High homocysteine levels prevent via H₂S the CoCl₂‐induced alteration of lymphocyte viability

Abstract

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 (H2S)‐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 AR). We observed that: (i) hypoxic conditions lowered the intracellular concentration of HCy by increasing adenosine production, which resulted in high A2 AR 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 H2S production; (iii) DL‐propargylglycine that inhibits H2S production abolished the HCy effect. Together, these data suggest that high HCy levels prevent, via H2S production and the resulting down‐regulation of A2 AR expression, the hypoxia‐induced adenosinergic alteration of lymphocyte viability. We point out the relevance of these mechanisms in the pathophysiology of cardiovascular diseases.

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

“…This relationship was confirmed in cellulo. These results are in agreement with our previous observation that A 2A R cell expression in hypoxia condition is down‐regulated by H 2 S produced from HCy via the transsulfuration pathway . In adenosinergic signalling, exoenzymes CD39 and CD73 are expressed in cancer exosomes where they produce adenosine, which inhibits T cell functions in tumour environment .…”

“…A 2A R expression in CEM T cells increased under stress hypoxia (42.4% pixels) and returned to control value in the presence of HCy (28.6% pixels). Hypoxia stress was confirmed by about fifty per cent of cell loss as compared to control (A 550 nm = 0.8 ± 0.1 vs 1.8 ± 0.2), which was reversed by HCy (A 550 nm = 1.7 ± 0.2) as assessed by the MTT assay . The highest content of A 2A R in EV (38.4% pixels) matched with the lowest cellular content of A 2A R (28.6% pixels) and, conversely, the lower one in EV was the highest in cells (30.2% vs 42.4% pixels, respectively).…”

“…Cells were seeded in 75‐cm 2 flasks (0.5 × 10 6 cells/mL, 50 mL/flask) and stimulated using phorbol myristate acetate (PMA, 50 ng/mL) and phytohemagglutinin (PHA, 5 µg/mL) for 24 hours in control condition. As previously reported in this cellular model, hypoxia condition was achieved by adding 100 µmol/L CoCl 2 to the culture medium and the effect of HCy on cells in hypoxia was obtained by adding 200 µmol/L HCy just prior to the addition of CoCl 2 …”

“…Cell viability was monitored using the 3‐(4,5‐dimethyl‐2‐thiazolyl)‐2,5‐diphenyl‐2H‐tetrazolium bromide (MTT) assay as previously described . MTT (0.5 mg in 100 µL of PBS, pH 7.3) was added to 24‐well plates containing 0.5 × 10 6 cells/mL cultured in the three conditions (Control, Hypoxia, Hypoxia + HCy) 3 hours prior to the end of the 24 hours incubation period.…”

“…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 . 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 …”