Diagnostic of Obliterative Arteriopathies

Abstract

The invention relates to a method of diagnosis or prognosis of an obliterative arteriopathy in a subject, comprising determining the expression level of adenosine A.sub.2A receptors expressed at the cell surface of peripheral blood mononuclear cells obtained from said subject. The invention also relates to an agonist-like monoclonal antibody directed to the human adenosine A.sub.2A receptor, useful for carrying out this method.

Claims

1. An in vitro method of diagnosis or prognosis of an obliterative arteriopathy in a subject, characterised in that it comprises the following steps: a) determining the expression level of adenosine A.sub.2A receptors (A.sub.2AR) expressed at the cell surface of peripheral blood mononuclear cells (PBMC) obtained from said subject, and b) comparing the expression level determined in step a) with expression levels of A.sub.2AR expressed at the cell surface of PBMC previously obtained from said subject or with the standard expression level of A.sub.2AR expressed at the cell surface of PBMC, an expression level determined in step a) lower than said expression level of A.sub.2AR expressed previously obtained from said subject or with said standard expression level of A.sub.2AR constituting a marker of an obliterative arteriopathy or of a predisposition to an obliterative arteriopathy.

2. The method according to claim 1, characterised in that the obliterative arteriopathy is selected from the group consisting of coronary artery disease and peripheral obliterative arteriopathy, preferably coronary artery disease.

3. The method according to claim 1, characterised in that the PBMC are obtained from a blood sample collected from brachial vein of said subject.

4. The method according to claim 1, characterised in that the expression level of A.sub.2AR is determined by Western blotting or flow cytometric assay.

5. The method according to claim 1, characterised in that it further comprises a step of determining the presence or the absence of spare adenosine A.sub.2A receptor (spare A.sub.2AR) on PBMC obtained from said subject, the presence of spare A.sub.2AR constituting a further marker of an obliterative arteriopathy or of a predisposition to an obliterative arteriopathy.

6. The method according to claim 4, characterised in that the expression level of A.sub.2AR and/or the presence of spare A.sub.2AR are determined with the agonist-like monoclonal antibody produced by the hybridoma deposited at CNCM under number I-4908.

7. The method according to claim 1, characterised in that it further comprises a step of determining the adenosine plasma concentration (APC) change during exercise stress testing (EST) in said subject from blood samples, an increase in APC during EST constituting a further marker of an obliterative arteriopathy or a predisposition to an obliterative arteriopathy.

8. The method according to claim 7, characterised in that the exercise stress testing is a treadmill exercise test.

9. The hybridoma producing an agonist-like monoclonal antibody directed to the human adenosine A.sub.2A receptor deposited at CNCM on Nov. 7, 2014, under the number I-4908.

10. The agonist-like monoclonal antibody directed to the human adenosine A.sub.2A receptor produced by the hybridoma of claim 9, or a fragment thereof.

11. An isolated fragment from the antibody of claim 10, comprising the three CDRs from said agonist-like monoclonal antibody and able to bind A.sub.2AR.

12. A polypeptide comprising one, two or the three CDRs from the antibody of claim 10 and able to bind A.sub.2AR.

13. Use of an agonist-like monoclonal antibody directed to the human adenosine A.sub.2A receptor or an isolated fragment of claim 11 wherein said isolated fragment is able to bind A.sub.2AR, for the in vitro diagnosis or prognosis of an obliterative arteriopathy in a subject.

14. Use according to claim 13, characterised in that the agonist-like monoclonal antibody directed to the human adenosine A.sub.2A receptor is an antibody directed to human adenosine A.sub.2A receptor.

15. Kit for the in vitro diagnosis or prognosis of coronary artery disease or peripheral obliterative arteriopathy in a subject comprising: an agonist-like monoclonal antibody directed to the human adenosine A.sub.2A receptor or an isolated fragment of claim 11 or 3 polypcptidc of claim 12 wherein said antibody, isolated fragment is able to bind A.sub.2AR, at least one positive control, consisting in PBMC lysate from a subject having a coronary artery disease or peripheral obliterative arteriopathy, and at least one negative control, consisting in PBMC lysate from an healthy subject.

Description

[0072] In addition to the above features, the invention further comprises other features which will emerge from the following description, which refers to an example illustrating the present invention, as well as to the appended figures.

[0073] FIG. 1 shows the adenosine plasma concentration (APC) at basal level (T0), at the end of exercise stress testing (T1), and after 10 min recovery (T2) in 30 patients. a: p<0.01 compared with T0.

