Background: Vascular endothelial injury during ischemia generates apoptotic cell death and precedes apoptosis of underlying tissues. We aimed at studying the role of extracellular adenosine triphosphate (ATP) on endothelial cells protection against hypoxia injury. Methods: In a hypoxic model on endothelial cells, we quantified the extracellular concentration of ATP and adenosine. The expression of mRNA (ectonucleotidases, adenosine, and P2 receptors) was measured. Apoptosis was evaluated by the expression of cleaved caspase 3. The involvement of P2 and adenosine receptors and signaling pathways was investigated using selective inhibitors. Results: Hypoxic stress induced a significant increase in extracellular ATP and adenosine. After a 2-h hypoxic injury, an increase of cleaved caspase 3 was observed. ATP anti-apoptotic effect was prevented by suramin, pyridoxalphosphate-6-azophenyl-2′,4′-disulfonic acid (PPADS), and CGS15943, as well as by selective A2A, A2B, and A3 receptor antagonists. P2 receptor-mediated anti-apoptotic effect of ATP involved phosphoinositide 3-kinase (PI3K), extracellular signal-regulated kinases (ERK1/2), mitoK_ATP, and nitric oxide synthase (NOS) pathways whereas adenosine receptor-mediated anti-apoptotic effect involved ERK1/2, protein kinase A (PKA), and NOS. Conclusions: These results suggest a complementary role of P2 and adenosine receptors in ATP-induced protective effects against hypoxia injury of endothelial. This could be considered therapeutic targets to limit the development of ischemic injury of organs such as heart, brain, and kidney.

Background: Several studies have reported the beneficial effects of anti-platelet drugs in cardioprotection against ischaemia–reperfusion injuries. To date, no studies have focused on the indirect cytoprotective effects of ticagrelor via adenosine receptor on the endothelium. Method: By evaluating cell viability and cleaved caspase 3 expression, we validated a model of endothelial cell apoptosis induced by hypoxia. In hypoxic endothelial cells treated with ticagrelor, we quantified the extracellular concentration of adenosine, and then we studied the involvement of adenosine pathways in the cytoprotective effect of ticagrelor. Results: Our results showed that 10 µM ticagrelor induced an anti-apoptotic effect in our model associated with an increase of extracellular adenosine concentration. Similar experiments were conducted with cangrelor but did not demonstrate an anti-apoptotic effect. We also found that A2B and A3 adenosine receptors were involved in the anti-apoptotic effect of ticagrelor in endothelial cells exposed to 2 h of hypoxia stress. Conclusion: we described an endothelial cytoprotective mechanism of ticagrelor against hypoxia stress, independent of blood elements. We highlighted a mechanism triggered mainly by the increased extracellular bioavailability of adenosine, which activates A2B and A3 receptors on the endothelium.

Regenerative vascular medicine research has positioned mesenchymal stromal cells (MSCs) as a leading candidate to treat ischemic diseases. Recent studies have highlighted the emerging role of small extracellular vesicles (sEVs) produced by MSCs in their own potential. This study explores a strategy to improve the angiogenic potential of MSCs through the acquisition of endothelial features. Umbilical cord Wharton's jelly MSCs were cultured in fetal bovine serum‐free endothelial growth medium under hypoxic conditions (SH‐MSCs). sEVs were characterised by a multimodal approach: visualisation, count and particle size distribution, sEVs surface antigen, proangiogenic potential and ability to internalise into recipient cells. Compared with MSCs, SH‐MSCs exhibited significant morphological and phenotypical change characterised by the up‐regulation of CD31 and CD144 mRNA as well as a marked increase in sEVs secretion. MSC‐ and SH‐MSC‐derived sEVs had the capacity to internalise into endothelial cells, myoblasts and macrophages; exhibited a strong proangiogenic effect in vitro, particularly in promoting endothelial cell proliferation and pseudotube formation, likely due to an enriched cargo of angiogenic factors. These results highlight the dual benefit of hypoxia conditioning and endothelial differentiation of MSCs to optimise the angiogenic potential of their secreted sEVs, thus paving the way for innovative regenerative therapies in ischemic diseases.