IntroductionPrevious studies have implicated extracellular nucleotide metabolites, predominantly adenosine, as triggers of endogenous protective mechanisms in a number of acute injury models. [1][2][3][4][5][6][7] Extracellular adenosine is derived primarily through phosphohydrolysis of adenosine 5Ј-monophosphate (AMP). Ecto-5Ј-nucleotidase (CD73), a ubiquitously expressed ectoenzyme, is the pacemaker of this reaction. 8 Studies on the role of CD73 in tissue-injury showed that cd73 Ϫ/Ϫ mice develop profound vascular leakage and pulmonary edema upon hypoxia exposure. 8 Once generated into the extracellular space, adenosine can signal through any of 4 G-protein coupled adenosine-receptors (ARs: A1AR/A2AAR/A2BAR/A3AR). All of these receptors are expressed on vascular endothelia 9 and have been implicated in tissue-protection in different models of injury. [1][2][3]7,[10][11][12][13][14][15][16][17][18] Changes in vascular barrier function closely coincide with tissue injury of many etiologies, and result in fluid loss, edema, and organ dysfunction. [19][20][21] The predominant barrier (ϳ90%) to movement of macromolecules across a blood vessel wall is presented by the vascular endothelium. 20,22 Under physiologic conditions, macromolecules such as albumin (molecular weight ϳ70 kD) can cross the endothelial monolayer via a paracellular route (eg, by passing between adjacent endothelia) with some contribution of transcellular passage. 23,24 Endothelial barrier function correlates inversely with the size of molecules that can gain entry into tissues and differs between tissues of different origins. Endothelial permeability is highly regulated and may increase markedly upon exposure to inflammatory stimuli (eg, lipopolysaccharide, bacteria, bacterial compounds, prostaglandins, reactive oxygen species, leukotrienes) or adverse conditions such as ischemia or hypoxia. 18,20,[25][26][27][28][29] Given that activation of ARs can lead to an elevation of intracellular cAMP, and that elevated cAMP in endothelia promotes barrier function, 20,30 we considered the possibility of endothelial AR-signaling to regulate vascular permeability. In contrast to previous studies that found tissue protection during hypoxia or inflammation through signaling pathways involving the A2AAR, 1,3,7,31,32 the present studies conclude that the A2BAR is central to the control of vascular leak in hypoxia. Methods Cell cultureHuman microvascular endothelial cells (HMEC)-1 were cultured as described previously. 9,18 For preparation of experimental HMEC-1 monolayers, confluent endothelial cells were seeded at approximately less than 10 5 cells/cm 2 onto either permeable polycarbonate inserts or 100-mm Petri dishes. Endothelial cell purity was assessed by phase microscopic "cobblestone" appearance and uptake of fluorescent acetylated low-density lipoprotein. Stable repression of AR expression by siRNAWith the help of the siRNA Wizard (www.sirnawizard.com; InvivoGen, San Diego, CA) the following primer sequences were chosen within the coding region of the g...

