Future Research Directions in Acute Lung Injury
NHLBI Workshop Summary
Summary of a National Heart, Lung, and Blood Institute
Published in Am J Respir Crit Care Med Vol 167. pp 1027-1035,
2003 Internet address: www.atsjournals.org
Michael A. Matthay, Guy A. Zimmerman, Charles Esmon, Jahar
Bhattacharya, Barry Coller, Claire M. Doerschuk, Joanna Floros,
Michael A. Gimbrone Jr, Eric Hoffman, Rolf D. Hubmayr, Mark
Leppert, Sadis Matalon, Robert Munford, Polly Parsons, Arthur
S. Slutsky, Kevin J. Tracey, Peter Ward, Dorothy B. Gail,
and Andrea L. Harabin
Cardiovascular Research Institute, University of California
at San Francisco, San Francisco, California; Program in Human
Molecular Biology and Genetics and Department of Human Genetics,
University of Utah, Salt Lake City, Utah; Cardiovascular and
Biological Research Program, Howard Hughes Medical Institute,
Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma;
Critical Care Service, Mayo Clinic and Foundation, Rochester;
Laboratory of Biomedical Science, North Shore-LIJ Research
Institute, Manhasset; Department of Medicine and Physiology,
Columbia University College of Physicians and Surgeons; Department
of Blood and Vascular Biology, Rockefeller University, New
York, New York; Department of Pediatrics, Rainbow Babies and
Childrens Hospital, Case Western Reserve University,
Cleveland, Ohio; Department of Cellular and Molecular Physiology
and Pediatrics, Pennsylvania State University, College of
Medicine, Hershey, Pennsylvania; Center for Excellence in
Vascular Biology, Brigham and Women's Hospital, Harvard Medical
School, Boston, Massachusetts; Research Center for Genetic
Medicine, Childrens National Medical Center, Washington,
DC; Department of Anesthesiology, University of Alabama at
Birmingham, Birmingham, Alabama; Department of Internal Medicine,
University of Texas, Southwestern Medical Center, Dallas,
Texas; Department of Pulmonary and Critical Care Medicine,
University of Vermont, Fletcher Allen Healthcare, Burlington,
Vermont; Department of Pathology, University of Michigan,
School of Medicine, Ann Arbor, Michigan; and Division of Lung
Diseases, National Heart, Lung, and Blood Institute, Bethesda,
Maryland; and Interdepartmental Division of Critical Care
Medicine and Department of Medicine, University of Toronto,
St. Michaels Hospital, Toronto, Ontario, Canada
Acute lung injury (ALI) and its more severe form, the
acute respiratory distress syndrome (ARDS), are syndromes
of acute respiratory failure that result from acute pulmonary
edema and inflammation. The development of ALI/ARDS is associated
with several clinical disorders including direct pulmonary
injury from pneumonia and aspiration as well as indirect pulmonary
injury from trauma, sepsis, and other disorders such as acute
pancreatitis and drug overdose. Although mortality from ALI/ARDS
has decreased in the last decade, it remains high. Despite
two major advances in treatment, low VT ventilation for ALI/ARDS
and activated protein C for severe sepsis (the leading cause
of ALI/ARDS), additional research is needed to develop specific
treatments and improve understanding of the pathogenesis of
these syndromes. The NHLBI convened a working group to develop
specific recommendations for future ALI/ARDS research. Improved
understanding of disease heterogeneity through use of evolving
biologic, genomic, and genetic approaches should provide major
new insights into pathogenesis of ALI. Cellular and molecular
methods combined with animal and clinical studies should lead
to further progress in the detection and treatment of this
Keywords: pulmonary inflammation; critical care;
genomics; pulmonary edema; acute respiratory distress syndrome
Acute lung injury (ALI) and the acute respiratory distress
syndrome(ARDS) are syndromes with a spectrum of increasing
severity of lung injury defined by physiologic and radiographic
criteria in which widespread damage to cells and structures
of the alveolar capillary membrane occurs within hours to
days of a predisposing insult. In this report we will consider
ALI and ARDS together, although we also specically refer to
ARDS because it has been studied as a defined entity with
exclusion of patients with less severe degrees of lung injury.
The time course of ALI/ARDS distinguishes these syndromes
of alveolar damage from most other lung diseases, whose natural
histories occur over a much longer duration, usually years.
ALI/ARDS is a major cause of acute respiratory failure with
high morbidity and mortality in critically ill patients (1).
Recent epidemiologic data indicate that the incidence of ARDS
defined by consensus physiologic criteria may account for
36,000 deaths per year in a country the size of the U.S. (2).
There is reason to believe that this number will increase
significantly in the future because of increasing frequency
of some predisposing conditions that precipitate ALI/ARDS,
such as sepsis (3). Although there is evidence that mortality
in patients with ALI/ARDS may have declined over the last
10 to 15 years, it remains high (30-40%), and it is an important
cause of pulmonary and nonpulmonary morbidity in patients
who leave the hospital (4). Until recently, there were no
specific measures that altered mortality in ALI/ARDS, and
management was exclusively expectant and supportive, reflecting
major deficiencies in our understanding of the cellular and
molecular nature, pathogenesis, and natural history of acute
alveolar injury. Recently, however, a prospective multicenter
clinical trial demonstrated that a lung-protective ventilatory
strategy could substantially reduce mortality (5). The results
of this clinical trial suggest that there are additional opportunities
to improve outcomes in ALI/ARDS if we can increase our fund
of knowledge at the basic, translational, and clinical levels.