[0074] FIG. 2 shows the APC at basal level (T0), at the end of EST (T1), and after 10 min recovery (T2) in patients with positive (+, n=15) or negative (−, n=15) EST and in 15 healthy subjects who served as controls. a: p<0.01 compared with T0+; b: p=0.12 compared with T0−.

[0075] FIG. 3 shows (A) the A.sub.2A adenosine receptor expression (A.sub.2AR) evaluated by Western blotting in patients (whole population of patients n=30) who underwent an EST as scheduled of their clinical follow-up and in 15 healthy subjects who served as controls. EST was positive in 15 patients and negative in 15 patients. A.sub.2AR expression as a function of positive or negative EST and in controls is also given. (B): example of Western blot of A.sub.2AR obtained from a patient with positive EST (EST+), negative EST (EST−) and in control subject.

EXAMPLE: DIAGNOSIS OF CORONARY ARTERY DISEASE

[0076] 1) Patients and Methods

[0077] Patients

[0078] It was performed a monocenter observational study including 33 consecutive patients: 25 men and 8 women, mean age 62.6±13.5 years, range 33-89. Among the patients, 16 had established coronary artery disease and underwent EST as part of their scheduled clinical follow-up and 17 underwent EST for the first time as part of their clinical investigations. Fifteen healthy subjects (9 men and 6 women, mean age 60±15.6 years, range 27-81) served as controls for basal biological parameters.

[0079] EST Procedure

[0080] All patients performed the treadmill exercise test according to the Bruce protocol (Bruce R A 1974). The EST was considered positive when the patient developed significant ST segment T wave changes (>2 mm) and ST segment horizontal or down looping depression and or typical chest pain.

[0081] APC Measurement

[0082] Blood samples (3 mL) were collected (obtained) and processed as described previously (Saadjian et al., 2002; Deharo et al., 2012), using laboratory-made tubes containing 2 mL of cold-stop solution under vacuum. The stop solution was composed of saline containing dipyridamole 0.2 mM; ethylene diamine tetracetic acid disodium (Na 2 EDTA 4 mM); erythro-9-(2-hydroxy-3-nonyl adenine (EHNA, 5 mM); alpha, beta-methyleneadenosine 5′ diphosphate (AOPCP, 79 mM); 2′ deoxycoformycin 10 μg/mL; and heparin sulfate 1 IU/mL.

[0083] This method allows whole blood to mix quickly with the stop solution in order to prevent red blood cell uptake and degradation of adenosine (Guieu et al., 1999, Saadjian et al., 2002, Deharo et al., 2012, Bonello et al., 2013). For patients, samples were collected at basal level, at the end of EST, and after 10 min recovery period.

[0084] APC dosage has been described (Guieu et al., 1999; Saadjian et al., 2002). In brief, after collection, samples were quickly centrifuged (4° C., 1500 g) and then deproteinized (perchloric acid, 70% 1/10V). After another centrifugation (2500 g for 10 mM) supernatants were pipetted off, mixed with 1 mL of phosphate buffer and analyzed by HPLC. A modular system with a diode array detector (Chrom System, Germany) was used. Samples were eluted with a methanol gradient (0% to 35% in 60 min) on a Merck LiChrospher C18 column (Nottingham, UK). Adenosine was identified using its elution time and spectrum, and quantified by comparison of peak areas with those of known quantities of adenosine. The sensitivity threshold was 2 nM of plasma matrix. The intra- and inter-assay coefficients of variation ranged from 1% to 3%.

[0085] A.sub.2AR Expression on Peripheral Blood Mononuclear Cells

[0086] PBMC was chosen because behavior of A.sub.2AR expressed at the surface of peripheral blood mononuclear cells (PBMC) mirrors the behavior of these receptors on cells of the cardiovascular system (Varani et al., 2003). The methodology has been described (Franceschi et al., 2013; Jacquin et al., 2013). In brief, blood samples from brachial vein of patients were collected at basal level into tubes containing sodium citrate, a polyester gel, and a density gradient liquid (BD Vacutainer CPT, Beckton Dickinson, Franklin Lakes, N.J.). PBMC were prepared according to the manufacturer's instructions prior to lysis using 62.5 mM Tris-HCl buffer, pH 8.3, containing 2% SDS, 10% glycerol, 0.01% bromophenol blue and 5% mercaptoethanol followed by 10 min sonication treatment at 47 kHz. A.sub.2AR expression in lysates was determined by Western blotting using the monoclonal antibody directed to A.sub.2AR named Adonis (By et al., 2009). After sonication, samples were submitted to standard electrophoresis in Mini Protean II system (BioRad, Hercules, Calif.). Separated proteins in 12% acrylamide minigel were electrotransferred onto a PVDF membrane that was then placed in the blot holder of the SNAP i.d. protein detection system (Millipore, Billerica, Mass.), saturated with non-fat dried milk, and incubated 20 min with Adonis (1 μg/ml). Blots were visualized by horseradish peroxidase-labeled anti-mouse antibodies and enhanced chemiluminescence substrate (SuperSignal West Femto, Pierce Biotechnology, Rockford, Ill.) using a Kodak system. Bands (45 kDa for A.sub.2AR) were measured densitometrically using Image J 1.42q software developed at the U.S. National Institutes of Health. Results were expressed as arbitrary units defined as pixels of the A.sub.2AR band versus blot background.

