Plasma A2AR Measurement Can Help Physicians Identify Patients Suspected of Coronary Chronic Syndrome: A Pilot Study.

The evaluation of suspected coronary artery disease (CAD) in the medical community is challenging. Patients with suspected coronary chronic syndrome (CCS) are referred by the medical community to be assessed by specialists for the performance of noninvasive tests that have high rates of false positives and false negatives. While troponins are the gold standard for evaluate myocardial injuries, there is no biomarker to assess myocardial ischemia in patient populations with negative electrocardiography or without an increase in troponin level. A2A adenosine receptors control the coronary blood flow through its vasodilating properties. It has been shown that patients with CAD have a lower A2AR expression on peripheral blood mononuclear cells, suggesting a link between A2AR production and the severity of CAD. Herein, we present a new and innovative method of inhibition ELISA for A2AR in the plasma of patients who permit the evaluation of the amount of soluble A2AR. For this analysis, the total study sample was 54, including 31 patients with CAD with stenosis > 50% and a significant fractional flow reserve (FFR < 0.8) (Group 1) and 23 patients with normal or non-obstructive coronary arteries (stenosis < 50% and nonsignificant FFR > 0.8) (Group 2). The % inhibition (which is linked to the presence of soluble receptors) with the plasma of patients with FFR < 0.8 was significantly lower than that of patients with FFR > 0.8 (median [range]: 68% [20.7−86.9] vs. 83% [67−88.4]; p < 0.001). The ROC curve indicated a good sensitivity/specificity ratio with a cut off of 72.5% and an area under the curve of 0.87. In conclusion, a rapid ELISA to assess soluble A2AR in the plasma shows promise to screen patients suspected of having CAD.

Homocysteine concentration and adenosine A2A receptor production by peripheral blood mononuclear cells in coronary artery disease patients.

Hyperhomocysteinemia is associated with coronary artery disease (CAD). The mechanistic aspects of this relationship are unclear. In CAD patients, homocysteine (HCy) concentration correlates with plasma level of adenosine that controls the coronary circulation via the activation of adenosine A2A receptors (A2A R). We addressed in CAD patients the relationship between HCy and A2A R production, and in cellulo the effect of HCy on A2A R function. 46 patients with CAD and 20 control healthy subjects were included. We evaluated A2A R production by peripheral blood mononuclear cells using Western blotting. We studied in cellulo (CEM human T cells) the effect of HCy on A2A R production as well as on basal and stimulated cAMP production following A2A R activation by an agonist-like monoclonal antibody. HCy concentration was higher in CAD patients vs controls (median, range: 16.6 [7-45] vs 8 [5-12] µM, P < 0.001). A2A R production was lower in patients vs controls (1.1[0.62-1.6] vs 1.53[0.7-1.9] arbitrary units, P < 0.001). We observed a negative correlation between HCy concentration and A2A R production (r = -0.43; P < 0.0001), with decreased A2A R production above 25 µM HCy. In cellulo, HCy inhibited A2A R production, as well as basal and stimulated cAMP production. In conclusion, HCy is negatively associated with A2A R production in CAD patients, as well as with A2A R and cAMP production in cellulo. The decrease in A2A R production and function, which is known to hamper coronary blood flow and promote inflammation, may support CAD pathogenesis.

Altered muscle membrane potential and redox status differentiates two subgroups of patients with chronic fatigue syndrome.

BACKGROUND: In myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS), altered membrane excitability often occurs in exercising muscles demonstrating muscle dysfunction regardless of any psychiatric disorder. Increased oxidative stress is also present in many ME/CFS patients and could affect the membrane excitability of resting muscles. METHODS: Seventy-two patients were examined at rest, during an incremental cycling exercise and during a 10-min post-exercise recovery period. All patients had at least four criteria leading to a diagnosis of ME/CFS. To explore muscle membrane excitability, M-waves were recorded during exercise (rectus femoris (RF) muscle) and at rest (flexor digitorum longus (FDL) muscle). Two plasma markers of oxidative stress (thiobarbituric acid reactive substance (TBARS) and oxidation-reduction potential (ORP)) were measured. Plasma potassium (K+) concentration was also measured at rest and at the end of exercise to explore K+ outflow. RESULTS: Thirty-nine patients had marked M-wave alterations in both the RF and FDL muscles during and after exercise while the resting values of plasma TBARS and ORP were increased and exercise-induced K+ outflow was decreased. In contrast, 33 other patients with a diagnosis of ME/CFS had no M-wave alterations and had lower baseline levels of TBARS and ORP. M-wave changes were inversely proportional to TBARS and ORP levels. CONCLUSIONS: Resting muscles of ME/CFS patients have altered muscle membrane excitability. However, our data reveal heterogeneity in some major biomarkers in ME/CFS patients. Measurement of ORP may help to improve the diagnosis of ME/CFS. Trial registration Ethics Committee "Ouest II" of Angers (May 17, 2019) RCB ID: number 2019-A00611-56.

