Endothelial Jagged1 antagonizes Dll4 regulation of endothelial branching and promotes vascular maturation downstream of Dll4/Notch1.

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 (eJag1OE and eJag1cKO). 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 eJag1cKO 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 (eDll4OE) 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 eJag1OE 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.

A computational and experimental study to develop E-selectin targeted peptides for molecular imaging applications.

Aim: E-selectin is overexpressed on angiogenic and inflamed endothelium. Molecules binding to E-selectin with high affinity and specificity enable its use as a molecular imaging biomarker. Material & methods: The interactions of four different peptides (i.e., Ac-P1 [Acetyl-IELLQAR-CONH2], H2N-P2 [H2N-DITWDQLWDLMK-CONH2], H2N-P3A5 [H2N-YRNWAGRW-CONH2], and Ac-P4 [Acetyl-YRNWDGRW-CONH2]) with E-selectin were analyzed by computational methodologies, surface plasmon resonance and in vitro using activated human umbilical vein endothelial cells. Poly(butyl cyanoacrylate) microbubbles were functionalized with the best candidates and evaluated as molecular ultrasound probes in cultured cells and explanted carotid arteries. Results: H2N-P3A5 and Ac-P4 peptides bound stronger to E-selectin than Ac-P1 and H2N-P2, but with lower specificity. H2N-P2 bound with higher specificity and affinity than Ac-P1. Conclusion: H2N-P2 is a good candidate for designing E-selectin-targeted molecular imaging agents.

Low-dosage inhibition of Dll4 signaling promotes wound healing by inducing functional neo-angiogenesis.

Recent findings regarding Dll4 function in physiological and pathological conditions indicate that this Notch ligand may constitute an important therapeutic target. Dll4 appears to be a major anti-angiogenic agent, occupying a central role in various angiogenic pathways. The first trials of anti-Dll4 therapy in mice demonstrated a paradoxical effect, as it reduced tumor perfusion and growth despite leading to an increase in vascular density. This is seen as the result of insufficient maturation of the newly formed vasculature causing a circulatory defect and increased tumor hypoxia. As Dll4 function is known to be closely dependent on expression levels, we envisioned that the therapeutic anti-Dll4 dosage could be modulated to result in the increase of adequately functional blood vessels. This would be useful in conditions where vascular function is a limiting factor for recovery, like wound healing and tissue hypoxia, especially in diabetic patients. Our experimental results in mice confirmed this possibility, revealing that low dosage inhibition of Dll4/Notch signaling causes improved vascular function and accelerated wound healing.