Circulating Microparticles Decrease After Cardiac Stress in Patients With Significant Coronary Artery Stenosis

ABSTRACT

Background

Cardiac stress leads to a dynamic increase of circulating microparticles (MPs) in healthy individuals that is diminished in individuals with vascular disease. The impact of coronary ischemia on circulating MP level is unknown. This study investigates the kinetics of circulating MPs during cardiac stress in patients with coronary artery stenosis.

Hypothesis

Patients with significant coronary stenosis show altered circulating MP levels after cardiac stress.

Methods

Eighty patients with stable coronary artery disease underwent dobutamine stress echocardiography (DSE) on the day before coronary angiography. Before, immediately after, at 4 hours, and at 24 hours after DSE, blood was drawn to determine CD144 ⁺ endothelial microparticles (EMPs), CD14 ⁺ CD16 ⁺ monocyte‐derived microparticles (MMPs), and CD31 ⁺ CD42b ⁺ platelet microparticles. A significant stenosis was defined as stenosis diameter ≥70% in a major native epicardial coronary artery with a diameter of ≥2.5 mm.

Results

Significant coronary artery stenoses were found in 41 patients. In these patients, CD144 ⁺‐EMP and CD14 ⁺ CD16 ⁺‐MMP concentrations decreased immediately after DSE. Stimulation of target endothelial cells with sera from patients with significant coronary artery stenoses significantly augmented endothelial capacity to take up EMPs, but not MMPs, in vitro. Serum‐induced enhancement of endothelial phosphatidylserine receptor expression was found as a potential mechanism of increased endothelial EMP uptake and subsequently reduced circulating EMP levels after cardiac stress.

Conclusions

Cardiac ischemia leads to reduced circulating MP levels under cardiac stress. Changes of endothelial MP uptake capacities could be one possible mechanism.

Introduction

Microparticles (MP) are subcellular plasma membrane vesicles ranging in size from 0.1 to 1 µm that are released from various cell types during activation by physical or chemical stress, including apoptosis.1, 2, 3, 4 These MPs can be identified by parent‐cell surface markers. Certain subtypes, particularly endothelial cell–derived MPs (EMPs), are increased in individuals with traditional cardiovascular risk factors and associated with cardiovascular prognosis.5, 6, 7, 8, 9

Although many studies have successfully addressed the role of circulating MPs as a biomarker and effector of cardiovascular diseases (CVDs) under resting conditions, their function under stress conditions is poorly investigated so far.

Importantly, a recent study demonstrated that a broad range of circulating MPs rapidly increases immediately after cardiac stress selectively induced by dobutamine stress echocardiography (DSE) in healthy individuals.10, 11 This presumably physiological response was diminished in patients with coronary artery disease (CAD) undergoing DSE, as levels of circulating MP surprisingly remained unchanged after cardiac stress.10

Coronary ischemia locally and systemically affects the function of endothelial cells, platelets, and monocytes, but its influence on circulating MP level is unknown. To explore circulating MPs as a tool for risk stratification in patients with stable CAD, we studied the kinetics of circulating MP levels during cardiac stress in patients with or without significant coronary artery stenoses.

Methods

Eighty patients with previously documented stable CAD and indication for coronary angiography due to typical symptoms were included into this prospective study after written informed consent. The day before invasive coronary angiography, all individuals underwent DSE with serial measurement of MP and biomarker levels (Figure 1). The study has been registered (Deutsches Register Klinischer Studien, Freiburg, DRKS‐ID: DRKS00000737) and was approved by the local ethics committee of the University of Bonn (No. 169/10).

Figure 1.

Study flow. According to the study protocol, study participants underwent DSE on the day before QCA. Blood was drawn before, immediately after, at 4 hours, and at 24 hours after DSE. Abbreviations: DSE, dobutamine stress echocardiography; MPs, microparticles; Neg, negative; NT‐proBNP, N‐terminal pro brain natriuretic peptide; Pos, positive; QCA, quantitative invasive coronary angiography.

