Overview of ventilator-induced lung injury mechanisms

Vincenzo Lionetti,a Fabio A. Recchiaa and V. Marco Ranierib

Purpose of review Mechanical ventilation is the main supportive therapy for patients with acute respiratory distress syndrome. As with any therapy, mechanical ventilation has side effects and may induce lung injury. This review will focus on stretchdependent activation of alveolar epithelial and endothelial cells and polymorphonuclear leukocytes, and apoptosis/necrosis balance. Recent findings The past year has seen important research in the area of mechanotransduction and lung native immunity, suggesting further mechanisms of lung inflammation and injury in ventilator-induced lung injury. Research in the past year has also stressed the importance of inflammatory response by alveolar cells and role of polymorphonuclear leukocytes in stretch-induced lung injury and has suggested a role for apoptosis in the maintenance of the alveolar epithelium. Summary The proportion of patients receiving protective ventilatory strategies remains modest. If efforts to minimize the iatrogenic consequences of mechanical ventilation are to succeed, there must be a greater understanding of the signal transduction mechanisms and the development of potential pharmacologic targets to modulate the molecular and cellular effects of lung stretch. Keywords inflammation, polymorphonuclear cells, apoptosis, necrosis, ventilator-induced lung injury
Curr Opin Crit Care 11:8286. 2005 Lippincott Williams & Wilkins.
a Laboratorio di Fisiologia e Biologia Molecolare, Classe di Scienze Sperimentali, Settore di Scienze Mediche, Pisa, Italy, and bDipartimento di Anestesiologia e ` Rianimazione, Universita di Torino, Ospedale S. Giovanni Battista-Molinette, Torino, Italy

Introduction
Mechanical ventilation is an important lifesaving tool in the management of respiratory failure. As with any therapy, mechanical ventilation has side effects and may result in ventilator-induced lung injury (VILI). Therefore, VILI has been established as a significant risk to patients receiving mechanical ventilation. The potential importance of VILI in the clinical treatment of critically ill patients was established by the recent trials performed by the Acute Respiratory Distress Syndrome Network [1,2]. The results of these large clinical trials showed a relative risk reduction of 22% in patients ventilated with the lower tidal volume. This indicates that mortality attributable to VILI is at least 9 to 10%. Several experimental and clinical studies lead to the hypothesis that the deleterious effects of mechanical ventilation may be mediated by localized inflammation and the systemic release of inflammatory cytokines (biotrauma) [1,36]. The clinical relevance of biotrauma is not only that it can aggravate ongoing lung injury but also that it may have important systemic consequences via spillover of lung-borne inflammatory mediators into the systemic circulation, and may explain why most patients with acute respiratory distress syndrome (ARDS) succumb to multiple organ failure [68]. Gurkan et al. [8] have suggested that mechanical ventilation strategy has differential effects on systemic inflammatory changes and may selectively predispose to systemic organ dysfunction. The authors emphasized that pulmonary and systemic release of inflammatory mediators occurs only if the lungs are primed by the primary inflammatory hit of acute lung injury (ALI), suggesting that biotrauma may occur only if mechanical stretch acts as a secondary hit to already inflamed lungs [3,5]. The onset of VILI can be linked to several possible mechanisms. Although the hypothesis that a series of proinflammatory and profibrogenetic processes may be involved in the course of VILI by activation of molecular mechanisms is well established, the cellular and molecular mechanisms underlying lung injury by mechanical stress have not been fully elucidated [9]. Full understanding of the mechanisms that mediate lung injury may permit potential strategies directed at preventing VILI and at reducing the incidence of VILI-induced multiple organ failure to be instituted early in the course of illness. In this review, we will highlight the most recent data on the mechanisms of VILI as they relate to alveolar epithelial and vascular endothelial mechanical stretch,

` Supported by Ministero dellUniversita e della Ricerca: COFIN 2003-2004 ` Correspondence to V. Marco Ranieri, Universita di Torino, Dipartimento di Anestesia, Ospedale S. Giovanni Battista-Molinette, Corso Dogliotti 14, 10126 Torino, Italy Tel: 39 011 633 4001; fax: 39 011 696 0448; e-mail: marco.ranieri@unito.it Current Opinion in Critical Care 2005, 11:8286 Abbreviations ALI ARDS NF-kB PMN VILI acute lung injury acute respiratory distress syndrome nuclear factor-kB polymorphonuclear leukocytes ventilator-induced lung injury

2005 Lippincott Williams & Wilkins. 1070-5295

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polymorphonuclear leukocyte (PMN) recruitment and activation, and apoptotic/necrotic balance.