Adenosine Receptor Profiling Reveals an Association between the Presence of Spare Receptors and Cardiovascular Disorders.

Adenosine and its receptors exert a potent control on the cardiovascular system. This review aims to present emerging experimental evidence supporting the existence and implication in cardiovascular disorders of specific adenosinergic pharmacological profiles, conforming to the concept of "receptor reserve", also known as "spare receptors". This kind of receptors allow agonists to achieve their maximal effect without occupying all of the relevant cell receptors. In the cardiovascular system, spare adenosine receptors appear to compensate for a low extracellular adenosine level and/or a low adenosine receptor number, such as in coronary artery disease or some kinds of neurocardiogenic syncopes. In both cases, the presence of spare receptors appears to be an attempt to overcome a weak interaction between adenosine and its receptors. The identification of adenosine spare receptors in cardiovascular disorders may be helpful for diagnostic purposes.

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

BACKGROUND: Altered 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. METHODS: Artery 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). RESULTS: A2AR 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. CONCLUSION: A2AR 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.

Adenosine Plasma Level and A2A Receptor Expression in Patients With Cardiogenic Shock.

OBJECTIVES: To investigate whether adenosine A2A receptors lead to vasodilation and positive inotropic function under stimulation and whether they play a role in the control of blood pressure in patients with cardiogenic shock. DESIGN: Prospective observational study. SETTING: Monocentric, Hopital Nord, Marseille, France. SUBJECTS: Patients with cardiogenic shock (n = 16), acute heart failure (n = 16), and acute myocardial infarction (n = 16). INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Arterial adenosine plasma level and A2A receptor expression on peripheral blood mononuclear cells were evaluated by mass spectrometry and Western blot, respectively, at admission and after 24 hours. Hemodynamic parameters, including systemic vascular resistance, were also assessed. Mean adenosine plasma level at admission was significantly higher in patients with cardiogenic shock (2.74 ± 1.03 µM) versus acute heart failure (1.33 ± 0.27) or acute myocardial infarction (1.19 ± 0.27) (normal range, 0.4-0.8 µM) (p < 0.0001). No significant correlation was found between adenosine plasma level and systemic vascular resistance. Mean adenosine plasma level decreased significantly by 24 hours after admission in patients with cardiogenic shock (2.74 ± 1.03 to 1.53 ± 0.68; p < 0.001). Mean A2A receptor expression was significantly lower in patients with cardiogenic shock (1.18 ± 0.11) versus acute heart failure (1.18 ± 0.11 vs 1.39 ± 0.08) (p = 0.005). CONCLUSIONS: We observed high adenosine plasma level and low A2A receptor expression at admission in patients with cardiogenic shock versus acute heart failure or acute myocardial infarction. This may contribute to the physiopathology of cardiogenic shock.

Specific Pharmacological Profile of A2A Adenosine Receptor Predicts Reduced Fractional Flow Reserve in Patients With Suspected Coronary Artery Disease.

