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Elevated levels of circulating microvesicles in coronary artery disease patients with

type 2 diabetes and albuminuria. Effects of exercise training.

Short title: Microvesicles in type 2 diabetes.

Vibeke Bratseth (VB)1,2, Gemma Chiva-Blanch (GCB)3, Rune Byrkjeland (RB)1,

Svein Solheim (SS)1, Harald Arnesen (HA)1,2, Ingebjørg Seljeflot(IS)1,2

1 Center for Clinical Heart Research, Department of Cardiology, Oslo University

Hospital Ullevål, Oslo, Norway

2 Faculty of Medicine, University of Oslo, Oslo, Norway

3 Cardiovascular Program – ICCC – IR Hospital Santa Creu I Sant Pau, Biomedical

Research Institute Sant Pau (IIB-Sant Pau), Barcelona, Spain

Corresponding Author: Vibeke Bratseth, MSc

Postal address: Center for Clinical Heart Research, Oslo University Hospital, Pb

4950 Nydalen, 0424 Oslo, Norway.

Phone: +47 91726687

Fax: +47 22119181

E-mail address: vibebra@studmed.uio.no

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Abstract

Objective: Circulating microvesicles (cMVs), released from activated/apoptotic cells are

involved in vascular-complications and may be looked upon as biomarkers. Albuminuria is

characteristic of disease-progression in type-2 diabetes-mellitus (T2DM). We aimed to

investigate quantitative and qualitative differences of cMVs in T2DM with and without

albuminuria, and if 12-months exercise-training influenced expression of cMVs.

Methods: Coronary artery disease (CAD) patients with T2DM (n= 75), of which 25 had

albuminuria were included. Annexin-V+ (AV+) cMVs were analyzed by flow-cytometry in

citrated plasma. The exercise-volume was 150 min per-week.

Results: In albuminuria-patients, cMVs from endothelial-(CD146+/CD62E+/AV+) and

endothelial-progenitor-(CD309+/CD34+/AV+) cells were significantly higher compared to

those without (p≤ 0.01, both). ROC-curve analysis of the endothelial cMVs shows an area-

under-the-curve of 0.704 (95 CI 0.57-0.84)(p= 0.004). Albuminuria-patients had more

cMVs derived from activated leukocytes and monocytes and monocytes carrying tissue-

factor (CD11b+/AV+, CD11b+/ CD14+/AV+, CD142+/CD14+/AV+, respectively, p≤ 0.05, all),

and higher number of cMVs from activated platelets (CD62P+/AV+). Within exercising

patients, cMVs from progenitor-cells increased (p= 0.023), however, not significantly

different from controls.

Conclusions: CAD-patients with T2DM and albuminuria had elevated number of cMVs

from activated blood- and vascular-cells, rendering them as potential predictors of disease-

severity. The cMVs were limitedly affected by long-term exercise-training in our population.

Keywords: Albuminuria, Exercise training, Flow cytometry, Microvesicles and Type-2

diabetes mellitus.

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Introduction

Circulating microvesicles (cMVs) are submicron fragments released from the plasma

membrane surface of different cell types during cell activation and/or apoptosis. 1 cMVs

share the antigenic profile and part of the cytoplasmic content including nucleic acids, of

their parent cell, and due to translocation of phosphatidylserine (PS) to the outer leaflet of

the membrane during cMVs formation, they have procoagulant properties. 2 In addition,

some cMVs also carry tissue factor (TF), the initiator of extrinsic coagulation.

cMVs are novel mediators of intercellular signaling and they are shown to be directly

involved in the pathogenesis of vascular disease. 3 As the number and variety of cMVs

increase in conditions like cardiovascular disease (CVD) and diabetes mellitus (DM), 4 they

might be useful as biomarkers of disease severity, procoagulant states, vascular activation

and also as therapeutic targets. 5 cMVs are mainly detected by flow cytometry, based on

their size, binding to annexin V (AV+), a high affinity ligand for PS, and also binding on

specific markers of cell lineage or cell activation.

Type-2 DM (T2DM) is characterized by hyperglycemia, insulin resistance, dyslipidemia,

inflammation and hypercoagulability, all devastating conditions for the endothelium,

contributing to enhanced development of microvascular diseases as well as

atherosclerosis and coronary artery disease (CAD). 6 The disease progression in DM is

characterized by albuminuria, categorized as micro- or macro albuminuria which is used

as a conventional marker of chronic kidney disease (CKD). 7

Endothelial cell injury is the main component of disruption of cardiovascular homeostasis

within the vasculature. Endothelial derived MVs (EMVs) are independent risk factors for

coronary heart disease 8 and increased amount is associated with endothelial dysfunction.

