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Contents lists available at ScienceDirect

Thrombosis Research

journal homepage: www.elsevier.com/locate/thromres

Full Length Article

High levels of endothelial and platelet microvesicles in patients with type 1

diabetes irrespective of microvascular complications

Karin Bergen

a,⁎

, Fariborz Mobarrez

b

, Gun Jörneskog

c

, Håkan Wallén

d

, Sara Tehrani

c

a Karolinska Institutet, Department of Clinical Sciences, Danderyd Hospital, Division of Nephrology, Danderyd University Hospital, Stockholm, Sweden b Department of Medical Sciences, Uppsala University, Uppsala, Sweden

c Karolinska Institutet, Department of Clinical Sciences, Danderyd Hospital, Division of Internal Medicine, Danderyd University Hospital, Stockholm, Sweden d Karolinska Institutet, Department of Clinical Sciences, Danderyd Hospital, Division of Cardiovascular Medicine, Danderyd University Hospital, Stockholm, Sweden

A R T I C L E I N F O Keywords: Type 1 diabetes Extracellular vesicles Microvesicles Microangiopathy Endothelial Platelet A B S T R A C T

Introduction: Patients with type 1 diabetes have high risk of developing microvascular complications, and mi-croangiopathy contributes to premature cardiovascular disease in this population. The role that microvesicles (MVs) may play in the development of microangiopathy in type 1 diabetes remains unclear.

Materials and methods: Plasma levels of endothelial MVs (EMVs) and platelet MVs (PMVs) in 130 patients with type 1 diabetes without microangiopathy, 106 patients with microangiopathy and 100 matched healthy controls were analyzed using flow cytometry. MV expression of procoagulant phosphatidylserine (PS) and proin-flammatory high mobility group box-1 protein (HMGB1) was also assessed.

Results: Patients with type 1 diabetes had markedly elevated levels of EMVs and PS+ EMVs as well as PMVs and PS+ PMVs compared to healthy controls (p < .001 for all). Furthermore, HMGB1+ EMVs and HMGB1+ PMVs were significantly increased in patients (p < .001 for all). After adjusting for potential confounders, there were no clear differences between patients with or without microvascular complications for any of the MV para-meters.

Conclusion: Type 1 diabetes is a prothrombotic and proinflammatory disease state that, regardless of the pre-sence of clinical microangiopathy, is associated with elevated levels of plasma MVs, in particular those of an endothelial origin. We have for the first time demonstrated that patients with type 1 diabetes have higher levels of HMGB1+ MVs. HMGB1 is an alarmin with potent proinflammatory effects that drive endothelial dysfunction, and it would therefore be of interest to further study the role of HMGB1+ MVs in the development of macro-vascular complications in type 1 diabetes.

1. Introduction

Type 1 diabetes is characterized by an altered vascular homeostasis with increased platelet reactivity and endothelial dysfunction [1–3]. Patients with type 1 diabetes are at increased risk of microvascular disease, including retinopathy, nephropathy, and neuropathy as well as myocardial and cerebral microangiopathy [4]. Altered hemostasis and vascular cell function may contribute to development of diabetes complications.

Extracellular vesicles (EVs) are a heterogeneous group of nano-sized vesicles released by platelets and endothelial cells into the circulation upon activation and apoptosis [5]. The term EVs includes both small size EVs of endosomal origin, often referred to as exosomes, as well as medium-large size EVs released through membrane budding,

henceforth referred to as microvesicles (MVs).

As reviewed elsewhere, a large body of research suggests that EVs play an important role as mediators and potential biomarkers in a range of prothrombotic and proinflammatory conditions, including type 2 diabetes [6–10]. Studies looking specifically at the role of MVs in mi-croangiopathy in patients with type 1 diabetes, however, have shown somewhat conflicting results [11–13]. Furthermore, the importance of EV exposure of procoagulant molecules such as the negatively-charged phospholipid phosphatidylserine (PS) [14,15] and inflammatory med-iators such as high mobility group box-1 protein (HMGB1) [17,18] to the development of microvascular disease remains unclear. PS-expres-sion on the EV surface may facilitate coagulation since positively coa-gulation factors bind to the negatively-charged phospholipid [14,15]. HMGB1 acts as an alarmin with a range of proinflammatory effects

https://doi.org/10.1016/j.thromres.2020.08.012

Received 16 April 2020; Received in revised form 2 August 2020; Accepted 6 August 2020

Corresponding author at: Karolinska Institutet, Department of Clinical Sciences, Danderyd Hospital, Division of Nephrology, Danderyd University Hospital, SE- 182 88 Stockholm, Sweden.

E-mail addresses: karin.bergen@ki.se, karin.bergen@sll.se (K. Bergen).

Available online 09 August 2020

0049-3848/ © 2020 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

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mediated by interaction with toll-like receptors (TLRs) and the receptor for advanced glycation endproducts (RAGE) [16]. Earlier studies have demonstrated that HMGB1 can be expressed by MVs [17,18], but this has not been studied in type 1 diabetes.