Dobutamine Stress Protocol

Study participants were asked to avoid agents that could antagonize the effects of dobutamine (eg, β‐blockers) prior to DSE. Intravenous dobutamine infusion was started at 5 µg/kg/min and increased by 10 µg/kg/min at 3‐minute intervals to a maximum of 40 µg/kg/min until 85% of maximum age‐predicted heart rate was reached.12 If the peak maximum heart rate was not reached with dobutamine, 1.0 mg atropine was given, if the patient had no contraindication. The test was terminated before the maximum heart rate was reached if the patient developed significant ischemia or arrhythmia; and, if necessary for clinical stability, β‐blockers were administered to reverse these conditions.

All DSE testing was conducted using a Philips iE33 system with X5‐1 transducer (Philips Medical Systems, The Netherlands). Echocardiographic imaging was performed from the parasternal long‐ and short‐axis, apical long‐axis, and apical 4‐ and 2‐chamber views. Images were recorded in resting condition, after each dobutamine infusion increase, at peak stress, and after cessation of stress. In the presence of obvious or suspected dyssynergy, a complete echocardiographic examination was performed and recorded from all employed approaches to allow optimal documentation of the presence and extent of myocardial ischemia. Analysis and scoring of the echocardiographic studies were performed using a 17‐segment model of the left ventricle and a 4‐grade scale of regional wall‐motion analysis (1 = normal; 2 = hypokinetic; 3 = akinetic; and 4 = dyskinetic).

Coronary Angiography

Coronary angiography was performed in all individuals according to European Society of Cardiology (ESC) guidelines. A significant stenosis was defined as diameter stenosis ≥70% (as determined by quantitative coronary angiography) in a major native epicardial coronary artery with a diameter of ≥2.5 mm. The interventional strategy was left to the discretion of the treating operator, who was blinded to MP and biomarker results, and standard practice in accordance with the ESC guidelines for myocardial revascularization.13

Laboratory Methods

Blood was drawn peripherally before, immediately after, at 4 hours, and at 24 hours after DSE. Circulating MPs were measured by flow cytometry from citrate‐buffered blood immediately after venipuncture according to an established protocol9, 14, 15: Blood was centrifuged at 1500 g for 15 minutes, followed by centrifugation at 13 000 g for 2 minutes to generate platelet‐deficient plasma. One hundred µL of platelet‐poor plasma was incubated with monoclonal antibodies against (1) CD144 (PE mouse anti‐human CD144, 4 µL; BD Pharmingen, San Diego, CA); (2) CD14 (PE mouse anti‐human CD14, 4 µL; BD Pharmingen) and CD16 (Alexa Fluor 647 mouse anti‐human CD16, 4 µL; BD Pharmingen); or (3) CD31 (PE mouse anti‐human CD31, 4 µL; BD Pharmingen) and CD42b (APC mouse anti‐human CD42b, 4 µL; BD Pharmingen) for 45 minutes at room temperature and protected from light. This step was followed by incubation with fluorescein isothiocyanate‐conjugated annexin V (FITC annexin V, 4 µL; BD Pharmingen) for 15 minutes at room temperature and protected from light, and finally by the addition of 200 µL sterile phosphate buffered saline (PBS; 10 mM HEPES pH 7.4, 140 mM NaCl, 2.5 mM CaCl₂). The CD144⁺ EMPs were defined as MPs positively labeled for annexin V and CD144; CD14⁺CD16⁺ monocyte‐derived microparticles (MMPs) as positively labeled for annexin V, CD 14, as well as CD16; and CD31 + CD42b + platelet microparticles (PMPs) as positively labeled for annexin V, CD31, as well as CD42b. Fluorescence‐activated cell sorting analysis was performed immediately after staining using a FACSCalibur flow cytometer (Becton Dickinson Biosciences, San Jose, CA). Microparticle concentration was assessed by comparison to flowcount calibrator beads from TruCOUNT tubes (Becton Dickinson Biosciences), as described previously.9, 14 Data were analyzed using Cellquest software (Becton Dickinson Biosciences). All MP analyses were performed blind to the patients' stress echocardiography and angiogram results. Unstained and isotype‐matched antibodies served as controls. Gating and staining strategies are demonstrated in Supporting Information, Figure 1, in the online version of this article.

