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1. Introduction
2. Anti-cytokine therapy
3. Endothelial targets
4. Coagulation factors
5. Growth factors
6. Gene therapy
7. Mesenchymal stem cells
8. Expert review
Review
Biological therapies in the acuterespiratory distress syndromeAndrew James Boyle, James Joseph McNamee & Daniel Francis McAuley†
†Queen’s University Belfast, Centre for Infection and Immunity, Belfast, UK
Introduction: The acute respiratory distress syndrome (ARDS) is characterised
by life-threatening respiratory failure requiring mechanical ventilation, and
multiple organ failure. It has a mortality of up to 30 -- 45% and causes a
long-term reduction in quality of life for survivors, with only approximately
50% of survivors able to return to work 12 months after hospital discharge.
Areas covered: In this review we discuss the complex pathophysiology of
ARDS, describe the mechanistic pathways implicated in the development of
ARDS and how these are currently being targeted with novel biological
therapies. These include therapies targeted against inflammatory cytokines,
mechanisms mediating increased alveolar permeability and disordered
coagulation, as well as the potential of growth factors, gene therapy and
mesenchymal stem cells.
Expert opinion: Although understanding of the pathophysiology of ARDS has
improved, to date there are no effective pharmacological interventions that
target a specific mechanism, with the only potentially effective therapies to
date aiming to limit ventilator-associated lung injury. However, we believe
that through this improved mechanistic insight and better clinical trial design,
there is cautious optimism for the future of biological therapies in ARDS, and
expect current and future biological compounds to provide treatment options
to clinicians managing this devastating condition.
Keywords: acute hypoxaemic respiratory failure, acute lung injury, acute respiratory distress
syndrome, biological therapies
Expert Opin. Biol. Ther. [Early Online]
1. Introduction
Despite recognition nearly 50 years ago [1], the acute respiratory distress syndrome(ARDS) has yet to lend itself to an effective pharmacological treatment [2]. Thedefinition of ARDS has recently been updated [3], with the term acute lung injuryno longer recognised, and instead ARDS is now divided into three categories basedon severity. ARDS causes significant mortality and morbidity, and is associated witha major health care burden. The long-term morbidity to patients is significant, withsurvivors experiencing significantly reduced exercise tolerance 5 years after intensivecare unit discharge as well as other physical and psychological sequelae [4]. There arelimited treatment options available; the mainstay of care is lung-protective mechan-ical ventilation [5] with other strategies aimed at limiting ventilation-associated lunginjury [6,7]. Despite improved compliance with lung-protective ventilation [8],mortality from ARDS has remained stable since 1994 [9], highlighting the needfor new therapies for ARDS.
ARDS is an inflammatory condition characterised by neutrophil [10] andmacrophage-mediated [11] injury, with excessive pro-inflammatory cytokine andprotease activity in the alveolar space, which is associated with alveolar epithelialand capillary endothelial injury. During this inflammatory process, the coagulationcascade is activated, producing micro-thrombi and promoting fibrin deposition thatfurther exacerbates the disordered alveolar structure [2]. Protein-rich inflammatory
10.1517/14712598.2014.905536 © 2014 Informa UK, Ltd. ISSN 1471-2598, e-ISSN 1744-7682 1All rights reserved: reproduction in whole or in part not permitted
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infiltrate accumulates within the alveolar space causingnon-cardiogenic pulmonary oedema, which impairs gaseousexchange.There is temporal overlap between this acute inflammatory
phase and the subsequent alveolar repair phase and how theacute inflammatory response interacts with the repair stageis largely unknown. As one example, temporal changes inMMPs, a group of proteases upregulated in ARDS, can pre-dict resolution of pulmonary oedema suggesting that proteaseactivity in the lung at some time points could contribute toalveolar-capillary repair [12,13]. As a result, biological therapiesthat can limit the severity of the initial inflammatory insultcould modify the process of cellular repair at a later timepoint.To date, the majority of pharmacological treatment
strategies that have been investigated have aimed to attenuatethe inflammatory response or improve the resolution ofpulmonary oedema without specific reference to timing [14].However, as ARDS is characterised by these overlapping acuteinflammatory and alveolar reparative phases, timing oftreatment may be more important than has been previouslyidentified, to permit targeting of the appropriate pathogenicprocess. This may, in part, explain the failure to establish aneffective therapy.As scientific understanding of the mechanisms underpin-
ning ARDS develops, a stratified medicine approach may bemore appropriate with biological therapies targeting processesexpressed either at a particular point in the course of theillness or in specific subgroups and this is increasingly possi-ble. Subsequent interest in biological therapies has led tonumerous potential therapies, and in the following review,the evidence to support a role for these biological agents is
presented. For the purpose of this review, a biological therapyis defined as a therapy of biological origin, that is, notchemically synthetic. We provide a summary of the biologicaltherapies discussed in Table 1.
2. Anti-cytokine therapy
2.1 IL-8IL-8 is a pro-inflammatory cytokine that plays a major role inthe recruitment, activation and influx of neutrophils to thealveolar space [15]. Neutrophil accumulation within the lungis an early pathogenic process in ARDS [10], and IL-8 is foundin significantly higher levels in broncho-alveolar lavage (BAL)fluid of at-risk patients who develop ARDS compared withthose who do not [16]. The importance of IL-8 to ARDS issupported by data showing higher BAL concentration ofIL-8 correlates with increased mortality [17,18].
