Ticagrelor Improves Endothelial Function by Decreasing Circulating Epidermal Growth Factor (EGF)

Abstract

Ticagrelor is one of the most powerful P2Y₁₂ inhibitor. We have recently reported that, in patients with concomitant Stable Coronary Artery Disease (SCAD) and Chronic Obstructive Pulmonary Disease (COPD) undergoing percutaneous coronary intervention (PCI), treatment with ticagrelor, as compared to clopidogrel, is associated with an improvement of the endothelial function (Clinical Trial NCT02519608). In the present study, we showed that, in the same population, after 1 month treatment with ticagrelor, but not with clopidogrel, there is a decrease of the circulating levels of epidermal growth factor (EGF) and that these changes in circulating levels of EGF correlate with on-treatment platelet reactivity. Furthermore, in human umbilical vein endothelial cells (HUVEC) incubated with sera of the patients treated with ticagrelor, but not with clopidogrel there is an increase of p-eNOS levels. Finally, analyzing the changes in EGF and p-eNOS levels after treatment, we observed an inverse correlation between p-eNOS and EGF changes only in the ticagrelor group. Causality between EGF and eNOS activation was assessed in vitro in HUVEC where we showed that EGF decreases eNOS activity in a dose dependent manner. Taken together our data indicate that ticagrelor improves endothelial function by lowering circulating EGF that results in the activation of eNOS in the vascular endothelium.

Keywords: EGF; chronic obstructive pulmonary disease (COPD); eNOS; endothelial function; epidermal growth factor (EGF); stable coronary artery disease (SCAD); ticagrelor.

Figure 1

(A) EGF levels in the serum of patients at baseline and after 1 month of treatment. Black circle: patient randomized to clopidogrel. White square: patient randomized to ticagrelor. **P < 0.01; (B) EGF levels of patients representation before and after treatments. Orange circle: patient randomized to clopidogrel. Blue squares: patient randomized to ticagrelor. **P < 0.01.

Figure 2

Correlation between on-treatment platelet reactivity at 1 month and EGF changes. On treatment platelet reactivity was measured by VerifyNow after and expressed as P2Y₁₂ reaction unit (PRU). EGF changes were expressed as ratio between serum EGF concentration at 1 month over serum EGF concentration at baseline. Correlation was assessed by Spearman correlation test: R = 0.350; p = 0.020.

Figure 3

p-eNOS in HUVEC treated with patient's sera at baseline and after 1 month of treatment. Black circle: patient randomized to clopidogrel. p-eNOS levels are expressed as OD at 450 nm. White square: patient randomized to ticagrelor. **P < 0.01; ***P < 0.001.

Figure 4

(A) Changes in EGF levels in the serum of patients (on the y-axis is shown the ratio between EGF levels after 1 month of treatment and at baseline). Black circle: patient randomized to clopidogrel. White square: patient randomized to ticagrelor. *P < 0.05. (B) Changes in p-eNOS levels in HUVEC treated with patient's sera (on the y-axis is shown the ratio between p-eNOS levels in HUVEC treated with patient's sera after 1 month of treatment and at baseline). Black circle: patient randomized to clopidogrel. White square: patient randomized to ticagrelor. *P < 0.05.

Figure 5

Correlation between changes in levels of circulating EGF (ratio between EGF levels after 1 month of treatment and at baseline) and of p-eNOS in serum treated HUVEC (ratio between p-eNOS levels in HUVEC treated with patient's sera after 1 month of treatment and at baseline) in patients treated with clopidogrel (A) or with ticagrelor (B). Correlation was assessed by Spearman correlation test. Clopidogrel: R = 0.100; p = 0.714. Ticagrelor: R = −0.571; p = 0.002.

Figure 6

Effect of exogenous EGF in HUVEC. (A) HUVECs were treated for 48 h with different concentrations of EGF ant then were lysed. Proteins were quantified by BCA assay and 10 μg of protein were used for each well in the ELISA. p-eNOS levels were expressed as absorbance (OD) at 450 nm. Results are expressed as mean ± SD of three independent experiments. ***P < 0.001 (comparison between control and treatment). (B) HUVECs were treated for 48 h with EGF. Lysates were electrophoresed and immunoblotted with total eNOS antibody, β-actin antibody was used to ensure equal loading. Images are representative of one of three independent experiments. (C) Densitometric analysis of western blot. *P < 0.05 (comparison between control and treatments).