Microparticle Uptake

To explore the uptake of EMPs and MMPs into the endothelium, sera from 10 randomly chosen patients with or without significant coronary stenosis collected before and immediately after DSE was used for ex vivo stimulation of human coronary artery endothelial cells (HCAECs). The HCAECs were incubated for 4 hours (37 °C) with 10% of conditioned serum samples (900 µL growth factor–deprived medium + 100 µL of patient's serum), as previously described.16, 17 Afterward, serum was removed and the cells were washed with sterile PBS 3 times. To explore whether stimulation of HCAECs with sera influences the uptake of EMP, 2 × 10⁶ EMPs were generated from starved HCAECs, labeled with PKH26, diluted in 1 mL PBS, and added to target cells as previously described, resulting in a concentration of 200 EMP/µL, which represented approximately the EMP level in patients.15 In‐depth characterization of size and surface markers of EMPs and MMPs was performed and published in previous articles from our group.18, 19, 20 After 4 hours of incubation at 37 °C, HCAEC were washed 3 times with PBS and fixed with 2% paraformaldehyde for 10 minutes at 37 °C. Blocking was performed with 0.5% bovine serum albumin (BSA) for 30 minutes. To stain endothelial cells, mouse–anti‐human PECAM‐1 (1:50; Sigma‐Aldrich, St. Louis, MO) was used as primary antibody, which was diluted in 0.5% BSA‐PBS and incubated overnight at 4 °C. IgG‐Cy2 (1:200; Sigma‐Aldrich) rat–anti‐mouse was used as a secondary antibody, which was incubated for 1 hour at room temperature. After staining using mounting medium containing DAPI (Vectashield; Vector Laboratories, Burlingame, CA), the total number of EMPs per cell were counted using fluorescence microscopy (Axiovert 200 M microscope; Zeiss, Jena, Germany) and AxioVision software (Zeiss). When indicated, soluble phosphatidylserine (O‐phospho‐L‐serine; Sigma‐Aldrich) was added to the cells prior to MP incubation.

For MMP uptake experiments, 2 × 10⁶ MMPs were generated from THP‐1 cells and labeled with calcein as previously described.16 Uptake experiments were performed as described for EMP.

Annexin V Enzyme‐Linked Immunosorbent Assay

Sera from 10 randomly chosen patients were selected for the detection of circulating annexin V. Annexin V levels were assessed using enzyme‐linked immunosorbent assay (Enzo Life Sciences, Farmingdale, NY) according to the company's suggestions.

Western Blot

The HCAECs were incubated for 4 hours (37 °C) with 10% of conditioned serum samples (900 µL growth factor–deprived medium + 100 µL of patient's serum). Afterward, cells were washed and homogenized with radioimmunoprecipitation assay (RIPA) buffer (150 mM NaCl, 1.0% Nonidet P‐40, 0.5% deoxycholate, 0.1% SDS, and 50 mM Tris, pH 8.0) containing 1 mM Na₃VO₄, 5 mM NaF and protease inhibitor cocktail (Roche Diagnostics, Indianapolis, IN) at 4 °C. Protein concentration was measured using Lowry protein assay (BioRad Laboratories, Hercules, CA). Equal amounts of proteins (30 µg) were loaded into 12% SDS electrophoresis, transferred onto PVDF membranes, and blocked with 5% BSA/PBS for 1 hour. Blots were incubated with the appropriate primary antibodies (anti‐PSR, Abcam; anti‐GAPDH, Hytest) followed by the correspondent HRP‐conjugated secondary antibodies, and proteins were revealed by chemiluminescence using the ECL kit (GE Healthcare, Wauwatosa, WI). GAPDH was used as the loading control.

Statistical Analysis

Data are presented as mean ± SD if normally distributed or as median and interquartile range (IQR) if not normally distributed. Continuous variables were tested for normal distribution with the use of the Kolmogorov‐Smirnov test. Categorical variables are given as frequencies and percentages. For continuous variables, the t test or Mann‐Whitney U test was used for comparison between 2 groups. For categorical variables, the χ² test was used for further analysis.

