrogenesis, attributable to increased TGF-ß activity because of
increased secretion and activation of the cytokine, combined with enhanced
TGF-ß binding; (5) release of interleukin-10, colony stimulating
factor, and leukocyte chemoattractants, including macrophage chemotactic
peptide-1, which amplify the inflammatory response; (6) loss of
retinoids caused by hydrolysis of retinyl esters; and (7) enhanced
degradation of basement-membrane matrix as a result of increased secretion of
matrix metalloproteinases and reduced production of tissue inhibitors of
metalloproteinases. Continuing progress in understanding the molecular events
driving stellate cell activation is likely to identify key pathways for further
exploration in pulmonary fibrosis.
TGF-ß and Its
evidence has identified a transforming growth factor(s) as a central regulator
of tissue fibrosis at multiple sites. With respect to pulmonary fibrosis,
studies using blocking antibodies (28), chimeric TGF-ß receptors (29),
and inhibitors of TGF-ß signaling have all been found to inhibit
bleomycin-induced fibrosis dramatically in rodent models. The three mammalian
TGF-ß isoforms, TGF-ß1, TGF-ß2, and TGF-ß3, are members
of a superfamily that includes more than 40 members, all of which signal
through heterodimeric transmembrane serine/threonine kinases. TGF-ß1,
TGF-ß2, and TGF-ß3 all bind to the same receptors, composed of the
TGF-ß type I receptor (Alk5) and the TGF-ß type II receptor, and
signal by activating the cytoplasmic transcription factors SMAD2 or SMAD3. When
phosphorylated by the type I receptor, these SMAD proteins bind a co-SMAD
(SMAD4) and translocate to the nucleus, where they bind to TGF-ß response
elements in regulatory regions of TGF-ß-responsive target genes. Recent
findings make it clear that different cells can employ multiple mechanisms to
modulate this canonical signaling pathway, including induction of
inhibitory SMAD (e.g., SMAD7) proteins that compete with SMAD2/3 for
interaction with activated receptors, and positive and negative regulators of
SMAD-mediated transcriptional activation and repression. The importance of
these modulatory signals is made clear by the dramatic differences in the
patterns of gene expression induced by TGF-ß in lung fibroblasts,
epithelial cells, and airway smooth muscle cells.
Although TGF-ß appears
to be a potent inducer of fibrosis in the lungs and other organs, large amounts
of TGF-ß protein are present in the lungs and other tissues of healthy
adults without apparent TGF-ß effect. This is due, in large part, to the
fact that TGF-ß isoforms are secreted as larger complexes that include
TGF-ß latency-associated peptide and latent TGF-ß binding proteins
(30). These latent complexes, often stored cross-linked to components of the
ECM, prevent TGF- from binding to its receptors and inducing subsequent
signals. One of the principal pathways for regulating TGF-ß effects is
the regulation of the activity of these latent complexes. Latent complexes can
be activated in vitro by extremes of temperature or pH, and by a variety
of proteases that cleave the latency-associated peptide, but the mechanisms
responsible for activation in vivo in health and disease have not been
fully elucidated. At least three mechanisms of activation have been implicated
in pulmonary fibrosis: binding to the matrix protein thrombospondin-1 (31, 32),
macrophage-mediated activation by plasmin (33), and binding of latent complexes
to the inducible epithelial integrin alpha v beta 6 (34). This last mechanism
may be especially useful as a therapeutic target, as alpha v beta 6-mediated
TGF-ß activation is not critical for developmental effects of TGF-ß
but appears to have a restricted role in the enhanced activation that
characterizes pulmonary and renal fibrosis.
Based on currently available
evidence, it is likely that systemic global inhibition of TGF-ß would be
effective in inhibiting pulmonary fibrosis but would be too toxic for long-term
treatment. Targeting mechanisms of TGF-ß activation or fibrosis or
pulmonary-specific downstream targets of TGF-ß might therefore be
attractive alternatives. To realize the potential of these strategies, it
will be important to identify the critical downstream targets and to develop
methods to block specifically TGF-ß activation pathways (e.g.,
thrombospondin-1 or integrin alpha v beta 6 binding) in vivo. Testing
these ideas (and even the feasibility of affecting ongoing fibrosis by blocking
TGF-ß) would be greatly facilitated by the development and
characterization of animal models in which pulmonary fibrosis is persistent
Apoptosis and Proliferation in the Development of Fibrosis
Apoptosis (i.e., programmed
cell death), necrosis, and proliferation are dynamic and coexisting processes
that are commonly noted in wound repair and in epithelial cells of the lung
after exposure to a number of toxicants leading to fibrosis. Alterations in
apoptosis and proliferation of myofibroblasts and fibroblasts have been noted
in isolated cells and in lung tissues of patients with IPF, although several
reports have yielded contradictory results (35, 36). Other data suggest the
importance of cross-talk between epithelial cells and fibroblasts/
myofibroblasts in both the development of proliferation and apoptosis. Recent
studies indicate that apoptosis of epithelial cells by bleomycin or Fas-Fas
ligand cross-linking is causally related to fibrosis (37, 38). Preventive or
therapeutic strategies aimed at inhibition of apoptosis in these cell types or
augmentation of apoptosis in myofibroblasts may be possible. However,
understanding the complex and critical pathways of proliferation and apoptosis
in each cell type in situ in models of fibrosis or patient tissues is
essential for targeting key regulatory pathways. More importantly, deciphering
the eventual outcomes of apoptotic and proliferative responses in terms of
either promotion and/or inhibition of lung inflammation and fibrosis is
necessary in well-defined models of disease.
