New Insights Into Acute Lung Injury

Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) were first identified over 40 years ago by Ashbaugh et al [1]. ALI and ARDS are forms of progressive respiratory failure characterized by an acute onset of dyspnea, decreased arterial oxygen pressure (hypoxemia), bilateral infiltrates on chest radiograms, and absence of clinical evidence of primary left heart failure. The American-European Consensus Conference on ARDS has led to a definition of ALI as a PaO₂/FiO₂ < 300 while ARDS is defined as <200 for ARDS [2, 3]. Thus, ARDS represents a subset of ALI patients with greater severity of symptoms. In ALI/ARDS, the integrity of the separation between the alveolus and the pulmonary circulation is compromised either by endothelial or epithelial injury or more commonly both. This damage leads to increased vascular permeability, alveolar flooding, and surfactant abnormalities (due to damage of type II pneumocytes). ALI/ARDS can occur in response to a number of insults that either directly or indirectly cause lung injury. The most common indirect insult leading to ALI is the release of lipopolysaccharide (LPS) from the outer cell wall of Gram-negative (G^-) bacteria producing sepsis [4]. Other common causes include severe trauma with shock, multiple transfusions, burn injury, pneumonia and aspiration of gastric contents. Based on a recent announcement from the National Institute of Health (NIH), ALI and its more severe form, acute respiratory distress syndromes (ARDS) affect approximately 150,000 people in the United States every year. Nearly 28,500 are likely to die. Some survivors recover completely. However, others may have lasting damage to their lungs and additional health problems. Sepsis represents the systemic inflammatory response to infection [5]. Severe sepsis is defined as sepsis complicated by organ dysfunction and hypotension (septic shock). Lungs are among the most frequently affected organs in severe sepsis leading to ALI and ARDS[6]. The incidence of sepsis has increased by 8.7% from 1979-2000 [6] and mortality ranges from 30-50% [7-9]. Clinical trials targeting inflammatory mediators have shown no survival benefit [10-15] and other strategies have failed to reduce morbidity associated with severe sepsis except for the survival benefit that has been identified with low tidal volume mechanical ventilation [16] and the use of recombinant activated protein C [17]. With an unacceptably high mortality rate up to 58% [18] it is evident that a clearer understanding of both the mechanisms involved in the pathogenesis of ALI/ARDS and the development of new therapies for the control of the disease are critical. Thus, this special issue has brought together five studies that are highlight the development of new reagents for the study of the mechanisms involved in ALI, elucidate new signaling pathways that may be involved in the pathophysiology of ALI, as well as evaluating potential new therapeutic agents to prevent the endothelial barrier disruption which is a hallmark of ALI. In this editorial I will briefly describe the major findings of each of the studies and attempt to highlight both the strengths and limitations of these individual studies and the field of ALI as a whole.

The initial site affected during the development of ALI/ARDS is the endothelial cell (EC) layer lining the micro-vessels in the lung. The vascular endothelium is a single-cell layer that acts as a semi-selective barrier between the plasma and interstitial fluid. This function is critical for normal vessel wall homeostasis. Endothelial permeability is regulated by the balance between the contractile machinery within the cell and the elements that oppose contraction. The latter include tethering complexes that are responsible for cell-cell and cell-substrate contacts and systems granting cell rigidity that prevent cell collapse, such as actin filaments, microtubules and intermediate filaments [19, 20]. One of the major limitations in the study of ALI/ARDS is the lack of a reproducible cell culture model that can be used to investigate how EC that line the micro-vessels in the pulmonary system are disrupted by ALI/ARDS. However, a significant number of published studies utilize pulmonary EC of either bovine or human origin but are isolated from the major vessels such as the pulmonary artery. These vessels are not normally affected by ALI/ARDS. Further, the commercially available EC isolated from human micro-vessels do not appear to maintain the archetypal properties of EC namely their cobblestone appearance. Further, the currently available EC lines require large quantities of agents such as LPS (∼100 endotoxin units (EU)/ml) for measureable barrier disruption to occur. However, Catravas et al in this issue describe a new method for harvesting and culturing of human lung microvascular endothelial cells (HLMVEC). Further, they present convincing data demonstrating the identity of these cells and their response to appropriate stimuli. These HLMVEC appear to be of superior quality to other available cells exhibiting small size, characteristic cobblestone appearance, and a contact-inhibited monolayer. Further, these characteristics are maintained over multiple passages. The HLMVEC exhibited a tight monolayer with excellent transendothelial resistance (TER) (∼1000MΩ) measured using an Applied Biosystems ECIS instrument. Most excitingly, freshly harvested HLMVEC demonstrated excellent sensitivity to a variety of barrier disrupting agents. For example as little as 1 EU/ml elicited a profound decrease in TER. These cells are likely to become the “gold-standard” for investigations into the mechanisms underlying the endothelial barrier disruption in ALI/ARDS.

