Data_Sheet_1_Shared gene characteristics and molecular mechanisms of macrophages M1 polarization in calcified aortic valve disease.ZIP

BackgroundCAVD is a common cardiovascular disease, but currently there is no drug treatment. Therefore, it is urgent to find new and effective drug therapeutic targets. Recent evidence has shown that the infiltration of M1 macrophages increased in the calcified aortic valve tissues, but the mechanism has not been fully elucidated. The purpose of this study was to explore the shared gene characteristics and molecular mechanisms of macrophages M1 polarization in CAVD, in order to provide a theoretical basis for new drugs of CAVD.MethodsThe mRNA datasets of CAVD and M1 polarization were downloaded from Gene Expression Omnibus (GEO) database. R language, String, and Cytoscape were used to analyze the functions and pathways of DEGs and feature genes. Immunohistochemical staining and Western Blot were performed to verify the selected hub genes.ResultsCCR7 and GZMB were two genes appeared together in hub genes of M1-polarized and CAVD datasets that might be involved in the process of CAVD and macrophages M1 polarization. CCR7 and CD86 were significantly increased, while CD163 was significantly decreased in the calcified aortic valve tissues. The infiltration of M1 macrophages was increased, on the contrary, the infiltration of M2 macrophages was decreased in the calcified aortic valve tissues.ConclusionThis study reveals the shared gene characteristics and molecular mechanisms of CAVD and macrophages M1 polarization. The hub genes and pathways we found may provide new ideas for the mechanisms underlying the occurrence of M1 polarization during CAVD process.</p

Knockdown of GAPDH attenuates Mst1 activation and cell apoptosis in response to hypoxia/reoxygenation.

<p><b>A</b>, NRVMs were transfected with either control siRNA or GAPDH siRNA. 72 hours after transfection, cells were treated with hypoxia for 12 hours and reoxygenation for 24 hours. Mst1 was then immunoprecipitated and its activity was determined by an in vitro kinase assay using histone H2B as a substrate. <b>B</b>, NRVMs were transfected with either control siRNA or GAPDH siRNA. 72 hours after transfection, cells were treated with hypoxia/reoxygenation and the cell apoptosis was determined by using the TUNEL staining kit (Roche). Values are means ± SEM obtained from 4 experiments.</p

Identification of Mst1 and GAPDH interaction sites.

<p><i>A,</i> schematic representation of Mst1 deletion mutants. <i>B,</i> flag-GAPDH expression vector in combination of either empty vector or expression vectors of Myc-Mst1 mutants were cotransfected into HEK293T cells. Extracted proteins were precipitated by anti-Myc antibody and then separated by 12% SDS-PAGE. The transferred membrane was immunoblotted with either HRP-conjugated anti-FLAG or HRP-conjugated anti-Myc antibody. Lysates were also immunoblotted with anti-Flag antibody to show the expression levels of Flag-GAPDH in HEK293T cells. <i>C,</i> schematic representation of GAPDH deletion mutants. <i>D,</i> Flag-Mst1 expression vector in combination of either empty vector or expression vectors of Myc-GAPDH mutants were cotransfected into HEK293T cells. Extracted proteins were precipitated by anti-Myc antibody and then separated by 12% SDS-PAGE. The transferred membrane was immunoblotted with either HRP-conjugated anti-FLAG or HRP-conjugated anti-Myc antibody. Lysates were also immunoblotted with anti-Flag antibody to show the expression of Flag-Mst1 in HEK293 cells.</p

Knockdown of GAPDH attenuates Mst1 activation and cell apoptosis in response to chelerythrine.

<p>A, Total RNA extracted from control siRNA (siCTL) and GAPDH siRNA transfected cells was analyzed for the expression of GAPDH in NRCMs by qRT-PCR. B, NRVMs were transfected with either control siRNA or GAPDH siRNA. 72 hours after transfection, cell lysates were then subjected to western blot analysis to detect the expression of GAPDH. C, NRVMs were transfected with either control siRNA or GAPDH siRNA. 72 hours after transfection, cells were treated with chelerythrine (5 µM) for 2 hours. Mst1 was then immunoprecipitated and its activity was determined by an in vitro kinase assay using histone H2B as a substrate. D, NRVMs were transfected with either control siRNA or GAPDH siRNA. 24 hours after siRNA transfection, cells were then transduced with either Ad-LacZ or Ad-Mst1 (MOI = 50). 48 hours after virus transduction, NRVMs were treated with chelerythrine (5 µM) for 2 hours and the cell apoptosis was determined by using the TUNEL staining kit (Roche). Values are means ± SEM obtained from 4 experiments.</p

Activation of Mst1 kinase by GAPDH.

<p>A, 0.1 µg active Mst1 was incubated with either 4 µg of recombinant GAPDH or MBP for 60 min in the presence of ³²P-ATP in an <i>in vitro</i> phosphorylation assay. Phosphorylation was detected by autoradiography. B, Mst1 immunoprecipitated from HEK293 cells transfected with either Myc-Mst1 or Myc-Mst1 plus Flag-GAPDH expression vectors was incubated with 2 µg MBP for the in vitro kinase assay. Kinase assay was carried out in the presence of ³²P-ATP for 60 min. Phosphorylation was detected by autoradiography. C, Phosphorylation levels of Mst1 and MBP were quantified by densitometry of autoradiograms. Values are means ± SEM obtained from 3 experiments.</p

Phosphorylation of GAPDH by Mst1.

