Ethical approval was obtained for human sample collection from the Ethics Committee at Guangzhou Women and Children's Medical Center (trial no. 077 2013), and written informed consent was obtained from all guardians. Blood samples from six KD patients with CAD were randomly selected according to the American Heart Association and the Japanese Ministry of Health and Welfare criteria. The patients underwent systematic examination in the hospital and were confirmed to have no other diseases. Blood samples from six healthy children were used as the control group. Blood samples were separated by centrifugation at 1000 g for 10 min. Serum aliquots were collected and stored at −80°C.

Exosomes were isolated from two pooled serum samples consisting of equal amounts of each of six experimental samples from KD patients and healthy children using ExoQuick precipitation (System Biosciences Inc, Mountain View, CA) following the manufacturer's instructions.7,8

The prepared pooled protein samples (200 μg protein) were mixed with rehydration buffer to a volume of 450 μl. Immobilized pH gradient (IPG) strips (pH 4–7, 24 cm, GE Healthcare) for the first dimension were used to isolate the altered proteins and the following parameters were set at 20°C: 300 V for 30 min, 700 V for 30 min, 1500 V for 1.5 h, 9900 V for 3 h, 9900 V for 6.5 h, 600 V for 20 h, and 8000 V constant voltage for a total of 56 kVh. After isoelectric focusing, the strips were equilibrated in two steps: 15 min in an IPG equilibration buffer (6 M urea, 2% SDS, 30% glycerol, 0.375 M Tris, 20 mg/ml DTT, and a trace of bromophenol blue), and then alkylated for 15 min. Subsequently, a 12.5% SDS-PAGE gel was applied. Electrophoresis was carried out at 20 mA per gel for 40 min and then at 30 mA per gel until the dye front reached the bottom. The protein spots were detected by either Coomassie Brilliant Blue G-250 staining or silver staining.

The gels were analyzed in ImageMaster 2D Platinum software (GE Healthcare). The normalized protein amount for each spot was calculated as the ratio of that spot volume to the total spot volume on the gel. A threshold of 2.0-fold (p<0.05) change was used to determine significant differences between groups.

Each gel piece was washed three times with deionized water, destained with 50% methanol for 30 min at 37°C, and dehydrated in 100 μl of acetonitrile (ACN) for 20 min at room temperature. The gel spots were then swollen in 50 μl of 100 mM NH4HCO3, dehydrated for a second time, and incubated in 1 μg/50 μl trypsin (Promega) for 30 min at 4°C. Next, cover solution (10% acetonitrile, 50 mM NH4HCO3, deionized water) was added and the samples were incubated for 16 h at 37°C. The peptide mixtures were extracted using 2.5% trifluoroacetic (TFA)/90% ACN for 30 min at room temperature and completely dried in a Speed Vac centrifuge.

After digestion, the tryptic peptides were dissolved in 1.5 μl solution containing deionized water, 50% ceric ammonium nitrate, and 0.1% trifluoroacetic acid. Then, 0.8 μl mixture was spotted onto a MALDI target plate using 0.5 μl HCCA (5 mg/ml a-Cyano-4-hydroxycinnamic acid) matrix. Peptide mass spectra were analyzed with a MALDI TOF mass spectrometer (Applied Biosystems, USA). The UV laser was operated at a 200 Hz repetition rate with an acceleration voltage of 20 kV and a wavelength of 355 nm.

The mass spectrometer data were matched to a corresponding virtual peptide mass database derived from GPS Explorer™ version 3.6, the UniProt database, and the Mascot search engine with the Homo sapiens taxonomy. Protein identification was performed by peptide mass fingerprint (PMF) using Mascot software (http://www.matrixscience.com). The search parameters used in PMF were as follows: species: Homo sapiens; fixed modifications: carbamidomethylation; enzyme: trypsin; variable modifications: oxidation (M). The function, gene name, and Gene Ontology category of each protein were determined with the UniProt Homo sapiens protein database and the Mascot v2.1 software protein database search engine.

Protein extracts from the exosomes of six KD patients and six healthy children serum samples were separated using SDS-PAGE, and then transferred onto PVDF membranes. The membranes were incubated with sex hormone-binding globulin (SHBG) (1:1000), leucine-rich alpha-2-glycoprotein (LRG1) (1:1000) and serotransferrin (TF) (1:1000) antibodies at 4°C overnight, followed by incubation with secondary antibodies at room temperature for 2 h. The bands were detected using the SuperSignal chemiluminescence detection system (Pierce, Rockford, IL).

