The Alveolar–Epithelial Barrier: A Target for Potential Therapy

Clin Chest Med 27 (2006) 655–669

The Alveolar–Epithelial Barrier: A Target for Potential Therapy G.R. Scott Budinger, MD, Jacob I. Sznajder, MD* Division of Pulmonary and Critical Care Medicine, Northwestern University, 240 East Huron Street, M300, Chicago, IL 60611, USA

In the lung, the alveolar–capillary barrier is responsible for controlling the volume and solute content of the thin layer of fluid that lines the alveolar space (epithelial lining fluid) [1–3]. This function is critical for the provision of an adequate surface area for gas exchange and for host defense against viral and bacterial pathogens. In performing this activity, the epithelium plays a dual role. First, the formation of tight junctions between alveolar epithelial cells is responsible for the majority of the resistance of the alveolar–capillary barrier to the movement of proteins [3–8]. Second, active transport of sodium through the concerted activity of apically located sodium channels and the Na,K-ATPase at the basolateral membrane determines the volume and ionic composition of the alveolar lining fluid [1,3,9] and the rate of reabsorption of alveolar edema in patients who have congestive heart failure and acute respiratory distress syndrome (ARDS). Acute lung injury is defined by impairment in one or both of these functions of the alveolar– capillary membrane. Increases permeability of the membrane to solutes or decreased ability of the membrane to remove sodium results in an increased volume of alveolar fluid (edema), which causes the appearance of pulmonary opacities detected on the chest radiograph and the development of an intrapulmonary shunt detected by a reduction in the ratio of partial pressure of

Supported in part by HL48129, PO171643 and ES013995. * Corresponding author. E-mail address: [email protected] (J.I. Sznajder).

arterial oxygen to fraction of inspired oxygen [10,11]. Hypoxemia and injury of the alveolar– capillary barrier might result in disordered repair of the alveolar epithelium and contribute to the development of pulmonary fibrosis, prolonged mechanical ventilation, and the multiple-organ dysfunction syndrome that is frequently the cause of death in patients who have acute lung injury [12–19]. The alveolar epithelium therefore is a critical target for interventions designed to reduce the nearly 40% mortality observed in the estimated 200,000 patients in the United States who develop acute lung injury and ARDS each year [19]. This article focuses on the current understanding of the cellular basis for epithelial cell dysfunction in ARDS and some of the emerging molecular therapies designed to attenuate injury.

Epithelial barrier function: paracellular transport Most patients who have ARDS have impaired clearance of fluid from the alveolar space. Patients who demonstrate impaired fluid clearance are more likely to die than those who have normal clearance [20]. It therefore is important to understand the mechanisms that underlie edema formation and clearance in the injured lung (Fig. 1). The solute and ionic composition of the fluid lining the alveolar space differ from that of serum [1–3]. To maintain this difference in concentration, solutes and anions are transported through tightly regulated intracellular pathways. The resulting electrochemical gradients drive the passive movement of molecules through the paracellular pathway [1,3,7,9,21–24]. This ‘‘leakiness’’ of the epithelium can be characterized by its electrical

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Normal

Injury Alveolar space

Na+

Na+

Na+ Na+ Na+ Na+

ClNa+ H2O

ClNa+ H2O Cl- channel (CFTR)

ENaC

Na+

Na+

Aquaporin ZO-1 Actin cytoskeleton

Cl- channel (CFTR)

ENaC

Tight junction

Tight junction dissolution

Aquaporin

Occludin Claudin JAM

ZO-1 Actin cytoskeleton

Viruses Bacterial proteins Apoptosis

K+

K+

K+

K+

K+

Na,K-ATPase

Na,K-ATPase Na+

Na+

Na+

Na+

Na+ Na+ Na+

Na+ Na+ Na+ Na+

Na+ Na+

Na+ Na+

Albumin, fibrinogen

Na+ Na+ Na+

Albumin, fibrinogen

Alveolar interstitium Fig. 1. Alveolar epithelium. (Left) Normal. (Right) Injured. ENaC, epithelial Naþ channel; CFTR, cystic fibrosis transmembrane conductance regulator; JAM, junction adhesion molecules.

