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1.  Adenosine promotes vascular barrier function in hyperoxic lung injury 
Physiological Reports  2014;2(9):e12155.
Abstract
Hyperoxic lung injury is characterized by cellular damage from high oxygen concentrations that lead to an inflammatory response in the lung with cellular infiltration and pulmonary edema. Adenosine is a signaling molecule that is generated extracellularly by CD73 in response to injury. Extracellular adenosine signals through cell surface receptors and has been found to be elevated and plays a protective role in acute injury situations. In particular, ADORA2B activation is protective in acute lung injury. However, little is known about the role of adenosine signaling in hyperoxic lung injury. We hypothesized that hyperoxia‐induced lung injury leads to CD73‐mediated increases in extracellular adenosine, which is protective through ADORA2B signaling pathways. To test this hypothesis, we exposed C57BL6, CD73−/−, and Adora2B−/− mice to 95% oxygen or room air and examined markers of pulmonary inflammation, edema, and monitored lung histology. Hyperoxic exposure caused pulmonary inflammation and edema in association with elevations in lung adenosine levels. Loss of CD73‐mediated extracellular adenosine production exacerbated pulmonary edema without affecting inflammatory cell counts. Furthermore, loss of the ADORA2B had similar results with worsening of pulmonary edema following hyperoxia exposure without affecting inflammatory cell infiltration. This loss of barrier function correlated with a decrease in occludin in pulmonary vasculature in CD73−/− and Adora2B−/− mice following hyperoxia exposure. These results demonstrate that exposure to a hyperoxic environment causes lung injury associated with an increase in adenosine concentration, and elevated adenosine levels protect vascular barrier function in hyperoxic lung injury through the ADORA2B‐dependent regulation of occludin.
e12155
Hyperoxic lung injury is characterized by cellular damage from high oxygen concentrations that lead to an inflammatory response in the lung with cellular infiltration and pulmonary edema, and extracellular adenosine has been found to be elevated and plays a protective role in acute injury situations; however, little is known about the role of adenosine signaling in hyperoxic lung injury. We hypothesized that hyperoxia‐induced lung injury leads to CD73‐mediated increases in extracellular adenosine, which is protective through ADORA2B signaling pathways. Our results demonstrate that exposure to a hyperoxic environment causes lung injury associated with an increase in adenosine concentration, and elevated adenosine levels protect vascular barrier function in hyperoxic lung injury through the ADORA2B‐dependent regulation of occludin.
doi:10.14814/phy2.12155
PMCID: PMC4270235  PMID: 25263205
Adenosine; hyperoxic lung injury; vascular barrier function
2.  Blockade of IL-6 Trans Signaling Attenuates Pulmonary Fibrosis 
Idiopathic pulmonary fibrosis (IPF) is a lethal lung disease with progressive fibrosis and death within 2–3 y of diagnosis. IPF incidence and prevalence rates are increasing annually with few effective treatments available. Inhibition of IL-6 results in the attenuation of pulmonary fibrosis in mice. It is unclear whether this is due to blockade of classical signaling, mediated by membrane-bound IL-6Rα, or trans signaling, mediated by soluble IL-6Rα (sIL-6Rα). Our study assessed the role of sIL-6Rα in IPF. We demonstrated elevations of sIL-6Rα in IPF patients and in mice during the onset and progression of fibrosis. We demonstrated that protease-mediated cleavage from lung macrophages was important in production of sIL-6Rα. In vivo neutralization of sIL-6Rα attenuated pulmonary fibrosis in mice as seen by reductions in myofibroblasts, fibronectin, and collagen in the lung. In vitro activation of IL-6 trans signaling enhanced fibroblast proliferation and extracellular matrix protein production, effects relevant in the progression of pulmonary fibrosis. Taken together, these findings demonstrate that the production of sIL-6Rα from macrophages in the diseased lung contributes to IL-6 trans signaling that in turn influences events crucial in pulmonary fibrosis.
doi:10.4049/jimmunol.1302470
PMCID: PMC4169999  PMID: 25172494
3.  Future Directions in Idiopathic Pulmonary Fibrosis Research. An NHLBI Workshop Report 
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.