All tests were 2‐sided, and a P value of <0.05 was considered statistically significant. Statistical analyses were conducted with PASW Statistics version 22.0.0 64‐bit (IBM Corp., Armonk, NY), MedCalc version 11.1.1.0 (MedCalc Software, Mariakerke, Belgium), and GraphPad Prism 5 (GraphPad Software, La Jolla, CA).

All authors vouch for the accuracy and completeness of the data and all analyses and confirm that the study was conducted according to the protocol.

Results

Baseline Characteristics

Eighty patients (mean age, 66.9 ± 10.9 years; 71% male) with stable CAD were enrolled from March 2011 to March 2012 in this prospective study (Figure 1). Quantitative invasive coronary angiography revealed a significant stenosis (as determined by QCA as ≥70% diameter stenosis of ≥1 major epicardial coronary artery with a diameter ≥2.5 mm) in 41 patients. Baseline characteristics of the study participants are shown according to the prevalence of a significant stenosis in the Table 1. Patient groups did not differ concerning cardiovascular risk factors and baseline levels of CD144⁺‐EMP, CD14⁺CD16⁺‐MMP, and CD31⁺CD42⁺‐PMP.

Table 1.

Baseline Characteristics According to Prevalence of Significant Coronary Artery Stenosis

Characteristics	Total, N = 80	Nonsignificant Stenosis, N = 39	Significant Stenosis, N = 41	P Value
Age, y	66.9 ± 10.9	67.4 ± 10.5	66.4 ± 11.4	0.67
Sex				0.70
F	23 (28.8)	12 (30.8)	11 (26.8)
M	57 (71.3)	27 (69.2)	30 (73.2)
Cardiovascular risk factors
Arterial hypertension	80 (100)	39 (100)	41 (100)	NA
Hyperlipoproteinemia	66 (82.5)	32 (82.1)	34 (82.9)	0.92
DM	19 (23.8)	6 (15.4)	13 (31.7)	0.09
Family history of CAD	25 (31.3)	14 (35.9)	11 (26.8)	0.38
Smoking	29 (36.3)	11 (28.2)	18 (43.9)	0.14
BMI, kg/m2	27.6 ± 4.0	27.0 ± 4.2	28.0 ± 3.9	0.41
Laboratory parameters
Total cholesterol, mg/dL	180.1 ± 38.9	178.8 ± 42.4	181.2 ± 36.0	0.79
LDL‐C, mg/dL	101.7 ± 28.1	103.2 ± 30.3	100.2 ± 25.8	0.65
HDL‐C, mg/dL	49.9 ± 13.3	50.2 ± 15.1	49.6 ± 11.6	0.84
Hb, g/dL	14.6 ± 1.5	14.5 ± 1.6	14.7 ± 1.3	0.54
Leucocytes, 109/L	7.2 ± 1.9	6.9 ± 1.9	7.4 ± 1.9	0.31
Serum Cr, mg/dL	1.02 ± 0.31	1.02 ± 0.28	1.04 ± 0.34	0.77
CRP, mg/L	1.50 (0.78/3.93)	1.55 (0.93/4.48)	1.50 (0.68/3.18)	0.23
CD144+ EMP, per µL	327.1 (102.4/781.6)	263.2 (93.9/582.3)	359.8 (128.1/1169.8)	0.22
CD14+CD16+ MMP, per µL	35.8 (9.9/88.8)	26.7 (7.8/58.2)	48.3 (11.6/134.7)	0.13
CD31+CD42b+ PMP, per µL	857.8 (304.6/2182.6)	1014.9 (280.3/2102.4)	715.5 (315.4/2825.4)	0.81
Medical history
Previous MI	28 (35.0)	10 (25.6)	18 (43.9)	0.09
Previous PCI	39 (48.8)	16 (41.0)	23 (56.1)	0.18
Previous stroke	2 (2.5)	0 (0)	2 (4.9)	0.16
COPD	7 (8.8)	6 (15.4)	1 (2.4)	0.04
PAD	10 (12.5)	3 (7.7)	7 (17.1)	0.21
CAD				0.04
1 vessel	19 (23.8)	14 (35.9)	5 (12.2)
2 vessels	21 (26.3)	9 (23.1)	12 (29.3)
3 vessels	40 (50.0)	16 (41.0)	24 (58.5)
DSE result				0.001
Positive	31 (38.8)	8 (20.5)	23 (56.1)
Negative	49 (61.2)	31 (79.5)	18 (43.9)
LVEF, %	57.0 ± 11.8	58.2 ± 10.8	55.9 ± 12.7	0.40
Medication on admission
ACEI or ARB	66 (82.5)	32 (82.1)	34 (82.9)	0.69
β‐Blocker	43 (53.8)	18 (46.2)	25 (61.0)	0.18
Statin	45 (56.3)	18 (46.2)	27 (65.9)	0.08
ASA	75 (93.8)	36 (92.3)	39 (95.1)	0.60

