Clinical/Translational Research
Dynamic Release and Clearance of Circulating
Microparticles During Cardiac Stress
Daniel Augustine,* Lisa V. Ayers,* Eduardo Lima, Laura Newton, Adam J. Lewandowski,
Esther F. Davis, Berne Ferry, Paul Leeson
Rationale: Microparticles are cell-derived membrane vesicles, relevant to a range of biological responses and
known to be elevated in cardiovascular disease.
Objective: To investigate microparticle release during cardiac stress and how this response differs in those with
vascular disease.
Methods and Results: We measured a comprehensive panel of circulating cell-derived microparticles by a
standardized flow cytometric protocol in 119 patients referred for stress echocardiography. Procoagulant, platelet,
erythrocyte, and endothelial but not leukocyte, granulocyte, or monocyte-derived microparticles were elevated
immediately after a standardized dobutamine stress echocardiogram and decreased after 1 hour. Twenty-five
patients developed stress-induced wall motion abnormalities suggestive of myocardial ischemia. They had similar
baseline microparticle levels to those who did not develop ischemia, but, interestingly, their microparticle levels
did not change during stress. Furthermore, no stress-induced increase was observed in those without inducible
ischemia but with a history of vascular disease. Fourteen patients subsequently underwent coronary angiography.
A microparticle rise during stress echocardiography had occurred only in those with normal coronary arteries.
Conclusions: Procoagulant, platelet, erythrocyte, and endothelial microparticles are released during cardiac
stress and then clear from the circulation during the next hour. This stress-induced rise seems to be a normal
physiological response that is diminished in those with vascular disease. (Circ Res. 2014;114:109-113.)
Key Words: dobutamine stress echocardiography
■
C
ell-derived microparticles are subcellular membrane vesicles,
ranging in size from 0.1 to 1.0 μm, that bud off during cell
activation or apoptosis.1 They are identifiable in the circulation by
their parent cell surface markers.2 Certain subtypes, particularly
platelet derived, increase after strenuous exercise in healthy individuals,3,4 which suggests that microparticles may have a physiological function to remove stress-induced cellular by-products.5,6
Such a response could be relevant to cardiovascular disease pathogenesis because failure to clear microparticles could lead to an
increase in the circulating levels of proatherogenic factors.7–9 In
addition, ineffective microparticle release could lead to localized
cell damage.10,11 We studied whether there is a stress-induced dynamic release and clearance of microparticles in patients exposed
to cardiac stress during dobutamine echocardiography.12 The
group included those with atypical symptoms in whom coronary
disease is excluded, as well as those with complex disease who
require risk stratification, which allowed study of variation in response between those with and without vascular disease.
microparticles, cell-derived
■
vascular diseases
In This Issue, see p 2
Editorial, see p 15
Methods
Expanded Methods can be found in the Online Data Supplement.
Sample Collection and Analysis
Participants were recruited sequentially from patients undergoing
dobutamine stress echocardiography (DSE) at the John Radcliffe
Hospital, Oxford. Citrate blood samples were taken peripherally
before, immediately after, and 1 hour after DSE.13 Circulating microparticles were measured by flow cytometry.14 Functional effect was
assessed by procoagulant assay.
Cardiovascular Disease Assessment
DSE identified patients with coronary disease sufficient to cause
acute myocardial ischemia to study microparticle response related to
ischemia. Medical history and coronary angiography identified patients with vascular disease to determine whether differences in microparticle response relate to vascular disease or ischemia.
Original received June 3, 2013; revision received October 16, 2013; accepted October 18, 2013. In September 2013, the average time from submission
to first decision for all original research papers submitted to Circulation Research was 13.2 days.
From the Oxford Cardiovascular Clinical Research Facility, University of Oxford, Oxford, United Kingdom (D.A., E.L., L.N., A.J.L., E.F.D., P.L.); and
Department of Clinical Immunology, Oxford University Hospitals NHS Trust, Oxford, United Kingdom (L.V.A., B.F.).
*These authors contributed equally to this work.
The online-only Data Supplement is available with this article at http://circres.ahajournals.org/lookup/suppl/doi:10.1161/CIRCRESAHA.
114.301904/-/DC1.
Correspondence to Paul Leeson, PhD, FRCP, Oxford Cardiovascular Clinical Research Facility, John Radcliffe Hospital, Oxford OX39DU, United
Kingdom. E-mail paul.leeson@cardiov.ox.ac.uk
© 2013 American Heart Association, Inc.