doi:10.1164/rccm.201306-1141WS
PMCID: PMC3983890  PMID: 24160862
idiopathic pulmonary fibrosis; alveolar epithelial cells; extracellular matrix; interstitial lung disease; inflammation
4.  Adenosine Signaling During Acute and Chronic Disease States 
Adenosine is a signaling nucleoside that is produced following tissue injury, particularly injury involving ischemia and hypoxia. The production of extracellular adenosine and its subsequent signaling through adenosine receptors plays an important role in orchestrating injury responses in multiple organs. There are four adenosine receptors that are widely distributed on immune, epithelial, endothelial, neuronal and stromal cells throughout the body. Interestingly, these receptors are subject to altered regulation following injury. Studies in mouse models and human cells and tissues have identified that the production of adenosine and its subsequent signaling through its receptors plays largely beneficial roles in acute disease states, with the exception of brain injury. In contrast, if elevated adenosine levels are sustained beyond the acute injury phase, adenosine responses can become detrimental by activating pathways that promote tissue injury and fibrosis. Understanding when during the course of disease adenosine signaling is beneficial as opposed to detrimental and defining the mechanisms involved will be critical for the advancement of adenosine based therapies for acute and chronic diseases. The purpose of this review is to discuss key observations that define the beneficial and detrimental aspects of adenosine signaling during acute and chronic disease states with an emphasis on cellular processes such as inflammatory cell regulation, vascular barrier function and tissue fibrosis.
doi:10.1007/s00109-013-0997-1
PMCID: PMC3606047  PMID: 23340998
adenosine receptors; inflammation; fibrosis; vascular barrier function; CD73; ADORA2B; ADORA2A; ADORA3; ADORA1; acute lung injury; remodeling; anti-inflammatory
5.  Alveolar Type II Epithelial Cell Dysfunction in Rat Experimental Hepatopulmonary Syndrome (HPS) 
PLoS ONE  2014;9(11):e113451.
The hepatopulmonary syndrome (HPS) develops when pulmonary vasodilatation leads to abnormal gas exchange. However, in human HPS, restrictive ventilatory defects are also observed supporting that the alveolar epithelial compartment may also be affected. Alveolar type II epithelial cells (AT2) play a critical role in maintaining the alveolar compartment by producing four surfactant proteins (SPs, SP-A, SP-B, SP-C and SP-D) which also facilitate alveolar repair following injury. However, no studies have evaluated the alveolar epithelial compartment in experimental HPS. In this study, we evaluated the alveolar epithelial compartment and particularly AT2 cells in experimental HPS induced by common bile duct ligation (CBDL). We found a significant reduction in pulmonary SP production associated with increased apoptosis in AT2 cells after CBDL relative to controls. Lung morphology showed decreased mean alveolar chord length and lung volumes in CBDL animals that were not seen in control models supporting a selective reduction of alveolar airspace. Furthermore, we found that administration of TNF-α, the bile acid, chenodeoxycholic acid, and FXR nuclear receptor activation (GW4064) induced apoptosis and impaired SP-B and SP-C production in alveolar epithelial cells in vitro. These results imply that AT2 cell dysfunction occurs in experimental HPS and is associated with alterations in the alveolar epithelial compartment. Our findings support a novel contributing mechanism in experimental HPS that may be relevant to humans and a potential therapeutic target.
doi:10.1371/journal.pone.0113451
PMCID: PMC4242631  PMID: 25419825
6.  Equilibrative nucleoside transporter (ENT)-1-dependent elevation of extracellular adenosine protects the liver during ischemia and reperfusion 
Hepatology (Baltimore, Md.)  2013;58(5):1766-1778.
Ischemia and reperfusion-elicited tissue injury contributes to morbidity and mortality of hepatic surgery or during liver transplantation. Previous studies had implicated extracellular adenosine signaling in liver protection. Based on the notion that extracellular adenosine signaling is terminated by uptake from the extracellular towards the intracellular compartment via equilibrative nucleoside transporters (ENTs), we hypothesized a functional role of ENTs in liver protection from ischemia. During orthotopic liver transplantation in humans, we observed higher expressional levels of ENT1 than ENT2, in conjunction with repression of ENT1 and ENT2 transcript and protein levels following warm ischemia and reperfusion. Treatment with the pharmacologic ENT inhibitor dipyridamole revealed elevations of hepatic adenosine levels and robust liver protection in a murine model of liver ischemia and reperfusion. Subsequent studies in gene-targeted mice for Ent1 or Ent2 demonstrated selective protection from liver injury in Ent1−/− mice. Treatment with selective adenosine receptor antagonists indicated a contribution of Adora2b receptor signaling in ENT-dependent liver protection. Taken together, these findings implicate ENT1 in liver-protection from ischemia and reperfusion injury and suggest ENT inhibitors in the prevention or treatment of ischemic liver injury.