Abbreviations: ACEI, angiotensin‐converting enzyme inhibitor; ARB, angiotensin receptor blocker; ASA, aspirin; BMI, body mass index; CAD, coronary artery disease; COPD, chronic obstructive pulmonary disease; Cr, creatinine; CRP, C‐reactive protein; DM, diabetes mellitus; DSE, dobutamine stress echocardiography; EMP, endothelial microparticles; F, female; Hb, hemoglobin; HDL‐C, high‐density lipoprotein cholesterol; LDL‐C, low‐density lipoprotein cholesterol; LVEF, left ventricular ejection fraction; M, male; MI, myocardial infarction; MMP, monocyte‐derived microparticles; NA, not applicable; NT‐proBNP, N‐terminal pro‐brain natriuretic peptide; PAD, peripheral arterial disease; PCI, percutaneous coronary intervention; PMP, platelet microparticles; SD, standard deviation.

Data are presented as n (%) or mean ± SD.

Microparticles and Significant Coronary Stenosis

Stratifying the study population according to the prevalence of a significant coronary artery stenosis, we found that the level of CD144⁺‐EMP (P = 0.04) and CD14⁺CD16⁺‐MMP (P = 0.007) significantly decreased in patients with significant stenosis immediately after DSE, whereas circulating MP levels in patients without significant stenosis remained unchanged (Figure 2). CD31⁺CD42⁺‐PMP did not show any significant changes when subdivided according to the prevalence of a significant coronary artery stenosis (data not shown).

Figure 2.

Microparticle levels according to the prevalence of significant coronary artery stenosis. Changes of MPs after dobutamine stress echocardiography (DSE) according to the prevalence of a significant coronary artery stenosis. Abbreviations: DSE, dobutamine stress echocardiography; MPs, microparticles.

In patients with significant coronary artery stenosis and positive DSE, we also found a significant MMP (P = 0.02) decrease and an even more pronounced EMP decrease (P = 0.02) immediately after the DSE.

Endothelial Microparticle Uptake

As we found reduced circulating EMP and MMP levels in patients with significant coronary artery stenosis after cardiac stress, and endothelial uptake is one mechanism to clear circulating MPs from the bloodstream, we tested whether the dropout of circulating EMP and MMP level is mediated by an augmented capacity of the endothelium to take up MP.

Uptake experiments in vitro revealed that endothelial cells stimulated with sera collected from patients with significant coronary artery stenosis after cardiac stress showed an increased uptake of EMPs, but not MMPs. Endothelial cells stimulated with sera from patients without significant coronary artery stenosis did not show any differences in MP uptake after DSE in vitro (Figure 3A). These data suggested that cardiac stress induces functional changes in endothelial cells regarding EMP uptake capacities, which is differentially regulated in patients with and without significant coronary artery stenosis.

Figure 3.

