Matrix metalloproteinase-9 (MMP9) has been involved in inflammatory and pathologic processes of coronary artery lesions (CAL) in Kawasaki disease (KD). Intravenous immunoglobulin (IVIG), a traditional treatment for Kawasaki disease, could decrease the expressions of MMP9. The purpose of this study was to investigate the protective effect of IVIG in chemotactic migration of monocyte and the regulation of MMP9 induced by tumor necrosis factor-α (TNF-α) in U937s. Studies were carried out with real time polymerase chain reaction (RT-PCR), zymographic, Western blotting and immunofluorescence. U937s' migration was enhanced by TNF-α stimulation, while was inhibited by IVIG pretreatment. MMP9 expression and activity in U937s were also significantly enhanced by TNF-α and inhibited by IVIVG pretreatment. During inflammatory stimulus, nuclear factor kappa B (NF-κB) and P38 Mitogenactivated protein kinase (P38 MAPK) pathways play a significant role in regulating MMP9 gene expression. TNF-α induced nuclear translocation of NF-κB and P38 MAPK activation in U937s were inhibited significantly by IVIG. Furthermore, we clarified that nuclear NF-κB and P38 MAPK pathways play pivotal roles in regulating U937s' migration and MMP9 expressions using PDTC and SB203580, which were specific inhibitors of NF-κB and p38 MAPK pathways. IVIG displays striking biological effects, notably promoting monocyte migration. These effects involve the NF-κB and p38 pathways, and increased MMP9 activity. It might be a crucial mechanism of IVIG reducing the occurrence of CAL that IVIG inhibited monocytes expressing MMP9 and decreased chemotactic migration of monocyte.

IVIG inhibits TNF-α-induced MMP9 expression and activity in monocytes by suppressing NF-κB and P38 MAPK activation

Background: Salvianolic acid B (Sal B) is a bioactive water-soluble compound of Salviae miltiorrhizae , a traditional herbal medicine that has been used clinically for the treatment of cardiovascular diseases. This study sought to evaluate the effect of Sal B on matrix metalloproteinase-9 (MMP-9) and on the underlying mechanisms in tumor necrosis factor-α± (TNF-α±)-activated human coronary artery endothelial cells (HCAECs), a cell model of Kawasaki disease. Methods: HCAECs were pretreated with 1–10 αμmol/L of Sal B, and then stimulated by TNF-α± at different time points. The protein expression and activity of MMP-9 were determined by Western blot assay and gelatin zymogram assay, respectively. Nuclear factor-κB (NF-κB) activation was detected with immunofluorescence, electrophoretic mobility shift assay, and Western blot assay. Protein expression levels of mitogen-activated protein kinase (c-Jun N-terminal kinase [JNK], extra-cellular signal-regulated kinase [ERK], and p38) were determined by Western blot assay. Results: After HCAECs were exposed to TNF-α±, 1–10 αμmol/L Sal B significantly inhibited TNF-α±-induced MMP-9 expression and activity. Furthermore, Sal B significantly decreased IκBα± phosphorylation and p65 nuclear translocation in HCAECs stimulated with TNF-α± for 30 min. In addition, Sal B decreased the phosphorylation of JNK and ERK1/2 proteins in cells treated with TNF-α± for 10 min. Conclusions: The data suggested that Sal B suppressed TNF-α±-induced MMP-9 expression and activity by blocking the activation of NF-κB, JNK, and ERK1/2 signaling pathways.

Salvianolic Acid B Down-regulates Matrix Metalloproteinase-9 Activity and Expression in Tumor Necrosis Factor-α-induced Human Coronary Artery Endothelial Cells

