Circulating endothelial microparticles in female migraineurs with aura

Abstract

Background and purpose

Endothelial microparticles (EMPs) are vesicles that are released from activated endothelial cells and serve as a surrogate for endothelial dysfunction (ED). ED may be involved in migraine pathophysiology and contribute to the increased risk of ischemic stroke, particularly in female migraineurs with aura (MA). We sought to determine whether EMPs are elevated in women with MA.

Methods

In this case-control study, EMPs were detected by analysing surface markers using fluorescence-activated cell sorting (FACS). Surface markers were measured covering the main cell lines relevant in cardiovascular disease like endothelial cells, platelets, monocytes and leucocytes. Microparticles (MPs) were identified in correlation to calibration by 1 -µm calibrator beads (Beckman Coulter). Arterial stiffness was assessed using fingertip tonometry and the heart rate-adjusted augmentation index (AI).

Results

We included 29 patients with MA and 29 matched controls. MA patients had significantly higher EMPs (CD62E⁺AnnexinV⁺: 5142/µl vs 1535/µl; p < 0.001; CD144⁺AnnexinV⁺: 6683/µl vs 3107/µl; p < 0.001), monocytic (CD14⁺AnnexinV⁺ 6378 vs 3161; p < 0.001), and platelet MPs (CD62P⁺CD42b⁺AnnexinV⁺ 5450 vs 3204; p < 0.001). Activated EMPs (CD62E⁺AnnexinV⁺) correlated with heart-rate adjusted AI (r = 0.46; p < 001).

Conclusion

EMP levels are significantly elevated in women with MA and correlated with increased AI. Our findings suggest that endothelial activation is present in women with MA. This might contribute to higher stroke risk in MA.

Keywords

Endothelial microparticle migraine endothelial dysfunction stroke risk

Introduction

Endothelial microparticles (EMPs) are vesicles that are released from endothelial cells (ECs) and serve as a surrogate for endothelial activation and dysfunction (ED) (1,2). ED may be involved in migraine pathophysiology and contribute to increased ischemic stroke risk, particularly in premenopausal women with migraine with aura (MA) (3,4). Studies investigating peripheral endothelial function in MA report controversial results, but there is mounting evidence that circulating markers for ED are elevated in MA (5). Furthermore, recent findings suggest that reduced numbers and function of endothelial progenitor cells (EPCs) in MA may be indicative of ED and the need for vascular repair (6). Higher levels of EMPs in the peripheral blood are associated with endothelial dysfunction and worse cardiovascular outcome. Thus, increased levels of EMPs have been described in several cardiovascular diseases that are associated with ED – namely, coronary artery disease and diabetes mellitus (2,7,8).

We sought to determine whether levels of EMP as a novel marker for ED are elevated in premenopausal women with MA.

Methods

Study population

This study was approved by the Charité – Universitätsmedizin, Berlin ethics committee and is registered with clinicaltrials.org (NCT01388699). Data on endothelial function and arterial stiffness measured with EndoPAT in the same study cohort were published previously (9). All patients and healthy volunteers provided written informed consent.

Migraine patients ≤50 years of age who fulfilled International Headache Society MA diagnostic criteria (second edition) and reported having MA at least once per month were recruited from the outpatient headache clinic over a three-month period. Healthy participants were randomly recruited from Charité hospital staff.

Exclusion criteria were severe infectious disease, history of cardiovascular disease (stroke, coronary heart disease, myocardial infarction), body mass index (BMI) <18 or >25 kg², arterial hypertension (systolic blood pressure >140 mmHg, diastolic blood pressure >90 mmHg), antihypertensive drugs, diabetes mellitus, pregnancy, drug abuse and medication of statins, anticoagulants, aspirin, clopidogrel or intake of triptans less than 24 hours prior.

All patients had a detailed clinical evaluation and a semi-structured interview about history of headache by an experienced neurologist. Healthy volunteers were matched with patients by age (±3 years) and blood pressure (±10 mmHg). Healthy volunteers were of similar age, body composition and main clinical characteristics, including lipid status, smoking habits and oral contraceptive intake. Carotid intima-media thickness (CIMT) was assessed for detection of subclinical atherosclerosis as previously recommended for case-control studies on endothelial function in migraine patients (10).

