Adenosine A2A Receptor Expression Research Articles

Asiaticoside attenuates bleomycin-induced pulmonary fibrosis in A2aR−/− mice by promoting the BMP7/Smad1/5 signaling pathway

Idiopathic Pulmonary fibrosis(PF)is a chronic progressive disease, which is a lack of effective treatment,and the pathogenesis of IPF is not fully elucidated. Asiaticoside(AS) is isolated from Centella asiatica and has the effect of promoting scar healing and reducing scar formation. However,its possible role in idiopathic pulmonary fibrosis remains unclear. Adenosine A2A receptor (A2AR) is reported a protective factor in pulmonary fibrosis, and the bone morphogenetic protein 7 (BMP7) signaling pathway plays a crucial role in fibrosis in multiple organs. But the impact of A2AR on the BMP7 pathway has not yet been reported. Therefore, we hypothesized AS may promote the expression of A2AR, and then influence the BMP7/Smad1/5 pathway to alleviate pulmonary fibrosis. A2AR−/− mice and wild-type (WT) mice were administered bleomycin (BLM) by intratracheal injection. AS (50 mg/kg/d) was given daily for 28 days. AS reduced collagen deposition in lung tissue, interstitial lung inflammation. Furthermore, AS promoted A2AR expression and BMP7 pathway. Collectively, AS may attenuate BLM-induced pulmonary fibrosis by upregulating the BMP7 signaling pathway through A2AR.

High expression of immune checkpoints is associated with the TIL load, mutation rate and patient survival in colorectal cancer.

Adoptive cell therapy with the use of tumor-infiltrating lymphocytes (TILs) is a very promising immunotherapeutic approach for the treatment of patients with colorectal cancer (CRC). However, within the tumor microenvironment, co-inhibitory immune checkpoints can inactivate TILs. The aim of the present study was to examine the association between the TIL load, the mutation rate and the clinical outcome in the immune landscape of patients with CRC. RNA-seq and whole exome seq data of 453 colon adenocarcinomas (COAD) and rectal adenocarcinomas (READ), along with the TIL load and clinicopathological information of each patient, were extracted from the TCGA GDC Data Portal and analyzed computationally. The expression of immune checkpoint molecules was compared between colon cancer and normal tissue. A total of 9 immune-related gene signatures were investigated in CRC. Spearman's correlation analysis was performed to examine the correlation between the TIL load with the expression of each immune checkpoint molecule. Indoleamine 2,3-dioxygenase 1 (IDO1) was found to be significantly overexpressed in CRC, whereas V-domain Ig suppressor of T cell activation (VISTA) and lymphocyte activating 3 (LAG3) were markedly downregulated. A high expression of cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), IDO1, programmed cell death 1 (PD-1) and T-cell immunoreceptor with Ig and ITIM domains (TIGIT), tended to be associated with a better overall survival of the patients. In COAD, the TIL load positively correlated with the expression of adenosine A2A receptor (ADORA2A), CTLA-4, hepatitis A virus cellular receptor 2 (HAVCR2), lymphocyte activating 3 (LAG3), programmed death-ligand PD-L1, PD-L2, TIGIT and VISTA, whereas in READ, such positive correlations were noted only between the TIL load and LAG3 or PD-L2. The 'central memory T-cell' and 'exhausted T-cell' gene signatures were significantly lower among the READ tumors. The expression of PD-1, PD-L1, PD-L2, CTLA-4 and IDO1 was significantly higher among COAD patients with a high mutation rate (>34 mutations/Mb) compared to those with a lower rate. Somatic mutations in PD-1, PD-L1, CTLA-4 and other checkpoint molecules did not seem to affect their expression levels. On the whole, the data of the present study highlight the association of immune checkpoint molecules with the TIL load, patient survival and a high mutation rate in CRC. The data corroborate that patients with colon cancer with higher PD1, PD-L1/2, CTLA-4 and IDO1 expression, and a high mutation rate, are the ones who will benefit more from the respective immune checkpoint inhibition therapies.

