Atherosclerosis 273 (2018) 67e74

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Atherosclerosis
journal homepage: www.elsevier.com/locate/atherosclerosis

Release of endothelial microparticles in patients with arterial

hypertension, hypertensive emergencies and catheter-related injury
Roberto Sansone a, Maximilian Baaken a, Patrick Horn a, Dominik Schuler a,
Ralf Westenfeld a, Nicolas Amabile c, Malte Kelm a, b, Christian Heiss a, d, e, *
a
Division of Cardiology, Pulmonology, and Vascular Medicine, Medical Faculty, University Duesseldorf, Duesseldorf, Germany
b
CARID-Cardiovascular Research Institute Duesseldorf, Medical Faculty, University Duesseldorf, Duesseldorf, Germany
c
Institut Mutualiste Montsouris, Paris, France
d
University of Surrey, Faculty of Health and Medical Sciences, Guildford, UK
e
Surrey and Sussex Healthcare NHS Trust, Redhill, UK

a r t i c l e i n f o a b s t r a c t

Article history: Background and aims: Circulating endothelial microparticles (EMPs) are increased in arterial hyperten-
Received 5 November 2017 sion. The role of physicomechanical factors that may induce EMP release in vivo is still unknown. We
Received in revised form studied the relationship of EMPs and physicomechanical factors in stable arterial hypertension and
7 April 2018
hypertensive emergencies, and investigated the pattern of EMP release after mechanical endothelial
Accepted 11 April 2018
injury.
Available online 12 April 2018
Methods: In a pilot study, 41 subjects (50% hypertensives) were recruited. EMPs were discriminated by
flow-cytometry (CD31þ/41-, CD62eþ, CD144þ). Besides blood pressure measurements, pulse-wave-
Keywords:
Cell-derived microparticles
analysis was performed. Flow-mediated dilation (FMD), nitroglycerin-mediated dilation (NMD), and
Endothelial function wall-shear-stress (WSS) were measured ultrasonographically in the brachial artery; microvascular
Arterial hypertension perfusion by laser-Doppler (Clinicaltrials.gov: NCT02795377). We studied patients with hypertensive
Endothelial function emergencies before and 4 h after BP lowering by urapidil (n ¼ 12) and studied the release of EMPs due to
Wall shear stress mechanical endothelial injury after coronary angiography (n ¼ 10).
Results: Hypertensives exhibited increased EMPs (CD31þ/41-, CD144þ, CD62eþ) as compared to nor-
motensives and EMPs univariately correlated with systolic BP (SBP), augmentation index, and pulse wave
velocity and inversely with FMD. CD31þ/41--EMPs correlated with diameter and inversely with WSS and
NMD. CD62eþ and CD144þ-EMPs inversely correlated with microvascular function. During hypertensive
emergency, only CD62eþ and CD144þ-EMPs were further elevated and FMD was decreased compared to
stable hypertensives. Blood pressure lowering decreased CD62eþ and CD144þ-EMPs and increased FMD.
CD31þ/41dEMPs, diameter, and WSS remained unaffected. Similar to hypertensive emergency, catheter-
related endothelial injury increased only CD144þ and CD62eþ-EMPs.
Conclusions: EMP release in hypertension is complex and may involve both physicomechanical endo-
thelial injury and activation (CD144þ, CD62eþ) and decreased wall shear stress (CD31þ/41-).
© 2018 Elsevier B.V. All rights reserved.

1. Introduction dysfunction, it is not entirely clear how it is linked to atheroscle-

Arterial hypertension is an important risk factor facilitating the
development and progression of atherosclerosis. While arterial
hypertension is associated with increased mechanical stress on the
arteries leading to macrovascular stiffening and microvascular
* Corresponding author. University of Surrey, Faculty of Health and Medical Sci-
ences, Section of Clinical Medicine and Ageing, Guildford, GU2 7XH, UK.
E-mail address: c.heiss@surrey.ac.uk (C. Heiss).
rosis progression [1]. Stiffness of conduit arteries leads to acceler-
ated PWV, which in turn enhances transmission of mechanical
forces to the microcirculation. While the larger arteries are exposed
to higher pulsatility leading to facture of load bearing wall com-
ponents and positive remodeling with increased diameters and
stiffness, the pulse reflection goes along with force transmission
that is associated with microvascular remodeling, rarefaction, and
microvascular dysfunction. It could be hypothesized that the me-
chanical stress may lead to endothelial injury and dysfunction

https://doi.org/10.1016/j.atherosclerosis.2018.04.012
0021-9150/© 2018 Elsevier B.V. All rights reserved.
68 R. Sansone et al. / Atherosclerosis 273 (2018) 67e74