In ARDS, a large proportion of IL-8 is bound to ananti-IL-8 IgG autoantibody [19], and this complex can interactwith the FcgRIIa receptor expressed by neutrophils to delayapoptosis [20] and prolong the inflammatory process. In addi-tion, IL-8:anti-IL-8 complexes have been shown to depositwithin the lung tissue itself in ARDS [21], which may act asa pro-inflammatory mediator to further the pathological pro-cess [22]. Indeed, signalling through the FcgRIIa receptor, andto a lesser extent the IL-8 receptor, has been shown to increaseneutrophil chemotaxis in ARDS [23]. The scientific rationalefor IL-8 as a key mediator in ARDS is supported clinicallywith evidence of higher levels of complexed IL-8 in patientswho die from ARDS [22].
The potential role for monoclonal antibodies that block theaction of IL-8 has been investigated in animal models ofARDS. Intravenous humanised anti-IL-8 monoclonalantibody was shown in a lipopolysaccharide (LPS) model ofARDS to reduce alveolar neutrophil infiltration, an effect inpart explained by reduced levels of BAL IL-1b and TNF-a.Histological scoring (evaluating neutrophil infiltration,capillary congestion, fibrin clot deposition, epithelial celldesquamation and alveolar septum width) was improved inthe anti-IL-8 treatment group [24]. This data suggest theremay be a role for anti-IL-8 therapy in ARDS; however,human studies are awaited and currently it is difficult to assesshow effective a therapy may be for human intervention.
2.2 TNFTNF is present in two forms, a and b, and both have an earlypathogenic role in ARDS, with their presence in plasmaassociated with the onset of ARDS during sepsis [25]. TNF-ais secreted by activated tissue macrophages and mediates theattraction and activation of neutrophils within the alveolarspace [26]. TNF-a mediates its effect through two cell-surfacereceptors, TNF-receptor (TNFR)-1 and TNFR-2, withTNFR-1 thought to primarily mediate pro-inflammatoryeffects. In the setting of murine models of ARDS, TNFR-1signalling has been shown to promote ventilator-induced
Article highlights.
. Acute respiratory distress syndrome (ARDS) causessignificant morbidity and mortality; the pathophysiologyis complex and to date, there is no effectivetherapeutic intervention.
. Many pharmacological interventions have beeninvestigated without success; biological therapies areincreasingly recognised as potential future therapies.
. Encouraging pre-clinical data has often been followedby disappointing results in clinical trials; this likelyreflects both the limitations of pre-clinical models andthe complex pathophysiology underlying ARDS andpossibly a need for improved clinical trial designin ARDS.
. Timing of therapies is of increasing importance, withtreatment targets often expressed at different periods ofillness in ARDS. Biological therapies may have anadvantage in this regard as they can actmore specifically.
. There is cautious optimism for the role of biologicaltherapies in ARDS, and in particular stem cell therapy, inthe management of this complex disorder.
This box summarizes the key points contained in the article.
A. J. Boyle et al.
2 Expert Opin. Biol. Ther. (2014) 14(7)
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Table
1.Summary
ofbiologicaltherapiesin
ARDS.
Biological
substrate
Mech
anismsin
ARDS
Therapeuticagent
Phase
ofclinical
development
Resu
lts
IL-8
Neutrophilrecruitmentto
thealveolar
space
Humanisedanti-IL-8monoclonal
antibody
Pre-clinical[24]
Reducedhistologicalevidence
ofARDS
TNF-a
Secretedbymacrophagesto
attract
neutrophils
toinjuredalveolus
Actionmediatedviapro-inflammatory
TNF-receptor1
Inhaledhumanisedanti-TNFR1
monoclonalantibodies
Phase
I(NCT01587807)
Resultsawaited
CD14
Cellsurface
receptorforLPSon
macrophagesandneutrophils
Inducespro-inflammatory
cytokinerelease
(e.g.,TNF-a)
Anti-CD14monoclonalantibodies
Pre-clinical[38]
Reductionsin
pulm
onary
oedema,
neutrophilmigrationandTNF-a
production
Phase
I(NCT00233207)
Term
inateddueto
poorrecruitment
CD73
Rate-lim
itingglycoprotein
inadenosine
synthesis.