Identified proteins were classified based on the Database for Annotation, Visualization and Integrated Discovery (DAVID) (http://david.abcc.ncifcrf.gov/). DAVID consists of an integrated biological knowledge base and a comprehensive set of analytic tools to extract biological themes from large gene/protein lists, such as those derived from genomic and proteomic studies. The significance cutoff parameter was estimated by Fisher's exact test.

The differentially expressed proteins were processed by the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) system (http://www.string-db.org). STRING is a database of known and predicted protein interactions, including direct (physical) and indirect (functional) associations.

Microvesicles were separated from the serum of healthy controls and KD patients with CAD with ExoQuick precipitation reagent. TEM and western blot were used in validation of exosomes. Under TEM, the morphological structures of the isolated microvesicles were 30–100 nm in diameter (Figure 1A). The expression levels of the three specific exosome biomarkers (CD9, CD81, and flotillin) were markedly higher in isolated microvesicles than in serum (Figure 1B). Both results confirmed that the microvesicles separated from serum were indeed exosomes.

Figure 1.

Characterization of exosomes in serum samples from Kawasaki disease patients with coronary artery dilatation (CAD) and healthy children by transmission electron microscopy and western blot. (A) Morphological characterization by transmission electron microscopy; (B) molecular characterization by western blot. Protein extracts prepared from sera (S) or exosomes (Ex) were examined using antibodies against exosomal protein markers (CD9, CD81 and flotillin).

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Pooled proteins from serum exosomes of six KD patients with CAD and six healthy children were separated by 2-DE (Figure 2A and 2B). After visual review, 52 differential protein spots with at least a 2.0-fold discrepancy between the two groups were selected for MALDI-TOF/TOF MS analysis. In total, 38 differentially expressed proteins (13 up-regulated and 25 down-regulated) were successfully identified (Table 1). The unidentified proteins likely had special physical and chemical properties, or ambiguous identifications, or low Mascot scores. Some proteins appeared as multiple spots in 2-DE, likely due to modifications and/or the presence of isoforms.

Figure 2.

Two-dimensional electrophoresis analysis of the serum exosome proteome. Gels were visualized by silver staining. Arrows indicate differentially expressed proteins in Kawasaki disease (KD) patients with coronary artery dilatation (CAD) and healthy controls. Spot numbers correspond to proteins in Table 1. (A) Healthy controls; (B) KD patients with CAD.

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To further confirm the proteomic results, western blot analysis was performed to examine the expression levels of SHBG, LRG1 and TF of serum exosomes from all 12 individuals (six KD patients with CAD and six healthy controls) used in the 2-DE analysis. The validation results were consistent with the 2-DE analysis (Figure 3).

Figure 3.

Western blot analysis for expression of SHBG, LRG1 and TF in six Kawasaki disease patients with coronary artery dilatation (CAD) and six healthy controls.

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To understand the biological relevance of the changes in protein expression in KD, DAVID software was used to categorize the identified proteins according to their molecular functions, biological processes and protein classes (Figure 4). The molecular functions of the identified proteins were mainly in endopeptidase inhibitor activity, peptidase inhibitor activity, and enzyme inhibitor activity (Figure 4A). These differentially expressed proteins are involved in the acute inflammatory response, response to wounding, defense response, complement activation, humoral immune response and positive regulation of immune response (Figure 4B). As for protein class, most belonged to the extracellular region, platelet alpha granule lumen, cytoplasmic membrane-bounded vesicle lumen and high-density lipoprotein particle classes (Figure 4C).

Figure 4.

Classification of differentially expressed proteins identified in Kawasaki disease patients with coronary artery dilatation. Categorization was based on information obtained from DAVID software. (A) Molecular functions; (B) biological processes; (C) protein classes.

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STRING analysis revealed the protein interaction networks in KD patients with CAD (Figure 5). The nodes with highest connectivity were alpha-1-antitrypsin (SERPINA1), serum albumin (ALB), serotransferrin (TF), clusterin (CLU), kininogen-1 (KNG1), and inter-alpha-trypsin inhibitor heavy chain H4 (ITIH4).

Figure 5.

The network of protein-protein interactions of Kawasaki disease patients with coronary artery dilatation.

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The authors declare that the procedures followed were in accordance with the regulations of the relevant clinical research ethics committee and with those of the Code of Ethics of the World Medical Association (Declaration of Helsinki).

The authors declare that they have followed the protocols of their work center on the publication of patient data.

The authors have obtained the written informed consent of the patients or subjects mentioned in the article. The corresponding author is in possession of this document.