Release of endothelial microparticles in patients with arterial hypertension, hypertensive emergencies and catheter-related injury

Atherosclerosis 273 (2018) 67e74

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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 CARID-Cardiovascular Research Institute Duesseldorf, Medical Faculty, University Duesseldorf, Duesseldorf, Germany Institut Mutualiste Montsouris, Paris, France d University of Surrey, Faculty of Health and Medical Sciences, Guildfort, UK e Surrey and Sussex Healthcare NHS Trust, Redhill, UK b c

a r t i c l e i n f o

a b s t r a c t

Article history: Received 5 November 2017 Received in revised form 7 April 2018 Accepted 11 April 2018 Available online 12 April 2018

Background and aims: Circulating endothelial microparticles (EMPs) are increased in arterial hypertension. The role of physicomechanical factors that may induce EMP release in vivo is still unknown. We studied the relationship of EMPs and physicomechanical factors in stable arterial hypertension and hypertensive emergencies, and investigated the pattern of EMP release after mechanical endothelial injury. 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-waveanalysis was performed. Flow-mediated dilation (FMD), nitroglycerin-mediated dilation (NMD), and wall-shear-stress (WSS) were measured ultrasonographically in the brachial artery; microvascular perfusion by laser-Doppler (Clinicaltrials.gov: NCT02795377). We studied patients with hypertensive emergencies before and 4 h after BP lowering by urapidil (n ¼ 12) and studied the release of EMPs due to mechanical endothelial injury after coronary angiography (n ¼ 10). Results: Hypertensives exhibited increased EMPs (CD31þ/41-, CD144þ, CD62eþ) as compared to normotensives 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, catheterrelated endothelial injury increased only CD144þ and CD62eþ-EMPs. Conclusions: EMP release in hypertension is complex and may involve both physicomechanical endothelial injury and activation (CD144þ, CD62eþ) and decreased wall shear stress (CD31þ/41-). © 2018 Elsevier B.V. All rights reserved.

Keywords: Cell-derived microparticles Endothelial function Arterial hypertension Endothelial function Wall shear stress

1. Introduction 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 Sciences, Section of Clinical Medicine and Ageing, Guildford, GU2 7XH, UK. E-mail address: [email protected] (C. Heiss). https://doi.org/10.1016/j.atherosclerosis.2018.04.012 0021-9150/© 2018 Elsevier B.V. All rights reserved.

dysfunction, it is not entirely clear how it is linked to atherosclerosis progression [1]. Stiffness of conduit arteries leads to accelerated 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 components 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 mechanical stress may lead to endothelial injury and dysfunction

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which, in turn, could drive arterial remodeling and plaque development [2,3]. According to the response to injury theory, mechanical injury and exposure to cardiovascular risk factors disrupt endothelial integrity driving the initiation and progression of atherosclerosis [4]. Endothelial microparticles (EMPs) can be viewed as circulating markers of endothelial injury and a compromised endothelial integrity that are released from activated and apoptotic endothelial cells as shown in vitro [5,6]. These EMPs are membrane particles of less than a micrometer in diameter and carry endothelial surface markers [5e7] and enzymes including eNOS [8]. Circulating levels of EMPs increase in plasma early in the atherosclerotic processes, correlate with the degree of endothelial dysfunction [8,9], and have been established as prognostic biomarkers that predict adverse CV outcome [10e12]. Cardiovascular risk factors may trigger EMP release [7,13]. While it was previously shown that arterial hypertension goes along with increases EMP levels in human subjects [14e16], the role of physicomechanical factors present in arterial hypertension that may induce mechanical endothelial injury and activation and shedding of EMPs in vivo is still unknown. In order to investigate this, we first studied in a cross-sectional pilot cohort how hemodynamic, structural, and functional characteristics that are chronically changed in stable hypertensives relate to circulating EMP concentrations. We then evaluated the relationships of arterial characteristics and the pattern of EMP dynamics during hypertensive emergencies and after consecutive blood pressure lowering. Finally, we determined the pattern of EMPs released by a prototypical mechanical endothelial injury as induced during arterial catheterization.

