Extracellular Adenosine Concentrations Research Articles

Mass Spectrometry Analysis of Neurotransmitter Shifting during Neurogenesis and Neurodegeneration of PC12 Cells.

Parkinson's disease (PD) affects movement; however, most patients with PD also develop nonmotor symptoms, such as hyposmia, sleep disorder, and depression. Dopamine levels in the brain have a critical influence on movement control, but other neurotransmitters are also involved in the progression of PD. This study analyzed the fluctuation of neurotransmitters in PC12 cells during neurogenesis and neurodegeneration by performing mass spectrometry. We found that the dopaminergic metabolism pathway of PC12 cells developed vigorously during the neuron differentiation process and that the neurotransmitters were metabolized into 3-methoxytyramine, which was released from the cells. The regulation of the intracellular and extracellular concentrations of adenosine indicated that adenine nucleotides were actively utilized in neural differentiation. Moreover, we exposed the differentiated PC12 cells to rotenone, which is a suitable material for modeling PD. The cells exposed to rotenone in the early stage of differentiation exhibited stimulated serotoninergic metabolism, and the contents of the serotoninergic neurotransmitters returned to their normal levels in the late stage of differentiation. Interestingly, the nondifferentiated cells can resist the toxicant rotenone and produce normal dopaminergic metabolites. However, when differentiated neuron cells were exposed to rotenone, they were seriously damaged, leading to a failure to produce dopaminergic neurotransmitters. In the low-dosage damage process, the amino acids that functioned as dopaminergic pathway precursors could not be absorbed by the cells, and dopamine and L-dopa were secreted and unable to be reuptaken to trigger the cell damage.

Extracellular adenosine oppositely regulates the purinome machinery in glioblastoma and mesenchymal stem cells.

Glioblastoma (GB) is a lethal brain tumor that rapidly adapts to the dynamic changes of the tumor microenvironment (TME). Mesenchymal stem/stromal cells (MSCs) are one of the stromal components of the TME playing multiple roles in tumor progression. GB progression is prompted by the immunosuppressive microenvironment characterized by high concentrations of the nucleoside adenosine (ADO). ADO acts as a signaling molecule through adenosine receptors (ARs) but also as a genetic and metabolic regulator. Herein, the effects of high extracellular ADO concentrations were investigated in a human glioblastoma cellular model (U343MG) and MSCs. The modulation of the purinome machinery, i.e., the ADO production (CD39, CD73, and adenosine kinase [ADK]), transport (equilibrative nucleoside transporters 1 (ENT1) and 2 (ENT2)), and degradation (adenosine deaminase [ADA]) were investigated in both cell lines to evaluate if ADO could affect its cell management in a positive or negative feed-back loop. Results evidenced a different behavior of GB and MSC cells upon exposure to high extracellular ADO levels: U343MG were less sensitive to the ADO concentration and only a slight increase in ADK and ENT1 was evidenced. Conversely, in MSCs, the high extracellular ADO levels reduced the ADK, ENT1, and ENT2 expression, which further sustained the increase of extracellular ADO. Of note, MSCs primed with the GB-conditioned medium or co-cultured with U343MG cells were not affected by the increase of extracellular ADO. These results evidenced how long exposure to ADO could produce different effects on cancer cells with respect to MSCs, revealing a negative feedback loop that can support the GB immunosuppressive microenvironment. These results improve the knowledge of the ADO role in the maintenance of TME, which should be considered in the development of therapeutic strategies targeting adenosine pathways as well as cell-based strategies using MSCs.

Translational Experimental Basis of Indirect Adenosine Receptor Agonist Stimulation for Bone Regeneration: A Review.

Bone regeneration remains a significant clinical challenge, often necessitating surgical approaches when healing bone defects and fracture nonunions. Within this context, the modulation of adenosine signaling pathways has emerged as a promising therapeutic option, encouraging osteoblast activation and tempering osteoclast differentiation. A literature review of the PubMed database with relevant keywords was conducted. The search criteria involved in vitro or in vivo models, with clear methodological descriptions. Only studies that included the use of indirect adenosine agonists, looking at the effects of bone regeneration, were considered relevant according to the eligibility criteria. A total of 29 articles were identified which met the inclusion and exclusion criteria, and they were reviewed to highlight the preclinical translation of adenosine agonists. While preclinical studies demonstrate the therapeutic potential of adenosine signaling in bone regeneration, its clinical application remains unrealized, underscoring the need for further clinical trials. To date, only large, preclinical animal models using indirect adenosine agonists have been successful in stimulating bone regeneration. The adenosine receptors (A1, A2A, A2B, and A3) stimulate various pathways, inducing different cellular responses. Specifically, indirect adenosine agonists act to increase the extracellular concentration of adenosine, subsequently agonizing the respective adenosine receptors. The agonism of each receptor is dependent on its expression on the cell surface, the extracellular concentration of adenosine, and its affinity for adenosine. This comprehensive review analyzed the multitude of indirect agonists currently being studied preclinically for bone regeneration, discussing the mechanisms of each agonist, their cellular responses in vitro, and their effects on bone formation in vivo.

