Vascular Pharmacology 52 (2010) 171–174

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Vascular Pharmacology

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Introduction

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.(1967). ALI and ARDS are forms of progressive respiratory failurecharacterized by an acute onset of dyspnea, decreased arterial oxygenpressure (hypoxemia), bilateral infiltrates on chest radiograms, andabsence of clinical evidence of primary left heart failure. TheAmerican-European Consensus Conference on ARDS has led to adefinition of ALI as a PaO2/FiO2b300while ARDS is defined as b200 forARDS (Bernard et al., 1994a; Bernard et al., 1994b). Thus, ARDSrepresents a subset of ALI patients with greater severity of symptoms.In ALI/ARDS, the integrity of the separation between the alveolus andthe pulmonary circulation is compromised either by endothelial orepithelial injury or more commonly both. This damage leads toincreased vascular permeability, alveolar flooding, and surfactantabnormalities (due to damage of type II pneumocytes). ALI/ARDS canoccur in response to a number of insults that either directly orindirectly cause lung injury. The most common indirect insult leadingto ALI is the release of lipopolysaccharide (LPS) from the outer cellwall of Gram-negative (G−) bacteria producing sepsis (Hudson et al.,1995). Other common causes include severe trauma with shock,multiple transfusions, burn injury, pneumonia and aspiration ofgastric contents. Based on a recent announcement from the NationalInstitute of Health (NIH), ALI and its more severe form, acuterespiratory distress syndromes (ARDS) affect approximately 150,000people in the United States every year. Nearly 28,500 are likely to die.Some survivors recover completely. However, othersmay have lastingdamage to their lungs and additional health problems. Sepsisrepresents the systemic inflammatory response to infection (Jacobi,2002). Severe sepsis is defined as sepsis complicated by organdysfunction and hypotension (septic shock). Lungs are among themost frequently affected organs in severe sepsis leading to ALI andARDS (Martin et al., 2003). The incidence of sepsis has increased by8.7% from 1979–2000 (Martin et al., 2003) and mortality ranges from30–50% (Angus et al., 2000; Annane et al., 2000; Rangel-Frausto et al.,1995). Clinical trials targeting inflammatory mediators have shownno survival benefit (Abraham et al., 1998; Abraham et al., 2001;Dhainaut et al., 1998; Fink, 1998; Fisher et al., 1994; McCloskey et al.,1994) and other strategies have failed to reduce morbidity associatedwith severe sepsis except for the survival benefit that has beenidentified with low tidal volume mechanical ventilation (Jain &DalNogare, 2006) and the use of recombinant activated protein C(Bernard et al., 2001). With an unacceptably high mortality rate up to58% (MacCallum & Evans, 2005) it is evident that a clearerunderstanding of both the mechanisms involved in the pathogenesisof ALI/ARDS and the development of new therapies for the control ofthe disease are critical. Thus, this special issue has brought togetherfive studies that are highlight the development of new reagents for

1537-1891/$ – see front matter © 2010 Elsevier Inc. All rights reserved.doi:10.1016/j.vph.2010.02.002

the study of the mechanisms involved in ALI, elucidate new signalingpathways that may be involved in the pathophysiology of ALI, as wellas evaluating potential new therapeutic agents to prevent theendothelial barrier disruption which is a hallmark of ALI. In thiseditorial I will briefly describe themajor findings of each of the studiesand attempt to highlight both the strengths and limitations of theseindividual studies and the field of ALI as a whole.