Aluminum-induced alterations of purinergic signalling in embryonic neural progenitor cells

The ubiquitous presence of aluminum in the environment leads to a high likelihood of human exposure. Neurotoxicity of the trivalent cationic form of this metal (Al3+) occurs in the central nervous system via accumulation of Al in cells of neural origin, including neural progenitor cells (NPCs). NPCs play a key role in the development and regeneration of the brain throughout life; therefore, this metal may contribute to neuropathological conditions. Here, we evaluated the effects of different Al3+ concentrations (0–50 μM) on the purinergic system of NPCs isolated from embryonic telencephalons, cultured as neurospheres. Al3+ adhered to the cell surface of neurospheres reducing extracellular ATP release, as well as ATP, ADP, and AMP hydrolysis by NTPDase and 5′-nucleotidase, respectively. In addition, impaired nucleotide release by Al3+ reduced P2Y1 and adenosine A2A receptors expression in differentiated neurospheres. These receptors are crucial for NPC proliferation during brain development and self-repair against external stimuli, such as metal exposure. Thus, Al3+ represents an environmental agent linked to neurodegeneration through alterations in the ATP-signalling pathway, proving to be a potential mechanism associated with NPC proliferation and brain degeneration.

Polydeoxyribonucleotide Exerts Therapeutic Effect by Increasing VEGF and Inhibiting Inflammatory Cytokines in Ischemic Colitis Rats.

Ischemic colitis is resulted from an inadequate blood supply to a segment or entire colon. Polydeoxyribonucleotide (PDRN), extracted from salmon sperm, has been reported to exert anti-inflammatory and anti-ischemic effects through the adenosine A2A receptor (A2AR). We investigated whether PDRN possesses therapeutic effectiveness on ischemic colitis rats. Ischemic colitis was induced by selective devascularization. The skin temperature on the ischemic colitis-induced region was determined. To assess the colonic damage score and collagen deposition, colonic tissue sections were stained with hematoxylin and eosin (H&E), and Masson trichrome staining was performed. Western blot analysis for A2AR, vascular endothelial growth factor (VEGF), cyclooxygenase-2 (COX-2), tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and IL-6, Bax, Bcl-2, and extracellular signal-regulated kinase 1/2 (ERK1/2) was performed. Skin temperature was increased and mucosal damage and collagen deposition were observed in the affected colonic tissues in the ischemic colitis rats. Expressions of inflammatory cytokines (TNF-α, IL-1β, and IL-6) and inflammatory mediator (COX-2) were upregulated in the ischemic colitis rats. Apoptosis was increased by decreasing the ratio of Bcl-2 to Bax and by suppressing the phosphorylated form of ERK1/2 expression in the ischemic colitis rats. Treatment with PDRN alleviated mucosal damage reduced the expressions of inflammatory cytokines and COX-2 and inhibited apoptosis in the ischemic colitis rats. PDRN treatment more enhanced the expressions of A2AR and VEGF in the ischemic colitis rats. PDRN showed therapeutic effectiveness on ischemic colitis by increasing VEGF expression and inhibiting inflammatory cytokines and COX-2 through enhancing A2AR expression.

Detection of G Protein-Coupled Receptor Complexes in Postmortem Human Brain by Proximity Ligation Assay.

Combining immunological and molecular biological methods, the antibody-based proximity ligation assay (PLA) has been used for more than a decade to detect and quantify protein-protein interactions, protein modification, and protein expression in situ, including in brain tissue. However, the transfer of this technology to human brain samples requires a number of precautions due to the nature of the specimens and their specific processing. Here, we used the PLA brightfield detection technique to assess the expression of dopamine D2 receptor and adenosine A2A receptor and their proximity in human postmortem brains, and we developed a systematic random sampling method to help quantify the PLA signals. © 2019 by John Wiley & Sons, Inc. Basic Protocol 1: Sample preparation and sectioning for PLA_BF Basic Protocol 2: PLA_BF staining of brain tissue Basic Protocol 3: Image acquisition and result analysis Support Protocol: Luxol fast blue/cresyl violet staining.

The protective effect of inosine against rotenone-induced Parkinson's disease in mice; role of oxido-nitrosative stress, ERK phosphorylation, and A2AR expression.

Parkinson's disease (PD) is a severe disabling syndrome in which neuroinflammation and various signaling pathways are believed to mediate dopaminergic neurodegeneration. Here, the possible disease-modifying effects of the purine nucleoside inosine were examined against rotenone-induced PD. Mice were allocated into six groups, namely, a normal control group receiving dimethylsulfoxide, a PD control group receiving rotenone, a standard treatment group receiving L-dopa/carbidopa together with rotenone, and three treatment groups receiving inosine in low, medium, and high doses together with rotenone. At the end of the experimental protocol, three behavioral tests were performed to assess PD motor manifestations, namely, wire-hanging test, wood-walking test, and stair test. After performing the behavioral study, mice striata were isolated for the colorimetric assay of hypoxanthine, the enzyme-linked immunosorbent assay of dopamine, tumor necrosis factor-α, interleukin-6 and nitrite, the Western blot estimation of total and phosphorylated extracellular signal-regulated kinase (tERK and pERK), the polymerase chain reaction estimation of adenosine A2A receptor (A2AR) expression, as well as the histopathological examination of substantia nigra and striatal tissue. Inosine protected against PD progression in a dose-dependent manner, with the effect comparable to the standard treatment L-dopa/carbidopa, evidenced by behavioral, biochemical, and histologic findings. The beneficial antiparkinsonian effect of inosine could be attributed to the ability of the drug to ameliorate neuroinflammation and oxido-nitrosative stress, together with suppression of ERK phosphorylation and down-regulation of A2AR expression. Inosine could therefore be considered as a disease-modifying agent against PD, but further studies are claimed to confirm such effects clinically.