Astrocytes in preoptic area regulate acute nociception-induced hypothermia through adenosine receptors.

The preoptic area (POA) of the hypothalamus, crucial in thermoregulation, has long been implicated in the pain process. However, whether nociceptive stimulation affects body temperature and its mechanism remains poorly studied. We used capsaicin, formalin, and surgery to induce acute nociceptive stimulation and monitored rectal temperature. Optical fiber recording, chemical genetics, confocal imaging, and pharmacology assays were employed to confirm the role and interaction of POA astrocytes and extracellular adenosine. Immunofluorescence was utilized for further validation. Acute nociception could activate POA astrocytes and induce a decrease in body temperature. Manipulation of astrocytes allowed bidirectional control of body temperature. Furthermore, acute nociception and astrocyte activation led to increased extracellular adenosine concentration within the POA. Activation of adenosine A1 or A2A receptors contributed to decreased body temperature, while inhibition of these receptors mitigated the thermo-lowering effect of astrocytes. Our results elucidate the interplay between acute nociception and thermoregulation, specifically highlighting POA astrocyte activation. This enriches our understanding of physiological responses to painful stimuli and contributes to the analysis of the anatomical basis involved in the process.

The Critical Role of Equilibrative Nucleoside Transporter-2 in Modulating Cerebral Damage and Vascular Dysfunction in Mice with Brain Ischemia-Reperfusion.

Cerebral vascular protection is critical for stroke treatment. Adenosine modulates vascular flow and exhibits neuroprotective effects, in which brain extracellular concentration of adenosine is dramatically increased during ischemic events and ischemia-reperfusion. Since the equilibrative nucleoside transporter-2 (Ent2) is important in regulating brain adenosine homeostasis, the present study aimed to investigate the role of Ent2 in mice with cerebral ischemia-reperfusion. Cerebral ischemia-reperfusion injury was examined in mice with transient middle cerebral artery occlusion (tMCAO) for 90minutes, followed by 24-hour reperfusion. Infarct volume, brain edema, neuroinflammation, microvascular structure, regional cerebral blood flow (rCBF), cerebral metabolic rate of oxygen (CMRO2), and the production of reactive oxygen species (ROS) were examined following the reperfusion. Ent2 deletion reduced the infarct volume, brain edema, and neuroinflammation in mice with cerebral ischemia-reperfusion. tMCAO-induced disruption of brain microvessels was ameliorated in Ent2-/- mice, with a reduced expression of matrix metalloproteinases-9 and aquaporin-4 proteins. Following the reperfusion, the rCBF of the wild-type (WT) mice was quickly restored to the baseline, whereas, in Ent2-/- mice, rCBF was slowly recovered initially, but was then higher than that in the WT mice at the later phase of reperfusion. The improved CMRO2 and reduced ROS level support the beneficial effects caused by the changes in the rCBF of Ent2-/- mice. Further studies showed that the protective effects of Ent2 deletion in mice with tMCAO involve adenosine receptor A2AR. Ent2 plays a critical role in modulating cerebral collateral circulation and ameliorating pathological events of brain ischemia and reperfusion injury.

Hypoxia-Evoked Spinal Adenosine Kinetics in Rats with Chronic Spinal Cord Injury

Therapeutic acute intermittent hypoxia (tAIH) is emerging as a simple and effective means of improving motor function after spinal cord injury (SCI) and ALS. For example, daily AIH applied one-week after C2 spinal hemisection (C2Hx) improves breathing in rats. Unfortunately, studies using the same protocol with chronic cSCI (>8 weeks) report less robust recovery, except when the rats are pretreated with an adenosine 2A receptor antagonist. Pro-inflammatory conditions, such as those experienced after SCI, increase CNS extracellular adenosine concentrations. Guided by increased understanding of adenosinergic signaling mechanisms constraining AIH-induced phrenic motor plasticity, we aimed to determine real-time in vivo dynamics of extracellular adenosine in response to hypoxia in the ventral cervical spinal cord in intact and spinally injured rats. We used enzyme-immobilized micro-biosensors for real time enzymatic detection of extracellular adenosine in the mid-cervical spinal cord of intact rats and rats with chronic C2Hx (>8 weeks post-injury) during 5-min 13% O2, 1-min 9% O2 and 1-min 9% O2 + 4% CO2. Extracellular adenosine exhibits rapid kinetics with a time-constant of seconds, regardless of condition. Rats with chronic C2Hx were classified post hoc into 2 groups: normotensive (systolic blood pressure > 120 mmHg) and hypotensive (systolic blood pressure < 120 mmHg). Normotensive C2Hx rats had baseline adenosine levels comparable to intact rats; hypoxia-induced increases in adenosine were slightly reduced versus intact rats. On the other hand, hypotensive C2Hx rats had elevated baseline adenosine levels and almost completely diminished hypoxia-evoked adenosine increases. Hypercapnic hypoxia had minimal impact on adenosine levels during exposures, but lowered adenosine following each hypercapnic hypoxic episode. These results suggest that changes in cardiovascular hemodynamics in chronic SCI rats can alter basal tissue adenosine levels, possibly undermining the therapeutic efficacy of tAIH in the condition of chronic SCI. Further, hypercapnic hypoxia appears to minimize inhibitory effects of adenosine on tAIH-induced phrenic/diaphragm motor plasticity. Supported by: NIH HL147554, HL148030 & OT2OD023854 and the University of Florida McKnight Brain Institute This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Sensory Neurons, PIEZO Channels and PAC1 Receptors Regulate the Mechanosensitive Release of Soluble Ectonucleotidases in the Murine Urinary Bladder Lamina Propria.