Our group has previously analyzed total circulating MV levels and PS+ MVs in a large group of well-characterized patients with type 1 diabetes compared to healthy controls and found that, although pa-tients had significantly higher circulating total MV levels and PS+ MV levels in plasma, there was no clear link to clinical microangiopathy [13]. We also found that women with type 1 diabetes may have a disproportionally prothrombotic MV profile [13]. For the present study, we wanted to investigate subpopulations of platelet and endothelial MVs (PMVs and EMVs), as well as their expression of PS and HMGB1, in the same study population. We were also interested in any potential sex differences between male and female patients and controls. We hy-pothesized that patients with type 1 diabetes would have higher levels of both PMVs and EMVs, as well as higher levels of MV subgroups ex-pressing PS and HMGB1. We also theorized that women with type 1 diabetes would have MV levels on par with men with type 1 diabetes, whereas female controls would have lower MV levels than male con-trols.

2. Materials and methods

2.1. Patients and controls

A total of 236 patients with type 1 diabetes were recruited for the study from the out-patient clinic at the Department of Endocrinology and Diabetology at Danderyd University Hospital, Stockholm, Sweden, a tertiary hospital setting. In order to be eligible for the study patients had to be between 20 and 70 years of age. Patients with a history of CVD, currently using non-steroidal anti-inflammatory drugs (NSAIDs) or anticoagulants as well as pregnant women were excluded from the study. 100 healthy controls matched for age, sex and body mass index (BMI) were recruited through the population registry in Stockholm and also included in the study. None of the controls were treated with any regular medications, except for one male control who used a proton pump inhibitor (PPI) daily. None of the female patients or controls were on oral contraceptives.

2.2. Blood samples

Following a 10-hour over-night fast, subjects arrived to the la-boratory in the morning. Patients had been instructed to not take their

morning insulin until after the blood sampling. After a resting period of at least 20 minutes (min), venous blood samples were drawn into ci-trated tubes (citrate concentration 0.109 M) using no or minimal stasis. Samples were centrifuged within 1 h at 2000g at room temperature (RT) for 20 min. 250 μL aliquots of platelet-poor plasma (PPP) were then stored at −80° and thawed only once immediately before analysis. 2.3. Clinical investigations

Brachial blood pressure was taken with subjects in a seated position following a period of 20 min rest. The mean blood pressure of three measurements by an oscillometric device (OMRON 705IT, OMON Healthcare, Kyoto, Japan) was registered. Medical records of all sub-jects were examined by a single person in order to document prevalence of microangiopathy, i.e. microvascular complications including retino-pathy, nephropathy and/or neuropathy. The presence of retinopathy was determined using funduscopic findings by ophthalmologists and categorized as a) no retinopathy (including simplex retinopathy, an early and reversible stage of retinopathy), b) mild-moderate retino-pathy, or c) severe retinoretino-pathy, defined as either laser treated non- proliferative retinopathy or proliferative retinopathy. Nephropathy was defined as an estimated glomerular filtration rate (eGFR) below 60 mL/ min/1,73m2 as calculated from plasma creatinine levels using the Lund- Malmö formula (well validated in the Swedish population [19]) and/or the prevalence of albuminuria using dipstick tests (Clinitek®, Bayer HealthCare LLC, USA). At least two instances of documented albumi-nuria were required for a diagnosis. Microalbumialbumi-nuria was defined as a urinary albumin-creatinine ratio of 3.4–33.9 mg/mmol and macro-albuminuria as a ratio above 33.9 mg/mmol. Neuropathy status of patients was determined clinically through examinations by physicians during patients' regular follow-up visits and defined as a documented decreased vibratory sensation when testing with a tuning fork (128 Hz), decreased sensation for monofilament (Semmes-Weinstein 5.07) testing and/or diminished patellar and achilles reflexes. Patients were divided into two groups for comparison in the study, those with no micro-angiopathy (other than simplex retinopathy) versus those with docu-mented microangiopathy, which entailed having docudocu-mented presence of microangiopathy in at least one organ system (i.e. retinopathy, neuropathy and/or nephropathy).

2.4. Biochemical analyses

Glycated hemoglobin (HbA1C) levels were assessed by the Mono S

method using high-performance liquid chromatography (Variant II;

Abbreviations

ACE-I angiotensin converting enzyme inhibitors ANCOVA analysis of covariance

ANOVA analysis of variances

ARBs angiotensin II receptor blockers BMI body mass index

CVD cardiovascular disease

DAMP damage-associated molecular pattern eGFR estimated glomerular filtration rate EMV(s) endothelial microvesicle(s) EV(s) extracellular vesicle(s) FMO fluorescence minus one HbA1C glycated hemoglobin HDL high-density lipoprotein

HMGB1 High mobility group box-1 protein hsCRP High-sensitivity C-reactive protein

IFCC International Federation of Clinical Chemistry IL-1 interleukin 1

IQR interquartile ranges LDL low-density lipoprotein min minutes

miRNA microRNA

MV(s) microvesicle(s)

NF-ĸB Nuclear Factor kappa-light-chain-enhancer of activated B cells

NSAIDs non-steroid anti-inflammatory drugs P-glucose plasma glucose levels

PMV(s) platelet microvesicle(s) PPP platelet poor plasma PPI proton pump inhibitor PS phosphatidylserine

RAGE receptor for advanced glycation endproducts RNA ribonucleic acid

RT room temperature SD standard deviations TLRs toll-like receptors

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Bio-Rad Laboratories, Hercules, CA, USA) and expressed in Interna-tional Federation of Clinical Chemistry (IFCC) standardization of HbA1c values of mmol/mol. Venous blood samples were sent to the central hospital laboratory for determination of plasma creatinine, plasma glucose levels (P-glucose), lipids including total cholesterol, triglycer-ides, high-density lipoprotein (HDL) and low-density lipoprotein (LDL), and platelet concentration. High-sensitivity C-reactive protein (hsCRP) was measured by a particle-enhanced immunoturbidimetric method (Beckman Inc., High Wycombe, UK) with a reference value of ≤3 mg/ L. RAGE was analyzed in plasma using ELISA (R&D Systems, Minnesota, USA) according to instructions from the manufacturer.

