Enhancing Extracellular Adenosine Levels Restores Barrier Function in Acute Lung Injury Through Expression of Focal Adhesion Proteins

Abstract

Background: Acute respiratory distress syndrome (ARDS) is a clinical presentation of acute lung injury (ALI) with often fatal lung complication. Adenosine, a nucleoside generated following cellular stress provides protective effects in acute injury. The levels of extracellular adenosine can be depleted by equilibrative nucleoside transporters (ENTs). ENT inhibition by pharmaceutical agent dipyridamole promotes extracellular adenosine accumulation and is protective in ARDS. However, the therapeutic potential of dipyridamole in acute lung injury has not yet been evaluated.Methods: Adenosine acts on three adenosine receptors, the adenosine A1 (Adora1), A2a (Adora2a), the A2b (Adora2b) or the adenosine A3 (Adora 3) receptor. Accumulation of adenosine is usually required to stimulate the low-affinity Adora2b receptor. In order to investigate the effect of adenosine accumulation and the contribution of epithelial-specific ENT2 or adora2b expression in experimental ALI, dipyridamole, and epithelial specific ENT2 or Adora2b deficient mice were utilized. MLE12 cells were used to probe downstream Adora2b signaling. Adenosine receptors, transporters, and targets were determined in ARDS lungs.Results: ENT2 is mainly expressed in alveolar epithelial cells and is negatively regulated by hypoxia following tissue injury. Enhancing adenosine levels with ENT1/ENT2 inhibitor dipyridamole at a time when bleomycin-induced ALI was present, reduced further injury. Mice pretreated with the ADORA2B agonist BAY 60-6583 were protected from bleomycin-induced ALI by reducing vascular leakage (558.6 ± 50.4 vs. 379.9 ± 70.4, p < 0.05), total bronchoalveolar lavage fluid cell numbers (17.9 ± 1.8 to 13.4 ± 1.4 e4, p < 0.05), and neutrophil infiltration (6.42 ± 0.25 vs. 3.94 ± 0.29, p < 0.05). While mice lacking Adora2b in AECs were no longer protected by dipyridamole. We also identified occludin and focal adhesion kinase as downstream targets of ADORA2B, thus providing a novel mechanism for adenosine-mediated barrier protection. Similarly, we also observed similar enhanced ADORA2B (3.33 ± 0.67 to 16.12 ± 5.89, p < 0.05) and decreased occludin (81.2 ± 0.3 to 13.3 ± 0.4, p < 0.05) levels in human Acute respiratory distress syndrome lungs.Conclusion: We have highlighted a role of dipyridamole and adenosine signaling in preventing or treating ALI and identified Ent2 and Adora2b as key mediators in important for the resolution of ALI.

The Hypoxia-Adenosine Link during Myocardial Ischemia—Reperfusion Injury

“…Due to its function as an ENT inhibitor, dipyridamole treatment has been used clinically for many decades during pharmacologic stress echocardiography, where it increases coronary adenosine levels, and can unmask coronary artery stenosis [ 148 , 149 ]. Importantly, for the hypoxia-adenosine link during myocardial injury, previous studies have shown that HIF functions to repress both ENT1 and ENT2 during conditions of hypoxia or inflammation, and thereby functions to increase extracellular adenosine levels [ 17 , 19 , 20 , 21 , 108 , 150 ]. Interestingly, mice with global deletion of Ent1 experience elevated plasma levels of adenosine, which can contribute to cardioprotection [ 151 ].…”

“…Stabilization of HIF activates a transcriptional program leading to the increased production of the extracellular signaling molecule adenosine [ 11 , 12 ]. These transcriptional changes include enhanced extracellular production of adenosine from precursor nucleotides [ 13 , 14 , 15 , 16 ], increased levels and signaling events through extracellular adenosine receptors [ 17 , 18 ], attenuated uptake of extracellular adenosine via adenosine transporters [ 19 , 20 , 21 ], and the attenuated metabolism of adenosine [ 22 ]. Adenosine exerts its cardioprotective effects during ischemia-reperfusion injury through multiple actions, such as vasodilation to increase blood flow and oxygen, decreasing myocardial oxygen consumption, preserving endothelial cell function, and attenuating inflammation.…”

Vitamin A-treated natural killer cells reduce interferon-gamma production and support regulatory T cell differentiation

“…Although blocking the adenosine receptor did not indicate its role in our co-culture system, this axis might support pleiotropic immunoregulatory functions of NK cells in vitamin A-enriched tissues. In the liver, where NK cells comprise the majority of lymphocytes (Gao, Jeong, & Tian, 2008), the adenosine pathway plays an important roles in regulating inflammation and immune responses in liver disease (W. Wang et al, 2021; Zimmerman et al, 2013). Liver tissue injury leads to concomitant release of intracellular ATP from damaged and necrotic cells and to the depletion of vitamin A storage from hepatic stellate cells (HSCs) (Blomhoff et al, 1990).…”

Probiotic-derived ecto-5'-nucleotidase produces anti-inflammatory adenosine metabolites in Treg-deficient scurfy mice

“…Samples were then neutralized with KHCO 3 /KOH, followed by acidi cation with NH 4 PO 4 and phosphoric acid. The supernatant was collected after centrifugation and analyzed by reverse-phase HPLC as described previously [50][51][52][53]. Representative adenosine, inosine, and AMP peaks were identi ed and quanti ed using standard external curves.…”