which, in turn, could drive arterial remodeling and plaque devel- including 50% patients with stable CAD. No dedicated matching
opment [2,3]. procedures were applied. Hemodynamics and endothelial function
According to the response to injury theory, mechanical injury of the brachial artery were assessed as flow-mediated dilatation
and exposure to cardiovascular risk factors disrupt endothelial (FMD). Pulse wave analyses including central blood pressure, pulse
integrity driving the initiation and progression of atherosclerosis wave velocity (PWV), and aortic augmentation index (AIX) were
[4]. Endothelial microparticles (EMPs) can be viewed as circulating performed by tonometry. In all subjects, EMP subpopulations
markers of endothelial injury and a compromised endothelial (CD31þ/41-, CD144þ, CD62eþ) were analyzed by flow-cytometry
integrity that are released from activated and apoptotic endothelial according to the expression of surface antigens. Exclusion criteria
cells as shown in vitro [5,6]. These EMPs are membrane particles of were manifest peripheral artery, or cerebrovascular disease, acute
less than a micrometer in diameter and carry endothelial surface inflammation (C-reactive protein [CRP] >0.6 mg/dl), kidney failure
markers [5e7] and enzymes including eNOS [8]. Circulating levels (estimated glomerular filtration rate [eGFR] <30 ml/min), malig-
of EMPs increase in plasma early in the atherosclerotic processes, nancies, and arrhythmias (heart rhythm other than sinus).
correlate with the degree of endothelial dysfunction [8,9], and have In a second study, we analysed 12 patients that were admitted to
been established as prognostic biomarkers that predict adverse CV the emergency department with acute symptoms of dyspnea or
outcome [10e12]. Cardiovascular risk factors may trigger EMP angina in the presence of blood pressure >180/120 mmHg,
release [7,13]. While it was previously shown that arterial hyper- troponin T value not exceeding the upper reference limit (14 ng/l) at
tension goes along with increases EMP levels in human subjects admission and 1 h excluding acute myocardial infarction and
[14e16], the role of physicomechanical factors present in arterial indicating hypertensive emergency [17]. Measurements of EMPs
hypertension that may induce mechanical endothelial injury and and vascular measurements were taken at admission (0 h) before
activation and shedding of EMPs in vivo is still unknown. and at 4 h and after lowering of BP by urapidil (Stragen, Denmark,
In order to investigate this, we first studied in a cross-sectional 10e50 mg i.v.). EMP subpopulations were analyzed by flow-
pilot cohort how hemodynamic, structural, and functional charac- cytometry and FMD measured by ultrasound. All patients were
teristics that are chronically changed in stable hypertensives relate discharged on the same day. Exclusion criteria were manifest pe-
to circulating EMP concentrations. We then evaluated the re- ripheral artery, or cerebrovascular disease, acute inflammation
lationships of arterial characteristics and the pattern of EMP dy- (CRP>0.6 mg/dl), kidney failure (eGFR<30 ml/min), malignancies,
namics during hypertensive emergencies and after consecutive arrhythmias (heart rhythm other than sinus), and troponin T value
blood pressure lowering. Finally, we determined the pattern of exceeding the upper reference limit (14 ng/l).
EMPs released by a prototypical mechanical endothelial injury as In a third series, we aimed at evaluating the pattern of EMP
induced during arterial catheterization. release due to a prototypical mechanical endothelial injury as
exerted by a catheter moved through large arteries. Therefore, we
studied EMPs release and vascular function before, at 1 h, at 4 h,
2. Materials and methods and at 24 h after elective transfemoral diagnostic coronary angi-
ography in n ¼ 10 stable CAD in-patients without arterial hyper-
2.1. Study subjects and protocol tension. EMP subpopulations were analyzed by flow-cytometry and
FMD measured by ultrasound. Inclusion criteria were normal left
In a first pilot study (study 1), we investigated circulating EMPs ventricular ejection fraction (>55%), stable CAD, and blood pressure
along with functional and mechanical characteristics of the arterial <140/90 mmHg. Exclusion criteria were manifest peripheral artery,
system in 41 consecutive male subjects, 20 subjects without arterial or cerebrovascular disease, acute inflammation (CRP>0.6 mg/dl),
hypertension and 21 subjects with arterial hypertension as defined kidney failure (eGFR<30 ml/min), malignancies, and arrhythmias
by office blood pressure 140/90 mmHg [17] or history of hyper- (heart rhythm other than sinus).
tension with ongoing antihypertensive medication (Table 1). The Measurements were always performed in the same order in one
patients were recruited from the out-patient clinic. As the presence session. We first took blood samples from the left arm. After a
of CAD might also affect circulating EMP values, we aimed at 15 min supine resting period in a quite air conditioned room, we
performed FMD measurements on the right arm, then laser
Doppler measurements on the left arm, followed by blood pressure
Table 1
measurements on the right arm, and, finally, performed applana-
Characteristics of study population (study 1).
tion tonometry on the neck and groin. The study protocol was
NT HT p-value approved by the ethics committee of the Heinrich Heine University
n 20 21 Duesseldorf and all patients and volunteers gave written informed
CAD 10 10 consent (Clinicaltrials.gov: NCT02795377).
Diabetes mellitus 0 0
Smoker 0 0
Age (y) 60 ± 4.7 59 ± 6.0 0.619 2.2. Characterization of EMP subpopulations by flow cytometry
BMI (kg/m2) 27.1 ± 3.6 29.2 ± 3.7 0.081
Height (m) 1.8 ± 0.6 1.8 ± 0.6 0.636 Citrated blood (6 ml) was drawn from the cubital vein and
Weight (kg) 86 ± 12 93 ± 12 0.081 processed within 2 h. Platelet-rich plasma was obtained by centri-
Creatinine (mg/dl) 0.9 ± 0.1 1.0 ± 0.1 0.591
Urea (mg/dl) 35 ± 11 33 ± 8 0.584
fugation of whole blood at 300  g over 15 min at room tempera-
Total cholesterol (mg/dl) 199 ± 43 191 ± 51 0.623 ture. Platelet-free plasma was obtained by 2 successive
LDL cholesterol (mg/dl) 138 ± 31 140 ± 40 0.866 centrifugations of platelet-rich plasma at 10,000  g for 5 min at
HDL cholesterol (mg/dl) 52 ± 15 55 ± 28 0.671 room temperature. Briefly, samples were incubated for 30 min with
Triglycerides (mg/dl) 134 ± 62 139 ± 34 0.746
fluorochrome-labeled antibodies or matching isotype controls and
Fasting plasma glucose (mg/dl) 102 ± 20 99 ± 10 0.506
HbA1c (%) 5.5 ± 0.5 5.5 ± 0.4 0.738 analyzed in a Canto II flow cytometer (Beckton Dickinson, Heidel-
CRP (mg/dl) 0.1 ± 0.2 0.1 ± 0.2 0.578 berg, Germany). Microbead standards (1.0 mm) were used to define
Hb (mg/dl) 14.5 ± 1.2 14.8 ± 1.5 0.410 MPs as <1 mm in diameter. The EMP subpopulations were defined
Leucocytes (1000/ml) 6.3 ± 1.3 7.4 ± 1.4 0.394 as CD31þ/CD41-, CD62eþ, or CD144þ events. The total number of
Values are mean and standard deviation; p-values refer to unpaired t-test. EMPs was quantified using flow-count calibrator beads (20 ml).