2.5. Plasma microvesicles

Circulating MVs were investigated by flow cytometry using a Beckman coulter Gallios flow cytometer. Frozen aliquots of PPP were thawed in a water bath (37 °C) and centrifuged at 2000g for 20 min at RT. The supernatant was then re-centrifuged at 13,000g for 2 min at RT. Subsequently, 20 μL of the supernatant was incubated for 20 min with 5 μL phalloidin-650 (Sigma-Aldrich St. Loiuse, MO, USA), 5 μL lac-tadherin-FITC (80 μM; Haematologic Technologies, VT, USA), 5 μL CD42a-PE (GPIX, Beckman Coulter, Brea, CA, USA), 5 μL CD144-APC (VE-Cadherin, AH diagnostics, Stockholm, Sweden) and 5 μL CD62E- APC (E-selectin, AH diagnostics, Stockholm, Sweden). The MV-gate was calibrated using Megamix-plus FSC beads (BioCytex, Marseille, France) with diameters of 0.1 μm, 0.3 μm, 0.5 μm and 0.9 μm. MVs were identified by both size (forward scatter) and complexity (side scatter) and defined as particles < 0.9 μm in size (threshold based on forward scatter). The setting of the MV-gate in all MV analyses was done by one skilled laboratory technician who was blinded to the origin of the samples. A representative flow cytometry plot of the gating is shown in Fig. 1 below. Lactadherin was used to detect expression of negatively- charged PS on MVs. A fluorescence minus one (FMO) was used as an in- house control of lactadherin. Our group has previously performed ex-periments where we defined the optimal concentration of lactadherin. Phalloidin, which binds to the exposed intracellular actin in cell frag-ments, was used to detect levels of cell fragmentation in our samples [20]. We consider samples with phalloidin-positive events less than 10% as acceptable [20]. Results of MV analyses are presented as total MV concentration (count per μL in original plasma samples). The intra- assay and inter-assay coefficients of variation for MV measurement were < 10% at our laboratory, calculated using 15 healthy individuals tested on two consecutive days and where each lab result was analyzed ten times in a row.

2.6. Statistical analysis

The size of the study was originally determined based on power analysis to detect differences in fibrin clot permeability between men and women with type 1 diabetes [21]. However, post hoc power cal-culations (2-sided t-tests) showed that the sample size exceeded that necessary to detect a 20% difference in levels of total circulating MVs and PS+ MVs between patients with and without microvascular com-plications, with a power level of 80% and a significance level of 0.05. Data are presented as means ± standard deviations (SD) for data with normal distributions, and as median with interquartile ranges (IQR) for skewed data. Normality was tested using Shapiro-Wilks tests and skewed data was log-converted and then checked once again for normality. Groups were compared using independent t-tests and ana-lysis of variances (ANOVA) with contrast analysis for normally dis-tributed data, and Mann-Whitney U tests and Kruskal-Wallis ANOVA for skewed data. Simple regression was used to test for correlations be-tween variables and analysis of covariance (ANCOVA) was used to control for potential confounders that might influence that ANOVA results. Results were considered significant if p < .05. All analyses were carried out using Statistica version 13 (TIBCO Software Inc.)

2.7. Ethical considerations

The study was conducted in accordance with the Declaration of Helsinki and approved by the local Ethics Committee at Karolinska University Hospital, Solna, Sweden. All subjects had given written in-formed consent prior to commencing the study.

3. Results

3.1. Subject characteristics

Baseline characteristics of the 236 patients and 100 matched healthy controls are presented in Table 1. Patients had significantly higher systolic- and diastolic blood pressure, HDL cholesterol, platelet concentration as well as fasting glucose but lower total and LDL cho-lesterol levels compared to healthy controls, whereas there was no difference in age, sex distribution, tobacco use or BMI. hsCRP tended to be higher among patients, but the difference did not reach statistical significance. Among the patients, 130 patients had no clinical signs of microvascular complications except for simplex retinopathy (an early and reversible stage of retinopathy) and 106 subjects had microvascular complications of various degrees. Patients with microvascular compli-cations were older, had longer diabetes duration, higher systolic blood pressure, worse glycemic control (P-glucose and HbA1C), lower eGFR and higher triglyceride level than patients without microvascular complications [13].

3.2. Endothelial microvesicles

Patients with type 1 diabetes had significantly higher levels of cir-culating VE-cadherin+ EMVs and E-selectin+ EMVs, as well as sig-nificantly higher levels of EMVs (both VE-cadherin+ and E-selectin+) expressing PS and HMGB1 (p < .001 for all, Figs. 2 and 3). Levels of EMV with different antigens are presented in Table 2.

As presented in Table 3, the differences between patients and con-trols remained significant in an ANCOVA model comparing the dif-ferent EMV subclasses between patients with type 1 diabetes and healthy controls. In the analyses, we controlled for current tobacco use

Fig. 1. Microvesicle gating in flow cytometric analyses.

