HIF1A up-regulates the ADORA2B receptor on alternatively activated macrophages and contributes to pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a deadly chronic lung disease. Extracellular accumulation of adenosine and subsequent activation of the ADORA2B receptor play important roles in regulating inflammation and fibrosis in IPF. Additionally, alternatively activated macrophages (AAMs) expressing ADORA2B have been implicated in mediating adenosine's effects in IPF. Although hypoxic conditions are present in IPF, hypoxia's role as a direct modulator of macrophage phenotype and identification of factors that regulate ADORA2B expression on AAMs in IPF is not well understood. In this study, an experimental mouse model of pulmonary fibrosis and lung samples from patients with IPF were used to examine the effects and interactions of macrophage differentiation and hypoxia on fibrosis. We demonstrate that hypoxia-inducible factor 1-α (HIF1A) inhibition in late stages of bleomycin-induced injury attenuates pulmonary fibrosis in association, with reductions in ADORA2B expression in AAMs. Additionally, ADORA2B deletion or pharmacological antagonism along with HIF1A inhibition disrupts AAM differentiation and subsequent IL-6 production in cultured macrophages. These findings suggest that hypoxia, through HIF1A, contributes to the development and progression of pulmonary fibrosis through its regulation of ADORA2B expression on AAMs, cell differentiation, and production of profibrotic mediators. These studies support a potential role for HIF1A or ADORA2B antagonists in the treatment of IPF.-Philip, K., Mills, T. W., Davies, J., Chen, N.-Y., Karmouty-Quintana, H., Luo, F., Molina, J. G., Amione-Guerra, J., Sinha, N., Guha, A., Eltzschig, H. K., Blackburn, M. R. HIF1A up-regulates the ADORA2B receptor on alternatively activated macrophages and contributes to pulmonary fibrosis.

Elevated adenosine signaling via adenosine A2B receptor induces normal and sickle erythrocyte sphingosine kinase 1 activity.

Erythrocyte possesses high sphingosine kinase 1 (SphK1) activity and is the major cell type supplying plasma sphingosine-1-phosphate, a signaling lipid regulating multiple physiological and pathological functions. Recent studies revealed that erythrocyte SphK1 activity is upregulated in sickle cell disease (SCD) and contributes to sickling and disease progression. However, how erythrocyte SphK1 activity is regulated remains unknown. Here we report that adenosine induces SphK1 activity in human and mouse sickle and normal erythrocytes in vitro. Next, using 4 adenosine receptor-deficient mice and pharmacological approaches, we determined that the A2B adenosine receptor (ADORA2B) is essential for adenosine-induced SphK1 activity in human and mouse normal and sickle erythrocytes in vitro. Subsequently, we provide in vivo genetic evidence that adenosine deaminase (ADA) deficiency leads to excess plasma adenosine and elevated erythrocyte SphK1 activity. Lowering adenosine by ADA enzyme therapy or genetic deletion of ADORA2B significantly reduced excess adenosine-induced erythrocyte SphK1 activity in ADA-deficient mice. Finally, we revealed that protein kinase A-mediated extracellular signal-regulated kinase 1/2 activation functioning downstream of ADORA2B underlies adenosine-induced erythrocyte SphK1 activity. Overall, our findings reveal a novel signaling network regulating erythrocyte SphK1 and highlight innovative mechanisms regulating SphK1 activity in normal and SCD.

Extracellular adenosine levels are associated with the progression and exacerbation of pulmonary fibrosis.

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.

Macrophage bone morphogenic protein receptor 2 depletion in idiopathic pulmonary fibrosis and Group III pulmonary hypertension.