Effects of pulsed electromagnetic fields on the A2A adenosine receptor in human degenerative nucleus pulposus cells

Objective To explore the expression of the A2A adenosine receptor and the inflammatory cytokines interleukin-1 beta (IL-1β) and tumor necrosis factor alpha (TNF-α) in human degenerative nucleus pulposus (NP) cells after they have been treated with a pulsed electromagnetic field (PEMF). Methods Human degenerative NP cells were cultured in vitro and treated using an 0.8mT PEMF with a pulse frequency of 50Hz. The pulse width was 150μs and the exposure time was 30min, repeated 5 times at 12 hour intervals. The expression of the A2A adenosine receptor in NP cells was determined using western blotting and reverse transcription polymerase chain reactions. The expression of the inflammatory cytokines IL-1β and TNF-α were detected using enzyme-linked immunosorbent assays (ELISA). The human degenerative NP cells were also treated with an antagonist and agonist of the A2A adenosine receptor, and the expression of IL-1β and TNF-α were also determined using ELISA. Results After the PEMF treatment the expression of the A2A adenosine receptor increased significantly, while the expression of IL-1β and TNF-α decreased significantly. However, the A2A adenosine receptor antagonist reversed the inhibitory effect of the PEMF on the expression of IL-1β and TNF-α, while the agonist played an opposite role. Conclusion A PEMF can significantly inhibit the expression of IL-1β and TNF-α in human degenerative NP cells, which could be related to up-regulation of the expression of the A2A adenosine receptor in those cells. Key words: Pulsed electromagnetic fields; Adenosine receptors; Nucleus pulposus cells; Inflammatory cytokines

Evaluation of the Prognostic Significance and Effect of NT5E-Adenosine Axis, a Novel Immune Checkpoint, in Diffuse Large B-Cell Lymphoma

