Eckle, T. et al. A2B adenosine receptor dampens hypoxia-induced vascular leak. Blood 111, 2024-2035

Departments of Anesthesiology and Intensive Care , Tübingen University Hospital, Tübingen, Germany.
Blood (Impact Factor: 10.45). 03/2008; 111(4):2024-35. DOI: 10.1182/blood-2007-10-117044
Source: PubMed


Extracellular adenosine has been implicated in adaptation to hypoxia and previous studies demonstrated a central role in vascular responses. Here, we examined the contribution of individual adenosine receptors (ARs: A1AR/A2AAR/A2BAR/A3AR) to vascular leak induced by hypoxia. Initial profiling studies revealed that siRNA-mediated repression of the A2BAR selectively increased endothelial leak in response to hypoxia in vitro. In parallel, vascular permeability was significantly increased in vascular organs of A2BAR(-/-)-mice subjected to ambient hypoxia (8% oxygen, 4 hours; eg, lung: 2.1 +/- 0.12-fold increase). By contrast, hypoxia-induced vascular leak was not accentuated in A1AR(-/-)-, A2AAR(-/-)-, or A3AR(-/-)-deficient mice, suggesting a degree of specificity for the A2BAR. Further studies in wild type mice revealed that the selective A2BAR antagonist PSB1115 resulted in profound increases in hypoxia-associated vascular leakage while A2BAR agonist (BAY60-6583 [2-[6-amino-3,5-dicyano-4-[4-(cyclopropylmethoxy)-. phenyl]pyridin-2-ylsulfanyl]acetamide]) treatment was associated with almost complete reversal of hypoxia-induced vascular leakage (eg, lung: 2.0 +/- 0.21-fold reduction). Studies in bone marrow chimeric A2BAR mice suggested a predominant role of vascular A2BARs in this response, while hypoxia-associated increases in tissue neutrophils were, at least in part, mediated by A2BAR expressing hematopoietic cells. Taken together, these studies provide pharmacologic and genetic evidence for vascular A2BAR signaling as central control point of hypoxia-associated vascular leak.