Kawasaki disease (KD) causes acute systemic vasculitis and has unknown etiology. Since the acute stage of KD is the most relevant, the aim of the present study was to identify hub genes in acute KD by bioinformatics analysis. We also aimed at constructing microRNA (miRNA)-messenger RNA (mRNA) regulatory networks associated with acute KD based on previously identified differentially expressed miRNAs (DE-miRNAs). DE-mRNAs in acute KD patients were screened using the mRNA expression profile data of GSE18606 from the Gene Expression Omnibus. The functional and pathway enrichment analysis of DE-mRNAs were performed with the DAVID database. Target genes of DE-miRNAs were predicted using the miRWalk database and their intersection with DE-mRNAs was obtained. From a protein-protein interaction (PPI) network established by the STRING database, Cytoscape software identified hub genes with the two topological analysis methods maximal clique centrality and Degree algorithm to construct a miRNA-hub gene network. A total of 1,063 DE-mRNAs were identified between acute KD and healthy individuals, 472 upregulated and 591 downregulated. The constructed PPI network with these DE-mRNAs identified 38 hub genes mostly enriched in pathways related to systemic lupus erythematosus, alcoholism, viral carcinogenesis, osteoclast differentiation, adipocytokine signaling pathway and tumor necrosis factor signaling pathway. Target genes were predicted for the up-regulated and down-regulated DE-miRNAs, 10,203, and 5,310, respectively. Subsequently, 355, and 130 overlapping target DE-mRNAs were obtained for upregulated and downregulated DE-miRNAs, respectively. PPI networks with these target DE-mRNAs produced 15 hub genes, six down-regulated and nine upregulated hub genes. Among these, ten genes (ATM, MDC1, CD59, CD177, TRPM2, FCAR, TSPAN14, LILRB2, SIRPA, and STAT3) were identified as hub genes in the PPI network of DE-mRNAs. Finally, we constructed the regulatory network of DE-miRNAs and hub genes, which suggested potential modulation of most hub genes by hsa-miR-4443 and hsa-miR-6510-5p. SP1 was predicted to potentially regulate most of DE-miRNAs. In conclusion, several hub genes are associated with acute KD. An miRNA-mRNA regulatory network potentially relevant for acute KD pathogenesis provides new insights into the underlying molecular mechanisms of acute KD. The latter may contribute to the diagnosis and treatment of acute KD.

In silico Identification of 10 Hub Genes and an miRNA-mRNA Regulatory Network in Acute Kawasaki Disease.

OBJECTIVE: To explore the correlation between genetic polymorphism of matrix metalloproteinase-9 (MMP-9) in patients with coronary artery disease (CAD) and cardiac remodeling. METHODS: A total of 272 subjects who received coronary angiography in our hospital from July 2008 to September 2013 were selected, including 172 CAD patients (CAD group) and another 100 ones (control group). Both groups were subjected to MMP-9 and ultrasonic detections to determine vascular remodeling and atherosclerotic plaques. C1562G polymorphism of MMP-9 gene was detected, and correlation with vascular remodeling and atherosclerotic plaque was analyzed. RESULTS: Serum MMP-9 level of CAD group (330.87±50.39 ng/ml) was significantly higher than that of control group (134.87±34.02 ng/ml) (P<0.05). Compared with control group, CAD group had significantly higher intima-media thickness, and significantly lower systolic peak velocity, mean systolic velocity and end-diastolic velocity (P<0.05). Total area of stenotic blood vessels was 67.34±22.98 mm(2), while that of control blood vessels was 64.00±20.83 mm(2). G/G, G/C and C/C genotype frequencies of MMP-9 differed significantly in the two groups (P<0.05). G and C allele frequencies of CAD group (70.9% and 29.1%) were significantly different from those of control group (50.0% and 50.0%) (P<0.05). G/G, G/C and C/C genotypes were manifested as lipid-rich, fibrous and calcified or ulcerated plaques respectively. Total area of stenotic blood vessels of G/G genotype significantly exceeded those of G/C and C/C genotypes (P<0.05), whereas the latter two had no significant differences. CONCLUSION: CAD promoted 1562C-G transformation of MMP-9 gene into genetic polymorphism, thus facilitating arterial remodeling and increasing unstable atherosclerotic plaques.

Correlation between genetic polymorphism of matrix metalloproteinase-9 in patients with coronary artery disease and cardiac remodeling.