2. Materials and methods 2.1. Study subjects and protocol In a first pilot study (study 1), we investigated circulating EMPs along with functional and mechanical characteristics of the arterial system in 41 consecutive male subjects, 20 subjects without arterial hypertension and 21 subjects with arterial hypertension as defined by office blood pressure 140/90 mmHg [17] or history of hypertension with ongoing antihypertensive medication (Table 1). The patients were recruited from the out-patient clinic. As the presence of CAD might also affect circulating EMP values, we aimed at

Table 1 Characteristics of study population (study 1).

n CAD Diabetes mellitus Smoker Age (y) BMI (kg/m2) Height (m) Weight (kg) Creatinine (mg/dl) Urea (mg/dl) Total cholesterol (mg/dl) LDL cholesterol (mg/dl) HDL cholesterol (mg/dl) Triglycerides (mg/dl) Fasting plasma glucose (mg/dl) HbA1c (%) CRP (mg/dl) Hb (mg/dl) Leucocytes (1000/ml)

NT

HT

p-value

20 10 0 0 60 ± 4.7 27.1 ± 3.6 1.8 ± 0.6 86 ± 12 0.9 ± 0.1 35 ± 11 199 ± 43 138 ± 31 52 ± 15 134 ± 62 102 ± 20 5.5 ± 0.5 0.1 ± 0.2 14.5 ± 1.2 6.3 ± 1.3

21 10 0 0 59 ± 6.0 29.2 ± 3.7 1.8 ± 0.6 93 ± 12 1.0 ± 0.1 33 ± 8 191 ± 51 140 ± 40 55 ± 28 139 ± 34 99 ± 10 5.5 ± 0.4 0.1 ± 0.2 14.8 ± 1.5 7.4 ± 1.4

0.619 0.081 0.636 0.081 0.591 0.584 0.623 0.866 0.671 0.746 0.506 0.738 0.578 0.410 0.394

Values are mean and standard deviation; p-values refer to unpaired t-test.

including 50% patients with stable CAD. No dedicated matching procedures were applied. Hemodynamics and endothelial function of the brachial artery were assessed as flow-mediated dilatation (FMD). Pulse wave analyses including central blood pressure, pulse wave velocity (PWV), and aortic augmentation index (AIX) were performed by tonometry. In all subjects, EMP subpopulations (CD31þ/41-, CD144þ, CD62eþ) were analyzed by flow-cytometry according to the expression of surface antigens. Exclusion criteria were manifest peripheral artery, or cerebrovascular disease, acute inflammation (C-reactive protein [CRP] >0.6 mg/dl), kidney failure (estimated glomerular filtration rate [eGFR] <30 ml/min), malignancies, and arrhythmias (heart rhythm other than sinus). In a second study, we analysed 12 patients that were admitted to the emergency department with acute symptoms of dyspnea or angina in the presence of blood pressure >180/120 mmHg, troponin T value not exceeding the upper reference limit (14 ng/l) at admission and 1 h excluding acute myocardial infarction and indicating hypertensive emergency [17]. Measurements of EMPs and vascular measurements were taken at admission (0 h) before and at 4 h and after lowering of BP by urapidil (Stragen, Denmark, 10e50 mg i.v.). EMP subpopulations were analyzed by flowcytometry and FMD measured by ultrasound. All patients were discharged on the same day. Exclusion criteria were manifest peripheral artery, or cerebrovascular disease, acute inflammation (CRP>0.6 mg/dl), kidney failure (eGFR<30 ml/min), malignancies, arrhythmias (heart rhythm other than sinus), and troponin T value exceeding the upper reference limit (14 ng/l). In a third series, we aimed at evaluating the pattern of EMP 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, and at 24 h after elective transfemoral diagnostic coronary angiography in n ¼ 10 stable CAD in-patients without arterial hypertension. EMP subpopulations were analyzed by flow-cytometry and FMD measured by ultrasound. Inclusion criteria were normal left ventricular ejection fraction (>55%), stable CAD, and blood pressure <140/90 mmHg. Exclusion criteria were manifest peripheral artery, or cerebrovascular disease, acute inflammation (CRP>0.6 mg/dl), kidney failure (eGFR<30 ml/min), malignancies, and arrhythmias (heart rhythm other than sinus). Measurements were always performed in the same order in one session. We first took blood samples from the left arm. After a 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 measurements on the right arm, and, finally, performed applanation tonometry on the neck and groin. The study protocol was approved by the ethics committee of the Heinrich Heine University Duesseldorf and all patients and volunteers gave written informed consent (Clinicaltrials.gov: NCT02795377). 2.2. Characterization of EMP subpopulations by flow cytometry Citrated blood (6 ml) was drawn from the cubital vein and processed within 2 h. Platelet-rich plasma was obtained by centrifugation of whole blood at 300  g over 15 min at room temperature. Platelet-free plasma was obtained by 2 successive centrifugations of platelet-rich plasma at 10,000  g for 5 min at room temperature. Briefly, samples were incubated for 30 min with fluorochrome-labeled antibodies or matching isotype controls and analyzed in a Canto II flow cytometer (Beckton Dickinson, Heidelberg, Germany). Microbead standards (1.0 mm) were used to define MPs as <1 mm in diameter. The EMP subpopulations were defined as CD31þ/CD41-, CD62eþ, or CD144þ events. The total number of EMPs was quantified using flow-count calibrator beads (20 ml).

