scholarly journals DNMT3A inhibits E2F1-induced arterial marker expression and impairs angiogenesis in human umbilical artery endothelial cells

2020 ◽  
Vol 52 (11) ◽  
pp. 1236-1246
Author(s):  
Kaiyue Su ◽  
Ningning Lin ◽  
Shouqiang Xie ◽  
Yabo Han ◽  
Zaiming Yang ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Dna Methyltransferase ◽  
Umbilical Artery ◽  
De Novo ◽  
Methylation Status ◽  
Tube Formation ◽  
Luciferase Reporter ◽  
Marker Genes ◽  
Cardiovascular Development ◽  
Human Umbilical Artery

Abstract Arterial marker genes EphrinB2 and HEY2 are essential for cardiovascular development and postnatal neovascularization. Our previous study confirmed that E2F1 could activate the transcription of EphrinB2 and HEY2 in human mesenchymal stem cells; however, the detailed mechanism has not been resolved yet. In this study, we focused on the interaction between E2F1 and DNMT3A, a de novo DNA methyltransferase, on regulating the expression of EphrinB2 and HEY2, and explored the potential mechanisms. Gain- and loss-of-function experiments implicated the positive effect of E2F1 on the expression of EphrinB2 and HEY2 and tube formation in human umbilical artery endothelial cells. Accumulation of DNMT3A decreased the levels of EphrinB2 and HEY2, and impaired tube formation induced by E2F1, while inhibiting DNMT3A by RNA interference augmented their expression and angiogenesis in E2F1-trasfected cells. We then asked whether the low expressions of EphrinB2 and HEY2 induced by DNMT3A are related to the methylation status of their promoters. Surprisingly, the methylation status of the CpG islands in the promoter region was not significantly affected by overexpression of exogenous DNMT3A. Furthermore, the interaction between E2F1 and DNMT3A was confirmed by co-immunoprecipitation. DNMT3A could inhibit the transcription of EphrinB2 and HEY2 promoters by affecting the binding of E2F1 to its recognition sequences as revealed by luciferase reporter assay and chromatin immunoprecipitation. These results identified a novel mechanism underlying the cooperation of DNMT3A with E2F1 on regulating target gene expression, and revealed their roles in the angiogenic process.

scholarly journals Infection with Human Immunodeficiency Virus Type 1 Upregulates DNA Methyltransferase, Resulting in De Novo Methylation of the Gamma Interferon (IFN-γ) Promoter and Subsequent Downregulation of IFN-γ Production

1998 ◽  
Vol 18 (9) ◽  
pp. 5166-5177 ◽  
Author(s):  
Judy A. Mikovits ◽  
Howard A. Young ◽  
Paula Vertino ◽  
Jean-Pierre J. Issa ◽  
Paula M. Pitha ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Dna Methyltransferase ◽  
De Novo ◽  
Methylation Status ◽  
Immunodeficiency Virus ◽  
Ifn Γ ◽  
Hiv 1 ◽  
De Novo Methylation

ABSTRACT The immune response to pathogens is regulated by a delicate balance of cytokines. The dysregulation of cytokine gene expression, including interleukin-12, tumor necrosis factor alpha, and gamma interferon (IFN-γ), following human retrovirus infection is well documented. One process by which such gene expression may be modulated is altered DNA methylation. In subsets of T-helper cells, the expression of IFN-γ, a cytokine important to the immune response to viral infection, is regulated in part by DNA methylation such that mRNA expression inversely correlates with the methylation status of the promoter. Of the many possible genes whose methylation status could be affected by viral infection, we examined the IFN-γ gene as a candidate. We show here that acute infection of cells with human immunodeficiency virus type 1 (HIV-1) results in (i) increased DNA methyltransferase expression and activity, (ii) an overall increase in methylation of DNA in infected cells, and (iii) the de novo methylation of a CpG dinucleotide in the IFN-γ gene promoter, resulting in the subsequent downregulation of expression of this cytokine. The introduction of an antisense methyltransferase construct into lymphoid cells resulted in markedly decreased methyltransferase expression, hypomethylation throughout the IFN-γ gene, and increased IFN-γ production, demonstrating a direct link between methyltransferase and IFN-γ gene expression. The ability of increased DNA methyltransferase activity to downregulate the expression of genes like the IFN-γ gene may be one of the mechanisms for dysfunction of T cells in HIV-1-infected individuals.