Although acute lung injury contributes significantly to critical illness, resolution often occurs spontaneously via activation of incompletely understood pathways. We recently found that mechanical ventilation of mice increases the level of pulmonary adenosine, and that mice deficient for extracellular adenosine generation show increased pulmonary edema and inflammation after ventilator-induced lung injury (VILI). Here, we profiled the response to VILI in mice with genetic deletions of each of the 4 adenosine receptors (ARs) and found that deletion of the A2BAR gene was specifically associated with reduced survival time and increased pulmonary albumin leakage after injury. In WT mice, treatment with an A2BAR-selective antagonist resulted in enhanced pulmonary inflammation, edema, and attenuated gas exchange, while an A2BAR agonist attenuated VILI. In bone marrow-chimeric A2BAR mice, although the pulmonary inflammatory response involved A2BAR signaling from bone marrow-derived cells, A2BARs located on the lung tissue attenuated VILI-induced albumin leakage and pulmonary edema. Furthermore, measurement of alveolar fluid clearance (AFC) demonstrated that A2BAR signaling enhanced amiloride-sensitive fluid transport and elevation of pulmonary cAMP levels following VILI, suggesting that A2BAR agonist treatment protects by drying out the lungs. Similar enhancement of pulmonary cAMP and AFC were also observed after β-adrenergic stimulation, a pathway known to promote AFC. Taken together, these studies reveal a role for A2BAR signaling in attenuating VILI and implicate this receptor as a potential therapeutic target during acute lung injury. IntroductionAcute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are life-threatening disorders that can develop in the course of different clinical conditions such as pneumonia, acid aspiration, major trauma, or prolonged mechanical ventilation, and contribute significantly to critical illness (1). Recent epidemiological studies show that each year 75,000 patients in the United States alone die from ARDS (2). The pathogenesis of these diseases is characterized by influx of a protein-rich edema fluid into the interstitial and intraalveolar spaces as a consequence of increased permeability of the alveolar-capillary barrier (1) in conjunction with excessive invasion of inflammatory cells - particularly polymorphonuclear neutrophils (3-6). At present, only little is known about how to target the alveolar-capillary barrier function or leukocyte trafficking therapeutically during ALI. In fact, to our knowledge, no such strategies have been translated into clinical practice, and we are unaware of any specific therapy currently available beyond mechanical ventilation and other supportive measures (1).Despite the large impact of ALI on morbidity and mortality in critically ill patients (1), many episodes are self-limiting and resolve spontaneously through unknown mechanisms. For example,

Background-Extracellular adenosine, generated from extracellular nucleotides via ectonucleotidases, binds to specific receptors and provides cardioprotection from ischemia and reperfusion. In the present study, we studied ecto-enzymatic ATP/ADP-phosphohydrolysis by select members of the ectonucleoside triphosphate diphosphohydrolase (E-NTPDase) family during myocardial ischemia. Methods and Results-As a first step, we used a murine model of myocardial ischemia and in situ preconditioning and performed pharmacological studies with polyoxometalate 1, a potent E-NTPDase inhibitor ( 01). Heightened levels of injury after myocardial ischemia and negligible preconditioning benefits in cd39Ϫ/Ϫ mice were corrected by infusion of the metabolic product (AMP) or apyrase. Moreover, apyrase treatment of wild-type mice resulted in 43Ϯ4.2% infarct size reduction (PϽ0.01). Conclusions-Taken together, these studies reveal E-NTPDase 1 in cardioprotection and suggest apyrase in the treatment of myocardial ischemia.

Pulmonary netrin-1 levels are repressed during ALI. This results in pronounced pulmonary damage, an increased infiltration of neutrophils, and increased pulmonary inflammation. Exogenous netrin-1 significantly dampens the extent of ALI through the adenosine 2B receptor.

Directed cell migration is a prerequisite not only for the development of the central nervous system, but also for topically restricted, appropriate immune responses. This is crucial for host defense and immune surveillance. Attracting environmental cues guiding leukocyte cell traffic are likely to be complemented by repulsive cues, which actively abolish cell migration. One such a paradigm exists in the developing nervous system, where neuronal migration and axonal path finding is balanced by chemoattractive and chemorepulsive cues, such as the neuronal repulsive guidance molecule-A (RGM-A). As expressed at the inflammatory site, the role of RGM-A within the immune response remains unclear. Here we report that RGM-A (i) is expressed by epithelium and leukocytes (granulocytes, monocytes, and T/B lymphocytes); (ii) inhibits leukocyte migration by contact repulsion and chemorepulsion, depending on dosage, through its receptor neogenin; and (iii) suppresses the inflammatory response in a model of zymosan-A-induced peritonitis. Systemic application of RGM-A attenuates the humoral proinflammatory response (TNF-α, IL-6, and macrophage inflammatory protein 1α), infiltration of inflammatory cell traffic, and edema formation. In contrast, the demonstrated anti-inflammatory effect of RGM-A is absent in mice homozygous for a gene trap mutation in the neo1 locus (encoding neogenin). Thus, our results suggest that RGM-A is a unique endogenous inhibitor of leukocyte chemotaxis that limits inflammatory leukocyte traffic and creates opportunities to better understand and treat pathologies caused by exacerbated or misdirected inflammatory responses.