Regulation of von Willebrand Factor Gene in Endothelial Cells That Are Programmed to Pluripotency and Differentiated Back to Endothelial Cells

Endothelial cells play a central role in physiological function and pathophysiology of blood vessels in health and disease. However, the molecular mechanism that establishes the endothelial phenotype, and contributes to its signature cell type‐specific gene expression, is not yet understood. We studied the regulation of a highly endothelial‐specific gene, von Willebrand factor (VWF), in induced pluripotent stem cells generated from primary endothelial cells (human umbilical vein endothelial cells [HUVEC] into a pluripotent state [HiPS]) and subsequently differentiated back into endothelial cells. This allowed us to explore how VWF expression is regulated when the endothelial phenotype is revoked (endothelial cells to HiPS), and re‐established (HiPS back to endothelial cells [EC‐Diff]). HiPS were generated from HUVECs, their pluripotency established, and then differentiated back to endothelial cells. We established phenotypic characteristics and robust angiogenic function of EC‐Diff. Gene array analyses, VWF chromatin modifications, and transacting factors binding assays were performed on the three cell types (HUVEC, HiPS, and EC‐Diff). The results demonstrated that generally cohorts of transacting factors that function as transcriptional activators, and those that contribute to histone acetylation and DNA demethylation, were significantly decreased in HiPS compared with HUVECs and EC‐Diff. In contrast, there were significant increases in the gene expression levels of epigenetic modifiers that function as methyl transferases in HiPS compared with endothelial cells. The results demonstrated that alterations in chromatin modifications of the VWF gene, in addition to expression and binding of transacting factors that specifically function as activators, are responsible for establishing endothelial specific regulation of the VWF gene. Stem Cells 2019;37:542–554 Model representing the mechanism of endothelial specific activation of the von Willebrand factor (VWF) promoter. (A): VWF promoter in HiPS (representative of embryonic stem cells) is modified by epigenetic modifying factors, including DNA methylases and repressive complexes that include histone deacetylases (HDACs). This results in methylation of DNA and deacetylation of histones, leading to maintenance of a chromatin structure that is transcriptionally inactive. (B): During differentiation toward endothelial cell phenotype, decreased levels of DNA methylases combined with increased levels of demethylase