Representative dot-plot of microvesicle gating together with gates demon-strating beads with diameter 0.3, 0.5 and 0.9 μm.

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Table 1

Background characteristics of patients with and without microangiopathy (MA) and healthy controls.

Patients with type 1 diabetes Healthy controls

Variable All patients

(n = 236) With MA (n = 106) No MA (n = 130) p-Value With vs without MA Controls (n = 100) p-Value Patients vs controls

Age (years) 44 ± 13 48 ± 13 42 ± 13 < 0.001 45 ± 10 0.86

Women (n, %) 107 (45) 45 (43) 62 (48) 0.42 46 (46) 0.91

BMI (kg/m2) 25.0 ± 3.8 25.5 ± 4.0 24.6 ± 3.5 0.06 25.0 ± 3.1 0.93

Current tobacco use (n, %) 66 (28) 30 (29) 36 (28) 0.84 20 (22) 0.26

Diabetes duration (years) 22 ± 14 28 ± 13 18 ± 13 < 0.001 n/a n/a

SBP (mmHg) 128 ± 17 133 ± 19 124 ± 14 < 0.001 121 ± 11 < 0.001 DBP (mmHg) 73 ± 9 74 ± 9 72 ± 8 0.16 78 ± 8 < 0.001 P-lipids (mmol/L) Cholesterol 4.5 (4.0–5.0) 4.5 (4.0–5.1) 4.4 (4.0–4.9) 0.15 5.1 (4.6–5.6) < 0.001 LDL 2.6 (2.1–3.0) 2.6 (2.1–3.0) 2.5 (2.1–2.9) 0.42 3.3 (2.8–3.8) < 0.001 HDL 1.5 (1.2–1.8) 1.4 (1.2–1.8) 1.6 (1.2–1.8) 0.22 1.3 (1.0–1.6) < 0.001 Triglycerides 0.7 (0.5–0.9) 0.7 (0.6–1.0) 0.6 (0.5–0.8) 0.001 0.7 (0.5–1.2) 0.11 eGFR (mL/min/1.73m2) 85 ± 13 83 ± 14 87 ± 12 0.003 87 ± 10.1 0.29

HbA1C (mmol/mol) 60 (52–71) 64 (55–72) 58 (51–68) 0.003 Not measured n/a

Plasma glucose (mmol/L) 10.3 ± 4.5 10.9 ± 4.7 9.7 ± 4.3 0.04 5.1 ± 0.5 < 0.001

Platelet count (×109/L) 223 (196–254) 221 (199–261) 223 (190–248) 0.10 206 (182–239) 0.001

hsCRP (mg/L) 1.0 (0.4–2.1) 1.0 (0.4–2.5) 0.9 (0.5–1.9) 0.39 0.7 (0.4–1.6) 0.05

RAGE (pg/mL) 1023 (825–1422) 1008 (755–1413) 1029 (866–1424) 0.25 1140 (871–1472) 0.15

Microangiopathy (n,%)

Retinopathy 84 (36) 84 (79) Not present n/a n/a n/a

Nephropathy 29 (12) 29 (27)

Neuropathy 43 (18) 43 (41)

Medications (n, %)

Betablockers 15 (6) 12 (11) 3 (2) – None n/a

Calcium antagonist 12 (5) 9 (9) 3 (2) ACE-I/ARBs 67 (28) 46 (43) 21 (16) < 0.001 Statins 85 (36) 51 (48) 34 (26) < 0.001 Aspirin 75 mg/day 2 (1) 1 (1) 1 (1) – Insulin intermittent 196 (83) 81 (76) 115 (89) 0.009 Insulin pump 39 (17) 25 (24) 14 (11) 0.009

Data presented as mean ± standard deviation (SD), median with interquartile range (IQR), number (n) of individuals or percentages (%). Statistically significant differences (p < .05) are highlighted in bold. Abbreviations used: MA, microangiopathy; BMI, body mass index; SBP, systolic blood pressure; DBP, diastolic blood pressure; P, plasma; LDL, low density lipoprotein; HDL, high density lipoprotein; eGFR, estimated glomerular filtration rate; HbA1C glycated hemoglobin; RAGE = receptor for advanced glycation endproducts; ACEeI, angiotensin converting enzyme inhibitor; ARBs, angiotensin II receptor blockers; n/a, not applicable; vs, versus.

Fig. 2. Plasma total EMV and PS+ EMV levels in patients with type 1 diabetes and healthy controls.

Patients with type 1 diabetes had significantly higher total EMV and PS+ (lactadherin+) EMV levels in plasma compared to healthy controls (p < .001 for all), but there were no significant differences between patients with and without microangiopathy. Figure shows median, 25–75% percentiles and non-outlier range.

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as well as the background characteristics that differed significantly between patients and healthy controls, including systolic- and diastolic blood pressure, plasma platelet concentrations, P-glucose, total cho-lesterol, LDL- and HDL cholesterol as well as the use of angiotensin converting enzyme inhibitors (ACEeI), angiotensin II receptor blockers (ARBs), and statins.

There were no significant differences in EMV parameters between patients with and without microvascular complications (Figs. 2 and 3). This held true even after controlling for the factors that differed sig-nificantly between patients with and without microvascular complica-tions, including age, systolic blood pressure, diabetes duration, eGFR, P- glucose, HbA1C and the use of ACEeI, ARBs, statins and insulin treatment modality (intermittent or pump therapy) (data not shown). No sex dif-ferences between men and women were found between any of the EMV parameters in either patients with type 1 diabetes or healthy controls.