On January 21-22, 2002 the NHLBI convened a working group
in Rockville, MD to consider future directions in research
in ALI/ARDS. Conference participants were charged with reviewing
the current state of knowledge, identifying major gaps in
information and understanding, identifying promising
opportunities for investigation and discovery, and developing
specific recommendations to be used by the NHLBI in planning
future research in ALI/ARDS. This article summarizes presentations
and discussions at the Working Group meeting. The conference
also considered trends and successes in other fields that
may be instructive and relevant to ALI/ARDS. As an example,
management of patients with acute coronary syndromes has undergone
major paradigm shifts in recent years because of insights
generated from fundamental, translational, and clinical investigation;
this has resulted in development of new interventional approaches
and biologic and pharmacologic strategies that address thrombosis
and inflammation in addition to a traditional focus on lipids
and vascular remodeling and has generated ongoing investigations
of these critical processes (6-8). Similarly, a new treatment
for severe sepsis, recombinant activated protein C, has recently
been developed based on molecular insights relevant to coagulation
and inflammation that were then explored in translational
and clinical studies (9-11). Sepsis is of particular interest
because it is a leading predisposing condition that initiates
ALI/ARDS, and it complicates ALI/ARDS induced by other causes
This report of the proceedings of the Working Group meeting
begins with a brief overview of the clinical features of ALI/ARDS
followed by sections that consider (1 ) gaps in our understanding
of basic mechanisms underlying development of ALI, (2 ) how
genomics and proteomics may advance research in ALI/ARDS,
(3 ) how genetic influences might be studied in patients with
ALI/ARDS, (4 ) the challenges posed by the heterogeneity of
the clinical phenotypes in ALI/ARDS, (5 ) how biological and
clinical indices of lung injury may be useful, and (6 ) how
animal models can be used to provide insights into lung injury.
The final section summarizes the major recommendations for
future research directions. The group did not attempt to provide
a comprehensive overview of the field, as there are many fine
ones available (1, 12); instead, citations outside of the
field are highlighted. The report reflects common opinions
of the participants. The challenges of ALI/ARDS remain substantial,
and modern and evolving approaches in fundamental science
(cell and molecular biology, genomics, proteomics, disease
gene discovery) together with new approaches to clinical investigation
pro-vide substantial opportunities to improve prevention,
treatment, and outcomes in ALI/ARDS.
OVERVIEW OF ALI/ARDS
ALI/ARDS is a cause of acute respiratory failure that develops
in patients of all ages from a variety of clinical disorders,
including sepsis (pulmonary and nonpulmonary), pneumonia (bacterial,
viral, and fungal), aspiration of gastric and oropharyngeal
contents, major trauma, and several other clinical disorders
including severe acute pancreatitis, drug over dose,and blood
products (1). Most patients require assisted ventilation with
positive pressure. The primary physiologic abnormalities are
severe arterial hypoxemia as well as a marked increase in
minute ventilation secondary to a sharp increase in pulmonary
dead space fraction. Patients with ALI/ARDS develop protein-rich
pulmonary edema resulting from exudation of fluid into the
interstitial and airspace compartments of the lung secondary
to increased permeability of the barrier. Additional pathologic
changes indicate that the mechanisms involved in lung edema
are complex and that edema is only one of the pathophysiologic
events in ALI/ARDS. One physiologic consequence is a significant
decrease in lung compliance that results in an increased work
of breathing (13), one of the reasons why assisted ventilation
is required to support most patients.
The clinical diagnosis of ALI is made by the presence of
ar terial hypoxemia (PaO2/FiO2 less than 300) and bilateral
pulmonary opacities on the chest radiograph. Current definitions
require exclusion of left atrial hypertension and overt heart
failure by clinical assessment. It is often difﬁcult
to precisely distinguish ALI/ARDS from other diffuse inﬂammatory
conditions of the lung and, in some cases, from high-pressure
pulmonary edema. New diagnostic measures to address this issue
would be useful. It is not yet clear how the physiologic alterations
that deﬁne the clinical diagnosis of ALI/ARDS correlate
with features that define the pathologic diagnosis, a constellation
of histologic findings termed diffuse alveolar damage (14).
Until recently, management of patients with ARDS was entirely
supportive with the objective of managing underlying infections
medically and surgically, providing nutritional support, and
employing mechanical ventilation for acute respiratory failure.
In a large prospective NHLBI-supported study, mortality was
reduced from 40 to 31% with the use of a ventilatory strategy
that employed low Vt (6 ml/kg) and limited plateau pressure
(<30 cm H2O) (5). The results of this trial demonstrated
that use of high Vt and high airway pressures exacerbated
the patient's ALI and raised interesting mechanistic questions
concerning interaction of lung stretch and systemic injury
that may provide additional therapeutic approaches. Also,
the results of this trial raise interesting questions regarding
how lung-protective ventilatory strategies work. For example,
is the protection a result of decreased mechanical injury
to the lung endothelium and epithelium, effects on the systemic
compartment, or is there also a down regulation of proinflammatory
stimuli for neutrophils and macrophages (15)? Also, are there
beneficial effects from the change in the carbon dioxide tension,
acid-base status, and effects on reactive oxygen and nitrogen
Despite this advance in ventilation of patients with ALI/ARDS,
considerable work is still needed to define the basic mechanisms
that (1 ) initiate lung injury before institution of positive
pressure ventilation, (2 ) mediate progression of ongoing
lung injury, (3 ) cause fibrosing alveolitis and pulmonary
hypertension with extensive obliteration of the lung circulation
in subsets of patients, and (4 ) mediate the propagation of
injury from the lung to other organs in many patients with
SYSTEMIC MANIFESTATIONS OF ALI/ARDS
A major feature of ALI/ARDS that distinguishes it from most
other lung diseases such as asthma, chronic obstructive pulmonary
disease, and idiopathic pulmonary fibrosis is that ALI/ARDS
frequently has systemic components. Several of the major triggering
conditions for ALI/ARDS, notably sepsis, nonpulmonary trauma,
and shock are systemic syndromes. Furthermore, diffuse injury
and infection of the lung are major causes of systemic sepsis
and the systemic inflammatory response syndrome and dysregulation
of cellular responses in the lung may result in circulating
cytokines and other inflammatory and thrombotic mediators,
as demonstrated in clinical studies and animal models (16-18).