Although extensive investigations have been carried out to delineate the mechanisms underlying the development of ALI/ARDS little of therapeutic value has resulted from these efforts. Thus, new signaling pathways may need to be elucidated and tested for their interventional potential. Thus, the remaining four studies in this series have begun to evaluate new mechanisms of endothelial protection in ALI/ARDS. In the first of these studies Sharma and colleagues have examined the role of protein nitration in the pathophysiology of ALI/ARDS. The nitration of tyrosine residues is mediated by reactive nitrogen species (RNS). Increased RNS production occurs when dysregulated nitric oxide (NO) production reacts with reactive oxygen species (ROS) such as superoxide. This reaction generates RNS, including peroxynitrite (ONOO-). ONOO-leads to tyrosine nitration, a covalent modification that adds a nitro group (-NO₂) to one ortho carbon of the phenolic ring of tyrosine to form 3-nitrotyrosine (3-NT). This introduces a net negative charge to the tyrosine, altering structural properties and catalytic activity of the protein. There has been increasing interest in the effects of tyrosine nitration on changes in protein structure in diverse pathologic conditions. The nitration of tyrosine residues to form 3-NT residues is widely used as a marker of ONOO- formation and Sharma et al have demonstrated that elevated levels of protein tyrosine nitration precedes the endothelial injury associated with the development of ALI in the LPS injected mouse. This increase in ONOO- appears to be related to eNOS uncoupling mediated via an increase in the levels of the endogenous NOS inhibitor asymmetric dimethyargine (ADMA). Further, the increases in ADMA occur secondary to a post-translational inhibition of the enzyme that degrades ADMA, dimethylarginine dimethylaminohydrolase (DDAH). Inhibition of DDAH without alteration of its gene expression is becoming more widely appreciated as Lin and colleagues have shown that elevated glucose raises endothelial ADMA levels by inhibiting DDAH activity via a mechanism involving oxidative stress [21]. Similarly, LPS significantly increases the levels of ADMA and decreased DDAH activity in cultured medium from human endothelial cells [22]. Of great clinical interest is a recent study indicating that ADMA levels are elevated in patients with septic shock [23] suggesting that the ADMA-DDAH pathway could be responsive to therapeutic intervention. Further, the studies of Church et al investigating the effect of phosphatase and tensin homologue deleted on chromosome 10 (PTEN) signaling on eNOS activity are intriguing with respect to DDAH activity. PTEN is a lipid phosphatase that functions as a negative regulator of the phosphoinositide-3-kinase (PI3K) pathway. Although most studies on PTEN have focused on its role in cancer progression where it is found in the advanced stages of a number of cancers [24] it is now becoming apparent that PTEN can also modulate the blood vessel structure and function [25-27]. In addition, some recent studies have shown a role for PTEN in the progression of ALI as both the pharmacological inhibition of PTEN and its conditional ablation in the epithelia of the lung have been shown to reduce the severity of ALI [28, 29]. The data indicating that enhancing PTEN signaling decreases NO generation from eNOS through the inhibition of Akt-mediated phosphorylation correlates well with the decrease in eNOS-derived NO that Sharma et al have shown to be an early event after I.P. LPS in the mouse lung. Although Church et al have focused on changes in seine phosphorylation to explain the decreases in NO induced by PTEN over-expression in COS-7 cells, the potential of PTEN to inhibit DDAH activity is an intriguing but unexplored possibility.

The Sharma study also highlights the limitations of current investigations: the lack of easily available means to identify post translational modifications of proteins. This is a major roadblock to ourunderstanding of the potential mechanistic contributions of these modifications to disease processes. New methodologies are needed that go beyond the limitations of current analytical approaches that focus on measuring either global changes in protein nitration (as in the Sharma study) or merely identifying individual proteins that are susceptible to nitration. Rather priority should be placed on moving towards identifying both the individual tyrosine residues targeted by nitration and the effect these nitration events have on the structure-function relationship of the nitrated protein.