Idiopathic pulmonary fibrosis (IPF) is a lethal lung disease of unknown etiology. The development of pulmonary hypertension (PH) is considered the single most significant predictor of mortality in patients with chronic lung diseases. The processes that govern the progression and development of fibroproliferative and vascular lesions in IPF are not fully understood. Using human lung explant samples from patients with IPF with or without a diagnosis of PH as well as normal control tissue, we report reduced BMPR2 expression in patients with IPF or IPF+PH. These changes were consistent with dampened P-SMAD 1/5/8 and elevated P-SMAD 2/3, demonstrating reduced BMPR2 signaling and elevated TGF-ß activity in IPF. In the bleomycin (BLM) model of lung fibrosis and PH, we also report decreased BMPR2 expression compared with control animals that correlated with vascular remodeling and PH. We show that genetic abrogation or pharmacological inhibition of interleukin-6 leads to diminished markers of fibrosis and PH consistent with elevated levels of BMPR2 and reduced levels of a collection of microRNAs (miRs) that are able to degrade BMPR2. We also demonstrate that isolated bone marrow-derived macrophages from BLM-exposed mice show reduced BMPR2 levels upon exposure with IL6 or the IL6+IL6R complex that are consistent with immunohistochemistry showing reduced BMPR2 in CD206 expressing macrophages from lung sections from IPF and IPF+PH patients. In conclusion, our data suggest that depletion of BMPR2 mediated by a collection of miRs induced by IL6 and subsequent STAT3 phosphorylation as a novel mechanism participating to fibroproliferative and vascular injuries in IPF.

Deletion of ADORA2B from myeloid cells dampens lung fibrosis and pulmonary hypertension.

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 (Adora2B(f/f)-LysM(Cre)) 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. Adora2B(f/f)-LysM(Cre) 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.

Hypoxia-induced deoxycytidine kinase expression contributes to apoptosis in chronic lung disease.

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 lacking 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.

Adenosine A2B receptor and hyaluronan modulate pulmonary hypertension associated with chronic obstructive pulmonary disease.

Chronic obstructive pulmonary disease (COPD) is the fourth leading cause of death worldwide. The development of pulmonary hypertension (PH) in patients with COPD is strongly associated with increased mortality. Chronic inflammation and changes to the lung extracellular matrix (ECM) have been implicated in the pathogenesis of COPD, yet the mechanisms that lead to PH secondary to COPD remain unknown. Our experiments using human lung tissue show increased expression levels of the adenosine A2B receptor (ADORA2B) and a heightened deposition of hyaluronan (HA; a component of the ECM) in remodeled vessels of patients with PH associated with COPD. We also demonstrate that the expression of HA synthase 2 correlates with mean pulmonary arterial pressures in patients with COPD, with and without a secondary diagnosis of PH. Using an animal model of airspace enlargement and PH, we show that the blockade of ADORA2B is able to attenuate the development of a PH phenotype that correlates with reduced levels of HA deposition in the vessels and the down-regulation of genes involved in the synthesis of HA.

Excess adenosine in murine penile erectile tissues contributes to priapism via A2B adenosine receptor signaling.

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 A2B adenosine receptor-mediated (A2BR-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 A2BR activation. Thus, we have shown that excessive adenosine accumulation in the penis contributes to priapism through increased A2BR 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 A2BR activation may prove beneficial in the treatment of this disorder.

Adenosine and osteopontin contribute to the development of chronic obstructive pulmonary disease.

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 investigate 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.

Enhanced airway inflammation and remodeling in adenosine deaminase-deficient mice lacking the A2B adenosine receptor.

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 A(2B)R antagonism can attenuate features of chronic lung disease, implicating this receptor in the progression of chronic lung disease. This study examines the contribution of A(2B)R signaling in this model by generating ADA/A(2B)R double-knockout mice. Our hypothesis was that genetic removal of the A(2B)R from ADA-deficient mice would lead to diminished pulmonary inflammation and damage. Unexpectedly, ADA/A(2B)R 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 A(2B)R signaling during acute stages of lung disease.

Downregulation of CFIm25 amplifies dermal fibrosis through alternative polyadenylation.

Systemic sclerosis (SSc; scleroderma) is a multisystem fibrotic disease. The mammalian cleavage factor I 25-kD subunit (CFIm25; encoded by NUDT21) is a key regulator of alternative polyadenylation, and its depletion causes predominantly 3'UTR shortening through loss of stimulation of distal polyadenylation sites. A shortened 3'UTR will often lack microRNA target sites, resulting in increased mRNA translation due to evasion of microRNA-mediated repression. Herein, we report that CFlm25 is downregulated in SSc skin, primary dermal fibroblasts, and two murine models of dermal fibrosis. Knockdown of CFIm25 in normal skin fibroblasts is sufficient to promote the 3'UTR shortening of key TGFß-regulated fibrotic genes and enhance their protein expression. Moreover, several of these fibrotic transcripts show 3'UTR shortening in SSc skin. Finally, mice with CFIm25 deletion in fibroblasts show exaggerated skin fibrosis upon bleomycin treatment, and CFIm25 restoration attenuates bleomycin-induced skin fibrosis. Overall, our data link this novel RNA-processing mechanism to dermal fibrosis and SSc pathogenesis.