Many patients with refractory ALI/ARDS die of multiple organ
dysfunction and/or sepsis, rather than isolated progressive
respiratory failure (1).
A consensus of the working group was that ALI/ARDS is a systemic
syndrome in virtually all cases. It is important to investigate
the cellular and molecular mechanisms that mediate pulmonary
injury responses to systemic insults, the factors that govern
the participation of blood cells and humoral agents that mediate
both pulmonary and systemic injury, and mechanisms that govern
compartmentalized injury in the lung versus dissemination
to systemic sites and distant organs. Systemic responses to
stress and injury are incompletely characterized but involve
neural, endocrine, and pro-and anti-inflammatory mechanisms
that may be either adaptive or pathologic (19). Animal studies
indicate that neural stimulation modifies systemic responses
to endotoxin and can differentially influence cytokine accumulation
in the lung and in systemic tissues; this phenomenon involves
vagal signaling of altered inflammatory cell function (20,
21). Molecular links that mediate interactions between the
acute inflammatory system (innate immune effector mechanisms)
and thrombotic and procoagulant responses are altered in stress
and injury, as are interactions between innate and acquired
immune responses (19).
Signaling pathways, such as those provided by cytokines and
their receptors, may have protective or injurious effects
depending on the site of challenge and whether the inflammation
is local or systemic (22). The unknown aspects of these complex
events are particularly relevant to ALI/ARDS that is induced
as a part of systemic disorders such as sepsis, multiple trauma,
and shock but also may be critical in order to understand
the pathologic responses to diffuse alveolar injury caused
by direct insults such as aspiration and microbial infection
of the lung.
CURRENT UNDERSTANDING AND SIGNIFICANT GAPS IN KNOWLEDGE
The Working Group identified many areas in which there are
significant knowledge gaps that must be addressed in order
to facilitate progress in understanding ALI/ARDS and for devising
new strategies for detection, prevention, management, and
The cells of the alveolar capillary membrane together with
cells of the innate immune and hemostatic systems are targets
of damage and effectors of injury in ALI/ARDS (1). The numbers
and morphologic phenotypes of these cells are altered in development
of ALI/ARDS and its progression or resolution (14). Although
much has been learned about the cell biology of the alveolar
capillary membrane since the clinical syndrome of ALI/ARDS
was described, the functional responses, biochemical pathways,
patterns of gene expression, molecular mechanisms of interaction
with other cells, and responses to injury remain incompletely
characterized and our understanding of these processes is
embryonic at best. New approaches to ALI/ARDS are likely to
require comprehensive understanding of the functions of key
interacting cells. New and evolving technologies and analytic
methods may provide opportunities to all knowledge gaps.
The important role of the alveolar epithelial barrier in
the pathogenesis and resolution of ALI/ARDS has been increasingly
appreciated, including the role it plays in the production
of surface-active material to maintain alveolar stability
as well providing salt and water transport pathways for the
resolution of alveolar edema. However, our understanding of
the responses of the alveolar epithelium to injury remains
incomplete. Disruption of alveolar epithelial integrity is
a major contributor to increased permeability and alveolar
flooding with protein-rich edema fluid, a hallmark of ALI/ARDS
(1). Understanding of molecular signaling pathways and mechanisms
of cell-cell interaction in alveolar epithelial cells at various
stages after injury is rudimentary at best. Although it is
clear that alveolar epithelial Type II cells re-populate the
injured, denuded alveolar barrier in ALI, very little is known
about the intracellular signaling pathways that are activated
or differential mechanisms of gene expression during injury,
proliferation, and differentiation. For example, we know that
epithelial cells produce cytokines in response to various
stimuli such as LPS or lung stretch (23), but the regulatory
features are not completely defined. As a second example,
specific mitogens such as keratinocyte growth factor can protect
the lung epithelium against experimental lung injury (24),
but the mechanisms for this cytoprotective effect are not
The responses of lung endothelial cells are also incompletely
defined even though they appear to be the first cells of the
lung to be altered in ALI/ARDS triggered by sepsis, trauma,
and other systemic conditions. The lung endothelium, in concert
with the epithelial barrier, mediates the initial change in
permeability and is also critical for repair and remodeling
of the alveolar capillary membrane. Endothelial heterogeneity
may be a factor in the lung's responses to pathologic stimuli
(25). Each short segment of a rodent lung capillary possesses
a few functionally distinct endothelial cells--the pacemaker
cells. These cells appear to regulate endothelial calcium
within the capillary segment by generating intercellular calcium
waves, and they may be the sites of inflammatory initiation
in the capillary (26). More needs to be learned about the
diversity of endothelial cells, particularly regarding how
they regulate the onset of lung inflammation. Activation of
endothelial cells, with functional changes that involve both
new gene expression and constitutive pathways that do not
require new gene products, occurs in both pulmonary and systemic
endothelium (27). We have an incomplete picture of the molecular
mechanisms that govern the responses of pulmonary endothelial
cells, their interaction with alveolar epithelium, and the
responses of systemic endothelium in ALI/ARDS.
