Adenosine A2A Receptor Activation Research Articles

Dual mechanisms of cholesterol-GPCR interactions that depend on membrane phospholipid composition

Cholesterol is a critical component of mammalian cell membranes and an allosteric modulator of G protein-coupled receptors (GPCRs), but divergent views exist on the mechanisms by which cholesterol influences receptor functions. Leveraging the benefits of lipid nanodiscs, i.e., quantitative control of lipid composition, we observe distinct impacts of cholesterol in the presence and absence of anionic phospholipids on the function-related conformational dynamics of the human A2A adenosine receptor (A2AAR). Direct receptor-cholesterol interactions drive activation of agonist-bound A2AAR in membranes containing zwitterionic phospholipids. Intriguingly, the presence of anionic lipids attenuates cholesterol's impact through direct interactions with the receptor, highlighting a more complex role for cholesterol that depends on membrane phospholipid composition. Targeted amino acid replacements at two frequently predicted cholesterol interaction sites showed distinct impacts of cholesterol at different receptor locations, demonstrating the ability to delineate different roles of cholesterol in modulating receptor signaling and maintaining receptor structural integrity.

Lateral septum adenosine A2A receptors control stress-induced depressive-like behaviors via signaling to the hypothalamus and habenula

Major depressive disorder ranks as a major burden of disease worldwide, yet the current antidepressant medications are limited by frequent non-responsiveness and significant side effects. The lateral septum (LS) is thought to control of depression, however, the cellular and circuit substrates are largely unknown. Here, we identified a subpopulation of LS GABAergic adenosine A2A receptors (A2AR)-positive neurons mediating depressive symptoms via direct projects to the lateral habenula (LHb) and the dorsomedial hypothalamus (DMH). Activation of A2AR in the LS augmented the spiking frequency of A2AR-positive neurons leading to a decreased activation of surrounding neurons and the bi-directional manipulation of LS-A2AR activity demonstrated that LS-A2ARs are necessary and sufficient to trigger depressive phenotypes. Thus, the optogenetic modulation (stimulation or inhibition) of LS-A2AR-positive neuronal activity or LS-A2AR-positive neurons projection terminals to the LHb or DMH, phenocopied depressive behaviors. Moreover, A2AR are upregulated in the LS in two male mouse models of repeated stress-induced depression. This identification that aberrantly increased A2AR signaling in the LS is a critical upstream regulator of repeated stress-induced depressive-like behaviors provides a neurophysiological and circuit-based justification of the antidepressant potential of A2AR antagonists, prompting their clinical translation.

Berberine attenuates epithelial mesenchymal transition in bleomycin-induced pulmonary fibrosis in mice via activating A2aR and mitigating the SDF-1/CXCR4 signaling

AimsBerberine is endowed with anti-oxidant, anti-inflammatory and anti-fibrotic effects. This study explored the role of adenosine A2a receptor (A2aR) activation and SDF-1/CXCR4 signaling suppression in the protective effects of berberine in bleomycin-induced pulmonary fibrosis in mice. Main methodsPulmonary fibrosis was generated in mice by injecting bleomycin (40 U/kg, i.p.) on days 0, 3, 7, 10 and 14. Mice were treated with berberine (5 mg/kg, i.p.) from day 15 to day 28. Key findingsSevere lung fibrosis and increased collagen content were observed in the bleomycin-challenged mice. Pulmonary A2aR downregulation was documented in bleomycin-induced pulmonary fibrosis animals and was accompanied by enhanced expression of SDF-1/CXCR4. Moreover, TGF-β1elevation and pSmad2/3 overexpression were reported in parallel with enhanced epithelial mesenchymal transition (EMT) markers expression, vimentin and α-SMA. Besides, bleomycin significantly elevated the inflammatory and pro-fibrogenic mediator NF-κB p65, TNF-α and IL-6. Furthermore, bleomycin administration induced oxidative stress as depicted by decreased Nrf2, SOD, GSH and catalase levels. Interestingly, berberine administration markedly ameliorated the fibrotic changes in lungs by modulating the purinergic system through the inhibition of A2aR downregulation, mitigating EMT and effectively suppressing inflammation and oxidative stress. Strikingly, A2aR blockade by SCH 58261, impeded the pulmonary protective effect of berberine. SignificanceThese findings indicated that berberine could attenuate the pathological processes of bleomycin-induced pulmonary fibrosis at least partially via upregulating A2aR and mitigating the SDF-1/CXCR4 related pathway, suggesting A2aR as a potential therapeutic target for the management of pulmonary fibrosis.