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9 On the other hand, endothelial progenitor cells (EPCs) which are immature cells

mobilized from the bone marrow in response to tissue ischemia and vascular injury might

facilitate endothelial repair. 10 Decreased numbers of circulating EPCs have been

associated with endothelial dysfunction and related to CVD risk, mortality and recurrent

events in patients with CVD. 11

Exercise is known to improve endothelial function and insulin sensitivity. 12, 13 However, the

effect of physical activity on levels of cMVs has been inconsistently reported, potentially

related to exercise- type and intensity. 14 Exercise might act beneficially by lowering the

number of cMVs derived from leukocytes and EMVs, 15 and by increased amount of

circulating EPCs. 16

We have previously reported on increased levels of pro-thrombotic markers in CAD

patients with T2DM and albuminuria. 17 The aims of the present study were to perform

quantitative and qualitative analyses of AV+ cMVs from the vascular compartment between

CAD patients with T2DM, with and without albuminuria. In addition, the effects of one year

exercise training on cMVs, with specific reference to the presence of albuminuria, were

studied. Our hypotheses were that patients having albuminuria express different pattern of

cMVs especially related to vascular dysfunction and thrombotic properties, compared to

non-albuminuria patients. Further, that exercise training would contribute beneficially to the

cMVs expression.

Methods

Study population

A subset of 75 patients from the Exercise training in patients with Coronary Artery Disease

and type 2 Diabetes (EXCADI) cohort of 137 patients, 18 was included. Of the 75 patients,

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25 presented with albuminuria and 50 age-matched patients were selected. All patients

were included at Department of Cardiology, Oslo University Hospital, Ullevål, Oslo,

Norway between August 2010 and March 2012. The main EXCADI study was a

randomized controlled clinical trial, exploring the effect of 12 months exercise training on

the development of atherosclerosis and glucometabolic control in patients with

angiographically verified CAD and T2DM. All study patients gave informed written consent

to participate and the study was conducted according to the Declaration of Helsinki. The

Regional Ethics Committee approved the trial and it is registered at

http://www.Clinicaltrials.gov, NCT 01232608.

The definition of albuminuria includes both micro- and macro albuminuria.

Microalbuminuria was defined as albumin/creatinine ratio in spot urine > 3mg/mmol and ≤

30mg/mmol, and macro albuminuria as levels above 30 mg/mmol. The updated

homeostatic model assessment 2 of insulin resistance (HOMA2-IR) was used to estimate

insulin resistance. 19

Blood sampling

Fasting venous blood was drawn before any morning medication between 08:00 and 10:00

AM at baseline and after 12 months exercise intervention. Tubes containing 3.8% sodium

citrate were used for cMV analysis. Blood cells were removed within 30 min by

centrifugation 2500×g for 20 min at 4°C, and plasma was immediately frozen and stored at

-80ºC until further preparation for analysis. Fasting glucose and serum lipids were

analyzed by conventional routine methods. HbA1c was measured by turbid metric

inhibition immunoassay (Roche, Basel, Switzerland), insulin by DELFIA method (Perkin

Elmer, Waltham, Massachusetts, USA) and C-peptide by electrochemiluminescence

immunoassay (ECLIA) (Roche Diagnostics).

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Flow Cytometry analysis of cMVs

The frozen plasma aliquots were thawed in an ice-bath, mixed and centrifuged at 2500xg

for 10 min at room temperature (RT) to ensure sedimentation of remnant platelets and

potential clots. Plasma from the upper part of the tube was transferred to a new tube and

the cMVs were washed and fractionated from plasma by a two-step high-speed

centrifugation, as previously described. 20 The final cMVs pellets were resuspended in

citrate-phosphate buffered saline (citrate-PBS) and prepared for triple-label flow cytometry

analysis. In brief, each combination of AV+ labeled with allophycocyanin (APC) (5 µL) with

two specific monoclonal antibodies (mAb, 5 µL each, see Supplementary Table 1)

conjugated to fluorescein isothiocyanate (FITC) or phycoerythrin (PE), or the isotype-

matched control antibodies, were diluted in Annexin Binding Buffer (ABB) (30 µL) and

mixed with the cMVs suspension (5 µL). After 20 min incubation in the dark at RT, the

labeling was stopped by adding ABB and the samples immediately analyzed by flow

cytometry. The Auto Collect mode and 96-well plates on an AccuriC6 flow cytometer (BD,

Accuri® Cytometers, Inc., San Diego, CA) were applied.

Every sample had 2 min of acquisition at a flow rate of 14 µL/min. Forward scatter (FSC),

side scatter (SSC) and fluorescence data were gathered with the settings in the

logarithmic scale. Cytometer settings were defined with the Megamix-Plus FSC, a mix of

beads with bead-equivalent sizes: 0.1 µm, 0.3 µm, 0.5 µm and 0.9 µm (BioCytex, Marseille,

France). The upper threshold for FSC and SSC was set to ≤ 1 (see Supplemental Figure

1) and according to beads signal, the lower limit of detection was placed as a threshold

above the electronic background noise of the flow cytometer for FSC, and approximately

at the second logarithm for SSC. In addition to the size criteria (>0.1 to ≤1 µm), cMVs were

identified and quantified based on their binding to AV+ and reactivity to cell-specific mAb

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(see Supplemental Figure 2). To classify positive marked events, thresholds of

fluorescence were set according to samples incubated with the isotype-matched control

antibodies (same final concentrations based on titration experiments). Fluorescence

signals acquired with MVs in a calcium-free buffer (PBS) were used to correct the AV+

binding for auto fluorescence. The use of fresh made buffers filtered through 0.2 µm pore

size (vacuum), contributed to reduce the background noise.