Several immunocytochemical and
molecular approaches are now available to study the sequence of apoptotic and
proliferative events in the lung, and these can be coupled with imaging
techniques to allow colocalization of signals in key cell types (39).
Complementary approaches such
as laser capture microdissection (LCM) and reverse
transcription-polymerase chain reaction (TaqMan), genomics, and proteomics
might be used to decipher critical genes and signaling proteins involved in
apoptosis and/or proliferation in pulmonary epithelial cells and
myofibroblasts. Finally, comprehensive studies using pharmacologic inhibitors
of specific signaling cascades and transgenic approaches are needed to
determine whether phenotypic and functional outcomes of the fibrotic response
can be modified in animal models of fibrosis.
Proteases and the ECM
The initiation and progression
of lung fibrosis is determined at least in part by how anchorage-dependent lung
cells interact with their surrounding ECM. In the setting of IPF, epithelial
cells and fibroblasts (or myofibroblasts) contribute to and respond to
provisional matrix deposition at sites of injury. Although the initiation of
injury is not clear, the outcome of provisional matrix turnover significantly
determines the extent of focal lung fibrosis. This is most clearly demonstrated
in mice with deficiency of plasminogen activator inhibitor-1, which is
protected from fibrotic responses, and is consistent with human studies showing
excessive plasminogen activator inhibitor-1 in the setting of IPF (40, 41).
Modifying the activation and inhibition of the set of "wound repair enzymes,"
which include the plasmin and thrombin systems (42), is an attractive
therapeutic approach to patients with IPF. However, realization of this
approach would require new tools that target successfully one or more of the
key determinants of provisional matrix resolution, especially plasminogen
activator inhibitor-1 and thrombin.
Although determinants of the
resolution of provisional ma-trices are reasonably well-defined, how lung
epithelial and fibroblastic cells respond to these matrix proteins is much less
clear. Whether activation of epithelial and/or fibroblastic cells is a key
initiator of remodeling and matrix modification is uncertain. A number of
cytokines, proteolytic enzymes, growth factors, and their receptors are
implicated in this pathobiology, but the key pathways to injury and fibrosis in
IPF remain uncertain. Better understanding of how cells coordinate their
signaling responses to matrix remodeling, stress, cytokines, and growth factors
is probably crucial to developing new insights into progressive fibrosis. In
part, this is best explored at the individual investigator level using modern
cell and molecular biology approaches and appropriate animal models. Such
recent studies of mouse knockouts have revealed presenilin 1 and matrix
metalloproteinase 7 as previously unsuspected proteolytic enzymes involved in
the pathobiology of pulmonary fibrosis (43, 44). However, application of modern
methodology for high-throughput pattern recognition at the DNA and protein
level to patients with IPF is also a particularly attractive approach to
developing relevant new hypotheses and to better understanding coordinated
responses at the whole cell level. This approach requires both an
infrastructure for high-throughput testing and a support network for the
ascertainment and assessment of patients and patient material. Such a network
could also serve as a coordinating mechanism for future clinical trials.