resistance. In epithelial cells, disruption of the tight junction dissipates the majority of the resistance, suggesting that the tight junction forms the major barrier to paracellular transport [7,25]. Direct measurements of the alveolar epithelial resistance in vivo are not yet technically feasible, and measurements of resistance in isolated epithelial cells in tissue culture tend to overestimate resistance [23]. Nevertheless, comparison of different epithelial cell populations in the lung suggests an increase in the leakiness of the membrane as one moves from the alveolar epithelium (O1000 U) to the bronchial epithelium (w 100 U) [23]. This increase is consistent with the need for the reabsorption of fluid as it moves from the alveolar space, with a surface area in excess of 70 m3, to the airways, where the surface area is substantially smaller [23,24]. The tight junction is a continuous bandlike structure typically located at the luminal end of the intracellular space [7]. In addition to its role in resisting paracellular transport, the tight junction defines cell polarity, separating lipids and proteins in the apical cell membrane from those in the basolateral membrane. It also serves a major structural and signaling function, anchoring the actin cytoskeleton and harboring a wide range of signaling proteins (reviewed in [4,6,7,25]). On

transmission electron microscopic images, the tight junction appears as a series of very close membrane appositions. Using freeze fracture replicas, these tight junctions have been revealed as interconnected networks of membrane proteins. Other junctional complexes between epithelial cells, for example desmosomes, adherens junctions, and hemidesmosomes, serve important functions for intracellular communication, scaffolding, and signaling but contribute little to the resistance to paracellular transport [4,6,7,25]. Nearly 40 proteins have been described that comprise the tight junctional complex. Most of the proteins are located intracellularly at the tight junction plaque and serve important structural and signaling functions [4,6,7,25]. The integral membrane components are comprised of three families of proteins, the occludins, claudins, and junction adhesion molecules. Occludins probably provide a primarily structural function, and the junction adhesion molecules seem to regulate the migration of inflammatory cells through the junction. A growing body of evidence suggests that the charge on the claudins is responsible for regulating the resistance of the junction to anions. Altering the charge or type of claudin expressed in isolated epithelial cells dramatically alters their electrical resistance [21–24]. Claudin knockout

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mice demonstrate phenotypes consistent with altered paracellular permeability, and defects in tissue specific claudins have been identified in patients who have inherited abnormalities in paracellular permeability [21,23].

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alveolar fluid reabsorption [34–37]. In addition, albumin and other macromolecules can be transported actively across the alveolar epithelium through the transcellular pathway [38].

Epithelial dysfunction during acute lung injury Epithelial barrier function: transcellular transport In the fetal lung, the alveolar epithelium secretes Cl into the alveolar space, and water follows passively through aquaporin channels [26]. Shortly after birth, increased expression of the Na,K-ATPase causes an increase in the vectorial transport of Naþ, with the passive reabsorption of water through aquaporin channels. Throughout life, Starling forces in the pulmonary vasculature favor the formation of a small amount of interstitial fluid that is cleared primarily by the pulmonary lymphatics and veins [1–3]. Despite the relatively high resistance of the alveolar epithelium to paracellular transport, some of this fluid leaks into the alveolar epithelial space and is cleared by active Naþ transport. The resulting alveolar epithelial lining fluid is typically 100 to 200 nM deep, has a low pH, and contains surfactant and proteins important for host defense [2]. The alveolar epithelium is comprised largely of two types of cells. Type II cells comprise up to 5% of the alveolar surface area. They are cuboidal cells that are clustered near the corners of alveoli and are recognized by the presence of lamellar bodies and expression of surfactant proteins [27]. Type II cells are responsible for surfactant production and secretion into the alveolus and are thought to proliferate and differentiate into type I cells in response to injury of the alveolar epithelium [28]. The transport of Naþ across the alveolar epithelium, and therefore edema clearance and the volume of epithelial lining fluid, is controlled by a host of signaling cascades that alter the protein abundance of the Na,K-ATPase at the basolateral membrane and the epithelial Naþ channel at the apical membrane (reviewed in [1,3,9,29]). Until recently, it was thought that active epithelial Naþ transport was performed primarily by type II cells. With advances in imaging and the successful isolation of primary type I cells, several reports have confirmed that these cells contribute the majority of the Naþ transport [3,30–33]. Increasing evidence suggests an additional contribution of Cl channels, particularly the cystic fibrosis transmembrane conductance regulator (CFTR) and, perhaps, aquaporins to