Background: The NT5E-adenosine axis constitutes one of the most promising immunosuppressive pathways in immune-oncology. NT5E is also named as CD73, which catalyzes the conversion of AMP to adenosine, and then the extracellular adenosine within tumor microenvironment bindings to A2a adenosine receptor (A2aR) to further dampen T cell-mediated immune responses and promote tumor immune escape. Several anti-CD73 or anti-A2aR antibodies are being evaluated in different types of malignancies in clinical trials. However, the prognostic significance and effect of NT5E-adenosine axis in diffuse large B-cell lymphoma (DLBCL) remain unclear. Methods: Multiplexed immunofluorescence staining, and a professionally and automatically assessed computer-assisted platform were applied to localize and quantify the markers from the DLBCL tumor and microenvironment cells. Gene expression status was analyzed according to the microarray data. The associations among marker expression patterns or their correlations with clinicopathological characteristics were estimated with the χ2 test and two-tailed Spearman analyses. Co-culture system of CD73 positive DLBCL cells and primary CD8 positive T cells was applied to evaluate the effect of NT5E-adenosine axis in DLBCL. Results: We found that CD73 was widely expressed on tumor and immune cells in DLBCL tissue. The CD73 positive rate on tumor cells was 52.31%, and CD73 expression on tumor cells was significantly associated with several clinicopathologic parameters, including IPI score (p = 0.020), LDH levels (p = 0.016) and Ki67 expression (p = 0.014). The A2aR positive rate on tumor-infiltrating lymphocytes (TILs) was 43.08%, and A2aR expression on TILs was significantly correlated with several clinicopathologic parameters, including IPI score (p = 0.036), clinical stage (p = 0.011), B symptom (p = 0.024) and Ki67 expression (p = 0.036). No significant difference in OS was observed in 233 DLBCL patients stratified by their CD73 gene expression status according to the microarray data (p = 0.267). There was also no significant effect of total CD73 protein expression on OS for the patients. However, a significant difference in OS was found when the patients were stratified by the CD73 expression on tumor cells, and the median survival times of the patients with CD73+/Pax-5+ and those with CD73-/Pax-5+ were 57.8 months (95% CI: 46.4-69.3) and 73.5 months (95% CI: 65.9-81.2), respectively. Patients with CD73+/Pax-5+ experienced significantly poorer outcomes than those with CD73-/Pax-5+ (p = 0.027). Furthermore, the median survival times of the patients with A2aR+ TILs and those with A2aR- TILs were respectively 53.3 months (95% CI: 40.6-66.0) and 74.5 months (95% CI: 67.5-81.5), and patients with A2aR+ TILs also had a significantly shorter survival time than those with A2aR- TILs (p = 0.003). Spearman's analysis revealed that CD73+/Pax-5+ tumor cells were positively correlated with A2aR+ TILs (R=0.395, p = 0.001). In a cohort, we found that 24 exhibited CD73-/Pax-5+ as well as A2aR- TILs, and 21 exhibited CD73+/Pax-5+ as well as A2aR+ TILs. Patients with CD73+/Pax-5+ and A2aR+TILs had poorer survival than those with CD73-/Pax-5+ and A2aR- TILs (p = 0.001). Residual patients were further classified into 2 groups, in which 7 exhibited CD73-/Pax-5+ as well as A2aR+ TILs, and 13 displayed CD73+/Pax-5+ as well as A2aR- TILs. There was a significant difference in survival among these four groups, and patients with CD73+/Pax-5+ and A2aR+ TILs had the worst outcome, with a 5-year OS rate of 57.1% (p = 0.017). We also found that CD73 positivity on tumor cells weakened the favorable prognosis for patients, which was correlated with the presence of CD8+ TILs. Patients with CD73+/Pax-5+ and CD8+ TILslow had the worst clinical outcome, with a 5-year OS rate of 50%. In contrast, patients with CD73-/Pax-5+ and CD8+ TILshigh had the best clinical outcome, with a 5-year OS rate of 100% (p = 0.002). Co-culture system displayed that CD73 being expressed on the DLBCL cells suppressed the growth of CD8 positive T cells through A2aR, but not A2aR negative T cells. Conclusions: Our findings uncovered that DLBCL patients with CD73+ on tumor cells as well as A2aR+ on TILs exhibited inferior survival and NT5E-adenosine axis inhibited the growth of CD8 positive T cells, supporting the potential combination strategies using CD73/A2aR immunosuppressive blockades as treatment options for DLBCL patients. Disclosures No relevant conflicts of interest to declare.

Extracellular vesicles with ubiquitinated adenosine A2A receptor in plasma of patients with coronary artery disease.

Extracellular vesicles (EV) can transfer cellular molecules for specific intercellular communication with potential relevance in pathological conditions. We searched for the presence in plasma from coronary artery disease (CAD) patients of EV containing the adenosine A2A receptor (A2AR), a signalling receptor associated with myocardial ischaemia and whose expression is related to homocysteine (HCy) metabolism. Using protein organic solvent precipitation for plasma EV preparation and Western blotting for protein identification, we found that plasma from CAD patients contained various amounts of EV with ubiquitin bound to A2AR. Interestingly, the presence of ubiquitinated A2AR in EV from patients was dependent on hyperhomocysteinemia, the amount being inversely proportional to A2AR expression in peripheral mononuclear cells in patients with the highest levels of HCy. CEM, a human T cell line, was also found to released EV containing various amounts of ubiquitinated A2AR in stimulated conditions depending on the hypoxic status and HCy level of culture medium. Together, these data show that ubiquitinated A2AR‐containing EV circulate in the plasma of CAD patients and that this presence is related to hyperhomocysteinemia. A2AR in plasma EV could be a useful tool for diagnosis and a promising drug for the treatment of CAD.

Upregulation of adenosine A2A receptor and downregulation of GLT1 is associated with neuronal cell death in Rasmussen's encephalitis.