doi:10.1002/hep.26505
PMCID: PMC3795856  PMID: 23703920
adenosine; equilibrative nucleoside tranporter; liver transplantation; liver surgery; inflammation; hypoxia; ischemia and reperfusion
7.  Enhanced Airway Inflammation and Remodeling in Adenosine Deaminase-Deficient Mice Lacking the A2B Adenosine Receptor1 
Adenosine is a signaling nucleoside that is generated in response to cellular injury and orchestrates the balance between tissue protection and the progression to pathological tissue remodeling. Adenosine deaminase (ADA)-deficient mice develop progressive airway inflammation and remodeling in association with adenosine elevations, suggesting that adenosine can promote features of chronic lung disease. Furthermore, pharmacological studies in ADA-deficient mice demonstrate that A2BR antagonism can attenuate features of chronic lung disease, implicating this receptor in the progression of chronic lung disease. This study examines the contribution of A2BR signaling in this model by generating ADA/A2BR double-knockout mice. Our hypothesis was that genetic removal of the A2BR from ADA-deficient mice would lead to diminished pulmonary inflammation and damage. Unexpectedly, ADA/A2BR double-knockout mice exhibited enhanced pulmonary inflammation and airway destruction. Marked loss of pulmonary barrier function and excessive airway neutrophilia are thought to contribute to the enhanced tissue damage observed. These findings support an important protective role for A2BR signaling during acute stages of lung disease.
doi:10.4049/jimmunol.0900515
PMCID: PMC3631106  PMID: 19494329
8.  Distinct Roles for the A2B Adenosine Receptor in Acute and Chronic Stages of Bleomycin-Induced Lung Injury 
Adenosine is an extracellular signaling molecule that is generated in response to cell injury where it orchestrates tissue protection and repair. Whereas adenosine is best known for promoting anti-inflammatory activities during acute injury responses, prolonged elevations can enhance destructive tissue remodeling processes associated with chronic disease states. The generation of adenosine and the subsequent activation of the adenosine 2B receptor (A2BR) is an important processes in the regulation of both acute and chronic lung disease. The goal of this study was to examine the contribution of the A2BR in models of bleomycin-induced lung injury that exhibit varying degrees of acute and chronic injury. Intratracheal bleomycin exposure results in substantial acute lung injury followed by progressive fibrosis. In this model, genetic removal of the A2BR resulted in enhanced loss of barrier function and increased pulmonary inflammation, with few differences in indexes of pulmonary fibrosis. These results support an anti-inflammatory role for this receptor in this model of acute lung injury. In contrast, systemic exposure of mice to bleomycin resulted in modest acute lung injury together with progressive pulmonary fibrosis. In this model, the effects of A2BR removal on acute lung injury were negligible; however, there were substantial reductions in pulmonary fibrosis, supporting a profibrotic role for this receptor. A2BR-dependent regulation of IL-6 production was identified as a potential mechanism involved in the diminished pulmonary fibrosis seen in A2BR knockout mice exposed to i.p. bleomycin. These studies highlight the distinct roles of A2BR signaling during acute and chronic stages of lung injury.
doi:10.4049/jimmunol.1002907
PMCID: PMC3607290  PMID: 21149612
9.  The p97-UFD1L-NPL4 Protein Complex Mediates Cytokine-Induced IκBα Proteolysis 
Molecular and Cellular Biology  2014;34(3):335-347.
IκBα is an inhibitor of NF-κB, a family of transcription factors that transactivate genes related to inflammation. Upon inflammatory stimuli, IκBα is rapidly degraded via the ubiquitin-proteasome pathway. While it is very clear that the SCFβ-TRCP ubiquitin ligase ubiquitinates IκBα upon stimulation, little is known about the postubiquitinational events of IκBα proteolysis. Here, we report that p97, a valosin-containing protein (also called VCP), plays an essential role in the postubiquitinational regulation of IκBα turnover after tumor necrosis factor alpha (TNF-α) or interleukin-1β (IL-1β) treatment. The ATPase activity of p97 is essential for its role in IκBα proteolysis. Moreover, we found that UFD1L and NPL4, two cofactors of p97, assist p97 to control the postubiquitinational regulation of IκBα. The p97-UFD1L-NPL4 protein complex specifically associates with ubiquitinated IκBα via the interactions between p97 and the SCFβ-TRCP ubiquitin ligase and between the polyubiquitin binding domain of UFD1L and polyubiquitinated IκBα. Furthermore, we observed that the postubiquitinational regulation of IκBα by the p97-UFD1L-NPL4 complex is important for NF-κB activation under stimuli.
doi:10.1128/MCB.01190-13
PMCID: PMC3911508  PMID: 24248593
10.  Muc5b Is Required for Airway Defense 
Nature  2013;505(7483):412-416.