Isolation of MPs and flow cytometry

Complete methods regarding MP isolation and flow cytometry has been described previously (11). Briefly, after centrifugation, platelet-poor plasma were incubated with fluorescent monoclonal antibodies (2 µl each): phycoerythrin (PE)-labelled anti-CD31 (BD Biosciences, San Jose, CA, USA), fluorescein isothiocyanate-labelled (FITC) anti-CD144 (R&D, Minneapolis, MN, USA), FITC-labelled anti-CD62E (BD Biosciences), FITC-labelled anti-CD14 (BD Biosciences), PE-labelled anti-CD45 (BD Biosciences), and allophycocyanin-labelled AnnexinV (AV) (BD Biosciences). Samples were analysed with flow cytometry (FACS Calibur, BD Biosciences). To delineate CD31-positive EMPs and not platelet-derived CD31-positive MPs, CD42b-negative MPs were analysed in platelet-free plasma.

Size calibration was performed using microbeads from an FACS size-calibration kit (LB-30, Sigma, Munich, Germany). We used samples that were not previously stained and isotype controls to discriminate events from background noise and to increase the specificity of MP detection. CD144 was chosen as a general marker for EMPs as CD144 is selectively expressed on ECs, in comparison with CD62E, which is increasingly expressed in activation and CD31, which is increased during apoptosis.

Platelet-derived MPs (PMPs) were quantified as CD62P⁺CD42b⁺AV⁺. Monocytic MPs (MMPs) were analysed as CD14⁺AV⁺. Leucocytic MPs (LMPs) were quantified as CD45⁺AV⁺.

Values were reported as counts in 1 µl platelet-poor or platelet-rich plasma (counts/500,000 events). MP and calibrator beads (CAL) (10 µm diameter) were visualised in a forward light scatter (FSC) and side-angle scatter (SSC) as previously plotted and reported (11). MPs were defined as events (size 0.1–1 µm, R1). MP concentration was assessed by comparison to flow count calibrator beads. Laboratory personnel were unaware of patients’ clinical or laboratory data.

Endothelial function, arterial stiffness and CIMT

Quantitative determination of endothelial function and arterial stiffness were performed with a non-invasive finger plethysmograph (EndoPAT2000; Itamar Medical, Caesarea, Israel). Endothelial function was assessed with peripheral arterial tonometry (PAT) during reactive hyperaemia of the forearm vascular bed as previously reported (9). Measurements were performed under standardised conditions after a) overnight fasting, b) a resting period of at least 15 minutes, and c) in a quiet room of comfortable temperature.

Arterial stiffness was assessed through finger tonometry-derived augmentation index (AI) and was calculated automatically by analysing the waveform of the PAT signal and averaged from all beat-to-beat signals of the finger arterial pulse-wave amplitude during the baseline period as previously described (9). AI was calculated as the difference between the second (P2) and first systolic peak pressure (P1) and expressed as percentage of the central pulse pressure (PP): AI (%) = [(P2 – P1)/PP]× 100. Because AI is influenced by changes in heart rate, all values of AI were corrected to 75 beats/minute (AI@75 bpm).

CIMT was assessed according to the Mannheim Intima Media Thickness Consensus (12). CIMT of the far wall was measured at 1 cm proximal to the carotid bulb using a standardised real-time measurement method as previously described (9).

Statistics analysis

Sample size estimation was based on a previous study that investigated differences in endothelial dysfunction between migraine and healthy controls (13). Data are expressed as mean ± SD for continuous variables and as frequencies and proportions for categorical variables. Continuous data of two groups were compared using the Wilcoxon singed-rank test or Student’s t test if data are normally distributed. Categorical variables were compared using the Pearson χ-test, for correlation the Spearman’s rank correlation was used. All tests were two tailed and statistical significance was determined at an alpha level of 0.05. Statistical analyses were performed with SPSS software (version 21, IBM). The p values shown in Table 1 are presented without adjustment for multiplicity.

