Search

Respiratory surfaces are exposed to billions of particulates and pathogens daily. A protective mucus barrier traps and eliminates them through mucociliary clearance (MCC). However, excessive mucus contributes to transient respiratory infections and to the pathogenesis of numerous respiratory diseases. MUC5AC and MUC5B are evolutionarily conserved genes that encode structurally related mucin glycoproteins, the principal macromolecules in airway mucus. Genetic variants are linked to diverse lung diseases, but specific roles for MUC5AC and MUC5B in MCC, and the lasting effects of their inhibition, are unknown. Here we show that mouse Muc5b (but not Muc5ac) is required for MCC, for controlling infections in the airways and middle ear, and for maintaining immune homeostasis in mouse lungs, whereas Muc5ac is dispensable. Muc5b deficiency caused materials to accumulate in upper and lower airways. This defect led to chronic infection by multiple bacterial species, including Staphylococcus aureus, and to inflammation that failed to resolve normally. Apoptotic macrophages accumulated, phagocytosis was impaired, and interleukin-23 (IL-23) production was reduced in Muc5b(-/-) mice. By contrast, in mice that transgenically overexpress Muc5b, macrophage functions improved. Existing dogma defines mucous phenotypes in asthma and chronic obstructive pulmonary disease (COPD) as driven by increased MUC5AC, with MUC5B levels either unaffected or increased in expectorated sputum. However, in many patients, MUC5B production at airway surfaces decreases by as much as 90%. By distinguishing a specific role for Muc5b in MCC, and by determining its impact on bacterial infections and inflammation in mice, our results provide a refined framework for designing targeted therapies to control mucin secretion and restore MCC.

The median survival of patients with idiopathic pulmonary fibrosis (IPF) continues to be approximately 3 years from the time of diagnosis, underscoring the lack of effective medical therapies for this disease. In the United States alone, approximately 40,000 patients die of this disease annually. In November 2012, the NHLBI held a workshop aimed at coordinating research efforts and accelerating the development of IPF therapies. Basic, translational, and clinical researchers gathered with representatives from the NHLBI, patient advocacy groups, pharmaceutical companies, and the U.S. Food and Drug Administration to review the current state of IPF research and identify priority areas, opportunities for collaborations, and directions for future research. The workshop was organized into groups that were tasked with assessing and making recommendations to promote progress in one of the following six critical areas of research: (1) biology of alveolar epithelial injury and aberrant repair; (2) role of extracellular matrix; (3) preclinical modeling; (4) role of inflammation and immunity; (5) genetic, epigenetic, and environmental determinants; (6) translation of discoveries into diagnostics and therapeutics. The workshop recommendations provide a basis for directing future research and strategic planning by scientific, professional, and patient communities and the NHLBI.

Idiopathic pulmonary fibrosis (IPF) is a deadly disease with a poor prognosis and few treatment options. Pathological remodeling of the extracellular matrix (ECM) is a key factor that drives the disease pathogenesis, although the underlying mechanisms remain unknown. Alternative polyadenylation (APA) has recently been shown to play a major role in cellular responses to stress by driving the expression of fibrotic factors through the alteration of miRNA sensitivity, but a connection to IPF has not been established. Here, we demonstrated that CFIm25, a global regulator of APA, was downregulated in the lungs of patients with IPF and mice with pulmonary fibrosis, with its expression selectively reduced in [alpha]-smooth muscle actin-positive ([alpha]-SMA-positive) fibroblasts. Following CFIm25 knockdown in healthy human lung fibroblasts, we identified 808 genes with shortened 3'-UTRs, including those involved in the TGF-[beta] signaling pathway, the Wnt signaling pathway, and cancer pathways. The expression of key profibrotic factors was suppressed by CFIm25 overexpression in IPF fibroblasts. Finally, we demonstrated that deletion of CFIm25 in fibroblasts or myofibroblast precursors using either the Col1a1 or the Foxdl promoter enhanced pulmonary fibrosis after bleomycin exposure. Collectively, our results identified CFIm25 downregulation as an important mechanism for elevating profibrotic gene expression in pulmonary fibrosis., Introduction Idiopathic pulmonary fibrosis (IPF) is a deadly disease that is initiated by repetitive epithelial and endothelial cell injury and progresses with rapid proliferation and differentiation of myofibroblasts. Myofibroblasts are [...]