Simple regression analysis showed that, in patients, none of the EMV parameters correlated significantly with BMI, diabetes duration, diastolic blood pressure, HbA1C, eGFR, or lipid levels. For controls, total

VE-cadherin+ EMV and PS+ VE-cadherin levels both correlated in-versely with age (r = −0.28, p = .01; r = −0.26, p = .01 respec-tively). There was an inverse correlation between P-glucose levels and total VE-cadherin+ EMVs (r = −0.27, p = .01) as well as total E- selectin+ EMVs (r = −0.22, p = .03). HMGB1+ VE-cadherin+ EMV correlated with P-glucose (r = −0.22, p = .03) and BMI (r = 0.21, p = .04). HMGB1+ E-selectin+ EMV levels correlated with total cholesterol (r = 0.26, p = .01) and LDL (r = 0.32, p < .001). In addition, we found several more statistically significant associations between different MV subgroups and other variables (hsCRP, plasma RAGE levels, P-glucose and systolic blood pressure). However, these associations were weak with r-values smaller than 0.2 and these data are not presented here.

3.3. Platelet microvesicles

Patients with type 1 diabetes had significantly higher plasma levels of total PMVs, PS+ PMVs (Fig. 4) and HMGB1+ PMVs (Fig. 5),

Fig. 3. Plasma HMGB1+ EMV levels in patients with type 1 diabetes and healthy controls.

Patients with type 1 diabetes had significantly higher HMGB1+ EMV levels (both CD144+ and CD62E+ EMVs) compared to healthy controls (p < .001 for all). There were no significant differences between patients with and without microangiopathy. Figure shows median, 25–75% percentiles and non-outlier range. HMGB1, high-mobility group box-1 protein; CD144, VE-cadherin; CD62E, E-selectin.

Table 2

Plasma microvesicle count in patients with type 1 diabetes and healthy controls.

Patients with type 1 diabetes Healthy controls

MV count/mL All patients Patients with

microangiopathy Patients without microangiopathy p-Value With vs without MA Controls p-Value Patients vs controls Platelet MVs Total PMV 11,345 (7288–19,489) 12,560 (7545–20,706) 11,117 (7042–19,327) 0.46 9230 (5679–12,103) < 0.001 PS+ PMV 7529 (4558–12,631) 7681 (4749–12,915) 7203 (4103–12,407) 0.37 5654 (3491–7357) < 0.001 HMGB1+ PMV 410 (141–920) 648 (165–1048) 343 (101–858) 0.04 65 (35–93) < 0.001 Endothelial MVs VE-cadherin + EMV Total VE-cadherin+ 1143 (490–1973) 1126 (567–1866) 1148 (369–2116) 1.00 39 (28–76) < 0.001 PS+ VE-cadherin+ 772 (306–1245) 783 (369–1234) 764 (260–1250) 1.00 28 (18–46) < 0.001 HMGB1+ VE-cadherin+ 214 (123–342) 222 (148–379) 200 (108–327) 0.67 28 (10–61) < 0.001 E-selectin + EMV Total E-selectin+ 3375 (1798–6227) 3358 (1785–6178) 3402 (1798–6260) 1.00 41 (23–79) < 0.001 PS+ E-selectin+ 1613 (763–2585) 1483 (765–2610) 1622 (743–2583) 1.00 34 (19–55) < 0.001 HMGB1+ E-selectin+ 285 (173–423) 300 (184–445) 284 (162–403) 0.90 25 (12–44) < 0.001

Data presented as median with interquartile range (IQR). Significant p-values (< 0.05) are highlighted in bold. Abbreviations used: MV, microvesicle; PMV, platelet microvesicle; EMV, endothelial microvesicle; PS, phosphatidylserine; HMGB1, High mobility group box-1 protein; MA = microangiopathy.

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compared to healthy controls (p-value < .001 for all). As presented in Table 3, these differences remained after controlling for current tobacco use, systolic- and diastolic blood pressure, plasma platelet concentra-tions, P-glucose, total cholesterol, LDL- and HDL cholesterol as well as the use of medications including ACEeI, ARBs, and statins.

There were no significant differences in total PMVs or PS+ PMVs between patients with and without microvascular complications (Fig. 4). ANCOVA analysis controlling for age, systolic blood pressure, diabetes duration, eGFR, P-glucose, HbA1C and the use of ACEeI, ARBs, statins and insulin treatment modality also did not demonstrate any significant differences in total PMVs or PS+ PMV levels between the groups (data not shown). Patients with microvascular complications had significantly higher levels of HMGB1+ PMVs than patients without microvascular complications (Fig. 5, p = .04, contrast analysis). However, after controlling for the aforementioned background char-acteristics that differed significantly between patients with and without complications, the significance did not remain (p = .42). Levels of PMVs with different antigens are presented in Table 2.

In controls, women had significantly lower levels of total PMVs and PS+ PMVs than men (p < .05 for both, data not shown), despite having significantly higher total platelet concentrations (221 ± 38 × 109/L for women vs 196 ± 42 × 109/L for men, p = .01). This sex difference in total PMV levels held true after cor-recting for the background characteristics that differed between male and female controls, including tobacco use, systolic blood pressure and age (p = .04). However, it no longer reached statistical significance for PS+ PMV (p = .09). In contrast, there were no significant differences in PMV levels between women and men in patients with type 1 diabetes either before or after controlling for those characteristics that differed between male and female patients, including systolic- and diastolic blood pressure, BMI, HbA1C, total cholesterol and HDL [21].