Mechanical stretch: alveolar epithelial cells and vascular endothelial cells

Alveolar epithelial cells and vascular endothelial cells undergo stretching during mechanical ventilation, undergoing different types of deformation-induced cellular response [1013]. Cyclic stretch stimulates alveolar epithelial cells through mechano-sensitive membrane-associated proteins and stretch-activated ion channels [11,12]. Fisher et al. [14] showed that the plasma membrane of alveolar epithelial cells expands during tonic stretch, not only through cell surface unfolding but also through recruitment of additional phospholipids, unaffected by F-actin disassembly, ATP depletion, and calcium deprivation. Such plasma membrane expansion would be protective during mechanical ventilation by reduced membrane tension and decreased stimulation of mechanically sensitive membrane proteins. These mechanisms are thought to play an important role in sensing and transducing cell deformation to biochemical pathways involved in cell viability and differentiation [15]. Several hypotheses have been advanced to link physical forces and intracellular signaling pathways, but in many cases the molecular mechanisms of mechanotransduction remain elusive. Recent data have established that compressive stress shrinks the lateral intercellular space surrounding epithelial cells, and it triggers cellular signaling via autocrine binding of epidermal growth factor family ligands to the epidermal growth factor receptor [16]. These important and original findings describe a mechanism by which mechanotransduction arises from an autocrine ligand-receptor circuit operating in a dynamically regulated extracellular volume, not requiring induction of force-dependent biochemical processes within the cell or cell membrane. Vlahakis and Hubmayr [17] conducted an overview of selected aspects of mechanotransduction and deformation-induced remodeling of pulmonary cells exposed to mechanical stretch. Several studies have shown that increased mechanical stretch during injurious mechanical ventilation may contribute to lung injury by an inflammatory process and influence pulmonary and systemic cellular lifespan, leading to end-organ epithelial cell apoptosis and organ dysfunction [18,19,20]. Vlahakis et al. [21] recently demonstrated that deformation per se can trigger an inflammatory response and that alveolar epithelial cells may be active participants in the alveolitis associated with VILI. However, it remains controversial whether injurious ventilation per se, without preceding lung injury, can initiate cytokine-mediated pulmonary inflammation. Therefore, Wilson et al. [22] have developed an in vivo mouse model of ALI produced by high tidal volume ventilation. High tidal volume, in the

absence of underlying injury, produced progressive lung injury with a decrease in respiratory system compliance, an increase in protein and cytokine concentration in lung lavage fluid, and lung pathologic changes showing hyaline membrane formation. In the past years, many investigators have supposed that the inflammatory response depends on the type of injurious stimulus and follows different molecular pathways. Uhlig et al. [23] have shown, in isolated perfused mouse lungs and in vivo, that hyperinflation causes nuclear translocation of nuclear factor-kB (NF-kB) and enhanced expression of interleukin-6 mRNA in alveolar macrophages and alveolar epithelial type II cells; inhibition of phosphoinositide 3-OH kinase prevented nuclear translocation of NF-kB and the subsequent release of interleukin-6 and macrophage inflammatory protein-2a in hyperinflated but not in endotoxic lungs. These findings show that resident cells contribute to the release of ventilation-induced proinflammatory mediators, and they show selective inhibition by a new specific phosphoinositide 3-OH kinase dependent mechanism that is NF-kB independent. The conversion of physical signals such as contractile forces or external mechanical perturbations into chemical signaling events is a fundamental cellular process that occurs at the cellextracellular matrix contact, known as focal adhesions, thus modifying cell viscoelastic properties, which may compromise the balance of forces in the alveolar epithelium [24]. Stretch-induced cell stiffening could compromise the balance of forces at the cell-cell and cell-matrix adhesions [25]. Mascarenhas et al. [26], in an in vitro study, showed that de novo synthesis of hyaluronan, a component of the extracellular matrix, increases proinflammatory cytokines in VILI. Moreover, Taylor et al. [27] demonstrated that hyaluronan released after lung injury can stimulate endothelial cells to produce cytokines by activation of a Toll-like receptor 4dependent mechanism, thus suggesting that endogenous components of the extracellular matrix can stimulate endothelial cells to trigger recognition of injury in the initial stages of the wound defense and repair response. Pulmonary endothelium is a component of the alveolarcapillary unit that is vulnerable to several injurious patterns, including mechanical stretch [28]. Many investigators have developed sophisticated in vitro deformation systems to more precisely and directly study the endothelial response to deformation [29]. Czarny et al. [30] recently explored the role of neutral sphingomyelinase concentrated at the endothelial cell surface in caveolae, and ceramides in mechanical signaling of stretched rat lung endothelium. Their study demonstrated that the neutral sphingomyelinase at the plasma membrane in caveolae may be an upstream initiating of the mechanical sensor, which acutely triggers mechanotransduction by