Ecto-5'-nucleotidase (CD73) is central to the generation of extracellular adenosine. Previous studies have highlighted a detrimental role for extracellular adenosine in cancer, as it dampens T cell-mediated immune responses. Here, we determined that, in contrast to other cancers, CD73 is markedly downregulated in poorly differentiated and advanced-stage endometrial carcinoma compared with levels in normal endometrium and low-grade tumors. In murine models, CD73 deficiency led to a loss of endometrial epithelial barrier function, and pharmacological CD73 inhibition increased in vitro migration and invasion of endometrial carcinoma cells. Given that CD73-generated adenosine is central to regulating tissue protection and physiology in normal tissues, we hypothesized that CD73-generated adenosine in endometrial carcinoma induces an innate reflex to protect epithelial integrity. CD73 associated with cell-cell contacts, filopodia, and membrane zippers, indicative of involvement in cell- cell adhesion and actin polymerization-dependent processes. We determined that CD73-generated adenosine induces cortical actin polymerization via adenosine A1 receptor (A1R) induction of a Rho GTPase CDC42-dependent conformational change of the actin-related proteins 2 and 3 (ARP2/3) actin polymerization complex member N-WASP. Cortical F-actin elevation increased membrane E-cadherin, β- catenin, and [Na.sup.+][K.sup.+] ATPase. Together, these findings reveal that CD73-generated adenosine promotes epithelial integrity and suggest why loss of CD73 in endometrial cancer allows for tumor progression. Moreover, our data indicate that the role of CD73 in cancer is more complex than previously described., Introduction Ecto-5'-nucleotidase (NT5E, referred to herein as CD73) is a cell surface glycosylphosphatidlinositol-anchored (GPI-anchored) glycoprotein that catalyzes 5'-adenosine monophosphate (5'-AMP) to adenosine (1). CD73 is overexpressed in a number of [...]

A complex biologic network regulates kidney perfusion under physiologic conditions. This system is profoundly perturbed following renal ischemia, a leading cause of acute kidney injury (AKI)--a life-threatening condition that frequently complicates the care of hospitalized patients. Therapeutic approaches to prevent and treat AKI are extremely limited. Better understanding of the molecular pathways promoting postischemic reflow could provide new candidate targets for AKI therapeutics. Due to its role in adapting tissues to hypoxia, we hypothesized that extracellular adenosine has a regulatory function in the postischemic control of renal perfusion. Consistent with the notion that equilibrative nucleoside transporters (ENTs) terminate adenosine signaling, we observed that pharmacologic ENT inhibition in mice elevated renal adenosine levels and dampened AKI. Deletion of the ENTs resulted in selective protection in [Ent1-/-] mice. Comprehensive examination of adenosine receptor-knockout mice exposed to AKI demonstrated that renal protection by ENT inhibitors involves the A2B adenosine receptor. Indeed, crosstalk between renal Ent1 and Adora2b expressed on vascular endothelia effectively prevented a postischemic no-reflow phenomenon. These studies identify ENT1 and adenosine receptors as key to the process of reestablishing renal perfusion following ischemic AKI. If translatable from mice to humans, these data have important therapeutic implications., Introduction Acute kidney injury (AKI) is clinically defined by an abrupt reduction in kidney function (e.g., a decrease in glomerular filtration rate [GFR]), occurring over a period of minutes to [...]

