These authors have contributed equally to this work
This article was submitted to Cardiovascular and Smooth Muscle Pharmacology, a section of the journal Frontiers in Pharmacology
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Coronary artery lesions (CALs) are severe complications of Kawasaki disease (KD), resulting in stenosis and thrombogenesis. Metabolomic profiling of patients’ plasma could assist in elucidating the pathogenesis of CALs and identifying diagnostic biomarkers, which are imperative for clinical treatment. The metabolic profiles between KD patients with CALs and without CALs (non-coronary artery lesion, or NCAL, group) indicated the most significantly differentially expressed metabolite, palmitic acid (PA), showed the most massive fold change at 9.879. Furthermore, PA was proven to aggravate endothelial cellular senescence by increasing the generation of reactive oxygen species (ROS) in KD, and those two phenotypes were confirmed to be enriched among the differentially expressed genes between KD and normal samples from GEO datasets. Collectively, our findings indicate that cellular senescence may be one of the mechanisms of vascular endothelial damage in KD. PA may be a biomarker and potential therapeutic target for predicting the occurrence of CALs in KD patients. All things considered, our findings confirm that plasma metabolomics was able to identify promising biomarkers and potential pathogenesis mechanisms in KD. To conclude, Palmitic acid could be a novel target in future studies of CALs in patients with KD.
Kawasaki disease (KD) is a febrile, self-limited illness that mainly affects children <5 years and has become the most common cause of acquired heart diseases in children from developed countries (
However, the pathogenesis and mechanism of CALs development in KD patients remain unknown. Due to the geographical differences indicating higher susceptibility in Asian children, numerous researches have shown the critical roles genetics played by genetics in disease susceptibility. Other than that, innate immune response, matrix metalloproteinases, microRNAs, and IL-1 signaling pathways are hypothesized to be the cellular and molecular circuitries associated with disease progression (
Metabolomic technologies such as ultra-high-performance liquid chromatography- mass spectrometry can detect and semi-quantitatively measure hundreds of distinct metabolites. Data regarding differentially expressed metabolites can be used to explore metabolic pathways. These approaches have been used to identify physiological and metabolic profiles of different disease states and meaningful diagnostic biomarkers and provide new insights into disease studies (
As one of the crucial components of triglycerides in adipose tissue, PA maintains excellent clinical diagnostic and prognostic values (
Overall, we further explored the probable mechanisms of the differentially expressed metabolite palmitic acid in children with KD complicated with CALs based on metabolomics to confirm the predictive value of PA as a molecular marker.
All patients were enrolled at the Second Affiliated Hospital and Yuying Children’s Hospital of Wenzhou Medical University (Zhejiang, China) between November 2014 and April 2016. Every patient and their parents received detailed information about the research and signed the consent form. This study was approved by the Ethics Committee of the Second Affiliated Hospital and Yuying Children’s Hospital of Wenzhou Medical University. All patients were clinically diagnosed as KD based on the American Academy of Pediatrics and the 2004 American Heart Association criteria (
Blood samples were collected in EDTA vacutainer tubes on day 7 after IVIG treatment, immediately placed on ice, and centrifuged (1000 g, 15 min) within 30 min. The supernatant was extracted and stored in a sterile tube at −80°C until required.
LC-MS/MS analyses were performed using an UHPLC system (1290, Agilent Technologies) with an UPLC BEH Amide column (1.7 μm, 2.1*100 mm, Waters) coupled to a TripleTOF 5600 (Q-TOF, AB Sciex). The mobile phase consisted of 25 mM NH4OAc and 25 mM NH4OH in water (pH = 9.75) (A) and acetonitrile (B), and an elution gradient was applied as follows: 0 min, 95% B; 7 min, 65% B; 9 min, 40% B; 9.1 min, 95% B; and 12 min, 95% B The flow rate was 0.5 ml min−1. The injection volume was 3 µL in the positive mode and 4 µL in the negative mode. The Triple TOF mass spectrometer was used for its ability to acquire MS/MS spectra on an information-dependent basis (IDA) during the LC/MS experiment. In this mode, the acquisition software (Analyst TF 1.7, AB Sciex) continuously evaluates the complete scan survey MS data as it collected and triggered the acquisition of MS/MS spectra, depending on preselected criteria. In each cycle, 12 precursor ions whose intensity was greater than 100 were chosen for fragmentation at collision energy (CE) of 30 V (15 MS/MS events with a production accumulation time of 50 msec each). ESI source conditions were set as follows: ion source gas 1, 60 Psi; ion source gas 2, 60 Psi; curtain gas, 35 Psi; source temperature, 650°C; and ion spray voltage floating (ISVF) 5000 V or −4000 V in the positive or negative mode, respectively. Parameters Processing such as retention time alignment, peak discrimination, filtration, alignment matching, and identification were performed by the XCMS package (Scripps Center for Metabolomics and Mass Spectrometry, La Jolla, California).
