Adrenomedullin/Cyclic AMP Pathway Induces Notch Activation and Differentiation of Arterial Endothelial Cells From Vascular Progenitors

Abstract

Objective— The acquisition of arterial or venous identity is highlighted in vascular development. Previously, we have reported an embryonic stem (ES) cell differentiation system that exhibits early vascular development using vascular endothelial growth factor (VEGF) receptor-2 (VEGFR2)-positive cells as common vascular progenitors. In this study, we constructively induced differentiation of arterial and venous endothelial cells (ECs) in vitro to elucidate molecular mechanisms of arterial-venous specification. Methods and Results— ECs were induced from VEGFR2 + progenitor cells with various conditions. VEGF was essential to induce ECs. Addition of 8bromo-cAMP or adrenomedullin (AM), an endogenous ligand-elevating cAMP, enhanced VEGF-induced EC differentiation. Whereas VEGF alone mainly induced venous ECs, 8bromo-cAMP (or AM) with VEGF supported substantial induction of arterial ECs. Stimulation of cAMP pathway induced Notch signal activation in ECs. The arterializing effect of VEGF and cAMP was abolished in recombination recognition sequence binding protein at the Jκ site deficient ES cells lacking Notch signal activation or in ES cells treated with γ-secretase inhibitor. Nevertheless, forced Notch activation by the constitutively active Notch1 alone did not induce arterial ECs. Conclusions— Adrenomedullin/cAMP is a novel signaling pathway to activate Notch signaling in differentiating ECs. Coordinated signaling of VEGF, Notch, and cAMP is required to induce arterial ECs from vascular progenitors.

Functional Arterial and Venous Fate Is Determined by Graded VEGF Signaling and Notch Status During Embryonic Stem Cell Differentiation

“…Yurugi-Kobayashi et al 17 did not report upregulation of arterial genes without cAMP activation, keeping in mind that they examined gene expression one day earlier compared with this study. FACS analysis show that CD31 ϩ cells at 6, 7, and 8 days of differentiation incorporate acetylated-LDL with high specificity indicative of functional endothelial population, allowing us to dissect the temporal linage commitment.…”

“…16 Using a similar approach Yurugi-Kobayashi et al recently found arterial specification to be critically dependent on Adrenomedullin-activated cAMP. 17 Our study shows a dual function for VEGF as a determinant of endothelial cell fate but also as a dose dependent inducer of lineage specificity. Blocking Notch signaling inhibited VEGF-induced arterial specification and promoted venous identity without effecting de novo endothelial cell formation.…”

Roles of TGF-β family signaling in stem cell renewal and differentiation

“…When Flk1 + hematopoieticendothelial progenitors isolated from differentiation cultures are re-differentiated in the presence of VEGF, lineages of both endothelial cells and mural cells (pericytes and vascular smooth muscle) develop from a common [46,47]. Endothelial differentiation has also been demonstrated in human ES cell differentiation cultures [48] We have been studying the roles of TGF-β family signals during vascular development from mouse ES cells.…”

In vitro and in vivo arterial differentiation of human multipotent adult progenitor cells

“…3 Indeed, arteriovenous (AV) specification of ECs is accomplished early in development and is associated with the expression of a specific complement of factors: venous endothelium is characterized by the expression of EphB4, 4 Lefty-1, 5 Lefty-2, 5 COUP-TFII, 6 and MYO1-␤, 5 and arterial ECs express high levels of Notch 1 and 4, 7 Dll-4, 8 EphrinB1 and EphrinB2, 4 Jagged-1 and -2, 7 connexin-40, and Hey-2 (gridlock zebrafish ortholog). 9,10 Studies in Xenopus, zebrafish, and mice have revealed that, besides blood flow, 11 vessel-intrinsic cues and-later in development-signals from outside the vasculature 12,13 are implicated in defining arterial or venous fate such as members of the TGF-␤ pathway, 14,15 VEGF isoforms, 13,[16][17][18] neuropilins, 17 angiopoietins, 19 the Notch pathway, 7,9,[20][21][22] the patched pathway, 20 and COUP-TFII, a member of the orphan nuclear receptor superfamily. 6 Although it has been shown that some of these pathways are well conserved from zebrafish to mouse, less information is available on whether they have a similar role in humans.…”