Adenosineisprotectivein
hypoxia,stim
ulatinganinnate
response
toinhibitadaptive
immunecells
IFN-b
(increasesCD73synthesisin
lungendothelialcells)
Phase
I/II[55]
Reducedmortality
ACE2
Promotesbreakdownofinjurious
AngiotensinII
RecombinantACE-2
Phase
I/II
(NCT01597635)
Resultsawaited
Adrenomedullin
Reducesvascularperm
eability
RecombinanthumanAdrenomedullin
Pre-clinical[67,68]
Reducedlunginjury
severity
TissueFactor
Initiatesextrinsiccoagulationcascade,
inducingpro-coagulantstate.Thiscan
propagate
fibrindeposition
Recombinantantibodyto
tissuefactoror
tissuefactor--factorVIIIcomplex
Phase
II(NCT00879606)
Resultsawaited
Activated
Protein
CReducedlevelsin
ARDS
Whenactivatedisapotentanti-coagulant
Syntheticactivatedprotein
CPhase
II[77]
Nosignificanteffect
upon
ventilator-freedays
Keratinocyte
growth
factor
PromotesalveolartypeIIepithelialcell
repair
Augments
AFC
RecombinanthumanKGF(paliferm
in)
Phase
II[85]
Resultsawaited
VEGF
Increasesperm
eability
ofalveolar-capillary
membrane
Bevacizumab(humanisedanti-VEGF
monoclonalantibody)
Phase
II(NCT01314066)
Term
inateddueto
poorenrolm
ent
GM-CSF
Augments
neutrophillifespanand
phagocytosis
Reducesalveolarepithelialcellapoptosis
RecombinanthumanGM-CSF
Phase
II[96]
Nobenefitin
ventilator-freedays
or
28-daymortality
GeneTherapy
Multiple
potentialtargets
Currenttherapieshave
targetedAFC
improvements
viaNa+,K
+-ATPase
andthe
b2-adrenergic
receptor
Vector-carriedgeneticmaterial
Pre-clinical
Improvements
inAFC
Stem
Cell
Cellcontact-dependentandindependent
mechanismsSecrete
soluble
mediators
(e.g.,antimicrobialpeptidesandgrowth
factors)Im
provedcellbioenergetics
Mesenchym
alstem
cells
Phase
I(NCT01775774,
NCT01902082)
Resultsawaited
AFC
:Alveolarfluid
clearance;LPS:lipopolysaccharide;KGF:
Keratinocyte
growth
factor;TNFR1:TNF-receptor1.
Biological therapies in the acute respiratory distress syndrome
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lung injury, with increases in pulmonary oedema, whileTNFR-2 signalling induces a protective effect [27]. Plasmalevels of both TNFR-1 and TNFR-2 act as biomarkers forthe activity for TNF-a during ARDS, and higher plasma levelsof both receptors are independently associated with anincreased mortality from ARDS [28].These data led to investigation of anti-TNF-a agents as a
therapeutic intervention for ARDS. Animal data showedthat pre-treatment with anti-TNF-a monoclonal antibodiesattenuated the development of ARDS with reductions inneutrophil migration across the endothelial border into thealveolar space [29]. Further animal studies using polyclonalanti-TNF-a antibodies highlighted benefits to gas exchangeand lung compliance [30]. Investigation of anti-TNF agentsin humans has been less effective, with two large human trialsshowing no benefit of anti-TNF therapy in the setting ofsepsis, a common cause of ARDS [31,32]. No clinical trialsspecifically investigating anti-TNF therapy in patients withARDS have been undertaken to date.However, more recent research has focussed on the differen-
tial effect of blocking TNFR1, aiming to reduce the pro-injurious impact of TNF-a, while simultaneously maximisingthe protective effects of signalling via TNFR2. Treatment withanti-TNFR1 monoclonal antibodies in an animal model ofARDS reduced deterioration in respiratory function andreduced alveolar-epithelial permeability [33]. An early-phaseclinical trial in a model of ARDS induced by LPS in healthyvolunteers, investigating inhaled antibodies against TNFR1,has recently concluded (NCT01587807) and results areawaited. If successful further investigation will need to focusearly in the course of ARDS, as the characteristics of TNFR1suggest that it is important in the early stages of ARDS butmay not have as much impact in later stages of illness.
2.3 CD14LPS is a major component of the cell wall of gram-negativebacteria, and it is recognised by host macrophages and neutro-phils through the cell surface receptor CD14 [34]. This processrequires a soluble protein, LPS-binding protein, before LPScan interact with CD14 [35]. Once activated, CD14 inducesintra-cellular signalling for the production of pro-inflammatorycytokines, including TNF-a, IL-1 and IL-6 [36], driving thepro-inflammatory response.In patients with ARDS, soluble CD14 is related to BAL total
protein (a measure of alveolar capillary permeability) andneutrophil count, but not clinical outcome [37]. Investigationof CD14 as a therapeutic target has utilised monoclonalantibodies antagonising the cell surface receptor. The use ofanti-CD14 monoclonal antibodies in murine LPS-models ofARDS reduced pulmonary oedema, neutrophil migration andTNF-a production by macrophages, supporting a potentialtherapeutic role in ARDS [38]. A human trial investigating therole of monoclonal antibodies against CD14 was terminatedin 2007 due to poor patient recruitment (NCT00233207).While these data suggest that CD14-dependant mechanisms
contribute to LPS-induced inflammation in ARDS, given therelatively weak scientific data and that this only targets ARDSwhere LPS is implicated in the development, we do not expectfurther investigation of CD14 as a therapeutic option in theforeseeable future.