The gene expression datasets were filtered from the GEO database, and three gene expression profiles (GSE18606, GSE68004, and GSE73463) were picked among 58 series of KD. All the data were freely available. The differentially expressed genes (DEGs) between KD and normal samples were detected by the GEO2R online analysis tool (
Palmitic acid was purchased from Sigma, BSA (Fatty Acid and IgG-Free, BioPremium), and was provided by Beyotime, and Acetylcysteine (NAC) was bought from MCE. In order to prepare the PA working solution, we first dissolved palmitic acid in DMSO to a storage concentration and then diluted in 1% BSA solution prepared with the corresponding culture medium to achieve a total palmitic acid concentration of 1000 μM. Subsequent different concentrations of palmitic acid were diluted on this sub-basis. This method was established on the Spector’s protocol (
Human umbilical vein endothelial cells (HUVECs) were bought from Lonza (United States), and Primary Human Aortic Smooth Muscle Cells; Normal (HASMCs) were purchased from ATCC (United States). HUVECs were cultured in an endothelium cell medium (ECM, Sciencell Research Laboratories) with 10% Fetal Bovine Serum (FBS, Gibico) and ECGs, and HASMCs were maintained in Dulbecco’s Modified Eagle Medium (DMEM) with 10% FBS, respectively, at 37°C in a humidified incubator under 5% CO2. When performing experiments, cells were seeded in 6-well plates at a density of 3×104 cells/well for HUVECs and 2×104 cells/well for HASMCs. Upon 80% confluence, HUVECs were incubated, after overnight starvation, in corresponding PA or KD serum for 24 h. Meanwhile, HASMCs were treated identically.
Cells were resuspended with different concentrations of PA and seeded in 96-well plates at a density of 3 × 103 per well. Cellular cytotoxicity was evaluated using the Cell Counting Kit-8 (CCK-8; Dojindo). After 24 h of treatment, the culture medium was changed to DMEM containing 10 μL per well of Cell Counting Kit-8 (CCK-8, Dojindo) solutions and incubated for a further 2 h at 37°C. The optical density (OD) value was measured at a 490 nm wavelength.
After treatment, the harvested cells were lysed by RIPA buffer (Byotime) containing a protease and phosphatase inhibitor cocktail (Beyotime). The protein content from each lysate was measured using the BCA Protein Assay Kit (Bio-Rad, Hercules, CA, United States) before loading equal amounts of protein (20 μg/lane) into 12% SDS-PAGE gels. After incubating with 5% non-fat milk for blocking about 2 h, specific antibody for 14h, and secondary antibodies conjugated to horseradish peroxidase for 2 h, the PVDF membrane with ECL developer was performed by the Bio-Rad gel imaging system. The following antibodies were involved in this experiment: p-RB (Abclonal, AP0089), P16(Proteintech, 10883-1-AP), LC3II(CST, 3868), SOD1 (Proteintech, 10269-1-AP), SOD2(Proteintech, 24127-1-AP), and GAPDH (Abways, AB0036).
The senescent status was demonstrated by the senescence-associated β-galactosidase (SA-β-gal) staining kit (Beyotime). Cells were fixed on the plate and incubated with the mixed staining solutions for 24 h at 37°C.
Intracellular ROS levels were evaluated by Dihydroethidium (DHE) fluorescence staining (Beyotime) and Malondialdehyde (MDA) assay kits (TBA method, Nanjing Jianchen Bioengineering Institute). After treatment with either PA or KD serum, cells were incubated with DHE (10 μM) in DMEM at 37°C for 40 min. Fluorescence was detected under excitation at 300 nm and emission at 610 nm. The experiments were performed in the dark conditions. In addition, the supernatant was collected for MDA assays, as per the manufacturer’s instructions. The optical density (OD) value was measured at a 532 nm wavelength.