Circulation Research is available at http://circres.ahajournals.org
DOI: 10.1161/CIRCRESAHA.114.301904
109
110 Circulation Research January 3, 2014
Nonstandard Abbreviations and Acronyms
DSE
EMP
dobutamine stress echocardiography
endothelial cell-derived microparticle
Statistical Analysis
Statistical analysis is detailed in the online-only Data Supplement.
Results
Microparticles and Cardiac Stress
One hundred nineteen participants took part, and 107 had
measurements 1 hour after DSE. Immediately after DSE, procoagulant, platelet, erythrocyte, and CD31+CD41− endothelial cell–derived microparticle (EMP) levels rose significantly.
One hour later, procoagulant and platelet microparticle levels
had returned to baseline. Erythrocyte-derived microparticles
had also started to decrease, and CD31+CD41− EMPs had
fallen below baseline (Figure 1). Other microparticles did
not change during stress (data not shown). The rise in microparticles was correlated with an increase in procoagulant
activity, consistent with a clinically relevant functional effect
(Figure 2C).
Microparticles and Myocardial Ischemia
There were 94 negative and 25 positive DSEs. Participants with
a positive DSE were significantly older and had slightly lower
cholesterol, but other clinical and echocardiographic variables
were similar (Online Table I). Baseline microparticle levels
did not differ between groups (Online Table II). However,
procoagulant, platelet, erythrocyte, and CD31+CD41− EMPs
increased in participants after a negative DSE (Figure 2A),
with no change up to 1 hour in those with a positive DSE.
Adjustment for risk factors and medication by regression analysis did not alter this finding.
Microparticles and Vascular Disease
To investigate whether this lack of response related to myocardial ischemia or underlying vascular disease, we compared microparticle release in 72 participants with a negative
DSE without evidence of vascular disease with microparticle release in 11 participants with known vascular disease.
Procoagulant, platelet, and erythrocyte-derived microparticles
had not increased in participants with vascular disease (Figure
2B). In 14 participants who subsequently underwent coronary
angiography, procoagulant, platelet, and erythrocyte-derived
microparticles had increased only in individuals with normal
coronary arteries (Figure 3A). Interestingly, a stress-induced
increase in procoagulant and erythrocyte-derived microparticles had occurred in a patient with a positive DSE subsequently found to have normal coronaries, and no change in
microparticles had occurred in a patient with a negative DSE
found to have coronary disease (Figure 3B).
Discussion
We demonstrate a dynamic rapid rise and fall in a broad range
of circulating microparticles in response to cardiac stress.
Interestingly, this response was not apparent in those who developed myocardial ischemia, which seemed to relate to their
underlying vascular disease because no increase was seen in
those with a negative stress echo but known vascular disease.
Subsequent analysis based on angiographic disease supported
these findings.
Our findings are consistent with previous reports of a trend
for platelet-derived microparticles to increase after DSE,15 as
well as significant rises in healthy individuals after exercise3,4
and a high-fat meal.16 Our analysis shows that procoagulant,
erythrocyte, and platelet-derived microparticle levels are related, and all elevate immediately after DSE. Microparticle
levels depend on speed of clearance mechanism, currently
thought to involve direct receptor binding of liver or spleen
phagocytes to phosphatidylserine or opsonization proteins
Figure 1. Dot plots (median with
interquartile range) of annexin V+
(A), platelet (B), erythrocyte (C), and
endothelial cell microparticles (MP;
D) at time points before dobutamine
stress echocardiography (DSE),
immediately after DSE, and 1 after DSE.
EMP indicates endothelial cell–derived
MP; and PMP, platelet-derived MP.
Augustine et al Cardiac Stress–Induced Microparticle Release 111
Figure 2. Dot plots (median with
interquartile range) of annexin V+,
platelet, erythrocyte, and endothelial
cell microparticles (MP) in negative
dobutamine stress echocardiography
(DSE) and positive DSE patients
before and immediately after DSE (A)
and in patients with a negative DSE
and no vascular disease or vascular
disease before and immediately after
DSE (B). Microparticle procoagulant
activity by Zymuphen assay (C). EMP
indicates endothelial cell–derived MP;
PMP, platelet-derived MP; and RBC, red
blood cell.
on the microparticles.17,18 Platelet-derived microparticle levels are known to fall rapidly after injection into rabbits19 and
within 30 minutes after infusion in mice,17 but few studies
have investigated human clearance.20 We found that, within
1 hour, elevated circulating procoagulant, platelet, and EMPs
are significantly reduced.