Apoptosis, Necrosis, and Fibrosis
A comprehensive understanding of the mechanisms of apoptosis
and necrosis in the initial injury and repair of lung epithelial
and endothelial cells and other key cells involved in ALI/ARDS
is lacking. These cellular processes are likely to be central
to imbalances between resolution and repair versus persistence
and progression in ALI/ARDS and can be influenced by biomechanical,
inflammatory, and thrombotic stimuli (28). The mechanisms
that govern apoptosis and necrosis in endothelial and epithelial
cells in the key phases of ALI/ARDS need definition. How apoptosis
and related cellular events influence fibrosing alveolitis,
which is an outcome in a subset of patients with ALI/ARDS,
is not known. Information is needed regarding signaling pathways,
patterns of gene and protein expression, and functional responses
in lung-broblasts and mesenchymal cells that lead to dysregulated
matrix remodeling and relentless fibrosis in some patients
It is unknown how lung endothelial and epithelial injury
modify the fibrogenic response in ALI/ARDS or if apoptosis
and necrosis affect the fibrotic process differently. Microarray
analysis and assays of new protein expression using isolated
cells in parallel with the study of animal models that include
fibrosis are likely to be revealing. New approaches to the
cell and molecular biology of fibrogenesis that are being
applied to the syndrome of idiopathic pulmonary fibrosis may
be useful in clarifying the nature of alveolar fibrosis in
ALI/ARDS. A caveat is that some early events in ALI/ARDS do
not involve new gene expression or changes in transcript level.
Similarly, dissection of the cellular and molecular processes
involved in endothelial death versus repair and other key
aspects of vascular wall remodeling will be critical to understanding
of the loss of pulmonary vessels and the resultant pulmonary
hypertension and the increase in alveolar dead space that
is also an outcome of a subset of patients with ALI/ARDS.
An approach for the future treatment of ALI/ARDS could involve
stem cells to replace dying lung cells. A similar strategy
has been proposed for brain, heart, and liver diseases. The
inflammatory response includes release of cells from the bone
marrow such as mature and immature neutrophils and stem cells.
Elegant bone marrow reconstitution studies have demonstrated
that these stem cells have the potential to differentiate
into endothelial, mesenchymal, and epithelial cells (29-31).
The proliferative potential of fully differentiated capillary
endothelial cells and epithelial Type I cells is very poor
in the lungs. The potential for stem cells to help in the
repair of injured lung tissue has not been explored and requires
a better understanding of stem cell release, targeting, proliferation,
and differentiation in lung tissue. Supplementing this release
by intravenous infusion of donor bone marrow-derived stem
cells might be useful in ALI/ARDS, as the pulmonary microvasculature
would be the first capillary bed that these cells would encounter,
enhancing their entrapment and accumulation. Newly implanted
endothelial cells may help restore the lung capillary network
after ALI, although considerable work will be needed to develop
approaches for delivery of viable cells to injured areas of
Mechanical Forces and Lung Injury
There is incomplete understanding of how normal lungs deform
during breathing and to what stresses key lung cells are exposed
(32).Lung injury may greatly alter these complex forces. Furthermore,
there is uncertainty about the molecular mechanisms involved
in mechanosensing and mechanotransduction (33). Recent observations
suggest that lung stretch induces local and/or systemic cytokine
release (23), yet it remains unclear if the proinflammatory
deformation response is a consequence of cellular stress failure
(34) or of stretch-mediated signal transduction. Integrated
studies examining molecular pathways, individual cellular
responses, and responses of the intact lung will be required
to determine how mechanical ventilation modifies the dynamic
interactions of lung cells in ALI/ARDS.
The effect of the mechanical stresses applied to the lung
may extend far beyond the thoracic structures to systemic
organs. Recent studies have demonstrated that mechanical ventilation
can release mediators (35) that may translocate into the systemic
circulation (23). This may contribute to the high prevalence
of multiple organ failure in patients with ALI/ARDS. This
hypothesis was strengthened by the recent low Vt study that
demonstrated a systemic component in ALI/ARDS that was altered
by the ventilatory strategy (5). Injurious ventilatory strategies
can lead to loss of compartmentalization in the lung and may
cause translocation of mediators, endotoxin, and bacteria
(36, 37) from the lung to the systemic circulation. Examination
of the physiological, biological, and genetic basis of stress-induced
injury to the lung in health and disease is thus a fertile
area of future research.
There is a lack of understanding of how cells of the innate
immune system (neutrophils, monocytes, macrophages, natural
killer cells, dendritic cells, and others) and cells involved
in hemostasis and thrombosis (platelets and endothelial cells,
together with interacting leukocytes) are dysregulated in
ALI/ARDS. Although it was originally believed that increased
permeability pulmonary edema and other features of ALI/ARDS
could be explained by endothelial and epithelial alterations
alone, it is now clear that acute inflammation, thrombosis,
and activation of the coagulation system are involved. There
is evidence that dysregulation of innate immune and thrombotic
cascades contribute to both the initiation and progression
of ALI/ARDS (1, 38). Recent studies of human leukocytes and
platelets have identified previously unrecognized surface
receptor interactions, intracellular signaling pathways, mechanisms
of gene product expression, and functional responses that
are directly relevant to ALI/ARDS and to triggering conditions
such as sepsis (39-43).