Histamine stimulation of prostaglandin and HETE synthesis in human endothelial cells

1988 ◽  
Vol 255 (2) ◽  
pp. C214-C225 ◽  
Author(s):  
G. E. Revtyak ◽  
M. J. Hughes ◽  
A. R. Johnson ◽  
W. B. Campbell
Keyword(s):  
Endothelial Cells ◽  
Arachidonic Acid ◽  
Umbilical Artery ◽  
Epoxyeicosatrienoic Acids ◽  
Human Endothelial Cells ◽  
Intact Cells ◽  
Histamine Stimulation ◽  
Human Umbilical Artery ◽  
A Minor ◽  
Stimulation Of

Endothelial cells (EC) cultured from human umbilical artery (UA) and vein (UV) metabolized [14C]arachidonic acid to prostaglandins (PGs), monohydroxyeicosatetraenoic acids (HETEs), and epoxyeicosatrienoic acids (EETs). Major radioactive products were identified as 6-keto-PGF1 alpha, PGE2, PGF2 alpha, 12-hydroxy heptadecatrienoic acid, 15-HETE, and 11-HETE. In addition, extracts from UV ECs contained 12-HETE, 5-HETE, 14,15-EET, and 5,6-EET as minor products, whereas extracts from UA ECs contained only 12-HETE as a minor product. UA ECs also produced metabolites comigrating with 14,15-EET, 11,12-EET, 8,9-EET, and 5,6-EET. Histamine increased the release of [14C]PGs and [14C]HETEs from [14C]arachidonic acid-labeled ECs. Indomethacin, aspirin, and nordihydroguauretic acid completely inhibited synthesis of both [14C]PGs and [14C]HETEs from exogenous [14C]arachidonic acid in these cells. Microsomes metabolized [14C]arachidonic acid to the same [14C]PGs and [14C]HETEs as intact cells. Pretreatment of microsomes with indomethacin completely inhibited formation of these products. These data indicate that UA ECs and UV ECs metabolize endogenous and exogenous arachidonic acid to both PGs and HETEs. Also 15-HETE and 11-HETE appear to be synthesized by a microsomal enzyme with the properties of cyclooxygenase.

Intragenic hypomethylation of DNMT3A in patients with myelodysplastic syndrome

2018 ◽  
Vol 56 (3) ◽  
pp. 485-491 ◽  
Author(s):  
Ying-Ying Zhang ◽  
Jing-Dong Zhou ◽  
Dong-Qin Yang ◽  
Pin-Fang He ◽  
Dong-Ming Yao ◽  
...  
Keyword(s):  
Myelodysplastic Syndrome ◽  
Dna Methyltransferase ◽  
De Novo ◽  
Methylation Status ◽  
World Health ◽  
Differentially Methylated Region ◽  
International Prognostic Scoring System ◽  
Aberrant Expression ◽  
Specific Pcr ◽  
Cell Counts

AbstractBackground:DNMT3Ais a DNA methyltransferase that acts inde novomethylation. Aberrant expression ofDNMT3Ahas been reported in several human diseases, including myelodysplastic syndrome (MDS). However, the pattern ofDNMT3Amethylation remains unknown in MDS.Methods:The present study was aimed to investigate the methylation status ofDNMT3Aintragenic differentially methylated region 2 (DMR2) using real-time quantitative methylation-specific PCR and analyze its clinical significance in MDS.Results:Aberrant hypomethylation ofDNMT3Awas found in 57% (51/90) MDS cases. There were no significant differences in age, sex, white blood cell counts, platelet counts, hemoglobin counts and World Health Organization, International Prognostic Scoring System and karyotype classifications betweenDNMT3Ahypomethylated andDNMT3Ahypermethylated groups. However, the patients withDNMT3Ahypomethylation had shorter overall survival time than those withoutDNMT3Ahypomethylation (11 months vs. 36 months, p=0.033). Multivariate analysis confirmed the independent adverse impact ofDNMT3Ahypomethylation in MDS.Conclusions:Our data suggest thatDNMT3ADMR2 hypomethylation may be a negative prognostic hallmark in MDS.

scholarly journals Exosomal miR-1290 promotes angiogenesis in hepatocellular carcinoma via targeting SMEK1

2020 ◽  
Author(s):  
Qiong Wang ◽  
Guanwen Wang ◽  
Lianjie Niu ◽  
Shaorong Zhao ◽  
Jianjun Li ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Endothelial Cells ◽  
Tumor Angiogenesis ◽  
Molecular Mechanisms ◽  
Target Genes ◽  
Primary Liver Cancer ◽  
Tumor Model ◽  
Tube Formation ◽  
Luciferase Reporter