Kawasaki disease (KD) is a leading cause of acquired heart disease predominantly affecting infants and young children. Intravenous immunoglobulin (IVIG) is applied as the most favorable treatment against KD, but IVIG resistant remains exist. Although several clinical scoring systems have been developed to identify children at highest risk of IVIG resistance, there is a need to identify sufficiently sensitive biomarkers for IVIG treatment. Some differentially expressed genes (DEGs) could be the promising potential biomarkers for IVIG-related sensitivity diagnosis. We employed a systematic and integrative bioinformatics framework to identify such kind of genes. The performance of the candidate genes was evaluated by hierarchical clustering, ROC analysis and literature mining. By analyzing three datasets of KD patients, 34 DEGs of the three groups have been found to be associated with IVIG-related sensitivity. A module of 12 genes could predict resistant group patients with high accuracy, and a module of ten genes could predict responsive group patients effectively with accuracy of 96 %. And three of them are most likely to serve as drug targets or diagnostic biomarkers in the future. Compared with unsupervised hierarchical clustering analysis, our modules could distinct IVIG-resistant patients efficiently. Two groups of DEGs could predict IVIG-related sensitivity with high accuracy, which are potential biomarkers for the clinical diagnosis and prediction of IVIG treatment response in KD patients, improving the prognosis of patients.

Identification of Differentially Expressed Genes in Kawasaki Disease Patients as Potential Biomarkers for IVIG Sensitivity by Bioinformatics Analysis.

Objective
To explore the changes of expression and biological activity of nuclear factor-kappa B (NF-κB) and matrix metalloproteinase-9 (MMP-9) after using intravenous immunoglobulin (IVIG) in a murine model of Kawasaki disease (KD) and elucidate the therapeutic mechanism of IVIG for the treatment of KD.


Methods
A total of 72 mice were categorized randomly into IVIG, KD and control groups.Lactobacillus casei cell wall extract (LCWE) was prepared and injected intraperitoneally into C57BL/6 mice to induce KD (0.5 mg single injection). IVIG group received an intraperitoneal injection of IVIG (2 mg/g) while KD model group had an intraperitoneal injection of normal saline. At Days 14, 28 and 56, the diameter of coronary artery was by echocardiography in 8 mice of each group. At the same time, the stains of hematoxylin & eosin and elastic fiber were used to observe the pathological damage of coronary artery. Western blot was used to evaluate the expressions of NF-κB and MMP-9, electrophoretic mobility shift assay (EMSA) was used to measure the activity of NF-κB and Gelatin zymography was used to evaluate the activity of MMP-9 in heart samples of murine model of KD.


Results
The local inflammatory infiltrate, composed predominantly of mononuclear lymphocytes, of coronary artery trunk and branches was observed at Days 14 and 28 while broken elastin was observed at Day 56. And the inflammatory cell infiltrate was less severe and no apparent broken elastin was observed in IVIG and control groups. On echocardiography, the average value of diameter of left coronary artery in KD model group was higher than that in IVIG and control groups (28 d: (0.48±0.07) vs (0.41±0.03) and (0.35±0.02) mm, all P<0.01). Compared with the other two groups, the result of Western blot showed that the expressions of NF-κB and MMP-9 in KD model group were markedly higher than those in IVIG treatment group and that in control group at each time point (28 d: (58±14) vs (25±14) & (19±11) μg/L, (100±41) vs (39±19) & (35±19) μg/L, all P<0.01). The activity of NF-κB by EMSA and the result from KD model group were much higher than those from the control and IVIG groups (28 d: (84 788±2 081) vs (27 220±4 990) & (50 192±1 586) μg/L, all P<0.01]. And it was in accord with the expression of NF-κB. The outcome of gelatin zymography demonstrated that the activity of MMP-9 had similar change with the expression of MMP-9(18 560±7 963) vs (9 112±3 398) & (11 834±4 996) μg/L, all P<0.05).


Conclusions
NF-κB/MMP-9 is overexpressed and over-activated in the heart of KD mouse models. IVIG may inhibit the inflammatory cell infiltration and alleviate coronary artery. And such a therapeutic effect is possibly achieved by a suppression of the overexpression and over-activation of NF-κB/MMP-9 pathway.


Key words: 
Mucocutaneous lymph node syndrome; gamma-Globulins; NF-kappa B; Matrix metalloproteinase 9; Mice

[Effects of intravenous immunoglobulin upon the overexpression and over-activation of nuclear factor-κB and matrix metalloproteinase-9 in murine model of Kawasaki disease].

Zhongdong Du

Papers

[Effects of intravenous immunoglobulin upon the overexpression and over-activation of nuclear factor-κB and matrix metalloproteinase-9 in murine model of Kawasaki disease].