Sub-millisecond conformational dynamics of the A2A adenosine receptor revealed by single-molecule FRET

The complex pharmacology of G-protein-coupled receptors (GPCRs) is defined by their multi-state conformational dynamics. Single-molecule Förster Resonance Energy Transfer (smFRET) is well suited to quantify dynamics for individual protein molecules; however, its application to GPCRs is challenging. Therefore, smFRET has been limited to studies of inter-receptor interactions in cellular membranes and receptors in detergent environments. Here, we performed smFRET experiments on functionally active human A2A adenosine receptor (A2AAR) molecules embedded in freely diffusing lipid nanodiscs to study their intramolecular conformational dynamics. We propose a dynamic model of A2AAR activation that involves a slow (>2 ms) exchange between the active-like and inactive-like conformations in both apo and antagonist-bound A2AAR, explaining the receptor’s constitutive activity. For the agonist-bound A2AAR, we detected faster (390 ± 80 µs) ligand efficacy-dependent dynamics. Our work establishes a general smFRET platform for GPCR investigations that can potentially be used for drug screening and/or mechanism-of-action studies.

Preservation of vascular endothelial glycocalyx and barrier by activation of adenosine A2A receptor (A2AR) improved renal dysfunction in cirrhotic rats

Cirrhosis-related hepatic and renal endothelial dysfunction is characterized by macrophage-endothelium adhesion-mediated inflammation, glycocalyx/barrier damage, and impaired vasodilation. Activation of adenosine A2A receptor (A2AR) protects cirrhotic rats from impairment of hepatic microcirculation post hepatectomy. This study evaluates the effects of A2AR activation on the cirrhosis-related hepatic and renal endothelial dysfunction in biliary cirrhotic rats receiving two weeks of A2AR agonist PSB0777 [bile duct ligated (BDL)+PSB0777] treatment.Endothelial dysfunction in cirrhotic liver, renal vessels, and kidney is characterized by downregulation of the A2AR expressions, decreased vascular endothelial vasodilatory (p-eNOS)/anti-inflammatory (IL-10/IL-10R)/barrier [VE-cadherin (CDH5) and β-catenin (CTNNB1)]/glycocalyx [syndecan-1 (SDC1) and hyaluronan synthase-2 (HAS2)] markers, and increased leukocyte-endothelium adhesion molecules (F4/80, CD68, ICAM-1, and VCAM-1). In BDL rats, PSB0777 treatment improves hepatic and renal endothelial dysfunction, ameliorates portal hypertension, and attenuates renal hypoperfusion by restoring of the vascular endothelial anti-inflammatory, barrier, glycocalyx markers and vasodilatory response as well as inhibiting the leukocyte-endothelium adhesion. In an in vitro study, conditioned medium (CM) of bone marrow-derived macrophage (BMDM) of BDL rats [BMDM-CM (BDL)] induced barrier/glycocalyx damage, which was reversed by the PSB0777 pre-treatment. The A2AR agonist is a potential agent that can simultaneously correct cirrhosis-related hepatic and renal endothelial dysfunction, portal hypertension, renal hypoperfusion, and renal dysfunction.

Regadenoson Reduces Soluble Receptor for Advanced Glycation End-Products in Lung Recipients

BackgroundThe selective adenosine A2A receptor (A2AR) agonist regadenoson reduces inflammation due to lung ischemia-reperfusion injury (IRI). The objective of this study was to investigate molecular and cellular mechanisms by which regadenoson reduces IRI in lung transplant recipients. MethodsFourteen human lung transplant recipients were infused for 12 hours with regadenoson and 7 more served as untreated controls. Plasma levels of high mobility group box 1 and its soluble receptor for advanced glycation end-products (sRAGE) were measured by Luminex. Matrix metalloproteinase (MMP) 2 and 9 were measured by gelatin zymography. Tissue inhibitor of metalloproteinase 1 was measured by mass spectroscopy. A2AR expression on leukocytes was analyzed by flow cytometry. MMP-9–mediated cleavage of RAGE was evaluated using cultured macrophages in vitro. ResultsRegadenoson treatment during lung transplantation significantly reduced levels of MMP-9 (P < .05), but not MMP-2, and elevated levels of tissue inhibitor of metalloproteinase 1 (P < .05), an endogenous selective inhibitor of MMP-9. Regadenoson infusion significantly reduced plasma levels of sRAGE (P < .05) during lung reperfusion compared with control subjects. A2AR expression was highest on invariant natural killer T cells and higher on monocytes than other circulating immune cells (P < .05). The shedding of RAGE from cultured monocytes/macrophages was increased by MMP-9 stimulation and reduced by an MMP inhibitor or by A2AR agonists, regadenoson or ATL146e. ConclusionsIn vivo and in vitro studies suggest that A2AR activation reduces sRAGE in part by inhibiting MMP-9 production by monocytes/macrophages. These results suggest a novel molecular mechanism by which A2AR agonists reduce primary graft dysfunction.