 R. Sansone et al. / Atherosclerosis 273 (2018) 67e74
2.3. Flow-mediated vasodilation (FMD)
Brachial artery (BA) FMD was measured by ultrasound (10 MHz
transducer; Vivid I, GE) in combination with an automated analysis
system (Brachial Analyzer, Medical Imaging Applications, Iowa City,
IO) in a 21  C temperature-controlled room after 15 min of supine
rest [18]. A forearm blood-pressure cuff was placed distal to the
cubital fossa and inflated to 250 mmHg for 5 min. Before cuff
inflation, the patients were instructed to keep the forearm muscles
relaxed during ischemia to avoid pain and all patients tolerated the
cuff inflation well. Diameter and Doppler-flow velocity were
measured at baseline and immediately after cuff deflation, at 20, 40,
60, and 80 s. FMD was expressed relative to baseline diameters as:
(diametermax - diameterbaseline)/diameterbaseline. The intra- and
inter-individual variability for FMD measurements established in
our laboratory are 0.9% (standard deviation [SD] of difference be-
tween repeated FMD measurements in n ¼ 20 middle aged healthy
subjects, unpublished) and 1% (SD within a group of healthy middle
aged subjects) [19]. Assuming a SD of difference between repeated
FMD measurements of 0.9%, intra-individual measurements in 10
experimental subjects would provide sufficient power to detect an
absolute change in FMD of 0.9% (two sided a of 5%, power ¼ 0.80).
Assuming a SD of FMD of 1%, 20 experimental and 20 control
subjects would provide sufficient power to detect an absolute
change in FMD of 0.9% (two sided a of 5%, power ¼ 0.80).
2.4. Wall shear stress (WSS)
WSS was assessed based on resting brachial artery diameter and
flow velocity measurements obtained during FMD measurements
and was calculated as 8 * m * mean flow velocity/mean diameter,
where blood viscosity (m) was assumed to be constant at 0.035 dyn/
sec * cm [2].
2.5. Microvascular function assessed by non-invasive laser doppler
imaging
All investigations were performed using a scanning laser
Doppler perfusion imager (PeriScan PIM III System, Perimed,
Sweden). The arm selected for measurements was immobilized to
avoid moving artifacts using a vacuum pillow containing poly-
urethane beads, which molds to the shape of the arm (Germa,
Sweden). The laser beam was positioned 15 cm above the forearm
scanning a field of 200 mm2 (region of interest [ROI] ¼ 8 $ 8 pixels;
3 s per scan) on the volar site of the forearm. Microvascular reac-
tivity was assessed during post-occlusive reactive hyperemia PORH.
Following the baseline perfusion (1 min; 20 images), a blood
pressure cuff located at the distal upper arm was inflated to
suprasystolic pressure over 5 min. After the cuff release, the
microvascular response on reactive hyperemia was recorded. Data
acquisition and analysis were performed by LDPI Win Software
(Perimed, Sweden) processing the perfusion as numerical values
and color-coded-images [20].
2.6. Hemodynamic monitoring
The Task Force Monitor (CN-Systems, Graz, Austria) was used for
continuous beat-to-beat assessment of cardiovascular variables,
including stroke volume, BP, heart rate, and total peripheral resis-
tance by impedance cardiography, which included ECG, phonocar-
diography, Finapres (Finapres-Medical-Systems, Amsterdam,
Netherlands), and BP monitoring system (Dynamap, Tampa, USA) at
the upper arm. We determined 24 h ambulatory BP measurements
on the day before study days. Central BP was derived from peripheral
pulse wave analysis (SphygmoCor®, AtCor-Medical, Australia).
69
2.7. Pulse wave analysis
Central blood pressure parameters including augmentation in-
dex (AIX) and pulse wave velocity (PWV) were measured by radial
applanation tonometry using the SphygmoCor® system in accor-
dance with the recommendations of the expert consensus on
arterial stiffness [21]. Via a proprietary generalized transfer func-
tion, the pressure waveform of the ascending aorta was synthe-
sized. PWV was determined from tonometry measurements taken
at the carotid and femoral artery.
2.8. Statistical methods and power analysis
Baseline characteristics of study subjects and results are
described as average and standard deviation. The sample size
estimation for between group comparisons was based on a probe
study in 10 healthy volunteers. The plasma concentrations of
CD31þ/41-, CD62eþ, and CD144þ were normally distributed, mean
values were 161, 611, and 146 Ev/ml, and the SDs were 116, 187, and
45 Ev/ml. Therefore, in parallel independent group comparisons as
presented in study 1, a group of n ¼ 20 (n ¼ 10) would provide
sufficient power to detect absolute differences in CD31þ/41-,
CD62eþ, and CD144þ mean values of 105 (154), 170 (248), and 41
(61) Ev/ml (two sided a of 5%, power ¼ 0.80). In these n ¼ 10 healthy
volunteers, we had also performed intra-individual repeated
measurements of EMP concentrations. The SDs of the average de-
viations were 43, 92, and 28 Ev/ml. Therefore, in a group of n ¼ 10
subjects intra-individual changes on repeated measurements in
CD31þ/41-, CD62eþ, and CD144þ of 43, 92, and 28 Ev/ml would be
detected with sufficient power (two sided a of 5%, power ¼ 0.80).
Comparisons between 2 groups were performed by indepen-
dent (Cross sectional study series 1) or paired t-tests when 2 groups
were compared (study 2), repeated measurements ANOVA when
three measurements were compared within subjects (study 3).
Subgroup analyses between 4 groups of patients were performed
with one-way ANOVA and Tukey post hoc test. Univarate correla-
tions were Pearson's r. Normal distribution was confirmed by
Kolmogorov-Smirnov test for all parameters except for subgroups
of EMP data. As a conservative approach and for consistency, we
performed decadic logarithmic transformation of all EMPs con-
centrations. P values of less than 0.05 were regarded as statistically
significant. All analyses were performed with SPSS 23 (IBM Corp.)
3. Results
3.1. Baseline characteristics of study subjects
See Supplemental Fig. 1 for study flow and Table 1 for detailed
characteristics of the 41 male subjects in study 1, comparing 20
patients with arterial hypertension (HT) with 21 normotensive
subjects at similar age (NT; 60.0 ± 4.7 and 59.1 ± 6.7 years). Within
each group, 10 subjects had a previous diagnosis of stable CAD but
were free of symptoms under normal daily life (Canadian Cardiac
Society [CCS]<¼1, New York Heart Association [NYHA] <¼II) and
had normal ejection fraction. With regard to routine clinical labo-
ratory parameters, the groups did not differ significantly. All pa-
tients were non-smokers and non-diabetic. The HT patients were
treated with the typical medication including aspirin (100%), clo-
pidogrel (60%), b-blockers (85%), angiotensin converting enzyme
inhibitors (57%) or angiotensin receptor blockers (43%), statins
(52%), as well as diuretics (29%) and calcium channel blockers
(66%).
As expected in HT, office systolic and diastolic office blood
pressure (122 ± 11 vs. 147 ± 5 mmHg, p < 0.001 and 81 ± 7 vs.
87 ± 11 mmHg, p < 0.001) and 24 h ambulatory blood pressure
70 R. Sansone et al. / Atherosclerosis 273 (2018) 67e74