The BD software (version 1.0.264.21, Accuri® Cytometers, Inc.) was applied to analyze

data. The number of cMVs per µL of plasma was calculated according to Nieuwland’s

formula, 21 based on the number of fluorescence-positive events (N), sample’s volume and

flow cytometer’s flow rate, as follows: cMVs/µL = N x (Vf/Va) x [Vt/(FRx 2)] x (1/Vi) [where

Vf(µL) = final volume of washed cMV suspension, Va(µL) = volume of washed cMV

suspension used for each labeling analysis, Vt(µL) = total volume of cMV suspension

before fluorescence-activated cell sorting analysis, FR(µL/min) = flow rate of the cytometer

(14 µl/min), 2 are the minutes of acquisition, 1 is the µL unit of volume, and Vi(µL) =

original volume of plasma used for microvesicle isolation].

Exercise intervention

The exercise intervention was planned and conducted in collaboration with Norwegian

School of Sport Sciences, Oslo, Norway, and consisted of a 12 months combined aerobic

and resistance training program. Detailed description has previously been reported.18 The

total exercise volume was 150 min per week, which included two group-based exercise

sessions of 60 min duration with qualified instructors and a third weekly home based

exercise session. All training sessions included intervals with high intensity, guided by

Borgs Scale of rated perceived exertion.

Statistics

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Data were analyzed with IBM© SPSS© statistics for windows, v 24.0 and 25.0 (IBM Corp.,

New York, NY, USA). Differences were considered statistically significant at p≤ 0.05. Due

to skewed distributed data in most variables, continuous data are presented by median

values with 25th and 75th percentiles if not otherwise stated. Categorical data are noted as

number and proportions. Between group differences were calculated by Mann–Whitney U

test, independent samples Student T-test, Chi-square test and Kruskal Wallis test, as

appropriate. To investigate changes within the intervention groups Wilcoxon’s test was

applied and the Mann-Whitney U test was applied for differences in changes between the

groups. Receiver operating characteristic (ROC) curve was used to evaluate the ability of

the EMVs as prognostic markers of albuminuria. The area under the curve (AUC) was

calculated. Adjustments for group differences in baseline characteristics were performed

by a logistic regression model. The correlation between EMVs and conventional vascular

biomarkers was analyzed with Spearman’s rho.

Results

Baseline characteristics of the total study population (n= 75) and according to having

albuminuria or not, are presented in Table 1.

Table 1. Baseline characteristics for the total population and subgroups with and

without albuminuria.

Characteristics All (n= 75)

Albuminuria (n= 25)

Non-Albuminuria

(n= 50)

p- value

Age* (years) 63.3 ± 5.6 62.8 ± 7.5 63.6 ± 4.5 0.63a

Male /Female 62 (83)/ 13 (17)

22 (88)/ 3 (12) 40 (80)/ 10 (20) 0.59

BMI (kg/m2) 29.2 (25.7, 31.9) 30.9 (27.8, 33.4) 28.1 (25.5, 30.9) 0.012

SBP* (mmHg) 140 ±17 144 ±16 139 ±17 0.16a

DBP* (mmHg) 79 ±9 80 ±9 78 ±8 0.27a

HT 60 (80) 23 (92) 37 (74) 0.13

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Previous AMI 33 (44) 15 (60) 18 (36) 0.08 Smokers 14 (19) 11 (44) 3 (6) <0.001 Years of Diabetes 10 (5, 15) 10 (5, 18) 10 (6, 15) 0.97 Glucose (mmol/l) 7.9 (6.7, 10.0) 7.7 (6.7, 10.0) 8.0 (6.7, 10.0) 0.80 HbA1c (%) 7.3 (6.7, 8.4) 7.1 (6.5, 8.9) 7.4 (6.8, 8.1) 0.98 Insulin (pmol/l) 63 (31, 105) 83 (27, 115) 55 (32, 115) 0.27 Cpeptide* (pmol/l) 1073 ±487 1129 ±613 1046 ±415 0.54a

HOMA2-IR 1.4 (0.7, 2.2) 1.9 (1.1, 3.3) 1.2 (0.7, 2.1) 0.050 Creatinine (µmol/l) 75 (66, 90) 80 (70, 95) 74 (66, 86) 0.26 Total Cholesterol (mmol/l) 3.9 (3.4, 4.6) 4.1 (3.7, 4.8) 3.7 (3.4, 4.4) 0.08 HDL Cholesterol (mmol/l) 1.11 (0.93, 1.33) 1.13 (0.88, 1.27) 1.11 (0.96, 1.39) 0.52

LDL Cholesterol 2.00 (1.60, 2.50) 2.15 (1.58, 2.83) 1.80 (1.55, 2.83) 0.30 Triglyceride 1.48 (1.04, 2.00) 1.52 (1.22, 2.13) 1.39 (1.04, 1.93) 0.17 Medication n (%) Insulin 16 (21) 10 (40) 6 (12) 0.013 Metformin 56 (75) 18 (72) 38 (76) 0.93 ACE-inhibitors or ARBs 50 (67) 19 (76) 31 (62) 0.34 Beta-blockers 52 (69) 19 (76) 33 (66) 0.54 Sulfonylurea 30 (40) 5 (20) 25 (50) 0.024 Gliptins 8 (11) 2 (8) 6 (12) 0.90 Statins 70 (93) 24 (96) 46 (92) 0.87 Platelet inhibitors 72 (96) 24 (96) 48 (96) 1.00 Data are presented as number (%) or median (25

th, 75

th percentiles) if not otherwise stated.

p- value refers to between group differences at baseline (Mann-Whitney U test for continuous and

Chi-Square for categorical variables).

