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

Abstract Extracellular vesicles (EV) can transfer cellular molecules for specific intercellular communication with potential relevance in pathological conditions. We searched for the presence in plasma from coronary artery disease (CAD) patients of EV containing the adenosine A2A receptor (A2AR), a signalling receptor associated with myocardial ischaemia and whose expression is related to homocysteine (HCy) metabolism. Using protein organic solvent precipitation for plasma EV preparation and Western blotting for protein identification, we found that plasma from CAD patients contained various amounts of EV with ubiquitin bound to A2AR. Interestingly, the presence of ubiquitinated A2AR in EV from patients was dependent on hyperhomocysteinemia, the amount being inversely proportional to A2AR expression in peripheral mononuclear cells in patients with the highest levels of HCy. CEM, a human T cell line, was also found to released EV containing various amounts of ubiquitinated A2AR in stimulated conditions depending on the hypoxic status and HCy level of culture medium. Together, these data show that ubiquitinated A2AR‐containing EV circulate in the plasma of CAD patients and that this presence is related to hyperhomocysteinemia. A2AR in plasma EV could be a useful tool for diagnosis and a promising drug for the treatment of CAD.

protein organic solvent precipitation for plasma EV preparation and Western blotting for protein identification, we found that plasma from CAD patients contained various amounts of EV with ubiquitin bound to A 2A R. Interestingly, the presence of Together, these data show that ubiquitinated A 2A R-containing EV circulate in the plasma of CAD patients and that this presence is related to hyperhomocysteinemia.
A 2A R in plasma EV could be a useful tool for diagnosis and a promising drug for the treatment of CAD.

K E Y W O R D S
adenosine A 2A receptor, coronary artery disease, extracellular vesicles, homocysteine, ubiquitin disorders and other human diseases. 5 In cardiovascular disease, EV represent one of the most intensely studied and rapidly growing areas of research. 6,7 EV were shown to exert diverse and sometimes discordant biological effects in different studies related to cardiovascular disease. For example, EV can play an atheroprotective or atherogenic role in several conditions accompanying atherosclerosis. 8 Adenosine greatly impacts the cardiovascular system via four specific G protein-coupled receptors, named respectively A 1 , A 2A , A 2B and A 3 . Among them, the A 2A receptor (A 2A R) is strongly expressed in coronary cells and its activation increases coronary blood flow, 9 partly through the production of cAMP in target cells. 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 A 2A R expressed on peripheral blood mononuclear cells (PBMC) of CAD patients reflect coronary tissue expression showing the systemic nature of the adenosinergic signalling. 15 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.

| Human materials
Fourteen patients (11 men and three women, 56-58 years old) with angiographically documented CAD were included in this pilot study (Table 1). The first group consisted of eight patients selected blind and the second group was six patients with moderate hyperhomocysteinemia. Controls were eight healthy individuals (six men and two women, 56-64 years old) with a normal level of HCy (Table 1) recruited from the research laboratory or hospital staff, without medical treatment or history of cardiovascular disease. The study was conducted in compliance with the principles of the Declaration of Helsinki and approved by the Ethics Committee for Human Research of our University Hospital. All participants provided written informed consent to participate.
Plasma and PBMC were obtained from blood collected by ve- Extracellular vesicles were isolated from fresh plasma by a simple, rapid and reliable solvent-based protein precipitation method as previously described. 17 Briefly, 200 µL of plasma was mixed with 800 µL cold acetone (−20°C) in 1.5 mL conical tube and centrifuged at 3000 × g for 1 minute. After centrifugation, 500 µL of EV-contain- Blots were then revealed using phosphatase alkaline-labelled secondary antibody by a BCIP ® /NBT-Purple liquid substrate system for membranes, (B3679, Sigma-Aldrich). Densitometric quantification of the blot was performed using the ImageJ software (https :// imagej. nih.gov). Briefly, a scanned blot image (in grey scale and TIFF format) was imported into ImageJ, areas of interest were selected and plots of pick profiles were generated. Lines were drawn to select the peaks of interest, and the peak areas were integrated and converted into pixel intensities. Results were given as per cent of total pixels of all the relevant bands on a same migration line on the blot.
The protein load was controlled by the reproducibility of the results, Western blots being done in triplicate.

| Homocysteine assay
Total HCy from the human plasma was quantified with the liquid chromatography-tandem mass spectrometry Clinmass ® apparatus using the dedicated kit (Homocysteine in plasma/serum, Recipe) according to the manufacturer's instructions. Results (in µmol/L) are the mean ± SD of duplicates. with 5% SDS, 5% 2-mercaptoethanol and a complete set of protease inhibitors (Roche) and heated for 5 minutes at 95°C. EV from culture supernatant and CEM T cells were analysed by Western blotting as described above for the human material.
The insoluble violet formazan crystals associated with the cell pellets were dissolved into pure dimethyl sulfoxide and absorbance was measured at 550 nm. Results are the mean ± SD of duplicates.

| Presence of ubiquitinated A 2A R in EV of CAD patients
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. 17 Freeze-dried EV were submitted to Western blot procedure using Adonis, a monoclonal antibody to the human A 2A R 18 widely used in previous studies [11][12][13][14][15] 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 1A). We found CD9 bands displaying similar intensi-   (Table 1).

| Comparison of EV and cell expression of A 2A R
We then confirmed the presence of heavy A 2A R (61 kD) in EV released by stimulated CEM T cells in culture medium and prepared using a commercial kit ( Figure 2B). Western blots were performed as above using EV from 10 mL of culture supernatant and 0.25 × 10 6

| Relationship between A 2A R export in EV and hyperhomocysteinemia
Finally, to further explore the relationship between the A 2A R export via EV and hyperhomocysteinemia, we compared on a same blot the A 2A R content of the EV from 6 healthy individuals (Reference Letters C-H) with normal level of HCy taken as controls with that of 6 CAD patients (Reference Numbers 9-14) with moderate hyperhomocysteinemia (Table1). The results shown in Figure 3 confirmed the previous data. The control samples lacked A 2A R, but those from CAD patients with moderate hyperhomocysteinemia all expressed the A 2A R band of a pixels intensity of 6.6 to 40.4% ( Figure 3A). The scatter plot in Figure 3B shows

| D ISCUSS I ON
We report here for the first time the presence of ubiquitinated A 2A R in EV isolated from plasma of CAD patients. Ubiquitinated A 2A R were also found in EV released in the culture supernatant of a stimulated human T cell line.
Plasma contains a mix of EV with different sizes derived from many different cells in varying proportions. Sorting of many membrane proteins into EV coincides with their association with tetraspanin membrane proteins. 20 We found that A 2A R expression in EV was related to a similar expression of EV assessed by CD9 and CD63 tetraspanins.
We also found here that A 2A R in EV was ubiquitinated. Ubiquitin  acting on the mitogen-activated protein kinase pathways 26,27 and cell signalling can be related to endocytosis and endosomal trafficking. 28 We therefore assume that HCy could act on the ubiquitination process so that A 2A R do not undergo proteosomal degradation but are exported into exosomes. In this case, hyperhomocysteinemia would favour the salvage pathway described above.
Exosomes contain ubiquitinated proteins that can serve as markers of exosomes 29 and ubiquitin plays a major role in the endosomal trafficking leading to exosomal release. 30

CO N FLI C T O F I NTE R E S T
None.

AUTH O R S ' CO NTR I B UTI O N
JR designed the study. FP provided the human samples. DV performed the experiments. JR, DV, FP and RG analysed the data. JR wrote the article with input from all authors.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.