A population of monocytes, known as Ly6Clo monocytes, patrol blood vessels by crawling along the vascular endothelium. Here we show that endothelial cells control their origin through Notch signalling. Using combinations of conditional genetic deletion strategies and cell-fate tracking experiments we show that Notch2 regulates conversion of Ly6Chi monocytes into Ly6Clo monocytes in vivo and in vitro, thereby regulating monocyte cell fate under steady-state conditions. This process is controlled by Notch ligand delta-like 1 (Dll1) expressed by a population of endothelial cells that constitute distinct vascular niches in the bone marrow and spleen in vivo, while culture on recombinant DLL1 induces monocyte conversion in vitro. Thus, blood vessels regulate monocyte conversion, a form of committed myeloid cell fate regulation.

Ischemia causes an inflammatory response that is intended to restore perfusion and homeostasis yet often aggravates damage. Here we show, using conditional genetic deletion strategies together with adoptive cell transfer experiments in a mouse model of hind limb ischemia, that blood vessels control macrophage differentiation and maturation from recruited monocytes via Notch signaling, which in turn promotes arteriogenesis and tissue repair. Macrophage maturation is controlled by Notch ligand Dll1 expressed in vascular endothelial cells of arteries and requires macrophage canonical Notch signaling via Rbpj, which simultaneously suppresses an inflammatory macrophage fate. Conversely, conditional mutant mice lacking Dll1 or Rbpj show proliferation and transient accumulation of inflammatory macrophages, which antagonizes arteriogenesis and tissue repair. Furthermore, the effects of Notch are sufficient to generate mature macrophages from monocytes ex vivo that display a stable anti-inflammatory phenotype when challenged with pro-inflammatory stimuli. Thus, angiocrine Notch signaling fosters macrophage maturation during ischemia.

Key Words: blood vessels Ⅲ development Ⅲ mouse, mutant strains Ⅲ branching Ⅲ tip cells Ⅲ Delta-like ligand 1 Ⅲ Notch B lood vessels form 3-dimensional branched networks whose architecture is tailored to serve specific physiological functions in different organs. 1 Retinal angiogenesis begins at birth with the formation of a superficial primary plexus (PP), which spreads by angiogenic sprouting from the center to the periphery on the surface of the ganglion cell layer. Subsequently, vessels branch vertically into the deeper layers of the retina to establish a secondary plexus, which supplies the retinal neurons. 2 During PP development, single endothelial cells (ECs) specialize into motile endothelial tip cells that locate at the leading edge of growing angiogenic sprouts and contribute to vessel branching by sensing guidance cues, such as vascular endothelial growth factor (VEGF) gradients, in the extracellular milieu. 3 Tip cell formation in the sprout is tightly regulated by the Notch signaling pathway, a cell contact-dependent signaling mechanism activated by binding of Notch receptors to ligands of the conserved Delta-like (Dll) and Jagged (Jag) gene families ( Figure 4E). 4 The ligand Dll4 is upregulated by VEGF in selected ECs in the angiogenic front, which behave as tip cells and activate Notch receptors in adjacent stalk ECs, thereby suppressing a tip cell phenotype and angiogenic sprouting. In contrast, the ligand Jag1 is strongly expressed in stalk ECs and antagonizes Dll4 function and tip cell suppression, which shows that endothelium-intrinsic Notch signaling regulates tip cell formation. 5 However, induction of Dll4 by VEGF is Notch dependent, which raises the possibility that extrinsic Notch ligands contribute to tip cell selection. 6 The Notch ligand Dll1Original received December 19, 2011; revision received January 4, 2012; accepted January 17, 2012. In December 2011, the average time from submission to first decision for all original research papers submitted to Circulation Research was 14.29 days.Brief UltraRapid Communications are designed to be a format for manuscripts that are of outstanding interest to the readership, report definitive observations, but have a relatively narrow scope. In This Issue, see p 523 MethodsVertical branching was studied in isolectin B4 -stained frontal cross sections from the posterior parts of the retina from isogenic 129S1/ SvImJ-background littermates. Detailed Methods are discussed in the online-only Data Supplement. ResultsIn mice with heterozygous Dll1 loss of function, which showed no overt developmental or postnatal phenotype (Online Figure I), tip cell numbers and plexus complexity were decreased significantly ( Figure 1A), and expansion of the PP was partially disturbed ( Figure 1B). Analysis of deep plexus angiogenesis in retina cross sections revealed that vertical branch development was delayed and strongly reduced in Dll1 ϩ/Ϫ retinas at all neonatal stages investigated ( Figures 1C and 1D). Furthermore, expansion of the deep plexus was also impa...

Arteriogenesis, the growth of natural bypass arteries, is triggered by hemodynamic forces within vessels and requires a balanced inflammatory response, involving induction of the chemokine MCP-1 and recruitment of leukocytes. However, little is known how these processes are coordinated. The MAP-kinase-activated-proteinkinase-2 (MK2) is a critical regulator of inflammatory processes and might represent an important link between cytokine production and cell recruitment during postnatal arteriogenesis. Therefore, the present study investigated the functional role of MK2 during postnatal arteriogenesis. In a mouse model of hindlimb ischemia (HLI) MK2-deficiency (MK2KO) significantly impaired ischemic blood flow recovery and growth of collateral arteries as well as perivascular recruitment of mononuclear cells and macrophages. This was accompanied by induction of endothelial MCP-1 expression in wildtype (WT) but not in MK2KO collateral arteries. Following HLI, MK2 activation rapidly occured in the endothelium of growing WT arteries in vivo. In vitro, inflammatory cytokines and cyclic stretch activated MK2 in endothelial cells, which was required for stretch- and cytokine-induced release of MCP-1. In addition, a monocyte cell autonomous function of MK2 was uncovered potentially regulating MCP-1-dependent monocyte recruitment to vessels: MCP-1 stimulation of WT monocytes induced MK2 activation and monocyte migration in vitro. The latter was reduced in MK2KO monocytes, while in vivo MK2 was activated in monocytes recruited to collateral arteries. In conclusion, MK2 regulates postnatal arteriogenesis by controlling vascular recruitment of monocytes/macrophages in a dual manner: regulation of endothelial MCP-1 expression in response to hemodynamic and inflammatory forces as well as MCP-1 dependent monocyte migration.

Regeneration of arterial endothelium after injury is critical for the maintenance of normal blood flow, cell trafficking, and vascular function. Using mouse models of carotid injury, we show that the transition from a static to a dynamic phase of endothelial regeneration is marked by a strong increase in endothelial proliferation, which is accompanied by induction of the chemokine CX 3 CL1 in endothelial cells near the wound edge, leading to progressive recruitment of Ly6Clo monocytes expressing high levels of the cognate CX 3 CR1 chemokine receptor. In Cx3cr1‐deficient mice recruitment of Ly6Clo monocytes, endothelial proliferation and regeneration of the endothelial monolayer after carotid injury are impaired, which is rescued by acute transfer of normal Ly6Clo monocytes. Furthermore, human non‐classical monocytes induce proliferation of endothelial cells in co‐culture experiments in a VEGFA‐dependent manner, and monocyte transfer following carotid injury promotes endothelial wound closure in a hybrid mouse model in vivo. Thus, CX 3 CR1 coordinates recruitment of specific monocyte subsets to sites of endothelial regeneration, which promote endothelial proliferation and arterial regeneration.