Roles of TGF-ß and
Connective Tissue Growth Factor in Pathological Scarring
Fibrosis, or pathological
scarring, occurs in many tissues, including the peritoneal cavity, the
eye, and the lung. Healing of injuries in the peritoneal cavity and eye appears
to share some important common molecular and cellular aspects with healing of
lung injury. For example, after an injury to the single layer of mesothelial
cells that line the peritoneal cavity, a fibrin clot forms on the injured
surface. If two injured areas come into contact, the surfaces adhere via a
provisional fibrin matrix. If the fibrin adhesion is not lysed in a few days by
plasmin, the fibroblasts in the underlying connective tissue will migrate into
the fibrin matrix and begin synthesizing collagen, which converts the
provisional fibrin matrix into a permanent scar (adhesion) that can cause
infertility, pelvic pain, or bowel obstruction (45). Experiments in animal
model of peritoneal adhesions have shown that TGF-ß is produced by
macrophages and fibroblasts that populate the injured areas. More importantly,
exogenous TGF-ß increases the formation of adhesions, whereas a single
injection of a 20-mer antisense oligonucleotide targeting TGF-ß or
repeated injections of neutralizing antibody to TGF-ß reduces the
formation of peritoneal adhesions (46). Similar observations have been reported
in animal models of glaucoma surgery to reduce intraocular pressure. In this
procedure, called trabeculectomy filtering surgery, a port is created through
the sclera and aqueous humor flows out of the anterior chamber into a filtering
bleb in the conjunctival tissue. Exogenous TGF-ß promotes rapid failure
of filtering blebs, and antisense oligonucleotides or neutralizing antibodies
to TGF-ß prolong bleb survival (47). A clinical trial is currently
underway in the United Kingdom that evaluates a humanized neutralizing
monoclonal antibody to TGF-ß in trabeculectomy patients that are at high
risk for bleb failure. Connective tissue growth factor is known to mediate
TGF-ß induction of collagen synthesis in cultured fibroblasts (48). Thus,
connective tissue growth factor may be a better gene to target for reducing
excessive scar formation than TGF-ß. In summary, excessive scarring that
leads to fibrosis in several different tissues appears to share some important
common pathways, including regulation by TGF-ß and connective tissue
growth factor, and agents that are designed to target these genes by
selectively reducing their expression may be beneficial in preventing fibrosis
from occurring. A similar approach in IPF might be beneficial.
Advances in our understanding
of the basic biology of tissue injury and repair and specific advances relevant
to lung injury and fibrosis suggest that epithelial injury and/or death,
induction and activation of TGF-ß and/or connective tissue growth factor,
and induction and activation of myofibroblasts are all likely to play critical
roles in the initiation and progression of pulmonary fibrosis. It is now
appropriate to begin translating these advances into improvements in treatment
of this currently devastating disease. To realize the potential of these
advances, it will be important to refine existing animal models and or to
design new ones that will specifically allow the evaluation of treatment
interventions during the sustained and/or progressive fibrotic stages of this
disease. It will also be important to encourage the rapid development of drugs
suitable for administration to animals and patients that will block the
molecular targets outlined previously here.
Several recommendations were
made that would address the issues raised related to investigating the
molecular mechanisms in fibrogenesis, including the following:
Provide an incentive to
encourage pharmaceutical companies to develop therapeutics for IPF by
establishing an infrastructure in which new treatments could be tested in
Develop animal models in
which interventions can be tested during disease progression or in a persistent
and durable fibrotic condition.
Encourage development of new
strategies for biopsy assessment, which could include laser capture dissection
and analysis of genetic variation in individual cell types using proteomics and
Use new technologies,
including susceptible and resistant mouse strains, to assess the effect of
genetic variation to fibrogenic stimuli.
ETIOLOGY AND DISEASE
Not since the central dogma of
modern biology was formulated and the genetic code elucidated has the
opportunity to catapult our understanding of human disease forward been
greater. This opportunity derives from timely advances in digital technology,
analytical biochemistry, structural chemistry, medicinal chemistry,
biostatistics, and sample acquisition, together with the sequencing of the
human genome. During the session "Etiology and Disease Heterogeneity," advances
in genomics, microarray analysis, and proteomics were reviewed, and the
application of these technologies in identifying informative differences
between biologic specimens (blood, body fluids, tissue) that are normal or
diseased was highlighted. Based on available published and emerging data, a set
of recommendations to use these powerful new approaches to understand and treat
IPF was developed. Emphasis was given to creating easily accessed, high-quality
core clinical and analytical resources and to funding mechanisms encouraging
high-risk exploratory studies of etiology.