Recently, attention has been given to the therapeutic potential of purinergic agonists in the treatment of cardiovascular and pulmonary diseases [30-32] and the study in this issue from Umapathy et al has evaluated the barrier protective properties of adenosine in vitro using cultured human PAEC. Extracellular purines can function as intercellular signaling molecules when released from different sources in the body [33] and accumulating experimental data suggest that purines could be barrier-protective agents against the effects of ALI, as they are present in the EC microenvironment in vivo and they decrease permeability in vitro. The dominant pathway modulating the levels of extracellular adenosine is the extracellular catabolism of ATP to adenosine through the progressive action of a number of ectonucleotidases [34, 35]. In this study Umapathy et al clearly demonstrate that the addition of adenosine in physiologically relevant concentrations (1-5μM) significantly increases the TER of cultured human PAEC. Further, using an elegant siRNA strategy they confirm that the mechanism of action is mediated via A2A receptors and a cAMP-dependent signaling pathway that produces changes in F-actin via the activation of myosin light chain phosphatase (MLCP). This report also emphasizes an important limitation in our current knowledge of the role of phosphatases in the pathophysiology of ALI. This is primarily due to the inherent technical difficulties in studying the action of protein phosphatases. MLCP is a type 1 Ser/Thr PPase and the holoenzyme is composed of 3 subunits: a catalytic subunit (CS1) of 38 kD that was identified as CS1δ isoform (currently CS1β) and two non-catalytic subunits of 20-21 and 110 -130 kD [36-39]. The 110-130 kD non-catalytic subunit, called myosin PPase targeting subunit 1 (MYPT1), binds to CS1 and this targets CS1 to MLC and provides substrate specificity [40, 41]. Human MYPT1 and its splice variants are encoded by a single gene on human chromosome 12q15-q21.2 [42]. It is well established that MLC is the major substrate for MLCP. However, recent data have revealed that MYPT1 can also bind directly to the ezrin/radixin/moesin (ERM) family of actin-binding proteins [43, 44]. ERM proteins act as linkers between the actin cytoskeleton and plasma membrane proteins, and as signal transducers in responses involving cytoskeletal remodeling [45] as Umapathy et al have shown with adenosine. Established a role of the ERMs in the barrier protective effect of adenosine could yield novel potential therapeutic targets for ALI. However, it should be noted that a major limitation of the Umapathy study is the fact that there was no in vivo confirmation of the barrier enhancement or protective effects in vivo. Hopefully these important confirmatory studies will be soon forthcoming.

One of the major limitations in our understanding of ALI/ARDS is that much of the work is carried out using LPS as a model of G^- sepsis ad studies on G⁺ infections are less common in the literature. Thus, the final study from Xiong et al is potentially very powerful and timely in that it has identified a new signaling mechanism and potential therapy for the treatment of G⁺ associated bacterial infections using the G⁺ pore forming toxin, Listeriolysin-O (LLO) isolated from Listeria monocytogenes, a bacterium that causes a severe food-borne disease in neonates characterized by meningitis and meningo-encephalitis. The data presented indicate that the oxidative stress associated with LLO is mediated via the activation of PKCα and that its pharmacologic inhibition using GÖ6976, attenuates the LLO-mediated decreases in TER in the HLMVEC isolated by Catravas et al. Further, and of significant therapeutic interest, the authors have shown that the lectin like domain of TNF-the TIP peptide- prevents the LLO-mediated increase in oxidative stress, at least in part, by preventing the up-regulation of Nox-4. Howver, the mechanism by which the TIP peptide exerts its barrier protective effect has been only partially elucidated as the TIP peptide prevented the LLO-mediated activation of PKCα but did not prevent the influx of intracellular calcium known to play a key role its activation. Similar to the report by Umapathy et al., this study was also in vitro and did not evaluate the barrier protective effects of the TIP peptide in vivo in LLO challenged mice. Again it is hoped that these studies will be forthcoming soon.

In conclusion, this special issue focuses on the role of the endothelium in ALI/ARDS and highlights the complexity of the disease process and the problems with developing single therapies targeted at alleviating the significant morbidity and mortality associated with ALI/ARDS. Several new signaling pathways that could have therapeutic potential (DDAH, PTEN, PKCα) are investigated in human microvascular endothelial cells that are isolated from human pulmonary microvessels and are significantly more relevant to this etiology of this disease. Finally, new agents are tested for their ability to prevent endothelial barrier disruption in vitro (adenosine, TIP peptide) and in vivo (peroxynitrite scavengers) against agents derived from both G^- and G⁺ bacteria. It is hoped that as these studies progress they will begin to elucidate mechanistic similarities and differences between the effects of G^- and G⁺ infections and that these mechanisms will provide a pathway to new and efficacious therapeutic strategies that integrates multiple approaches.