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Available from: Linda F Thompson, Aug 04, 2014
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    • "After blocking for 2 h at room temperature with 5% milk in Trisbuffered saline, pH 7.6, containing 0.1% Tween 20 (TBS-T), the membranes were incubated overnight at 4 °C with a rabbit anti- A 2B R antibody (1:250; sc-28996 from Santa Cruz Biotechnology, Santa Cruz, CA, USA). This particular antibody was selected as its selectivity has been previously been validated by eliminating its immunoreactivity upon neutralising A 2B R with a siRNA (Eckle et al., 2008). After four washing periods for 10 min with TBS-T containing 0.5% milk, the membranes were incubated with the alkaline phosphatase-conjugated anti-rabbit secondary antibody (1:2000; GE Healthcare) in TBS-T containing 1% milk for 90 min at room temperature. "
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    ABSTRACT: Adenosine is a neuromodulator mostly acting through A1 (inhibitory) and A2A (excitatory) receptors in the brain. A2B receptors (A2 B R) are Gs/q -protein-coupled receptors with low expression in the brain. As A2 B R function is largely unknown, we have now explored their role in the mouse hippocampus. We performed electrophysiological extracellular recordings in mouse hippocampal slices, and immunological analysis of nerve terminals and glutamate release in hippocampal slices and synaptosomes. Additionally, A2 B R-knockout (A2 B R-KO) and C57/BL6 mice were submitted to a behavioural test battery (open field, elevated plus-maze, Y-maze). The A2 B R agonist BAY60-6583 (300 nm) decreased the paired-pulse stimulation ratio, an effect prevented by the A2 B R antagonist MRS 1754 (200 nM) and abrogated in A2 B R-KO mice. Accordingly, A2 B R immunoreactivity was present in 73 ± 5% of glutamatergic nerve terminals, i.e. those immunopositive for vesicular glutamate transporters. Furthermore, BAY 60-6583 attenuated the A1 R control of synaptic transmission, both the A1 R inhibition caused by 2-chloroadenosine (0.1-1 μm) and the disinhibition caused by the A1 R antagonist DPCPX (100 nm), both effects prevented by MRS 1754 and abrogated in A2 B R-KO mice. BAY 60-6583 decreased glutamate release in slices and also attenuated the A1 R inhibition (CPA 100 nm). A2 B R-KO mice displayed a modified exploratory behaviour with an increased time in the central areas of the open field, elevated plus-maze and the Y-maze and no alteration of locomotion, anxiety or working memory. We conclude that A2 B R are present in hippocampal glutamatergic terminals where they counteract the predominant A1 R-mediated inhibition of synaptic transmission, impacting on exploratory behaviour. © 2015 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.
    Full-text · Article · Feb 2015 · European Journal of Neuroscience
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    • "This in turn leads to recruitment of immune cells to the lung and loss of pulmonary barrier function, producing the inflammation and pulmonary edema that are characteristics of hyperoxic lung injury. Adenosine is a nucleoside signaling molecule that has been recognized to play an important role in the regulation of inflammation following acute lung injury (Eckle et al. 2008a,b; Schingnitz et al. 2010; Karmouty-Quintana et al. 2013). The concentration of adenosine is normally low in the extracellular compartment, but following acute lung injury, extracellular adenosine concentrations rapidly increase (Volmer et al. 2006; Eckle et al. 2007). "
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    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.
    Full-text · Article · Sep 2014
    • "A number of studies demonstrated an increase of A2B receptor expression under hypoxic conditions in different cells: dendritic cells [38], bronchial smooth muscle cells [27], and fibroblasts [39]. Higher levels of A2B adenosine receptor was detected also in endothelial cells [9,13,17], macrophages [40], lymphocytes [41], and myocardial cells [42]. A2B adenosine receptors activate adenylate cyclase via G proteins leading to increased cAMP levels [43] which mediates intracellular signals [44]. "
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    ABSTRACT: Placental hypoxia is a result of abnormal and shallow trophoblast invasion and involved in the pathophysiology of preeclampsia. Hypoxia increases extracellular adenosine levels and plays an important role in the regulation of angiogenesis, proliferation, vascular tone, endothelial permeability and inflammation. It was shown that adenosine concentrations are higher in preeclamptic patients. We tested the hypothesis that hypoxia and A2B adenosine receptor activation influence cyclic adenosine monophosphate (cAMP) production, proliferation, invasion and cAMP-PKA-CREB signaling in trophoblast cells (HTR-8/SVneo). HTR-8/SVneo and human uterine microvascular endothelial cells (HUtMVEC) were used as model for experiments. We employed a cAMP assay, invasion assay, proliferation, RT-PCR and Western Blot. Statistical analyses were performed with ANOVA, Kruskal-Wallis-, Wilcoxon signed rank- or Mann-Whitney Test, as appropriate. Hypoxia (2% O2) in comparison to normoxia (21% O2) led to increased A2B mRNA levels (1.21 +/- 0.06 fold, 1 h 2% O2; 1.66 +/- 0.2 fold, 4 h 2% O2 and 1.2 +/- 0.04 fold, 24 h 2% O2). A2B adenosine receptor activation (NECA) stimulated trophoblast proliferation at 2% O2 (1.27 +/- 0.06 fold) and 8% O2 (1.17 +/- 0.07 fold) after 24 h and at 2% O2 (1.22 +/- 0.05 fold), 8% O2 (1.23 +/- 0.09 fold) and 21% O2 (1.15 +/- 0.04 fold) after 48 h of incubation. Trophoblast invasion into an endothelial monolayer was significantly expanded by activation of the receptor (NECA) at 8% O2 (1.20 +/- 0.07 fold) and 21% O2 (1.22 +/- 0.006 fold). A2B adenosine receptor stimulation (NECA) additionally led to increased CREB phosphorylation in trophoblast cells at 2% O2 (2.13 +/- 0.45 fold), 8% O2 (1.55 +/- 0.13 fold) and 21% O2 (1.71 +/- 0.34 fold). Blocking of CREB signaling resulted in reduced proliferation and CREB phosphorylation. These data expand the recent knowledge regarding the role of adenosine receptor A2B in human placental development, and may provide insight in mechanisms associated with pregnancy complications linked to impaired trophoblast invasion such as preeclampsia.
    No preview · Article · Jan 2014 · BMC Pregnancy and Childbirth
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