Respiratory surfaces are exposed to billions of particulates and pathogens daily. A protective mucus barrier traps and eliminates them via mucociliary clearance (MCC)1,2. However, excessive mucus contributes to transient respiratory infections and to the pathogenesis of numerous respiratory diseases1. MUC5AC and MUC5B are evolutionarily conserved genes that encode structurally related mucin glycoproteins, the principal macromolecules in airway mucus1,3. Genetic variants are linked to diverse lung diseases4-6, but specific roles for MUC5AC and MUC5B in MCC, and the lasting effects of their inhibition, are unknown. Here we show that Muc5b (but not Muc5ac) is required for MCC, for controlling infections in the airways and middle ear, and for maintaining immune homeostasis in the lungs. 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 normally7. Apoptotic macrophages accumulated, phagocytosis was impaired, and IL-23 production was reduced inMuc5b−/− mice. By contrast, in Muc5b transgenic (Tg) mice, 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 sputum1,8. However, in many patients, MUC5B production at airway surfaces decreases by as much as 90%9-11. 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.
doi:10.1038/nature12807
PMCID: PMC4001806  PMID: 24317696
12.  Elevated Ecto-5’-nucleotidase-Mediated Increased Renal Adenosine Signaling Via A2B Adenosine Receptor Contributes to Chronic Hypertension 
Circulation research  2013;112(11):10.1161/CIRCRESAHA.111.300166.
Rationale
Hypertension is the most prevalent life-threatening disease worldwide and is frequently associated with chronic kidney disease (CKD). However, the molecular basis underlying hypertensive CKD is not fully understood.
Objective
We sought to identify specific factors and signaling pathways that contribute to hypertensive CKD and thereby exacerbate disease progression.
Methods and Results
Using high-throughput quantitative reverse-transcription polymerase chain reaction profiling, we discovered that the expression level of 5′-ectonucleotidase (CD73), a key enzyme that produces extracellular adenosine, was significantly increased in the kidneys of angiotensin II–infused mice, an animal model of hypertensive nephropathy. Genetic and pharmacological studies in mice revealed that elevated CD73-mediated excess renal adenosine preferentially induced A2B adenosine receptor (ADORA2B) production and that enhanced kidney ADORA2B signaling contributes to angiotensin II–induced hypertension. Similarly, in humans, we found that CD73 and ADORA2B levels were significantly elevated in the kidneys of CKD patients compared with normal individuals and were further elevated in hypertensive CKD patients. These findings led us to further discover that elevated renal CD73 contributes to excess adenosine signaling via ADORA2B activation that directly stimulates endothelin-1 production in a hypoxia-inducible factor-α–dependent manner and underlies the pathogenesis of the disease. Finally, we revealed that hypoxia-inducible factor-α is an important factor responsible for angiotensin II–induced CD73 and ADORA2B expression at the transcriptional level.
Conclusions
Overall, our studies reveal that angiotensin II–induced renal CD73 promotes the production of renal adenosine that is a prominent driver of hypertensive CKD by enhanced ADORA2B signaling–mediated endothelin-1 induction in a hypoxia-inducible factor-α–dependent manner. The inhibition of excess adenosine-mediated ADORA2B signaling represents a novel therapeutic target for the disease.
doi:10.1161/CIRCRESAHA.111.300166
PMCID: PMC3886128  PMID: 23584256
adenosine; hypertension; chronic renal disease
13.  Adenosine signaling and the regulation of chronic lung disease 
Pharmacology & therapeutics  2009;123(1):105-116.
