Future Research Directions in Acute Lung Injury
NHLBI Workshop Summary
Summary of a National Heart, Lung, and Blood
Institute Working Group
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 complex disease.
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 (1).
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 species?
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 ALI/ARDS.
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 therapy.
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.
Lung Epithelium
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 well understood.
Lung Endothelium
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 with ALI/ARDS.
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 the lung.
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.
Innate Immunity
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 (55).
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 in
mice.
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 be informative.
- 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 studies.
Acknowledgment : The authors appreciate the
assistance of Rebecca Cleff and Beth Chernoff in the preparation of this
manuscript.
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Correspondence and requests for reprints should be
addressed to: 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 E-mail:
harabin@nih.gov
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