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

(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.


Blood handling
We only used citrate-anticoagulated blood for the analysis of extracellular vesicles (EV). In line with current recommendations (1), we performed two centrifugation steps: blood was centrifuged within 30 minutes after collection for the first time at room temperature with 2500xg for 20 minutes to generate platelet-poor plasma (PPP). PPP was collected and centrifuged again for 20 minutes at room temperature at 2500xg to generate platelet-free plasma (PFP). Aliquots of the PFP were immediately frozen at -80° Celsius until further use.

Labelling of extracellular vesicles
For EV analysis, PFP was thawed under controlled conditions in a water bath at 35° Celsius to avoid formation of ice crystals and reduce cryo-precipitation during this process.

Flowcytometry-based detection and characterization of extracellular vesicles
All analyses were performed on a Cytoflex LX flow cytometer (Beckman Coulter, Krefeld, Germany) and were analyzed using the CytExpert software version 2.2. The flow cytometer was equipped with four lasers (375nm, 405nm, 488nm, 638nm, 561nm and 375nm, respectively). Daily maintenance using manufacture beads, washing steps and instrument calibration were performed in accordance with the manufacturer's recommended protocols. Furthermore, we performed additional washing steps prior to EV analyses to assure a clean system.
In line with current recommendations (1) and previously established protocols (4,6), we used fluorescence-triggering of signals over conventional side-scatter triggering for all analyses as it refined the detection of especially small EVs (7). The trigger signal was set on a positive fluorescence signal in the Lactadherin or Calcein AM channels. In addition, we used 1000nm Silica beads (Ksiker Biotech, Steinfurt, Germany) to set an upper size limit to define EVs. We choose Silica beads as their refractive index is closer to biological material wherefore estimation of EV size is better compared to Polysterene beads (8). Beads were detected in a violet-side scatter and forward scatter plot (Figure 3).
Gates for all antibodies were set corresponding isotype control antibodies (Figure 3). To assure the measurement of EVs, we also stained PBS without sample with antibodies and Calcein AM and introduced 2-minute washing steps with sterile water to avoid spill over. Finally, EVs of stained samples were destroyed using triton. EV characterization of these samples was not possible anymore ( Figure 3).
Prior to characterization of all EVs, we performed dilution experiments to avoid swarm effects (9). In flowcytometry-based EV characterization, swarm effects may occur when several very small signals/particles/EVs pass the laser at the same time. The laser could not discriminate all the individual signals, wherefore one large signal would be detected. Finding the right dilution is, therefore, important prior to the analysis of the whole project. Finally, we kept the rate of flow on the flow cytometer at low speed and the number of detected events per second below 2000 as described previously (4).
A previously published study did not find differences regarding the enumeration of EVs comparing counting beads and the detected event rate on a Cytoflex S (4). Hence, we defined all EV populations as follow: CD31 + /CD54 + /CD146 + /CD41for endothelial EVs (EEV), CD41 + for platelet-derived EVs (PEV), CD14 + for monocyte-derived EVs (MEV), CD15 + for leukocyte-derived EVs (LEV) and CD66b + for granulocyte-derived EVs (GEVs), respectively. The gating strategy is also shown in Figure 1. EVs are given as number per µl of pure PFP.