Specific Pharmacological Profile of A _2A Adenosine Receptor Predicts Reduced Fractional Flow Reserve in Patients With Suspected Coronary Artery Disease

Abstract

BackgroundThe 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 (A2 AR)—characterized by a high dissociation constant/half maximal effective concentration (KD/EC 50) 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 A2 AR versus fractional flow reserve (FFR) in patients with suspected CAD.Methods and ResultsSixty 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; EC 50) on PBMC A2 AR 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/EC 50 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).ConclusionsIn patients with suspected CAD and non‐contributive electrocardiography and troponin, the absence of spare A2 AR on PBMC may help to rule out myocardial ischemia.Clinical Trial Registration URL: http://www.clinicaltrials.gov. Unique identifier: NCT03218007.

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

“…First, we tested plasma from four patients with CAD (Reference Numbers 1, 4, 6 and 7) for the presence of EV carrying A 2A R. EV were isolated from plasma using acetone to remove proteins by precipitation leaving purified EV suspended in the liquid phase . Freeze‐dried EV were submitted to Western blot procedure using Adonis, a monoclonal antibody to the human A 2A R widely used in previous studies and anti‐tetraspanin CD9 as a biomarker of EV with a low molecular weight (28 kD) migrating at high distance from the A 2A R to test both in same lanes of gel. Intriguingly, we found using Adonis various amounts of a band (intensity from 0% to 68.5% pixels depending of the patient) that migrated to a higher position than expected (45 kD for cellular A 2A R) between the 75 and 50 kD markers in three to four patients (Figure A).…”

“…In CAD, one could hypothesize that A 2A R circulate from donor cells to target cells as a salvage pathway to be stimulated by adenosine when vasodilation is required for oxygen delivery in failing artery tissues. This mechanism may explain the low expression of A 2A R with spare receptor characteristics (EC 50 < K D ) found in PBMC from CAD patients presenting a major cardiovascular risk by a release from the cells into the blood of EV carrying A 2A R, constituting a circulating pool of receptors.…”

“…Extracellular vesicles and PBMC were analysed by Western blotting as previously reported. [11][12][13][14][15] Briefly, freeze-dried EV corresponding to 100 µL of plasma and cell pellets corresponding to 0.25 × 10 6 PBMC were solubilized in 15 µL Laemmli sample buffer (BioRad) with 5% SDS, 5% 2-mercaptoethanol and a complete set of protease inhibitors (Roche), heated for 5 minutes at 95°C and loaded on 12% SDS-PAGE minigel (BioRad). Prestained molecular weight markers (BioRad) have always been loaded onto the gel.…”

“…10 A 2A R from patients with coronary artery disease (CAD) is poorly expressed and, consequently, produces low level of cAMP, two characteristics that are associated with myocardial ischaemia, as documented by positive exercise stress testing or reduced flow reserve. [11][12][13] The down-regulation of A 2A R expression in CAD patients is related to the homocysteine (HCy) metabolism via its degradation product H 2 S. 14 Circulating EV can be considered as a reserve of functional G protein-coupled receptors as previously suggested from data obtained on a mouse model of heart cellular stress for angiotensin II type 1 receptor. 16 Taking into account the major role of A 2A R in cardiovascular disease and the potential contribution of circulating EV in delivering cell receptor from donor to target cells, we searched for the presence of A 2A R in EV from plasma of patients with CAD and culture supernatant of human lymphoblastoid T cells cultured in CAD-like conditions.…”

“…Among them, the A 2A receptor (A 2A R) is strongly expressed in coronary cells and its activation increases coronary blood flow, partly through the production of cAMP in target cells . A 2A R from patients with coronary artery disease (CAD) is poorly expressed and, consequently, produces low level of cAMP, two characteristics that are associated with myocardial ischaemia, as documented by positive exercise stress testing or reduced flow reserve . The down‐regulation of A 2A R expression in CAD patients is related to the homocysteine (HCy) metabolism via its degradation product H 2 S .…”