The urinary bladder requires adequate concentrations of extracellular adenosine 5'-triphosphate (ATP) and other purines at receptor sites to function properly. Sequential dephosphorylation of ATP to ADP, AMP and adenosine (ADO) by membrane-bound and soluble ectonucleotidases (s-ENTDs) is essential for achieving suitable extracellular levels of purine mediators. S-ENTDs, in particular, are released in the bladder suburothelium/lamina propria (LP) in a mechanosensitive manner. Using 1,N6-etheno-ATP (eATP) as substrate and sensitive HPLC-FLD methodology, we evaluated the degradation of eATP to eADP, eAMP and eADO in solutions that were in contact with the LP of ex vivo mouse detrusor-free bladders during filling prior to substrate addition. The inhibition of neural activity with tetrodotoxin and ω-conotoxin GVIA, of PIEZO channels with GsMTx4 and D-GsMTx4 and of the pituitary adenylate cyclase-activating polypeptide type I receptor (PAC1) with PACAP6-38 all increased the distention-induced but not spontaneous release of s-ENTDs in LP. It is conceivable, therefore, that the activation of these mechanisms in response to distention restricts the further release of s-ENTDs and prevents excessive hydrolysis of ATP. Together, these data suggest that afferent neurons, PIEZO channels, PAC1 receptors and s-ENTDs form a system that operates a highly regulated homeostatic mechanism to maintain proper extracellular purine concentrations in the LP and ensure normal bladder excitability during bladder filling.

Leveraging the ATP-P2X7 receptor signalling axis to alleviate traumatic CNS damage and related complications.

The P2X7 receptor is an exceptional member of the P2X purinergic receptor family, with its activation requiring high concentrations of extracellularadenosine 5'-triphosphate(ATP) that are often associated with tissue damage and inflammation. In the central nervous system (CNS), it is highly expressed in glial cells, particularly in microglia. In this review, we discuss the role and mechanisms of the P2X7 receptor in mediating neuroinflammation and other pathogenic events in a variety of traumatic CNS damage conditions, which lead to loss of neurological and cognitive functions. We raise the perspective on the steady progress in developing CNS-penetrant P2X7 receptor-specific antagonists that leverage the ATP-P2X7 receptor signaling axis as a potential therapeutic strategy to alleviate traumatic CNS damage and related complications.

Effects of adenosine and dipyridamole on serum levels of some biochemical markers in rabbits: Running title: Biochemical effects of adenosine and dipyridamole

Adenosine is a nucleoside which occurs naturally in a diverse forms in all cells of the body and in most biological fluids. Under basal conditions, the extracellular adenosine concentration is maintained within certain limits. Dipyridamol inhibits adenosine reuptake by erythrocytes, endothelial cells and platelet increasing plasma levels of adenosine Aims: study the effects of adenosine and dipyridamole on serum levels of serum urea, creatinine, alkaline phosphatase(ALP), lactate dehydrogenase (LDH), Aspartate Aminotransferase (GOT), and Alanine Aminotransferase (GPT). Material and methods: Thirty-five male rabbits were included in the study. The animals were divided into 3 groups: Group one(5 animals): injected intraperitoneal (i.p) with 2 ml of distilled water/day (control group). Group two (15 animals): were treated by i.p injection of adenosine, they were divided into 3 sub groups (5 animals) according to adenosine dose:1 mg/kg, 2mg/kg and 4 mg/kg.Group 3 (15 animals): were treated by dipyridamole orally, they were divided into 3 sub-groups (5 animals) according to dipyridamole dose:4 mg/kg, 8 mg/kg and 12 mg/kg. Result: Significant differences among 3 groups were found in blood urea, LDH, GOT levels, and in GPT levels. While statistical analysis of serum levels of S. creatinine and ALP showed no significant differences among 3 study groups. Conclusion: both adenosine, dipyridamole cause Significant differences among 3 groups were found in blood urea, LDH, GOT levels, and in GPT levels in rabbit

EXTH-18. TARGETING THE ADENOSINERGIC IMMUNE SUPPRESSION PATHWAY IN HIGH GRADE GLIOMA SYNERGIZES WITH INNATE IMMUNE CHECKPOINT BLOCKADE