(123 ± 11 vs. 144 ± 6 mmHg, p < 0.001 and 81 ± 8 vs. 91 ± 8 mmHg,
p < 0.001) were significantly higher as compared to NT. Addition-
ally, central blood pressure was also increased in patients with
arterial hypertension (113 ± 12 vs. 136 ± 16, p < 0.001 and 76 ± 11 vs.
87 ± 11 mmHg, p < 0.002). HT was accompanied by increased AIX
(21 ± 7 vs. 28 ± 5%, p < 0.001), PWV (8 ± 1 vs. 11 ± 2 m/s, p < 0.001),
pulse pressure (PP, 36 ± 9 vs. 50 ± 14 mmHg, p ¼ 0.001), and
augmentation pressure (AP, 11 ± 6 vs. 17 ± 6 mmHg, p ¼ 0.002). The
heart rate did not differ between the two groups (69 ± 8 vs. 65 ± 6/
min, p ¼ 0.101).
The HT group exhibited increased BA diameter (4.5 ± 0.5 vs.
5.0 ± 0.3 mm, p ¼ 0.001) that went along with decreased resting
WSS (5.3 ± 1.2 vs. 3.9 ± 0.6 dyne/cm2, p < 0.001) and maximal WSS
during hyperemia (55.5 ± 10.7 vs. 45.0 ± 8.5 dyne/cm2, p ¼ 0.001).
The endothelial function as measured by FMD was significant lower
in the HT group (5.3 ± 1.2 vs 3.8 ± 1.3%, p ¼ 0.001). Furthermore, the
endothelium independent nitroglycerin-mediated vasodilation was
not different between NT and HT (10.7 ± 1.8 vs. 10.6 ± 1.0%,
p ¼ 0.95).
Microvascular function of the cutaneous microcirculation as
measured during reactive hyperemia was significantly lower in HT:
maximal perfusion (LDPImax: 177 ± 73 vs. 129 ± 43 PU, p ¼ 0.023),
amplitude (LDPIamp: 142 ± 57 vs. 95 ± 37 PU, p ¼ 0.005), and area
under the curve (LDPIAUC: 2064 ± 536 vs. 1496 ± 404 PU, p ¼ 0.001).
Elevation of EMPs in HT and correlations with blood pressure,
mechanical indices, micro- and macrovascular function.
HT exhibited increased concentrations of all three EMP groups,
logCD31þ/41-, logCD62eþ, and logCD144þ, as compared to NT
(Fig. 1AeC). As CAD may also affect EMP levels, we performed a
subgroup analysis of patients with and without CAD (see figures in
accompanying Data in Brief article [29]) that showed that EMP
concentrations were significantly larger in patients with HT
without CAD as compared to NT without CAD. Interestingly, the
presence of CAD was also accompanied by increased EMP levels
similar to HT.
All three EMP concentrations, logCD31þ/41-, logCD62eþ, and
logCD144þ, correlated with office SBP, ambulatory SBP, central SBP,
PWV, and AIX and inversely with FMD (Table 2). While only
logCD31þ/41- inversely correlated with BA diameter, NMD, resting
and maximal WSS, only logCD62eþ and logCD144þ inversely
correlated with microvascular function as measured by LDPI. Due to
the small number of subjects, no multiple regression analysis was
performed to identify independent predictors of EMP
concentrations.
3.2. Blood pressure lowering in hypertensive emergency decreases
MPs
During hypertensive emergency, logCD62eþ and logCD144þ
concentrations were elevated as compared to stable HT patients in
the cross sectional study 1 above. LogCD31þ/41- concentrations
were not significantly different from stable HT values. BA diameter,
resting and maximal WSS did not differ between the hypertensive
emergency group and stable HT group in series 1 (3.6 ± 0.6 vs.
3.9 ± 0.6 dyne/cm2, p ¼ 0.144). Importantly, the FMD in hyperten-
sive emergency at admission was significantly lower than in stable
HT (2.6 ± 0.8 vs. 3.8 ± 1.3%, p ¼ 0.001).
As shown in Fig. 2, SBP lowering was achieved within 4 h using

