Ticagrelor Induces Angiogenesis in Progenitor and Mature Endothelial Cells In Vitro: Investigation of the Possible Role of Adenosine

Abstract

Ticagrelor, a reversible platelet P2Y₁₂ receptor antagonist, exerts various pleiotropic actions, some of which are at least partially mediated through adenosine. We studied the ticagrelor and adenosine effect on the angiogenic properties of progenitor CD34⁺-derived endothelial colony-forming cells (ECFCs). Angiogenesis studies were performed in vitro using capillary-like tube formation and spheroid-based angiogenesis assays. The effects of adenosine receptor antagonists, including DPCPX (A₁ antagonist), SCH58621 (A_2A antagonist), MRS1706 (A_2B inverse agonist and antagonist), MRS1220 (A₃ antagonist) and adenosine deaminase (ADA), were also investigated. Ticagrelor, adenosine, and their combination increased capillary-like tube formation and spheroid sprout formation by ECFCs in a dose-dependent manner. This effect was significantly reduced by SCH58621, MRS1706, and their combination, as well as by ADA. By contrast, DPCPX and MRS1220 did not exhibit any inhibitory effects. Similar results were obtained when mature human umbilical vein endothelial cells (HUVECs) were studied. These results show that ticagrelor stimulates angiogenesis by progenitor and mature endothelial cells in an adenosine-dependent pathway in which the adenosine receptors A_2A and A_2B play major roles. The significance of these results at the clinical level in patients with atherothrombotic events and treated with ticagrelor needs to be investigated.

Keywords: adenosine; angiogenesis; endothelial colony-forming cells; endothelial progenitor cells; equilibrative nucleoside transporter-1; ticagrelor.

Figure 1

The dose-dependent effects of Vascular Endothelial Growth Factor (VEGF), ticagrelor, adenosine, and the combination of ticagrelor and adenosine on capillary-like tube formation by (A) Endothelial Colony-Forming Cells (ECFCs) and (Β) Human Umbilical Vein Endothelial Cells (HUVECs), and on spheroid sprout formation by (C) ECFCs and (D) HUVECs. The values represent the means ± SDs from at least 6 independent biological replicates each performed in 2 technical replicates. $ p < 0.02 and $$ p < 0.005 compared to BSA/PBS. # p < 0.05, * p < 0.02, ** p < 0.01, ## p < 0.005, and & p < 0.001 compared to DMSO. ^ p < 0.001 compared to 1.0 μM ticagrelor or 5.0 μM adenosine.

Figure 2

(A). The effects of Adenosine Deaminase (ADA) alone or in combination with ticagrelor or adenosine on capillary-like tube formation. (B). The effects of ADA (alone) or in combination with ticagrelor or adenosine on capillary-like tube formation on spheroid sprout formation. The values represent the means ± SDs from at least 6 independent biological replicates each performed in 2 technical replicates. (A). * p < 0.03 compared to ticagrelor alone, ^# p < 0.02 compared to adenosine, and ^$ p < 0.005 compared to the combination of ticagrelor with adenosine. (B). * p < 0.02 compared to ticagrelor alone, ^# p < 0.01 compared to adenosine, and ^$ p < 0.005 compared to the combination of ticagrelor with adenosine.

Figure 3

The effects of the adenosine receptor A_2A antagonist SCH58621, adenosine receptor A_2B inverse agonist, and antagonist MRS1706 on capillary-like tube formation by (A) ECFCs and (B) HUVECs, and on spheroid sprout formation by (C) ECFCs and (D) HUVECs, induced by ticagrelor, adenosine, and their combination. The values represent the means ± SDs from at least 6 independent biological replicates each performed in 2 technical replicates. * p < 0.02 and ** p < 0.01 compared to ticagrelor, adenosine, or their combination in the absence of SCH58621 and MRS1706. ^# p < 0.05 compared to ticagrelor, adenosine, or their combination in the presence of either SCH58621 or MRS1706. HUVECs: human umbilical vein endothelial cells, ECFCs: endothelial colony-forming cells.

Figure 4

Representative images of 2D capillary-like tube formation by ECFCs that were treated with ticagrelor, adenosine, and their combination in the presence of adenosine receptor A_2A antagonist SCH58621, adenosine receptor A_2B inverse agonist, and antagonist MRS1706. Scale bar 100 μΜ.

Figure 5

Representative images of 3D spheroid sprout formation by ECFCs that were treated with ticagrelor, adenosine, and their combination in the presence of adenosine receptor A_2A antagonist SCH58621, adenosine receptor A_2B inverse agonist, and antagonist MRS1706. Scale bar 200 μM.