Abstract Background: Hepatocellular carcinoma (HCC), the most common primary liver cancer, rely on the formation of new blood vessel for growth and frequent intrahepatic and extrahepatic metastasis. Therefore, it is important to explore the underlying molecular mechanisms of tumor angiogenesis of HCC. Recently, microRNAs have been shown to modulate angiogenic processes by modulating the expression of critical angiogenic factors. However, the potential roles of tumor-derived exosomal microRNAs in regulating tumor angiogenesis remain to be elucidated. Methods: MiRNome sequencing was performed to uncover the miRNAs that are dysregulated in HCC patient serum-derived exosomes. Expression levels of miR-1290 in tissues and cells were determined by quantitative real-time PCR. The effect of mir-1290 on proliferation was evaluated by CCK-8 assay. The angiogenic ability of cells were determined by transwell, wound-healing, tube formation and matrigel plug assays. SMMC-7721 xenograft tumor model was established in NOD-SCID nude mice using miR-1290 and NC antagomirs to determin the angiogenic effect of mir-1290 in vivo. Target protein expression was determined by western blotting. Dual luciferase reporter assay was performed to confirm the action of miR-1290 on downstream target genes including SMEK1. Results are reported as means ± S.D. and differences were tested for significance using 2-sided Student’s t-test.Results: In this study, our miRNome sequencing demonstrated that miR-1290 was overexpressed in HCC patient serum-derived exosomes, and we found that delivery of miR-1290 into human endothelial cells enhanced their angiogenic ability. Our results further revealed that SMEK1 is a direct target of miR-1290 in endothelial cells. MiR-1290 exerted its pro-angiogenic function, at least in part, by alleviating the inhibition of VEGFR2 phosphorylation done by SMEK1. Conclusions: Collectively, our findings provide evidence that miR-1290 is overexpressed in HCC and promotes tumor angiogenesis via exosomal secretion, implicating its potential role as a therapeutic target for HCC.

scholarly journals MicroRNA-132, Delivered by Mesenchymal Stem Cell-Derived Exosomes, Promote Angiogenesis in Myocardial Infarction

2018 ◽  
Vol 2018 ◽  
pp. 1-11 ◽  
Author(s):  
Teng Ma ◽  
Yueqiu Chen ◽  
Yihuan Chen ◽  
Qingyou Meng ◽  
Jiacheng Sun ◽  
...  
Keyword(s):  
Myocardial Infarction ◽  
Endothelial Cells ◽  
Real Time ◽  
Real Time Pcr ◽  
Target Gene ◽  
Tube Formation ◽  
Luciferase Reporter ◽  
Reporter Assay ◽  
Ischemic Diseases

Background. To cure ischemic diseases, angiogenesis needs to be improved by various strategies in ischemic area. Considering that microRNA-132 (miR-132) regulates endothelial cell behavior during angiogenesis and the safe and efficacious delivery of microRNAs in vivo is rarely achieved, an ideal vehicle for miR-132 delivery could bring the promise for ischemic diseases. As a natural carrier of biological molecules, exosomes are more and more developed as an ideal vehicle for miRNA transfer. Meanwhile, mesenchymal stem cells could release large amounts of exosomes. Thus, this study aimed to investigate whether MSC-derived exosomes can be used for miR-132 delivery in the treatment of myocardial ischemia. Methods. MSC-derived exosomes were electroporated with miR-132 mimics and inhibitors. After electroporation, miR-132 exosomes were labelled with DiI and added to HUVECs. Internalization of DiI-labelled exosomes was examined by fluorescent microscopy. Expression levels of miR-132 in exosomes and HUVECs were quantified by real-time PCR. The mRNA levels of miR-132 target gene RASA1 in HUVECs were quantified by real-time PCR. Luciferase reporter assay was performed to examine the targeting relationship between miR-132 and RASA1. The effects of miR-132 exosomes on the angiogenic ability of endothelial cells were evaluated by tube formation assay. Matrigel plug assay and myocardial infarction model were used to determine whether miR-132 exosomes can promote angiogenesis in vivo. Results. miR-132 mimics were effectively electroporated and highly detected in MSC-derived exosomes. The expression level of miR-132 was high in HUVECs preincubated with miR-132 mimic-electroporated exosomes and low in HUVECs preincubated with miR-132 inhibitor-electroporated exosomes. The expression level of RASA1, miR-132 target gene, was reversely correlated with miR-132 expression in HUVECs pretreated with exosomes. Luciferase reporter assay further confirmed that RASA1 was a direct target of miR-132. Exosomes loaded with miR-132, as a vehicle for miRNA transfer, significantly increased tube formation of endothelial cells. Moreover, subcutaneous injection of HUVECs pretreated with miR-132 exosomes in nude mice significantly increased their angiogenesis capacity in vivo. In addition, transplantation of miR-132 exosomes in the ischemic hearts of mice markedly enhanced the neovascularization in the peri-infarct zone and preserved heart functions. Conclusions. The findings suggest that the export of miR-132 via MSC-derived exosomes represents a novel strategy to enhance angiogenesis in ischemic diseases.

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