Role of A2B adenosine receptor signaling in adenosine-dependent pulmonary inflammation and injury.

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.

Adenosine and hyaluronan promote lung fibrosis and pulmonary hypertension in combined pulmonary fibrosis and emphysema.

Combined pulmonary fibrosis and emphysema (CPFE) is a syndrome that predominantly affects male smokers or ex-smokers and it has a mortality rate of 55% and a median survival of 5 years. Pulmonary hypertension (PH) is a frequently fatal complication of CPFE. Despite this dismal prognosis, no curative therapies exist for patients with CPFE outside of lung transplantation and no therapies are recommended to treat PH. This highlights the need to develop novel treatment approaches for CPFE. Studies from our group have demonstrated that both adenosine and its receptor ADORA2B are elevated in chronic lung diseases. Activation of ADORA2B leads to elevated levels of hyaluronan synthases (HAS) and increased hyaluronan, a glycosaminoglycan that contributes to chronic lung injury. We hypothesize that ADORA2B and hyaluronan contribute to CPFE. Using isolated CPFE lung tissue, we characterized expression levels of ADORA2B and HAS. Next, using a unique mouse model of experimental lung injury that replicates features of CPFE, namely airspace enlargement, PH and fibrotic deposition, we investigated whether 4MU, a HAS inhibitor, was able to inhibit features of CPFE. Increased protein levels of ADORA2B and HAS3 were detected in CPFE and in our experimental model of CPFE. Treatment with 4MU was able to attenuate PH and fibrosis but not airspace enlargement. This was accompanied by a reduction of HAS3-positive macrophages. We have generated pre-clinical data demonstrating the capacity of 4MU, an FDA-approved drug, to attenuate features of CPFE in an experimental model of chronic lung injury.This article has an associated First Person interview with the first author of the paper.

Cleavage factor 25 deregulation contributes to pulmonary fibrosis through alternative polyadenylation.

Idiopathic pulmonary fibrosis (IPF) is a chronic and deadly disease with a poor prognosis and few treatment options. Pathological remodeling of the extracellular matrix (ECM) by myofibroblasts is a key factor that drives 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 and ECMs through altering microRNA sensitivity, but a connection to IPF has not been established. Here, we demonstrate that CFIm25, a global regulator of APA, is down-regulated in the lungs of patients with IPF and mice with pulmonary fibrosis, with its expression selectively reduced in alpha-smooth muscle actin (α-SMA) positive fibroblasts. Following the knockdown of CFIm25 in normal human lung fibroblasts, we identified 808 genes with shortened 3'UTRs, including those involved in the transforming growth factor-ß signaling pathway, the Wnt signaling pathway, and cancer pathways. The expression of key pro-fibrotic factors can be suppressed by CFIm25 overexpression in IPF fibroblasts. Finally, we demonstrate that deletion of CFIm25 in fibroblasts or myofibroblast precursors using either the Col1a1 or the Foxd1 promoter enhances pulmonary fibrosis after bleomycin exposure in mice. Taken together, our results identified CFIm25 down-regulation as a novel mechanism to elevate pro-fibrotic gene expression in pulmonary fibrosis.

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 A(3) adenosine receptor (A(3)R) in a bleomycin model of pulmonary inflammation and fibrosis. Results demonstrated that A(3)R-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 A(3)R-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 A(3)R-deficient mice exposed to bleomycin, an observation suggesting that the A(3)R is necessary for eosinophil degranulation in this model. Despite an increase in inflammatory metrics associated with A(3)R-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)-beta1 expression, but not a concomitant increase in TGF-beta1 activity. This was associated with the loss of expression of matrix metalloprotease 9, an activator of TGF-beta1, in alveolar macrophages and airway mast cells in the lungs of A(3)R-deficient mice. Together, these results suggest that the A(3)R serves antiinflammatory functions in the bleomycin model, and is also involved in regulating the production of mediators that can impact fibrosis.

