Crucial Role for Ecto-5′-Nucleotidase (CD73) in Vascular Leakage during Hypoxia

Extracellular adenosine has been widely implicated in adaptive responses to hypoxia. The generation of extracellular adenosine involves phosphohydrolysis of adenine nucleotide intermediates, and is regulated by the terminal enzymatic step catalyzed by ecto-5′-nucleotidase (CD73). Guided by previous work indicating that hypoxia-induced vascular leakage is, at least in part, controlled by adenosine, we generated mice with a targeted disruption of the third coding exon of Cd73 to test the hypothesis that CD73-generated extracellular adenosine functions in an innate protective pathway for hypoxia-induced vascular leakage. Cd73 (−/−) mice bred and gained weight normally, and appeared to have an intact immune system. However, vascular leakage was significantly increased in multiple organs, and after subjection to normobaric hypoxia (8% O(2)), Cd73 (−/−) mice manifested fulminant vascular leakage, particularly prevalent in the lung. Histological examination of lungs from hypoxic Cd73 (−/−) mice revealed perivascular interstitial edema associated with inflammatory infiltrates surrounding larger pulmonary vessels. Vascular leakage secondary to hypoxia was reversed in part by adenosine receptor agonists or reconstitution with soluble 5′-nucleotidase. Together, our studies identify CD73 as a critical mediator of vascular leakage in vivo

Coordinated Adenine Nucleotide Phosphohydrolysis and Nucleoside Signaling in Posthypoxic Endothelium: Role of Ectonucleotidases and Adenosine A2B Receptors

Limited oxygen delivery to tissues (hypoxia) is common in a variety of disease states. A number of parallels exist between hypoxia and acute inflammation, including the observation that both influence vascular permeability. As such, we compared the functional influence of activated polymorphonuclear leukocytes (PMN) on normoxic and posthypoxic endothelial cells. Initial studies indicated that activated PMN preferentially promote endothelial barrier function in posthypoxic endothelial cells (>60% increase over normoxia). Extension of these findings identified at least one soluble mediator as extracellular adenosine triphosphate (ATP). Subsequent studies revealed that ATP is coordinately hydrolyzed to adenosine at the endothelial cell surface by hypoxia-induced CD39 and CD73 (>20-and >12-fold increase in mRNA, respectively). Studies in vitro and in cd39-null mice identified these surface ecto-enzymes as critical control points for posthypoxia-associated protection of vascular permeability. Furthermore, insight gained through microarray analysis revealed that the adenosine A2B receptor (AdoRA2B) is selectively up-regulated by hypoxia (>5-fold increase in mRNA), and that AdoRA2B antagonists effectively neutralize ATP-mediated changes in posthypoxic endothelial permeability. Taken together, these results demonstrate transcription coordination of adenine nucleotide and nucleoside signaling at the vascular interface during hypoxia

Antiinflammatory adaptation to hypoxia through adenosine-mediated cullin-1 deneddylation

A major adaptive pathway for hypoxia is hypoxic preconditioning (HPC), a form of endogenous protection that renders cells tolerant to severe challenges of hypoxia. We sought to define the antiinflammatory properties of HPC. cDNA microarray analysis of lung tissue from mice subjected to hypoxia or HPC identified a cluster of NF-κB–regulated genes whose expression is attenuated by HPC. Studies using an NF-κB luciferase reporter assay confirmed a significant suppression of NF-κB activation during HPC. HPC-elicited activity was conferrable, as a soluble supernatant from HPC-treated cells, and the active fraction was purified and identified as adenosine (Ado). Guided by recent studies demonstrating bacterial inhibition of NF-κB through cullin-1 (Cul-1) deneddylation, we found a dose-dependent deneddylation of Cul-1 by Ado receptor stimulation predominantly mediated by the Ado A2B receptor subtype. Further, siRNA-mediated repression of CSN5, a subunit of the COP9 signalosome responsible for deneddylation of Cul-1, partially reversed HPC-mediated inhibition of NF-κB. Cul-1 deneddylation was evident in a murine model of HPC and lost in animals lacking extracellular Ado (Cd73(–/–) mice). Taken together, these results demonstrate that HPC induces extracellular accumulation of Ado and suppresses NF-κB activity through deneddylation of Cul-1. These results define a molecular regulatory pathway by which Ado provides potent antiinflammatory properties