BACKGROUND: The rapid and reliable exclusion of myocardial revascularization is a major unmet clinical need in patients with suspected coronary artery disease (CAD) and non-contributive electrocardiography and troponin. Non-invasive tests have high rates of false positives and negatives, and there is no biomarker to assess myocardial ischemia. The presence of spare adenosine A2A receptors (A2AR)-characterized by a high dissociation constant/half maximal effective concentration (KD/EC50) ratio-expressed on peripheral blood mononuclear cells (PBMC) has been associated with ischemia during exercise stress testing in patients with CAD. In this work, we investigated the diagnostic accuracy of spare A2AR versus fractional flow reserve (FFR) in patients with suspected CAD. METHODS AND RESULTS: Sixty patients with suspected CAD, but non-contributive electrocardiography and troponin, were consecutively enrolled in this prospective study. The binding (KD), functional response (cyclic adenosine monophosphate [cAMP] production; EC50) on PBMC A2AR were compared with FFR results. Patients were divided into 3 groups: 17 (group 1) with normal coronary angiography (n=13) or stenosis <20% (n=4); 21 with CAD and non-significant FFR (group 2); and 22 with CAD and significant FFR (group 3). Median KD/EC50 was 6-fold higher in group 3 (4.20; interquartile range: 2.81-5.00) than group 2 (0.66; interquartile range: 0.47-1.25) and 7-fold higher than group 1 (0.60; interquartile range: 0.30-0.66). CONCLUSIONS: In patients with suspected CAD and non-contributive electrocardiography and troponin, the absence of spare A2AR on PBMC may help to rule out myocardial ischemia. CLINICAL TRIAL REGISTRATION: URL: http://www.clinicaltrials.gov. Unique identifier: NCT03218007.

Expressions of adenosine A2A receptors in coronary arteries and peripheral blood mononuclear cells are correlated in coronary artery disease patients.

BACKGROUND: Altered coronary blood flow occurs in patients with coronary artery disease (CAD). Adenosine strongly impacts blood flow mostly via adenosine A2A receptor (A2AR) expressed in coronary tissues. As part of a systemic regulation of the adenosinergic system, we compared A2AR expression in situ, and on peripheral blood mononuclear cells (PBMC) in CAD patients. METHODS AND RESULTS: Aortic and coronary tissues, and PBMC were sampled in 20 CAD patients undergoing coronary artery bypass surgery and consecutively included. Controls were PBMC obtained from 15 healthy subjects. Expression and activity of A2AR were studied by Western blotting and cAMP measurement, respectively. A2AR expression on PBMC was lower in patients than in controls (0.83±0.31 vs 1.2±0.35 arbitrary units; p<0.01), and correlated with A2AR expression in coronary and aortic tissues (Pearson's r: 0.77 and 0.59, p<0.01, respectively). Basal and maximal cAMP productions following agonist stimulation of PBMC were significantly lower in patients than in controls (120±42 vs 191±65 and 360±113 vs 560±215pg/106 cells, p<0.05, respectively). In CAD patients, the increase from basal to maximal cAMP production in PBMC and aortic tissues was similar (+300% and +246%, respectively). CONCLUSION: Expression of A2AR on PBMC correlated with those measured in coronary artery and aortic tissues in CAD patients, A2AR activity of PBMC matched that observed in aorta, and A2AR expression and activity in PBMC were found reduced as compared to controls. Measuring the expression level of A2AR on PBMC represents a good tool to address in situ expression in coronary tissues of CAD patients.

Spare Adenosine A2a Receptors Are Associated With Positive Exercise Stress Test In Coronary Artery Disease.

During exercise, cardiac oxygen-consumption increases and the resulting low oxygen level in myocardium triggers coronary vasodilation. This response to hypoxia is controlled notably by the vasodilator adenosine and its A2A receptor (A2AR). According to the "spare receptor" pharmacological model, a strong A2AR-mediated response can occur in the context of a large number of receptors remaining unoccupied, activation of only a weak fraction of A2AR (evaluated using KD) resulting in maximal cAMP production (evaluated using EC50), and hence in maximal coronary vasodilation. In coronary artery disease (CAD), myocardial ischemia limits adaptation to exercise, which is commonly detected using the exercise stress test (EST). We hypothesized that spare A2AR are present in CAD patients to correct ischemia. Seventeen patients with angiographically-documented CAD and 17 control subjects were studied. We addressed adenosine-plasma concentration and A2AR-expression at the mononuclear cell-surface, which reflects cardiovascular expression. The presence of spare A2AR was tested using an innovative pharmacological approach based on a homemade monoclonal antibody with agonist properties. EST was positive in 82% of patients, and in none of the controls. Adenosine plasma-concentration increased by 60% at peak exercise in patients only (p<0.01). Most patients (65%), and none of the controls, had spare A2AR (identified when EC50/KD≤0.1) and a low A2AR-expression (mean: -37% vs controls; p<0.01). All patients with spare A2AR had a positive EST whereas the subjects without spare A2AR had a negative EST (p<0.05). Spare A2AR are therefore associated with positive EST in CAD patients and their detection may be used as a diagnostic marker.