2.4 Additional cytokine targetsARDS develops as a result of a complex immune reactioninvolving many cytokines, often in combination. Inflamma-somes are intracellular molecules that can act as activators ofmultiple cytokines and propagate the immune response. InARDS, inflammasome gene expression of IL-18 is increased,while IL-18 itself correlates with disease severity and mortalityin critically ill patients [39]. Developing the ability to inactivatethe inflammasome may permit new therapeutic targets, and weexpect developments in this field as scientific understanding ofinflammasomes in ARDS improves.
IL-1b is produced by activated macrophages and epithelialcells and is a potent recruiter of neutrophils. It is elevated inBAL and plasma of ARDS patients [40], and increasesalveolar-capillary permeability; therefore, contributing to thedevelopment of inflammatory alveolar oedema [41]. Inaddition, IL-1b-dependant IL-6 induction has been shownto initiate fibroblast activity and is thought to contribute tothe fibrotic re-modelling stage of ARDS [42]. The mechanismsthrough which IL-1b is produced have been identified toinvolve the nucleotide-binding domain and leucine-richrepeat PYD-containing protein 3 (NLRP3) inflammasome,which is activated in hyperoxia [43,44]. In NLRP3-deficientmice, ARDS development was attenuated with reductions ininflammatory cell recruitment and TNF-a production [45],suggesting that strategies to target the NLRP3 inflammasomecould have many beneficial therapeutic effects.
As previously discussed, the activation and recruitment ofneutrophils to the site of lung injury is a key process in thepathophysiology of ARDS. IL-17 is primarily produced bylymphocytes and has been shown to attract neutrophils tothe lungs in a murine LPS-model of ARDS [46]. To affectthe recruitment of neutrophils, IL-17 acts in synergy withTNF-a to increase endothelial selectin expression; therefore,increasing the adhesion and transmigration of neutrophilsacross the endothelium [47]. Given the importance of thisprocess to the development of ARDS, IL-17 could become afuture biological target and may prove to be an effectivesynergistic target alongside TNFR-1 blockade.
IL-27 is produced by macrophages and dendritic cells, andis thought to have a predominantly anti-inflammatory role,although this has yet to be definitively clarified [48]. BALfrom ARDS patients contains significantly greater quantitiesof IL-27 compared to healthy controls, and correlates withseverity of disease [49]. In addition, murine models of ARDSsuggest that neutralising IL-27 may provide a protective effectto inflammation [49], and therefore, although its precise roleawaits clarification, IL-27 is showing promise as a potentialtreatment option for ARDS.
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3. Endothelial targets
3.1 CD73CD73 is a membrane-bound glycoprotein that is rate limitingfor the production of extracellular adenosine during hyp-oxia [50]. CD73 is activated by stretching of epithelial cells [51],and it hydrolyzes extracellular nucleoside monophosphatesinto bioactive adenosine. Adenosine is thought to be protec-tive in tissue hypoxia, forming part of an innate responsethat inhibits adaptive inflammatory responses. It reduces neu-trophil adhesion and TNF-a production, while macrophagesexhibit increased secretion of the anti-inflammatory cytokineIL-10, and reductions in the production of pro-inflammatoryIL-12 [52,53].
The depletion of CD73 reduces adenosine production, andthis has been shown to increase alveolar capillary leakage [51],suggesting that CD73-mediated adenosine release is protec-tive in ARDS. Additionally, CD73-deficient mice are unableto recover from ARDS [54] implicating adenosine in theresolution and recovery phase of ARDS.
IFN-b increases synthesis of CD73 in lung endothelialcells. In a Phase I study, intravenous administration ofIFN-b in ARDS (when compared with a contemporaneousbut non-randomised control group) was associated with amarked reduction in mortality [55]; however, larger clinicaltrials are required to confirm this therapeutic benefit.
3.2 ACE 2The renin--angiotensin system is best known for its role in theregulation of blood pressure homeostasis. ACE converts angio-tensin 1 to angiotensin 2 (Ang 2), a powerful endogenous vaso-pressor that also promotes inflammation and increasedvascular permeability. ACE2 is a homologue of ACE [56] andfunctions to break down Ang 2 [57], therefore, opposing theeffects of Ang 2 [58].
Natural variations in ACE activity occur between individu-als according to genetic phenotype. The ACE DD phenotypecauses greater ACE activity, and is associated with increasedmortality in ARDS [59]. To support the hypothesis that ACEis damaging in ARDS, and ACE2 may therefore be protective,animal models have found that both ACE deficiency andadministration of IV recombinant human ACE2 improvelung function and pulmonary oedema [60]. Recombinanthuman ACE2 has also been shown in animal models toreduce serum TNF-a levels and attenuate hypoxia [61]. Onthe basis of this data, a human Phase I/II clinical trial is cur-rently recruiting to investigate recombinant human ACE2 inpatients with early ARDS who are haemodynamically stable(NCT01597635).
3.3 AdrenomedullinAdrenomedullin (AM) is a multifunctional regulatory peptidethat has been implicated in a number of biological func-tions [62]. AM is produced by vascular smooth muscle,
vascular endothelial cells and macrophages in response toLPS, TNF-a and IL-1 [63-65]. AM signals through cAMP,preventing increased pulmonary vascular permeability [66]
and therefore reducing alveolar oedema.This ability to attenuate alveolar endothelial permeability
led to investigation of AM as a therapeutic interventionin ARDS. In a rodent model of ARDS induced by LPSintravenous infusion of recombinant-human AM reduced theseverity of lung injury [67], and similar findings were replicatedin a murine model of ventilator-induced lung injury [68]. TheEuropean Medicines Agency granted approval for thedevelopment of AM as a medicinal product for ARDS in2010 and the use of AM in clinical trials is awaited.
