Expressions of adenosine A2A receptors in coronary arteries and ...

International Journal of Cardiology 230 (2017) 427–431

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Expressions of adenosine A2A receptors in coronary arteries and peripheral blood mononuclear cells are correlated in coronary artery disease patients Vlad Gariboldi a,1, Donato Vairo b,1, Régis Guieu b,c,⁎,1, Marion Marlingue c, Eléonore Ravis d, David Lagier e, Alissa Mari b, Elsa Thery c, Frédéric Collart a, Marine Gaudry f, Laurent Bonello g, Franck Paganelli g, Jocelyne Condo b, Nathalie Kipson b, Emmanuel Fenouillet b, Jean Ruf b,2, Giovanna Mottola b,c,2 a

Department of Cardiac Surgery, Timone University Hospital, Marseille, France UMR MD2, Aix-Marseille University and IRBA, Marseille, France c Laboratory of Biochemistry, Timone University Hospital, Marseille, France d Department of Cardiology, Timone University Hospital, Marseille, France e Department of Anesthesia, Timone University Hospital, Marseille, France f Department of Vascular Surgery, Timone University Hospital, Marseille, France g Department of Cardiology, North University Hospital, Marseille, France b

a r t i c l e

i n f o

Article history: Received 11 July 2016 Received in revised form 15 November 2016 Accepted 16 December 2016 Available online 21 December 2016 Keywords: Coronary artery disease Adenosine A2A receptor Ischemia

a b s t r a c t 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 b 0.01), and correlated with A2AR expression in coronary and aortic tissues (Pearson's r: 0.77 and 0.59, p b 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 ± 215 pg/106 cells, p b 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. © 2016 Elsevier Ireland Ltd. All rights reserved.

1. Introduction The vasodilator adenosine is markedly released in the extracellular spaces by endothelial and muscle cells during inflammation [1] and ischemia [2–4]. Adenosine strongly impacts the cardiovascular system via four G-protein coupled receptors named A1R, A2AR, A2BR and A3R

⁎ Corresponding author at: UMR MD2, AMU, School of Medicine, Bvd P Dramard, F-13015, Marseille, France. E-mail address: [email protected] (R. Guieu). 1 The first three authors (V G, D V and RG) contributed equally to the work. 2 The last two authors (J R and GM) contributed equally to the work.

http://dx.doi.org/10.1016/j.ijcard.2016.12.089 0167-5273/© 2016 Elsevier Ireland Ltd. All rights reserved.

[5]. At the cellular level, A1R and A3R are coupled with G-proteins that inhibit cAMP production, while A2AR and A2BR are coupled with G-proteins that activate cAMP production in target cells [6,7]. In the cardiovascular system, activation of A1R and A3R leads to protection against myocardium/reperfusion injuries [8–10]. Adenosine also strongly acts on coronary blood flow (CBF) via A2AR [11] and A2BR [12–14]. Expression level of these receptors and their functional activity are therefore of paramount importance in CBF maintenance, and hence in the pathogenesis of CAD where myocardial oxygen delivery cannot match myocardial oxygen consumption due to altered CBF. It was shown that changes of A2AR expression on peripheral blood mononuclear cells (PBMC) occur in cardiovascular diseases [15], and that low A2AR levels

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are associated with adenosine metabolism abnormalities in CAD patients [16] raising the question whether such abnormal A2AR expression on PBMC may be representative of expression in altered coronary artery of CAD patients. The aim of this study was to compare A2AR expression in situ (coronary arteries and aortic tissues), and on PBMC in CAD patients to evaluate a possible systemic regulation of the adenosinergic response. We also evaluated A2AR activity (cAMP production) in aortic tissue and PBMC, and adenosine plasma level (APL).

Among the 20 patients consecutively included, 12 underwent coronary artery bypass graft using left or right internal thoracic artery grafts, which did not enable aortic tissue collection. Only 8 patients were treated using the saphenous vein graft, a condition that enabled us to collect a sample of the ascending aorta (median weight and range: 10.3 mg [8–16]). Tissue samples were stored in liquid nitrogen. Fresh tissue samples from aorta were tested extemporaneously in cAMP assays (see below). 2.6. Western blotting

