Indeed, by attenuating adenosine uptake, and concomitant increases of spontaneously formed extracellular adenosine levels, endogenously generated levels of extracellular

adenosine could become sufficient to trigger immunosuppressive adenosine receptor signaling events within the inflamed liver tissue microenvironments in vivo.[16, 17] In other words, the molecular concept is that pathophysiologically induced elevations of extracellular adenosine can provide better liver protection, if they are elevated over a longer time period, which can be achieved by adenosine uptake inhibitors, or by genetically targeting individual adenosine transporters. Therefore, we combined Crenolanib mw studies of ENT transcript and protein levels in biopsy samples obtained from patients undergoing liver transplantation with pharmacologic and genetic studies in a previously described model of murine partial hepatic ischemia and reperfusion.[8, 9, 18] These studies demonstrated a selective role for ENT1 in elevating hepatic adenosine levels and conveying liver protection from ischemia and reperfusion injury. Liver samples were obtained from patients undergoing orthotopic

liver transplantation (Supporting Table 1). Liver biopsies (I) were taken at the conclusion of cold ischemia time (CIT) during STAT inhibitor back table preparation of the cadaveric liver allograft (Fig. 1A). A second biopsy (R) was taken immediately prior to closure of the abdomen following

drain placement (Fig. 1A). Importantly, total reperfusion time is defined as the time from portal vein perfusion to abdominal closure at the conclusion of the procedure. All animal protocols were in accordance with the University of Colorado, Denver guidelines. this website Ent1 on the C57BL/6J strain were generated, validated, and characterized as described.[19] Ent2-deficient mice were obtained from Taconic Farms. Conditional hypoxia-inducible factor 1 alpha (HIF1α)loxP/loxP Albumin Cre+ mice were obtained by crossing HIF1αloxP/loxP with Albumin Cre+ mice (Jackson Laboratory). In all control experiments, age-, gender-, and weight-matched littermate controls were used. In an effort to avoid mesenteric congestion, a murine model of partial liver ischemia was employed using a hanging-weight system as described.[18] Ent1 and Ent2 transcript levels were measured by reverse-transcription polymerase chain reaction (RT-PCR) (iCycler, Bio-Rad Laboratories) as described.[20] In both human and mouse tissues Ent1 and Ent2 protein content was determined at different timepoints as described.[20] Liver preparation was performed as described in detail by Wei and colleagues.[21] IFN-γ, IL-6 (R&D Systems), and neutrophil sequestration was quantified according to the manufacturer’s instructions. Livers were removed and immediately snap-frozen after 45 minutes of liver ischemia without reperfusion. Adenosine was measured as described.