ƚ Mean ± SD.

ǂ Independent sample Students t-test.

BMI body mass index, SBP systolic blood pressure, DBP diastolic blood pressure, HT hypertension,

AMI acute myocardial infarction, ACE angiotensin converting enzyme, ARB angiotensin receptor II

blocker.

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Mean age was 63 years and the patients were overweight/obese according to body mass

index (BMI), which also was higher in those with albuminuria versus those without (30.9

(27.8, 33.4 kg/m2) versus 28.1 (25.5, 30.9 kg/m2)), (p= 0.012). Median diabetes duration

was ten years and about 20% were on insulin treatment, significantly more frequent in

patients with albuminuria (40% versus 12%), (p= 0.013). The latter patient group also

presented with more insulin resistance (1.9 (1.1, 3.3) versus 1.4 (0.7, 2.2)), (p=0.050) and

higher number of smokers (44% versus 6%), (p< 0.001). Use of sulfonylurea dominated in

the non-albuminuria patients (50% versus 20%), (p= 0.024). However, the above

mentioned differences affected the cMVs only to a limited degree. Also, none of the

glucometabolic variables measured was associated with cMVs (data not shown).

Almost all patients were on platelet inhibition and 93% on statin treatment. Of the 50

patients in the non-albuminuria group one patient was lacking citrated plasma for cMVs

analysis.

cMVs in patients with and without albuminuria.

Albuminuria patients compared to those without, presented with significantly elevated

levels of EMVs and cMVs originated from monocytes (MMVs), platelets (PMVs) and EPCs,

as shown in Figure 1a). When excluding patients with macro albuminuria (n= 6), thus

analyzing those with microalbuminuria (n= 19) compared to non-albuminuria (n= 49), only

EMVs (CD146+/AV+, CD62E+/AV+ and CD146+/CD62E+/AV+), were still significantly

elevated in the microalbuminuria group, as presented in Figure 1b). (Insert Figure 1).

ROC curve analysis of EMVs (CD146+/CD62E+/AV+) from activated endothelial cells, for

the presence of albuminuria shows an AUC of 0.704 (95 % confidence interval 0.57-

0.84)(p= 0.004), as visualized in Figure 2. (Insert Figure 2). When adjusting for the

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differences between the albuminuria groups as shown in Table 1, the results were limited

affected.

To confirm that the measured EMVs originate from endothelial cells, the different EMVs

were correlated to previously measured indices of vascular injury or dysfunction

(endothelial cell adhesion molecules and asymmetric dimethylarginine (ADMA). 22 As can

be seen (Supplemental Figure 3), significant correlations were found.

Effect of exercise training on cMVs

The number of patients with albuminuria in the exercise- and control group was equally

distributed (p= 1.000). At baseline no significant differences were observed between the

two randomized groups, regarding clinical characteristics (see Supplementary Table 2) or

cMVs (Table 2).

Table 2. cMVs in T2DM patients at baseline and after 12 months exercise

intervention.

Baseline 12 months

cMVs/µl PFP Exercise(n=38) Controls (n=36) Exercise (n=38) Controls (n=36) ΔP

Total AV+ 203.82 (167.31, 284.85)

217.64 (176.02, 279.18)

228.71 (167.32, 321.91)

261.62 (181.69, 316.65)

0.80

Platelet MVs

CD61+ 176.71 (120.05, 239.21)

168.42 (139.38, 233.82)

146.29 (120.58, 221.79)

187.78 (126.94, 239.35)

0.76

CD61+/CD142+ 3.32 (1.66, 4.56)

3.32 (1.66, 5.95)

3.32 (2.21, 3.99)

3.87 (2.21, 4.98)

0.59

CD42b+ 35.95 (30.33, 42.87)

31.92 (19.22, 40.38)

26.00 (18.25, 40.10)

26.83 (20.60, 36.50)

0.55

CD31+/CD42b+ 23.78 (13.87, 31.39)

20.74 (13.72, 31.39)

17.67 (11.75, 28.21)

18.53 (13.97, 22.71)

0.76

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CD62P+ 4.15 (2.21, 6.08)

4.15 (2.56, 5.95)

3.60 (2.21, 5.95)

3.32 (2.21, 5.95)

0.47

Endothelial MVs

CD146+ 1.11 (0.55, 2.21)

1.11 (0.55, 1.66)

1.11 (0.55, 1.86)

1.11 (0.55, 1.66)