2. Methods 2.1. Patients Fifteen men and 5 women (mean age [range]: 62[41–84] years) undergoing coronary artery bypass surgery were consecutively included. Controls were healthy subjects from the medical staff (8 women and 7 men; 60[39–72] years) without cardiac history and medication. The protocol was approved by the Ethics Committee of our institution (CPP Sud Méditerranée, Marseille, France). The study conformed to the standards set out in the 1983 Declaration of Helsinki. Written informed consent to participate in the study was obtained for all subjects. 2.2. Sample collection for adenosine measurement Samples were collected as previously described [17] before (basal state) and during coronary artery bypass surgery. Blood (3 ml) was taken using a syringe (5 mL) containing 3 ml of the cold stop solution (4 °C; 0.2 mM dipyridamole; 4.2 mM Na2 EDTA; 5 mM (9-erythro-3 nonyl) adenine (EHNA); 79 mM alpha-beta methylene adenosine 5′ diphosphates (AOPCP); 1UI/mL heparin sulfate; 0.9% NaCl) to prevent adenosine uptake by red blood cells, deamination into inosine, and ATP dephosphorylation by 5’nucleotidases. For healthy subjects, peripheral blood samples (3 ml) were similarly collected following veinipuncture. After collection, samples were immediately stored on ice prior to centrifugation. 2.3. Adenosine measurement APL was measured as described [18]. In brief, blood samples were centrifuged to remove cells, and plasma proteins were precipitated using 7% perchloric acid. After centrifugation the supernanatants were analyzed by chromatography using a modular system with a diode array detector (Chromsystem Germania®). Adenosine was identified by its elution time and spectrum, and quantified by comparison of peak areas vs those given by known quantities of adenosine (Sensitivity threshold: 3 pmol/mL of plasma matrix; intra- and inter-assay coefficients of variation: 3%–5%). 2.4. Peripheral blood mononuclear cells (PBMC) preparation PBMC were isolated from blood using the Vacutainer CPT system (Becton-Dickinson) following venipuncture from the brachial vein according to the manufacturer's instructions.

The procedure has been described [19,20]. Briefly, A2AR expression in PBMC and artery tissues was determined by Western blotting using Adonis, an agonist-like monoclonal antibody to human A2AR (By et al., 2009). PBMC (0.25 × 106) and tissue samples (0.5 mg) were solubilized using lysis buffer and sonication. Samples were then submitted to standard 12% gel electrophoresis prior to transfer onto a PVDF membrane. The filter was then incubated with Adonis (1 μg/ml). Blots were analyzed using horseradish peroxidase-labeled anti-mouse antibodies and enhanced chemiluminescence substrate. Antibody to the housekeeping protein Vinculin was used as an internal control for protein loading. Bands were submitted to densitometry analysis using the Image J 1.42q software (National Institutes of Health). Results were expressed in arbitrary units (AU) defined as pixels for the 45 kDa band (A2AR) / pixels for the 117 kDa band (Vinculin). 2.7. cAMP measurement The assay was performed on freshly prepared PBMC and aortic tissues that were collected during coronary artery bypass surgery. Samples were immediately placed into RPMI culture medium containing an inhibitor of phosphodiesterase (100 μM IBMX) and 1 μM Adonis. After a 2 h incubation at 37 °C, samples were centrifuged (1500 × g, 10 min) and supernatants were freezed (−80 °C) until cAMP quantitation. Frozen tissues (previously incubated, or not, with Adonis) were then homogenized in 6% trichloracetic acid at 4 °C to obtain a 10% (w/v) homogenate. Samples were centrifuged (2000 ×g, 15 min, 4 °C), supernatants were recovered and the pellets discarded. The supernatants were washed 4 times with 5 volumes of water-saturated diethyl ether and the upper organic layer from each wash was discarded. The aqueous extract was lyophilized and stored (− 80 °C) until their analysis. The cAMP concentration was determined using an immunoassay (cAMP Biotrak EIA, Amersham, Orsay, France) as described [18]. 2.8. Statistical analysis Patients' data were expressed either as median and range, or means and standard error of the mean (SEM) as stated in the text. Correlations between biological parameters were quantified and tested using Pearson rank correlation coefficient. Comparisons of biological parameters between patients and controls were performed using variance analysis (ANOVA two ways). Comparison of intra-individual biological parameters variations was performed using Nonparametric Wilcoxon pairedtest. All statistical tests were two-sided and p values b 0.05 were considered statistically significant. Analysis was performed with SPSS software (version 13.0 2004 SPSS Inc., Chicago, IL).

2.5. Cardiac tissues collection 3. Results We collected a small coronary artery tissue fragment at the site of distal anastomosis on the largest coronary artery found during the cross-clamp time. Samples (~ 1 mm3; median weight and range: 1.1 mg [0.8–1.4]) were taken with a cold knife blade, a procedure aimed to preserve tissue integrity. We considered that such lowvolume samples did not affect the quality and size of the anastomosis, and the surgery duration while they provided a sufficient amount of material to address A2AR expression only.