Abstract Immune response in the tumor microenvironment is modulated by the conversion of eATP to AMP to adenosine via the ecto-enzymes CD39 and CD73. Overexpression of CD73 in tumors leads to an increase in extracellular adenosine concentration and decrease in eATP, resulting in immune suppression. In this study, CD73 knockouts were created in adult and pediatric glioma cell lines (DIPG17, T3691, BT245) and used to quantify the effects of CD73 knockout on macrophage phagocytosis in the presence of extracellular AMP. In cell line DIPG17, CD73 knockout significantly increased phagocytosis with and without external AMP compared to the WT. In the WT condition, addition of AMP significantly reduced phagocytosis, while this decrease was not significant in the CD73-KO. The addition of anti-CD47 antibody significantly increased phagocytosis with external AMP in the WT condition. Similar results were obtained with cell line T3691. However, CD73 knockout combined with anti-CD47 treatment and external AMP showed significantly greater phagocytosis than the WT condition. This suggests that a combination of anti-CD73 and anti-CD47 treatment may be more effective in the tumor microenvironment. Lastly, in cell line BT245, CD73 knockout significantly increased phagocytosis as seen in the other cell lines. However, no significant difference was observed between WT and CD73-KO with extracellular AMP or anti-CD47. In vivo studies were conducted in orthotopic xenograft mouse models with DIPG17 CD73 knockout cells. Combination treatment with anti-CD47 antibody significantly decreased tumor burden and prolonged survival in the CD73-KO tumors compared to anti-CD47 treated DIPG17-WT tumors. We conclude that the CD73 adenosinergic pathway and the CD47-SIRPα pathway may present a target for immunotherapy in pediatric and adult gliomas.

Intermittent Hypoxia Differentially Regulates Adenosine Receptors in Phrenic Motor Neurons with Spinal Cord Injury

Cervical spinal cord injury (cSCI) impairs neural drive to the respiratory muscles, causing life- threatening complications such as respiratory insufficiency and diminished airway protection. Repetitive “low dose” acute intermittent hypoxia (AIH) is a promising strategy to restore motor function in people with chronic SCI. Conversely, “high dose” chronic intermittent hypoxia (CIH; ∼8 h/night), such as experienced during sleep apnea, causes pathology. Sleep apnea, spinal ischemia, hypoxia and neuroinflammation associated with cSCI increase extracellular adenosine concentrations and activate spinal adenosine receptors which in turn constrains the functional benefits of therapeutic AIH. Adenosine 1 and 2A receptors (A1, A2A) compete to determine net cAMP signaling and likely the tAIH efficacy with chronic cSCI. Since cSCI and intermittent hypoxia may regulate adenosine receptor expression in phrenic motor neurons, we tested the hypotheses that: 1) daily AIH (28 days) downregulates A2A and upregulates A1 receptor expression; 2) CIH (28 days) upregulates A2A and downregulates A1 receptor expression; and 3) cSCI alters the impact of CIH on adenosine receptor expression. Daily AIH had no effect on either adenosine receptor in intact or injured rats. However, CIH exerted complex effects depending on injury status. Whereas CIH increased A1 receptor expression in intact (not injured) rats, it increased A2A receptor expression in spinally injured (not intact) rats. The differential impact of CIH reinforces the concept that the injured spinal cord behaves in distinct ways from intact spinal cords, and that these differences should be considered in the design of experiments and/or new treatments for chronic cSCI.

The impaired distribution of adenosine deaminase isoenzymes in multiple sclerosis plasma and cerebrospinal fluid.

BackgroundAdenosine deaminase (ADA) via two isoenzymes, ADA1 and ADA2, regulates intra- and extracellular adenosine concentrations by converting it to inosine. In the central nervous system (CNS), adenosine modulates the processes of neuroinflammation and demyelination that together play a critical role in the pathophysiology of multiple sclerosis (MS). Except for their catalytic activities, ADA isoenzymes display extra-enzymatic properties acting as an adhesion molecule or a growth factor.AimsThis study aimed to explore the distribution and activity of ADA1 and ADA2 in the plasma and the CSF of MS patients as well as in the human brain microvascular endothelial cells (HBMEC), human brain vascular pericytes and human astrocytes.Methods and resultsThe enzyme assay following reverse phase-high performance liquid chromatography (HPLC) analysis was used to detect the ADA1 and ADA2 activities and revealed an increased ratio of ADA1 to ADA2 in both the plasma and the CSF of MS patients. Plasma ADA1 activity was significantly induced in MS, while ADA2 was decreased in the CSF, but significance was not reached. The brain astrocytes, pericytes and endothelial cells revealed on their surface the activity of ADA1, with its basal level being five times higher in the endothelial cells than in the astrocytes or the pericytes. In turn, ADA2 activity was only observed in pericytes and endothelial cells. Stimulation of the cells with pro-inflammatory cytokines TNFα/IL17 for 18 h decreased intracellular nucleotide levels measured by HPLC only in pericytes. The treatment with TNFα/IL17 did not modulate cell-surface ATP and AMP hydrolysis nor adenosine deamination in pericytes or astrocytes. Whereas in endothelial cells it downregulated AMP hydrolysis and ADA2 activity and upregulated the ADA1, which reflects the ADA isoenzyme pattern observed here in the CSF of MS patients.ConclusionIn this study, we determined the impaired distribution of both ADA isoenzymes in the plasma and the CSF of patients with MS. The increased ADA1 to ADA2 ratio in the CSF and plasma may translate to unfavorable phenotype that triggers ADA1-mediated pro-inflammatory mechanisms and decreases ADA2-dependent neuroprotective and growth-promoting effects in MS.