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generation of the lipid second messenger ceramide, which activates the Akt/endothelial nitric oxide synthase pathway [30]. Circumferential stretch in pulmonary microvessels is largely determined by the transmural pressure gradient, hence by both vascular perfusion and alveolar ventilation pressures [31]. Recent data from pulmonary microvascular endothelial cells and isolated perfused rat lungs underline the role of endothelial responses to stretch-inducing VILI [3234]. Kuebler et al. [33] showed that vascular stretch generated by ventilation increases nitric oxide by a signaling cascade involving phosphoinositide 3-OH kinase in lung endothelial cells, and that this response is independent from the mechanical factors causing vascular distension. Bhattacharya et al. [34] demonstrated that high tidal volume ventilation may induce focal adhesion formation and recruit leukocytes on the endothelial cells. Haseneen et al. [32] demonstrated the existence of a relation between stretched endothelial cells and lung remodeling by release of matrix metalloproteinases (MMP-2 and MMP-1) activated through a membrane type-1 MMP (MT1-MMP) mechanism.

strategy increased PMN influx and interleukin-8 production in the normal lung. Belperio et al. [41] demonstrated that PMN accumulation caused the change in microvascular permeability associated with high mechanical stretch and that this was associated with an increase in CXCR2 ligands, and evidence of IkB phosphorylation and degradation, suggesting an increase in NF-kBdependent gene transcription. Steinberg et al. [42] demonstrated that alveolar instability causes an early mechanical injury, initiating an inflammatory response independently of neutrophils, resulting in a secondary neutrophil-mediated proteolytic injury.

Role of the apoptosis/necrosis balance in ventilator-induced lung injury

Apoptosis is a process of controlled cell death, which is important in the development and remodeling of tissues that occur during the normal repair process, characterized by cytoplasmic and nuclear shrinkage, chromatin margination and fragmentation, and breakdown of the cell into spherical bodies that retain membrane integrity so that intracellular products are not spread into the extracellular space. By contrast, cell death by necrosis is characterized by a progressive loss of cytoplasmic membrane integrity and flux of Na+ and Ca2+ and water, resulting in cytoplasmic swelling and nuclear pyknosis, leading to cellular fragmentation and release of lysosomal and granular contents into the surrounding extracellular space. Whereas apoptosis occurs without corresponding inflammation of the surrounding tissue, necrosis is often associated with activation of the inflammatory response in the adjacent tissues. The same type of insult can induce either apoptosis or necrosis, but whether one mode of cell death is preferred over the other depends on the severity of the insult and the idiosyncrasy of the target cell [43]. Increasing evidence suggests a role for apoptosis in the maintenance of the alveolar epithelium under normal and pathologic conditions. Type II alveolar cells undergo apoptosis during normal development and maturation of the lung and in association with ALI [44,45]. Fischer et al. [46] reported that in ischemia-reperfusion after lung transplantation, low levels of lung injury were associated with high levels of apoptosis, whereas increased lung injury was associated with decreased apoptosis and increased necrosis. Imai et al. [20] showed that low levels of mechanical stretch caused high levels of pulmonary apoptosis, whereas high levels of mechanical stretch were associated with decreased apoptosis and increased necrosis. Mechanical stretch regulates pulmonary cell function and structure by mechanisms that include the expression of multiple genes. Although it is likely that these events are controlled by various signaling pathways, the stretch-induced activation of Akt-ERK1/2 via a G protein dependent pathway has been shown to play a key role in linking external signals to nuclear response [17]. Oudin and Pugin [47] exposed a bronchial epithelial cell culture