The initial site affected during the development of ALI/ARDS is theendothelial cell (EC) layer lining the micro-vessels in the lung. Thevascular endothelium is a single-cell layer that acts as a semi-selectivebarrier between the plasma and interstitial fluid. This function iscritical for normal vessel wall homeostasis. Endothelial permeabilityis regulated by the balance between the contractile machinery withinthe cell and the elements that oppose contraction. The latter includetethering complexes that are responsible for cell–cell and cell-substrate contacts and systems granting cell rigidity that preventcell collapse, such as actin filaments, microtubules and intermediatefilaments (Dudek et al., 2002; Birukov et al., 2002). One of the majorlimitations in the study of ALI/ARDS is the lack of a reproducible cellculture model that can be used to investigate how EC that line themicro-vessels in the pulmonary system are disrupted by ALI/ARDS.However, a significant number of published studies utilize pulmonaryEC of either bovine or human origin but are isolated from the majorvessels such as the pulmonary artery. These vessels are not normallyaffected by ALI/ARDS. Further, the commercially available EC isolatedfrom human micro-vessels do not appear to maintain the archetypalproperties of EC namely their cobblestone appearance. Further, thecurrently available EC lines require large quantities of agents such asLPS (∼100 endotoxin units (EU)/ml) for measureable barrierdisruption to occur. However, Catravas et al in this issue describe anew method for harvesting and culturing of human lung microvas-cular endothelial cells (HLMVEC). Further, they present convincingdata demonstrating the identity of these cells and their response toappropriate stimuli. These HLMVEC appear to be of superior quality toother available cells exhibiting small size, characteristic cobblestoneappearance, and a contact-inhibited monolayer. Further, thesecharacteristics are maintained over multiple passages. The HLMVECexhibited a tight monolayer with excellent transendothelial resis-tance (TER) (∼1000MΩ) measured using an Applied Biosystems ECISinstrument. Most excitingly, freshly harvested HLMVEC demonstratedexcellent sensitivity to a variety of barrier disrupting agents. Forexample as little as 1 EU/ml elicited a profound decrease in TER. Thesecells are likely to become the “gold-standard” for investigations intothe mechanisms underlying the endothelial barrier disruption in ALI/ARDS.

Although extensive investigations have been carried out todelineate the mechanisms underlying the development of ALI/ARDS

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little of therapeutic value has resulted from these efforts. Thus, newsignaling pathways may need to be elucidated and tested for theirinterventional potential. Thus, the remaining four studies in this serieshave begun to evaluate new mechanisms of endothelial protection inALI/ARDS. In the first of these studies Sharma and colleagues haveexamined the role of protein nitration in the pathophysiology of ALI/ARDS. The nitration of tyrosine residues is mediated by reactivenitrogen species (RNS). Increased RNS production occurs whendysregulated nitric oxide (NO) production reacts with reactiveoxygen species (ROS) such as superoxide. This reaction generatesRNS, including peroxynitrite (ONOO–). ONOO– leads to tyrosinenitration, a covalent modification that adds a nitro group (–NO2) toone ortho carbon of the phenolic ring of tyrosine to form 3-nitrotyrosine (3-NT). This introduces a net negative charge to thetyrosine, altering structural properties and catalytic activity of theprotein. There has been increasing interest in the effects of tyrosinenitration on changes in protein structure in diverse pathologicconditions. The nitration of tyrosine residues to form 3-NT residuesis widely used as a marker of ONOO– formation and Sharma et al havedemonstrated that elevated levels of protein tyrosine nitrationprecedes the endothelial injury associated with the development ofALI in the LPS injected mouse. This increase in ONOO– appears to berelated to eNOS uncoupling mediated via an increase in the levels ofthe endogenous NOS inhibitor asymmetric dimethyargine (ADMA).Further, the increases in ADMA occurs secondary to a post-translational inhibition of the enzyme that degrades ADMA, dimethy-larginine dimethylaminohydrolase (DDAH). Inhibition of DDAHwithout alteration of its gene expression is becoming more widelyappreciated as Lin and colleagues have shown that elevated glucoseraises endothelial ADMA levels by inhibiting DDAH activity via amechanism involving oxidative stress (Lin et al., 2002). Similarly, LPSsignificantly increases the levels of ADMA and decreased DDAHactivity in cultured medium from human endothelial cells (Xin et al.,2007). Of great clinical interest is a recent study indicating that ADMAlevels are elevated in patients with septic shock (O'Dwyer et al., 2006)suggesting that the ADMA-DDAH pathway could be responsive totherapeutic intervention. Further, the studies of Church et alinvestigating the effect of phosphatase and tensin homologue deletedon chromosome 10 (PTEN) signaling on eNOS activity are intriguingwith respect to DDAH activity. PTEN is a lipid phosphatase thatfunctions as a negative regulator of the phosphoinositide-3-kinase(PI3K) pathway. Although most studies on PTEN have focused on itsrole in cancer progressionwhere it is found in the advanced stages of anumber of cancers (Li et al., 1997) it is now becoming apparent thatPTEN can also modulate the blood vessel structure and function(Marsh et al., 1999; Zhong et al., 2000; Tsigkos et al., 2006). Inaddition, some recent studies have shown a role for PTEN in theprogression of ALI as both the pharmacological inhibition of PTEN andits conditional ablation in the epithelia of the lung have been shown toreduce the severity of ALI (Lai et al., 2009; Tiozzo et al., 2009). Thedata indicating that enhancing PTEN signaling decreases NO gener-ation from eNOS through the inhibition of Akt-mediated phosphor-ylation correlates well with the decrease in eNOS-derived NO thatSharma et al have shown to be an early event after I.P. LPS in themouse lung. Although Church et al have focused on changes in seinephosphorylation to explain the decreases in NO induced by PTENover-expression in COS-7 cells, the potential of PTEN to inhibit DDAHactivity is an intriguing but unexplored possibility.

