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Objective: Systemic sclerosis (SSc; scleroderma) is a chronic disease that affects the skin and various internal organs. Dermal fibrosis is a major component of this disease. The mechanisms that promote dermal fibrosis remain elusive. Elevations in tissue adenosine levels and the subsequent engagement of the profibrotic A2B adenosine receptor (ADORA2B) have been shown to regulate fibrosis in multiple organs including the lung, kidney, and penis; however, the role of ADORA2B in dermal fibrosis has not been investigated. We undertook this study to test our hypothesis that elevated expression of ADORA2B in the skin drives the development of dermal fibrosis. Methods: We assessed the involvement of ADORA2B in the regulation of dermal fibrosis using a well‐established mouse model of dermal fibrosis. Using an orally active ADORA2B antagonist, we demonstrated how inhibition of ADORA2B results in reduced dermal fibrosis in 2 distinct experimental models. Finally, using human dermal fibroblasts, we characterized the expression of adenosine receptors. Results: We demonstrated that levels of ADORA2B were significantly elevated in dermal fibrosis and that the therapeutic blockade of this receptor in vivo using an ADORA2B antagonist could reduce the production of profibrotic mediators in the skin and attenuate dermal fibrosis. Antagonism of ADORA2B resulted in reduced numbers of arginase‐expressing macrophages and myofibroblasts and in reduced levels of the extracellular matrix proteins fibronectin, collagen, and hyaluronan. Conclusion: These findings identify ADORA2B as a potential profibrotic regulator in dermal fibrosis and suggest that ADORA2B antagonism may be a useful approach for the treatment of SSc. [ABSTRACT FROM AUTHOR]

Abstract: Severe acute respiratory distress syndrome (ARDS) presents typically with an initializing event, followed by the need for mechanical ventilation. Most animal models of ALI are limited by the fact that they focus on a singular cause of acute lung injury (ALI) and therefore fail to mimic the complex, multifactorial pathobiology of ARDS. To better capture this scenario, we provide a comprehensive characterization of models of ALI combining two injuries: intra tracheal (i.t.) instillation of LPS or hypochloric acid (HCl) followed by ventilator‐induced lung injury (VILI). We hypothesized, that mice pretreated with LPS or HCl prior to VILI and thus receiving a (“two‐hit injury”) will sustain a superadditive lung injury when compared to VILI. Mice were allocated to following treatment groups: control with i.t. NaCl, ventilation with low peak inspiratory pressure (PIP), i.t. HCl, i.t. LPS, VILI (high PIP), HCl i.t. followed by VILI and LPS i.t. followed by VILI. Severity of injury was determined by protein content and MPO activity in bronchoalveolar lavage (BAL), the expression of inflammatory cytokines and histopathology. Mice subjected to VILI after HCl or LPS instillation displayed augmented lung injury, compared to singular lung injury. However, mice that received i.t. LPS prior to VILI showed significantly increased inflammatory lung injury compared to animals that underwent i.t. HCl followed by VILI. The two‐hit lung injury models described, resulting in additive but differential acute lung injury recaptures the clinical relevant multifactorial etiology of ALI and could be a valuable tool in translational research. [ABSTRACT FROM AUTHOR]