Rasmussen encephalitis (RE) is a severe pediatric inflammatory brain disease characterized by unilateral inflammation and atrophy of the cerebral cortex, drug-resistant focal epilepsy and progressive neurological and cognitive deterioration. The etiology and pathogenesis of RE remain unclear. Our previous results demonstrated that the adenosine A1 receptor (A1R) and the major adenosine-removing enzyme adenosine kinase play an important role in the etiology of RE. Because the downstream pathways of inhibitory A1R signaling are modulated by stimulatory A2AR signaling, which by itself controls neuro-inflammation, glial activation and glial glutamate homeostasis through interaction with glutamate transporter GLT-1, we hypothesized that maladaptive changes in adenosine A2A receptor (A2AR) expression are associated with RE. We used immunohistochemistry and Western blot analysis to examine the expression of A2ARs, glutamate transporter-I (GLT-1) and the apoptotic marker Bcl-2 in surgically resected cortical specimens from RE patients (n=18) in comparison with control cortical tissue. In lesions of the RE specimen we found upregulation of A2ARs, downregulation of GLT-1 and increased apoptosis of both neurons and astroglia. Double staining revealed colocalization of A2ARs and Bcl-2 in RE lesions. These results suggest that maladaptive changes in A2AR expression are associated with a decrease in GLT-I expression as a possible precipitator for apoptotic cell loss in RE. Because A2AR antagonists are already under clinical evaluation for Parkinson's disease, the A2AR might likewise be a tractable target for the treatment of RE.

Carbamazepine Attenuates Astroglial L-Glutamate Release Induced by Pro-Inflammatory Cytokines via Chronically Activation of Adenosine A2A Receptor

Carbamazepine (CBZ) binds adenosine receptors, but detailed effects of CBZ on astroglial transmission associated with adenosine receptor still need to be clarified. To clarify adenosinergic action of CBZ on astroglial transmission, primary cultured astrocytes were acutely or chronically treated with CBZ, proinflammatory cytokines (interferon γ (IFNγ) and tumor necrosis factor α (TNFα)), and adenosine A2A receptor (A2AR) agonist (CGS21680). IFNγ and TNFα increased basal, adenophostin-A (AdA)-evoked, and 2-amino-3-(3-hydroxy-5-methyl-isoxazol-4-yl)propanoic acid (AMPA)-evoked astroglial L-glutamate releases. In physiological condition, CGS21680 increased basal astroglial L-glutamate release but glutamate transporter inhibition prevented this CGS21680 action. CBZ did not affect basal release, whereas glutamate transporter inhibition generated CBZ-induced glutamate release. Furthermore, AdA-evoked and AMPA-evoked releases were inhibited by CBZ but were unaffected by CGS21680. Contrary to physiological condition, chronic administrations of IFNγ and TNFα enhanced basal, AdA-, and AMPA-evoked releases, whereas IFNγ and TNFα decreased and increased CGS21680-evoked releases via modulation A2AR expression. Both chronic administration of CGS21680 and CBZ suppressed astroglial L-glutamate release responses induced by chronic cytokine exposer. Especifically, chronic administration of CBZ and CGS21680 prevented the reduction and elevation of A2AR expression by respective IFNγ and TNFα. These findings suggest that A2AR agonistic effects of CBZ contribute to chronic prevention of pathomechanisms developments of several neuropsychiatric disorders associated with proinflammatory cytokines.

Effect of low androgen status on the expression of adenosine A 2A and A 2B receptors in rat penile corpus cavernosum

To investigate whether low androgen status affects erectile function by regulating the expression of adenosine A2A and A2B receptors in rat penile corpus cavernosum. Thirty-six 8-week-old male Sprague-Dawley rats were randomly divided into six groups: sham-operated group (4w-sham, 8w-sham), castration group (4w-cast, 8w-cast) and androgen replacement group (4w-cast+T, 8w-cast+T). The rats in the androgen replacement groups were subcutaneously injected with testosterone propionate (3mg/kg) every other day after castration. The maximum intracavernous pressure/mean arterial pressure (ICPmax/MAP), the expression of A2A , A2B , AKT and eNOS and the concentrations of cAMP and cGMP in the corpus cavernosum were detected at the 4th and 8th weeks after the operation. The serum testosterone level and the ratio of ICPmax/MAP decreased significantly in the castration group as compared to other groups (p<0.01). There was no significant difference in the expression of A2A receptor among groups, while the expression of A2B , AKT and eNOS and the concentrations of cAMP and cGMP in the castration group were significantly lower than in other groups (p<0.01). Low androgen status inhibits the AKT/eNOS/cGMP signalling pathways and the production of cAMP in the corpus cavernosum of castrated rats by down-regulating the expression of A2B receptor, and results in decreased of ICPmax/MAP.

Sequential Changes in Brain Glutamate and Adenosine A1 Receptors May Explain Severity of Adolescent Alcohol Withdrawal after Consumption of High Levels of Alcohol.