Adenosine A2A receptor activation reduces chondrocyte senescence.

Osteoarthritis (OA) pathogenesis is associated with reduced chondrocyte homeostasis and increased levels of cartilage cellular senescence. Chondrosenescence is the development of cartilage senescence that increases with aging joints and disrupts chondrocyte homeostasis and is associated with OA. Adenosine A2A receptor (A2AR) activation in cartilage via intra-articular injection of liposomal A2AR agonist, liposomal-CGS21680, leads to cartilage regeneration in vivo and chondrocyte homeostasis. A2AR knockout mice develop early OA isolated chondrocytes demonstrate upregulated expression of cellular senescence and aging-associated genes. Based on these observations, we hypothesized that A2AR activation would ameliorate cartilage senescence. We found that A2AR stimulation of chondrocytes reduced beta-galactosidase staining and regulated levels and cell localization of common senescence mediators p21 and p16 in vitro in the human TC28a2 chondrocyte cell line. In vivo analysis similarly showed A2AR activation reduced nuclear p21 and p16 in obesity-induced OA mice injected with liposomal-CGS21680 and increased nuclear p21 and p16 in A2AR knockout mouse chondrocytes compared to wild-type mice. A2AR agonism also increased activity of the chondrocyte Sirt1/AMPK energy-sensing pathway by enhancing nuclear Sirt1 localization and upregulating T172-phosphorylated (active) AMPK protein levels. Lastly, A2AR activation in TC28a2 and primary human chondrocytes reduced wild-type p53 and concomitantly increased p53 alternative splicing leading to increase in an anti-senescent p53 variant, Δ133p53α. The results reported here indicate that A2AR signaling promotes chondrocyte homeostasis in vitro and reduces OA cartilage development in vivo by reducing chondrocyte senescence.

Expression and Impact of Adenosine A3 Receptors on Calcium Homeostasis in Human Right Atrium.

Increased adenosine A2A receptor (A2AR) expression and activation underlies a higher incidence of spontaneous calcium release in atrial fibrillation (AF). Adenosine A3 receptors (A3R) could counteract excessive A2AR activation, but their functional role in the atrium remains elusive, and we therefore aimed to address the impact of A3Rs on intracellular calcium homeostasis. For this purpose, we analyzed right atrial samples or myocytes from 53 patients without AF, using quantitative PCR, patch-clamp technique, immunofluorescent labeling or confocal calcium imaging. A3R mRNA accounted for 9% and A2AR mRNA for 32%. At baseline, A3R inhibition increased the transient inward current (ITI) frequency from 0.28 to 0.81 events/min (p < 0.05). Simultaneous stimulation of A2ARs and A3Rs increased the calcium spark frequency seven-fold (p < 0.001) and the ITI frequency from 0.14 to 0.64 events/min (p < 0.05). Subsequent A3R inhibition caused a strong additional increase in the ITI frequency (to 2.04 events/min; p < 0.01) and increased phosphorylation at s2808 1.7-fold (p < 0.001). These pharmacological treatments had no significant effects on L-type calcium current density or sarcoplasmic reticulum calcium load. In conclusion, A3Rs are expressed and blunt spontaneous calcium release at baseline and upon A2AR-stimulation in human atrial myocytes, pointing to A3R activation as a means to attenuate physiological and pathological elevations of spontaneous calcium release events.

Activation of adenosine A2A receptors induces SUMOylation of HIF-1α.