The development of acute lung injury often is caused by the release of bacterial proteins or proinflammatory cytokines into the circulation. In response to these stimuli, the alveolar endothelium rapidly increases its permeability to ions, solutes, and even macromolecules (reviewed in [39]). Inhibition of this initial endothelial permeabilization has been shown to protect against the development of acute lung injury in some models [39–41]. Endothelial permeabilization without disruption of the alveolar epithelium should not result in the exudation of macromolecules into the alveoli because the size of pores in the tight junctional complexes (estimated to be approximately 0.4–0.8 nm) is roughly an order of magnitude smaller than many macromolecules in the serum [4,6,7]. For example, albumin and fibrinogen will not cross pores substantially smaller than 4.0 nm [42]. In this setting of modest injury with an intact epithelium, therefore, fluid with low protein content is likely to accumulate in the airspace where it could potentially be cleared through vectorial transcellular Naþ transport. Unfortunately, many of the same stimuli that cause lung injury and stimuli that are introduced into the injured lung as therapeutic interventions to maintain oxygen delivery impair alveolar fluid reabsorption [20]. For example Pseudomonas aeruginosa or Mycoplasma pneumoniae in the alveolar space [43,44], overdistension of the epithelium associated with mechanical ventilation [45,46], cytokines and chemokines including transforming growth factor-b (TGF-b) and interleukin -8 [47,48], hyperoxia [49–53], reactive nitrogen species [44,54], and acid [48] have all been shown to impair clearance of alveolar fluid. Although this impairment may be offset partially by the systemic release of catecholamines in response to hypotension [45,50,51,55–69], increased fluid accumulation in the alveolar interstitium and the alveolar space combined with impaired fluid reabsorption sets up a potentially vicious cycle of persistent pulmonary edema that impairs tissue oxygen delivery and exposes the alveolar epithelium to an environment with high concentrations of inflammatory cytokines, hypoxia or hyperoxia, and excessive

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stretch or shear that can further disrupt the alveolar epithelial barrier allowing the exudation of serum proteins into the alveolar space. Alveolar epithelial cell death and lung injury A defining pathologic feature of lung injury is loss of alveolar–capillary barrier integrity with the exudation of plasma proteins into the alveolar space resulting in the fibrin-rich exudates lining the alveoli characteristic of diffuse alveolar damage [11]. Loss of alveolar epithelial integrity might result from the disassembly of tight junction complexes in response to mechanical stimuli [70] or viral or bacterial proteins that target components of the tight junction. For example, adenoviruses [71], the coxsackie B virus [71], reoviruses [72], papillomaviruses [73], Clostridium perfringens exotoxin [74], and Helicobacter pylori [75] all bind to proteins in the tight junction and can cause their disassembly. Disruption of the alveolar epithelium also might result from death of alveolar epithelial cells [76]. To maintain tissue function in multicellular organisms, aged or damaged cells must die to be replaced by newly generated cells [77]. To achieve this goal, all the cells in the body contain a set of proteins that, when activated, kill the cell [77]. This highly coordinated cascade results in an organized form of death recognized morphologically as apoptosis (taken from the Greek meaning ‘‘falling leaves’’). Death by apoptosis is associated with the expression of surface receptors that allow clearance of the cells with minimal inflammation [78,79]. Cell death also can occur through necrosis with the spilling of intracellular contents, which can cause substantial inflammation. Previously, it was thought that necrotic cell death was unregulated, but it is increasingly recognized that similar upstream signaling events can result in either apoptotic or necrotic cell death [80]. Evidence for both apoptotic and necrotic cell death has been observed in many animal models of lung injury including exposure to hyperoxia [81–87], treatment with lipopolysaccharide (LPS) [88,89] or bleomycin [90–92], cecal ligation and puncture [93], ischemia reperfusion injury [94– 96], and ventilator-induced lung injury [97]. In these models, some of the cell death may be unregulated. For example, severe overdistention associated with mechanical ventilation might rupture the cell membrane [98,99]. In many cases, however, cell death is induced through activation of the apoptotic program. Apoptosis can occur