Polymorphonuclear leukocyte recruitment and activation

In an in vitro model using freshly isolated type II alveolar epithelial cells, Vanderbilt et al. [35] demonstrated that type II alveolar epithelial cells produce CXC chemokines (MIP-2) and their related receptor CXCR2 in response to lung injury. These observations support a central role for the type II cell as an immunologic effector cell in the alveoli. PMN are an important component of the inflammatory response that characterizes VILI [36]. In animals with intact circulating PMN, mechanical stretch increased alveolar-endothelial barrier permeability and caused hyaline membrane formation; the use of the same ventilatory strategy in neutrophil-depleted animals resulted a marked reduction in hyaline membrane formation and a decrease in alveolar-capillary permeability [37]. However, the specific cellular and molecular mechanisms that recruit leukocytes during the early phase of VILI have not been elucidated. Recently, Choudhury et al. [38] studied the effects of injurious ventilation in intact mice on early intravascular PMN sequestration. The authors demonstrated that mechanical stress initiates pulmonary PMN sequestration early in the course of VILI, and this phenomenon is associated with stretch-induced inflammatory events leading to circulating leukocytes stiffening, and is mediated by an L-selectindependent mechanism. PMN recruitment in VILI may be mediated by various chemoattractants and adhesion molecules derived from the several types of cells existing in the lung parenchyma and air spaces, such as epithelial and endothelial cells, alveolar macrophages, and fibroblasts [39]. Kotani et al. [40] demonstrated in intact animals that a noninjurious ventilatory

Ventilator-induced lung injury Lionetti et al. 85

to cyclic stretch and reported a strain dose-dependent increase in interleukin-8 messenger RNA and protein production; this deformation response could be inhibited by protein kinase inhibitors. Jo et al. [48] showed that mechanical stretch stimulates ERK1/2 rapidly and transiently in a dose-dependent manner through a Gai2 and tyrosine kinasemediated mechanism. Correa-Meyer et al. [49] reported that cyclic stretch of primary type II alveolar epithelial cells results in phosphorylation of ERK1/2 via G proteins. Akt and ERK1/2 are essential in regulating cell survival and cell death signals triggered by the injury via a G proteindependent pathway. Akt is a cytoplasmic protein that inhibits apoptosis by decreasing activation of caspase-3, caspase-9, Bad, and additional proapoptotic pathways [50]. ERK1/2 phosphorylation occurs through a receptor-mediated event; activated ERK1/2 can modulate the activity of several intracellular proteases involved in the inhibition of apoptosis including caspase-3 [51]. Several studies have demonstrated that inhibition of Akt and ERK1/2 results in enhancement of apoptosis [52]. Li et al. [53] recently demonstrated that inhibition of Erk1/2 in A549 cells exposed to cyclic stretch decreased apoptosis but did not affect the stretch-induced transcriptional regulation of interleukin-8 mRNA and interleukin-8 production. These observations may therefore lead to a novel hypothesis: mechanical deformation of pulmonary cells may trigger a G proteinmediated pathway that activates Akt and ERK1/2 and inhibits apoptosis, thus leading to cell death by necrosis; inhibition of this pathway turns down the Akt-ERK1/2 inhibitory effect, enhancing apoptosis and preserving the alveolar epithelium exposed to stretch. Further studies are required to explore this hypothesis.