Table 1.

Demographic, clinical and laboratory variables in women with migraine with aura and healthy women.

	Migraine with aura	Healthy subjects	p value
n	29	29
Age (mean, ± SD)	34 ± 10	33 ± 10	0.62
RR systolic in mmHg	118 ± 9	119 ± 6	0.82
MAP in mmHg	88.2 ± 7	90.8 ± 5	0.12
Heart rate	65 ± 10	67 ± 10	0.51
Weight	63.7 ± 12	64.0 ± 8	0.92
BMI	22.4 ± 3.4	21.9 ± 2.8	0.59
Total cholesterol in mmol/l	191.7 ± 31	190.9 ± 45	0.93
Triglycerides in mmol/l	81.5 ± 29	79.9 ± 37	0.34
LDL in mmol/l	99.7 ± 43	98.7 ± 31	0.91
Smoking (n, %)	4 (14)	3 (11)	0.52
Oral contraceptives (n, %)	11 (38)	10 (36)	0.54
IMT left in mm (mean, ± SD)	484 ± 119	506 ± 61	0.42
Common carotid artery diameter in cm	6.0 ± 0.6	6.1 ± 0.8	0.47
Augmentation index at 75 bpm,
HR averaged in % (median; IQR)	1.0 (17.5)	−9.5 (19.9)	0.002
PAT-ratio (median; IQR)	2.1 (1.0)	1.9 (1.4)	0.88
Microparticle profiles^a
in median (IQR)
CD144⁺AV⁺
(EMP)	6683 (937)	3107 (254)	<0.001
CD62E⁺AV⁺
(activated EMPs)	5142 (574)	1535 (141.5)	<0.001
CD31⁺AV⁺
(apoptotic EMPs)	1305 (430)	1533 (263)	0.1
CD62P⁺CD42b⁺AV⁺ (PMPs)	5450 (416)	3204 (274)	<0.001
CD14⁺AV⁺ (MMPs)	6378 (469)	3161 (331)	<0.001
CD45⁺AV⁺ (LMPs)	4383 (341)	4275 (484)	0.62

RR: respiratory rate; MAP: mean arterial pressure; BMI: body mass index; LDL: low-density lipoprotein; IMT: intima-media thickness; HR: heart rate; PAT: peripheral arterial tonometry; IQR: interquartile range; EMPs: endothelial microparticles; PMPs: platelet-derived microparticles; MMPs: monocytic microparticles; LMPs: leucocytic microparticles; AV: Annexin V. ^aShown as mean cell counts per 500,000 events.

Results

Demographics, clinical and laboratory data are shown in Table 1. Twenty-nine female migraineurs without comorbidities and 29 healthy participants were included. Four of the MA patients (13.8%) received prophylactic treatment (two with beta-blockers, one with lamotrigine, and one with topiramate). The frequency of migraine attacks per month was 3.2 ± 2.8 with 4.4 ± 3.5 days of migraine per month. Twenty-two patients (75.9%) had more than one attack per month. MA patients had significantly higher EMP (CD62E⁺AV⁺: 5142/µl vs. 1535/µl; p < 0.001; CD144⁺AV⁺: 6683/µl vs 3107/µl; p < 0.001), monocytic (CD14⁺AV⁺: 6378 vs 3161; p < 0.001), and platelet MP (CD62P⁺CD42b⁺AV⁺: 5450 vs 3204; p < 0.001) as shown in Figure 1 and are expressed as counts/500,000 events. Apoptotic EMP (CD31⁺AV⁺) and LMP (CD45⁺AV⁺) were not elevated in MA compared to controls.

Figure 1.

Bar charts (mean, SD) of circulating microparticles in women with migraine with aura (black) compared to age- and vascular risk factor-matched controls (white) for different subtypes of microparticles. EMPs were significantly increased in MA (EMP = CD144⁺AV⁺; p < 0.001; activated EMPs = CD62E⁺AV⁺; p < 0.001), monocytic MPs (CD14⁺AV⁺; p < 0.001) and platelet MPs (CD62P⁺CD42b⁺AV⁺; p < 0.001). No differences were found for apoptotic EMPs (CD31⁺AV⁺; p = 0.35) and leucocytic MPs (CD45⁺AV⁺; p = 0.23). Values were reported as counts in platelet-poor plasma as counts per 500,000 events.