through activation of death receptors on the cell surface that activate one or more of a family of cysteinyl aspartate–specific proteases (caspases) that bring about cell death [77]. Death receptor– ligand complexes implicated in animal models of lung injury include Fas/FasL, tumor necrosis factor-a/tumor necrosis factor-a receptor, TGFb1/TGF-bR, and LPS/Toll-like receptor 4 [100, 101]. Binding of these ligands with their receptors causes activation of caspase-8, which then activates effector caspases (primarily caspase-3 and caspase-7). Alternatively, cell death can be initiated through activation of the Bcl-2 family of proteins, resulting in permeabilization of the outer mitochondrial membrane with the resulting release of cytochrome c and a number of other pro-apoptotic proteins into the cytoplasm [77]. After their release, these proteins cause the activation of the effector caspases. In epithelial cells, the activation of caspase-8 often is insufficient to induce cell death without amplification through the Bcl-2 proteins. This amplification can be mediated by caspase-8–mediated cleavage of the proapoptotic Bcl-2 protein Bid to truncated Bid, which acts through the pro-apoptotic Bcl-2 proteins Bax or Bak to induce mitochondrial outer membrane permeabilization (Fig. 2) [102]. In rabbits and mice activation of the receptordependent apoptotic pathway by the instillation of FasL or an antibody that cross-links Fas can induce lung injury [89,103]. In patients at risk for or who have ARDS, the sFasL is present in bronchoalveolar lavage (BAL) fluid at concentrations that are sufficient to induce apoptosis in cultured alveolar epithelial cells [104,105]. Consistent with the importance of the receptor-dependent pathway in some forms of lung injury, mice lacking Fas are partially protected against lung injury induced by LPS and cecal ligation and puncture [89,106]. The importance of the mitochondrial-dependent apoptotic pathway has been observed in several different animal models including lung injury induced by hyperoxia [82–87,107,108], bleomycin [90,92,109], and ischemia reperfusion [94–96,110,111]. In all of these models, genetic strategies that prevent activation of mitochondrial-dependent apoptosis prevent or attenuate the lung injury.

Repair of the injured epithelium Despite the dramatic defects in the chest radiograph and oxygenation associated with alveolar fluid accumulation, death from acute

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Oxidative stress, growth factor withdrawal, radiation, anoxia, DNA damage Death receptor Fas, TNFαr Plasma membrane

BH3 protein

Procaspase- 8

Death ligand FasL, TNFα, TGF-β1,LPS

Death domain protein FADD, TRADD

Bid Bcl-XL

Bax or Bak

Caspase-8

Cytochrome c Apaf-1

Caspase-3/7

ATP apoptosis

Fig. 2. The apoptotic pathways. FADD, Fas-associating protein with death domain; LPS lipopolysaccharide; TGF-b1, transforming growth factor-beta 1; TNF-a, tumor necrosis factor-alpha.

hypoxemic respiratory failure is observed in approximately 15% of patients who succumb to ARDS [19]. More frequently, patients dying from ARDS develop the multiple-organ dysfunction syndrome several days or even weeks after the initial insult [19,112]. Although the immediate cause of death differs among patients, virtually all patients who have ARDS have persistent respiratory failure requiring mechanical ventilation at the time of death [19,112]. Typically, the reason for the respiratory failure is a marked decrease in respiratory compliance and an increase in the dead space fraction [113,114]. This change is paralleled by CT and pathologic evidence of increasing pulmonary fibrosis. In several cohorts of patients who have ARDS increased concentrations of procollagen III in the BAL fluid [18], the profibrotic cytokine TGF-b1 [12,13], and the ability of BAL fluid to stimulate procollagen transcription or the proliferation of fibroblasts in culture are associated with poorer outcomes [12,115,116]. Basic investigation has provided important insights into the mechanisms that underlie disordered repair of the alveolar epithelium that have implications for therapy. To understand the potential importance of alveolar cell death and regeneration in the pathophysiology of lung injury, it is useful to examine the bleomycin model of acute lung injury. The peritoneal or intratracheal instillation of bleomycin into rodents results in marked inflammation

