Ivan B. Lobov

Delta-like ligand 4 (Dll4) is induced by VEGF as a negative regulator of angiogenic sprouting

Genetic deletion studies have shown that haploinsufficiency of Delta-like ligand (Dll) 4, a transmembrane ligand for the Notch family of receptors, results in major vascular defects and embryonic lethality. To better define the role of Dll4 during vascular growth and differentiation, we selected the postnatal retina as a model because its vasculature develops shortly after birth in a highly stereotypic manner, during which time it is accessible to experimental manipulation. We report that Dll4 expression is dynamically regulated by VEGF in the retinal vasculature, where it is most prominently expressed at the leading front of actively growing vessels. Deletion of a single Dll4 allele or pharmacologic inhibition of Dll4/Notch signaling by intraocular administration of either soluble Dll4-Fc or a blocking antibody against Dll4 all produced the same set of characteristic abnormalities in the developing retinal vasculature, most notably enhanced angiogenic sprouting and increased endothelial cell proliferation, resulting in the formation of a denser and more highly interconnected superficial capillary plexus. In a model of ischemic retinopathy, Dll4 blockade also enhanced angiogenic sprouting and regrowth of lost retinal vessels while suppressing ectopic pathological neovascularization. Our data demonstrate that Dll4 is induced by VEGF as a negative feedback regulator and acts to prevent overexuberant angiogenic sprouting, promoting the timely formation of a well differentiated vascular network.

Nrarp Coordinates Endothelial Notch and Wnt Signaling to Control Vessel Density in Angiogenesis

When and where to make or break new blood vessel connections is the key to understanding guided vascular patterning. VEGF-A stimulation and Dll4/Notch signaling cooperatively control the number of new connections by regulating endothelial tip cell formation. Here, we show that the Notch-regulated ankyrin repeat protein (Nrarp) acts as a molecular link between Notch- and Lef1-dependent Wnt signaling in endothelial cells to control stability of new vessel connections in mouse and zebrafish. Dll4/Notch-induced expression of Nrarp limits Notch signaling and promotes Wnt/Ctnnb1 signaling in endothelial stalk cells through interactions with Lef1. BATgal-reporter expression confirms Wnt signaling activity in endothelial stalk cells. Ex vivo, combined Wnt3a and Dll4 stimulation of endothelial cells enhances Wnt-reporter activity, which is abrogated by loss of Nrarp. In vivo, loss of Nrarp, Lef1, or endothelial Ctnnb1 causes vessel regression. We suggest that the balance between Notch and Wnt signaling determines whether to make or break new vessel connections.

The Dll4/Notch pathway controls postangiogenic blood vessel remodeling and regression by modulating vasoconstriction and blood flow.

Blood vessel remodeling is crucial to the formation of the definitive vasculature, but little is known about the mechanisms controlling this process. We show that Delta-like ligand 4 (Dll4)/Notch pathway regulates vessel regression in normal pathologic conditions. Genetic and pharmacologic inhibition of Dll4/Notch prevented retinal capillary regression in the oxygen-induced retinopathy (OIR) model and during normal development. Deletion of the Notch-regulated ankyrin repeat protein, a negative regulator of the Notch pathway, produced an opposite phenotype. Inhibition of Dll4/Notch reduced vessel occlusion, maintaining blood flow that is essential for survival of microvessels. Dll4/Notch inhibition up-regulated the expression of vasodilators adrenomedullin and suppressed the expression of vasoconstrictor angiotensinogen. Angiotensin II induced rapid nonperfusion and regression of developing retinal capillaries, whereas Ace1 and AT1 inhibitors and adrenomedullin attenuated vasoobliteration in OIR, indicating that both pathways are involved in modulating vessel remodeling. In contrast, inhibition of vascular endothelial growth factor-A (VEGF-A) did not result in a pervasive loss of retinal capillaries, demonstrating that reduced expression of VEGF-A is not the proximate cause of capillary regression in OIR. Modulation of VEGF-A and Dll4/Notch signaling produced distinct changes in blood vessel morphology and gene expression, indicating that these pathways can have largely independent functions in vascular remodeling.