The Acute Respiratory Distress Syndrome Network: Ventilation with lower tidal volumes as compared with traditional tidal volumes for acute lung injury and the acute respiratory distress syndrome. N Engl J Med 2000; 342: 13011308. The Acute Respiratory Distress Syndrome Network: Higher versus lower positive end-expiratory pressures in patients with the acute respiratory distress syndrome. N Engl J Med 2004; 351:327336. Dos Santos CC, Slutsky AS: Protective ventilation of patients with acute respiratory distress syndrome. Crit Care 2004; 8:145147. Ranieri VM, Suter PM, Tortorella C, et al. Effect of mechanical ventilation on inflammatory mediators in patients with acute respiratory distress syndrome: a randomized controlled trial. JAMA 1999; 282:5461. Uhlig S, Ranieri M, Slutsky AS: Biotrauma hypothesis of ventilator-induced lung injury. Am J Respir Crit Care Med 2004; 169:314315. Offner PJ, Moore EE: Lung injury severity scoring in the era of lung protective mechanical ventilation: the PaO2/FIO2 ratio. J Trauma 2003; 55:285289. Slutsky AS, Tremblay LN: Multiple system organ failure: is mechanical ventilation a contributing factor? Am J Respir Crit Care Med 1998; 157:17211725. Gurkan OU, ODonnell C, Brower R, et al. Differential effects of mechanical ventilatory strategy on lung injury and systemic organ inflammation in mice. Am J Physiol Lung Cell Mol Physiol 2003; 285:L710L718.

3 4

5 6 7 8

Shimabukuro DW, Sawa T, Gropper MA: Injury and repair in lung and airways. Crit Care Med 2003; 31(8 Suppl):S524S531. This review highlights a tutorial approach to understanding the mechanisms that result in acute inflammation of the lung. The authors describe the potential mechanisms of injury and repair in the lung affected by ALI/ARDS. 9

10 Waters CM, Sporn PH, Liu M, et al. Cellular biomechanics in the lung. Am J Physiol Lung Cell Mol Physiol 2002; 283:L503L509. 11 Fisher JL, Margulies SS: Na(+)-K(+)-ATPase activity in alveolar epithelial cells increases with cyclic stretch. Am J Physiol Lung Cell Mol Physiol 2002; 283:L737L746. 12 Kumar A, Lnu S, Malya R, et al. Mechanical stretch activates nuclear factorkappaB, activator protein-1, and mitogen-activated protein kinases in lung parenchyma: implications in asthma. FASEB J 2003; 17:18001811. 13 Tschumperlin DJ, Oswari J, Margulies AS: Deformation-induced injury of alveolar epithelial cells: effect of frequency, duration, and amplitude. Am J Respir Crit Care Med 2000; 162:357362.

14 Fisher JL, Levitan I, Margulies SS: Plasma membrane surface increases with tonic stretch of alveolar epithelial cells. Am J Respir Cell Mol Biol 2004; 31:200208. Using an alveolar epithelial cell stretch system, confocal microscopy, and fluorescent markers, the authors demonstrated the importance of additional phospholipid recruitment in plasma membrane expansion during tonic stretch of the attached basal surface. They demonstrated that this plasma membrane remodeling preponderates in the changes in cell surface shape and size demanded by stretching the cell, and, by using specific inhibitors, they showed that phospholipid insertion is unaffected by F-actin disassembly, ATP depletion, and calcium deprivation. 15 Sanchez-Esteban J, Wang Y, Gruppuso PA, et al. Mechanical stretch induces fetal type II cell differentiation via an epidermal growth factor receptor-extracellular-regulated protein kinase signaling pathway. Am J Respir Cell Mol Biol 2004; 30:7683.

Conclusion
Young et al. [54] recently showed that although recent clinical trials demonstrated that protective ventilatory strategy improved survival in patients with ALI, the proportion of patients receiving protective ventilatory strategies remains modest [1,2]. We have reviewed key mechanisms of VILI, such as cellular response to mechanical stretch, PMN recruitment and activation, and apoptosis/necrosis balance. If we are to succeed in minimizing the iatrogenic consequences of mechanical ventilation, we must understand the signal transduction mechanisms and develop potential pharmacologic targets to modulate the molecular and cellular effects of lung stretch.