Uptake of endothelial and monocyte‐derived MPs into target endothelial cells. (A) Uptake of EMPs and MMPs into HCAECs was assessed after stimulation of cells with sera collected before and immediately after DSE. *P < 0.05, n = 10. (B) Circulating annexin V levels were analyzed using ELISA (n = 10–12). (C) Western blot was used to detect expression of PSR in endothelial cells after stimulation of cells with patient's sera. Results are demonstrated in relation to GAPDH. *P < 0.05, n = 4. (D) MP uptake was assessed after addition of labeled EMP to target cells. Zeiss Axiovert 200 M microscope and AxioVision software were used to visualize and count EMPs in target cells. Soluble PS (20 µg/mL) was added to the cells prior to MP incubation. *P < 0.05, ***P < 0.001, n = 8–10. Abbreviations: DSE, dobutamine stress echocardiography; ELISA, enzyme‐linked immunosorbent assay; EMPs, endothelial cell–derived microparticles; HCAECs, human coronary artery endothelial cells; MMPs, monocyte‐derived microparticles; MP, microparticle; PS, phosphatidylserine; PSR, phosphatidylserine receptor.

Further experiments were performed to elucidate a possible mechanism of augmented EMP uptake into target endothelial cells after serum stimulation from patients with significant coronary stenosis.

Phosphatidylserine (PS)‐expressing EMPs are taken up into the endothelium via the phosphatidylserine receptor (PSR).18 Circulating annexin V is a ligand for PS and inhibits the uptake of MPs into endothelial cells.21

The level of circulating annexin V was not different before and after cardiac stress in patients with or without significant coronary stenosis (Figure 3B), suggesting an annexin V–independent regulation of EMP uptake. In contrast, stimulation of target endothelial cells with sera collected from patients with coronary stenosis after cardiac stress augmented expression of endothelial PSR, a main regulator of EMP uptake (Figure 3C). Blockade of PSR using soluble PS abrogated serum‐induced enhancement of EMP uptake (Figure 3D). These findings indicate that cardiac stress in patients with significant coronary stenosis might induce endothelial PSR expression, resulting in an augmented endothelial EMP uptake and lower circulating EMP level (Figure 4).

Figure 4.

Proposed mechanism. Cardiac ischemia leads to increased endothelial PSR expression resulting in an augmented endothelial EMP uptake with subsequently diminished circulating EMP levels. Abbreviations: EMP, endothelial cell–derived microparticle; PSR, phosphatidylserine receptor.

Discussion

Our study confirms that circulating MP levels do not increase after cardiac stress induction by DSE in patients suffering from stable CAD. We were able to extend this finding by showing that EMP and MMP levels even decrease immediately after cardiac stress induced by DSE in patients with significant coronary artery stenosis.

Circulating MPs such as EMPs are increased in patients with CVD, associated with future cardiovascular prognosis, and potentially involved in sudden cardiac death.1, 2, 5, 6, 7, 8, 22 Augustine et al recently demonstrated that a rapid rise in a broad range of circulating MPs in patients undergoing a standardized cardiac stress protocol occurs. These MPs have then been cleared from the circulation within an hour. Surprisingly, this dynamic rise and fall was not apparent in patients with CAD that was predominantly diagnosed from medical history in this study and, when available, from QCA.10

Augustine et al suggested that MPs may have a physiological function to remove stress‐induced cellular byproducts and unwanted debris; and, therefore, a presumably physiological MP‐level elevation in response to strenuous cardiac exercise can be observed in healthy subjects.10 This response could be relevant to CVD pathogenesis because failure to clear MPs might lead to an increase in the circulating levels of proatherogenic factors.23 In addition, ineffective MP release could lead to localized cell damage. Stressed cells might expose PS on their plasma membranes and subsequently release PS via MP, in which the proapoptotic enzyme caspase 3 can be found, to relieve stress and avoid cell death.24

Our data extend these findings, because we were able to show that EMPs and MMPs decreased in patients with significant ischemia after cardiac stress.

Whereas previous studies showed an increase of diverse circulating MPs in patients after DSE,10, 25 we found a drop of EMPs and MMPs in patients with relevant coronary stenosis and no changes in patients with stable CAD. These heterogenous findings regarding circulating MP (cMP) levels after stress might be due to (1) patient collectives that are not comparable, (2) variances in the used MP quantification protocols, and (3) relatively low patient numbers. These points should be addressed in future studies to better understand the diagnostic potential of cMP in patients with CAD.