Lessons about Disease
Etiology/Pathobiology from Genetic Epidemiologic Studies
Complex disorders are
influenced by the actions of multiple genes and their interactions with each
other and with the environment. New statistical genetic methods have been
developed to facilitate the search for the genes influencing
susceptibility to these complex diseases (49, 50). In contrast to classic
genetic methods, these new techniques do not require specification of unknown
properties of the underlying genetic model, such as allele frequencies and
inheritance patterns at the disease loci. Using these methods, the genetic
influences on a complex trait can be quantified as heritabilities (51) or as
relative risk of disease in the family members of affected individuals as
compared with the general population (52). The results of such systematic
family studies can then be used to design linkage studies to localize the
specific genes influencing risk of disease (53, 54). Linkage methods for
complex traits generally use identity-by-descent allele sharing and are based
on finding regions of the genome where individuals who are phenotypically
similar share more identity-by-descent alleles than expected, given their
kinship. Once susceptibility loci have been localized to a particular
chromosomal region, association methods can be used to assess candidate genes
and polymorphisms within this region. More intensive statistical genetic
analyses may also be done to assess heterogeneity, gene-gene, or
IPF is currently at the very
beginning of this process. Although genetic studies are currently
underway with a subset of patients who appear to have simpler Mendelian forms
of the disease (see subsequent passages), no systematic assessment has
been made of the heritability of IPF in the general patient population or the
risk to relatives of affected individuals. Such analyses are a necessary first
step in determining whether genetic influences are important in general
pulmonary fibrosis and in designing future linkage studies. They may also be
used to investigate questions regarding the spectrum of phenotypes related to
IPF (e.g., whether other types of fibrosis appear in these families or whether
subclinical markers of disease susceptibility can be identified).
Experimental Approaches to
Searching for Fibrotic Genes
The following complementary
lines of logic strongly suggest that inherited genetic factors play a role in
the development of IPF:
Cases of familial pulmonary
fibrosis similar to IPF appear to be inherited as an autosomal dominant trait
with variable penetrance (55-57).
Pulmonary fibrosis is
associated with pleiotropic genetic disorders, such as Hermansky-Pudlak
syndrome, neurofibromatosis, tuberous sclerosis, Neimann-Pick disease,
Gaucher's disease, and familial hypocalciuric hypercalcemia.
Pulmonary fibrosis similar
to IPF is frequently observed in autoimmune diseases, including rheumatoid
arthritis and systemic sclerosis (58, 59).
Variable susceptibility is
evident among workers who are reported to be exposed occupationally to similar
concentrations of fibrogenic dusts.
Inbred strains of mice
differ in their susceptibility to fibrogenic agents.
Although complementary genetic
and pathogenic studies in animals may provide clues to the genetic determinants
of IPF in humans, it is now technologically feasible to apply the recent
advances in the human genome to the identification of fibrosis susceptibility
genes in humans. Using standard genetic methodology (linkage analysis), one can
investigate the distribution of polymorphisms for anonymous genetic markers in
individuals with familial pulmonary fibrosis and then use this information to
identify loci and genes that play a role in the susceptibility of IPF. Although
a traditional candidate gene approach is limited by our current understanding
of disease pathogenesis and is likely to pursue many unrelated genes, a
genome-wide search for linkage
is more comprehensive and may identify novel genes not currently considered to
be involved in the pathogenesis of IPF. Moreover, one could use these genetic
findings to develop new strategies for screening and diagnosis, to identify
novel therapeutic targets for this progressive life-threatening disorder, and
to understand further the pathogenesis of IPF. These genetic studies provide
obvious opportunities for collaboration with basic pathogenic research and
expression-based research projects in interstitial lung disease.
Use of Expression
Expression microarrays are
essentially a method to perform Northern blots on multiple genes simultaneously
using far less biologic material than that required by other techniques. Within
the next few years, it should be feasible to perform such analyses on all genes
in the human or murine genome. Despite the power of these methods, it is
important to keep in mind that they only measure mRNA abundance and that study
design (e.g., adequate repeats to accommodate expected biologic variability)
needs to be as rigorous as that employed for any other methods that measure
mRNA abundance. There are currently two widely used methods for microarray
analysis: one that employs a series of short oligonucleotide probes directly
synthesized onto slides by masked photolithography and the other that
robotically spots individual cDNA probes on slides or filters (60). The first
method has the advantage of being easy to use and commercially available in a
format that provides broad coverage of human or murine genomes, but has the
disadvantage of high cost. The second method can be done less expensively, but
access to high-throughput robots, difficulties in cDNA clone management, and
lack of specificity of long cDNAs for distinguishing closely related family
members and splice variants are all disadvantages. These latter problems can
probably be overcome by using spotted long synthetic oligonucleotides (e.g.,
50-70 mers), but these points need to be experimentally verified.