High homocysteine levels prevent via H2 S the CoCl2 -induced alteration of lymphocyte viability.

High homocysteine (HCy) levels are associated with lymphocyte-mediated inflammatory responses that are sometimes in turn related to hypoxia. Because adenosine is a potent lymphocyte suppressor produced in hypoxic conditions and shares metabolic pathways with HCy, we addressed the influence of high HCy levels on the hypoxia-induced, adenosine-mediated, alteration of lymphocyte viability. We treated mitogen-stimulated human lymphocytes isolated from healthy individuals and the human lymphoma T-cell line CEM with cobalt chloride (CoCl2 )to reproduce hypoxia. We found that CoCl2 -altered cell viability was dose-dependently reversed using HCy. In turn, the HCy effect was inhibited using DL-propargylglycine, a specific inhibitor of the hydrogen sulphide (H2 S)-synthesizing enzyme cystathionine-γ-lyase involved in HCy catabolism. We then addressed the intracellular metabolic pathway of adenosine and HCy, and the role of the adenosine A2A receptor (A2 A R). We observed that: (i) hypoxic conditions lowered the intracellular concentration of HCy by increasing adenosine production, which resulted in high A2 A R expression and 3', 5'-cyclic adenosine monophosphate production; (ii) increasing intracellular HCy concentration reversed the hypoxia-induced adenosinergic signalling despite high adenosine concentration by promoting both S-adenosylhomocysteine and H2 S production; (iii) DL-propargylglycine that inhibits H2 S production abolished the HCy effect. Together, these data suggest that high HCy levels prevent, via H2 S production and the resulting down-regulation of A2 A R expression, the hypoxia-induced adenosinergic alteration of lymphocyte viability. We point out the relevance of these mechanisms in the pathophysiology of cardiovascular diseases.

Effect of hyperoxic and hyperbaric conditions on the adenosinergic pathway and CD26 expression in rat.

The nucleoside adenosine acts on the nervous and cardiovascular systems via the A2A receptor (A2AR). In response to oxygen level in tissues, adenosine plasma concentration is regulated in particular via its synthesis by CD73 and via its degradation by adenosine deaminase (ADA). The cell-surface endopeptidase CD26 controls the concentration of vasoactive and antioxidant peptides and hence regulates the oxygen supply to tissues and oxidative stress response. Although overexpression of adenosine, CD73, ADA, A2AR, and CD26 in response to hypoxia is well documented, the effects of hyperoxic and hyperbaric conditions on these elements deserve further consideration. Rats and a murine Chem-3 cell line that expresses A2AR were exposed to 0.21 bar O2, 0.79 bar N2 (terrestrial conditions; normoxia); 1 bar O2 (hyperoxia); 2 bar O2 (hyperbaric hyperoxia); 0.21 bar O2, 1.79 bar N2 (hyperbaria). Adenosine plasma concentration, CD73, ADA, A2AR expression, and CD26 activity were addressed in vivo, and cAMP production was addressed in cellulo. For in vivo conditions, 1) hyperoxia decreased adenosine plasma level and T cell surface CD26 activity, whereas it increased CD73 expression and ADA level; 2) hyperbaric hyperoxia tended to amplify the trend; and 3) hyperbaria alone lacked significant influence on these parameters. In the brain and in cellulo, 1) hyperoxia decreased A2AR expression; 2) hyperbaric hyperoxia amplified the trend; and 3) hyperbaria alone exhibited the strongest effect. We found a similar pattern regarding both A2AR mRNA synthesis in the brain and cAMP production in Chem-3 cells. Thus a high oxygen level tended to downregulate the adenosinergic pathway and CD26 activity. Hyperbaria alone affected only A2AR expression and cAMP production. We discuss how such mechanisms triggered by hyperoxygenation can limit, through vasoconstriction, the oxygen supply to tissues and the production of reactive oxygen species.