The concept of targeting endothelial dysfunction, as acommon pathophysiological pathway seen in the majority ofpatients with ARDS, is a promising strategy.
4. Coagulation factors
4.1 Tissue factorIn addition to a pro-inflammatory state, there is also apro-coagulant state evident in the alveolar space of ARDSpatients, with tissue factor (TF) identified as a key moleculein this process [69]. TF is a potent initiator of the extrinsiccoagulation cascade and in ARDS alveolar epithelial cellscan express TF to stimulate the pro-coagulant state [70]; factorVIIa binds to TF to form a Factor X-binding complex, acti-vating the latter compound that cleaves prothrombin tothrombin and ultimately leads to fibrin formation. This isdamaging because fibrin can deposit within the alveolar space,increasing alveolar-capillary permeability and providing amatrix for disordered fibroblast repair. In addition to alveolarcell expression, TF can be produced in the alveolar space byneutrophils under TNF-a stimulation [71] and helps explainwhy BAL from ARDS patients has a greater concentrationof TF than that from patients with hydrostatic pulmonaryoedema [70].
On the basis of these data, attempts have been made todisrupt the coagulation cascade in ARDS. In primate modelsof sepsis, it has been shown that administration of inactivatedactivated Factor VII (FVIIa), which has higher affinity for TF,attenuated ARDS through a reduction in alveolar fibrindeposition, reduced neutrophil infiltration and alveolaroedema [72]. Two clinical trials have since taken place toestablish the impact of targeting TF as a treatment for ARDS.A Phase II study investigating inactivated recombinant FVIIain mechanically ventilated patients with ARDS for < 48 h wasdiscontinued prematurely because of excessive 28-day mortalityin one of the four treatment cohorts. A trend to increasedbleeding complications was seen with increasing doses oftreatment [73].
More recently, following a small Phase I trial of ALT-836(a recombinant antibody that binds to TF or TF--FactorVIII complex) to evaluate its pharmacokinetics and safety [74],a Phase II investigation (NCT00879606) in septic patients
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with ARDS is ongoing. The primary outcome is duration ofventilation, and results of this trial are awaited.
4.2 Activated protein COnce activated, protein C is a natural anticoagulant thatpromotes fibrinolysis and inhibits thrombosis. In the settingof sepsis there are decreased levels of protein C, while levelsof thrombomodulin (one of the endothelial cell surface recep-tors that activates protein C) are increased, reflecting a reducedbinding capacity and therefore promoting a pro-coagulantstate. In a similar manner to sepsis, ARDS is a pro-coagulantenvironment and plasma protein C in ARDS patients issignificantly reduced compared to normal controls, and thesereductions are associated with a higher mortality [75].Activated protein C (APC) is an anticoagulant therapy that
has been extensively investigated in sepsis with largely negativeresults [76]. In ARDS, a Phase II randomised, double-blindclinical trial of intravenous APC found no significant effecton ventilator-free days. There were however physiologicalimprovements in the treatment group, including improve-ments to the pulmonary dead-space fraction, suggesting thatAPC may restore the lung microcirculation and improveventilation-perfusion matching [77]. In contrast to these nega-tive findings, a recent small randomised placebo-controlledtrial of 27 patients has shown that APC in the setting ofnon-sepsis may attenuate lung injury, decreasing lung injuryscore and showing anti-coagulant properties without haemor-rhagic complications [78]. This suggests that there may yet berole for APC in a sub-group of non-septic ARDS patients.The role of APC is likely to remain limited because of the
negative results observed in large clinical trials investigatingits role as a treatment for sepsis [76], where no benefit wasfound in 28-day mortality. This has led to withdrawal ofAPC as a treatment, and therefore further investigation ofAPC in the setting of ARDS is unlikely to progress. Whiletargeting the coagulation cascade remains a potential strategy,the experience to date with APC is likely to reduce enthusiasmfor large clinical trials in this area.
5. Growth factors
5.1 Keratinocyte growth factorThere is interest in augmenting the resolution of inflamma-tory pulmonary oedema in ARDS. Disordered cellular repairmechanisms and impaired alveolar epithelial cell functionare characteristic of ARDS [79], and resolution of pulmonaryoedema and outcome in ARDS are determined by alveolarepithelial fluid transport.Keratinocyte growth factor (KGF) is an epithelial-derived
fibroblast growth factor produced exclusively by mesenchymalcells. The receptor is highly expressed on epithelial cells,although has recently been reported to also be present onmacrophages, augmenting phagocytosis [80,81]. Within thelung itself KGF has a variety of beneficial effects, including
stimulation of type II alveolar cell proliferation, which isfollowed by cell migration to repair the injured alveoli [82,83].