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2.3. Flow-mediated vasodilation (FMD)

2.7. Pulse wave analysis

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 between 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).

Central blood pressure parameters including augmentation index (AIX) and pulse wave velocity (PWV) were measured by radial applanation tonometry using the SphygmoCor® system in accordance with the recommendations of the expert consensus on arterial stiffness [21]. Via a proprietary generalized transfer function, the pressure waveform of the ascending aorta was synthesized. PWV was determined from tonometry measurements taken at the carotid and femoral artery.

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 polyurethane 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 reactivity 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 resistance by impedance cardiography, which included ECG, phonocardiography, 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).

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 deviations 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 independent (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 correlations 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 concentrations. 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 laboratory parameters, the groups did not differ significantly. All patients were non-smokers and non-diabetic. The HT patients were treated with the typical medication including aspirin (100%), clopidogrel (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

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(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. Additionally, 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 hypertensive 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 coefficients and p-values).

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Table 2 Univariate correlations in cross-sectional study 1. logCD31þ/41-

Office SBP (mmHg) Office DBP (mmHg) 24 h SBP (mmHg) 24 h DBP (mmHg) Central SBP (mmHg) Central DBP (mmHg) Heart rate (/min) PWV (m/s) FMD (%) AIX (%) LDPImax (PU) LDPIAUC (PU) NMD (%) BA diameter (mm) WSS (dyne/cm2) Maximal WSS (dyne/cm2)

logCD62eþ

logCD144þ

r

p

r

p

r

p

0.615 0.319 0.542 0.322 0.503 0.285 0.095 0.463 ¡0.475 0.336 0.232 0.239 ¡0.539 0.578 ¡0.402 ¡0.543

<0.001 0.042 <0.001 0.055 0.001 0.078 0.553 0.003 0.002 0.034 0.151 0.148 0.014 <0.001 0.009 0.001

0.453 0.341 0.382 0.220 0.291 0.239 0.110 0.456 ¡0.572 0.344 ¡0.530 ¡0.570 0.266 0.182 0.063 0.186

0.003 0.029 0.018 0.198 0.042 0.142 0.495 0.003 <0.001 0.030 0.001 <0.001 <0.257 0.255 0.697 0.245

0.497 0.082 0.423 0.109 0.393 0.037 0.192 0.492 ¡0.635 0.659 ¡0.419 ¡0.484 0.051 0.211 0.048 0.305

0.001 0.568 0.008 0.527 0.013 0.824 0.228 0.001 <0.001 <0.001 0.009 0.002 0.831 0.186 0.767 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 normotensive 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 significant 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 concentrations 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 correlated with brachial artery diameter and inversely with endotheliumindependent 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 hypertension 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 concentrations 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 function (FMD). While limited by the small n-value, observational nature, 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 endothelial 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 improvement correlated with EMP decrease is driven be large blood pressure 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 increase in CD144þ and late increase in CD62eþeEMPs after mechanical 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.

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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.

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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 diameters 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, individual 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 nvalue in particular of the pilot study 1, observational nature, restriction 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 performed 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 indicate for the first time a link between individual patterns of hemodynamic 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 indicate 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 physicomechanics. 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 Forschungsgemeinschaft (KE405/5-1 to MK; GRK 1902 TP9 and SFB1116 TP A07 and B06 to CH and MK) and the

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Forschungskommission of the Medical Faculty of the HeinrichHeine University Duesseldorf to CH, DS, and RS. MK was supported 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 analyses; 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 haemostasis 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 patients 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 microparticles 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 cardiovascular 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 depresses endothelial progenitor cells activity and endothelial function: sustained 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.

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