Abstract 3499: M1069 as dual A2A/A2B adenosine receptor antagonist counteracts immune-suppressive mechanisms of adenosine and reduces tumor growth in vivo

Abstract Under physiological conditions, the extracellular concentrations of adenosine are low, however, levels dramatically increase under metabolically stressful conditions, including inflammation and cancer. The regulatory functions of adenosine are mediated through four members of the adenosine receptor family: A1, A2A, A2B, and A3. While A2A was considered the major contributor to adenosine-mediated suppression of T cell, natural killer cell, and myeloid cell functions, A2B has also recently emerged as a potential modulator of these processes. Both A2A and A2B signal through the same Gs-mediated activation of adenylate cyclase, which can allow the lower affinity A2B receptor to compensate for the inhibition of A2A in an adenosine-rich tumor microenvironment (TME). A2B receptors are also reported to support tumor growth independent of A2A through the Gq subunit of G-protein-coupled receptor-mediated production of vascular endothelial growth factor (VEGF) by myeloid and tumor cells. Therefore, simultaneous targeting of both the A2A and A2B receptors may provide a higher potential for cancer immunotherapy in an adenosine-rich TME, where A2B can act in compensatory or complementary means to A2A. M1069 is a small-molecule, dual antagonist of the A2A and A2B adenosine receptors with a selectivity of >100 fold against the A1 and A3 receptors. In assays with primary human T cells, M1069 caused a dose-dependent suppression of 5′-N-ethylcarboxamide adenosine (stable analog of adenosine)-stimulated cyclic adenosine monophosphate (cAMP) and phosphorylated cAMP- response element binding protein (pCREB) induction and rescue of interleukin (IL)-2 production (A2A readout). M1069 also suppressed VEGF production from human macrophages (A2B readout) in adenosine-rich settings. M1069 exhibited superior suppression of protumorigenic cytokine secretion, including CXCL1, CXCL5 and granulocyte-colony stimulating factor, and the rescue of IL12 secretion from adenosine-differentiated dendritic cells, as compared to an A2Aselective antagonist. In addition, in a one-way mixed lymphocyte reaction assay, adenosine-differentiated dendritic cells treated with M1069 demonstrated superior T cell activation compared to adenosine-differentiated dendritic cells treated with an A2A-selective antagonist. These findings were further corroborated with the results from in vivo studies in a murine CD73hi/adenosine-rich 4T1 syngeneic breast tumor model, in which M1069, but not an A2A-selective antagonist, reduced tumor growth as a monotherapy and enhanced anti-tumor activity with chemotherapeutic agents. In summary, M1069 is a potent, dual A2A/A2B adenosine receptor antagonist, which is expected to counteract immune-suppressive mechanisms in the presence of high concentrations of adenosine and enhance the anti-tumor activity of chemotherapies. Citation Format: Rinat Zaynagetdinov, Kai Schiemann, Kalyan Nallaparaju, Natalya Belousova, Armine Matevossian, Zhouxiang Chen, Giorgio Kradjian, Meghana Pandya, Nemisha Dawra, Eva-Maria Krauel, Elissaveta Petrova, Oliver Poeschke, David Fischer, Marc Lecomte, Andree Blaukat, Bayard Huck, Jacques Moisan. M1069 as dual A2A/A2B adenosine receptor antagonist counteracts immune-suppressive mechanisms of adenosine and reduces tumor growth in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3499.

IMMU-21. Targeting the adenosinergic immune suppression pathway in high grade glioma synergizes with innate immune checkpoint blockade

Abstract Immune response in the tumor microenvironment is modulated by the conversion of eATP to AMP to adenosine via the ecto-enzymes CD39 and CD73. Overexpression of CD73 in tumors leads to an increase in extracellular adenosine concentration and decrease in eATP, resulting in immune suppression. In this study, CD73 knockouts were created in adult and pediatric glioma cell lines (DIPG17, T3691, BT245) and used to quantify the effects of CD73 knockout on macrophage phagocytosis in the presence of extracellular AMP. In cell line DIPG17, CD73 knockout significantly increased phagocytosis with and without external AMP compared to the WT. In the WT condition, addition of AMP significantly reduced phagocytosis, while this decrease was not significant in the CD73-KO. The addition of anti-CD47 antibody significantly increased phagocytosis with external AMP in the WT condition. Similar results were obtained with cell line T3691. However, CD73 knockout combined with anti-CD47 treatment and external AMP showed significantly greater phagocytosis than the WT condition. This suggests that a combination of anti-CD73 and anti-CD47 treatment may be more effective in the tumor microenvironment. Lastly, in cell line BT245, CD73 knockout significantly increased phagocytosis as seen in the other cell lines. However, no significant difference was observed between WT and CD73-KO with extracellular AMP or anti-CD47. In vivo studies were conducted in orthotopic xenograft mouse models with DIPG17 CD73 knockout cells. Combination treatment with anti-CD47 antibody significantly decreased tumor burden and prolonged survival in the CD73-KO tumors compared to anti-CD47 treated DIPG13-WT tumors. We conclude that the CD73 adenosinergic pathway and the CD47-SIRPα pathway may present a target for immunotherapy in pediatric and adult gliomas.

