Notch1 controls macrophage recruitment and Notch signaling is activated at sites of endothelial cell anastomosis during retinal angiogenesis in mice.

Notch is a critical regulator of angiogenesis, vascular differentiation, and vascular integrity. We investigated whether Notch signaling affects macrophage function during retinal angiogenesis in mice. Retinal macrophage recruitment and localization in mice with myeloid-specific loss of Notch1 was altered, as these macrophages failed to localize at the leading edge of the vascular plexus and at vascular branchpoints. Furthermore, these retinas were characterized by elongated endothelial cell sprouts that failed to anastomose with neighboring sprouts. Using Notch reporter mice, we demonstrate that retinal macrophages localize between Dll4-positive tip cells and at vascular branchpoints, and that these macrophages had activated Notch signaling. Taken together, these data demonstrate that Notch signaling in macrophages is important for their localization and interaction with endothelial cells during sprouting angiogenesis.

Angiogenesis versus arteriogenesis: neuropilin 1 modulation of VEGF signaling.

In development and disease, vascular endothelial growth factor (VEGF) regulates the expansion of the vascular tree. In response to hypoxia, VEGF promotes new capillary formation through the process of angiogenesis by inducing endothelial cell sprouting, proliferation, and migration. Wound healing, tissue regeneration, and tumor growth depend on angiogenesis for adequate nutrient and oxygen delivery. Under different conditions, VEGF promotes arterial growth, modulates lumen expansion, and induces collateral vessel formation, events collectively referred to as arteriogenesis. Induction of arteriogenesis after cardiac or cerebral arterial occlusion can reduce ischemia and improve disease outcome. Endothelial VEGF receptor 2 (VEGFR2) signaling governs both processes. However, modulation of downstream VEGF signaling effectors, such as extracellular-signal-regulated kinase (ERK) activation, differs in order to achieve angiogenic versus arteriogenic outcomes. Recent reports show that neuropilin 1 (NRP1), a VEGF receptor, can instill VEGF signaling outcomes that specifically regulate either angiogenesis or arteriogenesis. Here, we discuss how NRP1 functions as a VEGFR2 co-receptor in angiogenesis and a modulator of VEGFR2 trafficking in arteriogenesis. The unique role played by neuropilin in different endothelial processes makes it an exciting therapeutic target to specifically enhance angiogenesis or arteriogenesis in disease settings.

Combined deficiency of Notch1 and Notch3 causes pericyte dysfunction, models CADASIL, and results in arteriovenous malformations.

Pericytes regulate vessel stability and pericyte dysfunction contributes to retinopathies, stroke, and cancer. Here we define Notch as a key regulator of pericyte function during angiogenesis. In Notch1(+/-); Notch3(-/-) mice, combined deficiency of Notch1 and Notch3 altered pericyte interaction with the endothelium and reduced pericyte coverage of the retinal vasculature. Notch1 and Notch3 were shown to cooperate to promote proper vascular basement membrane formation and contribute to endothelial cell quiescence. Accordingly, loss of pericyte function due to Notch deficiency exacerbates endothelial cell activation caused by Notch1 haploinsufficiency. Mice mutant for Notch1 and Notch3 develop arteriovenous malformations and display hallmarks of the ischemic stroke disease CADASIL. Thus, Notch deficiency compromises pericyte function and contributes to vascular pathologies.

NOTCH decoys that selectively block DLL/NOTCH or JAG/NOTCH disrupt angiogenesis by unique mechanisms to inhibit tumor growth.