Chronic lung diseases such as asthma, chronic obstructive pulmonary disease and interstitial lung disease are characterized by inflammation and tissue remodeling processes that compromise pulmonary function. Adenosine is produced in the inflamed and damaged lung where it plays numerous roles in the regulation of inflammation and tissue remodeling. Extracellular adenosine serves as an autocrine and paracrine signaling molecule by engaging cell surface adenosine receptors. Preclinical and cellular studies suggest that adenosine plays an anti-inflammatory role in processes associated with acute lung disease, where activation of the A2AR and A2BR have promising implications for the treatment of these disorders. In contrast, there is growing evidence that adenosine signaling through the A1R, A2BR and A3R may serve pro-inflammatory and tissue remodeling functions in chronic lung diseases. This review discusses the current progress of research efforts and clinical trials aimed at understanding the complexities of this signaling pathway as they pertain to the development of treatment strategies for chronic lung diseases.
doi:10.1016/j.pharmthera.2009.04.003
PMCID: PMC2743314  PMID: 19426761
asthma; fibrosis; emphysema; COPD; G-protein coupled receptors; adenosine receptors; inflammation
14.  Interleukin-6 Underlies Angiotensin II-induced Hypertension and Chronic Renal Damage 
Hypertension  2011;59(1):10.1161/HYPERTENSIONAHA.111.173328.
Chronic kidney disease (CKD) is a prevalent life-threatening disease frequently associated with hypertension, progression to renal fibrosis and eventual renal failure. While the pathogenesis of CKD remains largely unknown, an increased inflammatory response is known to be associated with the disease and has long been speculated to contribute to disease development. However, the causative factors, the exact role of the increased inflammatory cascade in CKD and the underlying mechanisms for its progression remain unidentified. Here we report that interleukin-6 (IL-6) expression levels were significantly increased in the kidneys collected from CKD patients and further elevated in CKD patients characterized with hypertension. Functionally, we determined that angiotensin II (Ang II) is a causative factor responsible for IL-6 induction in the mouse kidney and that genetic deletion of IL-6 significantly reduced hypertension and key features of CKD including renal injury and progression to renal fibrosis in Ang II-infused mice. Mechanistically, we provide both human and mouse evidence that IL-6 is a key cytokine functioning downstream of Ang II signaling to directly induce fibrotic gene expression and preproendothelin-1 (prepro-ET-1) mRNA expression in the kidney. Overall, both the mouse and human studies reported here provide evidence that Ang II induces IL-6 production in the kidney and that, in addition to its role in hypertension, increased IL-6 may play an important pathogenic role in CKD by inducing fibrotic gene expression and ET-1 gene expression. These findings immediately suggest the IL-6 signaling is a novel therapeutic target to manage this devastating disorder affecting millions worldwide.
doi:10.1161/HYPERTENSIONAHA.111.173328
PMCID: PMC3842011  PMID: 22068875
Renin Angiotensin System; Cytokines; Hypertension; Chronic Kidney Disease; Endothelin-1
15.  A3 Adenosine Receptor Signaling Influences Pulmonary Inflammation and Fibrosis 
Adenosine is a signaling molecule produced during conditions that cause cellular stress or damage. This signaling pathway is implicated in the regulation of pulmonary disorders through the selective engagement of adenosine receptors. The goal of this study was to examine the involvement of the A3 adenosine receptor (A3R) in a bleomycin model of pulmonary inflammation and fibrosis. Results demonstrated that A3R-deficient mice exhibit enhanced pulmonary inflammation that included an increase in eosinophils. Accordingly, there was a selective up-regulation of eosinophil-related chemokines and cytokines in the lungs of A3R-deficient mice exposed to bleomycin. This increase in eosinophil numbers was accompanied by a decrease in the amount of extracellular eosinophil peroxidase activity in lavage fluid from A3R-deficient mice exposed to bleomycin, an observation suggesting that the A3R is necessary for eosinophil degranulation in this model. Despite an increase in inflammatory metrics associated with A3R-deficient mice treated with bleomycin, there was little difference in the degree of pulmonary fibrosis. Examination of fibrotic mediators demonstrated enhanced transforming growth factor (TGF)-β1 expression, but not a concomitant increase in TGF-β1 activity. This was associated with the loss of expression of matrix metalloprotease 9, an activator of TGF-β1, in alveolar macrophages and airway mast cells in the lungs of A3R-deficient mice. Together, these results suggest that the A3R serves antiinflammatory functions in the bleomycin model, and is also involved in regulating the production of mediators that can impact fibrosis.