The toll-like receptors of innate immune cells and endothelial
cells, which recognize pathogen-associated molecular patterns
in LPS and other microbial factors, are another group of factors
that are critical to inflammation and injury but were only
recently discovered. Toll-like receptors are linked by intracellular
signal transduction cascades to activation of the nuclear
factor-kB family of transcription factors and induction of
tumor necrosis factor alpha and other nuclear factor-kB-dependent
gene products. Their engagement is central to host defense
and sepsis, septic shock, and their complications including
ALI/ARDS (44). The recent explosion of information regarding
toll-like receptors illustrates the point that much remains
unknown regarding cellular responses and molecular signaling
in the innate immune system. Precise understanding of compartmentalized
versus dysregulated systemic inﬂammation will depend
on additional insights into the biology of these cells and
the molecular systems that regulate them, including biologically
active lipids, chemokines, and cytokines.
Thrombosis and Inflammation
Additional knowledge regarding the mechanisms that link
acute inflammatory responses to thrombosis and to activation
of the coagulation cascade is also important because there
is substantial evidence that microvascular thrombosis and
dysregulated intracellular and extracellular fibrin deposition
are early events in ALI/ARDS and in experimental ALI (1, 45).
Recombinant activated protein C, recently approved for treatment
of severe sepsis, interrupts both inflammatory and thrombotic
responses (10, 46, 47). Recombinant platelet activating factor
acetylhydrolase also may reduce mortality and organ failure
in sepsis and trauma and diminish inflammatory and thrombotic
signaling (47). The development of both of these therapies
can be traced to fundamental cell and molecular biology experiments
in parallel with preclinical animal studies.
FUNCTIONAL GENOMICS, PROTEOMICS, AND OTHER NEW METHODS
Modern approaches to cell and molecular biology provide
significant opportunities to generate new information relevant
to ALI/ARDS. In many cases, these methods can be applied or
adapted to experiments with lung or other complex tissues
as well as with isolated primary or surrogate cells and can
be used with appropriate modification in analysis of clinical
samples. Thus, there is the potential for rapid integration
of basic and clinical data in a translational fashion. Genomic
approaches using microarrays and other methods to display
multiple DNA sequences have rapidly become major tools in
biologic investigation and are increasingly being applied
in critical care medicine. Microarray analysis can be applied
to isolated cells and normal and diseased tissues (48-50).
Laser capture microdissection, real time polymerase chain
reaction, and additional analytic techniques enhance the power
of this technology (51). "Functional genomic" approaches are
increasingly being applied to questions in pulmonary and critical
care medicine (52) and have great potential to address many
unanswered questions related to changes in phenotypes of key
cells in ALI/ARDS. For example, injury mechanisms mediated
by release of granular enzymes or oxygen radicals by neutrophils,
aggregation of neutrophils, monocytes and platelets, and activation
of the clotting cascade use constitutive biochemical pathways
and do not require induction of new transcripts or expression
of new proteins. Furthermore, transcript abundance does not
unequivocally reflect abundance of corresponding proteins
or the physiological results. Critical messenger RNAs are
under stringent posttranscriptional control in many cases.
Constitutively expressed but repressed messenger RNAs can
be translated to proteins in response to cellular signals
that are relevant to ALI/ARDS and its systemic manifestations
(42, 43, 53). Thus, studies of acute constitutive cellular
and functional responses should not be abandoned in wholesale
fashion but should be used in a complementary fashion with
gene analysis strategies.
Evolving proteomic technologies have the potential to identify
each protein expressed in isolated cells or in complex tissues
such as injured lung and also to determine posttranslational
modifications, protein-protein interactions, and other critical
features that define phenotype and regulate cellular function
(54). Proteomic approaches can provide physiologic relevance
that is not inherent in measuring transcript levels and profiles
(53). New proteomic strategies based on isotope-coded affinity
tags can accurately quantify individual proteins in complex
mixtures, a capability that has been lacking in the past,
and can also provide sequence information linking descriptive
and quantitative proteomics (54). Proteomic analysis of clinical
samples may be useful in establishing molecular patterns that
are characteristic of individual diseases or disease stages
The most effective use of proteomic strategies, as with genomic
technology, will be in combination with traditional approaches
to determine the functions of key proteins and their effects
on cell and tissue behavior. In many cases these experimental
strategies will require new reagents, databases, and bioinformatic
tools to handle large amounts of complex data (50, 51, 54)
GENETICS OF ALI/ARDS
A major gap in our knowledge is the extent to which genetic
features interact with environmental variables to inﬂuence
susceptibility to ALI/ARDS. In sepsis, trauma, and other
triggering conditions, only a subset of patients develops
ALI/ARDS even among those in whom the pathologic stimuli are
apparently equivalent (1, 3, 5, 11), suggesting that there
are genetic features that may influence its onset. Similarly,
we do not know what genetic features influence the natural
history of ALI/ARDS once it is established or how genetic
variables interact with environmental factors to determine
resolution and repair versus persistence and progression of
acute lung inflammation and injury. Furthermore, we have little
understanding of genetic features that regulate compartmentalization
of inflammation and injury in one case and engender a systemic
response in another (19).
Human genetic syndromes are commonly classiﬁed as
simple Mendelian disorders or as multifactorial complex diseases.
In Mendelian diseases, a mutation in one or both alleles of
a single disease gene leads to a pathologic phenotype, whereas
complex genetic syndromes are generally caused by mutations
in more than one gene with significant contributions from
the environment usually influencing the manifestations of
the disease. Additional features of complex genetic diseases
include large phenotypic variation, incomplete penetrance,
late age of onset, the potential for locus heterogeneity,
and lack of easily identified large families because of a
complicated pattern of heritability. The frequency of alterations
in disease genes is variable in multi-factorial disorders,
potentially making them difficult to identify. Recent identification
of alleles of the NOD 2 gene as variants that predispose
to Crohn's disease (56, 57) illustrates many of these points.