The Sharma study also highlights the limitations of currentinvestigations: the lack of easily available means to identify posttranslational modifications of proteins. This is a major roadblock toour understanding of the potential mechanistic contributions of thesemodifications to disease processes. New methodologies are neededthat go beyond the limitations of current analytical approaches thatfocus on measuring either global changes in protein nitration (as inthe Sharma study) or merely identifying individual proteins that are

susceptible to nitration. Rather priority should be placed on movingtowards identifying both the individual tyrosine residues targeted bynitration 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 ofpurinergic agonists in the treatment of cardiovascular and pulmonarydiseases (Burnstock, 2008; Raju et al., 2008; Smyth et al., 2009;Kolosova et al., 2008) and the study in this issue from Umapathy et alhas evaluated the barrier protective properties of adenosine in vitrousing cultured human PAEC. Extracellular purines can function asintercellular signaling molecules when released from differentsources in the body (Burnstock & Williams, 2000) and accumulatingexperimental data suggest that purines could be barrier-protectiveagents against the effects of ALI, as they are present in the ECmicroenvironment in vivo and they decrease permeability in vitro.The dominant pathway modulating the levels of extracellularadenosine is the extracellular catabolism of ATP to adenosine throughthe progressive action of a number of ectonucleotidases (Eltzschig etal., 2004; Thomson et al., 2000). In this study Umapathy et al clearlydemonstrate that the addition of adenosine in physiologically relevantconcentrations (1–5 µM) significantly increases the TER of culturedhuman PAEC. Further, using an elegant siRNA strategy they confirmthat the mechanism of action is mediated via A2A receptors and acAMP-dependent signaling pathway that produces changes in F-actinvia the activation of myosin light chain phosphatase (MLCP). Thisreport also emphasizes an important limitation in our currentknowledge of the role of phosphatases in the pathophysiology ofALI. This is primarily due to the inherent technical difficulties instudying the action of protein phosphatases. MLCP is a type 1 Ser/ThrPPase and the holoenzyme is composed of 3 subunits: a catalyticsubunit (CS1) of 38 kD that was identified as CS1δ isoform (currentlyCS1β) and two non-catalytic subunits of 20–21 and 110–130 kD(Alessi et al., 1992; Shimizu et al., 1994; Shirazi et al., 1994;Hartshorne et al., 1998). The 110–130 kD non-catalytic subunit, calledmyosin PPase targeting subunit 1 (MYPT1), binds to CS1 and thistargets CS1 to MLC and provides substrate specificity (Hubbard &Cohen, 1993; Johnson et al., 1997). Human MYPT1 and its splicevariants are encoded by a single gene on human chromosome 12q15–q21.2 (Takahashi et al., 1997). It is well established that MLC is themajor substrate for MLCP. However, recent data have revealed thatMYPT1 can also bind directly to the ezrin/radixin/moesin (ERM)family of actin-binding proteins (Fukata et al., 1998; Kimura et al.,1998). ERM proteins act as linkers between the actin cytoskeleton andplasma membrane proteins, and as signal transducers in responsesinvolving cytoskeletal remodeling (Bretscher et al., 2002) as Uma-pathy et al have shownwith adenosine. Established a role of the ERMsin the barrier protective effect of adenosine could yield novel potentialtherapeutic targets for ALI. However, it should be noted that a majorlimitation of the Umapathy study is the fact that there was no in vivoconfirmation of the barrier enhancement or protective effects in vivo.Hopefully these important confirmatory studies will be soonforthcoming.