<p>A, 0.1 µg active Mst1 was incubated with different amounts of recombinant GAPDH for 60 min in the presence of ³²P-ATP in an <i>in vitro</i> phosphorylation assay. B, 0.1 µg active Mst1 was incubated with 4 µg bacterially expressed recombinant GAPDH for different time points in the presence of ³²P-ATP in an <i>in vitro</i> phosphorylation assay. Phosphorylation was detected by autoradiography. The data are representatives of 4 independent experiments.</p

Meta-Analysis of <em>RAGE</em> Gene Polymorphism and Coronary Heart Disease Risk

<div><h3>Background</h3><p>Recent data from human and animal studies have shown an upregulated expression of advanced glycosylation end product–specific receptor (RAGE) in human atherosclerotic plaques 1 and in retina, messangial, and aortic vessels, suggesting an important role of RAGE in the pathogenesis of atherothrombotic diseases. In the past few years, the relationship between <em>RAGE</em> polymorphisms (−429T/C, −374T/A, and G82S) and coronary heart disease (CHD) has been reported in various ethnic groups; however, these studies have yielded contradictory results.</p> <h3>Methods</h3><p>PubMed, ISI web of science, EMBASE and the Chinese National Knowledge Infrastructure databases were systematically searched to identify relevant studies. Data were abstracted independently by two reviewers. A meta-analysis was performed to examine the association between <em>RAGE</em> polymorphisms and susceptibility to CHD. Odds ratios (ORs) and 95% confidence intervals (95% CIs) were calculated.</p> <h3>Results</h3><p>A total of 17 studies including 4343 patients and 5402 controls were involved in this meta-analysis. Overall, no significant results were observed for −429T/C (OR  = 1.01, 95% CI: 0.92–1.12, P  = 0.78), −374T/A (OR  = 1.11, 95% CI: 0.98–1.26, P  = 0.09) and G82S (OR  = 1.12, 95% CI: 0.86–1.45, P  = 0.41) polymorphism. In the stratified analyses according to ethnicity, sample size, CHD endpoint and Hardy-Weinberg status, no evidence of any gene-disease association was obtained.</p> <h3>Conclusions</h3><p>This meta-analysis demonstrates that there is no association between the RAGE −429T/C, −374T/A and G82S polymorphisms and CHD.</p> </div

Overexpression of GAPDH enhances Mst1 mediated cardiomyocyte apoptosis.

<p>A, NRVMs were transduced with either Ad-LacZ or Ad-GAPDH (MOI = 30). 48 hr after transduction, cell lysates were subjected to western blot analysis to detect the expression of GAPDH. B, NRVMs were transduced with Ad-LacZ or Ad-GAPDH at 30 mois. Twenty-four hours after transduction, myocytes were transduced with Ad-LacZ or Ad-Mst1 at 30 mois. 48 hours after the second transduction, cytoplasmic accumulation of mono- and oligonucleosomes was quantitated by the Cell Death Detection ELISA. Values are means ± SEM obtained from 4 experiments. C, NRVMs were transduced with Ad-LacZ, Ad-GAPDH, or Ad-DNMST with different combinations (total 60 mois). 48 hours after the transduction, the cells were treated with chelerythrine (5 µM) for 2 hours, the cytoplasmic accumulation of mono- and oligonucleosomes was then quantitated by the Cell Death Detection ELISA. Values are means ± SEM obtained from 4 experiments.</p

Association of Mst1 with GAPDH in cardiomyocytes.

<p>A. Cell lysates obtained from NRVMs were immunoprecipitated with anti-Mst1 antibody and then separated by 12% SDS-PAGE. Transferred membrane was immunoblotted with either anti-GAPDH or Mst1 antibody. B, Tissue homogenates obtained from normal mouse heart and hypertrophic heart were immunoprecipitated with anti-Mst1 antibody and then separated by 12% SDS-PAGE. Transferred membrane was immunoblotted with either anti-GAPDH or Mst1 antibody.</p

Physical interaction between Mst1 and GAPDH by immunoprecipitation analysis.

<p>A, Myc-Mst1 expression vector in combination of either empty vector or pFlag-GAPDH were co-transfected into HEK293 cells. Extracted proteins were precipitated by anti-FLAG antibody and then separated by 12% SDS-PAGE. The transferred membrane was immunoblotted with either HRP conjugated anti-Myc or HRP conjugated anti-FLAG antibody. B, Flag-GAPDH expression vector in combination of either empty vector, pMT2-Myc-Mst1 were co-transfected into HEK293 cells. Extracted proteins were precipitated by anti-Myc antibody and then separated by 12% SDS-PAGE. The transferred membrane was immunoblotted with either HRP conjugated anti-Myc or HRP conjugated anti-FLAG antibody. C, HEK293 cells were transfected with Flag-Mst1. 48 hr after transfection, cells were then treated with etoposide (100 M) or TNF- (20 ng/ml) for 6 hrs. Extracted proteins were precipitated by anti-Flag antibody and then separated by 12% SDS-PAGE. The transferred membrane was immunoblotted with either anti-Mst1 or anti-Flag antibodies.</p