Fig. 1. Endothelial microparticles (EMPs) in arterial hypertension (n ¼ 41).

(AeC) Increased concentrations EMPs in patients with arterial hypertension (HT) as compared to normotensives (NT; [A] logCD31þ/41-, [B] logCD62eþ, and [C] logCD144þ). Bars are
mean ± SD, *p < 0.05 vs. NT (t-test). (DeF) Positive correlations between EMPs and office systolic blood pressure (SBP) and (GeI) inverse correlations with flow-mediated dilation
(FMD). [D,G] logCD31þ/41-, [E,H] logCD62eþ, and [F,I] logCD144þ). Open symbols are NT, closed symbols are HT. (all p < 0.01; see Table 2 for individual Pearson's correlation co-
efficients and p-values).
 R. Sansone et al. / Atherosclerosis 273 (2018) 67e74 71

Table 2
Univariate correlations in cross-sectional study 1.

logCD31þ/41- logCD62eþ logCD144þ

r p r p r p

Office SBP (mmHg) 0.615 <0.001 0.453 0.003 0.497 0.001

Office DBP (mmHg) 0.319 0.042 0.341 0.029 0.082 0.568
24 h SBP (mmHg) 0.542 <0.001 0.382 0.018 0.423 0.008
24 h DBP (mmHg) 0.322 0.055 0.220 0.198 0.109 0.527
Central SBP (mmHg) 0.503 0.001 0.291 0.042 0.393 0.013
Central DBP (mmHg) 0.285 0.078 0.239 0.142 0.037 0.824
Heart rate (/min) 0.095 0.553 0.110 0.495 0.192 0.228
PWV (m/s) 0.463 0.003 0.456 0.003 0.492 0.001
FMD (%) ¡0.475 0.002 ¡0.572 <0.001 ¡0.635 <0.001
AIX (%) 0.336 0.034 0.344 0.030 0.659 <0.001
LDPImax (PU) 0.232 0.151 ¡0.530 0.001 ¡0.419 0.009
LDPIAUC (PU) 0.239 0.148 ¡0.570 <0.001 ¡0.484 0.002
NMD (%) ¡0.539 0.014 0.266 <0.257 0.051 0.831
BA diameter (mm) 0.578 <0.001 0.182 0.255 0.211 0.186
WSS (dyne/cm2) ¡0.402 0.009 0.063 0.697 0.048 0.767
Maximal WSS (dyne/cm2) ¡0.543 0.001 0.186 0.245 0.305 0.053

Bold was used to highlight significant p-values <0.05.