UNLABELLED: A proangiogenic role for Jagged (JAG)-dependent activation of NOTCH signaling in the endothelium has yet to be described. Using proteins that encoded different NOTCH1 EGF-like repeats, we identified unique regions of Delta-like ligand (DLL)-class and JAG-class ligand-receptor interactions, and developed NOTCH decoys that function as ligand-specific NOTCH inhibitors. N110-24 decoy blocked JAG1/JAG2-mediated NOTCH1 signaling, angiogenic sprouting in vitro, and retinal angiogenesis, demonstrating that JAG-dependent NOTCH signal activation promotes angiogenesis. In tumors, N110-24 decoy reduced angiogenic sprouting, vessel perfusion, pericyte coverage, and tumor growth. JAG-NOTCH signaling uniquely inhibited expression of antiangiogenic soluble (s) VEGFR1/sFLT1. N11-13 decoy interfered with DLL1-DLL4-mediated NOTCH1 signaling and caused endothelial hypersprouting in vitro, in retinal angiogenesis, and in tumors. Thus, blockade of JAG- or DLL-mediated NOTCH signaling inhibits angiogenesis by distinct mechanisms. JAG-NOTCH signaling positively regulates angiogenesis by suppressing sVEGFR1-sFLT1 and promoting mural-endothelial cell interactions. Blockade of JAG-class ligands represents a novel, viable therapeutic approach to block tumor angiogenesis and growth. SIGNIFICANCE: This is the first report identifying unique regions of the NOTCH1 extracellular domain that interact with JAG-class and DLL-class ligands. Using this knowledge, we developed therapeutic agents that block JAG-dependent NOTCH signaling and demonstrate for the first time that JAG blockade inhibits experimental tumor growth by targeting tumor angiogenesis.

Endothelial Jagged-1 is necessary for homeostatic and regenerative hematopoiesis.

The bone marrow (BM) microenvironment is composed of multiple niche cells that, by producing paracrine factors, maintain and regenerate the hematopoietic stem cell (HSC) pool (Morrison and Spradling, 2008). We have previously demonstrated that endothelial cells support the proper regeneration of the hematopoietic system following myeloablation (Butler et al., 2010; Hooper et al., 2009; Kobayashi et al., 2010). Here, we demonstrate that expression of the angiocrine factor Jagged-1, supplied by the BM vascular niche, regulates homeostatic and regenerative hematopoiesis through a Notch-dependent mechanism. Conditional deletion of Jagged-1 in endothelial cells (Jag1((ECKO)) mice) results in a profound decrease in hematopoiesis and premature exhaustion of the adult HSC pool, whereas quantification and functional assays demonstrate that loss of Jagged-1 does not perturb vascular or mesenchymal compartments. Taken together, these data demonstrate that the instructive function of endothelial-specific Jagged-1 is required to support the self-renewal and regenerative capacity of HSCs in the adult BM vascular niche.

Notch signaling in developmental and tumor angiogenesis.

The discovery that Notch, a key regulator of cell fate determination, is functional in the vasculature has greatly improved our understanding of differentiation and specialization of vessels. Notch signaling has been proven to be critical for arterial specification, sprouting angiogenesis, and vessel maturation. In newly forming vascular sprouts, Notch promotes the distinction between the leading "tip" endothelial cell and the growing "stalk" cell, the endothelial cells that eventually form a new capillary. Notch signaling has also been implicated in vessel stability by regulating vascular mural cell function. More recently, macrophages carrying an activated Notch have been implicated in shaping the course of new sprout formation. Tumor vessels abide by similar principles and use Notch signaling in similar ways. An exciting discovery, made by several researchers, shows that blocking Notch function in tumor vasculature provides a means by which to suppress tumor growth. The authors discuss the developmental and physiological role of Notch in the vasculature and apply this knowledge to an overview of how Notch targeting in the tumor environment can affect tumor angiogenesis and growth.

Dissociation of the glucose and lipid regulatory functions of FoxO1 by targeted knockin of acetylation-defective alleles in mice.

FoxO1 integrates multiple metabolic pathways. Nutrient levels modulate FoxO1 acetylation, but the functional consequences of this posttranslational modification are unclear. To answer this question, we generated mice bearing alleles that encode constitutively acetylated and acetylation-defective FoxO1 proteins. Homozygosity for an allele mimicking constitutive acetylation (Foxo1(KQ/KQ)) results in embryonic lethality due to cardiac and angiogenesis defects. In contrast, mice homozygous for a constitutively deacetylated Foxo1 allele (Foxo1(KR/KR)) display a unique metabolic phenotype of impaired insulin action on hepatic glucose metabolism but decreased plasma lipid levels and low respiratory quotient that are consistent with a state of preferential lipid usage. Moreover, Foxo1(KR/KR) mice show a dissociation between weight gain and insulin resistance in predisposing conditions (high fat diet, diabetes, and insulin receptor mutations), possibly due to decreased cytokine production in adipose tissue. Thus, acetylation inactivates FoxO1 during nutrient excess whereas deacetylation selectively potentiates FoxO1 activity, protecting against excessive catabolism during nutrient deprivation.