Changes in Circulating Extracellular Vesicles in Patients with ST-Elevation Myocardial Infarction and Potential Effects of Remote Ischemic Conditioning-A Randomized Controlled Trial

Abstract

(1) Background: Extracellular vesicles (EVs) have been recognized as a cellular communication tool with cardioprotective properties; however, it is unknown whether cardioprotection by remote ischemic conditioning (RIC) involves EVs. (2) Methods: We randomized patients with ST-elevation myocardial infarction (STEMI) undergoing primary percutaneous coronary intervention (PCI) to additionally receive a protocol of RIC or a sham-intervention. Blood was taken before and immediately, 24 h, four days and one month after PCI. Additionally, we investigated EVs from healthy volunteers undergoing RIC. EVs were characterized by a high-sensitive flow cytometer (Beckman Coulter Cytoflex S, Krefeld, Germany). (3) Results: We analyzed 32 patients (16 RIC, 16 control) and five healthy volunteers. We investigated platelet-, endothelial-, leukocyte-, monocyte- and granulocyte-derived EVs and their pro-thrombotic sub-populations expressing superficial phosphatidylserine (PS⁺). We did not observe a significant effect of RIC on the numbers of circulating EVs, although granulocyte-derived EVs were significantly higher in the RIC group. In line, RIC had not impact on EVs in healthy volunteers. Additionally, we observed changes of PS⁺/PEV, EEVs and PS⁺/CD15⁺ EVs irrespective of RIC with time following STEMI. 4) Conclusion: We provide further insights into the course of different circulating EVs during the acute and sub-acute phases of STEMI. With respect to the investigated EV populations, RIC seems to have no effect, with only minor differences found for granulocyte EVs.

Keywords: cardioprotection; extracellular vesicles; flow cytometry; myocardial infarction; remote ischemic conditioning.

Figure 1

Methodological considerations for the detection of extracellular vesicles (EVs). (A) Setting the upper size limit using 1000 nm Silica beads in a violet-side-scatter (SSC-H)/forward scatter plot (FSC-H). Isotype controls for (Phycoerythrin Cyanin 7) PC7 (B) and (Phycoerythrin) PE (C) were used to set gates. (D) Washing steps with sterile water were performed before every measurement to assure a clean system and avoid spill-over. (E) Labelled EVs were destroyed using triton to confirm their presence (EV gate in absolute numbers: 155/µL plasma). In comparison, (F) provides a scatter-plot of a conventionally stained sample (EV gate absolute numbers: 1138 EVs/µL plasma). APC = Allophycocyanin

Figure 2

Number of circulating platelet-derived and endothelial EVs stratified by remote ischemic conditioning at all investigated times. (A) Provides results of all platelet-derived EVs, whereas (B) provided results for all pro-coagulatory platelet-derived EVs, which also express PS on their surface and their ratio is provided in (C). Endothelial EVs are provided in (D). All results are presented as mean ± standard deviation. * Marks statistically significant results at the specific time compared to “Pre PCI” derived from mixed-models. EV—extracellular vesicle; PCI—percutaneous coronary intervention; PS—phosphatidylserine; RIC—remote ischemic.

Figure 3

Number of circulating leukocyte-derived EVs stratified by remote ischemic conditioning at all investigated times. (A) Provides results of all monocyte-derived EVs, (B) shows the number granulocyte-derived EVs. The ratio of PS positive and negative granulocyte derived EVs is presented in (C). (D) Provides results of PS⁺ leukocyte (CD15⁺) EVs. All results are presented as mean ± standard deviation. * marks statistically significant results at the specific time compared to “Pre PCI” derived from mixed-models. # marks a statistically significant interaction term comparing RIC and control at the specific time. EV—extracellular vesicle; PCI—percutaneous coronary intervention; PS—phosphatidylserine; RIC—remote ischemic conditioning.