with the exudation of fluid and proteins into the airspace that is maximal 3 to 5 days after the instillation [117,118]. During this acute phase of the injury, there is marked inflammation with an increase in the numbers of neutrophils and mononuclear cells in the interstitium and alveolar space. Loss of epithelial integrity is demonstrated by a markedly increased permeability of the lung to macromolecules, including albumin [119]. Apoptosis of alveolar epithelial cells is evident during this acute phase of the illness [120,121]. In response to the acute injury, latent TGF-b1 in the alveolar interstitium is activated by exposure to an integrin expressed on the surface of alveolar epithelial cells (avb6) [119]. Active TGF-b1 is required for the subsequent development of fibrosis and acts by stimulating the transition of interstitial fibroblasts to myofibroblasts and by inducing alveolar epithelial apoptosis [109,122,123]. As a result of the activation of TGF-b1, fibrosis develops in the lung that is maximal 3 weeks after the instillation and then slowly resolves. Inhibition of the activation of TGF-b1 or the pro-apoptotic effects of TGF-b1 prevents the development of fibrosis but has no effect on the acute inflammatory response [109]. The bleomycin model parallels the pathology observed in patients who have lung injury in which an initial insult results in the delayed development of fibrosis that resolves slowly over time [113]. Active TGF-b1 has been observed in

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the BAL fluid of patients who have lung injury and has been associated with worsened clinical outcomes [12,13,124]. Similarly, other markers of fibrosis in the BAL fluid of patients who have ARDS portend a poor prognosis [18,114–116]. From the bleomycin model emerges the hypothesis that injury to the epithelium and endothelium allow an inflammatory exudate to accumulate in the alveolar space. Factors in this exudate or proteins expressed on the injured epithelium cause the activation of TGF-b1 and other profibrotic cytokines that induce further epithelial cell apoptosis and stimulate fibrosis. The resulting scar restores epithelial barrier function but decreases the surface of lung available for gas exchange and may impair lung host defense mechanisms, increasing the susceptibility to ventilator-associated pneumonia. According to this hypothesis, interventions that decrease the initial epithelial and endothelial injury would prevent both the initial injury and the subsequent fibrosis. Interventions that antagonize the pro-apoptotic effects of TGF-b1 in epithelial cells or the profibrotic effects of TGF-b1 in fibroblasts might prevent fibrosis after resolution of the acute inflammatory response. Plasma proteins that gain access to the alveolar space in the injured lung include fibrinogen and other factors in the coagulation cascade [11]. Activation of coagulation in the alveolar space results in the formation of a fibrin mesh lining the epithelial surface. The presence of fibrin in the alveolar space is recognized as a component of diffuse alveolar damage, the pathologic pattern observed in patients who have lung injury [11]. Repair of the injured lung must be accompanied by lysis and reabsorption of this fibrin meshwork. Strategies that promote the dissolution of fibrin therefore might benefit patients who have acute lung injury. Plasmin is a serine protease that is crucial for the dissolution of the fibrin network [125]. Plasmin is released upon cleavage of plasminogen, controlled by tissue plasminogen activator and urokinase plasminogen activator [125,126]. The activity of the latter two proteins is tightly negatively regulated by plasminogen activator inhibitor (PAI)-1 and to a lesser extent by PAI-2 [126]. The loss of PAI-1 is associated with increased plasmin activation. Mice lacking PAI-1 are protected against lung injury induced by exposure to hyperoxia [127] and ischemia-reperfusion [128] and have a blunted fibrotic response to bleomycin [129]. Plasmin is a relatively nonspecific protease and cleaves a number of other proteins, including membrane receptors and growth factors [126]. It therefore is

possible that the absence of PAI-1 protects against lung injury through alternative mechanisms. For example, mice lacking fibrinogen fail to show protection against bleomycin-induced pulmonary fibrosis (Fig. 3) [130].

Therapies to improve epithelial function: fluid clearance Particularly in the early phases of lung injury, or in regions of the lung where injury is less severe, removal of edema fluid from the alveolar space might improve gas exchange abnormalities, blunt the inflammatory response, and prevent regional worsening of epithelial and endothelial damage [1]. Control of epithelial transcellular Naþ transport is largely regulated by the maintenance of Na,K-ATPase on the basolateral membrane and the epithelial Naþ channel on the apical membrane. The protein abundance of the Na,KATPase at the plasma membrane is decreased in the injured lung through a number of mechanisms [1,3,131]. This decrease is associated with a decrease in the ability of the lung to clear edema fluid and adverse outcomes. For example, in a cohort of patients who had ARDS, impaired clearance of alveolar fluid was associated with decreased survival [20]. In cultured cells and in animals, decreased abundance of the Na,K-ATPase in the plasma membrane and the resulting reduction in fluid clearance in the injured lung can be reversed by treatment with catecholamines, for example dopamine and b-adrenergic agonists [1,3,131]. This observation led to a clinical trial of intravenous b-agonists to improve fluid clearance in patients who had ARDS [59]. In this trial, 40 patients were assigned randomly to receive placebo or intravenous salbutamol. Patients who received salbutamol had significant reductions in Tissue Factor-VIIa Coagulation cascade

Tissue Factor Pathway Inhibitor Activated protein C

AntiThrombin Thrombin Fibrin

Fibrin Degradation Products

Plasmin

Plasminogen Activator Inhibitor (PAI-1,PAI-2) Plasminogen Activator

Fig. 3. The coagulation cascade.

