Study population

From January 2000 to December 2019, 6582 Japanese subjects voluntarily participated in a health checkup at the Shimane Institute of Health Science. Of these, 1093 subjects who participated several times and were followed-up for at least 1 year after the initial examination were included in the present study. Medical, neurological, and psychiatric histories were collected from the subjects. Neurological examinations were performed by an experienced neurologist. Subjects also underwent head magnetic resonance imaging (MRI), and blood tests. To confirm whether subjects developed ischemic stroke during the study period, questionnaires were mailed to all subjects on an annual basis. Of 1093 subjects, 10 subjects (6 males and 4 females, mean age: 64.2 ± 3.9 years) who developed symptomatic ischemic stroke (classified as the future ischemic stroke group, mean 7.3 ± 4.4 years’ follow-up) and randomly selected 10 age- and sex-matched controls, without development of any brain lesions (classified as the control group, mean 6.7 ± 2.8 years’ follow-up) were investigated.

Our study was approved by the Ethics Committee of the Shimane University School of Medicine in Japan, and all participants provided written informed consent in accordance with the Declaration of Helsinki. The procedures followed were in accordance with institutional guidelines.

Blood collection and assessment of clinical variables

At the first visit, after overnight fasting, blood samples were drawn into tubes containing silica particles and an inert gel separator for serum preparation, and tubes containing sodium fluoride and EDTA for plasma preparation. Subsequently, the blood samples were immediately centrifuged at 3000 rpm × 5 min at room temperature, and serum total cholesterol (TC), high-density lipoprotein (HDL-C), low-density lipoprotein cholesterol (LDL-C), triglyceride (TG), fasting plasma glucose, creatinine (Cr), and hemoglobin A₁C levels were measured. Simultaneously, serum samples were stored at − 80 °C for isolation of EVs enriched fractions.

Hypertension was defined as systolic/diastolic blood pressure ≥ 140/90 mmHg or treatment with antihypertensive drugs. Diabetes mellitus was defined as a fasting blood glucose level ≥ 126 mg/dL, a random blood glucose level ≥ 200 mg/dL, or treatment with oral antidiabetic drugs or insulin. Hyperlipidemia was defined as a serum cholesterol level ≥ 220 mg/dL or treatment with lipid-lowering drugs. The estimated glomerular filtration rate (eGFR) was calculated using the following formula: eGFR (mL/min/1.73 m²) = 194 × Cr^−1.094 × age^−0.287 for men and 194 × Cr^−1.094 × age^−0.287 × 0.739 for women. Smoking and drinking status were defined as follows: a smoker was a person who habitually smoked at least 1 cigarette per day and a drinker was a person who consumed ≥ 180 mL of alcohol per day.

Imaging data

To define a control group without development of any brain lesions, head MRI was performed at the first and last visits. Brain infarction was defined as a focal hyperintense lesion ≥ 3 mm in diameter on the T2-weighted images. Cerebral microbleeds were defined as homogenous 2‒10-mm round foci of signal loss on gradient-echo T2*-weighted images. Periventricular hyperintensities and white matter hyperintensities were defined as hyperintense lesions in fluid-attenuated inversion recovery images. All MRI findings were read and determined separately by an experienced neurologist and a radiologist who was blinded to the patients’ profiles.

Isolation of EVs enriched fractions and protein extraction

EVs enriched fractions were isolated from serum at the first visit using the Exo Trap™ exosome isolation spin column kit (Cosmo Bio, Tokyo, Japan) following the manufacturer’s instructions. Briefly, 500 µL of serum was diluted three times with PBS and centrifuged at 12,000×g for 4 min. The supernatant was filtered through a 0.22-μm disc filter (STRALAB, Milton Keynes, UK) and was applied to the Exo Trap™ column and then centrifuged at 5000×g for 1 min. After washing with phosphate-buffered saline (PBS), exosomal protein from the column was eluted with urea lysis buffer (7 M urea, 0.1% Nonidet P-40, and 500 mM triethylammonium bicarbonate) for iTRAQ-based quantitative proteomics and with RIPA buffer (PBS, pH 7.4, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 10 mg/mL PMSF, and 1 mg/mL aprotinin) for enzyme-linked immunosorbent assay (ELISA). Protein concentration was determined using a bicinchoninic acid assay kit (Thermo Fisher Scientific, Waltham, MA, USA).

