ALBERT

Description: Extracellular levels of adenosine increase during hypoxia. While acute increases in adenosine are important to counterbalance excessive inflammation or vascular leakage, chronically elevated adenosine levels may be toxic. Thus, we reasoned that clearance mechanisms might exist to offset deleterious influences of chronically elevated adenosine. Guided by microarray results revealing induction of endothelial adenosine deaminase (ADA) mRNA in hypoxia, we used in vitro and in vivo models of adenosine signaling, confirming induction of ADA protein and activity. Further studies in human endothelia revealed that ADA-complexing protein CD26 is coordinately induced by hypoxia, effectively localizing ADA activity at the endothelial cell surface. Moreover, ADA surface binding was effectively blocked with glycoprotein 120 (gp120) treatment, a protein known to specifically compete for ADA-CD26 binding. Functional studies of murine hypoxia revealed inhibition of ADA with deoxycoformycin (dCF) enhances protective responses mediated by adenosine (vascular leak and neutrophil accumulation). Analysis of plasma ADA activity in pediatric patients with chronic hypoxia undergoing cardiac surgery demonstrated a 4.1 ± 0.6-fold increase in plasma ADA activity compared with controls. Taken together, these results reveal induction of ADA as innate metabolic adaptation to chronically elevated adenosine levels during hypoxia. In contrast, during acute hypoxia associated with vascular leakage and excessive inflammation, ADA inhibition may serve as therapeutic strategy.

Description: Nucleotides and nucleosides—such as adenosine triphosphate (ATP) and adenosine—are famous for their intracellular roles as building blocks for the genetic code or cellular energy currencies. In contrast, their function in the extracellular space is different. Here, they are primarily known as signaling molecules via activation of purinergic receptors, classified as P1 receptors for adenosine or P2 receptors for ATP. Because extracellular ATP is rapidly converted to adenosine by ectonucleotidase, nucleotide-phosphohydrolysis is important for controlling the balance between P2 and P1 signaling. Gene-targeted mice for P1, P2 receptors, or ectonucleotidase exhibit only very mild phenotypic manifestations at baseline. However, they demonstrate alterations in disease susceptibilities when exposed to a variety of vascular or blood diseases. Examples of phenotypic manifestations include vascular barrier dysfunction, graft-vs-host disease, platelet activation, ischemia, and reperfusion injury or sickle cell disease. Many of these studies highlight that purinergic signaling events can be targeted therapeutically.

Description: 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.

Description: Extracellular adenosine has been implicated in vascular adaptation to hypoxia. Based on the observation that increases in intracellular adenosine can effectively elevate extracellular adenosine, we studied the contribution of adenosine kinase (AK, intracellular conversion of adenosine to adenosine monophosphate [AMP]) to vascular adenosine responses. Initial in vitro studies of ambient hypoxia revealed prominent repression of endothelial AK transcript (85% ± 2% reduction), protein, and function. Transcription factor binding assays and hypoxia inducible factor 1-α (HIF-1α) loss- and gain-of-function studies suggested a role for HIF-1α in transcriptional repression of AK. Moreover, repression of AK by ambient hypoxia was abolished in conditional HIF-1α mutant mice in vivo. Studies of endothelial barrier function revealed that inhibition or siRNA repression of AK is associated with enhanced adenosine-dependent barrier responses in vitro. Moreover, in vivo studies of vascular barrier function demonstrated that AK inhibition with 5′-iodotubericidin (1 mg/kg prior to hypoxia) significantly attenuated hypoxia-induced vascular leakage in multiple organs and reduced hypoxia-associated increases in lung water. Taken together, our data reveal a critical role of AK in modulating vascular adenosine responses and suggest pharmacologic inhibitors of AK in the treatment of conditions associated with hypoxia-induced vascular leakage (eg, sepsis or acute lung injury).