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extravascular lung water measured using a singleindicator transpulmonary thermodilution system and a reduction in the plateau pressure. This study is limited by its small size and by the lack of detailed measures of hemodynamic parameters to exclude an effect of intravenous salbutamol on the pulmonary microvasculature. Studies examining the efficacy of inhaled beta agonists, which can be delivered in high concentrations to the injured lung, perhaps in combination with intravenous dopamine, will address these questions and are scheduled to be examined by the ARDSNet [3]. Prevention of the extravasation of fluid through the injured epithelium is theoretically attractive, particularly in patients at risk for the development of ARDS. Proteins in the tight junctional complex that regulate the formation, dissociation, and claudin content of tight junctions are attractive potential therapeutic targets. Investigation into the signaling events that regulate these processes is in its early stages; however, it seems likely that these processes could become important therapeutic targets. Overdistention of the alveolar epithelium during mechanical ventilation might contribute to increased permeability, providing a mechanism by which a strategy of lower tidal volume ventilation might improve mortality [132]. Other mechanisms, for example reduced release of inflammatory cytokines or prevention of alveolar epithelial cell death, might also explain this observation [133,134].

Therapies to improve epithelial function: prevention of cell death A number of small molecules and peptide antagonists that can prevent cell death are being developed [135]. These compounds are predicted to attenuate end-organ dysfunction in a variety of acute-injury models including stroke, myocardial infarction, acute liver injury, and acute lung injury. Caspase inhibitors are likely to be the first compounds to enter clinical trails. For example, a pan-caspase inhibitor, IDN6556 (Pfizer), is in phase II clinical trails for the prevention of acute liver injury induced by the hepatitis C virus and ischemia reperfusion injury during liver transplantation [136,137]. A selective inhibitor of caspase-1 (activation of which induces the generation of pro-inflammatory cytokines in macrophages), VX-740 (Vertex Pharmaceuticals), is in phase II studies in patients who have rheumatoid arthritis [136–138]. Although less developed, small

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molecule manipulation of the Bcl-2 family of proteins represents another strategy to attenuate cell death either through a gain of function in the antiapoptotic proteins or a loss of function in the proapoptotic proteins [139,140]. Examples of the latter include the recent description of small molecule or peptide antagonists to the pro-apoptotic Bcl-2 proteins Bax and Bid [140–142]. Growth factors, for example keratinocyte growth factor (KGF), hepatocyte growth factor, and fibroblast growth factor-10, have been proposed as potential therapies targeted at the alveolar epithelium [143]. In particular KGF has been shown to prevent alveolar epithelial cell apoptosis, improve alveolar fluid clearance, and prevent edema formation and fibrosis in a variety of animal models of lung injury [68,86,107,144– 160]. The protection conferred by KGF against hyperoxic lung injury in mice seems to be independent of its effects on cell growth, which are inhibited by hyperoxic exposure [86,144]. All of the growth factors reported to prevent lung injury activate the pro-survival protein Akt, which prevents cell death through a variety of mechanisms [161]. Overexpression of Akt prevents hyperoxiainduced lung injury, and inhibition of KGFmediated Akt activation abolishes its protective effects [86,144,162]. Although clinical trials of growth factors in patients who have lung injury have not yet begun, a synthetic version of KGF, palifermin, has recently been approved by the Food ad Drug Administration for the prevention of mucositis in patients receiving chemotherapy [163]. Therapies to prevent disordered alveolar epithelial repair Mortality in ARDS is driven by the development of multiorgan failure days to weeks after the initial insult [19,112]. At the time of death, virtually all patients who have ARDS remain on the mechanical ventilator [112]. Generally, these patients exhibit increased minute ventilation reflecting an increase in dead space ventilation [113,114]. The lungs of these patients exhibit extensive areas of fibrosis [15,164]. Therefore strategies that prevent the development of this fibrosis might reduce mortality in these patients. Inhibiting the activation of TGF-b in patients who have established lung injury might prevent the development of lung fibrosis. For example, the profibrotic effects of TGF-b require activation of c-abl, a serine/threonine kinase that is constitutively active in chronic myelogenous

