Abstract
Recent studies indicate that Endothelial cells (EC) are required for the engineering of all tissues/organs and their dysfunction mediates the pathogenesis of most cardiovascular diseases. However, EC production remains inefficient and identifying novel EC specification genes remains a priority. Dnmt1-/- embryonic stem cells (ESC) where found to have a higher propensity to differentiate into EC than wild type EC. In addition, endoglin (Eng), recently implicated in early vascular development and angiogenesis, was methylated and repressed in WT ESC, but was demethylated and expressed in Dnmt1-/- ESC. Interestingly, a CpG site located at a locus predicted to bind HIF1 and Sp1, the two main transcription factors regulating Eng transcription, was not methylated in Dnmt1-/- ESC, but was 80% methylated in WT ESC. We hypothesized that Eng may be an epigenetically regulated EC specification gene. Mouse ESC were treated with recombinant Eng or the Eng ORF, and differentiated in the presence or absence of BMP4. Expression of the mesoderm and EC marker genes, and known mediators of EC specification and their downstream targets was monitored by QRT-PCR, western, immunocytochemistry, and flow cytometry. Functionality of the differentiated EC was assessed by in vitro angiogenesis assay and the induction of ICAM-1 expression in response to TNF-α. The percentage of Tie2-positive cells was increased by recombinant Eng relative to vehicle-treated cells (17.1 ± 2.5 vs. 26.0 ± 1.5, p≤0.01). BMP4 also increased the percentage of Tie2-positive cells relative to controls (17.1 ± 2.5 vs. 26.4 ± 1.3, p≤0.01), and the percentage was further increased by combining BMP4 with recombinant Eng (26.4 ± 1.3 vs. 34.5 ± 2.6, p≤0.01). Similar results were obtained with forced Eng expression and with the other EC markers Flk1, vWF, and CD31. The Eng-induced differentiation was independent of known mediators of EC specification such as Indian Hedgehog and BMP4 or of BMP4/phospho-Smad1/5/8 pathway. The effect of CpG methylation at the HIF1/Sp1 binding site on the binding of these factors and transcriptional regulation of Eng are being investigated. The data indicates that Eng is a novel epigenetically regulated EC specification gene, which induces robust EC production in combination with BMP4.
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