Clearance rate differed by cellular origin so that erythrocyte-derived microparticles had not declined as much and
112 Circulation Research January 3, 2014
Figure 3. Dot plots (median with interquartile range) of annexin V+ microparticles (MPs), CD31+CD41+ platelet-derived MPs
(PMPs), glycophorin A+ erythrocyte-derived microparticles, and CD31+CD41− endothelial cell–derived MPs (EMPs) before and
immediately after dobutamine stress echocardiography (DSE) in participants with a confirmed diagnosis by angiogram (A).
Percentage change in microparticle levels during DSE in a false-positive and a false-negative participant (B). RBC indicates red blood cell.
CD31+CD41− EMPs fell below baseline. Because EMPs carry
von Willebrand factor,21 they present a potential thrombotic
risk, which might explain their apparently more rapid clearance
mechanism. However, EMP response to cardiac stress, in general, differed. CD31+CD41− EMPs, indicative of endothelial
apoptosis,22 increased, but with a relatively small proportional
change. There was no change in CD62E+ EMPs, a marker of
endothelial cell activation,22 or CD144+ EMPs, an independent
predictor of future cardiovascular events.23 Lower concentrations of CD31+CD41− EMPs or the standard negative staining
approach,24,25 which excludes platelet-derived microparticles
but may lead to inclusion of leukocyte-derived microparticles,
could explain this. Alternatively, CD31+CD41− EMP release
in response to stress may have distinct characteristics because
different EMP subpopulations are known to be released in response to different triggers.22 Although we found no difference
in EMP levels between those with and without vascular disease, EMP levels are an independent predictor of cardiovascular events in coronary patients26 and in those with end-stage
renal failure,27 so EMP stress response in vascular patients may
vary from other cellular microparticles.
This significant variability in the degree of change in microparticle levels between individuals after cardiac stress raises
the interesting possibility that this could be a predictive biomarker.28 One patient with a normal DSE subsequently had
an out-of-hospital arrest and had no microparticle rise during
stress. However, lack of microparticle release was an indicator
of vascular disease rather than ischemia. A larger study with
more extensive angiographic and clinical follow-up will be required to determine prognostic significance of this dynamic
response and allows for calculation of positive and negative
predictive values. Investigation of dynamic microparticle release from other cells, in particular cardiomyocytes, may be
of interest as markers of ischemic burden.
In summary, we demonstrate, in a large cohort, that procoagulant, platelet, erythrocyte, and endothelial-derived microparticles are released and cleared rapidly in response to
cardiac stress. This dynamic response was not evident in the
group of subjects with vascular disease. Although provisional
pending replication, these observations raise the possibility
that microparticle release is a protective mechanism to remove
cellular stress signals, which is diminished in those with vascular disease. Interventions to enhance release and clearance
of microparticles may be relevant for disease prevention.
Sources of Funding
The work was supported by grants to P. Leeson from the British Heart
Foundation (FS/06/024 and FS/11/65/28865). D. Augustine was supported by the Engineering and Physical Science Research Council,
and L.V. Ayers was supported by a National Institute of Health
Research Healthcare Science Research Fellowship and the Oxford
National Institute for Health Research Biomedical Research Centre.
Disclosures
None.
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Novelty and Significance
What Is Known?
• Microparticles are small membrane vesicles, which can be stimulated
to bud off from cells into the circulation and can trigger a range of
biological responses.
• The cellular origin and quantity of circulating microparticles can be
measured.
• Certain subtypes have been shown to be elevated in those with
cardiovascular disease but also in healthy individuals after exercise.
What New Information Does This Article Contribute?
• Cardiac stress induces a rapid dynamic increase in a broad range of
circulating microparticles.
• These microparticles are then cleared from the circulation during the
next hour.
• The stress-induced rise seems to be a normal physiological response
that is diminished in those with vascular disease.
Microparticles are subcellular membrane vesicles that bud off
from cells into the circulation in response to different stimuli
and can be identified, and quantified, based on markers on their
surface. Although circulating levels of certain microparticles are
increased in people with cardiovascular disease, exercise has
also been found to increase levels in healthy subjects. This study
investigated whether microparticle release is a normal phenomenon in response to cardiac stress, what type of microparticles are
released, how quickly they then clear from the circulation, and
whether vascular disease is associated with a different response.
A dynamic rapid rise in a broad range of circulating microparticles was demonstrated in patients undergoing a standardized
cardiac stress protocol. These microparticles then cleared from
the circulation within an hour. Interestingly, this dynamic rise
and fall was not apparent in those who had vascular disease,
diagnosed based on their medical history and, when available,
evidence from coronary angiography. These observations raise
the possibility that stress-induced microparticle release may be
a biomarker of a physiological response to remove cellular stress
signals, which is diminished in those with vascular disease.
Interventions to enhance both release and clearance of microparticles may be relevant for disease prevention.