Abstract
SummaryThe generation of functional arterial endothelial cells (aECs) from embryonic stem cells (ESCs) holds great promise for vascular tissue engineering. However, the mechanisms underlying their generation and the potential of aECs in revascularizing ischemic tissue are not fully understood. Here, we observed that hypoxia exposure of mouse ESCs induced an initial phase of HIF1α-mediated upregulation of the transcription factor Etv2, which in turn induced the commitment to the EC fate. However, sustained activation of HIF1α in these EC progenitors thereafter induced NOTCH1 signaling that promoted the transition to aEC fate. We observed that transplantation of aECs mediated arteriogenesis in the mouse hindlimb ischemia model. Furthermore, transplantation of aECs in mice showed engraftment in ischemic myocardium and restored cardiac function in contrast to ECs derived under normoxia. Thus, HIF1α activation of Etv2 in ESCs followed by NOTCH1 signaling is required for the generation aECs that are capable of arteriogenesis and revascularization of ischemic tissue.
Highlights
Stabilization of the transcription factor hypoxia-inducible factor 1a (HIF1a) in response to hypoxia induces expression of downstream targets regulating vasculogenesis, angiogenesis, and arteriogenesis (Semenza and Wang, 1992) (BoschMarce et al, 2007; Wang and Semenza, 1995)
Hypoxia increases the production of vascular endothelial growth factor (VEGF), basic fibroblast growth factor, angiopoietin-1, and platelet-derived growth factor, all of which are linked to EC generation (Shin et al, 2011)
(D and E) Representative immunoblot and protein quantification of VE-cadherin and PECAM1 on days 0, 3, 5, and 7 of endothelial differentiation (n = 3 independent experiments). (F and G) Increased percentage of VE-cadherin+/PECAM1+ population was detected by flow cytometry at day 4 to day 7 under hypoxia (1% O2) Data shown as percentage of VE-cadherin+/PECAM1+ population in normoxia and hypoxic differentiation (n = 3 independent experiments). (H) Enhanced VE-cadherin+/PECAM1+ population at day 8 of endothelial differentiation from human ESCs (hESCs) under hypoxia stimulus (n = 3 independent experiments). (I) Cell-cycle analysis of FLK1+ endothelial progenitor cells derived during normoxia; hypoxia did not augment proliferation of differentiating cells (n = 3 independent experiments)
Summary
Stabilization of the transcription factor hypoxia-inducible factor 1a (HIF1a) in response to hypoxia induces expression of downstream targets regulating vasculogenesis, angiogenesis, and arteriogenesis (Semenza and Wang, 1992) (BoschMarce et al, 2007; Wang and Semenza, 1995). 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 (Shin et al, 2011). 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 (Shin et al, 2011) Another mechanism of EC generation involves the induction of the Notch ligand Dll and Notch target genes Hey and Hey (Diez et al, 2007). Studies in zebrafish and mice showed that activation of Notch signaling was critical for specifying arterial EC (aEC) fate differentiation during embryogenesis (Lawson et al, 2001, 2002). Studies showed that Etv activation functioned through the expression of Dll, and contributed to aEC differentiation (Wythe et al, 2013)
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