urapidil (203 ± 10 to 155 ± 9 mmHg, p < 0.001). Blood pressure
lowering also led to significant lowering of logCD62eþ and
logCD144þ-EMPs (each p < 0.001) but not logCD31þ/41-. While
FMD increased significantly after BP lowering, the BA diameter
(4.8 ± 0.5 vs. 4.8 ± 0.4 mm, p ¼ 0.716) and WSS were not affected
(3.6 ± 0.6 vs. 3.4 ± 0.9 dyne/cm2, p ¼ 0.313). Univariate correlations
were found between the change in office SBP and decrease in
logCD62eþ and logCD144þ and inverse correlations between FMD
and logCD62eþ and logCD144þ-EMPs. Neither changes in SBP nor
FMD correlated with changes in logCD31þ/41-. Due to the small
number of subjects, no multiple regression analysis was performed.
3.3. Catheter-induced arterial injury mobilizes endothelial MPs
along with endothelial dysfunction
As a positive control to evaluate which subpopulations of EMPs
increase after mechanical endothelial injury, we studied normo-
tensive patients before, at 1, 4, and 24 h after diagnostic artery
catheterization where the intra-arterial insertion of the sheath,
repeated wire and catheter passage cause mechanical injury along
the arterial endothelium of iliofemoral arteries and aorta (Fig. 3).
Interestingly, only logCD144þ-EMPs (p ¼ 0.001) increased at 1e4 h
and logCD62eþ increased late at 4 and 24 h (p ¼ 0.001) while
logCD31þ/41--EMPs remained unaffected. Again the BA diameter
und WSS remained unaffected. In parallel, we measured a signifi-
cant decrease in FMD as measured in the right brachial artery at
1e24 h.
4. Discussion
The key findings of the present study are that (a) the concen-
trations of CD31þ/CD41-, CD62eþ, and CD144þ-EMPs are elevated in
plasma of patients with hypertension, (b) elevated levels of all EMP
subpopulations were related to increased systolic blood pressure,
AIX, PWV and decreased FMD, (c) only CD31þ/CD41--EMPs corre-
lated with brachial artery diameter and inversely with endothelium-
independent dilation and wall shear stress, (d) only CD62eþ and
CD144þ-EMPs inversely correlated with microvascular perfusion, (e)
CD144þ and CD62eþ but not CD31þ/CD41--EMPs were elevated
during hypertensive emergency and acutely decreased along with
blood pressure normalization at unchanged wall shear stress, and
that (f) catheter related endothelial injury led to an acute temporary
increase in CD144þ-EMPs and delayed increase in CD62eþ-EMPs
while CD31þ/CD41--EMPs were unchanged.
A few others have previously investigated EMPs in the context of
arterial hypertension [22e24]. For instance, Preston at al showed
that CD31þ/CD42--EMPs were elevated in mild and even more in
severe uncontrolled hypertension and that EMPs (univariately)
correlated with both systolic and diastolic office blood pressure
[23]. Amabile et al. investigated the association of circulating EMPs
with cardiometabolic risk factors in the Framingham Heart study
[22]. In a multivariable analysis, they demonstrated that hyper-
tension was associated with increased CD144þ but not CD31þ/41--
EMPs. Hu et al. reported in a research letter that CD144þ and
CD62eþ-EMPs were significantly elevated in hypertensives as
compared to healthy controls [24]. In the latter 2 studies [22,24], no
correlations between blood pressure and EMPs were analyzed. Our
current data confirm the overall finding that the levels of EMPs are
increased in hypertensive subjects and show that the concentra-
tions of CD31þ/CD41-, CD62eþ, and CD144þ-EMPs all not only
correlate with increased (systolic) blood pressure but also with
arterial stiffness (AIX, PWV) and inversely with endothelial func-
tion (FMD). While limited by the small n-value, observational na-
ture, and potential confounding due to concomitant medication of
the studies, the univariate correlations hinted at a distinct pattern
of CD31þ/41- (correlating with diameter, NMD, and WSS) on the
one side and CD62eþ and CD144þ on the other (correlating with
microvascular perfusion). In hypertensive emergency, both CD62eþ
and CD144þ-EMPs were increased at admission and decreased with
blood pressure lowering together with improvements in endothe-
lial function. Likely the endothelial damage is most pronounced
with very high blood pressure. The observation that the magnitude
of blood pressure decrease and endothelial functional improve-
ment correlated with EMP decrease is driven be large blood pres-
sure drops, but also different blood pressure lowering medication
used, may explain why others have previously not observed
endothelial function improvements after blood pressure lowering
in stable hypertensives at 2 h [25]. To compare this response with
microparticle mobilization induced via a prototypical mechanical
injury, we evaluated EMPs after diagnostic catheterization. Similar
to hypertensive emergencies, we observed an early temporal in-
crease in CD144þ and late increase in CD62eþeEMPs after me-
chanical endothelial injury. Taken together and integrating the
results from the individual studies, we believe that it is feasible to
hypothesize that high blood pressure may cause mechanical
endothelial injury, which in turn leads to endothelial activation and
CD144þ and CD62eþ-EMPs may be markers of the respective
processes.
72 R. Sansone et al. / Atherosclerosis 273 (2018) 67e74

Fig. 2. Response to blood pressure lowering with urapidil in hypertensive emergencies (n ¼ 12).
(A) Both systolic and diastolic blood pressures were significantly lowered at 4 h (Post) as compared to baseline (BL), while (B) flow-mediated dilation (FMD) significantly increased,
and (C) WSS remained unaffected. Symbols are mean ± SD, *p < 0.05 vs. BL (paired t-test). (DeG) The decrease in systolic blood pressure (DSBP) at 4 h significantly correlated with
the decrease in (D) logCD62eþ (r ¼ 0.65, p ¼ 0.022) and (E) logCD144þ EMPs (r ¼ 0.66, p ¼ 0.020) while the change in FMD inversely correlated with decrease in (F) logCD62eþ
(r ¼ 0.56, p ¼ 0.04) and (G) logCD144þ EMPs (r ¼ 0.60, p ¼ 0.041).