Idiopathic pulmonary fibrosis is a devastating lung disease with limited treatment options. The signaling molecule adenosine is produced in response to injury and serves a protective role in early stages of injury and is detrimental during chronic stages of disease such as seen in lung conditions such as pulmonary fibrosis. Understanding the association of extracellular adenosine levels and the progression of pulmonary fibrosis is critical for designing adenosine based approaches to treat pulmonary fibrosis. The goal of this study was to use various models of experimental lung fibrosis to understand when adenosine levels are elevated during pulmonary fibrosis and whether these elevations were associated with disease progression and severity. To accomplish this, extracellular adenosine levels, defined as adenosine levels found in bronchioalveolar lavage fluid, were determined in mouse models of resolvable and progressive pulmonary fibrosis. We found that relative bronchioalveolar lavage fluid adenosine levels are progressively elevated in association with pulmonary fibrosis and that adenosine levels diminish in association with the resolution of lung fibrosis. In addition, treatment of these models with dipyridamole, an inhibitor of nucleoside transporters that potentiates extracellular adenosine levels, demonstrated that the resolution of lung fibrosis is blocked by the failure of adenosine levels to subside. Furthermore, exacerbating adenosine levels led to worse fibrosis in a progressive fibrosis model. Increased adenosine levels were associated with elevation of IL-6 and IL-17, which are important inflammatory cytokines in pulmonary fibrosis. These results demonstrate that extracellular adenosine levels are closely associated with the progression of experimental pulmonary fibrosis and that this signaling pathway may mediate fibrosis by regulating IL-6 and IL-17 production. [ABSTRACT FROM AUTHOR]

Lung fibrosis is the hallmark of the interstitial lung diseases. Alveolar epithelial cell (AEC) injury is a key step that contributes to a profibrotic microenvironment. Fibroblasts and myofibroblasts subsequently accumulate and deposit excessive extracellular matrix. In addition to TGF-β, the IL-6 family of cytokines, which signal through STAT-3, may also contribute to lung fibrosis. In the current manuscript, the extent to which STAT-3 inhibition decreases lung fibrosis is investigated. Phosphorylated STAT-3 was elevated in lung biopsies from patients with idiopathic pulmonary fibrosis and bleomycin (BLM)-induced fibrotic murine lungs. C-188-9, a small molecule STAT-3 inhibitor, decreased pulmonary fibrosis in the intraperitoneal BLM model as assessed by arterial oxygen saturation (control, 84.4 ± 1.3%; C-188-9, 94.4 ± 0.8%), histology (Ashcroft score: untreated, 5.4 ± 0.25; C-188-9, 3.3 ± 0.14), and attenuated fibrotic markers such as diminished a-smooth muscle actin, reduced collagen deposition. In addition, C-188-9 decreased the expression of epithelial injury markers, including hypoxia-inducible factor-1a (HIF-1a) and plasminogen activator inhibitor-1 (PAI-1). In vitro studies show that inhibition of STAT-3 decreased IL-6- and TGF-β-induced expression of multiple genes, including HIF-1a and PAI-1, in AECs. Furthermore, C-188-9 decreased fibroblast-to-myofibroblast differentiation. Finally, TGF-β stimulation of lung fibroblasts resulted in SMAD2/SMAD3-dependent phosphorylation of STAT-3. These findings demonstrate that STAT-3 contributes to the development of lung fibrosis and suggest that STAT-3 may be a therapeutic target in pulmonary fibrosis.--Pedroza, M., Le, T. T., Lewis, K., Karmouty-Quintana, H., To, S., George, A. T., Blackburn, M. R., Tweardy, D. J., Agarwal, S. K. STAT-3 contributes to pulmonary fibrosis through epithelial injury and fibroblast-myofibroblast differentiation. [ABSTRACT FROM AUTHOR]

Idiopathic pulmonary fibrosis (IPF) is a lethal, fibroproliferative disease. Pulmonary hypertension (PH) can develop secondary to IPF and increase mortality. Alternatively, activated macrophages (AAMs) contribute to the pathogenesis of both IPF and PH. Here we hypothesized that adenosine signaling through the ADORA2B on AAMs impacts the progression of these disorders and that conditional deletion of ADORA2B on myeloid cells would have a beneficial effect in a model of these diseases. Conditional knockout mice lacking ADORA2B on myeloid cells (Adora2Bf/f-LysMCre) were exposed to the fibrotic agent bleomycin (BLM; 0.035 U/g body weight, i.p.). At 14,17,21,25, or 33 d after exposure, SpO2, bronchoalveolar lavage fluid (BALF), and histologic analyses were performed. On day 33, lung function and cardiovascular analyses were determined. Markers for AAM and mediators of fibrosis and PH were assessed. Adora2Bf/f-LysMCre mice presented with attenuated fibrosis, improved lung function, and no evidence of PH compared with control mice exposed to BLM. These findings were accompanied by reduced expression of CD206 and arginase-1, markers for AAMs. A 10-fold reduction in IL-6 and a 5-fold decrease in hyaluronan, both linked to lung fibrosis and PH, were also observed. These data suggest that activation of the ADORA2B on macrophages plays an active role in the pathogenesis of lung fibrosis and PH. [ABSTRACT FROM AUTHOR]