Hypoxia can act as an initial trigger to induce erythrocyte sickling and eventual end organ damage in sickle cell disease (SCD). Many factors and metabolites are altered in response to hypoxia and may contribute to the pathogenesis of the disease. Using metabolomic profiling, we found that the steady-state concentration of adenosine in the blood was elevated in a transgenic mouse model of SCD. Adenosine concentrations were similarly elevated in the blood of humans with SCD. Increased adenosine levels promoted sickling, hemolysis and damage to multiple tissues in SCD transgenic mice and promoted sickling of human erythrocytes. Using biochemical, genetic and pharmacological approaches, we showed that adenosine [A.sub.2B] receptor ([A.sub.2B]R)-mediated induction of 2,3-diphosphoglycerate, an erythrocyte-specific metabolite that decreases the oxygen binding affinity of hemoglobin, underlies the induction of erythrocyte sickling by excess adenosine both in cultured human red blood cells and in SCD transgenic mice. Thus, excessive adenosine signaling through the [A.sub.2B]R has a pathological role in SCD. These findings may provide new therapeutic possibilities for this disease., SCD is a devastating inherited hemolytic disorder that affects red blood cells (RBCs) and is caused by a point mutation that results in the substitution of valine for glutamic acid [...]

We have previously demonstrated that adenosine plus homocysteine enhanced endothelial basal barrier function and protected against agonist-induced barrier dysfunction in vitro through attenuation of RhoA activation by inhibition of isoprenylcysteine-O-carboxyl methyltransferase. In the current study, we tested the effect of elevated adenosine on pulmonary endothelial barrier function in vitro and in vivo. We noted that adenosine alone dose dependently enhanced endothelial barrier function. While adenosine receptor Al or A3 antagonists were ineffective, an adenosine transporter inhibitor, NBTI, or a combination of DPMX and MRS1754, antagonists for adenosine receptors [A.sub.2A] and [A.sub.2B], respectively, partially attenuated the barrier-enhancing effect of adenosine. Similarly, inhibition of both [A.sub.2A] and [A.sub.2B] receptors with siRNA also blunted the effect of adenosine on barrier function. Interestingly, inhibition of both transporters and [A.sub.2A]/[A.sub.2B] receptors completely abolished adenosine-induced endothelial barrier enhancement. The adenosine receptor [A.sub.2A] and [A.sub.2B] agonist, NECA, also significantly enhanced endothelial barrier function. These data suggest that both adenosine transporters and [A.sub.2A] and [A.sub.2B] receptors are necessary for exerting maximal effect of adenosine on barrier enhancement. We also found that adenosine enhanced Rac1 GTPase activity and over-expression of dominant negative Rac1 attenuated adenosine-induced increases in focal adhesion complexes. We further demonstrated that elevation of cellular adenosine by inhibition of adenosine deaminase with Pentostatin significantly enhanced endothelial basal barrier function, an effect that was also associated with enhanced Rac1 GTPase activity and with increased focal adhesion complexes and adherens junctions. Finally, using a non-inflammatory acute lung injury (ALI) model induced by [alpha]-naphthylthiourea, we found that administration of Pentostatin, which elevated lung adenosine level by 10-fold, not only attenuated the development of edema before ALI but also partially reversed edema after ALI. The data suggest that adenosine deaminase inhibition may be useful in treatment of pulmonary edema in settings of ALI. endothelium; adenosine receptors; adenosine transporters doi: 10.1152/ajplung.00330.2009.

