Retinal myeloid cells regulate tip cell selection and vascular branching morphogenesis via Notch ligand Delta-like 1.

During angiogenesis, single endothelial cells (EC) specialize into tip cells that guide vessel sprouting towards growth factor gradients and instruct the adjacent vessel stalk. The balance between tip and stalk cells is regulated by endothelial Notch signalling through the expression of Notch ligand Delta-like 4 (Dll4) in tip cells, which suppresses a tip cell fate in adjacent stalk cells. Here we show, using genetic reporter and conditional deletion strategies, that myeloid cells regulate tip cell numbers and Dll4 expression via the Notch ligand Dll1 during vascular development in the retina. Dll1 is selectively expressed by a subpopulation of retinal myeloid cells, which progressively localizes to the sprouting vascular network. Conditional, myeloid-specific deletion of Dll1 impairs endothelial Dll4 tip-stalk gradient resulting in an increase of endothelial tip cells and EC filopodia, accompanied by an increase in vascular density and branching. In vitro, co-culture of human EC with monocyte-derived macrophages induced Dll1 upregulation in macrophages and Dll4 upregulation and an endothelial tip cell signature in EC. Furthermore, culturing human EC on recombinant DLL1 induced endothelial Dll4 expression and a tip cell program, indicating that changes are Dll1-dependent. Thus, myeloid cells regulate tip cell fate and angiogenesis through expression of Notch ligand Dll1.

Multimodal and Multiscale Analysis Reveals Distinct Vascular, Metabolic and Inflammatory Components of the Tissue Response to Limb Ischemia.

Ischemia triggers a complex tissue response involving vascular, metabolic and inflammatory changes. METHODS: We combined hybrid SPECT/CT or PET/CT nuclear imaging studies of perfusion, metabolism and inflammation with multicolor flow cytometry-based cell population analysis to comprehensively analyze the ischemic tissue response and to elucidate the cellular substrate of noninvasive molecular imaging techniques in a mouse model of hind limb ischemia. RESULTS: Comparative analysis of tissue perfusion with [99mTc]-Sestamibi and arterial influx with [99mTc]-labeled albumin microspheres by SPECT/CT revealed a distinct pattern of response to vascular occlusion: an early ischemic period of matched suppression of tissue perfusion and arterial influx, a subacute ischemic period of normalized arterial influx but impaired tissue perfusion, and a protracted post-ischemic period of hyperdynamic arterial and normalized tissue perfusion, indicating coordination of macrovascular and microvascular responses. In addition, the subacute period showed increased glucose uptake by [18F]-FDG PET/CT scanning as the metabolic response of viable tissue to hypoperfusion. This was associated with robust macrophage infiltration by flow cytometry, and glucose uptake studies identified macrophages as major contributors to glucose utilization in ischemic tissue. Furthermore, imaging with the TSPO ligand [18F]-GE180 showed a peaked response during the subacute phase due to preferential labeling of monocytes and macrophages, while imaging with [68Ga]-RGD, an integrin ligand, showed prolonged post-ischemic upregulation, which was attributed to labeling of macrophages and endothelial cells by flow cytometry. CONCLUSION: Combined nuclear imaging and cell population analysis reveals distinct components of the ischemic tissue response and associated cell subsets, which could be targeted for therapeutic interventions.

The chemokine receptor CX3CR1 coordinates monocyte recruitment and endothelial regeneration after arterial injury.

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 CX3CL1 in endothelial cells near the wound edge, leading to progressive recruitment of Ly6Clo monocytes expressing high levels of the cognate CX3CR1 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, CX3CR1 coordinates recruitment of specific monocyte subsets to sites of endothelial regeneration, which promote endothelial proliferation and arterial regeneration.

Blood vessel control of macrophage maturation promotes arteriogenesis in ischemia.

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.Molecular mechanisms of macrophage-mediated regulation of artery growth in response to ischemia are poorly understood. Here the authors show that vascular endothelium controls macrophage maturation and differentiation via Notch signaling, which in turn promotes arteriogenesis and ischemic tissue recovery.

Regulation of monocyte cell fate by blood vessels mediated by Notch signalling.

A population of monocytes, known as Ly6C(lo) 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 Ly6C(hi) monocytes into Ly6C(lo) 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.

MAP-Kinase Activated Protein Kinase 2 Links Endothelial Activation and Monocyte/macrophage Recruitment in Arteriogenesis.

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.