Following pre-clinical studies suggesting benefits ofKGF [82], an ex-vivo lung perfusion (EVLP) model of ARDSshowed that KGF treatment improved lung endothelial andepithelial function, and augmented alveolar fluid clearance(AFC) [84]. This promising pre-clinical data prompted aPhase II randomised, double-blind placebo-controlled clinicaltrial investigating the effect of recombinant human KGF(palifermin) in ARDS patients [85]. Patients will receive IVpalifermin for up to 6 days and the primary outcome is oxy-genation index at day 7, a marker of ARDS severity. This trialis ongoing, with results likely to have major significance bothin relation to KGF and stem cell therapy in ARDS. Onepotential limitation of KGF as a therapy for ARDS is thatthe majority of pre-clinical data have only found it to beeffective when used prior to injury, which would limit its util-ity as a treatment for ARDS. However, it might be potentiallyuseful as a preventative therapy in patients known to be athigh risk of developing ARDS.
5.2 VEGFVEGF is a cytokine produced by a range of inflammatory andepithelial cells that significantly increases vascular permeabil-ity [86]. ARDS is characterised by increased permeability ofthe alveolar-capillary membrane, and VEGF is significantlyelevated in plasma of ARDS patients [87]. This led to thehypothesis that VEGF increases vascular permeability inARDS. However, BAL VEGF levels are significantly lowerin ARDS patients initially, and lower levels correlate withincreasing severity of lung injury, while recovery of BALVEGF levels after day 4 is associated with a recovery fromARDS [88]. It has been proposed that this apparent paradoxwith elevated plasma VEGF levels is due to BAL VEGF caus-ing increased permeability of the alveolar-capillary membraneand therefore allowing leakage of VEGF into the plasma. Thelow levels of VEGF in the alveolar space can also be explainedby other mechanisms. VEGF levels are similar betweenpatients with non-cardiogenic pulmonary oedema as seen inARDS, and those with hydrostatic pulmonary oedema [89],suggesting that changes in VEGF in the alveolar space repre-sent the severity of alveolar oedema rather than the severityof ARDS itself. In addition, an antagonist to VEGF, solubleVEGFR-1, has been identified as a naturally occurring antag-onist to VEGF in the BAL of patients with ARDS. It is foundin higher levels in BAL compared to plasma, and may explainwhy measurable VEGF levels are lower in the alveolarcompartment [90].
The potential significance of VEGF in the development ofARDS led to a Phase II clinical trial investigating IV Bevacizu-mab (humanised anti-VEGF monoclonal antibody) as a treat-ment for ARDS (NCT01314066). This trial was stoppedearly due to poor enrolment, and further trials evaluatingthe therapeutic role of anti-VEGF therapies are awaited.
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5.3 GM-CSFGM-CSF is a growth factor for haematopoietic cells andpromotes type 2 epithelial cell hyperplasia [91]. Additionally,GM-CSF has an important role in regulating alveolar macro-phage functioning and surfactant homeostasis, helping tomaintain innate immune defence mechanisms [92].
Higher concentration of BAL GM-CSF in ARDS isassociated with reduced mortality [93], and mechanisms toachieve this include augmenting type II epithelial cell andmacrophage proliferation [94]. In addition, murine models ofhyperoxic lung injury show that overexpression of GM-CSFpreserves alveolar permeability and reduces alveolar epithelialcell apoptosis, therefore maintaining structural integrity [95].
Based on this experimental data, a randomised, placebo-controlled Phase II clinical trial investigating recombinanthuman GM-CSF as a treatment for ARDS was undertaken.It showed no reduction in ventilator-free days, although thestudy was underpowered as it did not recruit the plannedsample size. However, there was a non-significant trendtowards reduction in 28-day mortality and an increase inorgan failure-free days. [96]. Given this unsuccessful Phase IItrial, we do not anticipate further investigation of GM-CSFas a treatment for ARDS.
6. Gene therapy
Gene therapy is the manipulation of genetic components(genes or nucleic acid sequences) into cells to replace adisordered gene. This can occur pre-fertilisation or througha somatic approach to manipulate mature cells. By manipulat-ing messenger RNA, specific therapies can be introducedwithout wider effects [97].
There are a number of considerations with regard to genetherapy. To overcome cell defence mechanisms a carrier, orvector, is required to transport the genes into the cell. Giventhe ease with which viruses can infiltrate epithelium, viral vec-tors have been investigated to deliver gene therapy in ARDSbecause of the efficiency they offer. The viral genome is editedto prevent replication and coupled with the gene of interest topermit insertion within the host cell [98]. A number of virusesincluding adenovirus have been investigated, although successhas been limited because of innate immune activation [99].
Non-viral vectors are less efficient in entering epithelialcells, but are thought to be less immunogenic [98]. These vec-tors involve the manipulation of DNA or RNA compoundsthat are complexed with plasmids or lipids to prevent degra-dation and allow entry of the gene into the cell. Non-viraldelivery mechanisms are generally better tolerated by theinnate immune system, producing a lower immunologicalresponse, but the high degradation rate is restricting [100].