Despite these caveats,
expression microarrays have already provided some useful insights into
molecular mechanisms of pulmonary fibrosis. In studies using genetically
identical mice that differed only in the expression of a single integrin gene
(the ß6 subunit), distinct clusters of genes were identified that were
predicted to regulate lung inflammation and pulmonary fibrosis differentially
(34, 61). In each case, roughly two-thirds of the genes in the cluster were
already known to regulate inflammation and fibrosis, respectively, providing an
additional group of genes that are candidates to regulate each of these
responses. The "fibrosis" cluster included a substantial number of genes
regulated by TGF-ß, providing further support for the central role of
this cytokine in the pathogenesis of pulmonary fibrosis. Furthermore, careful
analysis of the time course of induction of these genes identified unexpected
effects of TGF-ß in the early stages of the response to injury and led to
the identification of a role for this cytokine in the pulmonary edema that
follows acute lung injury (62), as well as the expected role in the subsequent
Application of Expression
Microarrays to Tissue Samples from Patients with IPF
Analysis of a small number of
such samples using informatics tools designed to evaluate the information
content of expression data for each gene on the array was reported at the
work-shop. Analysis of samples from five patients with well-characterized IPF
suggests that there are approximately 100 informative genes that can be
evaluated in more detail. At least one gene was identified for which the
encoded protein has now been shown to be functionally important in
bleomycin-induced fibrosis in mice. These preliminary results suggest that this
technology could identify unexpected molecular participants in IPF and might
help in the development of novel targets for improved treatment. The method may
also allow molecular fingerprinting that could improve the ability to identify
sub-classifications of pulmonary fibrosis that might be more informative than
the current classification based primarily on histologic and radiographic
patterns. Use of expression microarrays with microdissected samples containing
specific cell types from a number of patients with a range of histologic
diagnoses could be especially informative, as could analysis of samples before
and after therapeutic intervention. Optimal use of this technology will require
design and evaluation of a uniform set of oligonucleotide probes encompassing
the entire human and mouse genome, improved methods for unbiased amplification
of small amounts of input RNA, and access to appropriately sampled tissue from
a series of well-characterized patients.
Using Microarrays to Elucidate the
Pathobiology of Lung Disease and to Identify Molecular Targets for Drug
Microarrays have also been
useful in elucidating the patterns of gene expression in the lung in health and
disease, with the goal of identifying specific genes or pathways that play
critical roles in defending the lung and in modulating abnormalities in disease
states. An example presented at the workshop was an analysis of gene expression
in the human airway epithelium in normal nonsmokers compared with 20 pack/year
smokers with no evidence of lung dysfunction. The airway epithelium (106
to 107 cells per individual, more than 98% epithelium)
recovered by fiberoptic bronchoscopy and brushing was assessed for levels of
mRNA expression of 7,000 genes using the Affymatrix human chip.
Assessment of the right and
left lung in the same individual and among different individuals demonstrated
that there was more variation in gene expression from individual to
individual than within the same individual. Gene expression among smokers
was greater than that among nonsmokers, but the individual-to-individual
variation was greater than that among smokers; that is, the stress of cigarette
smoking was not major when assessed in a global fashion. Antioxidant genes were
assessed as an example of a class of genes relevant to how the lung defends
itself from the environment. Evaluation of airway epithelial expression of the
superoxide dismutase genes, catalase, and the many genes involved in the
glutathione pathway demonstrated only minor (1.5- to 2.5-fold) increases in
smokers compared with nonsmokers in some of these genes. However, when each of
the genes was assessed on an individual basis, it became apparent that,
for several genes, there was a subset of smokers that exhibited significant
elevation of gene expression, whereas others were in the same range as
nonsmokers. This observation leads to the interesting hypothesis that
there are genetic variations in the population with regard to the ability to
upregulate genes that function to protect the lung from environmental stress
such as cigarette smoke; that is, those capable of upregulating these genes are
protected from such stress, whereas those unable to do so are vulnerable to
environmental stress-induced injury.
These observations serve as a
paradigm to using array-based assessment of gene expression in humans relevant
to fibrotic lung disease. For IPF, the cells that can be readily obtained for
gene expression analysis by fiberoptic bronchoscopy include alveolar
macrophages (a major source of mediators that stress lung parenchymal cells in
the fibrotic disorders) and the airway epithelium (the cell source of
bronchogenic carcinoma, known to affect 10 to 20% of individuals with IPF). For
gene expression in the lung parenchyma, lung biopsy via video-assisted
thorascopy, together with laser capture dissection of specific foci in the
biopsy (e.g., foci of mesenchymal cells), provides a cell source of specific
groups of cells within the lung parenchyma that plays a central role in the
pathogenesis of the fibrotic state.
The strategy of array-based
assessment of gene expression represents a technology that is readily adaptable
to evaluation of the lung in health and disease and should lead to major new
insights into pathogenesis and therapy of the fibrotic disorders.