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.tips.2009.07.005 Byline: Riccardo Polosa (1), Michael R. Blackburn (2) Abstract: Asthma and chronic obstructive pulmonary disease (COPD) are pulmonary disorders characterized by various degrees of inflammation and tissue remodeling. Adenosine is a signaling molecule that is elevated in the lungs of patients with asthma and COPD. Adenosine elicits its actions by engaging cell surface adenosine receptors, and substantial preclinical evidence suggests that targeting these receptors will provide novel approaches for the treatment of asthma and COPD. Studies in animal models of airway disease suggest that there may be clinical benefit to the use of A.sub.1, A.sub.3 and A.sub.2B adenosine receptor antagonists in the treatment of features of asthma and/or COPD, while A.sub.2A agonists may also prove effective. Several adenosine receptor based pharmacologic agents have entered clinical development for the treatment of asthma and COPD; however, the studies have been limited and the efficacy of such approaches is not yet clear. Author Affiliation: (1) Dipartimento di Medicina Interna e Specialistica, Institute of Internal Medicine and Clinical Immunology, S. Marta Hospital, University of Catania, Via G. Clementi, 36, Catania 95124, Italy (2) Department of Biochemistry and Molecular Biology, The University of Texas-Houston Medical School. 6431 Fannin, Houston TX, USA

Fatty liver is commonly associated with alcohol ingestion and abuse. While the molecular pathogenesis of these fatty changes is well understood, the biochemical and pharmacological mechanisms by which ethanol stimulates these molecular changes remain unknown. During ethanol metabolism, adenosine is generated by the enzyme ecto-5'-nucleotidase, and adenosine production and adenosine receptor activation are known to play critical roles in the development of hepatic fibrosis. We therefore investigated whether adenosine and its receptors play a role in the development of alcohol-induced fatty liver. WT mice fed ethanol on the Lieber-DeCarli diet developed hepatic steatosis, including increased hepatic triglyceride content, while mice lacking ecto-5'-nucleotidase or adenosine [A.sub.1] or [A.sub.2B] receptors were protected from developing fatty liver. Similar protection was also seen in WT mice treated with either an adenosine [A.sub.1] or [A.sub.2B] receptor antagonist. Steatotic livers demonstrated increased expression of genes involved in fatty acid synthesis, which was prevented by blockade of adenosine [A.sub.1] receptors, and decreased expression of genes involved in fatty acid metabolism, which was prevented by blockade of adenosine [A.sub.2B] receptors. In vitro studies supported roles for adenosine [A.sub.1] receptors in promoting fatty acid synthesis and for [A.sub.2B] receptors in decreasing fatty acid metabolism. These results indicate that adenosine generated by ethanol metabolism plays an important role in ethanol-induced hepatic steatosis via both [A.sub.1] and [A.sub.2B] receptors and suggest that targeting adenosine receptors may be effective in the prevention of alcohol-induced fatty liver., Introduction Fatty liver is the most common and earliest response of the liver to heavy alcohol consumption and may develop into alcoholic hepatitis and fibrosis. Although fatty liver is a [...]

Trehalose 6,6'-dimycolate (TDM) is a glycolipid component of the mycobacterial cell wall that causes immune responses in mice similar to Mycobacterium tuberculosis (MTB) infection, including granuloma formation with production of proinflammatory cytokines. The precise roles of tumour necrosis factor (TNF)-[alpha], complement C5 and interleukin (IL)-6 in the molecular events that lead to the initiation and maintenance of the granulomatous response to TDM have not been fully elucidated. Macrophage proinflammatory responses from wild-type and complementdeficient mice after infection with MTB were assessed, and compared to responses from organisms in which surface TDM had been removed. Removal of TDM abolished proinflammatory responses, markedly so in the complement-deficient macrophages. Mice deficient in TNF-[alpha], C5a and IL-6, along with wild-type C5?BL/6 controls, were intravenously injected with TDM in a water-in-oil emulsion, and analysed for histological response and cytokine production in lungs. Wild-type C57BL/6 mice formed granulomas with increased production of IL-1/[beta], IL-6, TNF-[alpha], macrophage inflammatory protein-1[alpha] (MIP-1[alpha]), IL-12p40, interferon-[gamma] (IFN-[gamma]), and IL-10 protein and mRNA. TNF-[alpha]-deficient mice failed to produce a histological response to TDM, with no increases in cytokine production following TDM administration. While C5a-deficient mice exhibited inflammation, they did not form structured granulomas and initially had decreased production of proinflammatory mediators. IL-6-deficient mice initiated granuloma formation, but failed to maintain the granulomas through day 7 and demonstrated decreased early production of proinflammatory mediators in comparison to wild-type mice. These data suggest that TNF-[alpha] is critical for initiation of the granulomatous response, C5a is necessary for formation of cohesive granulomas, and IL-6 plays a key role in the granuloma maintenance response to mycobacterial TDM.