There is an excellent correlation between the age when alcohol consumption begins and the likelihood of lifelong problems with alcohol abuse. Alcohol use often begins in adolescence, a time marked by brain development and maturation of numerous brain systems. Rats are an important model, wherein the emergence of alcohol withdrawal symptoms serves as a gauge of dependency following chronic alcohol consumption. Previous work has shown that adolescent Long-Evans rats consume high levels of alcohol and develop a severe alcohol withdrawal syndrome when fed alcohol as part of a liquid diet. Acutely, alcohol inhibits two important excitatory receptors for glutamate (NMDA and AMPA) and may further decrease glutamate activity through modulatory adenosine receptors. The present study focuses on potential adaptive changes in expression of these receptors that may create a receptor imbalance during chronic alcohol consumption and lead to severe overexcitation of the adolescent brain during alcohol withdrawal. Levels of brain expression of NMDA, AMPA, and adenosine A1 and A2a receptors were determined by Western blotting after adolescent rats consumed an alcohol-containing liquid diet for 4, 11, or 18 days. Severity of alcohol withdrawal was also assessed at these time points. Levels increased for both AMPA and NMDA receptors, significant and approaching maximal by day 11. In contrast, A1 receptor density showed a slow decline reaching significance at 18 days. There were no changes in expression of adenosine A2a receptor. The most severe withdrawal symptoms appear to coincide with the later downregulation of adenosine A1 receptors coming on top of maximal upregulation of excitatory AMPA and NMDA glutamate receptors. Thus, loss of adenosine “brakes” on glutamate excitation may punctuate receptor imbalance in alcohol-consuming adolescents by allowing the upregulation of the excitatory receptors to have full impact during early alcohol withdrawal.

The abnormal function of CD39+ regulatory T cells could be corrected by high-dose dexamethasone in patients with primary immune thrombocytopenia.

Primary immune thrombocytopenia is an autoimmune disease, characterized with decreased platelet and increased risk of bleeding. Recent studies have shown the reduction and dysfunction of regulatory T (Treg) cells in ITP patients. CD39 is highly expressed on the surface of Treg cells. It degrades ATP to AMP and CD73 dephosphorylates AMP into adenosine. Then adenosine binds with adenosine receptor and suppresses immune response by activating Treg cells and inhibiting the release of inflammatory cytokines from effector T (Teff) cells. Adenosine receptor has several subtypes and adenosine A2A receptor (A2AR) plays a crucial role especially within lymphocytes. The CD39+ Treg cells and the expression of A2AR showed abnormality in some autoimmune disease. But knowledge of CD39+ Treg cells and A2AR which are crucial in the adenosine immunosuppressive pathway is still limited in ITP. Thirty-one adult patients with newly diagnosed ITP were enrolled in this study. CD39 and A2AR expression was measured by flow cytometry and RT-PCR. The function of CD39 was reflected by the change of ATP concentration detected by CellTiter-Glo Luminescent Cell Viability Assay. CD39 expression within CD4+CD25+ Treg cells in ITP patients was decreased compared to normal controls. After high-dose dexamethasone therapy, response (R) group showed increased CD39 expression within Treg cells while non-response (NR) group did not show any difference in contrast to those before treatment. The expression of A2AR in CD4+CD25- Teff and CD4+CD25+ Treg cells was both lower in ITP patients than that of normal controls. After therapy, CD4+CD25- Teff cells had higher A2AR expression while CD4+CD25+ Treg cells did not show any difference in comparison to that before treatment. The enzymatic activity of CD39 was damaged in ITP patients and improved after high-dose dexamethasone therapy. In ITP, there was not only numerical decrease but also impaired enzymatic activity in CD39+ Treg cells. After high-dose dexamethasone treatment, these two defects could be reversed. Our results also suggested that ITP patients had reduced A2AR expression in both CD4+CD25+ Treg cells and CD4+CD25- Teff cells. CD4+CD25- Teff cells had increased A2AR expression after treatment.

Pharmacological profile of adenosine A2A receptors in patients with lower extremity peripheral artery disease and associated coronary artery disease: A pilot study