Description: Erythropoiesis is an extremely dynamic process finely regulated by cytokines, hormones, and growth factors at transcriptional and translational levels. Stress-induced erythropoiesis is defined as a stimulated basal erythropoiesis with expansion of the erythroid progenitor pool, associated with reticulocytosis and splenomegaly. Stress erythropoiesis is stimulated under the condition of insufficient oxygen availability such as high altitude, blood loss, infection, and anemia. Thus, stress erythropoiesis is an important stress adaptive response for survival. Although stress erythropoiesis has been long speculated to be linked with increased metabolic requirements, until recent two years with innovative metabolomics profiling and state of art isotopically labelled metabolic flux approaches, the filed has evolved and revealed that enhanced glucose and glutamine metabolism is essential for stress erythropoiesis. However, molecular basis underlying metabolic reprogramming to enhance glucose metabolism and subsequently stress erythropoiesis remains unclear. To address this question, we conducted both human and mouse studies. First, we found that plasma adenosine is rapidly induced and associated with stress erythropoiesis features including increased hematocrit (HCT), hemoglobin (Hb) mass and reticulocytes in healthy human volunteers at high altitude and in mice exposed to hypoxia mimicking high altitude. Follow-up mouse genetic studies showed that activation of adenosine signaling via erythroid ADORA2B promotes the survival and expansion of proerythroblasts both in spleen and bone marrow and in this way contributes to hypoxia-induced stress erythropoiesis independent of erythropoietin. Using unbiased high-throughput metabolic profiling, we identified that erythroid ADORA2B contributes to an overall hypoxia metabolic reprogramming with substantial increased glycolysis in proerythroblast progenitors in mice. Finally, using primary human CD34+ hematopoietic stem cells culture, we showed that adenosine analogue and ADORA2B agonist promote the survival and expansion of erythroid progenitors in a time and dose-dependent manner. Taken together, both human and mouse studies identify that adenosine ADORA2B is a previously unrecognized purinergic signaling underlying hypoxia-induced erythropoiesis by facilitating expansion and survival of proerythroblasts, and highlight that enhancing this pathway is a potential strategy to induce erythropoiesis. Disclosures No relevant conflicts of interest to declare.

Description: Using a nonbiased high throughput metabolomic screen, coupled with genetic and pharmacological approaches, recent studies demonstrated that excessive adenosine signaling through the A2B adenosine receptor triggers sickling by induction of 2,3-bisphosphoglycerate (2,3-BPG), an erythroid specific metabolite that induces O2 release from hemoglobin. Adenosine is a signaling nucleoside that elicits numerous physiological and pathological effects by engaging membrane receptors. Notably, equlibrative nucleoside transporters (ENTs) on erythrocytes have been long speculated to regulate extracellular adenosine concentrations under hypoxic conditions. Thus, we hypothesize that ENT is likely a key molecule responsible for elevated circulating adenosine levels and protects tissues from hypoxia induced injury. To test this hypothesis, we first conducted in vivo Carbon-14 labeled adenosine (C14-Ado) injection and in vitro functional C14-Ado uptake assays. We found that erythrocyte plays a key role in regulation of circulating adenosine. We then conducted western blot analysis to compare expression profiles of ENTs on erythrocyte. We found that ENT1 is the major ENT expressed on both mouse and human erythrocytes. Using genetic approach, we successfully generated an erythrocyte ENT1 knockout mouse model. Using this genetic model and pharmacological approach combined with in vivo C14-Ado injection and in vitro C14-Ado uptake assay, we demonstrated that ENT1 1) is the major adenosine transporter in erythrocyte and 2) erythrocyte is the major cell type involved in regulating circulating adenosine levels through ENT1’s function. Using erythrocyte ENT1 knockout mouse model, we found that, during acute hypoxia treatment, the loss of erythrocyte ENT1 can cause faster increase in circulating adenosine level, subsequently promoting 2,3-BPG production, triggering oxygen release, and protecting acute hypoxia-mediated tissue injury. Mechanistically, we demonstrated that hypoxia regulates ENT1 activity through adenosine-ADORA2B-PKA signaling pathway. Overall, our studies demonstrate that 1) ENT1 is a major adenosine transporter expressed by erythrocytes and erythrocytes are the major cell type responsible for regulating circulating adenosine. 2) Hypoxia regulates ENT1 activity through adenosine-ADORA2B-PKA signaling pathway. 3) Inhibition or deletion of erythrocyte ENT1 results in enhanced adenosine-mediated 2,3-BPG induction and hemoglobin deoxygenation in RBCs when hypoxia is encountered. Thus, our findings suggest that erythrocyte ENT1 and ADORA2B are novel targets to prevent hypoxia-mediated tissue injury. Disclosures No relevant conflicts of interest to declare.