As cMP level is a result of MP production and clearance, exploring MP clearance mechanisms is of high importance. Microparticles have a relatively short half‐life in the circulation.23 One study examined the role of the PS binding protein lactadherin in the clearance of platelet‐derived PS⁺ MPs. They found that the spleen was involved in the clearance of these MPs.26 Clearance of tumor‐derived human TF⁺ MPs was examined in control and splenectomized mice.27 In control mice, peak levels of TF⁺ MPs were observed at 30 minutes, and none were detected at 120 minutes; whereas in the splenectomized mice, significant levels of TF⁺ MPs were observed at 120 minutes. These results support the conclusion that the spleen is the major site for the clearance of PS⁺ MPs with or without TF. Besides the spleen, the endothelium has been shown to be a major route for MP clearance from the circulation.15, 28 Other potential mechanisms of MP clearance include uptake of MP from the circulation by liver Kupffer cells29 and phagocytosis of MPs by splenocytes.30

We found that stimulation of target endothelial cells in vitro with sera from patients with significant coronary stenosis collected after cardiac stress significantly increased endothelial PSR expression resulting in an augmented endothelial EMP uptake ability. As endothelial PSR expression is induced by hypoxia,31 it seems conclusive that under conditions of insufficient coronary perfusion and subsequent hypoxia, endothelial PSR expression is upregulated. The level of circulating annexin V was not different before and after cardiac stress in patients with or without significant coronary stenosis, suggesting an annexin V–independent regulation of EMP uptake. As all samples were processed identically, we can exclude an influence on sample processing on the obtained results.

Endothelial PSR expression mediates the uptake of EMP into the endothelium. Taken up by endothelial cells, EMPs promote endothelial cell migration and proliferation, both representing critical steps in endothelial repair.18 This hypothesis is supported by the fact that EMPs contain, among others, microRNA (miR)‐126, which regulates the target protein sprouty‐related, EVH1 domain‐containing protein 1 (SPRED1) and promotes vascular endothelial repair.15 As EMPs have been shown to protect target cells from apoptosis and initiate regeneration processes,15, 18 one may speculate that the endothelium, in conditions of insufficient oxygen supply and subsequent stress, increases its EMP uptake capacity for self‐protection and prevention of further damage. This concept is in line with a study from da Rosa et al, who showed that circulating miR‐126 is consumed most probably from the endothelium during the passage of the culprit lesion in patients with an acute coronary syndrome.32 Considering that miR‐126 is highly expressed and mainly transported within EMPs,15 these findings support the concept that the ischemic endothelium increases its abilities to take up EMPs to prevent cell damage and death.

Although reduced EMP levels could be explained in part by an augmented uptake of EMPs into the endothelium, endothelial uptake of MMPs was unchanged. That suggests that MMPs might be cleared by a different route than EMPs (eg, phagocytosis by Kupffer cells in the liver or splenocytes), and further research is needed to investigate these findings in detail. Additional studies are required to determine the use of MPs in the diagnosis and treatment of CVD and to fully understand how MPs are regulated in both health and disease.

Study Limitations

The sensitivity of DSE for the detection of a significant coronary artery stenosis (despite acceptable specificity) might have been limited by poor image quality, antianginal medication, or the high pretest probability in this study cohort with previously documented CAD; however, it reflects daily clinical routine in a tertiary center. Furthermore, the exact pathomechanism of MP decrease in patients with significant coronary stenosis needs further investigation (ie, which factors in sera from patients with significant coronary stenosis induce endothelial PSR expression and by which mechanisms MMPs are cleared). As conventional flow cytometer is not able to detect small‐sized MPs, their kinetics in cardiac stress in patients with or without significant coronary stenosis is unclear. Finally, the use of MP evaluation in this clinical setting to provide information contributing to treatment decisions in a time‐ and cost‐effective manner will require standardized procedures and reagents, and appropriate flow cytometric instruments, as outlined previously.33

Conclusion

Our findings demonstrate that EMPs and MMPs decrease immediately after cardiac stress induction in CAD patients with significant coronary ischemia.

Supporting information

Appendix S1. Online Supplemental figure legend

Appendix S2. Online supplemental methods