An emerging theme in many biological therapies is themost effective way to deliver the treatment to the injuredairways. Given the systemic effects many therapies couldhave upon the immune system, local delivery is an
increasingly popular option. Aerosolised delivery of genetherapy has been investigated in cystic fibrosis, a conditionwhere genetic defects lead to disordered cell membrane trans-port mechanisms, without clinical benefit [101]. Intravasculardelivery direct to the pulmonary vasculature has beenemployed successfully in pre-clinical studies using non-viralvectors [102], but has not yet faced the scrutiny of human trials.
The challenge of gene therapy in ARDS is not limited tothe delivery of genes, but crucially which genes to target.Several genes have been identified that predispose patients todeveloping ARDS, and their functions range from endothelialbarrier regulation to cytokine response and many more [103].All of these are potential therapeutic targets, but to dateprimary interest has related to genes that may impact AFCand those that can limit the development of ARDS from com-mon insults [100]. The most promising pre-clinical data relatesto Na+,K+-ATPase, a cell surface compound that regulatesfluid transport across the cell membrane. Plasmid delivery ofgenetic sequences coding Na+,K+-ATPase were delivered in amouse model of ARDS and showed protection for the devel-opment of ARDS. Of further relevance, however, was thefinding that the same treatment improved lung injury onceit was established, suggesting that genetic transfer of Na+,K+-ATPase may improve AFC [104].
Although gene therapy shows promise, current interventionis limited to animal models. The use of nanoparticles todeliver b2-adrenergic receptors showed reduction in ARDSseverity and improved AFC in mice with establishedARDS [105], while viral vector delivery of genes coding forextracellular superoxide dismutase (superoxide is producedby neutrophils in response to inflammation) reduced severityof ARDS [106]. These therapies remain experimental however,and it remains unclear the role that gene therapy will havein ARDS.
7. Mesenchymal stem cells
Stem cells are cells that have an infinite ability to self-renewand can differentiate into several cell types (pluripotent),effectively providing a repair mechanism for cells withinthe body. The bone marrow is a rich source of mesenchymalstem cells (MSCs), alongside adipose and neonatal tissues(e.g., placenta, umbilical cord), and these cells can beextracted for therapeutic use.
There are several mechanisms of action that make MSCs anappealing therapeutic concept in ARDS. MSCs are thought tobe anti-inflammatory, secreting multiple mediators thatdown-regulate the inflammatory process [107], and can alsosecrete growth factors, including KGF, that offer the benefitspreviously described. In addition, it has been suggested MSCsmay have the ability to repair the injured alveolar epithelium,differentiating into alveolar cells and replacing injured cells,although evidence to support this mechanism is limited [108].
Initial studies in animal models of sepsis showed that MSCtreatment increased IL-10 and improved organ function and
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survival [109]. In addition, in an Escherichia coli model ofpneumonia several anti-microbial compounds, includingLL-37 and lipocalin 2, were found to mediate the actions ofMSCs, inhibiting bacterial growth and augmenting microbialclearance [110,111]. In a murine model of gram-negative perito-neal sepsis treated with human MSCs, survival was improvedin the treatment group with improvements noted in plateletcount and augmentation of monocyte phagocytosis [112].MSCs have also been investigated specifically in ARDS.
A rodent model of ventilator-induced lung injury showedthat MSCs can reduce lung inflammation and enhance cellu-lar repair, reducing levels of the pro-inflammatory cytokineTNF-a, mediated at least in part through KGF secretion [113].An additional mechanism of action of MSCs has been
demonstrated in an animal ARDS model, where direct cell-dependant contact allowed MSCs to mediate their protectiveeffect by adhering to alveoli and transferring their mitochon-dria to improve cell bioenergetics [114]. In a human EVLPmodel of ARDS induced by E coli, treatment with humanMSCs improved AFC, an effect that was mediated in partby secreted KGF [81], further highlighting the therapeuticpotential of MSCs and their secreted products.In addition to EVLP models of ARDS induced by LPS and
E. coli [81,84], a recent EVLP model using natively injuredhuman lungs rejected for transplant suggests that MSCs canrestore AFC in the setting of in vivo lung injury [115]. In a sim-ilar manner to E. coli injured models, this effect was mediatedin part by KGF, and provides further evidence that MSCsmay prove an effective therapy in ARDS.Given these pre-clinical data [116], research has progressed
and currently there are two early-phase studies recruiting toassess the impact of MSCs in ARDS (NCT01775774,NCT01902082). It is anticipated that there will be manyfurther developments in this field.
8. Expert review
Despite half a century of research investigating therapies forARDS [1], specific treatments remain limited to supportivecare involving protective mechanical ventilation strategiesand conservative fluid management. The development of bio-logical therapies is viewed as a potential option for improvingoutcomes in ARDS.ARDS can be caused by a wide variety of insults, including
pneumonia and non-pulmonary sepsis, aspiration, pancreati-tis and trauma [117]. The incidence of, and mortality from,ARDS varies with the associated risk factor. For example,sepsis is associated with the highest incidence and mortality,while in contrast, trauma has the lowest incidence and mortal-ity. Inflammatory cytokines and biomarkers of cell injury areknown to differ by the clinical risk for ARDS, suggesting thatthe pathophysiology may differ by clinical risk factor [117-120].To date, most trials have recruited a heterogeneous cohort ofpatients with ARDS regardless of the aetiology, rather thantesting a specific therapy for a specific cause. It is possible
that this may also help explain why pharmacological interven-tions tested to date have been unsuccessful if the mechanismbeing targeted by a therapy is not actually expressed in all ofthe population recruited. Therefore, it may be appropriateto target a given biological therapy only to patients wherethe target is expressed, for example, in patients associatedwith a specific risk factor.