Description: To function effectively in O2 uptake, transport and delivery, erythrocytes rely on sophisticated regulation of hemoglobin (Hb)-O2 affinity by endogenous allosteric modulators. One of the best know allosteric modulators is 2,3-bisphophosphoglycerate (2,3-BPG), a metabolic byproduct of glycolysis synthesized primarily in erythrocytes for the purpose of regulating Hb-O2 affinity. Earlier studies demonstrated that erythrocyte 2,3-BPG levels are elevated at a high altitude and in patients with sickle cell disease (SCD). We have recently shown that adenosine, a molecule well known to be induced under hypoxic conditions, is significantly elevated in SCD and contributes to increased 2,3-BPG induction in SCD erythrocytes. However, whether adenosine contributes to increased erythrocyte 2,3-BPG production at high altitude is unknown. To address this question we recruited 24 individuals who normally lived at sea level, and placed them at high altitude for different time points. Similarly, blood was collected from 45 SCD patients and 50 controls (at sea level). Here we report that 1) Plasma adenosine and erythrocyte 2,3-BPG levels were significantly elevated in normal individuals at high altitude after 24 hours compared to sea level; 2) The elevations of both molecules are further enhanced at high altitude after 16 days; 3) Elevated circulating adenosine levels significantly correlated with increased erythrocyte 2,3-DPG levels in normal individuals at high altitude and in SCD patients. These results suggest that adenosine is a common factor responsible for elevated 2,3-BPG as a function in normal individuals and in SCD patients. To test this hypothesis we took pharmacologic and genetic approaches. We found that NECA, a stable adenosine analog, significantly increased 2,3-BPG production in cultured erythrocytes from normal and SCD mice and humans. Additional genetic and pharmacologic experiment showed that adenosine-induced 2,3-BPG production was mediated by ADORA2B receptor activation and downstream signaling by protein kinase A. To examine the in vivo physiological relevance of our findings we used adenosine deaminase (ADA)-deficient mice that accumulate high levels of circulating adenosine as a result of their enzyme deficiency. We found that elevated adenosine correlated to elevated erythrocyte 2,3-BPG and decreased Hb-O2 affinity. Experimental strategies to lower plasma adenosine or inhibit ADORA2B signaling in ADA-deficient mice resulted in reduced 2,3-BPG production and increased Hb-O2 affinity in erythrocytes. These studies provide in vivo evidence that elevated adenosine signaling via ADORA2B contributes to elevated of 2,3-BPG production and thereby triggers O2 release from erythrocytes. Taken together, our studies show that adenosine is a common factor regulating erythrocyte 2,3-BPG induction as a function of altitude and in patients with sickle cell disease. Our studies reveal numerous approaches to enhance oxygen release from oxyHb to physiologically or environmentally induced conditions of hypoxia. Disclosures: No relevant conflicts of interest to declare.

Description: Hypoxia is a well-documented inflammatory stimulus and results in tissue polymorphonuclear leukocyte (PMN) accumulation. Likewise, increased tissue adenosine levels are commonly associated with hypoxia, and given the anti-inflammatory properties of adenosine, we hypothesized that adenosine production via adenine nucleotide metabolism at the vascular surface triggers an endogenous anti-inflammatory response during hypoxia. Initial in vitro studies indicated that endogenously generated adenosine, through activation of PMN adenosine A2A and A2B receptors, functions as an antiadhesive signal for PMN binding to microvascular endothelia. Intravascular nucleotides released by inflammatory cells undergo phosphohydrolysis via hypoxia-induced CD39 ectoapyrase (CD39 converts adenosine triphosphate/adenosine diphosphate [ATP/ADP] to adenosine monophosphate [AMP]) and CD73 ecto-5′-nucleotidase (CD73 converts AMP to adenosine). Extensions of our in vitro findings using cd39- and cd73-null animals revealed that extracellular adenosine produced through adenine nucleotide metabolism during hypoxia is a potent anti-inflammatory signal for PMNs in vivo. These findings identify CD39 and CD73 as critical control points for endogenous adenosine generation and implicate this pathway as an innate mechanism to attenuate excessive tissue PMN accumulation. (Blood. 2004;104:3986-3992)