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 C57 BL6, 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. [ABSTRACT FROM AUTHOR]

Priapism is featured with prolonged and painful penile erection and is prevalent among males with sickle cell disease (SCD). The disorder is a dangerous urological and hematological emergency since it is associated with ischemic tissue damage and erectile disability. Here we report that phosphodiesterase-5 (PDE5) gene expression and PDE activity is significantly reduced in penile tissues of two independent priapic models: SCD mice and adenosine deaminase (ADA)-deficient mice. Moreover, using ADA enzyme therapy to reduce adenosine or a specific antagonist to block A2B adenosine receptor (ADORA2B) signaling, we successfully attenuated priapism in both ADA-/- and SCD mice by restoring penile PDE5 gene expression to normal levels. This finding led us to further discover that excess adenosine signaling via ADORA2B activation directly reduces PDE5 gene expression in a hypoxia-inducible factor-1α (HIF-1α)-dependent manner. Overall, we reveal that excess adenosine-mediated ADORA2B signaling underlies reduced penile PDE activity by decreasing PDE5 gene expression in a HIF-1α-dependent manner and provide new insight for the pathogenesis of priapism and novel therapies for the disease. [ABSTRACT FROM AUTHOR]

The signaling molecule adenosine has been implicated in attenuating acute lung injury (ALI). Adenosine signaling is terminated by its uptake through equilibrative nucleoside transporters (ENTs). We hypothesized that ENT-dependent adenosine uptake could be targeted to enhance adenosine-mediated lung protection. To address this hypothesis, we exposed mice to high-pressure mechanical ventilation to induce ALI. Initial studies demonstrated time-dependent repression of ENT1 and ENT2 transcript and protein levels during ALI. To examine the contention that ENT repression represents an endogenous adaptive response, we performed functional studies with the ENT inhibitor dipyridamole. Dipyridamole treatment (1 mg/kg; EC50=10 µM) was associated with significant increases in All survival time (277 vs. 395 min; P<0.05). Subsequent studies in gene-targeted mice for Ent1 or Ent2 revealed a selective phenotype in Ent2-/- mice, including attenuated pulmonary edema and improved gas exchange during ALI in conjunction with elevated adenosine levels in die bronchoalveolar fluid. Furthermore, studies in genetic models for adenosine receptors implicated the A2B adenosine receptor (Adora2b) in mediating ENT-dependent lung protection. Notably, dipyridamole-dependent attenuation of lung inflammation was abolished in mice with alveolar epithelial Adora2b gene deletion. Our newly identified crosstalk pathway between ENT2 and alveolar epithelial Adora2b in lung protection during ALI opens possibilities for combined therapies targeted to tins protein set. [ABSTRACT FROM AUTHOR]