LCM: Obtaining and
Analyzing Tissue Samples Exhibiting Heterogeneity of Disease Expression
LCM is a powerful method for
procuring pure cells from specific microscopic regions of tissue sections (63,
64) that has been under commercial development since 1997 through collaborative
efforts among the National Institutes of Health (NIH) and
Arcturus Engineering Inc.
(http://www.arctur.com). A description of LCM, the tissue processing protocols,
the facilities at the NIH and opportunities for interacting with NIH staff and
use of the facilities are available through
links online at
http://dir.nichd.nih.gov/lcm/lcm.htm. LCM has been developed to automate and
standardize microdissection and has greatly increased reproducibly and accuracy
of selecting specific, targeted cells from a complex tissue for subsequent
Most tissues are heterogeneous
complicated structures with many different cell types locked in morphologic
units exhibiting strong adhesive interactions with adjacent cells,
connective stroma, blood vessels, glandular and muscle components,
adipose cells, and inflammatory or immune cells. In normal or developing
organs, specific cells express different genes and undergo complex molecular
changes both in response to internal control signals, signals from adjacent
cells, and humoral stimuli. In disease pathologies, the diseased cells of
interest, such as precancerous cells or invading groups of cancer cells, are
surrounded by these heterogeneous tissue elements. Cell types undergoing
similar molecular changes, such as those thought to be most definitive of the
disease progression, may constitute less than 5% of the volume of the tissue
biopsy sample. Therefore, microdissection is essential to apply molecular
analysis methods to study evolving disease lesions in the context of intact
organs. The cDNA libraries generated from microdissected samples approximate
the true pattern of gene expression of the pure cell subpopulations in their
actual tissue context.
The process of LCM involves
use of a special transfer film that is applied to the surface of the tissue
section. Under the microscope, the thin tissue section is viewed through the
glass slide on which it is mounted and microscopic clusters of cells are chosen
to study. When the cells of choice are in the center of the field of view, the
operator pushes a button, which activates a near infrared laser diode
integral with the microscope optics. The pulsed laser beam activates a precise
spot on the special transfer film (either 30 or 60 micrometer in diameter),
which melts and fuses with the underlying cells of choice. When the film is
removed, the chosen cell(s) is tightly held within the focally expanded
polymer, and the rest of the tissue is left behind. The process allows for
multiple homogeneous samples within the tissue section or cytologic preparation
to be targeted and pooled for extraction of molecules and analysis. In the
commercial system, the process of handling the transfer film has been
simplified and automated, and computer software to control the tissue
microdissection process and to store data records, including digital images of
the microdissected cells before and after transfer, has been developed.
This technique has proved
useful in cancer research in demonstrating that genotype precedes phenotype and
cancer arises in discreet tissue fields, and in discovering new genes such as
tumor suppressors. Coupled with the use of protein microarray analysis and
artificial intelligence, it has been possible to identify proteomics patterns
that can be used to distinguish normal and precancerous tissue.
The power of LCM is the
ability to obtain DNA, RNA, or protein from selected pure cells that are not
damaged by the process. In the case of IPF, this technique could be very
informative in examining the expression of DNA, RNA, or proteins in
various cells (e.g., fibroblasts, epithelial cells) in the lesion, for example,
to look for molecules important in cell-cell interaction. A comparison to
levels in cells in normal tissue, either from the patient lung or from
nonfibrogenic lung from another source, could be used to identify those that
are expressed at increased or decreased levels in fibrotic lesions.
Another encouraging new
technology is the identification of proteomics signature patterns, discovery of
patterns of expression of new low molecular weight molecules, and
identification of serum protein patterns associated with disease. These
strategies could be readily applied to IPF as new approaches to diagnosis and
responses to therapy.
During the discussion of this
portion of the meeting, several suggestions were made that would address
the issues raised related to the etiology and disease heterogeneity in IPF,
which include the following:
research-supporting novel hypotheses that are not yet supported by extensive
preliminary data directed at etiology.
research that is directed at investigating leading hypotheses in disease
pathogenesis (e.g., epithelial cell versus matrix as potential targets;
biomechanical properties that lead to the unusual topographic features of the
disease; viruses [including prions] as a potential etiology; what does the
heterogeneity of the disease actually mean; what is the relationship between
inflammation and fibrosis; and whether fibrosis [or inflammation] is bad).
between institutions-this would be a natural extension of the Specialized
Center of Research (SCOR), Program Project Grant (PPG), and Program in Genomic
Applications (PGA) programs, but could be designed to specifically include
investigators outside of these programs (it might also provide a mechanism to
include investigators who study fibrosis in other organ systems or linking
genetic discoveries to pathogenic mechanisms of disease).