Extracellular cAMP-Adenosine Pathway Signaling: A Potential Therapeutic Target in Chronic Inflammatory Airway Diseases.

Adenosine is a purine nucleoside that, via activation of distinct G protein-coupled receptors, modulates inflammation and immune responses. Under pathological conditions and in response to inflammatory stimuli, extracellular ATP is released from damaged cells and is metabolized to extracellular adenosine. However, studies over the past 30 years provide strong evidence for another source of extracellular adenosine, namely the “cAMP-adenosine pathway.” The cAMP-adenosine pathway is a biochemical mechanism mediated by ATP-binding cassette transporters that facilitate cAMP efflux and by specific ectoenzymes that convert cAMP to AMP (ecto-PDEs) and AMP to adenosine (ecto-nucleotidases such as CD73). Importantly, the cAMP-adenosine pathway is operative in many cell types, including those of the airways. In airways, β2-adrenoceptor agonists, which are used as bronchodilators for treatment of asthma and chronic respiratory diseases, stimulate cAMP efflux and thus trigger the extracellular cAMP-adenosine pathway leading to increased concentrations of extracellular adenosine in airways. In the airways, extracellular adenosine exerts pro-inflammatory effects and induces bronchoconstriction in patients with asthma and chronic obstructive pulmonary diseases. These considerations lead to the hypothesis that the cAMP-adenosine pathway attenuates the efficacy of β2-adrenoceptor agonists. Indeed, our recent findings support this view. In this mini-review, we will highlight the potential role of the extracellular cAMP-adenosine pathway in chronic respiratory inflammatory disorders, and we will explore how extracellular cAMP could interfere with the regulatory effects of intracellular cAMP on airway smooth muscle and innate immune cell function. Finally, we will discuss therapeutic possibilities targeting the extracellular cAMP-adenosine pathway for treatment of these respiratory diseases.

Inhibitors of ectonucleotidases have paradoxical effects on synaptic transmission in the mouse cortex.

Extracellular adenosine plays prominent roles in the brain in both physiological and pathological conditions. Adenosine can be generated following the degradation of extracellular nucleotides by various types of ectonucleotidases. Several ectonucleotidases are present in the brain parenchyma: ecto-nucleotide triphosphate diphosphohydrolases 1 and 3 (NTPDase 1 and 3), ecto-nucleotide pyrophosphatase/phosphodiesterase 1 (NPP 1), ecto-5'-nucleotidase (eN), and tissue non-specific alkaline phosphatase (TNAP, whose function in the brain has received little attention). Here we examined, in a living brain preparation, the role of these ectonucleotidases in generating extracellular adenosine. We recorded local field potentials evoked by electrical stimulation of the lateral olfactory tract in the mouse piriform cortex in vitro. Variations in adenosine level were evaluated by measuring changes in presynaptic inhibition generated by adenosine A1 receptors (A1Rs) activation. A1R-mediated presynaptic inhibition was present endogenously and was enhanced by bath-applied AMP and ATP. We hypothesized that inhibiting ectonucleotidases would reduce extracellular adenosine concentration, which would result in a weakening of presynaptic inhibition. However, inhibiting TNAP had no effect in controlling endogenous adenosine action and no effect on presynaptic inhibition induced by bath-applied AMP. Furthermore, contrary to our expectation, inhibiting TNAP reinforced, rather than reduced, presynaptic inhibition induced by bath-applied ATP. Similarly, inhibition of NTPDase 1 and 3, NPP1, and eN induced stronger, rather than weaker, presynaptic inhibition, both in endogenous condition and with bath-applied ATP and AMP. Consequently, attempts to suppress the functions of extracellular adenosine by blocking its extracellular synthesis in living brain tissue could have functional impacts opposite to those anticipated.