Conditionals by inversion provide a universal method for the generation of conditional alleles

Significance We describe conditional by inversion (COIN), a new design for conditional alleles that uses an optimized conditional gene trap module (COIN module) inserted into the target gene in an orientation opposite to the gene’s direction of transcription. Activation by Cre recombinase inverts the COIN module, resulting in expression of a reporter and termination of transcription, thereby inactivating the target gene while marking the cells where the conditional event has occurred. Creation of COIN alleles for more than 20 genes showed that it is a robust and universal method—applicable to any gene regardless of exon–intron structure—that overcomes the limitations of previous conditional approaches. Conditional mutagenesis is becoming a method of choice for studying gene function, but constructing conditional alleles is often laborious, limited by target gene structure, and at times, prone to incomplete conditional ablation. To address these issues, we developed a technology termed conditionals by inversion (COIN). Before activation, COINs contain an inverted module (COIN module) that lies inertly within the antisense strand of a resident gene. When inverted into the sense strand by a site-specific recombinase, the COIN module causes termination of the target gene’s transcription and simultaneously provides a reporter for tracking this event. COIN modules can be inserted into natural introns (intronic COINs) or directly into coding exons as part of an artificial intron (exonic COINs), greatly simplifying allele design and increasing flexibility over previous conditional KO approaches. Detailed analysis of over 20 COIN alleles establishes the reliability of the method and its broad applicability to any gene, regardless of exon–intron structure. Our extensive testing provides rules that help ensure success of this approach and also explains why other currently available conditional approaches often fail to function optimally. Finally, the ability to split exons using the COIN’s artificial intron opens up engineering modalities for the generation of multifunctional alleles.

Delta-like Ligand 4/Notch Pathway in Tumor Angiogenesis

The process of angiogenesis, be it physiological or pathological, requires the coordinated interplay of a variety of vascular growth factor systems. Many preclinical models, and more recently several clinical trials, have shown that vascular endothelial growth factor (VEGF) is an essential mediator of developmental and pathological angiogenesis. Recent evidence suggests that another pathway—the Delta/ Notch pathway, and the Delta-like ligand 4 (Dll4) in particular—also plays a specific and critical role in angiogenesis, acting in part to restrain VEGF-mediated angiogenesis. Perturbation of this Dll4-mediated restraint can result in excessive non-productive vessel growth. For example, during embryogenesis, genetic deletion of even one allele of Dll4 in mice results in profound vascular defects and significant embryonic lethality at approx E10.5. The vascular defects include abnormal vascular remodeling in the yolk sac and reduced vascular invasion of the placental labyrinth, poor formation of the major arteries in the embryo, and excessive sprouting/branching in certain vessel beds. In genetic backgrounds that permit survival of Dll4 heterozygous mice, other vascular defects are found, including abnormal maturation of the vascular bed in the developing post natal retina. Dll4 also plays a fundamental role in pathological angiogenesis, as blockers of the Dll4 / Notch pathway result in decreased tumor growth, even for tumors resistant to anti-VEGF therapies. This reduced tumor growth is associated with markedly increased tumor vascularity, enhanced angiogenic sprouting, and more vessel branching. However, the increased vascularity is disorg anized and non-productive, as evidenced by poor perfusion and increased tumor hypoxia. The current model is that VEGF induces Dll4 as a negative feedback regulator of angiogenesis, thus helping to coordinate VEGF-induced sprouting and promoting the functional specialization of the endothelial cells (ECs) in a network. Although Dll4 is clearly induced by VEGF and helps regulate VEGF-mediated vascular growth, it appears to also have functions that are independent of VEGF, as blockade of both VEGF and Dll4 can show more potent anti-tumor effects than blockade of either pathway alone. Thus, blockade of Dll4 in tumors presents a novel therapeutic approach, even for tumors resistant to anti-VEGF therapies.