Optimize the use of
microarray technology by supporting the design and evaluation of a uniform set
of oligonucleotide probes encompassing the entire human and mouse genome,
improved methods for unbiased amplification of small amounts of input RNA, and
access to appropriately sampled tissue from a series of well-characterized
nonhypothesis driven grants to use these new technologies to study the etiology
and pathogenesis of IPF (e.g., the National Science Foundation commonly does
this with astronomy grants when new more powerful telescopes become available).
Develop easy access to core
equipment and informatics support for traditional R01 and P series
Develop high-quality patient
databases and sample banks that are readily accessible by funded investigators
and by investigators proposing new grants.
Encourage studies on genetic
models of pulmonary fibrosis with single gene inheritance.
Employ new methods
(genotyping, proteomics, microarrays) to assess etiology and pathogenesis in
Identify molecular targets
Establish a mechanism for
systematic phenotype and genotype characterization of patients and family
members in association with a standardized collection of medical and
environmental exposure histories for use in gene-environment investigations of
methods and establish mechanisms for rapid dissemination of phenotypic and
genotypic information to the scientific community in a format that is user
DIAGNOSIS AND TREATMENT
Because it appears that IPF is
associated, in part, with a dysregulated reparative response of the lung
resulting in the relentless accumulation of ECM, it is logical to propose that
the fibroproliferative process occurring in the distal airspaces represents a
promising molecular therapeutic target for treatment of IPF. Although it is
likely that in some cases of IPF a genetic predisposition exists, in the
majority of cases, the inciting injury remains unknown. Moreover,
corticosteroid and immunosuppressive therapy, although still recommended, does
not prevent ECM accumulation in the majority of affected individuals (65).
Hepatitis C virus produces
both liver inflammation and fibrosis (66). Treatment with antiviral drugs and
alpha-interferon can prevent and even reverse the fibrosis induced by hepatitis
C virus (67). The myofibroblast (hepatic stellate cell) is thought to play a
central role in the development of cirrhosis, as it does in IPF. Although
lessons can be learned from hepatic fibrosis in terms of potential
antifibroproliferative therapies, there are clear distinctions from IPF. The
cause of IPF is unknown. Inflammation, a prominent feature of hepatitis C, is
variable in IPF. The fibroblastic foci, essential features for progression in
IPF, are not present in hepatic fibroblastic proliferation.
There are several approaches
to the treatment of IPF. Interferon-gama, which has been shown in laboratory
studies to induce myofibroblast apoptosis and to suppress the
myofibroblast phenotype, has been evaluated in a small number of patients
with IPF (68). The results of a large placebo-controlled multicenter trial are
There is evidence that
TGF-ß may be central to the fibroproliferative response in skin wound
healing, cirrhosis, and IPF (69-71). TGF-ß is a product of inflammatory
and damaged epithelial cells. Intermittent injury to lung epithelial cells,
which is presumed to occur in IPF, may lead to enhanced local levels of
TGF-ß, which is capable of multiple profibrotic actions described earlier
in this report. Potential approaches to anti-TGF-ß therapy include the
use of neutralizing anti-bodies, soluble receptors, and decorin, a naturally
occurring matrix protein capable of antagonizing TGF-ß actions (33, 34,
72). Antibodies to connective tissue growth factor, another fibroproliferative
cytokine, are also being developed (68, 73, 74). Endothelin-1, in addition to
its vasoactive properties, also has potent fibroproliferative potential,
particularly as a myofibroblast inducer (75). If relevant, endothelin-1
receptor antagonists, some that are approved, and others that are
undergoing trials in primary pulmonary hypertension, could eventually be
available for IPF treatment trials.
Another interesting approach
is the development inhibitors to enzymes necessary for collagen synthesis, such
as prolyl hydroxylase (76). Studies have shown that inhibition of specific
enzymatic functions prevents excessive collagen accumulation in models of
dermal and cardiac scarring. Studies in lung models of fibrosis are underway.
Data suggest that arachidonic
acid metabolites can act to increase or decrease pulmonary fibrosis.
