DNA methyltransferase inhibition induces mouse embryonic stem cell differentiation into endothelial cells.
Understanding endothelial cell (EC) differentiation is a step forward in tissue engineering, controlling angiogenesis, and endothelial dysfunction. We hypothesized that epigenetic activation of EC lineage specification genes is an important mediator of embryonic stem cell (ESC) differentiation into EC. Mouse ESC was differentiated by removing leukemia inhibitory factor (LIF) from the maintenance media in the presence or absence of the specific DNA methyltransferase (DNMT) inhibitor 5'-aza-2'-deoxycytidine (aza-dC). Expression of EC specification and marker genes was monitored by quantitative PCR, western, immunocytochemistry, and flow cytometry. Functionality of differentiated EC was assessed by angiogenesis assay. The methylation status in the proximal promoter CpGs of the mediators of EC differentiation VEGF-A, BMP4, and EPAS-1 as well as of the mature EC marker VE-cadherin was determined by bisulfite sequencing. ESC differentiation resulted in repression of OCT4 expression in both the absence and presence of aza-dC treatment. However, significant increase in angiogenesis and expression of the mediators of EC differentiation and EC-specific genes was only observed in aza-dC-treated cells. The DNMT inhibition-mediated increase in EC specification and marker gene expression was not associated with demethylation of these genes. These studies suggest that DNMT inhibition is an efficient inducer of EC differentiation from ESC.
Banerjee
Saswati
S
Cardiovascular Research Institute, Morehouse school of Medicine, 720 Westview Drive, Atlanta, GA 30310, USA.
Bacanamwo
Methode
M
D042683:Angiopoietin-1 / Q000235:genetics; D000818:Animals; D015703:Antigens, CD / Q000235:genetics / Q000378:metabolism; D001374:Azacitidine / Q000031:analogs & derivatives / Q000494:pharmacology; D051792:Basic Helix-Loop-Helix Proteins / Q000235:genetics; D055415:Bone Morphogenetic Protein 4 / Q000235:genetics; D015820:Cadherins / Q000235:genetics / Q000378:metabolism; D002454:Cell Differentiation / Q000187:drug effects* / Q000502:physiology; D002460:Cell Line; D002478:Cells, Cultured; D018899:CpG Islands / Q000235:genetics; D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000037:antagonists & inhibitors*; D019175:DNA Methylation / Q000235:genetics; D000077209:Decitabine; D015536:Down-Regulation / Q000235:genetics; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D042783:Endothelial Cells / Q000166:cytology* / Q000378:metabolism; D015870:Gene Expression / Q000187:drug effects / Q000235:genetics; D051379:Mice; D018919:Neovascularization, Physiologic / Q000187:drug effects; D050814:Octamer Transcription Factor-3 / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics; D042787:Receptor, TIE-2 / Q000235:genetics / Q000378:metabolism; D015854:Up-Regulation / Q000235:genetics; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics; D014841:von Willebrand Factor / Q000235:genetics / Q000378:metabolism; D000099152:Cadherin 5; D000099162:Endothelial PAS Domain-Containing Protein 1
D042683:Angiopoietin-1; D015703:Antigens, CD; D001374:Azacitidine; D051792:Basic Helix-Loop-Helix Proteins; D055415:Bone Morphogenetic Protein 4; D015820:Cadherins; D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases; D000077209:Decitabine; D050814:Octamer Transcription Factor-3; D042787:Receptor, TIE-2; D042461:Vascular Endothelial Growth Factor A; D014841:von Willebrand Factor; D000099152:Cadherin 5; D000099162:Endothelial PAS Domain-Containing Protein 1; C467628:Angpt1 protein, mouse; C525810:Bmp4 protein, mouse; C467480:vascular endothelial growth factor A, mouse