Clinical characteristics of patients are given in Table 1. Optimal medical treatment could not be carried out in several cases: 9 patients with myalgia and increased creatine kinase were statin intolerant and 2 patients with cough were angiotensin-converting enzyme inhibitor intolerant. Sixty percent of the patients had tri-troncular coronary lesions. None of the patients had cardiovascular events during surgery or hospitalization. APL at basal state was higher in patients compared with


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Table 1 Clinical data of patients included in the study.a*, b***, ** Gender (M/F) Age (years) Weight (kg) Height (cm) Body mass index Euroscore 2 (%)

15/5 61[40–83]⁎ 76[67–98] 170[160–182] 26[23–35] 1.8[0.5–6.9]

Hypertension Dyslipidemia Diabetes Atrial fibrillation Smoker

n n n n n

Coronary lesion category Single vessel disease Double vessel disease Triple vessel disease

n = 3 (15%) n = 5 (25%) n = 12 (60%)

= = = = =

10 (50%) 12 (60%) 3 (15%) 5 (25%) 12 (60%)

Systolic blood pressure⁎⁎ Hypertensive (mm Hg) Non hypertensive (mm Hg)

130[110–140] 122[110−130]

LVEF % Creatinin (μM) Hgb before surgery (g/L) Hgb end of surgery (g/L) Lactates (mM) CBP duration (min) Clamp duration (min) Total surgery duration (min)

60[30–72] 87[47–116] 141[109–177] 114[81–144] 1.9[1.3–3.1] 66[53–113] 52[36–87] 180[120−210]

Treatment Aspirin Beta blockers Statins Fibrates ACE inhibitors Calcium inhibitors Amiodarone Acenocoumarol⁎⁎⁎ Rivaroxaban⁎⁎⁎

n n n n n n n n n

= = = = = = = = =

12(60%) 12(60%) 11(55%) 1(5%) 5(25%) 5(25%) 2(10%) 3(15%) 2(10%)

LVEF: left ventricular ejection fraction; Hgb: hemoglobin; CBP: coronary bypass; ACE : angiotensin converting enzyme. a Median[range]. b Stopped 3 days before surgery. ⁎⁎ At the time on hospital admission (hypertensive vs non hypertensive, p N 0.05).

controls (mean ± SEM: 0.9 ± 0.2 μM vs 0.6 ± 0.1 μM; p b 0.01), and the level increased during the surgical procedure (2.1 ± 0.6 μM, p b 0.01; Fig. 1). A2AR expression measured in arbitrary units (AU) on PBMC was lower in patients compared with controls (0.83 ± 0.31 vs 1.20 ± 0.35 AU, p b 0.01; Fig. 2), and levels of A2AR expression in coronary arteries and aortic tissues were found similar (0.85 ± 0.23 and 0.92 ± 0.22 AU, respectively). Most importantly, A2AR expression on PBMC was correlated with the level measured in coronary (Fig. 3A) and aortic (Fig. 3B) tissues of patients. Interestingly, the three patients with the lowest coronary lesions (two patients with monotroncular and one with bitroncular coronary lesions) displayed the highest A2AR expression levels in the three types of patient's tissues (Fig. 3). The basal cAMP production levels were addressed in PBMC samples and found to be lower in patients vs controls (120 ± 42 vs 191 ± 65 pg/106 cells, p b 0.05). PBMC of patients and controls were then treated using a saturating dose (1 μM) of Adonis and the resulting maximal cAMP productions were also found to be lower in patients than in controls (360 ± 113 vs 560 ± 215 pg/106, p b 0.05; Fig. 4A), the increase following stimulation being similar in both groups (300% vs 293%; Fig. 4A). The amount of aortic tissues was enough in 8 patients to test cAMP production before and after agonist stimulation of A2AR. We observed

Fig. 1. Adenosine plasma level. APL was measured in CAD patients (n = 20) that underwent coronary artery bypass surgery and in healthy subjects (n = 15). Samples were collected before (basal) and during surgery. Results are individual values in μM with mean ± SEM in each group; a: p b 0.01 compared with controls; b: p b 0.01 compared with basal state.

that A2AR stimulation of aortic tissues also strongly increased cAMP production (Basal vs maximal values: 302 ± 98 vs 742 ± 285 pg/mg of tissue, p b 0.05; Fig. 4B), and it is worth noting that this increase (+246%, Fig. 4B) was in the same range as the one established using PBMC of patients (300%; Fig. 4A).

4. Discussion We found that i) A2AR expression and cAMP production were lower in PBMC of patients than in controls; ii) A2AR expression in coronary and aortic tissues, and PBMC are correlated in CAD patients, and iii) A2AR in PBMC and aortic tissues of patients displayed a similar capacity to induce cAMP production following agonist stimulation. While surface expression of A2AR on PBMC was previously found to reflect A2AR expression by the left ventricle in cardiac transplant recipient [15], the coronary and aortic expression of A2AR has never been evaluated in CAD patients.