doi:10.1165/rcmb.2007-0419OC
PMCID: PMC2586045  PMID: 18587054
pulmonary fibrosis; adenosine receptors; inflammation; eosinophil; extracellular matrix
16.  Sustained Adenosine Exposure Causes Lung Endothelial Barrier Dysfunction via Nucleoside Transporter–Mediated Signaling 
Previous studies by our group as well as others have shown that acute adenosine exposure enhances lung vascular endothelial barrier integrity and protects against increased permeability lung edema. In contrast, there is growing evidence that sustained adenosine exposure has detrimental effects on the lungs, including lung edema. It is well established that adenosine modulates lung inflammation. However, little is known concerning the effect of sustained adenosine exposure on lung endothelial cells (ECs), which are critical to the maintenance of the alveolar–capillary barrier. We show that exogenous adenosine plus adenosine deaminase inhibitor caused sustained elevation of adenosine in lung ECs. This sustained adenosine exposure decreased EC barrier function, elevated cellular reactive oxygen species levels, and activated p38, JNK, and RhoA. Inhibition of equilibrative nucleoside transporters (ENTs) prevented sustained adenosine-induced p38 and JNK activation and EC barrier dysfunction. Inhibition of p38, JNK, or RhoA also partially attenuated sustained adenosine-induced EC barrier dysfunction. These data indicate that sustained adenosine exposure causes lung EC barrier dysfunction via ENT-dependent intracellular adenosine uptake and subsequent activation of p38, JNK, and RhoA. The antioxidant N-acetylcysteine and the NADPH inhibitor partially blunted sustained adenosine-induced JNK activation but were ineffective in attenuation of p38 activation or barrier dysfunction. p38 was activated exclusively in mitochondria, whereas JNK was activated in mitochondria and cytoplasm by sustained adenosine exposure. Our data further suggest that sustained adenosine exposure may cause mitochondrial oxidative stress, leading to activation of p38, JNK, and RhoA in mitochondria and resulting in EC barrier dysfunction.
doi:10.1165/rcmb.2012-0012OC
PMCID: PMC3547102  PMID: 22744860
adenosine deaminase; equilibrative nucleoside transporters; oxidative stress; MAP kinases; RhoA
17.  A Semiautomated Framework for Integrating Expert Knowledge into Disease Marker Identification 
Disease markers  2013;35(5):513-523.
Background. The availability of large complex data sets generated by high throughput technologies has enabled the recent proliferation of disease biomarker studies. However, a recurring problem in deriving biological information from large data sets is how to best incorporate expert knowledge into the biomarker selection process. Objective. To develop a generalizable framework that can incorporate expert knowledge into data-driven processes in a semiautomated way while providing a metric for optimization in a biomarker selection scheme. Methods. The framework was implemented as a pipeline consisting of five components for the identification of signatures from integrated clustering (ISIC). Expert knowledge was integrated into the biomarker identification process using the combination of two distinct approaches; a distance-based clustering approach and an expert knowledge-driven functional selection. Results. The utility of the developed framework ISIC was demonstrated on proteomics data from a study of chronic obstructive pulmonary disease (COPD). Biomarker candidates were identified in a mouse model using ISIC and validated in a study of a human cohort. Conclusions. Expert knowledge can be introduced into a biomarker discovery process in different ways to enhance the robustness of selected marker candidates. Developing strategies for extracting orthogonal and robust features from large data sets increases the chances of success in biomarker identification.
doi:10.1155/2013/613529
PMCID: PMC3809975  PMID: 24223463
18.  A3 Adenosine Receptor Signaling Contributes to Airway Mucin Secretion after Allergen Challenge 
Mucin hypersecretion is a prominent feature of obstructive airway diseases such as asthma. Clara cells conditionally produce mucin in response to inflammatory signals in a process termed mucous metaplasia. This can be followed by mucin secretion stimulated by various signaling molecules. The cellular and molecular mechanisms that regulate mucin production and secretion are not well understood. Adenosine is a signaling nucleoside that has been implicated in airway diseases in which mucus obstruction is prominent. Furthermore, the A3 adenosine receptor (A3AR) is upregulated in mucin-producing goblet cells of the airway, thereby implicating it in processes involved in mucous cell biology. Here we use genetic approaches to investigate the contribution of A3AR signaling to mucus production and secretion in a mouse model of allergen-induced pulmonary disease. We found that the degree of mucin production in response to allergen is similar in wild-type and A3AR-deficient mice, and that overexpression of this receptor in Clara cells neither induces mucin production itself, nor enhances mucin production in response to allergen challenge. Collectively, these experiments demonstrate that the A3AR is neither necessary nor sufficient for mucous cell metaplasia. In contrast to the lack of effect on mucin production, agonist-induced mucin secretion was increased in goblet cells overexpressing the A3AR, and was absent in A3AR-deficient mice. Thus, the A3AR contributes to mucin secretion in allergen-induced metaplasia. Signaling through this receptor may contribute to mucus airway obstruction seen in pulmonary disorders in which adenosine levels are elevated.