Complex human disorders may also result from multiple rare
variant mutations in a single gene leading to similar or identical
phenotypes (58). Finally, many traits of clinical significance
in complex diseases have continuous variation rather than
discrete phenotypes; interacting genes that influence variation
in these traits are located in chromosomal regions termed
quantitative trait loci. Mapping quantitative trait loci to
identify genes involved in polygenic traits has been used
for analysis of complex human diseases, including asthma,
arteriosclerosis, and diabetes (59).
In most cases, susceptibility to ALI/ARDS, and the natural
history of these syndromes once they are established, is likely
governed by genetic features together with environmental variables
in the complex fashion outlined previously. Studies of large
families, evaluation of large archives of small families,
and analysis of affected sibling pairs are major gene discovery
approaches that have been used in asthma and other complex
diseases; however, these investigational tools are not available
for ALI/ARDS. Furthermore, there is substantial heterogeneity
in ALI/ARDS (see next section), and there are no unique, easily
measured markers 1031 or combinations of markers that can
yet be used to define the syndromes in general or to more
precisely characterize subsets or phenotypes (60). Heterogeneity
is a particularly confounding problem in establishing genetic
and environmental contributions to complex diseases.
There is suggestive evidence for genetic variations in ALI/
ARDS and in predisposing conditions such as sepsis. Polymorphisms
in genes related to inflammatory markers (tumor necrosis factor
alpha, surfactant proteins, and interleukin-6), angiotensin
converting enzyme, and pathogen receptors (CD14, toll-like
receptors) appear to correlate with incidence and/or outcome
of sepsis (61, 62) and/or ALI/ARDS (63-65). These observations
used case-control association studies. Although association
studies pose significant logistic challenges in critically
ill patient populations and have limitations, they are the
most immediately applicable approach to ALI/ARDS. Family-based
association studies and case-controlled association studies
may be beneficial to explore in well-defined subgroups of
patients with ALI/ARDS.
Positional cloning has been used with success in the study
of complex diseases. However, availability of the sequence
of the human genome and new technologies that link gene discovery
to biologic function will likely provide accelerated insight
into multifactorial syndromes such as ALI/ARDS and will use
new approaches (66). These include identification and establishment
of the significance of single-nucleotide polymorphisms and
other variations in the human genome; methods to further characterize
quantitative trait loci; identification of new pathways of
cell function and regulation and refined analysis of known
metabolic pathways based on coregulated genes; new analysis
of mechanisms of transcriptional and posttranscriptional regulation,
protein-protein interactions, and cross talk between signaling
cascades; genotype-phenotype correlations that identify gene-gene
interactions and other modifying features that lead to variations
in the clinical phenotypes; and new approaches to dissecting
interactions between genes and environment involving epidemiologic
studies of carefully characterized populations. Robust databases
that link disease records, family records, and other data
sets that can be integrated for biomedical research will be
important tools. Application of the new genetic approaches
will also require parallel refinement of the clinical phenotypes
of diseased patients and control populations, a particular
challenge in the case of ALI/ARDS.
Postgenomic approaches will continue to depend on genetically
modified mice and other model systems. Mapping of traits,
generation of knockouts and transgenics, and mutational strategies
with genome-wide analysis are highly tractable in mouse models
and can circumvent some of the problems of human patient populations,
including heterogeneity. Genetic and genomic approaches using
mice and other model species have promise for dissection of
the complexity of cardiovascular diseases (67) and other disorders.
Some of these approaches have recently been applied in mouse
models of lung injury (68).
HETEROGENEITY OF PHENOTYPE IN ALI/ARDS
The heterogeneity of inciting factors and outcomes in ALI/ARDS
creates complexity and uncertainty in the study of this syndrome.
In order to better understand specific mechanisms of ALI under
different clinical conditions as well as to probe genetic
and environmental influences, it is important that more information
be obtained regarding characterization of subgroups of patients
with ALI/ARDS. For example, the incidence, natural history,
and outcome of ALI/ARDS induced by sepsis and trauma are quite
different in some senses, but the factors contributing to
this heterogeneity are not clear (1).
Patients dvelop pneumonia from several different etiologies,
including gram-positive or gram-negative bacteria, viruses,
or even fungi or parasites, particularly in the presence of
immunosuppression, and the signaling pathways that regulate
the inflammatory response to any particular organism depend
on the initial molecular interactions between the host cells
and the organism (39, 44). A better understanding of bacterial
genetics and the specific virulence factors of infecting bacteria
should provide new approaches to understanding susceptibility
to pneumonia and ALI/ARDS both as a cause of injury and as
a cause of nosocomial pneumonia in patients with ALI/ARDS
(17). A better understanding of why some patients with sepsis
develop ALI is needed. Is there a host susceptibility to sepsis
so that patients who are more likely to upregulate gene expression
for proinflammatory cytokines are therefore more likely to
develop ALI? This hypothesis is plausible but other factors
may also be involved, such as the ability of the host to confine
an infection to a localized area. For example, the ability
to mount a local proinflammatory response may be important
in containing bacterial infection to one portion of the lung,
thus preventing dissemination of cytokines and bacterial products
to the circulation that could result in multiple organ failure
and diffuse lung injury if not locally contained (19). A more
detailed understanding of the pathogenesis of clinical lung
injury, including biochemical markers, studies of lung pathology,
and integrated studies using genomics and proteomics may provide
more insight into individual patient susceptibility and potentially
identify ALI/ARDS subgroups. It is possible that one or more
speciﬁc markers may apply to one phenotypic ALI/ARDS
subgroup but not to another. Similar approaches are being
used in other human diseases (55).