Hypoxia is an established driver of numerous deleterious cell processes, including immunosuppression and tumor growth. Hypoxia-inducible factor 1α (HIF-1α) accumulates during periods of sustained hypoxia due to the action of multiple post-translational modifications, including the Small Ubiquitin-like modifier (SUMO) pathway. Although enhanced activity of the SUMO-pathway is upregulated during hypoxia, the mechanistic basis for this effect remains unclear. It has been proposed that the Gαs protein-coupled adenosine A2A receptor (A2AR), which is also activated during sustained hypoxia, mediates SUMOylation at a number of target proteins. We sought to determine if activation of A2AR by hypoxia induces SUMOylation of HIF-1α and which of three potential SUMO-sites on HIF-1α are modified by this mechanism. HEK293T cells expressing A2AR and treated with an A2AR agonist (CGS) showed significantly increased HIF-1α accumulation compared to untreated controls after exposure to hypoxia. Similarly, CGS and hypoxia enhanced SUMOylation of HIF-1α in human Jurkat T cells. Using site-directed mutagenesis and Förster resonance energy transfer (FRET), we determined that A2AR-mediated SUMOylation of HIF-1α occurs at lysine 532. Finally, we show that (1) this mechanism of regulation increases the mRNA levels of multiple HIF-1α transcriptional targets; and (2) this mechanism can be precluded by treating cells with a small molecule inhibitor of the SUMO pathway. Together, these data highlight a central role for A2AR-mediated SUMOylation of HIF-1α in mediating the effects of chronic hypoxia in Jurkat T cells.

Enhancement of adenosine A2A signaling improves dopamine D2 receptor antagonist-induced dyskinesia via β-arrestin signaling.

Repeated administration of dopamine D2 receptor (D2R) antagonists, which is the treatment for psychosis, often causes tardive dyskinesia (TD). Despite notable clinical demand, effective treatment for TD has not been established yet. The neural mechanism involving the hyperinhibition of indirect pathway medium spiny neurons (iMSNs) in the striatum is considered one of the main causes of TD. In this study, we focused on adenosine A2A receptors (A2ARs) expressed in iMSNs and investigated whether pharmacological activation of A2ARs improves dyskinetic symptoms in a TD mouse model. A 21-day treatment with haloperidol increased the number of vacuous chewing movements (VCMs) and decreased the number of c-Fos+/ppENK+ iMSNs in the dorsal striatum. Haloperidol-induced VCMs were reduced by acute intraperitoneal administration of an A2AR agonist, CGS 21680A. Consistently, haloperidol-induced VCMs and decrease in the number of c-Fos+/ppENK+ iMSNs were also mitigated by intrastriatal injection of CGS 21680A. The effects of intrastriatal CGS 21680A were not observed when it was concomitantly administered with a β-arrestin inhibitor, barbadin. Finally, intrastriatal injection of an arrestin-biased D2R agonist, UNC9994, also inhibited haloperidol-induced VCMs. These results suggest that A2AR agonists mitigate TD symptoms by activating striatal iMSNs via β-arrestin signaling.

Mitochondrial Dysfunction of Astrocytes Mediates Lipid Accumulation in Temporal Lobe Epilepsy.

Lipid-accumulated reactive astrocytes (LARAs) have recently been confirmed to be a pivotal cell type present in temporal lobe epilepsy (TLE) lesions. These cells not only induce anomalous lipid accumulation within the epileptic foci but also decrease the seizure threshold by employing upregulated activation of the adenosine A2A receptor (A2AR). Furthermore, disturbances in mitochondrial oxidative phosphorylation (OxPhos) have been noted as significant drivers of lipid accumulation in astrocytes. Moreover, the deficiency of OxPhos in astrocytes can induce severe neuroinflammation, which can worsen the progression of TLE. Accordingly, further exploration of the correlation between mitochondrial dysfunction, LARAs-mediated lipid accumulation, and A2AR activation within epilepsy lesions is warranted. It could potentially elucidate the vital role of mitochondrial dysfunction in the pathogenesis of TLE.

Increased ATP Release and Higher Impact of Adenosine A2A Receptors on Corticostriatal Plasticity in a Rat Model of Presymptomatic Parkinson’s Disease

Extracellular ATP can be a danger signal, but its role in striatal circuits afflicted in Parkinson’s disease (PD) is unclear and was now investigated. ATP was particularly released at high stimulation intensities from purified striatal nerve terminals of mice, which were endowed with different ATP-P2 receptors (P2R), although P2R antagonists did not alter corticostriatal transmission or plasticity. Instead, ATP was extracellularly catabolized into adenosine through CD73 to activate adenosine A2A receptors (A2AR) modulating corticostriatal long-term potentiation (LTP) in mice. In the presymptomatic phase of a 6-hydroxydopamine rat model of PD, ATP release from striatal nerve terminals was increased and was responsible for a greater impact of CD73 and A2AR on corticostriatal LTP. These observations identify increased ATP release and ATP-derived formation of extracellular adenosine bolstering A2AR activation as a key pathway responsible for abnormal synaptic plasticity in circuits involved in the onset of PD motor symptoms. The translation of these findings to humans prompts extending the use of A2AR antagonists from only co-adjuvants of motor control in Parkinsonian patients to neuroprotective drugs delaying the onset of motor symptoms.