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 A(1) adenosine receptor (A(1)AR) 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 A(1)AR were elevated in the lungs of ADA-deficient mice, in which expression was localized predominantly to alveolar macrophages. Genetic removal of the A(1)AR 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 A(1)AR plays an anti-inflammatory and/or protective role in the pulmonary phenotype seen in ADA-deficient mice, which suggests that A(1)AR 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.

Enhanced CXCL1 production and angiogenesis in adenosine-mediated lung disease.

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.

The Antifibrotic Effect of A2B Adenosine Receptor Antagonism in a Mouse Model of Dermal Fibrosis.

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.

Genetic deletion of the A1 adenosine receptor limits myocardial ischemic tolerance.

Adenosine receptors may be important determinants of intrinsic ischemic tolerance. Genetically modified mice were used to examine effects of global A1 adenosine receptor (A1AR) knockout (KO) on function and ischemic tolerance in perfused mouse hearts. Baseline contractile function and heart rate were unaltered by A1AR KO, which was shown to abolish the negative chronotropic effects of 2-chloroadenosine (A1AR-mediated) without altering A2 adenosine receptor-mediated coronary dilation. Tolerance to 25 minutes global normothermic ischemia (followed by 45 minutes reperfusion) was significantly limited by A1AR KO, with impaired contractile recovery (reduced by 25%) and enhanced lactate dehydrogenase (LDH) efflux (increased by 100%). Functional effects of A1AR KO involved worsened systolic pressure development with little to no change in diastolic dysfunction. In contrast, cardiac specific A1AR overexpression enhanced ischemic tolerance with a primary action on diastolic dysfunction. Nonselective receptor agonism (10 micromol/L 2-chloroadenosine) protected wild-type and also A1AR KO hearts (albeit to a lesser extent), implicating protection via subtypes additional to A1ARs. However, A1AR KO abrogated effects of 2-chloroadenosine on ischemic contracture and diastolic dysfunction. These data are the first demonstrating global deletion of the A1AR limits intrinsic myocardial resistance to ischemia. Data indicate the function of intrinsically activated A1ARs appears primarily to be enhancement of postischemic contractility and limitation of cell death.

Effects of adenosine deaminase and A1 receptor deficiency in normoxic and ischaemic mouse hearts.

OBJECTIVE: Adenosine deaminase (ADA) may be multifunctional, regulating adenosine levels and adenosine receptor (AR) agonism, and potentially modifying AR functionality. Herein we assess effects of ADA (and A1AR) deficiency on AR-mediated responses and ischaemic tolerance. METHODS: Normoxic function and responses to 20 or 25 min ischaemia and 45 min reperfusion were studied in isolated hearts from wild-type mice and from mice deficient in ADA and/or A1ARs. RESULTS: Neither ADA or A1AR deficiency significantly modified basal contractility, although ADA deficiency reduced resting heart rate (an effect abrogated by A1AR deficiency). Bradycardia and vasodilation in response to AR agonism (2-chloroadenosine) were unaltered by ADA deficiency, while A1AR deficiency eliminated the heart rate response. Adenosine efflux increased 10- to 20-fold with ADA deficiency (at the expense of inosine). Deletion of ADA improved outcome from 25 min ischaemia, reducing ventricular diastolic pressure (by 45%; 21+/-4 vs. 38+/-3mm Hg) and lactate dehydrogenase (LDH) efflux (by 40%; 0.12+/-0.01 vs. 0.21+/-0.02 U/g/min ischaemia), and enhancing pressure development (by 35%; 89+/-6 vs. 66+/-5mm Hg). Similar protection was evident after 20 min ischaemia, and was mimicked by the ADA inhibitor EHNA (5 microM). Deletion of ADA also enhanced tolerance in A1AR deficient hearts, though effects on diastolic pressure were eliminated. CONCLUSIONS: Deficiency of ADA does not alter sensitivities of cardiovascular A1 or A2ARs (despite markedly elevated [adenosine]), but significantly improves ischaemic tolerance. Conversely, A1AR deficiency impairs ischaemic tolerance. Effects of ADA deficiency on diastolic pressure appear solely A1AR-dependent while other ARs or processes additionally contribute to improved contractile recovery and reduced cell death.