In patients, no correlations were found between PMV parameters and diastolic blood pressure, BMI, P-glucose, HbA1C, eGFR, hsCRP, blood platelet concentration or lipid levels. In controls, total PMV levels correlated weakly with age (r = 0.27, p = .01), BMI (r = 0.21, p = .03), diastolic blood pressure (r = 0.27, p = .01), and RAGE (r = −0.24, p = .02); PS+ PMV correlated weakly with age (r = 0.22, p = .03) and diastolic blood pressure (r = 0.23, p = .03); and HMGB1+ PMV correlated with age (r = 0.22, p = .03).

4. Discussion

The results of this study of 236 well-characterized patients with type 1 diabetes and 100 healthy matched controls showed that patients with

type 1 diabetes have significantly higher levels of circulating EMVs and PMVs compared to healthy controls. In addition, we have demonstrated that patients with type 1 diabetes have significantly higher levels of HMGB1 expressing EMVs and PMVs compared to healthy controls. 4.1. EMV and PMV levels in patients with type 1 diabetes compared to healthy controls

Our results of significantly higher levels of circulating EMVs and PMVs in patients with type 1 diabetes compared to healthy controls are in agreement with previous studies [11,12,22]. In the present study, for PMV levels the difference between patients and controls was relatively modest in size whereas patients had markedly elevated levels of all subclasses of both VE-cadherin+ and E-selectin+ EMVs compared to healthy controls (Table 2). Patients with type 1 diabetes had median VE-cadherin+ EMVs levels around 1100 counts/mL and E-selectin+

Table 3

Results of ANCOVA comparing microvesicle count between patients with type 1 diabetes and healthy controls.

Variable ANCOVA results Adjusted means and 95% C.I. (MV count/μL)

F value (1284) p-Value Partial Eta2 All patients Healthy controls

Platelet MVs Total PMV 7.9 0.005 0.03 14,447 (13165–15,729) 9357 (8205–10,510) PS+ PMV 8.6 0.004 0.03 9262 (8433–10,091) 5706 (5031–6380) HMGB1+ PMV 31.1 < 0.001 0.10 581 (512–649) 65(55–75) Endothelial MVs VE-cadherin + EMV Total VE-cadherin+ 44.5 < 0.001 0.14 1407 (1236–1578) 84 (60–109) PS+ VE-cadherin+ 59.7 < 0.001 0.17 822 (745–898) 56 (39–73) HMGB1+ VE-cadherin+ 57.4 < 0.001 0.17 250 (228–273) 44 (36–53) E-selectin + EMV Total E-selectin+ 62.0 < 0.001 0.18 4316 (3879–4754) 74 (55–92) PS+ E-selectin+ 76.6 < 0.001 0.21 1750 (1588–1912) 47 (36–58) HMGB1+ E-selectin+ 78.1 < 0.001 0.22 325 (296–355) 40 (29–51)

Covariates corrected for include current tobacco use, systolic- and diastolic blood pressure, plasma platelet concentrations, plasma glucose, total cholesterol, LDL- and HDL cholesterol, as well as the use of angiotensin converting enzyme inhibitors (ACEeI), angiotensin II receptor blockers (ARBs), and statins. Adjusted means are presented as means with 95% confidence intervals. Significant p-values (< 0.05) are highlighted in bold. Abbreviations used: MV, microvesicle; PMV, platelet microvesicle; EMV, endothelial microvesicle; PS, phosphatidylserine; HMGB1, High mobility group box-1 protein; C.I., confidence intervals.

Fig. 4. Total PMV and PS+ PMV levels in patients with type 1 diabetes and

healthy controls.

Patients with type 1 diabetes had significantly higher total PMV and PS+ PMV levels in plasma compared to healthy controls (p < .001 for all), but there was no significant differences between patients with and without microangiopathy. Figure shows median, 25–75% percentiles and non-outlier range. CD42a, gly-coprotein IX; Lac, lactadherin.

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EMV levels around 3300 counts/mL, compared to 39 and 41 counts/mL for healthy controls, respectively. For comparison, a previous study of EMV levels in antiphospholipid syndrome, a condition known for its extreme prothrombogenicity, showed VE-cadherin+ EMV levels around 6000 counts/mL [23]. Since VE-cadherin is a critical component of endothelial adherence junctions and helps insure the integrity of the endothelial barrier, its expression on EMVs suggest potential implica-tions for vessel wall permeability [24,25]. E-selectin expression on EMVs, on the other hand, is believed to be indicative of endothelial activation, since E-selectin expression is induced during inflammatory conditions and involved in endothelial interaction with leukocytes [26]. This distinct elevation of all subclasses of EMV compared to normal physiological condition is therefore suggestive of both an abnormally high degree of endothelial activation as well as endothelial dysfunction including disrupted endothelial integrity in type 1 diabetes.

Patients also had significantly higher levels of PS-expression on EMVs and PMVs compared to healthy controls. These subclasses of MVs have been shown to be able to promote thrombus formation in vitro [27,28]. Increased levels of PS+ MVs in patients with type 1 diabetes would therefore theoretically put them at increased risk of future thrombotic complications.