CD73-Mediated Formation of Extracellular Adenosine Is Responsible for Adenosine A2A Receptor-Mediated Control of Fear Memory and Amygdala Plasticity.

Adenosine A2A receptors (A2AR) control fear memory and the underlying processes of synaptic plasticity in the amygdala. In other brain regions, A2AR activation is ensured by ATP-derived extracellular adenosine formed by ecto-5'-nucleotidase or CD73. We now tested whether CD73 is also responsible to provide for the activation of A2AR in controlling fear memory and amygdala long-term potentiation (LTP). The bilateral intracerebroventricular injection of the CD73 inhibitor αβ-methylene ADP (AOPCP, 1 nmol/ventricle/day) phenocopied the effect of the A2AR blockade by decreasing the expression of fear memory, an effect disappearing in CD73-knockout (KO) mice and in forebrain neuronal A2AR-KO mice. In the presence of PPADS (20 μM) to eliminate any modification of ATP/ADP-mediated P2 receptor effects, both AOPCP (100 μM) and the A2AR antagonist, SCH58261 (50 nM), decreased LTP magnitude in synapses of projection from the external capsula into the lateral amygdala, an effect eliminated in slices from both forebrain neuronal A2AR-KO mice and CD73-KO mice. These data indicate a key role of CD73 in the process of A2AR-mediated control of fear memory and underlying synaptic plasticity processes in the amygdala, paving the way to envisage CD73 as a new therapeutic target to interfere with abnormal fear-like emotional processing.

PDE1 inhibition and adenosine A2A receptor activation synergistically potentiates spontaneous calcium waves and transients in mouse ventricular myocytes

Abstract Background The phosphodiesterase-1C (PDE1C) and the adenosine A2A receptor (A2AR) are located in a macromolecular cluster and expected to modulate cAMP-dependent regulation of the intracellular calcium homeostasis in cardiac myocytes. In this contect, atrial fibrillation has been associated with a high incidence of spontanoeus arrhythmogenic calcium waves caused by excessive A2AR activation. On the other hand, inhibition of PDE1C and A2AR activation has also been reported to produce a synergistic and protective effect on cardiomyocyte survival, but little is known about the corresponding effects on the intracellular calcium homeostasis. Purpose This study, therfore, aimed to investigate how PDE1 inhibition and/or A2AR activation modulate sarcoplasmic reticulum (SR) calcium homeostasis in mouse ventricular myocytes. Methods Mouse ventricular myocytes (47 cells from 20 mice) were loaded with the calcium indicator Rhod-2 and spontaneous calcium release events at rest or caffeine induced calcium transients were visualized using resonance-scanning confocal microscopy at a frame rate of 150 Hz. The SR calcium content was estimated as the ratio of the peak fluorescence and the fluoresence the end of a 10 mM caffeine pulse (20s). Calcium leak was estimated as ratio of the flourescence emmision at baseline before and at the end of the caffeine pulse. Results The incidence of spontaneous calcium waves or transients increased from 0.7±0.3 events/min in control to 1.1±0.5 events/min and 3.4±1.0 events/min (p&amp;lt;0.01) after inhibition of PDE1 with the selective antagonist ITI-214 and the A2AR agonist CGS-21680 respectively. Concomitant PDE1 inhibition and A2AR activation synergistically increased the event frequency to 8.4±2.3 events/min (p&amp;lt;0.05). There were no significant differences in the corresponding SR calcium contents (4.4±0.3, 4.5±0.4, 4.7±0.4 and 4.1±0.2 for control, ITI-214, CGS-21680 and ITI-214+CGS-21680 respectively). However, there was a parallel increase in calcium leak from 8.7±4.2% in control to 21.8±9.8% with ITI-214, 17.6±7.2% with CGS-21680 and 37.5±11.1% when myocytes were exposed to both compounds (p&amp;lt;0.05), suggesting that the higher incidence of spontaneous calcium waves and transients observed upon PDE1 inhibition and/or A2AR activation is likely caused by a lowering of the threshold for spontaneous SR calcium release rather than by SR calcium overloading. Conclusion PDE1 inhibition exacerbates the incidence of spontaneous arrhythmogenic SR calcium release events in myocytes with high levels of A2AR activation, suggesting that PDE1 inhibition under these conditions increases the risk of arrhythmia. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Spanish Ministry of Science and Innovation