0.91

CD62E+ 12.72 (8.85, 17.70)

13.00 (9.96, 15.90)

13.55 (8.30, 19.08)

11.34 (9.96, 16.87)

0.38

CD146+/CD62E+ 1.11 (0.55, 1.80)

1.11 (0.55, 1.66)

1.11 (0.55, 1.66)

0.55 (0.10, 1.66)

0.60

CD309+ 9.13 (6.64, 13.27)

9.40 (6.22, 13.83)

8.57 (7.05, 14.10)

8.85 (6.46, 10.92)

0.34

CD309+/CD34+ 8.02 (6.08, 11.34)

7.19 (5.34, 11.34)

7.74 (6.08, 11.20)

7.19 (5.12, 9.40)

0.59

CD31+/CD42b- 23.51 (15.35, 35.40)

26.83 (18.67, 37.33)

24.06 (18.11, 34.43)

24.61 (15.49, 30.63)

0.24

Platelet and Endothelial MVs

CD31+ 48.67 (30.42, 69.69)

48.40 (32.25, 66.80)

42.05 (31.88, 58.08)

45.91 (31.25, 58.59)

0.60

Leukocyte MVs

CD45+ 87.39 (71.76, 112.83)

85.45 (69.07, 114.35)

85.58 (73.29, 118.22)

93.47 (70.80, 112.28)

0.28

CD15+ 9.60 (5.39, 17.15)

11.89 (6.64, 16.59)

11.56 (6.50, 19.63)

14.38 (8.85, 19.33)

0.59

CD45+/CD15+ 10.23 (5.99, 17.56)

11.62 (7.19, 18.25)

11.62 (6.08, 19.50)

14.10 (8.43, 19.33)

0.76

CD14+ 3.32 (2.21, 5.12)

4.12 (2.21, 5.78)

3.32 (2.21, 4.94)

4.42 (2.77, 6.08)

0.57

CD14+/CD11b+ 2.21 (1.11, 3.46)

2.35 (1.11, 4.63)

2.21 (1.52, 2.90)

3.32 (1.66, 4.84)

0.83

CD14+/CD142+ 2.21 (1.11, 4.01)

3.11 (1.66, 4.42)

2.19 (1.11, 3.18)

3.32 (1.76, 5.39)

0.44

cMVs from Pluripotent and Stemcells

CD34+ 57.25 (44.39, 82.41)

54.10 (42.04, 75.94)

62.50*

(54.07, 79.21) 59.73 (42.17, 69.83)

0.17

cMVs from activated cells

CD62L 8.30 (5.39, 12.17)

8.30 (5.12, 13.41)

9.13 (5.39, 12.19)

6.91 (4.98, 12.17)

0.48

CD142+ 7.19 7.74 7.47 9.13 0.53

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(4.84, 10.65) (5.53, 8.82) (4.98, 10.59) (6.64, 11.48)

CD11b+ 5.53 (2.77, 8.85)

6.27 (4.42, 8.16)

6.08 (4.42, 11.64)

7.19 (4.01, 8.30)

0.09

Results are expressed as median (25, 75 percentiles).

* p= 0.023, refers to intragroup change after one year in the exercise group. Δp refers to differences

in changes from baseline to 12 months between the randomized groups.

cMVs circulating microvesicles, PFP platelet free plasma, PMVs platelet derived microvesicles,

EMVs endothelial derived microvesicles.

Intervention results for the total population (n= 74) are presented in Table 2. During the

intervention period no significant differences in changes between the exercise- and control

group in any cMVs were observed, neither in the total population nor in the group with

albuminuria.

Nevertheless, within the exercise group the level of cMVs from progenitor cells

(CD34+/AV+) were significantly increased after twelve months (57.25 (44.39, 82.41 cMVs/µl

PFP) versus 62.50 (54.07, 79.21 cMVs/µl PFP)), (p= 0.023), and in patients with

albuminuria (n= 13) a significant reduction after exercise training were observed in levels

of PMVs carrying TF (CD61+/CD142+/AV+) and von Willebrand Factor (vWF)

(CD31+/CD42b+/AV+), and overall cMVs carrying vWF (CD42b+/AV+), (p< 0.05, all), as

shown in Figure 3. No significant changes were observed within the control group. (Insert

Figure 3).

Discussion

In the present study on patients with combined CAD and T2DM, significantly higher levels

of circulating endothelial related MVs (CD146+/AV+, CD62E+/AV+, CD146+/CD62E+/AV+),

monocyte derived MVs (CD14+/CD11b+/AV+, CD14+/CD142+/AV+), platelet derived MVs

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(CD62P+/AV+) and cMVs from endothelial progenitor cells (CD309+/CD34+/AV+), were

found in patients presenting with albuminuria compared to non-albuminuria patients. The

AUC of 0.704 for the EMVs to predict albuminuria may reflect enhanced chronic

endothelial cell activation in the advanced states of diabetes, when albuminuria occurs.

EMVs were significantly elevated also in the subgroup with microalbuminuria only. The

albuminuria patients included more smokers, higher BMI, more severe insulin

resistance/use of insulin and less intake of sulfonylurea compared to the non-albuminuria

group. However, no significant associations between these characteristics and the cMVs

were present. Also, no significant associations between the measured cMVs and

glucometabolic variables were observed.