16 Tschumperlin DJ, Dai G, Maly IV, et al. Mechanotransduction through growthfactor shedding into the extracellular space. Nature 2004; 429:8386. The authors demonstrated for the first time, in isolated perfused mouse lung model and normal human bronchial epithelial cells, that compressive stress shrinks the lateral intercellular space surrounding epithelial cells and triggers cellular signaling via autocrine binding of epidermal growth factor family ligands to the epidermal growth factor receptor, operating in a dynamically regulated extracellular volume. This establish a novel mechanotransduction system that is independent of forcedependent biochemical processes within the cell or cell membrane. In addition, they demonstrated by mathematical analysis that this mechanism is predictable. 17 Vlahakis NE, Hubmayr RD: Response of alveolar cells to mechanical stress. Curr Opin Crit Care 2003; 9:28. This is a comprehensive review of the effects of mechanical stretch on alveolar cells. The authors focused on the intracellular signal transduction pathways, the surfactant system, and cell injury and repair. 18 Bailey TC, Martin EL, Zhao L, et al. High oxygen concentrations predispose mouse lungs to the deleterious effects of high stretch ventilation. J Appl Physiol 2003; 94:975982.

References and recommended reading

Papers of particular interest, published within the annual period of review, have been highlighted as: of special interest of outstanding interest

19 Hammerschmidt S, Kuhn H, Grasenack T, et al. Apoptosis and necrosis induced by cyclic mechanical stretching in alveolar type II cells. Am J Respir Cell Mol Biol 2004; 30:396402.

20 Imai Y, Parodo J, Kajikawa O, et al. Injurious mechanical ventilation and endorgan epithelial cell apoptosis and organ dysfunction in an experimental

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model of acute respiratory distress syndrome. JAMA 2003; 2330;289: 21042112. In an in vivo rabbit model, the authors showed for the first time that an injurious ventilatory strategy may lead to end-organ epithelial cell apoptosis, Fas-dependent and organ dysfunction (kidney and small intestine), with elevation of biochemical markers. They found a significant correlation between changes in soluble Fas ligand and changes in biochemical markers in patients with ARDS. This may partially explain the high rate of multiple organ dysfunction observed in patients with ARDS. 21 Vlahakis NE, Schroeder MA, Limper AH: Stretch induces cytokine release by alveolar epithelial cells in vitro. Am J Physiol 1999; 277:L167L173. 22 Wilson MR, Choudhury S, Goddard ME, et al. High tidal volume upregulates intrapulmonary cytokines in an in vivo mouse model of ventilator- induced lung injury. J Appl Physiol 2003; 95:13851393. 23 Uhlig U, Fehrenbach H, Lachmann RA, et al. Phosphoinositide 3-OH kinase inhibition prevents ventilation-induced lung cell activation. Am J Respir Crit Care Med 2004; 169:201208. Using an isolated perfused mouse lung model and an in vivo rat model, the authors focused on cell types and specific signaling mechanisms that are activated by ventilation with increased pressure/volume. This study provides evidence that after overventilation there is increased expression of interleukin-6 mRNA and NF-kB activation in alveolar macrophages and alveolar epithelial type II cells that is selectively inhibited by phosphoinositide 3-OH kinase inhibitor.

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39 Jafari B, Ouyang B, Li LF, et al. Intracellular glutathione in stretch-induced cytokine release from alveolar type-2 like cells. Respirology 2004; 9:43 53. 40 Kotani M, Kotani T, Ishizaka A, et al. Neutrophil depletion attenuates interleukin-8 production in mild-overstretch ventilated normal rabbit. Crit Care Med 2004; 32:514519. 41 Belperio JA, Keane MP, Burdick MD, et al. Critical role for CXCR2 and CXCR2 ligands during the pathogenesis of ventilator-induced lung injury. J Clin Invest 2002; 110:17031716.

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26 Mascarenhas MM, Day RM, Ochoa CD, et al. Low molecular weight hyaluronan from stretched lung enhances interleukin-8 expression. Am J Respir Cell Mol Biol 2004; 30:5160.

42 Steinberg JM, Schiller HJ, Halter JM, et al. Alveolar instability causes early ventilator-induced lung injury independent of neutrophils. Am J Respir Crit Care Med 2003; Oct 2 (Epub ahead of print). Using an in vivo pig model of VILI and in vivo video microscopy, the authors provide evidence for the first time that unstable alveoli are subjected to VILI by surfactant-dependent and neutrophil-independent mechanism. The magnitude of alveolar instability was quantified by computer image analysis. 43 Majno G, Joris I: Apoptosis, oncosis, and necrosis: an overview of cell death. Am J Pathol 1995; 146:315. 44 Nishino H, Nemoto N, Lu W, et al. Significance of apoptosis in morphogenesis of human lung development: light microscopic observation using in situ DNA end-labeling and ultrastructural study. Med Electron Microsc 1999; 32: 5761.