The results were expressed as the mean ± SD or median (interquartile range, IQR) for continuous variables according to whether the variables conformed to a normal distribution or as a number (percentage) for categorical variables. The Student’s t-test was used to determine statistical significance for mean ± SD results. In comparison, one-way ANOVA was used for median (IQR) results, and X2 or Fisher’s exact test was used for results expressed as numbers (percentage). Principal component analysis (PCA) and orthogonal projections to latent structures discriminant analysis (OPLS-DA) were applied using SIMCA version 14.0.1 (Umetrics AB, Umea, Sweden). MetaboAnalyst (
79 patients were enrolled; none of them had a prior history of KD, and all were clinically diagnosed as typical during the study. There were 40 patients in CAL group and 39 in NCAL group. Based on the AHA KD guidelines (
Baseline characteristics and drug treatment of patients.
CAL (N = 40) | NCAL (N = 39) | P value | |
---|---|---|---|
Age, mon | 21.7 (8.2–30.9) | 23.7 (9.0–40.0) | 0.372 |
Sex, male | 29 (72.5%) | 28 (71.8%) | 0.944 |
Medication | |||
IVIG, 2 g/kg*1 d | 35 (87.5%) | 38 (97.4%) | 0.096 |
IVIG, 1 g/kg*2 days | 5 (12.5%) | 1 (2.6%) | 0.096 |
Aspirin | 38 (95%) | 38 (97.4%) | 1.000 |
Persantine | 29 (72.5%) | 15 (38.5%) | 0.002 |
Complications | 25 (62.5%) | 25 (64.1%) | 0.883 |
Liver function damage | 7 (17.5%) | 7 (17.9%) | 0.958 |
Granulocytopenia | 8 (20%) | 5 (12.8) | 0.390 |
URI | 3 (7.5%) | 4 (10.3%) | 0.972 |
LRI | 8 (20%) | 4 (10.3%) | 0.228 |
WBC, 109/L | 16.4 ± 6.8 | 15.8 ± 7.2 | 0.368 |
PLT, 109/L | 392.7 ± 115.9 | 374.2 ± 118.1 | 0.704 |
CRP, mg/L | 75.94 ± 53.6 | 71.1 ± 51.9 | 0.395 |
ESR, mm/h | 33.0 ± 12.0 | 31.8 ± 10.3 | 0.443 |
BNP, pg/mL | 1505.0 (455.0–3557.5) | 833.5 (318.5–2315.0) | 0.192 |
ALT, U/L | 50.0 (17.0–115.0) | 54.0 (22.0–109.0) | 0.734 |
AST, U/L | 35.5 (23.8–51.0) | 29.0 (25.0–35.0) | 0.294 |
TG,mmol/L | 1.29 ± 0.3 | 1.3 ± 0.46 | 0.972 |
TC,mmol/L | 3.56 ± 0.73 | 3.61 ± 0.63 | 0.701 |
HDL-C,mmol/L | 1.8 ± 0.75 | 2.35 ± 0.55 | 0.043 |
LDL-C.mmol/L | 2 ± 0.84 | 1.21 ± 0.83 | 0.000 |
Data are presented as the mean ± SD, n (%) or medians (interquartile ranges). CAL, coronary artery lesion; NCAL, non-coronary artery lesion; IVIG, intravenous immunoglobulin; URI, upper respiratory infection; LRI, lower respiratory infection; WBC, white blood cell; PLT, platelet; CRP, C-reactive protein; ESR, erythrocyte sedimentation rate; BNP, brain natriuretic peptide; ALT, alanine transaminase; AST, aspartate aminotransferase; TG, triglycerides; TC, total cholesterol; HDL-C, high density lipoprotein; LDL-C, low density lipoprotein.
The plasma metabolic profiles of patients in the CAL and NCAL groups were measured using UHPLC-QT-MS in both positive and negative modes. After peak alignment and removal of missing values (
Comprehensive Metabolomic Characterization of CALs in KD.
The ions confirmed by MS/MS spectra and mapped in the database with variable importance in the projection (VIP) values >1.0 based on OPLS-DA models were considered to be potential differentially expressed metabolites.
