Background-Extracellular adenosine, generated from extracellular nucleotides via ectonucleotidases, binds to specific receptors and provides cardioprotection from ischemia and reperfusion. In the present study, we studied ecto-enzymatic ATP/ADP-phosphohydrolysis by select members of the ectonucleoside triphosphate diphosphohydrolase (E-NTPDase) family during myocardial ischemia. Methods and Results-As a first step, we used a murine model of myocardial ischemia and in situ preconditioning and performed pharmacological studies with polyoxometalate 1, a potent E-NTPDase inhibitor ( 01). Heightened levels of injury after myocardial ischemia and negligible preconditioning benefits in cd39Ϫ/Ϫ mice were corrected by infusion of the metabolic product (AMP) or apyrase. Moreover, apyrase treatment of wild-type mice resulted in 43Ϯ4.2% infarct size reduction (PϽ0.01). Conclusions-Taken together, these studies reveal E-NTPDase 1 in cardioprotection and suggest apyrase in the treatment of myocardial ischemia.

Complement activation plays an important role in local and remote tissue injury associated with gastrointestinal ischemia-reperfusion (GI/R). The role of the classical and lectin complement pathways in GI/R injury was evaluated using C1q-deficient (C1q KO), MBL-A/C-deficient (MBL-null), complement factor 2- and factor B-deficient (C2/fB KO), and wild-type (WT) mice. Gastrointestinal ischemia (20 min), followed by 3-h reperfusion, induced intestinal and lung injury in C1q KO and WT mice, but not in C2/fB KO mice. Addition of human C2 to C2/fB KO mice significantly restored GI/R injury, demonstrating that GI/R injury is mediated via the lectin and/or classical pathway. Tissue C3 deposition in C1q KO and WT, but not C2/fB KO, mice after GI/R demonstrated that complement was activated in C1q KO mice. GI/R significantly increased serum alanine aminotransferase, gastrointestinal barrier dysfunction, and neutrophil infiltration into the lung and gut in C1q KO and WT, but not C2/fB KO, mice. MBL-null mice displayed little gut injury after GI/R, but lung injury was present. Addition of recombinant human MBL (rhuMBL) to MBL-null mice significantly increased injury compared with MBL-null mice after GI/R and was reversed by anti-MBL mAb treatment. However, MBL-null mice were not protected from secondary lung injury after GI/R. These data demonstrate that C2 and MBL, but not C1q, are necessary for gut injury after GI/R. Lung injury in mice after GI/R is MBL and C1q independent, but C2 dependent, suggesting a potential role for ficolins in this model.

Gastrointestinal ischemia/reperfusion (IR) injury significantly contributes to the morbidity and mortality of critical illness. In this study, we hypothesized a protective role for extracellular adenosine signaling in intestinal IR injury. Initial profiling studies of mucosal scrapings following murine IR demonstrated selective induction of the A2B adenosine receptor (A2BAR) transcript. Moreover, genetargeted mice for the A2BAR showed more profound intestinal IR injury compared with controls. In contrast, A2AAR؊/؊ mice exhibited no differences in intestinal injury compared with littermate controls. In addition, selective inhibition of the A2BAR resulted in enhanced intestinal inflammation and injury during IR. Furthermore, A2BAR agonist treatment (BAY 60-6583) protected from intestinal injury, inflammation, and permeability dysfunction in wild-type mice, whereas the therapeutic effects of BAY 60-6583 were abolished following targeted A2BAR gene deletion. Taken together, these studies demonstrate the A2BAR as a novel therapeutic target for protection during gastrointestinal IR injury.

Human islet expansion in monolayer culture leads to loss of function and senescence. By maintaining the 3-D configuration of islets in fibrin gels, it is feasible to expand ␤-cells in response to hepatocyte growth factor (HGF) while preserving physiologic glucose responsiveness both in vitro and in vivo after transplantation into nude mice. Islets were cultured free floating with or without growth factors and nicotinamide and in fibrin gels with the same conditions. Proliferation was observed only in islets cultured in fibrin gels and the cocktail; total insulin increased by threefold, with a concomitant increase in ␤-cell mass by morphometry. Insulin release after glucose challenge was also preserved. Islets in fibrin gels gave rise in vivo to large grafts rich in insulin and glucagon, and grafts from free-floating islets were smaller with fewer endocrine cells. Circulating human C-peptide levels were higher than in the mice receiving free-floating islets. In summary, fibrin allows for HGF-mediated cell proliferation while preserving glucose responsiveness in an environment that preserves cell-cell contacts. Limited islet ex vivo expansion under these conditions may improve recipient-donor tissue ratios to equal the functional results of whole-organ transplants. Diabetes 51: 3435-3439, 2002

Intestinal ischemia/reperfusion injury (IR) is characterized by intermittent loss of perfusion to the gut, resulting in dramatic increases in morbidity and mortality. Based on previous studies indicating an anti-inflammatory role for hypoxia-inducible factor (HIF)-1–elicited enhancement of extracellular adenosine production via ecto-5′-nucleotidase (CD73) and signaling through the A2B adenosine receptor (A2BAR), we targeted HIF-1 during IR using pharmacological or genetic approaches. Initial studies with pharmacological HIF activation indicated attenuation of intestinal injury with dimethyloxallyl glycine (DMOG) treatment during murine IR. Although DMOG treatment was associated with induction of CD73 transcript and protein, DMOG protection was abolished in cd73−/− mice. Similarly, DMOG treatment enhanced A2BAR transcript and protein levels, whereas DMOG protection was abolished in A2BAR−/− mice. Finally, studies of mice with conditional HIF-1α deletion in intestinal epithelia or pharmacological inhibition of HIF-1 with 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin revealed enhanced tissue injury during IR. These studies indicated a tissue-protective role of HIF-dependent enhancement of intestinal adenosine generation and signaling during intestinal IR.