ALBERT

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: Abstract 3232 Sickle cell disease (SCD) is a severe genetic disorder with a high morbidity and mortality. Understanding the molecular basis responsible for sickling, a central pathogenesis of SCD, is critical for developing new therapeutic strategies. 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 many physiological 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 contributing to pathophysiology of SCD. To test this hypothesis, we first conducted western blot analysis to compare expression profiles of ENTs on normal and sickle erythrocytes. We found that ENT1 is the major ENT expressed on both mouse and human erythrocytes. Unexpectedly, ENT1 levels were significantly reduced in sickle erythrocytes compared to normal erythrocytes in both humans and mice, suggesting that ENT1 may contribute to increased adenosine levels seen in SCD. Next, we performed pharmacological studies to determine the exact role of ENT in normal and sickle erythrocytes. We found that treatment with dipyridamole or an ENT1 specific inhibitor (NBMPR) enhanced adenosine-induced elevation of 2,3-BPG in cultured mouse RBCs. Using Hemox Analyzer, we found that co-treatment of adenosine with either dipyridamole or NBMPR resulted in a further right shift of oxygen equilibrium curve (OEC) and further increase in P50 compared to the cells treated with adenosine alone. Similar to our pharmacological studies, we found that genetic deletion of ENT1 further enhanced adenosine-induced 2,3-BPG production in cultured erythrocytes, additional right shift of OEC and increased P50. Extending mouse studies to human, we demonstrate that co-treatment of adenosine with either dipyridamole or NBMPR further enhanced the adenosine alone-mediated 2,3-BPG induction in cultured erythrocytes isolated from normal individuals and SCD patients. Finally, we found that dipyridamole treatment significantly enhanced hypoxia-mediated 2,3-BPG production, right shift of OEC and substantial sickling in cultured erythrocytes isolated from SCD patients. Overall, our studies demonstrate that 1) ENT1 is a major transporter expressed by RBCs and that inhibition or deletion of ENT1 results in enhanced adenosine-mediated 2,3-BPG induction and deoxygenation in normal RBCs; 2) Decreased ENT1 expression in sickle erythrocytes is responsible for elevated circulating adenosine and thereby contributes to sickling by promoting 2,3-BPG production and triggering deoxygenation. Therefore, our findings reveal a previously unrecognized role of ENT1 in erythrocyte physiology, add a new insight to the pathophysiology of SCD and open up new therapies for the disease. 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: 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.