NF-κB enhances hypoxia-driven T-cell immunosuppression via upregulation of adenosine A(2A) receptors.

Hypoxia affects inflammation by modulating T-cell activation via the adenosinergic system. We supposed that, in turn, inflammation influences cell hypoxic behavior and that stimulation of T-cells in inflammatory conditions involves the concerted action of the nuclear factor κB (NF-κB) and the related hypoxia-inducible factor 1α (HIF-1α) on the adenosinergic system. We addressed this hypothesis by monitoring both transcription factors and four adenosinergic signaling parameters - namely adenosine, adenosine deaminase (ADA), adenosine A2A receptor (A2AR) and cAMP - in T-cells stimulated using phorbol myristate acetate and phytohemagglutinin and submitted to hypoxic conditions which were mimicked using CoCl2 treatment. We found that cell viability was more altered in stimulated than in resting cells under hypoxia. Detailed analysis showed that: i) NF-κB activation remained at basal level in resting hypoxic cells but greatly increased following stimulation, stimulated hypoxic cells exhibiting the higher level; ii) HIF-1α production induced by hypoxia was boosted via NF-κB activation in stimulated cells whereas hypoxia increased HIF-1α production in resting cells without further activating NF-κB; iii) A2AR expression and cAMP production increased in stimulated hypoxic cells whereas adenosine level remained unchanged due to ADA regulation; and iv) the presence of H2S, an endogenous signaling molecule in inflammation, reversed the effect of stimulation on cell viability by down-regulating the activity of transcription factors and adenosinergic immunosuppression. We also found that: i) the specific A2AR agonist CGS-21680 increased the suppressive effect of hypoxia on stimulated T-cells, the antagonist ZM-241385 exhibiting the opposite effect; and ii) Rolipram, a selective inhibitor of cAMP-specific phosphodiesterase 4, and 8-Br-cAMP, a cAMP analog which preferentially activates cAMP-dependent protein kinase A (PKA), increased T-cell immunosuppression whereas H-89, a potent and selective inhibitor of cAMP-dependent PKA, restored cell viability. Together, these data indicate that inflammation enhances T-cell sensitivity to hypoxia via NF-κB activation. This process upregulates A2AR expression and enhances cAMP production and PKA activation, resulting in adenosinergic T-cell immunosuppression that can be modulated via H2S.

Ticagrelor increases adenosine plasma concentration in patients with an acute coronary syndrome.

OBJECTIVES: This study aimed to investigate the impact of ticagrelor on adenosine plasma concentration (APC) in acute coronary syndrome (ACS) patients. BACKGROUND: Ticagrelor is a direct-acting P2Y12-adenosine diphosphate receptor blocker. The clinical benefit of ticagrelor compared with clopidogrel in ACS patients suggests that the drug has non-platelet-directed properties. Animal and in vitro models suggested that the "pleiotropic" properties of ticagrelor may be related to an interaction with adenosine metabolism. METHODS: We prospectively randomized 60 ACS patients to receive ticagrelor or clopidogrel. The APC was measured by liquid chromatography. To assess the mechanism of APC variation, we measured adenosine deaminase concentration, adenosine uptake by red blood cells, and cyclic adenosine monophosphate production by cells overexpressing adenosine receptors. The P2Y12-adenosine diphosphate receptor blockade was assessed by the vasodilator-stimulated phosphoprotein index. RESULTS: Patients receiving ticagrelor had significantly higher APC than patients receiving clopidogrel (1.5 µM [interquartile range: 0.98 to 1.7 µM] vs. 0.68 µM [interquartile range: 0.49 to 0.78 µM]; p < 0.01). The APC was not correlated with vasodilator-stimulated phosphoprotein (p = 0.16). Serum-containing ticagrelor inhibited adenosine uptake by red blood cells compared with clopidogrel or controls (p < 0.01 for both comparisons). Adenosine deaminase activity was similar in serum of patients receiving clopidogrel or ticagrelor (p = 0.1). Ticagrelor and clopidogrel had no direct impact on adenosine receptors (p = not significant). CONCLUSIONS: Ticagrelor increases APC in ACS patients compared with clopidogrel by inhibiting adenosine uptake by red blood cells.