Summary The bone marrow (BM) microenvironment is composed of multiple niche cells that, by producing angiocrine 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, while quantification and functional assays demonstrate that loss ofJagged-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.

Epicardial cells on the heart’s surface give rise to coronary artery smooth muscle cells (caSMCs) located deep in the myocardium. However, the differentiation steps between epicardial cells and caSMCs are unknown as are the final maturation signals at coronary arteries. Here, we use clonal analysis and lineage tracing to show that caSMCs derive from pericytes, mural cells associated with microvessels, and that these cells are present in adults. During development following the onset of blood flow, pericytes at arterial remodeling sites upregulate Notch3 while endothelial cells express Jagged-1. Deletion of Notch3 disrupts caSMC differentiation. Our data support a model wherein epicardial-derived pericytes populate the entire coronary microvasculature, but differentiate into caSMCs at arterial remodeling zones in response to Notch signaling. Our data are the first demonstration that pericytes are progenitors for smooth muscle, and their presence in adult hearts reveals a new potential cell type for targeting during cardiovascular disease.DOI: http://dx.doi.org/10.7554/eLife.10036.001

A pro-angiogenic role for Jagged-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 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 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 anti-angiogenic sVEFGFR-1/sFlt-1. 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 sVEGFR-1/sFlt-1 and promoting mural/endothelial cell interactions. Blockade of JAG-class ligands represents a novel, viable therapeutic approach to block tumor angiogenesis and growth.

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. The role of Notch signaling in macrophages in the developing retina has not previously been assessed.In this study, we show that Notch signaling is important for macrophage recruitment and localization in the developing retina. We also found an increased frequency of elongated sprouts that did not anastomose with neighboring sprouts in retinas in mice with myeloid-specific loss of Notch1. Furthermore, we show Notch signal activation in macrophages that interact with Dll4-positive tip cells, and that macrophages with Notch signaling are found predominately at the vascular front and in association with vascular branchpoints. These data suggest a novel way that Notch signaling regulates retinal angiogenesis. MethodsNotch1 mutant mice have been described. 5 Mice with a conditional allele of Notch1 (Notch1 flox ) 6 and the myeloid-specific Cre recombinase driver line (LysMCre) 7 were obtained from The Jackson Laboratory. Transgenic Notch reporter mice (TNR), harboring an enhanced GFP sequence under the control of 4 tandem copies of the CBF1 binding site consensus sequence 8 were also obtained from The Jackson Laboratory. All procedures were carried out according to approved protocols and guidelines established by the Columbia University Institutional Animal Care and Use Committee. Eyes from postnatal day 5 mice were fixed for 2 hours in 4% paraformaldehyde. Retinas were permeabilized in 1% BSA and 0.5% Triton X-100 overnight at 4°C and washed in PBLEC buffer (1% Triton X-100, 0.1mM MgCl 2 , 0.1mM CaCl 2 , 0.1mM MnCl 2 in PBS pH 6.8), then incubated overnight in PBLEC plus isolectin-B4 (Sigma-Aldrich), anti-F4/80 (Abcam), anti-GFP (Invitrogen), or anti-Dll4 (R&D Systems). After washing, retinas were incubated with Alexa Fluor-conjugated secondary antibodies (Invitrogen), washed, and mounted on slides with Vectashield (Dako) mounting medium for visualization using a LSM Meta 510 or Nikon A1R MP Multiphoton confocal microscope. Results/discussionPrevious studies have shown that loss of Dll4 in mice leads to excessive sprouting during retinal angiogenesis. 3,4 We first investigated whether decreased expression of Notch1 would lead to a similar defect. We found increased vascular density in retinas from Notch1 ϩ/Ϫ mice compared with control littermates ( Figure 1A,C and data not shown), supporting the model where Notch1 and Dll4 in endothelial cells regulate sprouting. Because macrophages are present in the retina and are known to express Notch1, 9,10 we assessed macrophage r...

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.