AI@75bpm, a surrogate for arterial stiffness, moderately correlated with activated EMPs (r = 0.46; p < 0.001). No correlation was observed between activated EMPs and PAT ratio (r = 0.3; p = 0.1).

Discussion

The primary findings of our study were that 1) EMPs isolated from peripheral blood were elevated in women with MA compared to matched controls; 2) levels of activated EMs correlate with arterial stiffness; and 3) PMPs and MMPs were increased in MA.

Though mounting evidence suggests that systemic ED might be associated with migraine, very few studies have assessed the relationship between soluble endothelial activation biomarkers and MA (4). One study performed by Tietjen et al. found that patients with migraine had increased levels of endothelial activation biomarkers and that those patients with MA specifically had higher levels of endothelial activation markers for oxidative stress (total nitrite/nitrate concentration), inflammation (high-sensitivity C-reactive protein (hs-CRP)) and coagulation (plasma tissue plasminogen activator (t-PA), von-Willebrand-factor (vWF) activity) than those without aura (5). Our findings are in accordance with those of Tietjen et al. and support the hypothesis that endothelial activation, a component of ED, is associated with MA in women. Studies that have assessed systemic ED non-invasively (using, for instance, flow-mediated dilation) have yielded more conflicting results. Though some studies have found positive relationships to exist, others have not found any association between ED and MA (9,10).

According to observational studies, women with MA are at higher risk for ischemic stroke (3). ED is independently associated with an increased risk of cerebro- and cardiovascular events (14,15). Dreier et al. (16) were the first to describe an in vivo rat model that induced cortical spreading depression, the pathophysiological correlate to MA. In this model, endothelin-1, a vasoconstrictor and mediator of ED, was used to induce the cortical spreading depression, thus lending credence to the argument that ED may play a role in MA. Moreover, a number of genes associated with ED (e.g. angiotensin I-converting enzyme deletion/deletion (ACE DD) polymorphism or the methylenetetrahydrofolate reductase –677 TT polymorphism) have been shown to be associated with MA risk (4). Taken together, this evidence suggests that ED may at least in part explain why MA patients seem to be at a higher risk for developing ischemic stroke.

To the best of our knowledge, this is the first study to examine EMP levels in female MA patients. EMPs are thought to play a causative role in ED and have been used as a surrogate marker for ED (17,18). EMPs are membrane-shed vesicles that are released in response to endothelial activation and acute endothelial damage and are thought to provide biological information between cells. However, their specific role is not clear yet. Recent studies have shown that higher levels of MPs derived from ECs, inflammatory cells and platelets are found in cardiovascular diseases and in patients with vascular risk factors like diabetes and hypertension (19,20). Thus, MP levels are considered to be biomarkers for vascular injury and inflammation in vascular diseases (e.g. acute myocardial infarction, ischemic stroke, metabolic syndrome and preeclampsia) (21). Furthermore, levels of EMPs correlate with established endothelial function measures like quantitative coronary angiography during intracoronary acetylcholine infusion, which is considered the gold standard for measurement of endothelial function (2). Boulanger et al., for instance, found that EMPs from patients with myocardial infarction suppressed endothelium-dependent relaxation in isolated arteries (22). Moreover, higher levels of EMPs have been found to be associated with reduced peripheral endothelial function (21).

We previously found no association between peripheral endothelial function, e.g. vascular reactivity in MA compared to healthy controls using the EndoPAT device (9). Furthermore, we did not find elevated apoptotic EMP levels in MA in this study. One possible explanation might be that there is a preclinical endothelial activation in MA patients leading to ED rather than a manifest endothelial damage compared to e.g. patients with coronary artery disease (CAD) where high levels of apoptotic EMP (CD31⁺AV⁺) are described (8). Thus, EMP might be a more sensitive marker for an early ED state with endothelial irritation than clinical assessment methods such as flow-mediated vasodilation (FMD) or EndoPAT (21). However, the discrepancy between endothelial activation and vascular reactivity may also be due to the fact that vascular reactivity is being measured peripherally, rather than cerebrally, where endothelial activation might be more present in MA patients.