As we develop our understanding of the mechanismsunderlying ARDS we identify new challenges, including iden-tifying the correct target and timing of intervention. ARDS isa complex illness characterised by an initial inflammatoryphase, followed by a resolution and repair phase, althoughthese phases overlap. Many of the failed pharmacologicaltherapies investigated in ARDS have been used early in thecourse of ARDS, without targeting a specific mechanism. Itmay be more appropriate for a therapy to be used accordingto when its target is expressed during the course of ARDS.For example, blocking the action of pro-inflammatory cyto-kines is likely to be more effective in the early course of illnesswhere limiting the initial insult is the aim of therapy, whereasthe use of growth factors may prove most beneficial at a laterstage, to promote the resolution and repair phase. Currently,interventions are usually investigated at the earliest possibleonset of injury, which may not be suitable if the intendedtarget is expressed at different time points of injury.
There remain a number of challenges to implementing astrategy of delivering novel biological therapeutics accordingto when a specific target is expressed. Defining a therapy bya single biological target could have limitations. Given theredundancy that exists in the complex mechanisms causingARDS, targeting a single pathway may not be beneficial andin fact agents with pleiotropic effects may be more advanta-geous. For example, KGF and MSCs have anti-inflammatoryproperties that are beneficial in the acute inflammatory phase,while their ability to support cellular repair is likely to bebeneficial in the later stages of ARDS. Another challenge isdefining the phase of illness. The inflammatory and reparativephases of ARDS can have significant overlap and it is there-fore not possible to be specific with regard to the stage ofillness. Unlike myocardial infarction, for example, there areno clear biomarkers to guide clinicians as to the stage ofillness, when specific targets are expressed and therefore it isnot yet possible to confidently indicate when an interventionwould be appropriate. However, recognising that a treatmentintervention has a specific target should drive us in the futureto define when that target is expressed and clarify both howand when we can manipulate it. Biological therapies maylend themselves well to this treatment strategy.
Pre-clinical research currently informs whether newtherapies are progressed to human trials. Animal models ofARDS may fail to fully replicate the complex nature of thehuman syndrome [121]. The use of more clinically relevantmodels of ARDS such as the human EVLP models as wellas models of pulmonary inflammation induced by inhaledLPS in healthy volunteers is an attempt to improve the
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translation of pre-clinical findings; however these models stillhave limitations. Recognising that the translation from benchto bedside has many hurdles is important when interpretingpre-clinical research in the setting of ARDS.
There are many promising biological therapies currentlyunder investigation, and of these MSC therapy may providethe most potential for an effective treatment. MSCs havenumerous effects, including limiting the acute injury throughan anti-inflammatory action, promoting resolution of injuryand augmenting repair. This ability of MSCs to act through-out the course of ARDS provides the rationale explaining whyMSCs may overcome the challenges of appropriate timing oftreatment that could explain previous failed therapies. Cur-rently, there are two Phase I clinical trials recruiting withmore planned, and we expect significant progress in thisarea. Aligned with stem cell therapy is the use of growthfactors that are also being tested in Phase II studies [85].With over-lapping properties, it is anticipated that thesemay become effective therapies in the management ofARDS and we expect to see more clinical trials in the future.
In summary, although there have been many failed thera-pies to date [14], new biological therapies based on improvedunderstanding of the mechanisms implicated in the develop-ment of ARDS are emerging and are at various stages of
development, which could provide an effective interventionfor patients with ARDS. It is possible that the future ofARDS treatment may involve a stratified medicine approachwhere specific targets expressed in different populations or atdifferent time points are targeted with a range of biologicaltherapies.
Declaration of interest
D McAuley has performed paid consultancy work and hasbeen a member of advisory boards on ARDS for Glaxo-SmithKline. This author’s institution has been paid for theauthor to undertake bronchoscopy as part of a clinical trialfunded by GlaxoSmithKline. He has also received fees for lec-turing for AstraZenica, and has a patent submitted for a noveltreatment for ARDS (unrelated to the work described in thisreview). D McAuley has received funding from the NorthernIreland Public Health Agency Research and DevelopmentDivision Translational Research Group for Critical Care.The authors have no other relevant affiliations or financialinvolvement with any organisation or entity with a financialinterest in or financial conflict with the subject matter ofmaterials discussed in the manuscript apart from thosedisclosed.
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AffiliationAndrew James Boyle1,2,
James Joseph McNamee2 &
Daniel Francis McAuley†1,2
†Author for correspondence1Queen’s University Belfast, Centre for Infection
and Immunity, Belfast, UK
E-mail: d.f.mcauley@qub.ac.uk2Royal Victoria Hospital,
Regional Intensive Care Unit,
274 Grosvenor Road, Belfast BT12 6BA, UK
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