Shufeng Jiedu capsule inhibits inflammation and apoptosis by activating A2AAR and inhibiting NF-κB to alleviate LPS-induced ALI

Ethnopharmacological relevanceShufeng Jiedu capsule (SFJDC) is a pure form of traditional Chinese medicine (TCM) that contains eight medicinal plants. Known for its anti-inflammatory and antipyretic effects, it is mostly used to treat upper respiratory tract infections and other infectious diseases, such as colds, pharyngitis, laryngitis, and tonsillitis. Both acute lung injury (ALI) and COVID-19 are closely related to lung damage, primarily manifesting as lung inflammation and epithelial cell damage. However, whether SFJDC can improve ALI and by what mechanism remain unclear. The purpose of this study was to explore whether SFJDC could be used as a prophylactic treatment for COVID-19 by improving acute lung injury. Aim of the studyThe purpose of this study was to determine whether SFJDC could protect against ALI caused by lipopolysaccharide (LPS), and we wanted to determine how SFJDC reduces inflammation and apoptosis pharmacologically and molecularly. Materials and methodsPreadministering SFJDC at 0.1 g/kg, 0.3 g/kg, or 0.5 g/kg for one week was followed by 5 mg/kg LPS to induce ALI in mice. Observations included the study of lung histomorphology, tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6) secretion, as well as the ratio of lung wet/dry weights. In addition, RAW264.7 cells were treated for 24 h with 1 μg/mL LPS after being pretreated for 1 h with 0.5 mg/mL SFJDC. In the samples, we detected TNF-α, IL-1β, and IL-6. Cell apoptosis was detected by stimulating A549 cells for 24 h with RAW264.7 supernatant. Both in vitro and in vivo, the levels of A2A adenosine receptor (A2AAR), PKA, IκB, p-IκB, NF-κB P65 (P65), p–NF–κB P65 (p-P65), cleaved caspases-3 (Cc3), Bcl-2 associated X protein (Bax), and B-cell lymphoma-2 (Bcl-2) proteins were determined using Western blot analysis. ResultsLung tissue morphology was improved as SFJDC decreased cytokine secretion, the ratio of lung wet/dry weights, and lung tissue secretion of proinflammatory cytokines. The expression of A2AAR was increased by SFJDC, and the phosphorylation of NF-κB was inhibited. TUNEL staining and flow cytometry showed that SFJDC inhibited apoptosis by reducing the expression of Cc3 and the ratio of Bax/Bcl-2. ConclusionsAccording to the results of this study, SFJDC can reduce inflammation and inhibit apoptosis. A2AAR activation and regulation of NF-κB expression are thought to make SFJDC anti-inflammatory and anti-apoptotic. A wide range of active ingredients may result in an anti-inflammatory and antipyretic effect with SFJDC.

Abstract P2037: Activation Of A2A Adenosine Receptor Reduces Cardiac Dysfunction Resulting From Pulmonary Hypertension In Rats

Pulmonary hypertension (PH) is characterized by extensive pulmonary vascular remodeling, leading to right ventricle (RV) hypertrophy and dysfunction. This work evaluates the hypothesis that the activation of adenosine A2A receptor (AR-A2) by LASBio-1900 could interfere with the cardiac and vascular dysfunction on monocrotaline (MCT)-induced PH in rats. After 14 days of PH induction using a single injection of MCT (60 mg/kg i.p.), twelve male Wistar rats were randomly divided in groups and treated orally either with vehicle or LASSBio-1900 (180 μmol/kg/day). Hemodynamic parameters were obtained using the echocardiography and summarized in table 1. LASSBio-1900 reduced RV hypertrophy observed in PH because the Fulton index altered from 55.4 ± 2.3 to 35.3 ± 5.9% (p&lt;0.05). HP increased RV systolic and diastolic pressure from 19.2 ± 2.0 and 4.7 ± 1.0 to 51.5 ± 5.2 and 11.9 ± 1.3 mmHg, respectively. Ventricular dysfunction was recovered with LASSBio-1900 treatment reducing pressures to 28.4 ± 4.0 and 6.5 ± 0.8 mmHg, indicating improvement of RV dysfunction. Pulmonary arteriole (PA) muscularization was evaluated by alpha- smooth muscle actin stained and vessel medial wall area was expressed as the percentage of the portion positively stained relative to total transversal area. The increase of the wall thickness of distal PA from 61.7 ± 1.1 to 83.24 ± 1.82 % was reduced by LASSBio-1900 (75.2 ± 3.4 %). Perivascular collagen in PA was evaluated through picrosirius red staining and interstitial fibrosis was measured by obtaining the total collagen area per arteriole area. PH increased the perivascular collagen deposition in 15.1 ± 1.3% however, treatment with LASSBio-1900 reduced to 8.7 ± 1.4% (p&lt;0.05). Intense inflammation process on PH was confirmed with the increase of iNOS expression from 4.5 ± 0.3 (control) to 19.0 ± 0.9% (p&lt;0.05) with reduction to 8.7 ± 1.4% when treated with LASSBio-1900. In conclusion, the agonist of AR-A2, named LASSBio-1900 improved RV function and pulmonary structural alteration in PH, indicating a new alternative for treatment of PH-induced RV dysfunction.