doi:10.1165/rcmb.2006-0060OC
PMCID: PMC2643274  PMID: 16763221
mucin; mucous cell metaplasia; secretion; adenosine receptors; allergic lung disease
19.  Adenosine and dopamine receptors co-regulate photoreceptor coupling via gap junction phosphorylation in mouse retina 
Gap junctions in retinal photoreceptors suppress voltage noise and facilitate input of rod signals into the cone pathway during mesopic vision. These synapses are highly plastic and regulated by light and circadian clocks. Recent studies have revealed an important role for connexin36 (Cx36) phosphorylation by protein kinase A (PKA) in regulating cell-cell coupling. Dopamine is a light-adaptive signal in the retina, causing uncoupling of photoreceptors via D4 receptors (D4R), which inhibits adenylyl cyclase (AC) and reduces PKA activity. We hypothesized that adenosine, with its extracellular levels increasing in darkness, may serve as a dark signal to co-regulate photoreceptor coupling through modulation of gap junction phosphorylation. Both D4R and A2a receptor (A2aR) mRNAs were present in photoreceptors, inner nuclear layer neurons, and ganglion cells in C57BL/6 mouse retina, and showed cyclic expression with partially overlapping rhythms. Pharmacologically activating A2aR or inhibiting D4R in light-adapted daytime retina increased photoreceptor coupling. Cx36 among photoreceptor terminals, representing predominantly rod-cone gap junctions but possibly including some rod-rod and cone-cone gap junctions, was phosphorylated in a PKA-dependent manner by the same treatments. Conversely, inhibiting A2aR or activating D4R in daytime dark-adapted retina decreased Cx36 phosphorylation with similar PKA dependence. A2a-deficient mouse retina showed defective regulation of photoreceptor gap junction phosphorylation, fairly regular dopamine release, and moderately down-regulated expression of D4R and AC type I mRNA. We conclude that adenosine and dopamine co-regulate photoreceptor coupling through opposite action on the PKA pathway and Cx36 phosphorylation. In addition, loss of the A2aR hampered D4R gene expression and function.
doi:10.1523/JNEUROSCI.2807-12.2013
PMCID: PMC3711184  PMID: 23407968
20.  Role of A2B adenosine receptor signaling in adenosine-dependent pulmonary inflammation and injury  
The Journal of Clinical Investigation  2006;116(8):2173-2182.
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 A2B adenosine receptor (A2BAR) results in proinflammatory and profibrotic effects relevant to the progression of lung diseases; however, in vivo data supporting these observations are lacking. Adenosine deaminase–deficient (ADA-deficient) mice develop pulmonary inflammation and injury that are dependent on increased lung adenosine levels. To investigate the role of the A2BAR in vivo, ADA-deficient mice were treated with the selective A2BAR antagonist CVT-6883, and pulmonary inflammation, fibrosis, and airspace integrity were assessed. Untreated and vehicle-treated ADA-deficient mice developed pulmonary inflammation, fibrosis, and enlargement of alveolar airspaces; conversely, CVT-6883–treated ADA-deficient mice showed less pulmonary inflammation, fibrosis, and alveolar airspace enlargement. A2BAR antagonism significantly reduced elevations in proinflammatory cytokines and chemokines as well as mediators of fibrosis and airway destruction. In addition, treatment with CVT-6883 attenuated pulmonary inflammation and fibrosis in wild-type mice subjected to bleomycin-induced lung injury. These findings suggest that A2BAR signaling influences pathways critical for pulmonary inflammation and injury in vivo. Thus in chronic lung diseases associated with increased adenosine, antagonism of A2BAR-mediated responses may prove to be a beneficial therapy.
doi:10.1172/JCI27303
PMCID: PMC1501110  PMID: 16841096
21.  Interleukin-6 Contributes to Inflammation and Remodeling in a Model of Adenosine Mediated Lung Injury 
PLoS ONE  2011;6(7):e22667.
Background
Chronic lung diseases are the third leading cause of death in the United States due in part to an incomplete understanding of pathways that govern the progressive tissue remodeling that occurs in these disorders. Adenosine is elevated in the lungs of animal models and humans with chronic lung disease where it promotes air-space destruction and fibrosis. Adenosine signaling increases the production of the pro-fibrotic cytokine interleukin-6 (IL-6). Based on these observations, we hypothesized that IL-6 signaling contributes to tissue destruction and remodeling in a model of chronic lung disease where adenosine levels are elevated.
