Endothelial Jagged1 Antagonizes Dll4 Regulation of Endothelial Branching and Promotes Vascular Maturation Downstream of Dll4/Notch1

Abstract

Objective— Notch signaling controls cardiovascular development and has been associated with several pathological conditions. Among its ligands, Jagged1 and Dll4 were shown to have opposing effects in developmental angiogenesis, but the underlying mechanism and the role of Jagged1/Notch signaling in adult angiogenesis remain incompletely understood. The current study addresses the importance of endothelial Jagged1-mediated Notch signaling in the context of adult physiological angiogenesis and the interactions of Jagged1 and Dll4 on angiogenic response and vascular maturation processes. Approach and Results— The role of endothelial Jagged1 in wound healing kinetics and angiogenesis was investigated with endothelial-specific Jag1 gain-of-function and loss-of-function mouse mutants ( eJag1 OE and eJag1c KO). To study the interactions between the 2 Notch ligands, genetic mouse models were combined with pharmacological inhibition of Dll4 or Jagged1, respectively. Jagged1 overexpression in endothelial cells increased vessel density, maturation, and perfusion, thus accelerating wound healing. The opposite effect was seen in e Jag1 cKO animals. Interestingly, Dll4 blockade in these animals led to an increase in vascular density but induced a greater decrease in perivascular cell coverage. However, Jagged1 inhibition in Dll4 gain-of-function (e Dll4 OE) mutants, with reduced angiogenesis, further diminished angiogenic growth and hampered perivascular cell coverage. Our findings suggest that as Dll4 blocks endothelial activation through Notch1 signaling, it also induces Jagged1 expression. Jagged1 then blocks Dll4 signaling through Notch1, allowing endothelial activation by vascular endothelial growth factor and endothelial layer growth. Jagged1 also initiates maturation of the newly formed vessels, possibly by binding and activating endothelial Notch4. Importantly, mice administered with a Notch4 agonistic antibody mimicked the mural cell phenotype of e Jag1 OE mutants without affecting angiogenic growth, which is thought to be Notch1 dependent. Conclusions— Endothelial Jagged1 is likely to operate downstream of Dll4/Notch1 signaling to activate Notch4 and regulate vascular maturation. Thus, Jagged1 not only counteracts Dll4/Notch in the endothelium but also generates a balance between angiogenic growth and maturation processes in vivo.

The developmental biology of genetic Notch disorders

“…More recently, it has been proposed that the perivascular coverage of newly formed vessels by vSMCs and pericytes is facilitated by Jag1-induced expression of integrin αvβ3, which provides binding to a basement membrane-specific von Willebrand factor protein (Scheppke et al, 2012). In adulthood, Jag1 instead acts downstream of Dll4/Notch1 signaling to promote maturation of vSMCs after injury through P27 kip1 -mediated repression of proliferation (Boucher et al, 2013;Pedrosa et al, 2015). Jag1 also regulates sprouting during angiogenesis; both gain-and loss-of-function experiments in endothelial cells show that Jag1 promotes the sprouting of new tip cells during retinal angiogenesis (High et al, 2008; for review, see Benedito and Hellström, 2013).…”

Selective regulation of Notch ligands during angiogenesis is mediated by vimentin

“…Dll4 and Jagged ligands have opposite functions during angiogenesis, which makes angiogenesis an interesting process for evaluating the physiological effects of ligand-specific regulation. Blocking Dll4 or inhibiting Notch signaling by γ-secretase inhibitors (GSIs) leads to hyperbranching, whereas Jagged inhibition reduces branching and attenuates angiogenesis (31,38,56). As vimentin is important for efficient Jagged signaling, loss of vimentin could disturb the equilibrium between proangiogenic Jagged and antiangiogenic Dll4 signaling.…”

“…In contrast to Dll4, Jagged-Notch signaling promotes tip cell selection and sprouting by antagonizing Dll4-Notch signaling (31,(33)(34)(35)(36). How the competition between the ligands is balanced and how ligand-receptor signaling specificity is achieved is under intense investigation (31,33,34,37,38). Modification of the Notch receptor by the Fringe family of glycosaminyltransferases has been shown to enhance Dll4-Notch signaling and to suppress sprouting (31).…”

Ehmt2/G9a controls placental vascular maturation by activating the Notch pathway

“…5B). Finely tuned regulation of the Notch pathway is required for vascular maturation (Pedrosa et al, 2015;Ubezio et al, 2016). In addition, expression of the main effector of the Notch pathway, Rbpj, in developing endothelial cells is required for vascular maturation in extra-embryonic tissues (Copeland et al, 2011).…”

“…Evidence in vitro and in the mouse retina suggests that modulating the Notch pathway in endothelial cells favors vessel enlargement (Pedrosa et al, 2015;Ubezio et al, 2016). Notch signaling is required for vascular development and remodeling (Roca and Adams, 2007), and the main Notch effector, the transcription factor recombination signal binding protein for immunoglobulin kappa J (Rbpj), is required for extra-embryonic blood vessel maturation (Copeland et al, 2011).…”