4.2. EMV and PMV levels and microangiopathy

Contrary to our expectation, levels of EMV and PMV, as well as those subclasses expressing PS, did not differ significantly between patients with and without microvascular complications, including re-tinopathy, nephropathy and neuropathy. In other words, even though patients without microangiopathy were younger, had shorter diabetes duration and better metabolic control, they had EMV and PMV levels comparable to patients with established microangiopathy. From a clinical perspective this is important, because it suggests that type 1 diabetes per say, regardless of the presence of clinical signs of micro-angiopathy, is a procoagulant condition with an abnormally high de-gree of platelet and endothelial activation and high expression of PS.

The lack of correlation between MV levels and clinical micro-angiopathy in our study was somewhat surprising given previous an-imal and in vitro studies that had suggested a pathophysiological role

for EMV and PMVs in the development of microvascular disease in type 1 diabetes. Increased levels of proinflammatory MVs originating from platelets, leucocytes and endothelial cells have been shown in strepto-zotocin-induced diabetic rats compared to healthy rats [16,32]. These MVs were associated with production of reactive oxygen species, de-creased nitric oxide availability, inhibited endothelial nitric oxide synthase, increased externalization of PS and were able induce micro-vessel inflammation. Endothelial cells exposed to hyperglycemic con-ditions in vitro increase their EMV formation and the EMVs formed show increased pro-oxidative and pro-coagulant activity, have the ca-pacity to inhibit endothelial-dependent vascular relaxation and express unique proteins with functions related to coagulation and immune cell activation, which may contribute to endothelial dysfunction [29].

On the other hand, previous human studies on MVs and micro-angiopathy in patients with type 1 diabetes, have shown conflicting results. Sabatier et al. conducted a small study of 24 patients with type 1 diabetes and showed that patients with microvascular complications had higher mean CD51+ EMP levels than patients without complica-tions, but the number of patients with vascular complications was not sufficient to draw any firm statistical conclusions and PMV levels showed no significant correlation to microvascular complications [11]. Results published previously by our own group looking at the same patient population as in the current study showed that patients with type 1 diabetes had elevated total circulating MVs in plasma compared to healthy controls, but with no difference in MV levels between pa-tients with and without microvascular complications [13]. This finding is now strengthened by the current study again failing to find a sig-nificant relationship between PMV and EMV levels and microangio-pathy. In contrast, Salem et al. looked at CD41+ PMVs in 80 young patients with type 1 diabetes (mean age 10) and found that PMV levels were higher in patients with microvascular complications, thus drawing the conclusion that PMV levels have the potential to be used as a bio-marker for microangiopathy [12]. However, they studied PMV levels in whole blood rather than plasma, used a different PMV marker as well as a different flow cytometer from our group, making it difficult to com-pare results.

There are several possible explanations for the lack of correlation between EMV and PMV levels and diabetes microangiopathy in our study. PMV and EMV levels in plasma and their expression of PS and HMGB1 may not be specific enough to distinguish patients with mi-crovascular disease from those without, given that MV levels are also influenced by a range of other factors including age, obesity, systolic blood pressure and inflammatory conditions. This could also be an explanation for the discrepancy between the results of Salem et al. [12] and our study, since maybe it is only possible to use MV levels as a marker of microangiopathy at a very young age before patients with type 1 diabetes develop other comorbidities that may affect MV levels. Even though we attempted to correct for some of these potential con-founders using ANCOVA, there might also be unknown factors that influenced the MV levels in the patients.

Another potential explanation is that the study was underpowered to detect small differences in EMV and PMV levels between patients with and without microvascular complications. However, given how similar the EMV and PMV levels were in patients with and without microvascular complications in our study, even if a larger study would have been able to detect a small difference in MV levels between pa-tients with and without microangiopathy, the magnitude of the differ-ence is unlikely to be sufficient to use EMV and PMV levels in any meaningful way as a biomarker for microangiopathy in the clinic. 4.3. HMGB1+ MVs

HMGB1 is a nuclear DNA-binding protein released either passively upon necrosis or apoptosis as well as actively upon activation of monocytes. It is also stored in platelets and released into the circulation upon their activation [30]. To the best of our knowledge, we have for

Fig. 5. Plasma HMGB1+ PMV levels in patients with type 1 diabetes and

healthy controls.

Patients with type 1 diabetes had higher HMGB1+ PMV levels (CD42a+) in plasma compared to healthy controls (p < .001) and patients with micro-vascular complications had higher levels of HMGB1+ PMV than patients without microvascular complications (p = .04). Figure shows median, 25–75% percentiles and non-outlier range. HMGB1, high mobility group box-1 protein.

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the first time demonstrated that patients with type 1 diabetes have significantly higher levels of HMGB1 expressing EMVs and PMVs compared to healthy controls (Figs. 3 and 5). A growing body of in-vitro studies, animal studies and human studies have implicated HMGB1 as an important mediator in the development of vascular complications in both type 1 and type 2 diabetes [16,31]. Our study is in line with a recently published study showing elevated serum levels of extracellular HMGB1 in 96 young patients (median age 14) with recently diagnosed type 1 diabetes compared to healthy controls [32]. In contrast, a smaller study by Skrha et al. looking at HMGB1 in 45 adult patients with type 1 diabetes (median age 47) found only low serum HMGB1 levels that did not differ significantly from that of healthy controls [33]. The reason for this discrepancy is unclear but could perhaps be related to the use of different ELISA kits for their analyses.