During the 12 months intervention period, levels of cMVs derived from progenitor cells

increased within the exercise group, and within the albuminuria patients a significant

decrease in PMVs carrying TF and vWF and overall cMVs loaded with vWF were

observed. However, these changes did not differ significantly from the changes in the

control group.

The most striking findings were cMVs shed from activated endothelial cells to be

significantly elevated in patients with albuminuria, and also significantly elevated in those

with microalbuminuria only. High levels of EMVs might reflect progression of

atherosclerosis and vascular injury in T2DM, particularly in patients with diabetes

associated nephropathy.9 We have previously reported on levels of vascular parameters in

the EXCADI study, 22 and as shown (Supplemental Figure 3), there were significant

correlations between EMVs and other indices of vascular injury, verifying that the

measured EMVs originate from the endothelium.

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In line with our results, Rodrigues et al demonstrated elevated levels of EMVs (CD51+/AV+)

in a cohort of T2DM patients with nephropathy, 23 and EMVs (CD31+/AV+) were found

increased in patients with micro- or macro albuminuria, compared to those without

albuminuria. 24 Further, increased ratio of EMVs to EPCs was shown to be associated with

reduced glomerular filtration rate (GFR), indicating that an imbalance in endothelial

damage and repair capacity may in part explain reduced renal function. 25 Ongoing

inflammation, thrombin generation and hyperglycemia are all factors contributing to

accelerated release of EMVs, reflecting the state of the parent cell, 26 which might explain

the elevated levels observed in the present study.

In patients presenting with albuminuria cMVs derived from EPCs (CD309+/CD34+/AV+)

were significantly elevated, potentially reflecting increased vascular damage and the need

for repair. Increased mobilization of CD34+/CD133+ have been demonstrated in diabetic

patients on insulin therapy, 27 in consistency with our data of more frequent use of insulin

in the albuminuria patients. Insulin resistance and hyperglycemia were shown to attenuate

migration from the bone-marrow, differentiation and functionality of EPCs. 10 Of note, in the

latter study the number of EPCs per se was measured, while we investigated EPC-

derived cMVs. However, recent findings suggest that EPCs mediate vascular repair via

paracrine mechanisms, e.g. releasing cMVs containing micro-RNA’s that can promote

vascular repair. 28 Therefore, our albuminuria patients may have the same or lower levels

of circulating EPCs than non- albuminuria patients, despite their higher levels of EPC-

derived cMVs.

PMVs reflect chronic platelet activation and are the most abundant in the circulation. 29

Elevated levels of PMVs containing the receptor for P-selectin (CD62P+) have been shown

in diabetes patients. 30 We could demonstrate significantly higher levels of CD62P+ PMVs

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in our patients with combined CAD and T2DM with albuminuria compared to those without.

In line with our findings, Almquist et al found increased levels of PMVs, EMVs and MMVs

in patients with CKD compared to DM patients with normal glomerular filtration rate. 31

Further, in diabetic patients with an acute myocardial infarction (MI), significantly higher

amounts of CD62P+ PMVs were demonstrated and an early increase of these MVs after

an MI was associated with higher risk of CVD events during 2 year follow up period. 32

PMVs carrying P-selectin mediate leukocyte recruitment and aggregation via P-selectin

glycoprotein-1, contributing to atherothrombosis.

Activated monocytes and MMVs are key players in atherothrombosis. We could show

significantly elevated numbers of MMVs (CD14+/CD11b+) and MMVs expressing TF

(CD14+/CD142+) in our albuminuria patients. Sulfonylurea, which has been associated with

reduced expression of TF and reduced number of MMVs in vitro, 33 was far more common

in the non- albuminuria group. Our results are in accordance with previous studies

showing significantly higher levels of MMVs in T2DM patients who developed diabetic

complications, especially nephropathy and both MMVs and PMVs to be correlated to

microvascular damage. 34, 35 MMVs are also suggested to be involved in kidney disease

through effects on the podocytes, causing glomerular inflammation and increased

glomerular permeability. 36

The 12 months combined aerobic and resistance exercise training did not result in any

significant differences in changes from controls in any of the measured cMVs. There are

very limited data on the effect of long-term exercise on cMVs. In a study on healthy

individuals, acute exercise of moderate intensity reduced the number of CD62E+ MVs,

compared to high intensity interval training, 37 and accelerated production of PMVs was

observed after strenuous exercise. 38 More consistent with our data is a study on physically

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fit men with stable CHD, which showed no changes in EMVs and PMVs after high- and

moderate intensity training. 39

Within the exercise group we could, nevertheless, observe a significant increase in

progenitor cell derived cMVs, which might suggest a beneficial effect of exercise on the

vascular endothelium, supporting previous reports regarding recruitment and survival of

EPC after exercise. 40

Further, within patients with albuminuria that exercised, a favorable effect on the

hypercoagulable state by a decreased number of cMVs with procoagulant activity was

noted. However, precautions have to be taken due to low numbers.