Priapism, abnormally prolonged penile erection in the absence of sexual excitation, is associated with ischemia-mediated erectile tissue damage and subsequent erectile dysfunction. It is common among males with sickle cell disease (SCD), and SCD transgenic mice are an accepted model of the disorder. Current strategies to manage priapism suffer from a poor fundamental understanding of the molecular mechanisms underlying the disorder. Here we report that mice lacking adenosine deaminase (ADA), an enzyme necessary for the breakdown of adenosine, displayed unexpected priapic activity. ADA enzyme therapy successfully corrected the priapic activity both in vivo and in vitro, suggesting that it was dependent on elevated adenosine levels. Further genetic and pharmacologic evidence demonstrated that [A.sub.2B] adenosine receptor--mediated ([A.sub.2B]R-mediated) cAMP and cGMP induction was required for elevated adenosine--induced prolonged penile erection. Finally, priapic activity in SCD transgenic mice was also caused by elevated adenosine levels and [A.sub.2B]R activation. Thus, we have shown that excessive adenosine accumulation in the penis contributes to priapism through increased [A.sub.2B]R signaling in both Ada-/- and SCD transgenic mice. These findings provide insight regarding the molecular basis of priapism and suggest that strategies to either reduce adenosine or block [A.sub.2B]R activation may prove beneficial in the treatment of this disorder., Introduction Priapism is a condition of persistent penile erection in the absence of sexual excitation (1), (2). This condition is common among males with sickle cell disease (SCD), 40% of [...]

Adenosine is generated at sites of tissue injury where it serves to regulate inflammation and damage. Adenosine signaling has been implicated in the regulation of pulmonary inflammation and damage in diseases such as asthma and chronic obstructive pulmonary disease; however, the contribution of specific adenosine receptors to key immunoregulatory processes in these diseases is still unclear. Mice deficient in the purine catabolic enzyme adenosine deaminase (ADA) develop pulmonary inflammation and mucous metaplasia in association with adenosine elevations making them a useful model for assessing the contribution of specific adenosine receptors to adenosine-mediated pulmonary disease. Studies suggest that the [A.sub.2A] adenosine receptor ([A.sub.2A]R) functions to limit inflammation and promote tissue protection; however, the contribution of [A.sub.2A]R signaling has not been examined in the ADA-deficient model of adenosine-mediated lung inflammation. The purpose of the current study was to examine the contribution of [A.sub.2A]R signaling to the pulmonary phenotype seen in ADA-deficient mice. This was accomplished by generating ADA/[A.sub.2A]R double knockout mice. Genetic removal of the [A.sub.2A]R from ADA-deficient mice resulted in enhanced inflammation comprised largely of macrophages and neutrophils, mucin production in the bronchial airways, and angiogenesis, relative to that seen in the lungs of ADA-deficient mice with the [A.sub.2A]R. In addition, levels of the chemokines monocyte chemo-attractant protein-1 and CXCL1 were elevated, whereas levels of cytokines such as TNF-[alpha] and IL-6 were not. There were no compensatory changes in the other adenosine receptors in the lungs of ADA/[A.sub.2A]R double knockout mice. These findings suggest that the [A.sub.2A]R plays a protective role in the ADA-deficient model of pulmonary inflammation. inflammation; chemokines; asthma; adenosine receptors

Adenosine has been implicated in the pathogenesis of chronic lung diseases such as asthma and chronic obstructive pulmonary disease. In vitro studies suggest that activation of the [A.sub.2B] adenosine receptor [...]