Preparation and study of 3D printed dipyridamole/β-tricalcium phosphate/ polyvinyl alcohol composite scaffolds in bone tissue engineering

Critical-sized bone defects caused by trauma or infection require timely and effective treatment to restore the shape and function of the damaged area as soon as possible. We have prepared β-tricalcium phosphate (β-TCP)/polyvinyl alcohol (PVA)/dipyridamole by composite scaffolds 3D printing technique to improve the osteoinductive potential of bone substitutes, where dipyridamole can increase the extracellular adenosine concentration by hindering cells to uptake it to promote osteogenesis. For the evaluation of these prepared scaffolds, the experimental design includes mechanical strength test, the porosity measurement, the microscopic morphology observation by SEM, the release profile of loaded drugs, the biocompatibility of scaffolds with osteogenic stem cells, and the intracellular alkaline phosphatase (ALP) assay to verify the ability in osteogenesis. The results have shown that the composite scaffolds' mechanical strength, porosity, and microscopic morphology do not change significantly before and after drug addition. The reaction of cells proves that the new drug-loaded scaffolds have good biocompatibility. Moreover, the higher ALP activity of the cells seeded on the drug-loaded group has confirmed the truth of active osteogenic activities. So, we conclude that β-TCP/PVA/dipyridamole composite scaffolds have brilliant potential in new bone formation as a suitable alternative.

Adenosine Signaling Perturbs Erythropoiesis and Promotes Myeloid Differentiation

BackgroundAdenosine is a major signaling nucleoside that activates cellular signaling pathways through a family of four different G protein-coupled adenosine receptors (ARs), A 1, A 2A, A 2B, and A 3. At steady state conditions, extracellular levels of adenosine remain low (10 to 200 nM) either through its rapid cellular uptake by specialized nucleoside transporters, mainly through the equilibrative nucleoside transporter 1 (ENT1), or its degradation by adenosine deaminases. However, the extracellular levels of adenosine can be rapidly elevated up to 100 μM in response to hypoxia, inflammation, or tissue injury. Under pathophysiological conditions, adenosine signaling is involved in modulating inflammation, fibrosis, and ischemic tissue injury. In sickle cell disease (SCD), adenosine signaling is enhanced and contributes to the pathophysiology of the disease. Despite the importance of adenosine signaling in regulating cell proliferation, and stem cell regeneration, as well as in red blood cell functions and adaptation to hypoxia, very little is known about its implication in hematopoiesis, and more specifically during erythropoiesis. Here, we aimed to investigate the effects of high extracellular adenosine on the erythroid commitment and differentiation of hematopoietic progenitors, and to decipher the implication of ARs in these processes.ResultsTo investigate the role of high extracellular adenosine in regulating erythroid commitment and differentiation of hematopoietic progenitors, we performed ex vivo erythropoiesis of healthy CD34 + cells in the presence or absence of increased extracellular adenosine concentrations ranging from 10 to 200 µM. Our results showed that adenosine decreases erythroid proliferation in a dose dependent manner. High adenosine levels (>50μM) inhibited the proliferation of erythroid precursors and increased apoptosis via a cell cycle arrest in G1. Accordingly, western blots revealed the accumulation of p53 and its downstream target p21, a well-known mediator of G1 cell-cycle arrest, in adenosine-treated cells.Moreover, adenosine treatment led to the persistence of a non-erythroid GPA neg subpopulation expressing myeloid markers (CD18, CD11a, CD13, CD33). May-Grünwald Giemsa staining of this subset revealed granular cells at different stages of differentiation. The culture of FACS-sorted CD36 + and CD36 - cells suggested that this adenosine-induced GPA neg population originates from the survival of CD36 - myeloid progenitors even in the presence of erythropoietin. Importantly, these effects were specific to adenosine as neither guanosine, uridine nor cytidine affected the proliferation and differentiation of erythroid precursors. Furthermore, we have recently shown that ENT1-mediated adenosine uptake is essential for optimal erythroid differentiation. Therefore, we suggested that elevated extracellular adenosine perturbs erythropoiesis via its signaling upon ARs activation. To confirm this hypothesis, we assessed the effect of ARs activation during erythropoiesis. Given that A 2B and A 3 are the only known ARs expressed in human hematopoietic progenitors and erythroid precursors, we used BAY60-6583 and CI-IB-MECA, two highly selective agonists for A 2B and A 3 receptors, respectively. Both BAY60-6583 and CI-IB-MECA increased apoptosis and decreased erythroblast maturation and enucleation, while only Cl-IB-MECA led to the upregulation of CD33 and CD11a myeloid markers and promoted the differentiation of a GPA neg myeloid subpopulation.ConclusionOverall, our results place adenosine signaling as a new player in hematopoiesis regulation. Adenosine signaling via A 3 perturbs erythropoiesis and promotes the survival and differentiation of myeloid progenitors even in an erythroid favoring environment. While the activation of A 2B hampers optimal erythropoiesis without impacting the myeloid differentiation. As both ineffective erythropoiesis and increased leucocyte counts are reported in SCD, and given the detrimental role of high adenosine levels in its pathophysiology, further studies are ongoing to address the impact of adenosine signaling on hematopoiesis in this disease. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Astrocyte Ca2+-evoked ATP release regulates myelinated axon excitability and conduction speed.