Description: To function effectively in O2 uptake, delivery and release, erythrocytes rely on sophisticated regulation of hemoglobin (Hb)-O2 affinity by allosteric modulators, especially 2,3-bisphophosphoglycerate (2,3-BPG). Earlier studies had shown that elevated erythrocyte 2,3-BPG level is correlated to increased availability of oxygen to tissues in high altitudes. However, nothing is known how 2,3-BPG is induced in high altitude. Recent study revealed that plasma adenosine is increased in patient and mice with sickle cell disease and elevated adenosine signaling via A2B receptor (ADORA2B) induces 2,3-BPG and thereby promotes deoxy-sickle hemoglobin and subsequent sickling. However, whether adenosine is induced and its effect in 2,3-BPG induction in high altitude are unknown. To address this question, we recruited 24 low-land volunteers and placed them in high altitude for different time points. Here we report that: 1) plasma adenosine, a molecule well known to be induced under hypoxia, is increased and its elevation is correlated to elevated erythrocyte 2,3-BPG levels and decreased Hb-O2 affinity; and 2) elevated plasma adenosine is correlated to increased circulating ecto-5’-nucleotidase (CD73), a key enzyme responsible for generation of extracellular adenosine. Similar to humans, increased circulating CD73 activity, plasma adenosine levels and erythrocyte 2,3-BPG and decreased Hb-O2 binding affinity were observed in wild type (WT) mice under hypoxia condition mimicking our human high altitude studies. Moreover, we found that hypoxia-induced increased plasma adenosine, erythrocyte 2,3-BPG and decreased Hb-O2 binding affinity were significantly attenuated in CD73-deficient mice (CD73-/-). As such, hypoxia-induced tissue injury and cell apoptosis were significantly elevated in CD73-/- mice compared to WT mice. Mechanistically, we provide both in vitro and in vivo genetic evidence that erythrocyte adenosine A2B receptor (ADORA2B) is essential for 2,3-BPG induction and subsequent O2 release. This finding led us to further discover that AMP-Activated Protein Kinase (AMPK) functions downstream of ADORA2B underlying adenosine-induced 2,3-BPG induction and O2 release by direct phosphorylation of 2,3-BPG mutase, a key enzyme for 2,3-BPG production. Finally, we demonstrated that treatment of metformin, a FDA approved potent AMPK agonist, induced erythrocyte 2,3-BPG levels and triggered O2 release and thereby prevented hypoxia-induced tissue injury in both CD73-/- mice and erythrocyte specific ADORA2B-deficient mice. Overall, our studies have revealed that erythrocyte ADORA2B-mediated AMPK activation is a novel mechanism underlying hypoxia-induced 2,3-BPG levels and thereby highlight its beneficial role to prevent hypoxia-induced tissue injury. Disclosures No relevant conflicts of interest to declare.

Description: Hypoxia is common to several inflammatory diseases, where multiple cell types release adenine-nucleotides (particularly adenosine triphosphate/adenosine diphosphate). Adenosine triphosphate/adenosine diphosphate is metabolized to adenosine through a 2-step enzymatic reaction initiated by CD39 (ectonucleoside-triphosphate-diphosphohydrolase-1). Thus, extracellular adenosine becomes available to regulate multiple inflammatory endpoints. Here, we hypothesized that hypoxia transcriptionally up-regulates CD39 expression. Initial studies revealed hypoxia-dependent increases in CD39 mRNA and immunoreactivity on endothelia. Examination of the human CD39 gene promoter identified a region important in hypoxia inducibility. Multiple levels of analysis, including site-directed mutagenesis, chromatin immunoprecipitation, and inhibition by antisense, revealed a critical role for transcription-factor Sp1 in hypoxia-induction of CD39. Using a combination of cd39−/− mice and Sp1 small interfering RNA in in vivo cardiac ischemia models revealed Sp1-mediated induction of cardiac CD39 during myocardial ischemia. In summary, these results identify a novel Sp1-dependent regulatory pathway for CD39 and indicate the likelihood that CD39 is central to protective responses to hypoxia/ischemia.