One of the major limitations in our understanding of ALI/ARDS isthat much of the work is carried out using LPS as a model of G− sepsisad studies on G+ infections are less common in the literature. Thus,the final study fromXiong et al is potentially very powerful and timelyin that it has identified a new signaling mechanism and potentialtherapy for the treatment of G+ associated bacterial infections usingthe G+ pore forming toxin, Listeriolysin-O (LLO) isolated from Listeriamonocytogenes, a bacterium that causes a severe food-borne disease inneonates characterized by meningitis and meningo-encephalitis. Thedata presented indicate that the oxidative stress associated with LLOis mediated via the activation of PKCα and that its pharmacologicinhibition using GÖ6976, attenuates the LLO-mediated decreases inTER in the HLMVEC isolated by Catravas et al. Further, and ofsignificant therapeutic interest, the authors have shown that the

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lectin like domain of TNF-the TIP peptide- prevents the LLO-mediatedincrease in oxidative stress, at least in part, by preventing the up-regulation of Nox-4. However, the mechanism by which the TIPpeptide exerts its barrier protective effect has been only partiallyelucidated as the TIP peptide prevented the LLO-mediated activationof PKCα but did not prevent the influx of intracellular calcium knownto play a key role its activation. Similar to the report by Umapathy etal., this study was also in vitro and did not evaluate the barrierprotective 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 theendothelium in ALI/ARDS and highlights the complexity of the diseaseprocess and the problemswith developing single therapies targeted atalleviating the significant morbidity and mortality associated withALI/ARDS. Several new signaling pathways that could have therapeu-tic potential (DDAH, PTEN, PKCα) are investigated in humanmicrovascular endothelial cells that are isolated from humanpulmonary microvessels and are significantly more relevant to thisetiology of this disease. Finally, new agents are tested for their abilityto prevent endothelial barrier disruption in vitro (adenosine, TIPpeptide) and in vivo (peroxynitrite scavengers) against agentsderived from both G− and G+ bacteria. It is hoped that as thesestudies progress they will begin to elucidate mechanistic similaritiesand differences between the effects of G− and G+ infections and thatthese mechanisms will provide a pathway to new and efficacioustherapeutic strategies that integrates multiple approaches.

Acknowledgements

This work was supported in part by grants HL60190 (to SMB),HL67841, HL72123, HL70061, HL084739, and R21HD057406 all fromthe National Institutes of Health, by a Transatlantic NetworkDevelopment Grant from the Fondation Leducq, and by a Program-matic Development award from the from the CardiovascularDiscovery Institute of the Medical College of Georgia.

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Stephen M. BlackProgram in Pulmonary Disease, Vascular Biology Center,

1459 Laney Walker Blvd, CB3201B, Medical College of Georgia, Augusta,GA 30912, USA

E-mail address: sblack@mcg.edu.