Chronic obstructive pulmonary disease (COPD) is characterized by persistent inflammation and tissue remodeling and is a leading cause of death in the United States. Increased apoptosis of pulmonary epithelial cells is thought to play a role in COPD development and progression. Identification of signaling pathways resulting in increased apoptosis in COPD can be used in the development of novel therapeutic interventions. Deoxyadenosine (dAdo) is a DNA breakdown product that amplifies lymphocyte apoptosis by being phosphorylated to deoxyadenosine triphosphate (dATP). dAdo is maintained at low levels by adenosine deaminase (ADA). This study demonstrated that mice lacldng ADA developed COPD manifestations in association with elevated dAdo and dATP levels and increased apoptosis in the lung. Deoxycitidine kinase (DCK), a major enzyme for dAdo phosphorylation, was up-regulated in mouse and human airway epithelial cells in association with air-space enlargement. Hypoxia was identified as a novel regulator of DCK, and inhibition of DCK resulted in diminished dAdo-mediated apoptosis in the lungs. Our results suggest that activating the dAdo-DCK-dATP pathway directly results in increased apoptosis in the lungs of mice with air-space enlargement and suggests a novel therapeutic target for the treatment of COPD. [ABSTRACT FROM AUTHOR]

Development of pulmonary hypertension is a common and deadly complication of interstitial lung disease. Little is known regarding the cellular and molecular mechanisms that lead to pulmonary hypertension in patients with interstitial lung disease, and effective treatment options are lacking. The purpose of this study was to examine the adenosine 2B receptor (A2BR) as a regulator of vascular remodeling and pulmonary hypertension secondary to pulmonary fibrosis. To accomplish this, cellular and molecular changes in vascular remodeling were monitored in mice exposed to bleomycin in conjunction with genetic removal of the A2BR or treatment with the A2BR antagonist GS-6201. Results demonstrated that GS-6201 treatment or genetic removal of the A2BR attenuated vascular remodeling and hypertension in our model. Furthermore, direct A2BR activation on vascular cells promoted interleukin-6 and endothelin-1 release. These studies identify a novel mechanism of disease progression to pulmonary hypertension and support the development of A2BR antagonists for the treatment of pulmonary hypertension secondary to interstitial lung disease. [ABSTRACT FROM AUTHOR]

Pulmonary fibrosis, characterized by excess deposition of extracellular matrix by myofibroblasts, is a serious component of chronic lung diseases. Cadherin-11 (CDH11) is increased in wound healing and fibrotic skin. We hypothesized that CDH11 is increased in pulmonary fibrosis and contributes its development. CDH11 expression was assessed in lung tissue from idiopathic pulmonary fibrosis patients. The role of CDH11 in lung fibrosis was determined using the bleomycin model of pulmonary fibrosis, and in vitro analyses were performed on A549 cells during the process of epithelial to mesenchymal transition (EMT). Immunohistochemical studies demonstrated CDH11 expression on fibroblasts, epithelial cells, and alveolar macrophages of patients with pulmonary fibrosis and mice given bleomycin. Interestingly, CDH11-deficient mice had decreased fibrotic endpoints in the bleomycin model of pulmonary fibrosis compared to wild-type mice. Furthermore, anti-CDH11-neutralizing monoclonal antibodies successfully treated established pulmonary fibrosis induced by bleomycin. TGF-β levels were reduced in bronchoalveolar lavage (BAL) fluid, BAL cells, and primary alveolar macrophages from CDH11-deficient mice. Mechanistic studies demonstrated that TGF-β up-regulated CDH11 expression on A549 cells, and inhibition of CDH11 expression using siRNA reduced TGF-β-induced EMT. Together, these results identify CDH11 as a novel therapeutic target for pulmonary fibrosis. [ABSTRACT FROM AUTHOR]