Almost all patients in our up to date medical treated population were on statins and aspirin

therapy, both been associated with reduced numbers of EMVs and PMVs, 41 which to

some degree may explain the lack or limited effects of the intervention. We have

previously also reported that the intervention program did not have any impact on the

procoagulant state in the present population. 17

In conclusion, CAD patients with T2DM and albuminuria had elevated number of cMVs

derived from activated blood and vascular cells. More specifically, cMVs from endothelial

cells seem to be present in the early stages of albumin excretion rendering them as

potential markers of disease severity, and cMVs from EPCs suggested to undertake repair

of damaged endothelium were increased in patients with albuminuria. The measured

cMVs were only limitedly affected by long-term exercise training in our population, having

albuminuria or not.

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Acknowledgements

The authors want to thank Ida U Njerve and Sissel Åkra for valuable contributions to the

study.

Authors’ contributions

All authors have contributed significantly and in accordance with the latest guidelines of

the International Committee of Medical Journal Editors. VB was involved in the design of

the study, analysis and interpretation of the results and in the drafting of the manuscript.

GCB contributed to the interpretation of the results, drafting and revising of the manuscript.

RB was responsible for the main randomized trial involving recruitment and follow-up of

the study participants, acquisition and interpretation of clinical and laboratory data, as well

as revising of the manuscript. SS was involved in the conception of the trial and revising of

the manuscript. IS and HA contributed to the conception and design of the study,

interpretation of the results, drafting and revising of the manuscript. All authors are in

agreement of the content and approved the final manuscript before submission.

Funding

The authors received no financial support from any funding agency in the public,

commercial, or not-for-profit sectors for the work with this article.

Declaration of conflicting interests

The authors declare that they have no conflicts of interests.

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Supplementary Materials

Raw data related to the paper is available on request.

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31. Almquist T, Mobarrez F, Jacobson SH, et al. Effects of lipid-lowering treatment on circulating microparticles in patients with diabetes mellitus and chronic kidney disease. Nephrol Dial Transplant 2016; 31: 944-952. 2015/09/24. DOI: 10.1093/ndt/gfv337. 32. Christersson C, Thulin A and Siegbahn A. Microparticles during long-term follow-up after acute myocardial infarction. Association to atherosclerotic burden and risk of cardiovascular events. Thromb Haemost 2017; 117: 1571-1581. 2017/04/21. DOI: 10.1160/th16-11-0837. 33. Henriksson CE, Hellum M, Haug KB, et al. Anticoagulant effects of an antidiabetic drug on monocytes in vitro. Thromb Res 2011; 128: e100-106. 2011/08/25. DOI: 10.1016/j.thromres.2011.07.007. 34. Omoto S, Nomura S, Shouzu A, et al. Detection of monocyte-derived microparticles in patients with Type II diabetes mellitus. Diabetologia 2002; 45: 550-555. 2002/05/29. DOI: 10.1007/s00125-001-0772-7. 35. Ogata N, Nomura S, Shouzu A, et al. Elevation of monocyte-derived microparticles in patients with diabetic retinopathy. Diabetes Res Clin Pract 2006; 73: 241-248. 2006/04/06. DOI: 10.1016/j.diabres.2006.01.014. 36. Eyre J, Burton JO, Saleem MA, et al. Monocyte- and endothelial-derived microparticles induce an inflammatory phenotype in human podocytes. Nephron Experimental nephrology 2011; 119: e58-66. 2011/08/19. DOI: 10.1159/000329575. 37. Shill DD, Lansford KA, Hempel HK, et al. Effect of exercise intensity on circulating microparticles in men and women. Exp Physiol 2018; 103: 693-700. 2018/02/23. DOI: 10.1113/ep086644. 38. Chaar V, Romana M, Tripette J, et al. Effect of strenuous physical exercise on circulating cell-derived microparticles. Clin Hemorheol Microcirc 2011; 47: 15-25. 2011/02/16. DOI: 10.3233/ch-2010-1361. 39. Guiraud T, Gayda M, Juneau M, et al. A single bout of high-intensity interval exercise does not increase endothelial or platelet microparticles in stable, physically fit men with coronary heart disease. Can J Cardiol 2013; 29: 1285-1291. 2013/07/09. DOI: 10.1016/j.cjca.2013.03.024. 40. Laufs U, Werner N, Link A, et al. Physical training increases endothelial progenitor cells, inhibits neointima formation, and enhances angiogenesis. Circulation 2004; 109: 220-226. 2003/12/24. DOI: 10.1161/01.Cir.0000109141.48980.37. 41. Bulut D, Becker V and Mugge A. Acetylsalicylate reduces endothelial and platelet-derived microparticles in patients with coronary artery disease. Can J Physiol Pharmacol 2011; 89: 239-244. 2011/05/05. DOI: 10.1139/y11-013.

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Figure 1a and 1b.

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Figure 2.

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Figure 3.

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Supplemental Figure 1. Gate limits for microvesicle analysis with the Megamix-Plus FSC beads for cytometer settings in microvesicle analysis.

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Supplemental Figure 2. Circulating microvesicles identification and

characterization with the AccuriC6 flow cytometer.