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leukemia [165,166]. Inhibition of this kinase with imatinib mesylate, approved for the treatment of chronic myelogenous leukemia, prevents bleomycin-induced fibrosis in mice [166]. A phase II trial of imatinib in patients who had idiopathic pulmonary fibrosis has been completed recently, but the results have not yet been published [167]. The use of this or similar drugs in patients who have established lung injury, particularly those who have evidence of high levels of active TGF-b, may prevent the development of fibrosis. Another potential strategy aimed at preventing disordered repair is the use of thrombolytic therapy to remove the fibrin network that develops in the alveolar space. Strategies that accelerate thrombolysis, for example activated protein C, plasminogen activators, and inhibitors of PAI-1, might be beneficial in patients who have lung injury but may increase the risk of serious bleeding events. A phase II trial of activated protein C in patients who have acute lung injury is underway to test this hypothesis [168]. It has been hypothesized that adult hematopoietic stem cells infused into the blood localize to the lung and differentiate to repair the alveolar– capillary barrier [169]. If so, collection and reinfusion of these stem cells could provide a new therapy for patients who have lung injury. However, the frequency with which these stem cells localize to the lung is unclear. Several groups of investigators reported that in lethally irradiated animals that underwent bone marrow transplantation with labeled adult hematopoietic stem cells, approximately 5% to 14% of alveolar epithelial cells were of donor origin [170–173]. With injury induced by LPS, bleomycin, or radiation, the percentage of cells of donor origin increased [170,172–175]. The suggestion that early reports of the localization of stem cells to areas of myocardial infarction may have represented artifacts induced by the use of immunofluorescence raised questions about the validity of reports in the lung, many of which also relied on this technique [176,177]. Using a highly sensitive assay, Kotton and colleagues [178] failed to detect any evidence of donor cells in the lung even after bleomycininduced lung injury. Examination of the lungs of human recipients of gender-mismatched allogeneic bone marrow transplants have yielded similar disparate results with reports of between zero and 8% of alveolar epithelial cells originating from the donor [179–182]. Because similar technical considerations limit these studies, advances in technology will probably be necessary to address

the efficacy of this strategy in patients who have lung injury.

Gene therapy for the alveolar epithelium Overexpression of proteins that promote alveolar fluid clearance, prevent cell death, stimulate cell growth, or prevent fibrosis have all demonstrated efficacy in protecting against the sequelae of acute lung injury in animals. Currently, technical limitations prevent the use of gene therapy in human clinical trials [183,184]. For efficient gene expression to occur, DNA must cross the cell membrane, be transported to the nucleus, and be transcribed by the nuclear machinery [185]. Replication-deficient adenoviruses target the alveolar epithelium through the coxsackie and adenovirus receptor (CAR) receptor in the tight junction and lead to high-level expression of the transgene in these cells [183]. However, inflammation induced by the viral infection might exacerbate pre-existing lung injury. Expression of the virusencoded transgene is relatively short (days to weeks), further limiting its utility. The use of adeno-associated viruses or other viral vectors, for example lentiviruses, might partially overcome these problems [186]. It is unclear, however, whether viruses can be rendered sufficiently safe for use in patients who have lung injury. The problems with viral-mediated gene delivery have prompted the evaluation of a variety of chemical and mechanical techniques to deliver transgenes to the alveolar epithelium [184]. Intravenous administration of liposome-encapsulated DNA can result in relatively high-level expression in the liver and the lung [187]. The delivery of a modest electrical voltage creates transient pores in the alveolar epithelial cell membrane allowing entry of plasmid DNA (electroporation) [184]. This technique has been used successfully to increase expression of a functional b subunit of the Na,K-ATPase in the lungs of rats and mice without detectable levels of inflammation [188,189]. Further studies are required to evaluate the efficacy of these strategies in patients who have lung injury.

Summary Alveolar-capillary barrier dysfunction is a defining feature of acute lung injury. Evolving therapies developed in animal models of lung injury have resulted in the generation of an array of techniques, drugs, and novel small molecules that

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