[0087] Statistical Analysis

[0088] Quantitative variables were expressed using medians and interquartile or means and standard deviations (SD). Variance analysis (two-way ANOVA) was used to compare APC and A.sub.2AR expression. A Wilcoxon test was used for pair-wise comparisons with Bonferroni correction to account for the inflation in type I error. All statistical tests were two-sided and P-values less than 0.05 were considered statistically significant. Analyses were performed with Prism® software.

[0089] 2) Results

[0090] Clinical characteristics of the patients are given in Table 1 below. Results of exercise stress testing are given in Table 2 below.

TABLE-US-00001 Patient baseline characteristics n = 30. Demographics Age years (mean, range) 62.6 [33-89] Men/women 22/8 Cardiac Risk Factors Diabetes N = 8 Current or former smoker N = 12 Hyperlipidemia N = 13 Cardiac disease Documented CAD N = 16 Prior MI None Prior PCI N = 14 Prior CABG N = 0 CABG: Coronary artery bypass graft; CAD: Coronary artery disease; PCI: Percutaneous coronary intervention

TABLE-US-00002 TABLE 2 Stress test results. Data are presented as median and interquartile range. EST Performance Duration (min)  6.4 [6.2-8.8] Maximum Workload (Mets) 120 [75-135] Peak HR (b.p.m) 141 [125-144] Peak SBP (mmHg) 194 [185-211] Chest Pain No N = 27 Yes N = 3

[0091] Fifteen EST were positive and fifteen were negative, one was equivocal and was excluded from further investigations. One patient was unable to reach maximal heart rate.

[0092] APC (μM) (median [25th-75th percentile]) for the whole population of patients (n=30) was 0.6 [0.5-0.7] at basal level, which was not significantly different from APC of controls: 0.6 [0.5-0.7]; p=0.7. Basal APC of EST− or EST+ patients did not differ significantly from controls: EST−: 0.65 [0.55-0.7] vs controls; p=0.7, EST+: 0.6[0.5-0.6] vs controls p=0.7; and see FIG. 2. During EST, APC increased significantly in the whole population of patients, 0.6 [0.5-0.7] vs 0.8 [0.7-0.9], p=0.0002. However, this increase was limited to EST+ patients, 0.6[0.5-0.6] vs 0.80 [0.75-0.9]; p<0.01. APC did not increase significantly in the EST− group 0.65 [0.55-0.7] vs 0.70 [0.65-0.9]; p=0.12.; see FIG. 2. APC returned to basal values after 10 min recovery, 0.52 [0.31-0.63] and see FIG. 1.

[0093] A.sub.2AR expression (arbitrary units, AU) was lower than controls in the whole population of patients, 22.5 [19-29] vs 30 [26.75-32.25]; p=0.005 (see FIG. 3). However, the low A.sub.2AR expression was limited to EST+ patients, 19 [16-21.25]; p<0.0001 compared with controls). A.sub.2AR expression of EST− patients was not significantly different from controls, 29 [26-32.25]; p=0.75 and see FIG. 3.

[0094] 3) Conclusion

[0095] These results show that patients with a positive treadmill test had lower A.sub.2AR expression compared with negative treadmill test and a significant increase in APC at peak exercise compared to patients with a negative treadmill test. Such an increase is likely related to a myocardial ischemia process that occurs during EST in patients with obstructive coronary artery disease.

[0096] The present study suggests low basal A.sub.2AR expression and increase in APC during treadmill is associated with positive EST and coronary artery disease.

[0097] Since 100% of the patients having a CAD also have low basal A.sub.2AR expression, the present study suggests that low A.sub.2AR expression level of mononuclear cells is associated with CAD. The measure of basal A.sub.2AR expression of mononuclear cells can therefore be useful to screen CAD patients.

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