Normal penile erection is under the control of multiple factors and signaling pathways. Although adenosine signaling is implicated in normal and abnormal penile erection, the exact role and the underlying mechanism for adenosine signaling in penile physiology remain elusive. Here we report that shear stress leads to increased adenosine release from endothelial cells. Subsequently, we determined that ecto-5'-nucleotidase (CD73) is a key enzyme required for the production of elevated adenosine from ATP released by shear-stressed endothelial cells. Mechanistically, we demonstrate that shear stress-mediated elevated adenosine functions through the adenosine A2B receptor (A2BR) to activate the PI3K/AKT signaling cascade and subsequent increased endothelial nitric oxide synthase (eNOS) phosphorylation. These in vitro studies led us to discover further that adenosine was induced during sustained penile erection and contributes to PI3K/AKT activation and subsequent eNOS phosphorylation via A2BR signaling in intact animal. Finally, we demonstrate that lowering adenosine in wild-type mice or genetic deletion of A2BR in mutant mice significantly attenuated PI3K/AKT activation, eNOS phosphorylation, and subsequent impaired penile erection featured with the reduction of ratio of maximal intracavernosal pressure to systemic arterial pressure from 0.49 ± 0.03 to 0.41 ± 0.05 and 0.38 ± 0.04, respectively (both P<0.05). Overall, using biochemical, cellular, genetic, and physiological approaches, our findings reveal that adenosine is a novel molecule signaling via A2BR activation, contributing to penile erection via PI3K/AKT-dependent eNOS activation. These studies suggest that this signaling pathway may be a novel therapeutic target for erectile disorders. [ABSTRACT FROM AUTHOR]

ABSTRACT Priapism is a condition of persistent penile erection in the absence of sexual excitation. Of men with sickle cell disease (SCD), 40% display priapism. The disorder is a dangerous and urgent condition, given its association with penile fibrosis and eventual erectile dysfunction. Current strategies to prevent its progression are poor because of a lack of fundamental understanding of the molecular mechanisms for penile fibrosis in priapism. Here we demonstrate that increased adenosine is a novel causative factor contributing to penile fibrosis in two independent animal models of priapism, adenosine deaminase (ADA)-deficient mice and SCD transgenic mice. An important finding is that chronic reduction of adenosine by ADA enzyme therapy successfully attenuated penile fibrosis in both mouse models, indicating an essential role of increased adenosine in penile fibrosis and a novel therapeutic possibility for this serious complication. Subsequently, we identified that both mice models share a similar fibrotic gene expression profile in penile tissue (including procollagen I, TGF-β1, and plasminogen activator inhibitor-1 mRNA), suggesting that they share similar signaling pathways for progression to penile fibrosis. Thus, in an effort to decipher specific cell types and underlying mechanism responsible for adenosine-mediated penile fibrosis, we purified corpus cavernosal fibroblast cells (CCFCs), the major cell type involved in this process, from wild-type mice. Quantitative RT-PCR showed that the major receptor expressed in these cells is the adenosine receptor A2BR. Based on this fact, we further purified CCFCs from A2BR-deficient mice and demonstrated that A2BR is essential for excess adenosine-mediated penile fibrosis. Finally, we revealed that TGF-β functions downstream of the A2BR to increase CCFC collagen secretion and proliferation. Overall, our studies identify an essential role of increased adenosine in the pathogenesis of penile fibrosis via A2BR signaling and offer a potential target for prevention and treatment of penile fibrosis, a dangerous complication seen in priapism. [ABSTRACT FROM AUTHOR]

Chronic obstructive pulmonary disease (COPD) is a major health concern. Adenosine, a signaling molecule generated in response to cell stress, contributes to the pathogenesis of COPD. An established model of adenosine-mediated lung injury is the adenosine deaminase-deficient (Ada-/-) mouse. Osteopontin (OPN) is a chemokine that is produced following injury and is implicated in a variety of human pathologies, but its expression and role in the pathogenesis of COPD have not been examined. To investi- gate the role of OPN in a model of COPD, Ada-/- double-knockout mice were generated, and inflammation and air-space enlargement endpoints were examined. Results demonstrate that Ada-/- mice exhibit OPN-dependent neutrophilia, alveolar air-space enlargement, and increases in mediators of air-space enlargement. Furthermore, we demonstrate that patients with COPD have increased OPN expression within distal airways in association with clinical airway obstruction. These results suggest that OPN represents a novel biomarker and therapeutic target for patients with COPD. [ABSTRACT FROM AUTHOR]