To further explore the differentially expressed metabolites, the Student’s t-test was used to identify ion peak intensities with a P-value < 0.05. As a result, 16 metabolites were identified (
Differentially expressed metabolites.
Differential metabolite | Mass-to-charge ratio | Retention time (sec) | Fold change | P value |
---|---|---|---|---|
Higher concentration in CAL | ||||
Palmitic acid | 274.273 | 52.950 | 9.879 | <0.001 |
N-Acetylneuraminic acid |
290.086 | 514.804 | 1.554 | 0.003 |
Dodecanoic acid | 218.210 | 92.196 | 1.499 | 0.009 |
cis-9-Palmitoleic acid | 296.258 | 36.189 | 1.361 | 0.036 |
N-Acetylneuraminic acid | 292.102 | 515.098 | 1.322 | 0.019 |
N-Acetyllactosamine | 366.139 | 515.061 | 1.259 | 0.030 |
trans-Vaccenic acid | 265.251 | 35.104 | 1.223 | 0.002 |
Lower concentration in CAL | ||||
1-Stearoyl-sn-glycerol |
417.319 | 35.321 | 0.162 | <0.001 |
1-Stearoyl-sn-glycerol | 341.305 | 35.328 | 0.239 | <0.001 |
Heptadecanoic acid | 288.289 | 73.946 | 0.244 | <0.001 |
L-Phenylalanine | 331.165 | 254.527 | 0.316 | <0.001 |
Phenylethylamine | 281.137 | 36.312 | 0.549 | <0.001 |
Behenic acid | 358.366 | 139.662 | 0.608 | 0.046 |
Linoleic acid | 298.273 | 111.232 | 0.734 | 0.010 |
7-Ketocholesterol | 461.362 | 34.234 | 0.795 | 0.011 |
Sphingosine | 300.289 | 109.074 | 0.800 | 0.040 |
Metabolites were confirmed by negative-mode analysis.
To identify potential biomarkers for CALs in patients with KD, a cut-off P-value < 0.001 and FC (fold change) > 2 were used in both positive and negative mode. The volcano plots of these metabolites are shown in
Due to a lack of datasets concerning the gene expressions between CALs and NCALs, we chose three gene expression series (GSE18606, GSE68004, and GSE73463) to analyze the functional enrichment of DEGs between the KD and normal groups in this study. Among these three groups, there were a total of 242 KD samples and 101 normal ones (
Functional enrichment analyses of DEGs between KD and Normal group.
The functional enrichment analyses, including GO function, KEGG, and Reactome pathway enrichment analyses of DEGs, were performed
Based on previous studies, PA can induce oxidative stress and apoptosis as a saturated fatty acid (
PA affected cell activity by inducing autophagy and cell senescence
We further tested whether palmitic acid worsened endothelial cell senescence in KD. First, we tested the expression of cellular senescence-associated proteins cyclin-dependent kinase inhibitor 2A (P16) and Phospho-RB-S811 (pRB), as well as SA-β-gal with specific staining for cellular senescence. Our results showed that compared with the BSA control group, the expression level of P16 increased after stimulation by KD serum. In contrast, the expression level of pRB decreased, implying that KD serum also induced endothelial cell senescence. Based on that, the addition of PA further exacerbated cellular senescence. (
PA aggravated cellular senescence of endothelial cells in Kawasaki disease.
At the same time, when endothelial cells become senescent, some studies have shown that endothelial cell function is also hindered, and proliferative and migrative abilities are weakened. Therefore, we used the endothelial cell scratch test to analyze the changes in migration ability (
Based on the GEO analysis, the reactive oxygen species metabolic process was also enriched in the KD group. Therefore, we further explored whether PA, a metabolite enriched in KD metabolites, activated the production of reactive oxygen species in endothelial cells. We used immunofluorescence staining of superoxide anion probe DHE to detect the endothelial cells pre-stained with DHE and observed that PA or KD serum treated alone increased ROS generation. At the same time, the co-treatment of PA and KD serum further increased the production of ROS compared with the group treated with KD serum alone (
PA also increased intracellular ROS accumulation in endothelial cells in KD.