CLINICAL AND BIOCHEMICAL PREDICTORS OF ALI
The search for biochemical and clinical predictors or markers
of ALI has been hampered by a number of factors, including
(1 ) the lack of correlation between the clinical diagnosis
and the pathogenesis, (2 ) the recognition that patients at
risk for and with ALI are heterogeneous, (3 ) the continuous
discovery of new mediators and modulators of inﬂammation,
(4 ) the recognition that the development of ALI is likely
the result of a balance of mediators and modulators of inflammation,
and (5) the increasing awareness of the importance of correlating
biochemical markers with physiologic variables. Although the
recognition of these factors has complicated the quest for
markers in ALI/ARDS, each of these factors provides unique
new opportunities for their study.
Patients with a higher severity of illness score are more
likely to develop ALI/ARDS and die of lung injury. However,
the search for a pulmonary-specific variable has been challenging.
Indices of hypoxemia have not been predictive of clinical
outcome (5). One recent study established that an elevated
dead space fraction has an independent predictive power for
identifying patients more likely to die (13). This finding
needs to be explored in conjunction with detailed biochemical
studies and other methods to determine the pathologic and
physiologic basis for a high dead space fraction. Procoagulant
mechanisms are activated both in the circulation and the distal
airspaces of the lung in patients with early ALI and may ultimately
provide markers of alveolar capillary damage or vascular occlusion
(45). More work is needed to evaluate products of endothelial
injury as well as mechanisms that reflect endothelial, neutrophil,
and thrombotic interactions. Markers of endothelial injury
such as von Willebrand factor that reflect pulmonary and systemic
endothelial injury may prove useful.
Although measurement of soluble biochemical markers in plasma,
edema fluid, and bronchoalveolar lavage fluid has provided
insights into mechanisms of lung injury (69), they may not
necessarily reflect critical molecular events at the cell
surface. In juxtacrine signaling between cells, the signaling
molecule remains associated with cellular plasma membranes
and is not released into solution (27). Thus, it may not be
detected in bronchoalveolar lavage or other fluid samples
and/or its concentration in fluid samples may bear little
relationship to its signaling actions. Also, antigenic measurement
of cytokines in edema fluid or bronchoalveolar lavage may
not correlate well with biological activity. Both interleukin-beta
and tumor necrosis factor alpha can be detected by immunoassays
in the distal airspaces of the lung in patients with early
ALI, but interleukin-1-beta has more biological activity than
tumor necrosis factor alpha (70).
ANIMAL MODELS OF LUNG INJURY
Animal studies that have attempted to mimic human ALI/ARDS
have been useful and will likely continue to provide valuable
observations regarding both the mechanisms underlying the
pathogenesis, progression, and resolution of this syndrome
and ways in which its course can be modulated therapeutically
(39, 68, 7176). The lack of an animal model that unequivocally
mimics key aspects of human ALI/ARDS has been limiting in
mechanistic studies and in providing meaningful and rapid
extrapolations to the clinical syndrome. Furthermore, there
is uncertainty as to which of the many available animal models
best reflects the human clinical syndrome. Animal models are
usually monitored over a shorter term than the human syndrome,
which requires hours or days to develop. Most animal models
do not include ventilation and fluid management interventions,
features that may be crucial factors in determining outcome
in humans. A major question is whether the pathogenesis of
human ALI/ARDS varies with risk factors, such as trauma and
sepsis. This has led to studies comparing and contrasting
the mechanisms of injury in animal models of endotoxin and
hemorrhagic shock-associated ALI (71). Administration of LPS
alone does not completely mimic the systemic or pulmonary
effects of bacteremia or endotoxemia, contributing to the
use of more complex models such as cecal ligation and puncture
(72). Furthermore, many workshop participants suggested that
two-hit models (73-76) might be more appropriate to reflect
common comorbidities and risk factors commonly present in
these patients. Continued study of the heterogeneity of precipitating
causes of ALI/ARDS is needed.
The traditional knockout and transgenic approaches to delete
or add a gene will remain useful, but more sophisticated aproaches
to modify genes and to conditionally express or knock out
genes are needed to better understand the role of particular
molecules and pathways (67). In combination, these studies
will elucidate interactions and redundancies in pathways and
molecular functions. Furthermore, cell-and organ-specific
approaches, either through bone marrow reconstitution or selection
and manipulation of promoter function, will also prove valuable
for understanding the complexities of cell function and interaction
in the lungs and to provide potential therapeutic targets.
Furthermore, animal studies can also facilitate understanding
of the heterogeneity of phenotype using chemical mutagenesis
or gene mapping and linkage studies in mice of specific genetic
backgrounds that display variable responses to experimental
lung injury. Finally, the availability of the rat genome will
be extremely useful (77), as opportunities for comprehensive
physiologic studies of lung injury are better in rats than
Animal models that facilitate "fast track" analysis of therapeutic
leads and combinations of therapeutic agents would be expected
to accelerate clinical application of basic discoveries. One
approach that may prove useful for identifying clinical targets
would involve gene profiling and proteomic evaluation of tissue
from one or more animal models of lung injury that reﬂect
phenotypic subgroups of ALI/ARDS. The effect of agents having
the potential to diminish the severity of systemic and pulmonary
injury on genomic and proteomic variables could then provide
mechanistic insights to determine if similar pathways were
being altered in human ALI/ARDS and help to narrow the field
of candidate genes for human ALI/ARDS. Animal models could
and should focus on both systemic and pulmonary injury and
may also provide insights about the interactions between them.
For example, recent studies of an intraperitoneal sepsis model
in rats using a limited gene microarray revealed interesting
results regarding the host response in lung, kidney, brain,
and lymphoid tissues (72). Studies in animal models with clinical
relevance to ALI/ARDS would facilitate the design of clinical
trials focused on diminishing clinical lung injury and the
associated nonpulmonary organ dysfunction, including renal
failure and hepatic and hematologic dysfunction, as well as
the consequences of shock states produced by sepsis and trauma.