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45 Bardales RH, Xie SS, Schaefer RF, et al. Apoptosis is a major pathway responsible for the resolution of type II pneumocytes in acute lung injury. Am J Pathol 1996; 149:845852. 46 Fischer S, Cassivi SD, Xavier AM, et al. Cell death in human lung transplantation: apoptosis induction in human lungs during ischemia and after transplantation. Ann Surg 2000; 231:424431. 47 Oudin S, Pugin J: Role of MAP kinase activation in interleukin-8 production by human BEAS-2B bronchial epithelial cells submitted to cyclic stretch. Am J Respir Cell Mol Biol 2002; 27:107114. 48 Jo H, Sipos K, Go YM, et al. Differential effect of shear stress on extracellular signal-regulated kinase and N-terminal Jun kinase in endothelial cells. Gi2and Gbeta/gamma-dependent signaling pathways. J Biol Chem 1997; 272: 13951401. 49 Correa-Meyer E, Pesce L, Guerrero C, et al. Cyclic stretch activates ERK1/2 via G proteins and EGFR in alveolar epithelial cells. Am J Physiol Lung Cell Mol Physiol 2002; 282:L883L891. 50 Cardone MH, Roy N, Stennicke HR, et al. Regulation of cell death protease caspase-9 by phosphorylation. Science 1998; 282:13181321. 51 Bonni A, Brunet A, West AE, et al. Cell survival promoted by the Ras-MAPK signaling pathway by transcription-dependent and -independent mechanisms. Science 1999; 286:13581362. 52 Flaherty DM, Hinde SL, Monick MM, et al. Adenovirus vectors activate survival pathways in lung epithelial cells. Am J Physiol Lung Cell Mol Physiol 2004; 287:L393L401. 53 Li LF, Ouyang B, Choukroun G, et al. Stretch-induced IL-8 depends on c-Jun NH2-terminal and nuclear factor-kappaB-inducing kinases. Am J Physiol Lung Cell Mol Physiol 2003; 285:L464L475. 54 Young MP, Manning HL, Wilson DL, et al. Ventilation of patients with acute lung injury and acute respiratory distress syndrome: has new evidence changed clinical practice? Crit Care Med 2004; 32:12601265.

30 Czarny M, Schnitzer JE: Neutral sphingomyelinase inhibitor scyphostatin pre vents and ceramide mimics mechanotransduction in vascular endothelium. Am J Physiol Heart Circ Physiol 2004; 287:H1344H1352. In an in vitro model using pulmonary endothelial cells, the authors extended their previous work focusing on the role of neutral sphingomyelinase and ceramides in mechanosignaling after endothelial deformation. In this study they established that after endothelial distension there is an activation of caveolar neutral sphingomyelinase as well as downstream tyrosine and mitogen-activated protein kinases by generation of the lipid second messenger ceramide. 31 Moore JE Jr, Burki E, Suciu A, et al. A device for subjecting vascular endothelial cells to both fluid shear stress and circumferential cyclic stretch. Ann Biomed Eng 1994; 22:416422. 32 Haseneen NA, Vaday GG, Zucker S, et al. Mechanical stretch induces MMP-2 release and activation in lung endothelium: role of EMMPRIN. Am J Physiol Lung Cell Mol Physiol 2003; 284:L541L547. 33 Kuebler WM, Uhlig U, Goldmann T, et al. Stretch activates nitric oxide production in pulmonary vascular endothelial cells in situ. Am J Respir Crit Care Med 2003; 168:13911398. 34 Bhattacharya S, Sen N, Yiming MT, et al. High tidal volume ventilation induces proinflammatory signaling in rat lung endothelium. Am J Respir Cell Mol Biol 2003; 28:218224.

35 Vanderbilt JN, Mager EM, Allen L, et al. CXC chemokines and their receptors are expressed in type II cells and upregulated following lung injury. Am J Respir Cell Mol Biol 2003; 29:661668.