Fig. 3. EMPs release after endothelial injury during coronary angiography (n ¼ 10).
While (A) logCD31þ/41- did not change, (B) logCD62eþ increased late at 4e24 h and (C) logCD144þ early and temporarily increased at 1e4 h, and (D) FMD was decreased early at 1 h
and remained decreased up to 24 h; Bars are mean ± SD, *p < 0.05 vs. BL, #p < 0.05 vs. 1 h.
 R. Sansone et al. / Atherosclerosis 273 (2018) 67e74
The mobilization of CD31þ/41--EMPs appears to be less linked
with acute blood pressure changes or mechanical endothelial
injury as they were not affected by acute blood pressure changes
and catheter-mediated mechanical injury. However, endothelial
dyfunction in arterial hypertension was previously linked with
larger arteries [26] and may be in part due to decreased wall shear
stress. In the present study, we observed that larger arterial di-
ameters and lower wall shear stress correlated with CD31þ/41--
EMPs. One interpretation is that chronic arterial hypertension leads
to macrovascular arterial enlargement, microvascular rarefaction,
and decreases athero-protective WSS on the arterial endothelium
and thereby leads to increased CD31þ/41--EMPs. Supporting this,
an inverse correlation between WSS and EMPs has been previously
shown using different EMP markers [27,28]. Taken together, indi-
vidual EMP subpopulations may be released in response to
different stimuli and, therefore, likely via different mechanisms
potentially involving endothelial injury, activation, and decreased
athero-protective wall shear stress.
4.1. Limitations
The major limitations of the present work are the limited n-
value in particular of the pilot study 1, observational nature, re-
striction to male subjects, and potential confounding due to
concomitant medication of the subjects in the studies. Due to the
small number of subjects, no multivariate analysis could be per-
formed to identify independent predictors of EMP concentrations.
Furthermore, correlations are no proof of causality and some of our
results could also be explained by potential bioactivities of EMPs.
4.2. Conclusion and perspectives
While previous studies have indicated that arterial hypertension
goes along with increased concentrations of EMPs, our data indi-
cate for the first time a link between individual patterns of he-
modynamic and physicomechanical changes associated with
hypertension with distinct patterns of EMPs. Our findings provide
novel mechanistic insight that could serve as food for thought with
regard to future work. Particularly, the striking similarities between
hypertensive emergencies and catheter-induced denudation indi-
cate that hypertension leads to mechanical endothelial ‘injury’
(CD144þ-EMPs) and consecutive activation in ‘response to injury’
(CD62eþ-EMPs). CD31þ/41--EMPs may rather indicate the response
to more chronic changes i.e. chronic pathological phys-
icomechanics. Besides the potential of EMPs as biomarkers of
distinct processes in the pathophysiology of arterial hypertension,
future work should address the signaling properties of EMPs in vivo.
This is currently hurdled by problems with regard to isolation and
purification of EMPs to be used in mechanistic experiments e.g. in
mice. However with a larger perspective, the measurement of EMPs
may help to understand and manage the degree of injury caused by
hypertension and endovascular procedures, evaluate endothelial
‘response to injury’, and serve as integrative therapeutic targets in
hypertension therapy.
Conflicts of interest
The authors declared they do not have anything to disclose
regarding conflict of interest with respect to this manuscript.
Financial support
This study was funded by the Deutsche For-
schungsgemeinschaft (KE405/5-1 to MK; GRK 1902 TP9 and
SFB1116 TP A07 and B06 to CH and MK) and the
73
Forschungskommission of the Medical Faculty of the Heinrich-
Heine University Duesseldorf to CH, DS, and RS. MK was sup-
ported by the Susanne Bunnenberg Stiftung at the Duesseldorf
Heart Center.
Author contributions
RS, MB, DS, and PH performed clinical exams; CH, MK, and NA
planned the studies; MB, RW, PH, and NA participated in EMP an-
alyses; CH and RS wrote the manuscript; MB, DS, RS, CH, PH, and NA
performed data analyses; MK, NA, and RW revised the manuscript.
Appendix A. Supplementary data
Supplementary data related to this article can be found at
https://doi.org/10.1016/j.atherosclerosis.2018.04.012.
References
[1] M.F. O'Rourke, J. Hashimoto, Mechanical factors in arterial aging: a clinical
perspective, J. Am. Coll. Cardiol. 50 (2007) 1e13.
[2] E.L. Schiffrin, J.B. Park, H.D. Intengan, et al., Correction of arterial structure and
endothelial dysfunction in human essential hypertension by the angiotensin
receptor antagonist losartan, Circulation 101 (2000) 1653e1659.
[3] H.C. Stary, D.H. Blankenhorn, A.B. Chandler, et al., A definition of the intima of
human arteries and of its atherosclerosis- prone regions. A report from the
committee on vascular lesions of the council on arteriosclerosis, American
Heart Association, Circulation 85 (1992) 391e405.
[4] R. Ross, The pathogenesis of atherosclerosis: a perspective for the 1990s,
Nature 362 (1993) 801e809.