BackgroundAltered blood flow occurs in patients with low extremity peripheral artery disease (LE-PAD). LE-PAD is mostly associated with coronary artery disease (CAD). Adenosine is an endogenous nucleoside that affects both coronary and limb artery blood flow, mostly via the adenosine A2A receptor (A2AR). We evaluated A2AR expression and function in peripheral blood mononuclear cells (PBMCs) and the femoral artery tissues of patients with LE-PAD. MethodsArtery tissues and PBMCs were sampled in 24 patients with intermittent claudication, and compared with PBMCs in 24 healthy subjects. Expression and function of A2AR was studied, using a A2AR monoclonal antibody with agonist properties, allowing determination of A2AR affinity (KD) and cAMP production (ie.EC50). ResultsA2AR expression on PBMCs was lower in patients than controls (median1.3 [range 0.6–1.8] vs 1.75 [1.45–2.1] arbitrary units; P < 0.01), and correlated with A2AR expression in artery tissues (Pearson's r = 0.71; P < 0.01). Basal and maximally stimulated cAMP production of PBMCs was lower in patients vs controls: 172 [90–310] vs 244 [110–380] pg/106 cells (P < 0.05) and 375 [160–659] vs 670 [410–980] pg/106 cells (P < 0.05), respectively. A high KD/EC50 ratio, characteristic of spare receptors, was observed in CAD with inducible-myocardial-ischemia. ConclusionA2AR expression in the arteries of patients, correlated with their expression in PBMCs. A2AR expression was lower in patients than in controls. A single blood sample (for measurement of A2AR expression on PBMCs) may help to screen patients with LE-PAD, whereas the presence of spare receptors may help with risk stratification before vascular surgery in CAD patients with high risk of myocardial ischemia.

Caffeine programs hepatic SIRT1-related cholesterol synthesis and hypercholesterolemia via A2AR/cAMP/PKA pathway in adult male offspring rats

Clinical and animal studies have indicated that hypercholesterolemia has intrauterine developmental origin. Our previous studies showed that prenatal caffeine exposure (PCE) increased the serum total cholesterol (TCH) levels in adult offspring rats. This study investigates the intrauterine programming mechanism of PCE male offspring rats susceptible to adult hypercholesterolemia. Pregnant Wistar rats were intragastrically administered caffeine (30, 60, and 120 mg/kg∙d) from gestational days (GD) 9 to 20. Male offspring were sacrificed under anesthesia at GD20 and postnatal week (PW) 12, and the serum and liver were collected. The effects of caffeine (0–100 μM, 24 h) on the expression of cholesterol synthesis related genes and their epigenetic mechanisms were confirmed in L02 cells. The results showed that PCE induced higher levels of serum TCH, LDL-C and higher ratios of TCH/HDL-C and LDL-C/HDL-C. Furthermore, the high levels of histone acetylation (via H3K14ac and H3K27ac) and the expression of genes (Srebf2, Hmgcr, Hmgcs1) were responsible for cholesterol synthesis. The results of PCE offspring in utero and the data in vitro exhibited similar changes, and accompanied by the reduced expression of adenosine A2A receptor (A2AR), cyclic adenosine monophosphate (cAMP), sirtuin1 and protein kinase A (PKA). These changes could be reversed by A2AR agonist (CGS-21680), cAMP agonist (forskolin) and sirtuin1 agonist (resveratrol). Therefore, our results confirmed that caffeine could enhance histone acetylation and expression levels of genes responsible for cholesterol synthesis via inhibiting the A2AR/cAMP/PKA pathway and down-regulating sirtuin1, which continued throughout adulthood and elevated hepatic cholesterol synthesis and hypercholesterolemia in the male offspring rats.

Blockade of microglial adenosine A2A receptor suppresses elevated pressure-induced inflammation, oxidative stress, and cell death in retinal cells.

Glaucoma is a retinal degenerative disease characterized by the loss of retinal ganglion cells and damage of the optic nerve. Recently, we demonstrated that antagonists of adenosine A2A receptor (A2AR) control retinal inflammation and afford protection to rat retinal cells in glaucoma models. However, the precise contribution of microglia to retinal injury was not addressed, as well as the effect of A2AR blockade directly in microglia. Here we show that blocking microglial A2AR prevents microglial cell response to elevated pressure and it is sufficient to protect retinal cells from elevated pressure‐induced death. The A2AR antagonist SCH 58261 or the knockdown of A2AR expression with siRNA in microglial cells prevented the increase in microglia response to elevated hydrostatic pressure. Furthermore, in retinal neural cell cultures, the A2AR antagonist decreased microglia proliferation, as well as the expression and release of pro‐inflammatory mediators. Microglia ablation prevented neural cell death triggered by elevated pressure. The A2AR blockade recapitulated the effects of microglia depletion, suggesting that blocking A2AR in microglia is able to control neurodegeneration in glaucoma‐like conditions. Importantly, in human organotypic retinal cultures, A2AR blockade prevented the increase in reactive oxygen species and the morphological alterations in microglia triggered by elevated pressure. These findings place microglia as the main contributors for retinal cell death during elevated pressure and identify microglial A2AR as a therapeutic target to control retinal neuroinflammation and prevent neural apoptosis elicited by elevated pressure.