A2A adenosine receptor activation prevents neutrophil aging and promotes polarization from N1 towards N2 phenotype.

Extracellular adenosine is a biologically active signaling molecule that accumulates at sites of metabolic stress in sepsis. Extracellular adenosine has potent immunosuppressive effects by binding to and activating G protein-coupled A2A adenosine receptors (A2AARs) on the surface of neutrophils. A2AAR signaling reproduces many of the phenotypic changes in neutrophils that are characteristic of sepsis, including decreased degranulation, impaired chemotaxis, and diminished ability to ingest and kill bacteria. We hypothesized that A2AARs also suppress neutrophil aging, which precedes cell death, and N1 to N2 polarization. Using human neutrophils isolated from healthy subjects, we demonstrate that A2AAR stimulation slows neutrophil aging, suppresses cell death, and promotes the polarization of neutrophils from an N1 to N2 phenotype. Using genetic knockout and pharmacological blockade, we confirmed that A2AARs decrease neutrophil aging in murine sepsis induced by cecal ligation and puncture. A2AARs expression is increased in neutrophils from septic patients compared to healthy subject but A2AAR expression fails to correlate with aging or N1/N2 polarization. Our data reveals that A2AARs regulate neutrophil aging in healthy but not septic neutrophils.

Abstract 2064: Targeting Cblb-Notch1 axis as a novel strategy for cancer immunotherapy

Abstract Background: Tumor-induced immunosuppression is a way in which cancers evade the host immune response. In recent years, a great effort has been made in designing immunotherapies that can boost the immune system response against cancer. However, not all tumors respond to these therapies. Therefore, there remains an unmet need for immunotherapies that are able to circumvent tumor-induced immunosuppression. Adenosine is an immunosuppressive metabolite which is overproduced in hypoxic tumor microenvironments and dampens T-cell anti-tumor immune responses. Adenosine A2A receptor (A2AR) activation was shown to downregulate Notch1, a key regulator of T-cell functions, in CD8+ T-cells, leading to immunosuppression. Notch1 signaling appears to protect T-cells against immunosuppressive signals, as Notch1 overexpressing T-cells were shown to be resistant to tumor-induced immunosuppression mediated by myeloid-derived suppressor cells (MDSC), as well as adenosine. We hypothesize that rescuing Notch1 from downregulation will make T-cells resistant to tumor-induced immunosuppression. We applied several functional assays in primary CD8+ T-cells and tumor-derived organoids to study how Notch1 is regulated by A2AR and explore strategies to target this pathway for cancer immunotherapy. Results: Our results indicate that the ubiquitin ligase Cbl-b, a negative regulator of T-cell functions, is responsible for the ubiquitination and degradation of Notch1 in CD8+ T-cells. A2AR signaling leads to Notch1 downregulation by promoting Cbl-b-mediated Notch1 degradation. Inhibition of Cbl-b using small molecule compounds restores Notch1 and T-cell functions in CD8+ T-cells in the presence of adenosine, making them resistant to A2AR-mediated immunosuppression. Cbl-b inhibitors show anti-tumor activity in tumor-derived organoids from pre-clinical models and enhance immune-checkpoint immunotherapy, by promoting T-cells anti-cancer responses. Conclusions: Our work suggests that promoting Notch1 signaling by blocking Cbl-b-mediated degradation results in increased T-cell responses and resistance to immunosuppression. Targeting Cbl-b-Notch1 axis represents a promising novel strategy to boost anti-cancer T-cell responses. Citation Format: Giulia Monticone, Fred Csibi, Silvana Leit, Jermaine E. Austin, Deniz A. Ucar, Fokhrul M. Hossain, Samarpan Majumder, Barbara A. Osborne, Christine Loh, Lucio Miele. Targeting Cblb-Notch1 axis as a novel strategy for cancer immunotherapy [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 2064.