[5] L. Burnier, P. Fontana, B.R. Kwak, et al., Cell-derived microparticles in hae-
mostasis and vascular medicine, Thromb. Haemostasis 101 (2009) 439e451.
[6] M.C. Martinez, A. Tesse, F. Zobairi, et al., Shed membrane microparticles from
circulating and vascular cells in regulating vascular function, Am. J. Physiol.
Heart Circ. Physiol. 288 (2005) H1004eH1009.
[7] P.E. Rautou, A.C. Vion, N. Amabile, et al., Microparticles, vascular function, and
atherothrombosis, Circ. Res. 109 (2011) 593e606.
[8] P. Horn, M.M. Cortese-Krott, N. Amabile, et al., Circulating microparticles carry
a functional endothelial nitric oxide synthase that is decreased in patients
with endothelial dysfunction, J. Am. Heart Assoc. 2 (2012) e003764.
[9] N. Werner, S. Wassmann, P. Ahlers, et al., Circulating CD31þ/annexin Vþ
apoptotic microparticles correlate with coronary endothelial function in pa-
tients with coronary artery disease, Arterioscler. Thromb. Vasc. Biol. 26 (2006)
112e116.
[10] J.M. Sinning, J. Losch, K. Walenta, et al., Circulating CD31þ/Annexin Vþ mi-
croparticles correlate with cardiovascular outcomes, Eur. Heart J. 32 (2011)
2034e2041.
[11] S.T. Lee, K. Chu, K.H. Jung, et al., Circulating CD62Eþ microparticles and car-
diovascular outcomes, PLoS One 7 (2012) e35713.
[12] N. Amabile, A.P. Guerin, A. Tedgui, et al., Predictive value of circulating
endothelial microparticles for cardiovascular mortality in end-stage renal
failure: a pilot study, Nephrol. Dial. Transplant. 27 (2012) 1873e1880.
[13] E. Stepien, E. Stankiewicz, J. Zalewski, et al., Number of microparticles
generated during acute myocardial infarction and stable angina correlates
with platelet activation, Arch. Med. Res. 43 (2012) 31e35.
[14] C. Heiss, N. Amabile, A.C. Lee, et al., Brief secondhand smoke exposure de-
presses endothelial progenitor cells activity and endothelial function: sus-
tained vascular injury and blunted nitric oxide production, J. Am. Coll. Cardiol.
51 (2008) 1760e1771.
[15] C. Gordon, K. Gudi, A. Krause, et al., Circulating endothelial microparticles as a
measure of early lung destruction in cigarette smokers, Am. J. Respir. Crit. Care
Med. 184 (2011) 224e232.
[16] N.M. Navasiolava, F. Dignat-George, F. Sabatier, et al., Enforced physical
inactivity increases endothelial microparticle levels in healthy volunteers, Am.
J. Physiol. Heart Circ. Physiol. 299 (2010) H248eH256.
[17] G. Mancia, R. Fagard, K. Narkiewicz, et al., 2013 ESH/ESC guidelines for the
management of arterial hypertension: the Task force for the management of
arterial hypertension of the European society of hypertension (ESH) and of the
European society of cardiology (ESC), Eur. Heart J. 34 (2013) 2159e2219.
[18] C. Heiss, S. Jahn, M. Taylor, et al., Improvement of endothelial function with
dietary flavanols is associated with mobilization of circulating angiogenic cells
in patients with coronary artery disease, J. Am. Coll. Cardiol. 56 (2010)
218e224.
[19] P. Horn, N. Amabile, F.S. Angeli, et al., Dietary flavanol intervention lowers the
levels of endothelial microparticles in coronary artery disease patients, Br. J.
Nutr. 111 (2014) 1e8.
[20] S. Keymel, J. Sichwardt, J. Balzer, et al., Characterization of the non-invasive
assessment of the cutaneous microcirculation by laser Doppler perfusion
scanner, Microcirculation 17 (2010) 358e366.
74 R. Sansone et al. / Atherosclerosis 273 (2018) 67e74
[21] L.M. Van Bortel, S. Laurent, P. Boutouyrie, et al., Expert consensus document
on the measurement of aortic stiffness in daily practice using carotid-femoral
pulse wave velocity, J. Hypertens. 30 (2012) 445e448.
[22] N. Amabile, S. Cheng, J.M. Renard, et al., Association of circulating endothelial
microparticles with cardiometabolic risk factors in the Framingham Heart
Study, Eur. Heart J. 35 (2014) 2972e2979.
[23] R.A. Preston, W. Jy, J.J. Jimenez, et al., Effects of severe hypertension on
endothelial and platelet microparticles, Hypertension 41 (2003) 211e217.
[24] S.S. Hu, H.G. Zhang, Q.J. Zhang, et al., CD144(þ)EMPs/CD62E(þ)EMPs: a couple
of new biomarkers to monitor endothelial function in hypertension with
hyperlipidemia involved, Int. J. Cardiol. 175 (2014) 203.
[25] L. Ghiadoni, Y. Huang, A. Magagna, et al., Effect of acute blood pressure
reduction on endothelial function in the brachial artery of patients with
essential hypertension, J. Hypertens. 19 (2001) 547e551.
[26] T. Lauer, C. Heiss, J. Balzer, et al., Resting microvascular resistance and conduit
artery tone: relevance to endothelium-dependent flow-mediated dilation,
Eur. J. Cardiovasc. Prev. Rehabil. 15 (6) (2008) 677e682.
[27] A.C. Vion, B. Ramkhelawon, X. Loyer, et al., Shear stress regulates endothelial
microparticle release, Circ. Res. 112 (2013) 1323e1333.
[28] P. Horn, D. Stern, V. Veulemans, et al., Improved endothelial function and
decreased levels of endothelium-derived microparticles after transcatheter
aortic valve implantation, EuroIntervention 10 (2015) 1456e1463.
[29] R. Sansone, M. Baaken, P. Horn, D. Schuler, R. Westenfeld, N. Amabile, M.
Kelm, C. Heiss, Endothelial microparticles and vascular parameters and in
subjects with and without arterial hypertension and coronary artery disease,
Submitted.