Embryonic Stem Cell Differentiation to Functional Arterial Endothelial Cells through Sequential Activation of ETV2 and NOTCH1 Signaling by HIF1α

摘要

class="head no_bottom_margin" id="sec1title">IntroductionStabilization of the transcription factor hypoxia-inducible factor 1α (HIF1α) in response to hypoxia induces expression of downstream targets regulating vasculogenesis, angiogenesis, and arteriogenesis () (, ). Hif1α^−/− mice are embryonically lethal whereas Hif1α heterozygotes (Hif1α^+/−), although viable, are defective in expressing the angiogenic factor vascular endothelial growth factor (VEGF) and fail to induce adaptive arteriogenesis in the mouse hindlimb ischemia model (). Hypoxia participates in the EC fate commitment of ESCs (, ). Studies showed that hypoxia-primed embryoid bodies more efficiently differentiated into ECs than normoxic bodies (). Multiple mechanisms are thought to be involved in the generation of ECs. Hypoxia increases the production of VEGF, basic fibroblast growth factor (bFGF), angiopoietin-1, and platelet-derived growth factor, all of which are linked to EC generation (). Another mechanism of EC generation involves the induction of the Notch ligand Dll4 and Notch target genes Hey1 and Hey2 (). Studies in zebrafish and mice showed that activation of Notch signaling was critical for specifying arterial EC (aEC) fate differentiation during embryogenesis (, ). Ets transcriptional factor family members can also regulate vasculogenesis and angiogenesis (, ) () (), indicative of their role in specifying EC fate. Deletion of the Ets family member Etv2 in particular impaired vasculogenesis in mice (href="#bib19" rid="bib19" class=" bibr popnode">Lee et al., 2009). Studies showed that Etv2 activation functioned through the expression of Dll4, and thus contributed to aEC differentiation (href="#bib39" rid="bib39" class=" bibr popnode">Wythe et al., 2013).While HIF-1α and ETV2 signaling induce differentiation of ESCs to ECs, the mechanisms specifying the differentiation to aECs and whether aEC generation requires sequential activation of a specific set of signaling pathways have not been addressed. Here, we demonstrate that hypoxia-mediated EC differentiation from ESCs occurs in a biphasic manner, initially inducing HIF1α-dependent Etv2 activation that promotes ESC commitment to the EC lineage and then directing EC progenitors to the aEC fate via activation of NOTCH1 signaling. Upon transplantation, aECs were capable of inducing arteriogenesis in the mouse hindlimb ischemia model as well as engrafting and restoring the function of ischemic myocardium in mice. Thus, activation of the HIF1α-ETV2-NOTCH1 signaling axis in ESCs is an essential mechanism of differentiation to aECs that are capable restoring arterial perfusion and function of ischemic tissue.