Spironolactone inhibits endothelial-mesenchymal transition via the adenosine A2A receptor to reduce cardiorenal fibrosis in rats

AimsThe mechanisms underlying cardiorenal syndromes are complex and not fully understood; Fibrosis seems to be a primary driver of the diseases' pathophysiology. Spironolactone can reduce cardiac or renal fibrosis by inhibiting endothelial-mesenchymal transition (EndMT). Spironolactone protection may rely on activation of adenosine receptors, but the role of the adenosine A2A receptor (A2AR) is unclear. We hypothesize that spironolactone may modulate A2AR to suppress EndMT and reduce cardiorenal remodeling. Main methodsA model of renal injury followed by heart failure was established by subcutaneous administration of isoprenaline (Iso) to rats. Assessment of cardiac and renal function, fibrosis, EndMT markers, adenosine and A2AR expression was performed. TGF-β was used to induce EndMT in primary human umbilical vein endothelial cells (HUVECs). Rats or cells were divided into four groups: those that treated with spironolactone alone or in combination with A2AR antagonist ZM241385 or neither, and compared to normal controls. Key findingsIsoprenaline-treated rats exhibited cardiac and renal fibrosis, impaired cardiac and renal function, enhanced EndMT, and lower A2AR expression. Spironolactone significantly up-regulated A2AR expression and inhibited EndMT in vivo and in vitro. Moreover, spironolactone improved cardiorenal remodeling and reduced dysfunction. These changes were exacerbated by administration of ZM241385. Together, these findings show that spironolactone up-regulated A2AR to reduce EndMT and ameliorate cardiorenal fibrosis. SignificanceThe anti-fibrotic effects of spironolactone may partly depend on the up-regulation of A2AR, and that A2AR might be a potential therapeutic target for the treatment of cardiorenal syndrome.

Prenatal caffeine damaged learning and memory in rat offspring mediated by ARs/PKA/CREB/BDNF pathway.

Prenatal exposure to caffeine can cause developmental problems. This study determined chronic influence of prenatal caffeine at relatively higher doses on cognitive functions in the rat offspring. Pregnant Sprague-Dawley rats (4-month-old) were exposed to caffeine (20 mg/kg, twice a day) for whole pregnancy from gestational day 4. Fetal and offspring body and brain weight was measured. Learning and memory were tested in adult offspring with Morris water maze. Learning and memory-related receptors were measured. The exposure to prenatal caffeine not only caused fetal growth restriction, but also showed long-term effects on learning and memory in the offspring. The caffeine offspring exhibited longer escape latency and path length in navigation testing. The number of passing the target was significantly reduced in those offspring. The expression of adenosine A(1) and A(2A) receptors, nuclear PKA C(alpha), C(beta) subunits, and pCREB were significantly increased in the fetal and neonatal brain, and suppressed in the hippocampus of the adult offspring. The expression of BDNF and TrkB were reduced regardless of various ages. The results suggest that intrauterine programming dysfunction of adenosine receptors and the down-stream of cAMP/PKA/pCREB system may play an important role in prenatal caffeine induced cognition disorders in the adult offspring.

Resveratrol-mediated reversal of changes in purinergic signaling and immune response induced by Toxoplasma gondii infection of neural progenitor cells.

The effects of Toxoplasma gondii during embryonic development have not been explored despite the predilection of this parasite for neurons and glial cells. Here, we investigated the activation of the purinergic system and proinflammatory responses during congenital infection by T. gondii. Moreover, neuroprotective and neuromodulatory properties of resveratrol (RSV), a polyphenolic natural compound, were studied in infected neuronal progenitor cells (NPCs). For this study, NPCs were isolated from the telencephalon of infected mouse embryos and subjected to neurosphere culture in the presence of EGF and FGF2. ATP hydrolysis and adenosine deamination by adenosine deaminase activity were altered in conditions of T. gondii infection. P2X7 and adenosine A2A receptor expression rates were augmented in infected NPCs together with an increase of proinflammatory (INF-γ and TNF-α) and anti-inflammatory (IL-10) cytokine gene expression. Our results confirm that RSV counteracted T. gondii-promoted effects on enzymes hydrolyzing extracellular nucleotides and nucleosides and also upregulated P2X7 and A2A receptor expression and activity, modulating INF-γ, TNF-α, and IL-10 cytokine production, which plays an integral role in the immune response against T. gondii.