To define the factors that control the tissue effects of IL-4, we compared the effects of Tg IL-4 in Balb/c and C57BL/6 mice. In the former, IL-4 caused modest eosinophilic [...]

Adenosine, a signaling nucleoside, exhibits tissue-protective and tissue-destructive effects. Adenosine levels in tissues are controlled in part by the enzyme adenosine deaminase (ADA). ADA-deficient mice accumulate adenosine levels in multiple tissues, including the lung, where adenosine contributes to the development of pulmonary inflammation and chronic airway remodeling. The present study describes the development of pulmonary fibrosis in mice that have been genetically engineered to possess partial ADA enzyme activity and, thus, accumulate adenosine over a prolonged period of time. These partially ADA-deflcient mice live for up to 5 mo and die from apparent respiratory distress. Detailed investigations of the lung histopathology of partially ADA-deficient mice revealed progressive pulmonary fibrosis marked by an increase in the number of pulmonary myofibroblasts and an increase in collagen deposition. In addition, in regions of the distal airways that did not exhibit fibrosis, an increase in the number of large foamy macrophages and a substantial enlargement of the alveolar air spaces suggest emphysemic changes. Furthermore, important proinflammatory and profibrotic signaling pathways, including IL-13 and transforming growth factor-[beta]1, were activated. Increases in tissue fibrosis were also seen in the liver and kidneys of these mice. These changes occurred in association with pronounced elevations of lung adenosine concentrations and alterations in lung adenosine receptor levels, supporting the hypothesis that elevation of endogenous adenosine is a proinflammatory and profibrotic signal in this model. adenosine receptor; cytokine; chemokine

Author(s): Jianfa Zhang [1, 3]; Krista Kaasik [2, 3]; Michael R. Blackburn [1]; Cheng Chi Lee (corresponding author) [1] Environmental light is the 'zeitgeber' (time-giver) of circadian behaviour [1]. Constant [...]

Chronic lung diseases are associated with persistent lung inflammation and damage. The mechanisms that govern the chronic nature of these disorders are not known. Adenosine is a signaling nucleoside that is generated in hypoxic environments such as that found in the inflamed lung, which suggests that it might serve a regulatory role in chronic lung diseases. Support for this hypothesis comes from studies in adenosine-deaminase-deficient mice where lung adenosine levels accumulate in association with increased lung inflammation and damage. Furthermore, lowering adenosine levels or antagonizing adenosine receptors can reverse pulmonary phenotypes in this model, suggesting that chronic adenosine elevations can affect signaling pathways that mediate aspects of chronic lung disease.

Gene expression profiling in inflammatory airway disease associated with elevated adenosine. Am J Physiol Lung Cell Mol Physiol 282: L169-L182, 2002; 10.1152/ajplung.00243.2001.--Adenosine has been implicated as a modulator of inflammatory processes central to asthma. However, the molecular mechanisms involved are poorly understood. We used Atlas mouse cDNA arrays to analyze differential gene expression in association with lung inflammation resulting from elevated adenosine in adenosine deaminase (ADA)-deficient mice. We report that of the 1,176 genes on the array, the expression patterns of 280 genes were consistently altered. Of these genes, the steady-state levels of 93 genes were upregulated and 29 were downregulated. We also show that lowering adenosine levels with ADA enzyme therapy has striking effects on gene expression that may be associated with resolution of pulmonary eosinophilia. In addition, we confirmed the nucleic acid and protein expression of vascular endothelial growth factor and monocyte chemoattractant protein-3, two candidate genes that may be regulated by adenosine. In conclusion, high-throughput profiling of gene expression by cDNA array hybridization has provided an overview of critical regulatory genes involved in airway inflammation in ADA-deficient mice. These mice will serve as a useful in vivo model for characterizing molecular mechanisms of adenosine-mediated lung damage. complementary deoxyribonucleic acid; adenosine deaminase; vascular endothelial growth factor; monocyte chemoattractant protein-3