Antibody array

The immunoblotting analyses of the EVs-specific markers were performed to validate the separated EVs using a commercial Exo-Check™ Exosome Antibody Array (Neuro) Mini (System Biosciences, Palo Alto, CA, USA). The same amount of EVs enriched fractions (30 μg) was added to the membrane-based blot array according to the manufacturer’s instructions. Briefly, the EVs enriched fractions were lysed by a lysis buffer, and labelled using the labelling reagent. Following incubation for 30 min, the labelled sample was washed through the column and blocked by 5 mL blocking buffer. The membrane was incubated with 5 mL labelled EVs lysate/blocking buffer mixture at 4 °C overnight with shaking. Next day, the membrane was washed with a wash buffer and incubated using a detection buffer followed by development with enhanced chemiluminescence (ECL; Cytiva, Tokyo, Japan); the chemiluminescent signals were captured with the Amersham ImageQuant 800 (Cytiva, Tokyo, Japan).

ELISA

To evaluate the extraction efficiency of EVs enriched fractions between the two groups, the absolute expression levels of CD9 were measured in all samples from each group, using a commercial ELISA kit (CUSABIO, Wuhan, China) according to the manufacturer’s instructions. Briefly, the EVs enriched fractions were diluted with sample diluent, and 25 μL of the sample or standards were added per well. After the reaction, the optical density of each well was determined immediately, using a microplate reader set to 450 nm.

iTRAQ labeling and strong cationic exchange fractionation

To minimize biological variation, 10 samples were pooled in each group. Samples from the future ischemic stroke group were labeled with 114 iTRAQ labels, and samples from the control group were labeled with 115 iTRAQ labels. iTRAQ labeling and strong cationic exchange fractionation were performed as previously described¹¹.

Nano liquid chromatography and mass spectrometry analysis

Peptide fractions were first separated by nano liquid chromatography (LC; DiNa nanoLC system; KYA Tech, Tokyo, Japan) as described previously¹¹. Then, fractionated peptides were analyzed on a MALDI time-of-flight (TOF)/TOF 5800 MS/MS Analyzer with TOF/TOF Series software (version 4.0) (AB Sciex). The MS was performed in positive ion mode across the mass range of 800–4000 m/z. Duplicate LC–MS/MS runs per fractions were performed.

Database research

For protein identification, the Paragon algorithm¹², which was integrated with Protein Pilot 3.0 software, was analyzed using the UniProt/International Protein Index protein sequence database for Human. Proteins were identified according to the following criteria: i) a false discovery rate (FDR) < 5% (FDR was estimated by “decoy database searching” using Protein Pilot software); and ii) protein confidence > 95% (“unused ProtScore” > 1.3). The Unused ProtScore was defined as − log (1 − % confidence/100). Comparisons of differential protein expression in the two groups were made with the statistical significance level set at p < 0.05, and a 1.2-fold change (> 1.20, or < 0.83) threshold.

Statistical analysis

Clinical characteristics of study participants were compared between the two groups using Student’s t-test or Fisher’s exact test. The expression levels of CD9 obtained by ELISA were compared using Student’s t test. All analyses were performed using SPSS version 23 (IBM Corp., Armonk, NY, USA). Statistical significance was set at p < 0.05.

To confirm that the sample size was large enough to detect significant differences, a post-hoc power analysis was conducted using G ∗ Power v3.1.9.

Ethics approval

All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and national research committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards. The study was approved by the Bioethics Committee of the Shimane University (No. 20160217-1).