Fig. 2. A2AR expression. A2AR expression was evaluated by Western blotting (WB) in PBMC and in coronary and aortic tissues of patients (n = 20). PBMC of healthy subjects established the control values (n = 15). Results are individual values in arbitraty units (AU) defined as A2AR/vinculin pixels ratio) with mean ± SEM in each group. Insert is a representative blot of A2AR and vinculine obtained from each kind of sample.

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Fig. 3. Correlation curves. Pearson's correlation analysis of A2AR expression evaluated by Western blotting in PBMC and in coronary (A) and aortic tissues (B) of patients (n = 20). Values in AU are those of Fig. 2.

Low expression of A2AR on PBMC of CAD patients has been previously reported [16] and we additionally found here that A2AR were similarly expressed on PBMC and in coronary and aortic tissues of patients. Interestingly, although weakly expressed, A2AR were found similarly functional when stimulated for cAMP production in aortic tissues and PBMC of CAD patients. Because A2AR expression measured in PBMC correlated with that measured in coronary and aortic tissues, their low expression suggested a defective function in arterial tissues, especially in coronary arteries. Such decreased A2AR expression may participate in the pathophysiology of CAD by lowering the myocardial blood flow through coronary microcirculation [21]. In animal studies, both A1R and A2AR were expressed in the left anterior descending arteries but only A2AR was detected in arterioles [22]. Here, we found that A2AR were also expressed in human aortic tissues. It was shown that aorta vasodilation implicates A2AR [23]. Thus, A2AR is involved in the control of blood pressure and high levels of A2AR expression on PBMC are associated with high blood pressure [24]. Here, probably due to their treatment that normalized blood pressure, treated hypertensive patients displayed the same A2AR expression as non hypertensive patients (Table 1). It was also reported that diabetes and obesity increase A2AR expression, which was attributed to the inflammatory process [25]. Our cohort is too small however to address in the present work a difference between diabetic vs non diabetic patients, only 3 patients having type 2 diabetes mellitus. Stimulation of A1R and A3R in the heart was shown to be associated with cardioprotection and ischemic preconditioning [26] while activation of A2AR in coronary smooth muscle cells, endothelial cells, and mononuclear cells results in vasodilation [27], neoangiogenesis [28], and increase in anti-inflammatory cytokines level [29,30]. Regarding the role of adenosine receptors on PBMC in CAD patients, it was reported that upregulation of A2AR results in adenosinergic T-cell

Fig. 4. Production of cAMP. Production of cAMP by PBMC in controls (n = 15) and patients (n = 20) (A) and by aortic tissues (patients n = 8) (B) prior to (−), or after (+) Adonis (1 μM) stimulation. Results are individual values of cAMP (pg) produced adjusted by cell number or mg tissue with mean ± SEM in each group; a: p b 0.05 compared without Adonis; b: p b 0.05 compared with controls.

immunosuppression during hypoxia [31], and conversely downregulation of A2AR in PBMC of CAD patients may therefore promote inflammation [32]. This hypothesis is consistent with the observation that activation of A2AR on CD4+ T-cells inhibits inflammation and reduces the size of myocardial infarction [33,34]. Another systemic regulation shared by heart tissues and white blood cells relates to the myocardial β-adrenergic receptor and its G-protein coupled receptor kinase-2 [35,36], and it was recently demonstrated that measurement of this lymphocytic kinase can be used to assess disease severity and predict patient mortality [37]. In agreement with previous data [15,38,39], we found here high APL in CAD patients. Adenosine is released during myocardial ischemia and/or cardiac failure and APL greatly increase during surgery, which is probably due to the hypoxemia status and inflammatory processes during the cardiopulmonary bypass procedure [40,41]. It may be also that the high APL found in basal conditions participates in the downregulation of A2AR we observed here in cardiovascular tissues and PBMC of patients. Finally, PBMC are exposed to the blood flow and communicate with coronary tissues via the lymphatic network [42]. That PBMC participate in the atherosclerosis process and the acute phase of myocardial ischemia is therefore consistent with our present finding that the A2AR signaling in PBMC and coronary and aortic tissues follows the same pattern. 5. Conclusion Expression of A2AR in PBMC of CAD patients correlated with those measured in coronary artery and aortic tissues, and A2AR activity in PBMC matched that observed in aorta. Furthermore, A2AR expression and activity were found reduced in PBMC of CAD patients as compared to controls. Measuring the expression level of A2AR on PBMC may represent a useful and convenient tool to address in situ expression in


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