We additionally found that activated EMP levels correlated with arterial stiffness (AS). This finding confirms previous findings that identified an association between increased AS and migraine and supports the hypothesis that profound vascular alterations are coupled both with structural and functional changes in MA (23). Moreover, elevated MPs in the peripheral blood might be a more generalised marker for overall cardiovascular risk than cerebrovascular risk as suggested by the correlation between activated EMPs and heart rate-adjusted AI.

We also found that MA patients had higher levels of PMPs and MMPs than controls. These findings suggest that MA might be a pro-inflammatory and hypercoagulable state as a possible downstream result of endothelial activation. PMPs and MMPs are thought to be involved in the regulation of endothelial integrity, interact with ECs and blood cells, and trigger endothelial inflammation by release of cytokines such as interleukin (IL)-6 and IL-8. In vitro experimental studies have shown that PMPs can induce the up-regulation and release of intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1) and E-selectin, which are used as biomarkers for ED in clinical studies (24). MMP has been shown to promote inflammatory processes in EC via extracellular signal-regulated protein kinases (ERK)1/2 and kappa-light-chain-enhancer of activated B-cells (NFkB)-dependent pathways (21).

Our study has the following strengths and limitations. To the best of our knowledge, this is the first study that investigated MP profiles in migraine patients. Furthermore, we employed strict matching criteria (e.g. assessment of CIMT for detection of subclinical atherosclerosis, lipid profiles) to avoid possible changes in EMP values caused by confounding factors.

Because of our small sample size in this study, the generalisability of our results has to be interpreted with caution. Further evidence is needed from larger observational studies. Currently, there are no international guidelines for the assessment of MP levels, possibly making it difficult to compare studies that have analysed MP levels. However, consensus statements on the evaluation on endothelial functions measures comprising endothelial MP do exist (e.g. from the European Society of Cardiology Working Group on Peripheral Circulation) (25).

In conclusion, our data provide the first evidence that higher levels of EMPs, MMPs and PMPs are present in women with MA. These findings suggest that endothelial activation is present in MA, which may contribute to the stroke risk in MA patients.

Clinical implications

The primary findings of our study were that 1) endothelial microparticles (EMPs) isolated from peripheral blood were elevated in women with migraine with aura (MA) compared to matched controls; 2) levels of activated EMPs correlate with arterial stiffness; and 3) platelet MPs and monocytic MPs were increased in MA.

Footnotes

Funding

This work was supported by the Federal Ministry of Education and Research via the grant Center for Stroke Research Berlin (01 E0 0801), from the Volkswagen Foundation (Lichtenberg program to Matthias Endres), the German Center for Cardiovascular Research (DZHK) Charité (to Matthias Endres), the DFG (NeuroCure), and EU (European Stroke Network).

Conflict of interest

TGL, KB-W, JR and MB have nothing to declare.

ME has received grant support from AstraZeneca and Sanofi, has participated in advisory board meetings of Bayer, Boehringer Ingelheim, Bristol-Myers Squibb, MSD, Pfizer and Sanofi and has received honoraria from Astra Zeneca, Bayer, Berlin Chemie, Bristol-Myers Squibb, Boehringer-Ingelheim, Desitin, Eisei, Ever, GlaxoSmithKline, MSD, Novartis, Pfizer, Sanofi, Takeda, Trommsdorff. UR has participated in advisory board meetings of PharmAllergan, Co-Lucid, Amgen and Autonomic Technologies and also has received honoraria from PharmAllergan, St Jude Medical, Berlin Chemie, Almirall, MSD, Janssen-Cilag and GlaxoSmithKline. LN has received travel grants and speaking fees from PharmAllergan, St Jude Medical, Janssen-Cilag and MSD.

Acknowledgement

We thank Christopher O. Leonards for proofreading the manuscript.

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