Print Page SummaryIntroductionAstrocytes support neuronal function throughout the central nervous system. In the grey matter they regulate synapse number during development, remove synaptically-released neurotransmitters to terminate their action and prevent excitotoxicity, control the extracellular potassium concentration to prevent hyperexcitability, regulate blood flow to ensure an adequate energy supply, provide lactate to neurons for energy, and respond to rises of intracellular calcium concentration ([Ca2+]i) by releasing ATP and other gliotransmitters that act on neuronal receptors to modulate information processing. However, their role is unclear in the white matter, which transmits information rapidly between grey matter areas using axons wrapped with capacitance-reducing myelin, although they have been suggested to regulate myelination during development and during normal function.RationaleRecently it has been suggested that learning and memory may reflect, not only changes of synaptic function in the grey matter, but also changes of white matter function. In particular, neural circuit function might be regulated by changes in the conduction speed of myelinated axons that result in an altered arrival time of action potentials at a distant neuron. These speed changes might be brought about by alterations of the properties of the passively conducting myelinated internodes, or of the intervening excitable nodes of Ranvier where the action potential is generated. We applied immunohistochemistry to assess how astrocytes interact with myelinated axons, used neuronal stimulation and light evoked calcium uncaging in astrocytes to evoke Ca2+-dependent release of gliotransmitters, and employed electrophysiology and pharmacology to characterise how astrocyte-released substances might affect the axon initial segment (AIS) and nodes of Ranvier of myelinated neurons. Measurements of conduction velocity and computer modelling allowed us to interpret the results.ResultsAstrocytes closely approach the axons of myelinated neurons in layer V of the cerebral cortex. Uncaging Ca2+ within astrocytes, or stimulating spike trains in neurons, evoked a rise of astrocyte [Ca2+]i that triggered the release of ATP-containing vesicles from these cells. This evoked an inward current in the AIS and nodes of Ranvier of the pyramidal neurons. Pharmacology showed that this was mediated by the activation of Gs-linked adenosine A2a receptors, implying that the released ATP was converted to adenosine by extracellular enzymes. The A2a receptors raise the intracellular concentration of cyclic AMP, which activates HCN channels mediating the inward hyperpolarization-activated current Ih, and thus depolarizes the cell. In the AIS the activation of A2a receptors alters excitability and hence action potential generation, while in the nodes of Ranvier it decreases the conduction speed of the action potential along the axon.ConclusionAs in the grey matter, astrocyte [Ca2+]i regulates the release of ATP into the extracellular space in the white matter. After conversion to adenosine, this regulates the excitability and conduction speed of myelinated axons. The changes in excitability at the AIS will lead to changes in the relationship between the synaptic input and action potential output of the cell. The altered conduction speed of the myelinated axon may change neural circuit function by changing the action potential arrival time at the cell’s output synapses, thus altering the integration of signals in postsynaptic neurons. Variations in astrocyte-derived adenosine level can occur between wake and sleep states, and the extracellular adenosine concentration rises during energy deprivation conditions. These changes of adenosine level could thus control white matter information flow and neural circuit function.Astrocytes regulate myelinated axon excitability and conduction speed.For cortical neurons with myelinated axons crossing the corpus callosum (left), astrocytes regulate axon initial segment (AIS) excitability and axonal conduction speed (middle). Increases of astrocyte [Ca2+]i release ATP which, after conversion to adenosine (Ado) extracellularly, activates A2a receptors that raise the intracellular cyclic AMP concentration, and thus generate an inward current via HCN2 channels in the AIS and nodes of Ranvier (right). Created with BioRender.com.In the brain’s grey matter, astrocytes regulate synapse properties, but their role is unclear for the white matter where myelinated axons rapidly transmit information between grey matter areas. We found that, in rodents, neuronal activity raised [Ca2+]i in astrocyte processes located near action potential generating sites in the axon initial segment (AIS) and nodes of Ranvier of myelinated axons. This released ATP which was converted extracellularly to adenosine and thus, via A2a receptors, activated HCN2-containing cation channels that regulate two aspects of myelinated axon function: excitability of the AIS and speed of action potential propagation. Variations in astrocyte-derived adenosine level, between wake and sleep states or during energy deprivation, could thus control white matter information flow and neural circuit function.

P2X Receptor-Dependent Modulation of Mast Cell and Glial Cell Activities in Neuroinflammation.

Localisation of mast cells (MCs) at the abluminal side of blood vessels in the brain favours their interaction with glial cells, neurons, and endothelial cells, resulting in the activation of these cells and the release of pro-inflammatory mediators. In turn, stimulation of glial cells, such as microglia, astrocytes, and oligodendrocytes may result in the modulation of MC activities. MCs, microglia, astrocytes, and oligodendrocytes all express P2X receptors (P2XRs) family members that are selectively engaged by ATP. As increased concentrations of extracellular adenosine 5′-triphosphate (ATP) are present in the brain in neuropathological conditions, P2XR activation in MCs and glial cells contributes to the control of their communication and amplification of the inflammatory response. In this review we discuss P2XR-mediated MC activation, its bi-directional effect on microglia, astrocytes and oligodendrocytes and role in neuroinflammation.