CHANGES OF VASCULAR REACTIVITY TO ADENOSINE A1 AND A2A RECEPTOR AGONISTS AFTER HYPERBARIC OXYGENATION

Objective: Hyperbaric oxygenation (HBO) affects vascular reactivitiy, which plays an important role in the pathophysiology of hypertension and other cardiovascular diseases. New data suggest important effects of HBO on oxidative stress and alteration of production of vasoactive metabolites in blood vessels; however, the role of adenosine A1 and A2a receptors is still underinvestigated.This study aimed to determine the functional role of adenosine A1 and A2a receptors in changes of vascular reactivity in isolated middle cerebral arteries (MCA) of Spague-Dawley rats, after acute and intermittent HBO. Design and method: Male and female healthy Sprague-Dawley rats aged 8–10 weeks were divided into 3 groups: CTRL (control non HBO), A-HBO (single animals exposed to HBO for 2 hours) and 4D-HBO (animals exposed to HBO at a pressure od 2 bars for 2 hours daily for 4 days, with vascular experiments conducted on the fifth day). The rats were anesthetized with ketamine-chloride (3 ml/kg) and midazolam (0,5 ml/kg) and decapitated. The MCA were isolated, cannulated and pressurized for 60’at 80 mmHg to assess basal diameter using pressure myograph Danish Myo Technology. A1 adenosin receptor selective agonist CCPA or A2a adenosin receptor agonist CGS in concentrations from 10–5 to 10–10 M were added to the vessel chamber, and blood vessel diameter was measured after incubation of the drug for 15 minutes. Statistical analyses were performed with Two-way ANOVA tests; p &lt; 0.05 was considered significant. Results: The vasoconstriction in response to A1 agonist administration was similar between the CTRL and 4D-HBO group, while it was significantly reduced in A-HBO compared to the CTRL and 4D-HBO groups. The vasodilation in response to A2a stimulation was significantly reduced in rats exposed to A-HBO and I-HBO compared to CTRL at higher drug concentrations (10–7 to 10–5 M). Conclusions: Acute exposure of rats to HBO leads to decrease in the vasoconstrictive response to adenosine A1 receptor stimulation, while acute and intermittent exposure of rats to HBO is associated with a decrease in the vasodilatory response of MCA to adenosine A2a receptor activation, suggesting that HBO alters sensitivity to or expression of receptors.

Adenosine A2a Receptor Regulates Autophagy Flux and Apoptosis to Alleviate Ischemia-Reperfusion Injury via the cAMP/PKA Signaling Pathway.

Exploring effective methods to lessen myocardial ischemia-reperfusion injury still has positive significance. The adenosine A2a receptor (A2aR) has played a crucial part in cardiac ischemia-reperfusion injury. Previous studies revealed that the adenosine A2a receptor regulated autophagy, but the specific mechanism in myocardial ischemia-reperfusion injury was still unclear. We established an ischemia-reperfusion model (30 min of ischemia and 2 h of reperfusion) in vivo and a model with oxygen-glucose deprivation for 6 h and reoxygenation for 18 h (OGDR) in vitro. The ischemia-reperfusion injury resulted in prolonged QTc interval, left ventricular systolic dysfunction, and myocardial infarction. In vitro model, we found that the OGDR-induced autophagosomes and apoptosis caused myocardial cell death, as evidenced by a significant increase in the generation of lactate dehydrogenase and creatine kinase-MB. Furthermore, overactivated autophagy with rapamycin showed an anti-apoptotic effect. The interaction between autophagy and apoptosis in myocardial ischemia-reperfusion injury was complex and variable. We discovered that the activation of adenosine A2a receptor could promote the expression of Bcl-2 to inhibit the levels of Beclin-1 and LC3II. The number of autophagosomes exceeded that of autolysosomes under OGDR, but the result reversed after A2aR activation. Activated A2aR with its agonist CGS21680 before reperfusion saved cellular survival through anti-apoptosis and anti-autophagy effect, thus improving ventricular contraction disorders, and visibly reducing myocardial infarction size. The myocardial protection of adenosine A2a receptor after ischemia may involve the cAMP-PKA signaling pathway and the interaction of Bcl-2-Beclin-1.