Methodology/Principal Findings
We tested this hypothesis by neutralizing or genetically removing IL-6 in adenosine deaminase (ADA)-deficient mice that develop adenosine dependent pulmonary inflammation and remodeling. Results demonstrated that both pharmacologic blockade and genetic removal of IL-6 attenuated pulmonary inflammation, remodeling and fibrosis in this model. The pursuit of mechanisms involved revealed adenosine and IL-6 dependent activation of STAT-3 in airway epithelial cells.
Conclusions/Significance
These findings demonstrate that adenosine enhances IL-6 signaling pathways to promote aspects of chronic lung disease. This suggests that blocking IL-6 signaling during chronic stages of disease may provide benefit in halting remodeling processes such as fibrosis and air-space destruction.
doi:10.1371/journal.pone.0022667
PMCID: PMC3143181  PMID: 21799929
22.  A protective role for the A1 adenosine receptor in adenosine-dependent pulmonary injury 
Adenosine is a signaling nucleoside that has been implicated in the regulation of asthma and chronic obstructive pulmonary disease. Adenosine signaling can serve both pro- and anti-inflammatory functions in tissues and cells. In this study we examined the contribution of A1 adenosine receptor (A1AR) signaling to the pulmonary inflammation and injury seen in adenosine deaminase–deficient (ADA-deficient) mice, which exhibit elevated adenosine levels. Experiments revealed that transcript levels for the A1AR were elevated in the lungs of ADA-deficient mice, in which expression was localized predominantly to alveolar macrophages. Genetic removal of the A1AR from ADA-deficient mice resulted in enhanced pulmonary inflammation along with increased mucus metaplasia and alveolar destruction. These changes were associated with the exaggerated expression of the Th2 cytokines IL-4 and IL-13 in the lungs, together with increased expression of chemokines and matrix metalloproteinases. These findings demonstrate that the A1AR plays an anti-inflammatory and/or protective role in the pulmonary phenotype seen in ADA-deficient mice, which suggests that A1AR signaling may serve to regulate the severity of pulmonary inflammation and remodeling seen in chronic lung diseases by controlling the levels of important mediators of pulmonary inflammation and damage.
doi:10.1172/JCI200522656
PMCID: PMC539198  PMID: 15630442
23.  Effect of A2B Adenosine Receptor Gene Ablation on Proinflammatory Adenosine Signaling in Mast Cells1 
Pharmacological studies suggest that A2B adenosine receptors mediate proinflammatory effects of adenosine in human mast cells in part by up-regulating production of Th2 cytokines and angiogenic factors. This concept has been recently challenged by the finding that mast cells cultured from bone marrow-derived mast cells (BMMCs) of A2B knockout mice display an enhanced degranulation in response to FcεRI stimulation. This finding was interpreted as evidence of anti-inflammatory functions of A2B receptors and it was suggested that antagonists with inverse agonist activity could promote activation of mast cells. In this report, we demonstrate that genetic ablation of the A2B receptor protein has two distinct effects on BMMCs, one is the previously reported enhancement of Ag-induced degranulation, which is unrelated to adenosine signaling; the other is the loss of adenosine signaling via this receptor subtype that up-regulates IL-13 and vascular endothelial growth factor secretion. Genetic ablation of A2B receptors had no effect on A3 adenosine receptor-dependent potentiation of Ag-induced degranulation in mouse BMMCs, but abrogated A2B adenosine receptor-dependent stimulation of IL-13 and vascular endothelial growth factor secretion. Adenosine receptor antagonists MRS1706 and DPCPX with known inverse agonist activity at the A2B subtype inhibited IL-13 secretion induced by the adenosine analog NECA, but did not mimic the enhanced Ag-induced degranulation observed in A2B knockout BMMCs. Thus, our study confirmed the proinflammatory role of adenosine signaling via A2B receptors and the anti-inflammatory actions of A2B antagonists in mouse BMMCs.
PMCID: PMC3628765  PMID: 18490720
24.  ADENOSINE RECEPTORS AS TARGETS FOR THERAPEUTIC INTERVENTION IN ASTHMA AND CHRONIC OBSTRUCTIVE PULMONARY DISEASE 
Trends in pharmacological sciences  2009;30(10):528-535.
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 A1, A3 and A2B adenosine receptor antagonists in the treatment of features of asthma and/or COPD, while A2A 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.
doi:10.1016/j.tips.2009.07.005
PMCID: PMC2778268  PMID: 19762093
respiratory diseases; COPD; asthma; fibrosis; G-Protein coupled receptors; inflammation

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