4.4. Sex differences in MV levels

Our group has previously found that PS+ MV levels are sig-nificantly lower in healthy women compared to corresponding men, whereas there is no difference between men and women with type 1 diabetes [13]. In the present study investigating subclasses of MVs, we found that while EMV levels were similar between the sexes, healthy women had significantly lower total levels of PMVs and PS+ PMVs in plasma than healthy men, despite having a significantly higher plasma platelet concentration. This sex difference was absent in patients with type 1 diabetes. This seems to indicate that women with type 1 diabetes have increased levels of platelet activation, on par with men, which is interesting given previous studies indicating that women with type 1 diabetes lose the normal female protection against CVD found in non- diabetic subjects [34–36].

4.5. Strengths and limitations

This study has several key strengths. To our knowledge, this con-stitutes the largest study to date looking at PMV and EMV levels in plasma in relation to microvascular complications in type 1 diabetes. Both the patient group and the controls are well characterized and matched for age, BMI and sex, thus limiting the contributions of these factors towards differences in MV levels between the groups. Efforts have also been made in the statistical analysis to try to control for potential confounding factors that differed significantly between pa-tients and controls, as well as between papa-tients with and without mi-crovascular complications.

There are, however, several important limitations in the study that need to be acknowledged. First of all, the centrifugation protocol used in the flow cytometry method does not live up to current guidelines recommending two centrifugations before freezing [37], since blood samples were drawn and MV levels analyzed before these guidelines were published. However, a standardized protocol has been used to analyze all blood samples, ensuring that samples from both the control and the patient group have all been handled in the same way and minimizing the effect of preanalytical confounders. Another limitation is that we did not test our isolated MVs for the presence of co-isolated proteins such as lipoproteins. However, blood samples in the study were all taken in fasting subjects, thus limiting the presence of chylomicrons. Although guidelines recommend analyzing MV levels in fresh samples, this is practically not feasible in a large clinical study such as this, and freezing and thawing could theoretically have affected the number of MVs and their exposure of PS in our study [38]. However, all samples were frozen and thawed only once, which has previously been shown to have minimal effect [39]. Also, samples were re-centrifuged upon thawing in order to remove cell debris, and only samples with < 10% phalloidin (a cell-debris marker) were deemed of sufficient quality [20]. It is also worth noting that because of the detection limit of our flow cytometer; our study was not able to detect the smallest EVs. If microangiopathy would be associated with a disproportionately large

increase in small EVs compared to medium-large EVs, this difference would therefore not have been detected.

With regards to HMGB1, one limitation is that our methodology relies on measuring the levels of HMGB1+ MVs, but we cannot be certain of the biological activity of this antigen on the MVs. Extracellular HMGB1 may exist in both an active and inactive form, as regulated by post-translational modifications involving the redox state of three key cysteine chains [40]. We also did not measure the soluble protein HMGB1 in plasma, which could have been of interest to put HMGB1+ expression on MVs into perspective.

4.6. Future directions

Since endothelial dysfunction is linked to the development of CVD [41] it would be of great interest to follow up our patients in a future study to see if baseline EMV levels can predict future heart disease and stroke. Given the important role of HMGB1 as a mediator of in-flammation and endothelial dysfunction [16,42], the elevated levels of HMGB1+ MVs in our patients could also be an important risk factor for progression of CVD, however a prospective study would be needed to properly assess this risk. Also, although we have clearly demonstrated that patients with type 1 diabetes have elevated levels of HMGB1+ PMVs and EMVs, more research is needed to assess the bioactivity and biological significance of such MVs. Furthermore, it could be mean-ingful to focus more in general on the content of PMVs and EMVs rather than their total number. As shown by Garcia-Contreras et al., plasma exosomes isolated from patients with type 1 diabetes display a distinct micro RNA (miRNA) signature compared to those from healthy controls [43]. Unfortunately, their study did not report if there was any corre-lation between the exosome miRNA signature and clinically relevant parameters such as microvascular complications in the patients. Ur-inary EV miRNA could be another promising area for future research into suitable liquid biomarkers for microvascular disease in type 1 diabetes since studies indicate a link to albuminuria [44,45]. 4.7. Conclusions

In conclusion, this large study of 236 patients with type 1 diabetes and 100 healthy matched controls showed that type 1 diabetes per say, regardless of the presence of microangiopathy, is associated with higher PMV and, in particular, markedly elevated EMV levels. We have for the first time demonstrated that patients with type 1 diabetes have sig-nificantly higher levels of HMGB1+ PMVs and EMVs compared to healthy controls, which could potentially aggravate endothelial dys-function in these patients and contribute to future CVD.

Funding

This study was supported by independent grants from the Berth von Kantzow Foundation, Swedish Diabetes Foundation, Wallenius Foundation, Swedish Heart-Lung Foundation and Foundation of Women and Health. Sara Tehrani was supported by the Stockholm County Council (clinical postdoctoral appointment). None of the funding sources played any role in the design or conduct of the study, nor in the preparation or submission of this article.

Acknowledgements

The authors would like to especially thank diabetes nurses Lena Gabrielsson and Kerstin Bergqvist as well medical laboratory assistants Ann-Christin Salomonsson and Katherina Aguilera Gatica at Karolinska Institutet, Department of Clinical Sciences, Danderyd University Hospital, Stockholm, Sweden, for data collection and management.

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Declaration of competing interest

None.

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