Excessive production of oxygen free radicals will further attack the polyunsaturated fatty acids in the cell membrane, trigger lipid peroxidation, and form lipid peroxides, which will trigger the cross-linking and polymerization of macromolecular nucleic acids and proteins, resulting in cytotoxicity. Therefore, we chose one of the lipid peroxides, MDA, to detect the cytotoxicity caused by reactive oxygen species, proving that PA significantly aggravated the cytotoxicity caused by reactive oxygen species in endothelial cells after KD serum treatment (
As the research on the mechanism of aging in KD is unclear, currently recognized cellular senescence triggers include DNA damage, shortening and damage of telomerase, and activation of oncogenes. Reports have outlined that reactive oxygen species not only attack lipids but also damage mitochondria and nuclear DNA. Therefore, we chose acetylcysteine (NAC), an inhibitor of reactive oxygen species, to further verify whether PA mediates the increase in ROS production and leads to cellular senescence in the KD model. As displayed in
PA aggravates cell senescence by mediating ROS production.
Palmitic acid, a critical metabolite, aggravates cell senescence through reactive oxygen species generation in Kawasaki disease.
In summary, we analyzed the differences in plasma metabolites between the CAL and NCAL groups
As the first study to identify the differences in circulating metabolites between the CAL and NCAL groups with the application of UHPLC-QTOFMS, a key result of metabolomics was PA showing the most massive fold change among all the significantly differently expressed metabolites. Alongside the metabonomics findings, we further demonstrated the potential role played by PA in the pathogenesis of KD patients with CALs. Our results signaled that PA exacerbates endothelial cell senescence by increasing the production of reactive oxygen species in endothelial cells, and cellular senescence may be one of the mechanisms of coronary artery lesions in KD.
The metabolic profiles demonstrated significant pattern differences between patients in the CAL and NCAL groups in the acute phase. Moreover, the alterations in metabolites suggested that several metabolic pathways were disrupted in KD patients with CALs, including the Phenylalanine metabolism, Linoleic acid metabolism, and Fatty acid biosynthesis pathways. A panel of biomarkers showed excellent predictive values in distinguishing between KD patients with CALs and those without CALs. PA, heptadecanoic acid and 1-stearoyl-sn-glycerol are fatty acids. Among these three fatty acids, PA had the highest fold change (FC = 9.88) and high relative connections in patients with CALs compared with patients in the NCAL group. At the same time, the other two showed moderate fold changes (FC = 4.09 for heptadecanoic acid, and FC = 4.17 for 1-stearoyl-sn-glycerol) and low relative connections in patients with CALs. Therefore, we speculate that PA is involved in the development of CALs in patients with KD.
Numerous studies have affirmed that PA can accelerate artery damage.
Initial studies have imposed that as one of the characteristic histological features, endothelial cell senescence is closely related to atherosclerosis.
Considering the severe effects of CALs in patients with KD, we advocate a new method to predict the risk of CALs in the early stage of the disease, which contributes to these patients’ treatment. 1-Stearoyl-sn-glycerol, palmitic acid, heptadecanoic acid, and phenylethylamine had sufficient power to distinguish between patients in the CAL and NCAL groups individually (the AUC values ranged from 0.913 to 0.956). Our findings may also provide a novel vision for elucidating the pathogenesis of KD patients with CALs. In summary, we will further explore whether the other three biomarkers, excluding PA can exacerbate CALs in KD patients and verify our findings in CAWS models in the next stage of our study. In addition, the triggering factors of cellular senescence in endothelial cells will be further analyzed.
The original contributions presented in the study are included in the article/
The studies involving human participants were reviewed and approved by The Ethics Committee of the Second Affiliated Hospital and Yuying Children’s Hospital of Wenzhou Medical University. Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin.
TW and RW designed the experiments and completed the reanalysis of article data. QZ and QD performed the experiments and wrote the manuscript. XW and TX collected the patients’ samples and performed the Metabolomic experiments. YF and QW analyzed the results.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.
The Supplementary Material for this article can be found online at:
The typical peak intensity chromatograms of quality control samples under negative and positive modes.
The presence of SA-β-gal under PA treated in HASMCs. HASMCs were treated with PA under the concentrations of 250 and 500 μM. The staining of SA-β-gal activity in HASMCs seldom indicated that cellular senescence was detected.