CONCLUSIONS AND RECOMMENDATIONS
There are major unanswered questions about ALI/ARDS that
will require continued research efforts at the basic, translational,
and clinical levels. Recent successes in the treatment of
critically ill patients provide an excellent opportunity for
rapid progress, as do new and evolving biologic, genomic and
genetic approaches. Investigations and collaborative efforts
that include cellular and mechanistic studies combined with
animal and clinical studies will be the key to improving detection
and treatment of this complex syndrome.
- Continued support for high quality collaborative multi-center
clinical trials, as well as individual investigator-initiated
studies, is crucial for continued progress in prevention
and treatment of ALI/ARDS. Studies of multiple drug treatments
for this complex syndrome may be needed. Future studies
could be broadened to include common comorbid conditions
such as renal failure and sepsis. Support for mechanistic/pathogenic
investigations in conjunction with clinical trials is essential.
The smaller clinical investigations conducted at single
centers continue to have value because of complex patient
populations and to test new treatments for evidence to support
further evaluation in larger multicenter trials.
- Strategies for robust and facile analysis of genomic
and proteomic data from studies in animal models, including
genome sequencing databases, tissue-and species-specific
microarrays, online archiving of serial microarray and other
data, and other resources should facilitate translation
of animal experiments to clinical relevance.
- Studies that correlate biochemical and biological markers
with clinical variables should continue in order to address
the issue of heterogeneity, to more precisely define the
phenotypes of patients with ALI/ARDS, to permit genetic
analysis, and to facilitate clinical trials of new candidate
therapies. Panels of biological and biochemical markers,
in conjunction with clinical indices, may be needed to generate
a prognostic index in ALI/ARDS. Markers that reflect systemic
events (coagulation and inflammatory markers) as well as
those that reflect alterations in lung endothelial and epithelial
cells and fibrotic processes should be included. Pulmonary
edema fluid, circulating cells, DNA samples, and plasma
should be obtained in clinical studies.
- Accrual and collections of well-characterized lung tissue
samples from patients with ALI/ARDS will facilitate application
of modern analytic techniques (laser capture microdissection,
microarray interrogation, proteomic analysis) to define
the molecular phenotype of key lung cells and structures.
- Future research should build in part on the recently
demonstrated new treatments for ALI/ARDS and sepsis. More
basic research is needed to detail the cellular, molecular,
and physiologic mechanisms that protect the lung from biomechanical
stress and injury and potentially modulate systemic inflammation.
More studies are needed to explore the role of the inflammatory
and coagulation pathways in inducing organ injury in sepsis
and other injury states and in modulating ALI. Both basic
and clinical studies are needed. Although ARDS is a uniquely
human disease, animal models are relevant to several different
features of clinical ALI. Better understanding is needed
to determine which animal models are most relevant to the
human disease. "Two hit" models may better reflect comorbid
conditions and other pre-existing variables and may be more
informative in dissecting the roles of the innate immune
system and specific mechanisms of lung injury. Animal models
that focus on systemic as well as pulmonary injury may prove
to be particularly relevant to human ALI. Genetically altered
mouse models will also be useful. Lung-and tissue-specific
conditional knockouts, overexpression of candidate genes,
and other genetic models should be of value in dissecting
the host response to pathological conditions that model
human lung injury.
- Because infection is the most frequent cause of ALI/ ARDS,
the influence of bacterial and viral genetics on host response
that influence lung injury needs to be better understood.
- Research to study and improve the resolution of ALI,
including mechanisms to modulate clearance of edema fluid,
soluble and insoluble proteins, inflammatory cells, and
remodeling of the lung is needed. The potential for enhancement
of lung repair with stem cell therapy should be explored.
- More emphasis is needed on understanding cell-to-cell
communication in the lung, including the mechanisms that
transmit signals between endothelial and epithelial cells
and mechanisms of cell-to-cell interaction in inflammatory
and thrombotic responses. The signals and mechanisms that
determine normal cell turnover and influence apoptosis and
necrosis in lung injury need to be elucidated. Advanced
imaging modalities, such as microcomputerized tomography
and confocal microscopy, should be refined to generate data
on cell and parenchymal microstrains in intact lungs, so
that molecular research on mechanosensing and mechanotransduction
can be interpreted in an appropriate context. Similarly,
refined imaging techniques that are applicable to inflammatory
cell trafficking and cell-cell interactions will likely
- Studies that use cell biological approaches including
single and multiple cell type cultures should be integrated
with animal and human studies.
- More knowledge is needed to study the extent to which
genetic factors inﬂuence the development, severity
and time course of development and resolution of ALI/ARDS
as well as the development of multiple organ failure and
other systemic sequelae. Development of genetic tools and
resources specifically relevant to ALI/ARDS, triggering
conditions, and systemic manifestations will likely facilitate
characterization of genetic and environmental variables
that influence ALI. Case-control association studies, although
of lesser power, may be useful.
- Interaction of the new NHLBI Specialized Centers of Clinically
Oriented Research programs in ALI with the ARDS clinical
trials network should be encouraged. Progress will be greatly
enhanced with vertically and horizon tally integrated collaborative
Acknowledgment : The authors appreciate the assistance of
Rebecca Cleff and Beth Chernoff in the preparation of this
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Correspondence and requests for reprints should be addressed
Andrea L. Harabin, Ph.D.
Division of Lung Diseases
National Heart, Lung, and Blood Institute
6701 Rockledge Drive, Suite 10018, MSC-7952
Bethesda, MD 20892-7952