Prostaglandin E2 inhibits fibroblast proliferation (77, 78)
and fibroblast collagen synthesis (79, 80). Its level in pulmonary fibrosis
patients' lungs is reduced (81), and lung fibroblasts from pulmonary fibrosis
patients are less able to produce it in vitro (81). In addition, levels
of leukotriene B4 are higher in pulmonary fibrosis patients' lungs
(82), and elimination of leukotriene production has been shown to protect
against pulmonary fibrosis (83). Thus, inhibition or stimulation of the
appropriate metabolite is a possible point for intervening in this disease
A different approach to IPF
therapy includes strategies to promote matrix resorption by enhancing the
activity of certain matrix metalloproteinases, the interstitial collagenases,
whose levels are reduced in IPF (36, 85). This could be accomplished by
augmenting their activity either endogenously (e.g., interferon-gama) or
exogenously by the administration of supplemental enzymes. There is an
imbalance between the natural inhibitors of the matrix metalloproteinases
(tissue inhibitors of metalloproteinases) and the matrix metalloproteinases
favoring matrix accumulation rather than degradation in IPF. Tissue
inhibitors of metalloproteinases are antagonized by anti-TGF-ß
therapeutic strategies, thereby increasing collagenolytic activity. The
development of antagonists to the tissue inhibitors of metalloproteinases
should be considered.
There are approaches to
identifying therapeutic targets and therapeutics that provide a conceptual
framework that could be applied to IPF. The molecular target must be accessible
to chemical genetics using chemistry, structural biology, and library screening
techniques to design small agonists or antagonists for the molecular target.
There must be in vitro and in vivo assay systems for
evaluating the effectiveness of potential therapeutics. The potential toxicity,
pharmacokinetics, and pharmacodynamics of potential therapeutics should be
considered at the beginning of the process of discovery.
Within the area of IPF, it
should be possible to identify potential therapeutic targets that, if
stimulated or blocked by bioprobes, could interfere with or reverse
fibrogenesis. In order to develop such bioprobes, it will be necessary to
establish cell-free and cell-based assays for testing their agonistic or
antagonistic behavior. By identifying libraries of privileged structures that
have high potential to be used as nontoxic bioprobes in vitro and in
vivo, a wide range of potential therapeutics can be screened in the
appropriate animal models in which their effect on at least one aspect of the
disease can be addressed. Collaborations between clinical investigators and
molecular targeting programs (academic and industrial) would facilitate
During the discussion, several
suggestions were made that would address the issues raised related to diagnosis
and treatment in IPF, including the following:
Identify new molecular
targets for interfering with fibrogenesis and design treatments around agonists
or antagonists of the targets.
Develop the infrastructure
for conducting clinical trials in IPF. Because of the paucity of patients at
any one site, a multicenter organization will be required. Such "Centers for
Excellence in IPF" could also provide the framework for capturing clinical,
medical history, and environmental exposure data, DNA and biopsy specimens, and
radiographic images in a standardized way. The collection of such data would be
used to test specific hypotheses.
Develop drug inhalation
delivery systems that target IPF treatment to lungs. This is especially
important where systemic administration would be too toxic.
investigators to develop new chemical interventions in a way that industry will
have an interest in them because of their proprietary status.
After discussion of the
suggestions that arose during the meeting, the following overall
recommendations of the Working Group to the NHLBI were formulated:
Consider development of a
mechanism to encourage high-risk studies to elucidate the etiology of IPF.
Develop novel diagnostic
techniques and molecular diagnostics (e.g., molecular fingerprinting, serum
profiles) to allow early detection of IPF, elucidation of the pathogenesis of
lung fibrosis and/or etiology, and monitoring of treatment approaches.
Consider development of a
"molecular targets for drug discovery program" based on an attractive menu of
available targets (e.g., TGF-ß, platelet-derived growth factor, signaling
pathways) that regulate fibroblast and epithelial cell fate.
Foster investigation using
new technologies to define differences in the genetic repertoire, mRNA
expression, protein expression, and signaling patterns among normal cell types
(epithelial cells, endothelial cells, and mesenchymal cells) and their
counterparts from fibrotic lesions. Such technologies would include, but are
not limited to, LCM coupled with the use of data from the human genome project,
microarrays, and mass spectroscopy analysis of proteins.
Develop a mechanism to
establish a national consortium of Clinical Centers of Excellence to provide
well-characterized patients and patient-derived materials for these new
technological applications. The Clinical Centers of Excellence would conduct
coordinated laboratory and clinical research and clinical trials in IPF. Such
centers would conduct investigations into the etiology, pathogenesis, and
treatment of IPF through standardized collection and coordinated use of medical
and exposure history, clinical data, blood and tissue samples, and DNA. This
Center of Excellence program would include within it, or as a separate
component, a bioinformatics platform that links molecular data to clinical
data. This platform would include a user-friendly mechanism for depositing data
and readily available portals of access to obtain and use the data.
Establish a program for
identification of molecular targets for IPF treatment and identification and
synthesis of small molecular agonists or antagonists whose action would inhibit
fibrogenesis or cause the remission of existing fibrogenic foci.
Stimulate research to
develop animal models of durable (persistent) pulmonary fibrosis that can be
used to evaluate new intervention approaches.
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