APC denotes allophycocyanin; FITC indicates fluorescein isothiocyanate; and PE phycoerythrin.

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Supplemental Figure 3. Baseline correlations in the total population between

markers of EMVs and vascular parameters.

r= Coefficient of Correlation.

p= p-value refers to Spearman’s rho.

AV+ Annexin V positive, cMVs circulating microvesicles, PFP platelet-free plasma, CD cluster of

differentiation, CD62E+ E-selectin positive cMVs, CD146

+/CD62E

+ cMVs from activated endothelial

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cells, CD309+ and CD31

+_noCD42b

+ markers of endothelial cells, ICAM-1 intercellular adhesion

molecule-1, VCAM-1 vascular cell adhesion molecule-1, ADMA asymmetric dimethylarginine.

Supplementary Table 1. Cell surface molecules for circulating microvesicle

identification and characterization.

mAb Alternative name Expression

Annexin V PS-binding protein Widely expressed

CD142 Tissue Factor Widely expressed

CD61 β3-integrin Platelets

CD31 Platelet endothelial cell adhesion molecule Platelets, Endothelial cells

CD62P P-Selectin Activated Platelets

CD42b receptor for von Willebrand factor Activated Platelets

CD146 Melanoma Cell Adhesion Molecule Endothelial Cells

CD62E E-Selectin Endothelial Cells

CD34 Mucosialin Progenitor and Stem Cells

CD309 vascular endothelial growth factor receptor-2 Endothelial Cells

CD45 Leukocyte Common Antigen Leukocytes

CD11b Lymphocyte function-associated antigen 1 Leukocytes

CD62L L-Selectin Leukocytes

CD14 LPS-receptor Macrophages, monocytes

CD15 Lewis X Neutrophils, eosinophils and monocytes

mAb indicates monoclonal antibody; PS, phosphatidylserine; and LPS, lipopolysaccharide.

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Supplementary Table 2. Baseline Characteristics for the intervention groups.

Characteristics Exercise (n= 39) Controls (n=36) p value

Age ƚ (years) 64.2 ±5.6 62.3 ±5.6 0.156ǂ

Male /Female 34 (87) /5 (13) 28 (78) /8 (22) 0.442

BMI (kg/m2) 29.5 (25.5, 31.9) 28.9 (26.7, 32.1) 0.721

Weight ƚ (kg) 88.2 ±13.9 91.4 ±18.4 0.391ǂ

Waist (cm) 106.0 (95.4, 115.0) 105.0 (99.1, 113.4) 0.897

SBP ƚ (mmHg) 136.0 (129.0, 151.0) 141.4 ±15.4 0.630ǂ

DBP ƚ (mmHg) 76.0 (72.0, 82.0) 80.7 ±8.6 0.076ǂ

HT 29 (74) 31 (86) 0.326

Previous AMI 16 (41) 17 (47) 0.759

Smokers 8 (21) 6 (17) 0.896

Years of Diabetes 11 (6, 15) 10 (4, 15) 0.409

Albuminuria 13 (33) 12 (33) 1.000

Glucose (mmol/l) 7.8 (6.7, 10.1) 8.0 (6.7, 9.7) 0.991

HbA1c (%) 7.4 (6.8, 8.8) 7.2 (6.6, 7.9) 0.367

Insulin (pmol/l) 54 (27, 95) 75 (32, 118) 0.170

Cpeptide ƚ (pmol/l) 982 ±413 1172 ±546 0.092ǂ

HOMA2-IR 1.1 (0.7, 1.9) 1.7 (0.8, 2.4) 0.169

Creatinine (µmol/l) 74.0 (69.0, 90.0) 82.0 (65.3, 90.0) 0.996

Total Cholesterol (mmol/l) 3.8 (3.4, 4.5) 4.1 (3.4, 4.8) 0.308

HDL Cholesterol (mmol/l) 1.11 (0.94, 1.31) 1.12 (0.92, 1.40) 0.966

LDL Cholesterol 1.80 (1.48, 2.53) 2.10 (1.70, 2.60) 0.270

Triglyceride 1.48 (1.22, 2.04) 1.43 (0.85, 1.94) 0.324

Medication n (%)

Insulin 8 (21) 8 (22) 1.000

Metformin 32 (82) 24 (67) 0.206

ACE-inhibitors or ARBs 24 (62) 26 (72) 0.327

Beta-blockers 28 (72) 24 (67) 0.818

Sulfonylurea 19 (49) 11 (31) 0.171

Gliptins 4 (10) 4 (11) 1.000

Statins 37 (95) 33 (92) 0.926

Platelet inhibitors 37 (95) 35 (97) 0.604

Data are presented as number (%) or median (25th, 75

th percentiles) if not otherwise stated.

p- value refers to between group differences at baseline (Mann-Whitney)

ƚ Mean ± SD

ǂ Independent sample Students t-test

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BMI body mass index, SBP systolic blood pressure, DBP diastolic blood pressure, HT hypertension,

AMI acute myocardial infarction, ACE angiotensin converting enzyme, ARB angiotensin receptor II

blocker.