Angiogenesis is a feature of chronic lung diseases such as asthma and pulmonary fibrosis; however, the pathways controlling pathological angiogenesis during lung disease are not completely understood. Adenosine is a signaling molecule that has been implicated in the exacerbation of chronic lung disease and in the regulation of angiogenesis; however, the relationship between these factors has not been investigated. The current study utilized adenosine deaminase (ADA)-deficient mice to determine whether chronic elevations in adenosine in vivo result in pulmonary angiogenesis. Results demonstrate substantial angiogenesis in the tracheas of ADA-deficient mice in association with adenosine elevations. ADA replacement enzyme therapy resulted in a lowering of adenosine levels and reversal of tracheal angiogenesis, indicating that the increases in vessel number are dependent on adenosine elevations. Levels of the angiogenic chemokine CXCL1 (mouse functional homologue of human IL-8) were found to be elevated in an adenosine-dependent manner in the lungs of ADA-deficient mice. Neutralization of CXCL1 and its receptor, CXCR2, resulted in the inhibition of angiogenic activity, which suggests that CXCL1 signaling through the CXCR2 receptor mediated the observed increases in angiogenesis. Our findings suggest that adenosine plays an important role, via CXCL1, in the induction of pulmonary angiogenesis. [ABSTRACT FROM AUTHOR]

While inhibition of ischaemic contracture was one of the first documented cardioprotective actions of exogenously applied adenosine, it is not known whether this is a normal function of endogenous adenosine generated during ischaemic stress. Additionally, the relevance of delayed contracture to postischaemic outcome is unclear. We tested the ability of endogenous versus exogenous adenosine to modify contracture (and postischaemic outcomes) in C57/Bl6 mouse hearts. During ischaemia, untreated hearts developed peak contracture (PC) of 85 ± 5 mmHg at 8.9 ± 0.8 min, with time to reach 20 mmHg (time to onset of contracture; TOC) of 4.4 ± 0.3 min. Adenosine (50 μm) delayed TOC to 6.7 ± 0.6 min, as did pretreatment with 10 μm 2-chloroadenosine (7.2 ± 0.5 min) or 50 nm of A1 adenosine receptor (AR) agonist N6-cyclohexyladenosine (CHA) (6.7 ± 0.3 min), but not A2AAR or A3AR agonists (20 nm 2-[4-(2-carboxyethyl) phenethylamino]-5′ N-methylcarboxamidoadenosine (CGS21680) or 150 nm 2-chloro-N6-(3-iodobenzyl)-adenosine-5′-N-methyluronamide (Cl-IB-MECA), respectively). Adenosinergic contracture inhibition was eliminated by A1AR gene knockout (KO), mimicked by A1AR overexpression, and was associated with preservation of myocardial [ATP]. This adenosine-mediated inhibition of contracture was, however, only evident after prolonged (10 or 15 min) and not brief (3 min) pretreatment. Ischaemic contracture was also insensitive to endogenously generated adenosine, since A1AR KO, and non-selective and A1AR-selective antagonists (50 μm 8-sulphophenyltheophylline and 150 nm 8-cyclopentyl-1, 3-dipropylxanthine (DPCPX), respectively), all failed to alter intrinsic contracture development. Finally, delayed contracture with A1AR agonism/overexpression or ischaemic 2,3-butanedione monoxime (BDM; 5 μm to target Ca2+ cross-bridge formation) was linked to enhanced postischaemic outcomes. In summary, adenosinergic inhibition of contracture is solely A1AR mediated; the response is ‘supraphysiological’, evident only with significant periods of pre-ischaemic AR agonism (or increased A1AR density); and ischaemic contracture appears insensitive to locally generated adenosine, potentially owing to the rapidity of contracture development versus the finite time necessary for expression of AR-mediated cardioprotection. [ABSTRACT FROM AUTHOR]